Oxetanyl Amino Acids for Peptidomimetics

Article abstract of DOI:10.1021/acs.orglett.7b00745

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.

Full text of DOI:10.1021/acs.orglett.7b00745

Letter
pubs.acs.org/OrgLett
Oxetanyl Amino Acids for Peptidomimetics
Guido P. Moller,† Steffen Muller,† Bernd T. Wolfstadter,†,‡ Susanne Wolfrum,† Dirk Schepmann,§
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Bernhard Wunsch,§ and Erick M. Carreira*,†
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†Laboratorium fur Organische Chemie, ETH Zurich, Vladimir-Prelog-Weg 3, 8093 Zurich, Switzerland
‡Competence Center for Systems Physiology and Metabolic Diseases, Schorenstrasse 16, 8603 Schwerzenbach, Switzerland
§Institut fur Pharmazeutische und Medizinische Chemie, WWU Munster, Corrensstrasse 48, 48149 Munster, Germany
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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.
eptides and protein-based drugs currently hold a 10%
Pestimated 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 develop-
ment 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
Figure 1. (A) 3-Aminooxetanes as amide mimics; (B) oxetanyl
dipeptides as Leu-enkephalin analogues with affinity for the rat δ-
opioid receptor; see the Supporting Information.
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
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
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
Received: March 13, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.7b00745
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Letter
of the H-bond donor/H-bond acceptor properties of the amide
bond (Figure 1A).9 Moreover, the replacement of a peptide
CO 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 pepti-
des,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.
Scheme 1. Synthesis of Oxetanyl Diamines 9−17
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
aminooxetane building blocks. Analogues of L-alanine (15), L-
leucine (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 D-
amino 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
B
DOI: 10.1021/acs.orglett.7b00745
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Organic Letters
Letter
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
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
a
Table 1. Synthesis of Dipeptidic Building Blocks
amine
X
R2
R3
yield/%
9
Br
H
Bn
Bn
Bn
Bn
tBu
Bn
tBu
Me
Me
Bn
Bn
Me
Bn
Bn
Bn
Bn
Et
90
62
10
10
10
11
11
11
12
13
14
15
16
18
18
18
20
21
21
OTf
OTf
OTf
Br
iBu
b
(S)-sec-Bu
40
Bn
67
98
52
50
87
72
79
H
iBu
iBu
OTf
OTf
Br
■
●
;
Figure 3. Stability of Leu-enkephalin (2a, ) and its analogues (2b,
⧫
▲
▼
2c, ; 2d, ; 2e, ) in human serum. Relative concentration as mean
H
SEM against time is displayed.
Br
H
OTf
OTf
OTf
Br
Me
iPr
b
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 half-
lives. 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.
56
CH2(C6H4)-4-OCbz
73
75
87
78
39
60
87
H
OTf
OTf
OTf
OTf
Br
Bn
Me
Bn
Bn
H
Bn
a
Conditions: oxetane (1.0 equiv), triflate or bromide (2.0 equiv),
b
iPr2NEt (2.2 equiv), 60 °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 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 nano-
We next investigated the incorporation of the novel
dipeptidomimetic building blocks into larger peptides. The δ-
opioid 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. Leu-
enkephalin (2a) is an efficient analgesic with affinity for the μ-
and δ-opioid receptors (δ>μ).17 In total, four analogues (2b−e)
molar 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
C
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Organic Letters
Letter
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 heat-
exposure 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.
Zurich Postdoctoral Fellowship Program and the Marie Curie
Actions for People COFUND Program for a scholarship.
̈
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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 Leu-
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* Supporting Information
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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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AUTHOR INFORMATION
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Corresponding Author
*E-mail: carreira@org.chem.ethz.ch.
ORCID
Erick M. Carreira: 0000-0003-1472-490X
Notes
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The authors declare no competing financial interest.
́
ACKNOWLEDGMENTS
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E. Pharmacology 2003, 67, 6.
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We thank ETH Zurich (Grant No. ETH-15 10-2) for financial
̈
(21) Half-life times were estimated by an exponential decay fit with
support. We are grateful to Dr. B. Schweizer, Dr. N. Trapp, and
c(t) = c(0)e−λt
.
M. Solar of the ETH-Zurich/DCHAB Small Molecule
̈
(22) Affinity values are given as mean SEM (n = 3).
Crystallography Center for X-ray analysis. We acknowledge B.
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107, 385. (b) Dutta, A. S.; Gormley, J. J.; Hayward, C. F.; Morley, J. S.;
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Frehland (WWU Munster) for technical assistance with the
̈
affinity assay. Dr. D. Peleg-Raibstein (Laboratory of Transla-
tional Nutrition Biology/ETH Zurich) is kindly acknowledged
̈
for assistance with the hot-plate test. S.M. thanks the ETH
D
DOI: 10.1021/acs.orglett.7b00745
Org. Lett. XXXX, XXX, XXX−XXX