Biaryl-bridged macrocyclic peptides: Conformational constraint via carbogenic fusion of natural amino acid side chains

Article abstract of DOI:10.1021/jo202105v

A general method for constraining peptide conformations via linkage of aromatic sidechains has been developed. Macrocyclization of suitably functionalized tri-, tetra- and pentapeptides via Suzuki-Miyaura cross-coupling has been used to generate side chain to side chain, biaryl-bridged 14- to 21-membered macrocyclic peptides. Biaryl bridges possessing three different configurations, meta-meta, meta-ortho, and ortho-meta, were systematically explored through regiochemical variation of the aryl halide and aryl boronate coupling partners, allowing fine-tuning of the resultant macrocycle conformation. Suzuki-Miyaura macrocyclizations were successfully achieved both in solution and on solid phase for all three sizes of peptide. This approach constitutes a means of constraining peptide conformation via direct carbogenic fusion of side chains of naturally occurring amino acids such as phenylalanine and tyrosine, and so is complementary to strategies involving non-natural, for example, hydrocarbon, bridges.

Full text of DOI:10.1021/jo202105v

Article
pubs.acs.org/joc
Biaryl-Bridged Macrocyclic Peptides: Conformational Constraint via
Carbogenic Fusion of Natural Amino Acid Side Chains
Falco-Magnus Meyer,† James C. Collins,† Brendan Borin,† James Bradow,§ Spiros Liras,§
Chris Limberakis,§ Alan M. Mathiowetz,§ Laurence Philippe,§ David Price,§ Kun Song,§ and Keith James*,†
†Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States
§Pfizer Worldwide R&D, Eastern Point Road, Groton, Connecticut 06340, United States
S
* Supporting Information
ABSTRACT: A general method for constraining peptide
conformations via linkage of aromatic sidechains has been
developed. Macrocyclization of suitably functionalized tri-,
tetra- and pentapeptides via Suzuki−Miyaura cross-coupling
has been used to generate side chain to side chain, biaryl-
bridged 14- to 21-membered macrocyclic peptides. Biaryl
bridges possessing three different configurations, meta−meta,
meta−ortho, and ortho−meta, were systematically explored through regiochemical variation of the aryl halide and aryl boronate
coupling partners, allowing fine-tuning of the resultant macrocycle conformation. Suzuki−Miyaura macrocyclizations were
successfully achieved both in solution and on solid phase for all three sizes of peptide. This approach constitutes a means of
constraining peptide conformation via direct carbogenic fusion of side chains of naturally occurring amino acids such as
phenylalanine and tyrosine, and so is complementary to strategies involving non-natural, for example, hydrocarbon, bridges.
these cases, it is typically not envisaged that the newly created
bridge is part of the bioactive peptide pharmacophore, but
rather a means of forming the macrocyclic ring, thereby
influencing the conformation of a peptidic region elsewhere in
the macrocycle.
INTRODUCTION
■
Peptide hormones play a key role in mammalian regulatory
processes, and so in principle represent attractive points of
therapeutic intervention in dysregulated biological systems.1
However, their poor pharmacokinetic properties usually limit
their direct utility as therapeutic agents.2 Consequently,
strategies for stabilizing the bioactive conformation of
therapeutically important peptides, while limiting their
metabolic clearance, are of considerable interest.3 It has been
known for many years that macrocyclic peptides can exhibit
improved pharmacological and pharmacokinetic properties over
their acyclic counterparts.4 These advantages stem from the
conformational preorganization imposed by the macrocyclic
framework, which can be exploited in stabilizing the bioactive
peptide conformation and reducing susceptibility to protease
cleavage. Multiple opportunities for the macrocyclization of
linear peptides can be envisaged,5 involving linkages between
N- and C-termini, between termini and side chains, or between
side chains. The latter approach has the advantage of not
disrupting potential interactions between the N- or C-termini
and the target receptor. These side chain to side chain macro-
cyclization strategies have been widely explored and can be
used to stabilize specific conformational motifs such as α-
helices.6
As a result of our interest in bioactive peptides such as
glucagon-like peptide 1 (GLP-1),7 somatostatin,8 and the
enkephalins,9 all of which feature noncontiguous aromatic
amino acids which are potentially proximal in space, as
illustrated in 1, we envisaged a complementary peptide
macrocyclization strategy, whereby side chain to side chain
bridges comprised of naturally occurring amino acids were an
integral component of the bioactive pharmacophore. Thus, the
resultant biaryl-bridged macrocyclic peptides, such as 2, would
possess both a constrained peptide backbone and a
preorganized lipophilic, aromatic region for potential inter-
action with the relevant receptor. Intriguingly, biaryl peptide
motifs such as these are found widely in biologically active
natural products,10 such as the biphenomycins (e.g., 3),11
arylomycins (e.g., 4),12 and RP 66453 (5),13 supporting our
hypothesis that the profile of biologically active peptides could
be modulated through this type of ‘natural side-chain bridging’
strategy. We therefore chose to develop a flexible synthetic
approach to the construction of biaryl-bridged peptide
macrocycles, which would in due course allow systematic ex-
ploration of this approach to the constraint of bioactive peptides.
Since, in principle, all three of the unsubstituted positions on
the aromatic ring of a phenylalanine side chain (o-, m-, and p-),
Common synthetic strategies for generating macrocyclic
peptides via side chain to side chain linkages have included:
ring closing olefin metathesis (RCM) reactions between side
chains bearing terminal alkene groups;6a amide-coupling
reactions, for example between lysine and aspartic acid;6b and
copper-catalyzed azide−alkyne cycloaddition (CuAAC) reac-
tions between alkyne- and azide-substituted side chains.6c In
Received: October 14, 2011
Published: February 21, 2012
© 2012 American Chemical Society
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and either of the unsubstituted positions on the aromatic ring
of a tyrosine side chain (o- and m-) could be linked to a second
aromatic amino acid side chain, there are a number of possible
configurations of such a biaryl-bridge. All three natural product
classes referred to above feature a m,m-biaryl bridge on a
tripeptide backbone. Since modeling studies suggested that
changes in configuration of the biaryl bridge (and hence
macrocycle ring size) would have a marked effect on peptide
conformation, we saw a benefit in extending the bridge
permutations beyond the m,m-systems found in these natural
products to include also m,o- and o,m-bridged systems. In
addition, since pairs of aromatic amino acid residues are present
in the peptide hormones of interest at i/i + 2, i/i + 3, and i/i + 4
positions on the peptide chain, we sought approaches to con-
structing macrocycles of each bridge configuration for tri-,
tetra-, and pentapeptide backbones (6) employing alanines as
intervening amino acid units for simplicity.
It is known that introduction of additional substituents at the
α-position of amino acids can bias conformation by restricting
access to regions of the Ramachandran Φ/Ψ dihedral surface.14
We therefore wanted to ensure that any synthetic methodology
we developed would be compatible with such substitution
patterns. Consequently, we incorporated into our program
selected examples of α-methylated, that is, quaternary, amino
acids. By virtue of their i/i + 4 biaryl-bridges, we recognized the
possibility that the proposed pentapeptide systems had the
potential for helical conformations, by analogy with a number
of reported helix stabilization approaches.6 Finally, we also
wanted to demonstrate that substituents could be incorporated
successfully into either of the bridging aromatic rings, for
example, p-hydroxy-substituents to mimic tyrosine residues.
We envisaged that closure of the macrocyclic ring via
Suzuki−Miyaura cross-coupling reaction between a borylated
phenylalanine and an appropriately placed halogenated phenyl-
alanine residue would provide the most flexible strategy for
constructing libraries of biaryl-bridged systems.15 The requisite
macrocyclization precursors could thus be constructed either in
solution or on solid phase by standard peptide coupling appro-
aches. Key to our strategy is our recently reported methodology
using iridium-catalyzed borylation chemistry on substituted
phenylalanines to form the corresponding arylboronates.16 We
therefore anticipated having ready access to the necessary
regiochemical variants of borylated and halogenated phenyl-
alanine derivatives, either from our methodology, from Miyaura
borylation of halogenated phenylalanine derivatives, or from
commercial sources. Herein, we report the realization of this
strategy and the successful construction of a diverse set of
biaryl-bridged macrocyclic peptides. During the course of our
program, a related and complementary study was reported.17
RESULTS AND DISCUSSION
■
1. Solution-Phase Synthesis of m,m-Bridged Biaryl
Macrocyclic Peptides. We elected to explore the synthesis of
meta−meta-bridged systems initially via a solution-phase
synthesis, in order that we could determine the optimum
conditions for the key Suzuki−Miyaura cross-coupling, without
the reaction-monitoring complications associated with resin-
bound substrates and products. Furthermore, we chose to
construct macrocyclic tri-, tetra-, and pentapeptides which
represented a carbogenic fusion of a phenylalanine at the i
position with a tyrosine at the i + 2, i + 3, and i + 4 positions,
respectively, since these coupling reactions entailed use of an
o-substituted aryl halide; we presumed that optimized condi-
tions for these systems would then be generally applicable to un-
hindered systems. For consistency, we decided to incorporate
an N-terminal acetyl and a C-terminal primary amide in all the
macrocyclic peptides we synthesized.
The synthesis of the m,m-bridged tripeptide macrocycle is
outlined in Scheme 1a. The m-borylated phenylalanine
derivative 7 can be prepared regioselectively on a 20 g scale
using our previously described methodology.16 This could be
hydrolyzed selectively to yield the carboxylic acid 8, for
coupling with the appropriate peptide fragment. Iodotyrosine 9
was protected as its O-benzyl ether methyl ester 10, and then
coupled to Boc-(L)-alanine to yield the dipeptide 11.
Transformation of this key intermediate to the macro-
cyclization substrate was accomplished by deprotection to
yield hydrochloride 12, followed by coupling with 8 to yield
tripeptide 13. This material proved unstable toward chroma-
tography, and so was submitted directly to the Suzuki−Miyaura
macrocyclization. A screen of conditions for this reaction
demonstrated that use of Pd(dppf)Cl2·CH2Cl2 as catalyst,
together with CsF as base in degassed dioxane, yielded
optimum results. Thus, after heating at 90 °C for 18 h, at a
concentration of 0.02 M, the macrocycle 14 was isolated in 60%
yield for the three steps from 11. Macrocycle 14 was converted
to the corresponding acetamide 15, which yielded the desired
product 16 in a one-pot procedure involving hydrogenolytic
cleavage of the chloro-substituent and benzyl protecting group,
hydrolysis of the methyl ester, and amide formation.
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Scheme 1. Solution-Phase Synthesis of (a) m,m-Bridged Biaryl Tripeptide Macrocycles and (b) m,m-Bridged Biaryl Tetra- and
a
Pentapeptide Macrocycles
a
Reaction conditions: (a) LiOH·H2O (aq), MeOH, rt, 40 min (aq); (b) NaOH (aq), CuSO4 (aq), MeOH (aq), 60 °C, 10 min, BnBr, 12 h; (c)
SOCl2, MeOH, rt, 2 h; (d) Boc-(L)-alanine, PyBOP, NEt3, rt, 3 h; (e) HCl, dioxane, rt, 5 h; (f) PyBOP, DIPEA, DMF, rt, 12 h; (g)
Pd(dppf)Cl2·CH2Cl2, CsF (aq), dioxane 90 °C, 18 h; (h) HCl, dioxane, rt, 5 h; (i) Ac2O, DIPEA, DMF, rt, 12 h; (j) Pd(OH)2/C, NH4OH (aq) H2,
40 °C, 12 h, then LiOH·H2O (aq), MeOH, rt, 5 h, then PyBOP, NH3, DMF, CH2Cl2, rt, 5 h.
Several aspects of this synthetic sequence are noteworthy.
The chloro-substituent carried through the synthesis had served
in effect as a regiochemical directing group, ensuring selective
borylation to yield the requisite 3,5-disubstituted phenylalanine 7,
but not appearing in the product 16. However, it also proved
possible to conduct a selective hydrogenolysis of the benzyl
protecting group in macrocycle 14, thereby retaining the
chloro-substituent as a point of future diversification (results
not shown). Additionally, although the product of the
macrocyclization, 14, was converted to the simple N-acetyl,
primary amide derivative 16, its protection regime renders it
suitable for embedding within larger peptide sequences as a
conformational constraint element.
The corresponding biaryl-bridged tetra- and pentapeptide
macrocycles were prepared using an analogous synthetic
sequence, as depicted in Scheme 1b. Coupling of amine
hydrochloride 12 to a further Boc-(L)-alanine fragment yielded
tripeptide 17, which, following deprotection to yield the amine
hydrochloride 18, could be coupled with 8 to yield the
tetrapeptide macrocyclization precursor 19. This was again
subjected directly to the optimized Suzuki−Miyaura conditions
at 0.02 M concentration to yield macrocycle 20 in 51% yield
over the three steps from 17. Conversion to the macrocycle
product was achieved via formation of the N-terminal
acetamide 21, and then one-pot deprotection/amidation to
yield 22. This compound was observed to exist as a 1.0:0.4
mixture of conformers by NMR, reflecting the conformational
restraint imposed by the macrocyclic ring. Similarly, the
tripeptide hydrochloride 18 could be intercepted and converted
to the tetrapeptide 23, which yielded the pentapeptide
macrocycle precursor 25 following deprotection to 24 and
coupling to 12. In this case, macrocyclization yielded the 21-
membered system 26 in 34% overall yield from 23, which could
be converted to the N-terminal acetamide 27 and then to the
C-terminal primary amide 28. In this case, the molecule existed
1
as a 1.0:0.2 mixture of conformers by H NMR.
Although this solution-phase approach proved successful and
allowed straightforward optimization of the macrocyclization
step, the synthesis of these macrocyclic peptides proved
challenging from a practical standpoint. Thus, the routes
involved multiple purification steps, and the yields of the final
steps were variable, primarily because of the lower solubility of
these larger systems and the attendant difficulties in purifying
them chromatographically. Consequently, for the other
members of the library, we decided to adopt a solid-phase
synthesis strategy, whereby the Suzuki−Miyaura macrocycliza-
tion step would be conducted on a resin-bound substrate.
2. Solid-Phase Synthesis of m,m-Bridged Biaryl
Macrocyclic Pentapeptides. The synthesis of a prototypical
m,m-biaryl bridged pentapeptide is shown in Scheme 2. MBHA
resin was used together with a Boc-protection strategy, so that
release of the macrocyclic product from the resin would yield a
C-terminal primary amide directly. The m-borylated phenyl-
alanine derivative 8 was incorporated into the peptide chain
and the resultant pentapeptide capped with an acetyl group to
yield the solid phase-supported (SPS) substrate 29. This was
subjected to Suzuki−Miyaura macrocyclization under essen-
tially the same conditions as the analogous solution phase-
reaction. In the absence of a straightforward means of
monitoring reaction progress, the formation of resin-bound
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Scheme 2. Solid-Phase Synthesis of m,m-Bridged Biaryl
Macrocyclic Peptides
Table 1. Examples of m,m-Bridged Biaryl Macrocyclic
Pentapeptides
a
macrocyclic peptides 33 and 34 containing an additional α-
methyl group at the i + 2 and i + 1 positions, respectively,
yielded two isomeric products in each case, which were
separable by HPLC. To determine whether these isomer pairs
were diastereoisomers (resulting from epimerization of a
stereocenter during the synthesis) or atropisomers (resulting
from conformational constriction and therefore inability to
undergo conformational exchange at room temperature), each
product was subjected to a variable temperature NMR study.
Thus, NMR spectra were obtained for each isomer in both pairs
(33a and 33b, and 34a and 34b) in d6-DMSO at 400 MHz,
over the temperature range from 30 to 110 °C, in 20 °C
increments. A final spectrum was obtained after the temper-
ature had returned to 30 °C. In all cases, the final spectrum at
30 °C, after heating, was identical to the original spectrum at
30 °C, before heating, indicating that there had been no
interconversion between isomers within each pair. Since it
seems unlikely that atropisomers would be resistant to inter-
conversion at 110 °C, we concluded that the isomer pairs are
diastereoisomers, resulting from an epimerization during one of
the coupling steps in the solid-phase synthesis.
To prepare macrocycle 35, featuring a novel, quaternary
amino acid which placed an additional methyl group at the α-
carbon of the i position in the peptide, it was necessary to
generate the novel boronic acid 36, which was prepared via the
route shown in Scheme 3. This sequence is based upon an
established method for constructing homochiral, quaternary
amino acids,18 which utilized a suitably protected (S)-alanine
derivative 37, from which the homochiral oxazolidinone 38 can
be prepared. We adapted this approach by alkylating 38 to yield
the 3-iodobenzyl substituted system 39, which could be cleaved
with potassium trimethylsilanolate,19 to yield the quaternary
amino acid 40, with the correct stereochemical configuration.
Amino acid 40 was then converted to amido methyl ester 41,
which was subjected to a Miyaura borylation to yield 42.
Hydrolysis of 42 delivered the requisite boronic acid 36, which
was used directly in the peptide synthesis.
a
Reaction conditions: (a) N-Boc-(L)-3-bromophenylalanine, PyBOP,
HOAt, DIPEA, DMF, rt, 90 min; (b) TFA, DCM, 1 × 5 min, 1 ×
20 min, rt; (c) N-Boc-(L)-alanine, PyBOP, HOAt, DIPEA, DMF, rt,
90 min; (d) repeated deprotection and amino acid coupling; (e) Ac2O,
DIPEA, DMF; (f) Pd(OAc)2, dppf, dioxane, CsF, H2O, 90 °C, 16 h;
(g) pentamethylbenzene, TFA, HBr, rt, 2 h.
macrocycle 30 was presumed to be complete in a comparable
reaction time. The product was cleaved from the resin using a
TFA/HBr mixture and purified by HPLC to yield the desired
macrocycle 31 in 12.5% overall yield, based upon the theo-
retical maximum resin loading.
We were pleased to confirm that the macrocyclization
reaction could be accomplished on an SPS-substrate, as was
demonstrated by Planas and colleagues.17 As illustrated,
bromoaryl as well as iodoaryl systems also underwent macro-
cyclization. Consequently, having confirmed that the approach
was viable, we adopted SPS-synthesis as our standard strategy
for constructing these biaryl-peptide macrocycles. Since we had
previously demonstrated the ability to remove the chloro-
substituent by hydrogenolysis, no further chemistry was
conducted on the macrocycle product 31. To demonstrate
that this SPS−Suzuki−Miyaura macrocyclization strategy was
compatible with other conformational constraint elements, we
prepared a series of macrocyclic peptides, 32−35, bearing
additional methyl substituents at i + 4, i + 3, i + 2, and i + 1
positions, respectively, as shown in Table 1.
We did not prepare the final potential member of the series,
which would possess an α-methyl group at the i + 4 position of
the peptide, but based upon the results with other members of
the series, we are confident that this would be accessible if
required.
Macrocycles 32−34 were accessible via introduction of Boc-
protected aminoisobutyric acid (Aib) units at the appropriate
point in the sequence depicted in Scheme 2. The syntheses of
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a
As with the m,m-bridged series, we wanted to establish
whether it was also possible to incorporate substituents at the
α-position of selected amino acid units, in order to further
constrain peptide conformation. However, this route failed to
deliver any products when α-methyl substituents were
incorporated at the i and i + 4 positions. At this juncture, we
were uncertain whether the principal issue was an inability to
close the macrocyclic ring, or failure to cleave the product from
the resin under the harsh conditions employed. To better
understand this issue, we decided to adopt an Fmoc-based SPS-
strategy instead, since this offered a much milder resin cleavage
regime.
Scheme 3. Synthesis of Quaternary Amino Acid 36
4. Solid-Phase Synthesis of m,o-Biaryl-Bridged Macro-
cyclic Pentapeptides Using an Fmoc-Protection Strategy.
The revised synthetic sequence is illustrated in Scheme 4.
a
Reaction conditions: (a) (dimethoxymethyl)benzene, ZnCl2, SOCl2,
THF, 0 °C, 4 h; (b) 3-iodo-benzyl bromide, LiHMDS, THF, −30 °C,
1 h; (c) KOSiMe3, THF, 75 °C, 2.5 h; (d) SOCl2, MeOH, 0−25 °C,
2.5 h, then Ac2O, DIPEA, DMAP, DMF, 0−25 °C, 12 h; MeONa,
MeOH, reflux, 3 h; (e) B2pin2, Pd(dppf)Cl2·CH2Cl2, KOAc, degassed
DMSO, 85 °C, 6 h; (f) LiOH·H2O (aq), MeOH, rt, 12 h; product
used directly in next step.
Scheme 4. Solid-Phase Synthesis of m,o-Bridged
Biaryl Macrocyclic Peptides Using an Fmoc-Protection
a
Strategy
3. Solid-Phase Synthesis of m,o-Biaryl-Bridged Macro-
cyclic Pentapeptides Using a Boc-Protection Strategy.
An analogous synthetic strategy was initially adopted for synthesis
of the second series of macrocyclic peptides containing a
meta,ortho-configuration at the biaryl bridge. The synthetic
approach described in Scheme 2 was modified accordingly, by
loading the resin with N-Boc-(L)-2-iodophenylalanine. This
permitted the synthesis of the biaryl bridged macrocyclic tri-,
tetra-, and pentapeptides 43, 44, and 45, respectively. By virtue of
the m,o-configuration, these systems possess a macrocyclic ring
which is one atom smaller than the m,m-series, which represents a
significant increase in strain for the 14-membered macrocyclic
tripeptide 43. Furthermore, the macrocyclization reaction entails a
more sterically hindered coupling reaction. Nevertheless, it is still
apparently possible to close these macrocycles using this SPS−
Suzuki−Miyaura methodology. Compound 44 was not isolated,
but the C-terminal carboxylic acid was isolated instead, presumably
due to an unexpected hydrolysis during the cleavage step or during
the acetylation procedure.
a
Reaction conditions: (a) N-Fmoc-(L)-2-iodophenylalanine, PyBOP,
HOAt, DIPEA, DMF, rt, 90 min; (b) piperidine, DMF, rt, 2 × 10 min;
(c) N-Fmoc-(L)-alanine, PyBOP, HOAt, DIPEA, DMF, rt, 90 min; (d)
repeated deprotection and amino acid coupling; (e) Pd(OAc)2, dppf,
dioxane, CsF, H2O, 90 °C, 16 h; (f) TFA/H2O (95:5), rt, 3 h; (g)
AcOH, PyBOP, HOAt, DIPEA, DMF, rt, 3 h.
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Rink amide MBHA resin was selected once again to provide
the C-terminal amide directly upon final peptide cleavage. The
resin was loaded with N-Fmoc-(L)-2-iodophenylalanine and
the peptide chain built using repetitive deprotection/peptide
coupling steps. The Boc-protected amino acid 8 was used as an
N-terminal residue since it was readily available. However, we
recognized that this complicated the closing stages of each
synthesis because it was no longer possible to selectively
deprotect the peptide N-terminus in order to add an acetyl
group while the peptide was still bound to the resin. Therefore,
following Suzuki−Miyaura macrocyclization of the precursor
46 to yield the resin-bound macrocycle 47, cleavage was
effected with TFA to yield a product with a free N-terminus
which was then acetylated in solution to yield the target
macrocycles. Using this approach, it was possible to generate
both desired macrocycles, 48 and 49, bearing an α-methyl
substituent at the i + 4 and i + 1 positions, respectively, which
were purified by HPLC.
Scheme 6. Solid-Phase Synthesis of o,m-Bridged Biaryl
Macrocyclic Peptides Using an Fmoc-Protection Strategy
a
To prepare macrocycle 48, featuring a novel quaternary
amino acid which places an additional methyl group at the α-
carbon of the (i + 5)-position in the peptide, it was necessary to
generate the novel Fmoc-protected boronic acid derivative 50,
which was prepared via the route shown in Scheme 5. Thus,
a
Scheme 5. Synthesis of Quaternary Amino Acid 50
a
Reaction conditions: (a) 2-iodo-benzyl bromide, LiHMDS, THF,
−30 °C, 1 h, then rt, 3 h; (b) KOSiMe3, THF, 75 °C, 2.5 h; (c)
TMSCl, CH2Cl2, 60 °C, 6 h, then FmocCl, DIPEA, 0−25 °C, 30 h.
using the previously described homochiral oxazolidinone 38,18
alkylation with 2-iodobenzyl bromide to afford quaternary
substituted oxazolidinone 51, followed by hydrolysis,19 yielded
the parent amino acid 52. Fmoc protection of 52 then provided
the requisite quaternary amino acid 50 for incorporation into
the solid-phase synthesis.
Thus, across these two series of m,m-bridged and o,m-bridged
systems, we have shown it is possible to incorporate additional
α-substituents at every position along the macrocyclic peptide
chain.
5. Solid-Phase Synthesis of o,m-Biaryl-Bridged Macro-
cyclic Pentapeptides Using an Fmoc-Protection Strategy.
Having determined that an Fmoc-protection SPS-strategy
offered the most effective approach to construction of these
biaryl-bridged macrocyclic peptides, we adopted this approach
for the final series of o,m-bridged systems we had designed. The
synthesis is outlined in Scheme 6 and differs from earlier series
in the use of an o-borylated phenylalanine derivative at the
N-terminal position of the chain. It was found that a further
improvement could be made in the synthesis by conducting the
intramolecular Suzuki−Miyaura coupling of the resin-bound
peptide 53 under microwave conditions to yield resin-bound
macrocycle 54. The benefits of conducting the Suzuki−Miyaura
coupling under microwave conditions was also highlighted by
Planas and colleagues.17 Macrocycle 54 could be cleaved from
the resin and acetylated to yield the desired macrocyclic
product 55, which was purified by HPLC. This general strategy
a
Reaction conditions: (a) N-Fmoc-(L)-3-bromophenylalanine,
PyBOP, HOAt, DIPEA, DMF, rt, 90 min; (b) piperidine, DMF, rt,
2 × 10 min; (c) N-Fmoc-amino acid, PyBOP, HOAt, DIPEA, DMF, rt,
90 min; (d) repeated deprotection and amino acid coupling; (e)
Pd(PPh3)4, K2CO3 (aq), DME, 140 °C, 20 min; (f) TFA/H2O (95:5),
rt, 3 h; (g) Ac2O, DIPEA, DMF, rt, 3 h; (h) Pd/C (10 mol%), DMF,
rt, 24 h.
could be used to prepare the related pentapeptides 56−59,
tripeptide 60, and tetrapeptide 61. As shown in Scheme 6, the
Cbz-protected pentapeptides 58 and 59 could be further
deprotected via hydrogenolysis to yield the lysine-containing
pentapeptides 62 and 63.
The key o-borylated phenylalanine derivative 64 could
be prepared as shown in Scheme 7. Thus, esterification of
o-bromophenylalanine 65 yielded fully protected system 66,
which was subjected to a Miyaura-borylation,20 to yield the
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and macrocyclic ring sizes, via both solution-phase and solid-
phase approaches, using a Suzuki−Miyaura cross-coupling
methodology. In addition to constructing biaryl-bridged
macrocycles with the m,m-configuration commonly found in
natural products, we have shown that m,o- and o,m-systems are
accessible via this approach. These complement the p,p- and
m,p-systems described recently by Planas et al.,17 and suggest
that the remaining biaryl configurations are likely to be
accessible also, providing that ring strain in the product is not
excessive. We have also shown that it is possible to construct
biaryl-bridged macrocyclic peptides that incorporate additional
elements of steric constraint, such as α-methyl-substituted amino
acids. We have provided examples where such substituents are
featured at each of the possible positions in a pentapeptide chain.
Although we explored both solution-phase and solid-phase
approaches (with two different protection regimens), we
eventually concluded that a solid-phase approach, using an
Fmoc-protection strategy, represented the most practical
method of constructing these macrocyclic peptides. However,
our initial studies of the key Suzuki−Miyaura macrocyclization
in solution provided a straightforward means for us to directly
monitor reaction outcome across a panel of diverse reaction
conditions. It therefore constitutes a good initial strategy for
future studies of this type, where reaction optimization is likely
to be necessary but direct monitoring methods for solid-phase
supported substrates/products are limited. The stepwise
solution-phase approach also allowed us to determine that
the combined yields for the three steps up to and including the
key Suzuki−Miyaura macrocyclization in the m,m-biaryl series
were in the 40−67% range. The best overall yields we were able
to achieve with the solid-phase approach were with the lysine-
derived pentapeptides 62 (48%) and 63 (50%) in the o,m-biaryl
series. These represent averages of 95% per step over the 15-
step sequence. Assuming ∼99% efficiency for the 14 other
steps, this would also imply a yield for the solid-phase Suzuki−
Miyaura cross-coupling of ∼58%, which is consistent with the
solution-phase studies. Most of the other examples gave much
lower isolated yields, even though crude HPLC traces indicated
a single major product. We attribute this difference to the more
challenging physical properties of these nonbasic systems,
resulting in material loss during HPLC purification through, for
example, adherence to surfaces. This is also consistent with the
increased solubility of the lysine-derived macrocyclic peptides
in aqueous buffer in comparison with the low solubility and
tendency to aggregation observed in many other examples.
It appears from CD analysis that the biaryl-bridged
macrocyclic peptides adopt distinct conformations in solution,
rather than behaving as a random coil. However, it was not
possible to recognize specific secondary structural motifs such
as turns or helices. It did appear that these systems were prone
to aggregation in aqueous solution, which might be a con-
sequence of their amphiphilic nature. This tendency com-
plicated interpretation of CD and NMR. Nevertheless, good
solubility in aqueous buffer could be achieved via introduction
of lysine residues.
borylated derivative 67. Ester hydrolysis yielded derivative 64,
which was used directly in the solid-phase peptide synthesis.
a
Scheme 7. Synthesis of o-Borylated Amino Acid 64
a
Reaction conditions: (a) MeI, NaHCO3, DMF, rt, 12 h; (b)
Pd(dppf)Cl2·CH2Cl2, B2pin2, KOAc, degassed dioxane, 85 °C, 3 h; (c)
LiOH·H2O (aq), MeOH, rt, 50 min; product used directly in next
step.
The borylation step was slow and required recharging several
times with additional aliquots of catalyst in order to drive the
reaction to completion, presumably because of the hindrance
from the adjacent ortho-substituent.
6. Spectroscopic Analysis of Macrocyclic Peptides.
Although our principal objective was to be able to constrain
peptides via side chain to side chain bridges that were an
integral component of the bioactive pharmacophore, rather
than to investigate the stabilization of specific secondary
structural motifs, we examined the macrocyclic systems
described above for any evidence of any secondary structure.
We looked initially by circular dichroism (CD). Measurements
were taken in buffered aqueous solution at approximately
100 μM concentration, and in most cases, the resultant spectra
were unlike those expected for turn, sheet, or helical
conformations.21 However, NMR experiments performed
later suggested the presence of aggregated forms of the
peptides, which can interfere with CD measurements. In the
cases of peptides 56 and 57, although the spectra did not match
an ideal helical profile, they did possess maxima and minima in
the appropriate regions of the spectra. We therefore examined
1
their conformations more closely by H NMR. This, together
with their physical form in aqueous buffer, further supported
the presence of aggregated species. More soluble analogues
of o,m-bridged pentapeptides, 62 and 63, containing a
lysine residue at the i + 3 position were therefore prepared
(Scheme 6). These peptides did indeed show enhanced
solubility, but again appeared to aggregate. This phenomenon
could be a general property of these amphiphilic macrocyclic
biaryl-bridged systems, which possess both a polar, peptidic face
and a lipophilic biaryl face. CD measurements in aqueous buffer
represent a stringent test for the presence of secondary
structural motifs, such as helices. It is possible that measure-
ments in nonaqueous systems would increase the likelihood of
observing secondary structure. This will be examined in future
studies.
The methodology we have established offers the prospect of
constraining the conformations of biologically active peptides,
which possess phenylalanine or tyrosine side chains within 1−3
residues of each other, via direct carbogenic fusion of their
aromatic rings. Future studies will examine the structures and
activities of such systems, created by embedding these
macrocyclic motifs at relevant points within the biologically
active peptide sequence.
CONCLUSION
Our studies demonstrate that biaryl-bridged macrocyclic
peptides can be generated with a range of biaryl configurations
■
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The sealed microwave tube was stirred at 90 °C for 16 h. After the
reaction, the resin was filtered, washed (3 × 5 mL i-PrOH, 5 × 5 mL
DMF, 5 × 5 mL CH2Cl2). and dried.
EXPERIMENTAL SECTION
■
General Procedures. All reactions were carried out under an
argon atmosphere with dry solvent under anhydrous conditions, unless
otherwise noted.
General Procedure C2 for Suzuki Coupling. A microwave vial
fitted with a magnetic stirrer bar was charged with the peptide resin
(0.1−0.25 mmol), degassed DME (2 mL), degassed 2 M K2CO3 (0.5
mL), and Pd(PPh3)4 (5 mol %). The suspension was heated in a
microwave to 140 °C for 10 min. A further 5 mol % Pd(PPh3)4 was
added and the suspension heated to the same temperature for a further
10 min. The resin was filtered, washed (3 × 5 mL H2O, 3 × 5 mL
CH2Cl2), and dried.
General Procedure D for Cleavage from the Resin (Boc). The
peptide-resin was placed in a round-bottom flask with a stirring bar. A
solution of pentamethylbenzene (593 mg, 4.00 mmol), TFA (6.3 mL),
and HBr (30% in AcOH, 0.37 mL) was added to the peptide-resin and
stirred for 2 h at rt. The resin was removed by filtration and rinsed
with TFA (2 × 2 mL). The filtrate was concentrated to about 0.5 mL
and then added to cold MTBE (10 mL). The precipitated resin was
centrifuged. The residue was washed with MTBE (10 mL) and
centrifuged two more times. The crude peptide was submitted to
HPLC purification.
General Procedure E for Cleavage from the Resin (Fmoc).
The peptide-resin was placed in a round-bottom flask with a stirring
bar. A solution of TFA/H2O (95:5, 10.0 mL) was added to the
peptide-resin and stirred for 3 h at rt. The resin was removed by
filtration and rinsed with TFA (2 × 2 mL). The filtrate was
concentrated to about 0.5 mL and then added to cold MTBE (10 mL).
The precipitated resin was centrifuged. The residue was washed with
MTBE (10 mL) and centrifuged two more times.
General Procedure F1 for Acetylation. The final peptides
amino group was capped with AcOH (1.1 equiv), PyBOP (1.1 equiv),
HOAt (1.1 equiv), and DIPEA (3 equiv) in DMF (6 mL). The crude
reaction mixture was concentrated in vacuo and submitted to HPLC
purification.
General Procedure F2 for Acetylation. The precipitated peptide
was dissolved in DMF (1−2 mL) before addition of Ac2O (1.5−3
equiv) and DIPEA (3−6 equiv). The reaction mixture was stirred at rt
for 1−3 h before the reaction mixture was concentrated. In some cases,
the resulting acetylated peptide could be partially purified by
precipitation from cold Et2O. The residue or precipitate was then
submitted to HPLC purification.
General Procedures G1−6 for HPLC Purification. Compounds
were screened against a standard HPLC screening panel which
includes reverse phase and normal phase HPLC columns and then
purified using DAD monitoring at 210−360 nm and mass
spectrometer detection in APCI mode positive scanning from 175
to 900 Da, using one of the following methods.
Method G1. Reverse phase conditions on a 150 mm × 21.2 mm
5 μm column with a gradient of 5−100% B over 8.5 min with a flow
rate of 28 mL/min. Mobile phase A was 0.1% formic acid in water, and
mobile phase B was 0.1% formic acid in methanol.
Method G2. Normal phase conditions on a 250 mm × 21.2 mm
5 μm column with a gradient of 5−100% B over 8.5 min with a flow
rate of 28 mL/min. Mobile phase A was n-heptane, and mobile phase
B was ethanol.
Solvents. Dry toluene, diethyl ether (Et2O), and methylene
chloride (CH2Cl2) were obtained by passing commercially available
predried, oxygen-free formulations through activated alumina columns.
Tetrahydrofuran was distilled from sodium. Anhydrous N,N-dimethyl
formamide (DMF) and methanol (MeOH) were purchased in
anhydrous form. Hexanes (HPLC grade), water (HPLC grade),
n-heptane (HPLC grade), methanol (HPLC grade), SDA3A denatured
ethanol (HPLC grade), formic acid 96.0%+ (reagent grade), and
ammonium hydroxide (reagent grade) were used as supplied.
Chromatography. Column chromatography was performed using
an automated flash chromatography system. Preparative thin layer
chromatography was performed on precoated glass-backed plates
(Whatman Partisil PK6F Silica Gel 60 Å 1000 μm) and visualized by
ultraviolet radiation (λ = 254 nm). Analytical thin layer chromatog-
raphy was performed on precoated glass-backed plates (Merck
Kieselgel 60 F254) and visualized by ultraviolet radiation (λ = 254
nm) or acidic potassium permanganate solutions as appropriate.
Solvents for chromatography were used as supplied.
CD Measurements. Peptides were dissolved in a buffer of 25 mM
Na2HPO4, pH 7, to a concentration of approximately 100 μM. Peptide
and buffer blank solutions were placed in a 2 mm cell, and CD spectra
were acquired over a range of 260−190 nm, with a 0.5 nm step size
and a 3 s averaging time, and each spectrum is an average over 3 scans.
Peptide NMR Studies. NMR samples were prepared by dissolving
peptides in 25 mM Na2HPO4, pH 5 (90% H2O/10% D2O). A small
amount of DSS was added as an internal reference. Experiments were
performed on a 500 MHz spectrometer at 298 K. For all peptides, a
1D proton spectrum was recorded with 4096 complex points over a
sweep-width of 9 ppm and 128 scans. Selected peptides were further
characterized by recording 2D TOCSY and ROESY spectra. TOCSY
spectra were acquired with 4096 × 128 points, 16 scans per increment,
and a 50 ms mixing time. ROESY spectra were acquired with 2048 ×
128 points, 64 scans per increment, and 300 ms mixing time. Spectra
were processed with NMRPipe,21 or MestReNova. A 90° phased-
shifted sine bell or squared sine bell window functions were applied in
both dimensions, followed by zero-filling to twice the original size and
Fourier transformation. Chemical shifts were referenced to the internal
DSS standard at 0.00 ppm.
General Procedure A for Boc SPPS Chemistry. Peptides were
prepared on 0.20 mmol scale by manual stepwise solid-phase peptide
synthesis using PyBOP/HOAt/DIPEA activation on MBHA resin.
Amino acid (4 equiv), PyBOP (4 equiv), HOAt (4 equiv), and DIPEA
(8 equiv) in DMF (4 mL) were employed in each coupling step (90
min). Boc deprotections were achieved with TFA/CH2Cl2 (1:1, 4 mL)
for 5 and 20 min. The peptide-resin was neutralized with TEA/
CH2Cl2 (1:9, 4 mL) for 2 × 10 min. Capping of the resin was per-
formed using Ac2O (50 equiv) and DIPEA (50 equiv) in DMF (5 mL).
Coupling yields were monitored by quantitative ninhydrin assay.
General Procedure B for Fmoc SPPS Chemistry. Peptides were
typically prepared on 0.20 mmol scale by manual stepwise solid-phase
peptide synthesis using PyBOP/HOAt/DIPEA activation on Rink
Amide MBHA resin. Amino acid (4 equiv), PyBOP (4 equiv), HOAt
(4 equiv), and DIPEA (5 equiv) in 4 mL of DMF were employed in
each coupling step (90 min). For couplings using synthesized or
expensive amino acids, only 1.5−2 eqiv of these reagents were used,
with a correspondingly longer reaction time (4−16 h). Fmoc
deprotections were achieved with piperidine/DMF (1:4, 4 mL) for
2 × 10 min. Coupling yields were monitored by quantitative ninhydrin
assay.
Method G3. Normal phase conditions on a 250 mm × 21.2 mm
5 μm silica column with a gradient of 5−100% B over 8.5 min with a
flow rate of 28 mL/min. Mobile phase A was n-heptane, and mobile
phase B was ethanol.
Method G4. Normal phase conditions on a 21.2 mm × 250 mm
5 μm cellulose column with a gradient of 5−100% B over 8.5 min with
a flow rate of 28 mL/min. Mobile phase A was n-heptane, and mobile
phase B was ethanol.
Method G5: Reverse phase conditions on a 21.2 mm × 150 mm
5 μm pentafluorophenyl column with a gradient of 5−100% B over 8.5
min with a flow rate of 28 mL/min. Mobile phase A was 0.1% formic
acid in water, and mobile phase B was 0.1% formic acid in methanol.
Method G6. Reverse phase conditions on a 21.2 mm × 150 mm
5 μm C18 column with a gradient of 5−100% B over 8.5 min with a
flow rate of 28 mL/min. Mobile phase A was 0.1% ammonium hydroxide
General Procedure C1 for Suzuki Coupling. A vial fitted with a
magnetic stirring bar was charged with Pd(OAc)2 (4.5 mg, 0.02
mmol), dppf (33 mg, 0.06 mmol), and degassed dioxane (2 mL). The
suspension was heated to 60 °C for 10 min and then transferred to a
10 mL microwave tube containing the peptide-resin (0.20 mmol), CsF
(3 M in H2O, 0.20 mL, 0.60 mmol). and degassed dioxane (10 mL).
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in water, and mobile phase B was 0.1% ammonium hydroxide in
methanol.
(dd, J = 8.3, 2.1 Hz, 1H), 6.76 (d, J = 8.3 Hz, 1H), 6.59 (br s, 1H),
5.12 (s, 2H), 4.91 (br s, 1H), 4.77 (dd, J = 13.1, 5.8 Hz, 1H), 4.18−
4.09 (m, 1H), 3.72 (s, 3H), 3.08 (dd, J = 14.0, 5.8 Hz, 1H), 2.98 (dd,
J = 14.0, 5.7 Hz, 1H), 1.44 (s, 9H), 1.33 (d, J = 7.1 Hz, 3H); 13C NMR
(125 MHz, CDCl3) δ 172.2, 171.4, 156.3, 140.3, 140.2, 136.4, 130.2,
130.1, 128.5, 127.8, 126.9, 112.5, 86.7, 70.8, 53.2, 52.3, 36.4, 28.2, 18.1;
HRMS (ESI) calcd for C25H32IN2O6 583.1300; found, 583.1300.
Boc-(Cyclo-m,m)-[(3-Cl)FAY]-CO2Me (14). (S)-Methyl 3-(4-
(benzyloxy)-3-iodophenyl)-2-((S)-2-((tert-butoxycarbonyl)amino)-
propanamido)propanoate (11) (146 mg, 0.250 mmol) was dissolved
in 4 M HCl in dioxane (8 mL, 32.0 mmol) and stirred at rt for 5 h.
The suspension was concentrated in vacuo. The resultant hydro-
chloride salt (12) of the dipeptide was suspended in CH2Cl2 (12.0
mL). To this suspension, DIPEA (0.131 mL, 0.75 mmol), PyBOP
(169 mg, 0.325 mmol), and (S)-2-((tert-butoxycarbonyl)amino)-3-(3-
chloro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-
propanoic acid (8) (138 mg, 0.325 mmol) were added and the
reaction mixture was stirred at rt for 12 h. The reaction mixture was
diluted with EtOAc (80 mL) and washed with 1 M aqueous HCl (2 ×
30 mL), saturated aqueous NaHCO3 (25 mL), and brine (40 mL).
The organic layer was dried (Na2SO4) and concentrated in vacuo to
give tripeptide 13. Because of the instability of this material upon
purification, the crude product was submitted to the next reaction. In a
100 mL flask were the tripeptide 13, Pd(dppf)Cl2·CH2Cl2 (10.2 mg,
0.013 mmol), and CsF (1 M in H2O, 1.5 mL, 1.5 mmol) in degassed
dioxane (62.5 mL). The flask was sealed and heated to 90 °C for 18 h.
The reaction mixture was diluted with EtOAc (150 mL) and washed
with water (2 × 25 mL) and brine (2 × 25 mL). The organic layer was
dried (Na2SO4) and concentrated in vacuo, the residue was purified by
an automated system (KP-Sil 25 g column; hexanes/EtOAc 80:20 to
hexanes/EtOAc 0:100) leading to cyclic peptide 14 (96 mg, 0.151
mmol, 60%): 1H NMR (600 MHz, CDCl3) δ 7.45 (s, 1H), 7.38−7.29
(m, 3H), 7.28−7.24 (m, 3H), 7.00 (s, 2H), 6.90 (s, 1H), 6.87 (d, J =
8.3 Hz, 1H), 6.78 (s, 1H), 6.69 (d, J = 8.3 Hz, 1H), 5.55 (d, J = 8.0 Hz,
1H), 4.93−4.82 (m, 3H), 4.76−4.73 (m, 1H), 4.52−4.48 (m, 1H),
3.80 (s, 3H), 3.19 (dd, J = 14.3, 7.2 Hz, 1H), 2.91 (d, J = 13.2 Hz, 1H),
2.80 (d, J = 13.2 Hz, 1H), 2.49 (dd, J = 14.3, 8.4 Hz, 1H), 1.48 (s, 9H),
1.36 (d, J = 6.9 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 171.8, 171.7,
170.3, 155.2, 154.0, 139.8, 137.7, 136.9, 132.7, 131.3, 129.7, 129.3,
129.1, 128.5, 128.4, 128.2, 128.0, 127.5, 126.6, 112.3, 79.8, 70.0, 54.5,
53.4, 52.6, 49.0, 37.6, 36.5, 28.3, 19.0; HRMS (ESI) calcd for
C34H39ClN3O7 636.2471; found, 636.2459.
Ac-(Cyclo-m,m)-[(3-Cl)FAY]-CO2Me (15). (m,m)-Cyclo Boc-F(3-
Cl)AY-CO2Me 14 (50 mg, 0.079 mmol) was dissolved in 4 M HCl in
dioxane (2 mL, 8.0 mmol) and stirred at rt for 3 h. The suspension
was concentrated in vacuo. The hydrochloride salt of the tripeptide
was suspended in DMF (2.0 mL). To this suspension, DIPEA (0.138
mL, 0.79 mmol) and Ac2O (0.075 mL, 0.790 mmol) were added and
the reaction mixture was stirred at rt for 12 h. The reaction mixture
was diluted with EtOAc (50 mL)and washed with 1 M aqueous HCl
(2 × 20 mL), saturated aqueous NaHCO3 (2 × 20 mL), and brine (20
mL). The organic layer was dried (Na2SO4) and concentrated in
vacuo. The residue was recrystallized leading to cyclic peptide 15 (38
mg, 0.066 mmol, 83%): 1H NMR (600 MHz, d6-DMSO) δ 9.01 (d, J =
9.3 Hz, 1H), 8.76 (d, J = 8.5 Hz, 1H), 7.63−7.60 (m, 2H), 7.43−7.34
(m, 5H), 7.30 (t, J = 7.0 Hz, 1H), 7.22 (s, 1H), 7.19 (d, J = 8.4 Hz,
1H), 7.09 (d, J = 8.4 Hz, 1H), 6.99 (s, 1H), 6.88 (s, 1H), 5.16 (d, J =
12.1 Hz, 1H), 5.08 (d, J = 12.1 Hz, 1H), 4.75−4.65 (m, 2H), 4.62 (t,
J = 9.9 Hz, 1H), 3.71 (s, 3H), 3.09 (d, J = 14.8 Hz, 1H), 2.99 (d, J =
4.2 Hz, 2H), 2.91 (dd, J = 14.8, 10.8 Hz, 1H), 1.89 (s, 3H), 1.24 (d, J =
7.0 Hz, 3H); 13C NMR (150 MHz, d6-DMSO) δ 172.3, 171.5, 168.8,
153.6, 139.1, 138.9, 136.9, 131.3, 131.3, 129.9, 129.4, 129.2, 128.2,
128.0, 127.9, 127.5, 127.1, 127.0, 112.5, 69.4, 52.3, 52.3, 52.2, 47.4,
37.4, 35.1, 22.4, 18.6; HRMS (ESI) calcd for C31H33ClN3O6 578.2052;
found, 578.2060.
(S)-Methyl 2-((tert-Butoxycarbonyl)amino)-3-(3-chloro-5-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-
propanoate (7). A 500 mL round-bottomed flask fitted with a reflux
condenser and magnetic stirring bar was charged with (S)-methyl 2-
((tert-butoxycarbonyl)amino)-3-(3-chlorophenyl)propanoate (10.3 g,
32.7 mmol), bis(pinacolato)diboron (12.5 g, 49.0 mmol), [Ir(OMe)-
COD]2 (0.217 g, 0.327 mmol), and 4,4′-di-tert-butyl-2,2′-bipyridine
(dtbpy) (0.176 g, 0.654 mmol). Hexanes (163 mL) were added and
the reaction was heated to reflux for 16 h. Subsequent removal of
residual solvent in vacuo, the residue was purified by an automated
system (FLASH 65iTM column; hexanes/EtOAc 95:5 to hexanes/
1
EtOAc 80:20) leading to 3,5-isomer 7 (12.8 g, 29.1 mmol, 89%): H
NMR (500 MHz, CDCl3) δ 7.63 (s, 1H), 7.42 (s, 1H), 7.18 (s, 1H),
5.02 (d, J = 7.5 Hz, 1H), 4.58−4.59 (m, 1H), 3.71 (s, 3H), 3.12 (dd,
J = 13.5, 5.3 Hz, 1H), 2.98 (dd, J = 13.5, 6.2 Hz, 1H), 1.41 (s, 9H),
1.32 (s, 12H); 13C NMR (125 MHz, CDCl3) δ 171.9, 154.8, 137.5,
133.9, 133.7, 133.0, 131.9, 84.0, 79.9, 54.3, 52.2, 37.7, 28.2, 24.8;
HRMS (ESI) calcd for C21H32BClNO6 440.2006; found, 440.1998.
(S)-2-((tert-Butoxycarbonyl)amino)-3-(3-chloro-5-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propanoic Acid
(8). To a stirred solution of (S)-methyl 2-((tert-butoxycarbonyl)-
amino)-3-(3-chloro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
phenyl)propanoate (7) (141 mg, 0.3 mmol) in MeOH (3 mL) was
added LiOH·H2O (84 mg, 2.00 mmol) in H2O (2 mL) at rt. The
mixture was stirred at the same temperature for 40 min. The reaction
mixture was acidified with 1 M aqueous HCl to pH ∼ 2. The aqueous
layer was extracted with EtOAc (3 × 50 mL). The combined organic
layers were dried (Na2SO4), filtered, and concentrated in vacuo
providing crude acid 8 (138 mg). Because of the instability of this
material, it was carried forward without further purification.
(S)-Methyl 2-Amino-3-(4-(benzyloxy)-3-iodophenyl)-
propanoate Hydrochloride (10). 3-Iodo-L-tyrosine 9 (2.5 g, 8.14
mmol) was dissolved in water (2.5 mL) and 2 M NaOH (9 mL).
CuSO4 (1.02 g) was added and the resulting solution was warmed to
60 °C for 10 min. The reaction changed from blue to green during that
time. The solution was cooled to rt and charged with MeOH (35 mL)
followed by BnBr (1.16 mL, 9.77 mmol). The reaction was stirred for
12 h during which time the product precipitated as a white solid. The
solid was filtered and washed sequentially with water (50 mL) and 1 M
HCl (50 mL) then dried in vacuo, resulting in a tan powder (2.9 g,
6.71 mmol, 82%). This material was carried forward without further
purification. To a cooled solution of MeOH (30 mL) was added
dropwise SOCl2 (4.63 mL, 63.4 mmol) followed by the addition of the
HCl salt of H2NTyr(3-I)(Bn)−OH (2.75 g, 6.34 mmol). The reaction
mixture was warmed to rt and stirred for 2 h. The reaction mixture was
concentrated in vacuo and washed with cold Et2O (2 × 10 mL)
providing the methyl ester 10 as a pure yellow powder (2.46 g, 5.50
1
mmol, 87%): H NMR (500 MHz, CD3OD) δ 7.71 (s, 1H), 7.49 (d,
J = 7.5 Hz, 2H), 7.37 (t, J = 7.5 Hz, 2H), 7.30 (t, J = 7.2 Hz, 1H), 7.20
(d, J = 7.5 Hz, 1H), 6.99 (d, J = 8.3 Hz, 1H), 5.17 (s, 2H), 4.29−4.22
(m, 1H), 3.80 (s, 3H), 3.17 (dd, J = 14.4, 5.8 Hz, 1H), 3.07 (dd, J =
14.4, 7.3 Hz, 1H); 13C NMR (125 MHz, CD3OD) δ 170.1, 158.2,
141.3, 137.8, 131.8, 129.4, 128.8, 128.2, 114.3, 87.6, 72.0, 55.2, 54.0,
36.0; HRMS calcd for C17H19INO3 412.0404; found, 412.0396.
(S)-Methyl 3-(4-(Benzyloxy)-3-iodophenyl)-2-((S)-2-((tert-
butoxycarbonyl)amino)propanamido)propanoate (11). (S)-
Methyl 2-amino-3-(4-(benzyloxy)-3-iodophenyl)propanoate hydro-
chloride (10) (322 mg, 0.78 mmol) was suspended in CH2Cl2 (10
mL). To this suspension, PyBOP (530 mg, 1.02 mmol), NEt3 (0.142
mL, 1.02 mmol), and Boc-Ala-OH (178 mg, 0.94 mmol) were added
and the reaction mixture was stirred at rt for 3 h. The reaction mixture
was poured into water (15 mL) and the aqueous layer was extracted
with CH2Cl2 (3 × 20 mL). The combined organic layers were dried
(Na2SO4) and concentrated in vacuo, the residue was purified by an
automated system (Flash 40+S column; hexanes/EtOAc 91:9 to
hexanes/EtOAc 0:100) leading to dipeptide 11 (396 mg, 0.68 mmol,
Ac-(Cyclo-m,m)-[FAY]-NH2 (16). To a suspension of palladium
hydroxide on carbon (16.8 mg, 20 wt %, 0.024 mmol) in MeOH
(4 mL), (m,m)-cyclo Ac-F(3-Cl)AY-CO2Me 15 (69 mg, 0.119 mmol)
and NH4OH (30% in H2O, 0.310 mL, 2.39 mmol) were added. The
reaction mixture was flushed with hydrogen gas and the reaction
1
87%): H NMR (500 MHz, CDCl3) δ 7.53 (d, J = 2.1 Hz, 1H), 7.48
(d, J = 7.0 Hz, 2H), 7.42−7.37 (m, 2H), 7.32 (t, J = 7.3 Hz, 1H), 7.02
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mixture was stirred under hydrogen for 12 h at 40 °C. The crude
reaction mixture was filtered through a short plug of Celite (MeOH)
and concentrated in vacuo. Carrying this material forward without
further purification, the newly formed intermediate was dissolved in
THF (1.8 mL), MeOH (0.37 mL), and H2O (0.18 mL). LiOH (57
mg, 2.38 mmol) was added and the reaction mixture was stirred at rt
for 5 h. The reaction mixture was acidified with 1 M aqueous HCl to
pH ∼ 2. The aqueous layer was extracted with EtOAc (5 × 15 mL).
The combined organic layers were dried (Na2SO4), filtered, and
concentrated in vacuo. Carrying this material forward without further
purification, the newly formed intermediate was dissolved in DMF
(1 mL) and CH2Cl2 (5 mL). To this solution, PyBOP (93 mg, 0.179
mmol) was added. After ammonia gas was bubbled through the
solution for 5 min, the reaction mixture was stirred at rt for 5 h. The
reaction mixture was concentrated in vacuo and the residue was
purified by an automated system (KP-Sil 10 g column; CH2Cl2/
MeOH 95:5 to CH2Cl2/MeOH 80:20) leading to cyclic peptide 16
(35 mg, 0.08 mmol, 67%): 1H NMR (600 MHz, d6-DMSO) δ 9.35 (s,
1H), 8.66 (d, J = 9.0 Hz, 1H), 8.63 (d, J = 9.0 Hz, 1H), 7.56 (d, J = 7.6
Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.39 (s, 1H), 7.21 (t, J = 7.6 Hz,
1H), 7.14−7.09 (m, 2H), 6.96−6.90 (m, 3H), 6.79 (d, J = 8.2 Hz,
1H), 4.78−4.71 (m, 1H), 4.69−4.64 (m, 1H), 4.47 (dt, J = 8.9, 3.5 Hz,
1H), 3.03 (dd, J = 13.7, 6.3 Hz, 1H), 2.96 (dd, J = 13.7, 2.7 Hz, 1H),
2.89−2.81 (m, 2H), 1.88 (s, 3H), 1.22 (d, J = 7.0 Hz, 3H); 13C NMR
(150 MHz, d6-DMSO) δ 173.1, 171.8, 168.9, 168.6, 152.6, 138.2,
136.5, 130.0, 129.9, 129.3, 129.0, 127.7, 127.2, 127.1, 126.7, 115.1,
53.4, 52.6, 47.4, 37.6, 36.5, 22.5, 19.0; HRMS (ESI) calcd for
C23H27N4O5 439.1976; found, 439.1994.
(6S,9S,12S)-Methyl 12-(4-(Benzyloxy)-3-iodobenzyl)-2,2,6,9-
tetramethyl-4,7,10-trioxo-3-oxa-5,8,11-triazatridecan-13-oate
(17). A solution of (S)-methyl 3-(4-(benzyloxy)-3-iodophenyl)-2-((S)-
2-((tert-butoxycarbonyl)amino)propanamido)propa-noate (11) (14.0 g,
24.0 mmol) in ethyl acetate (100 mL) at 0 °C was treated with 4 M
HCl in ethyl acetate (100 mL, 400 mmol) and stirred at rt for 5 h. The
suspension was concentrated in vacuo to yield the hydrochloride salt
12 of the dipeptide. To a solution of Boc-Ala-OH (5.46 g, 28.9 mmol)
and DIPEA (12.6 mL, 72.2 mmol) in DMF (70 mL) at 0 °C was
added EDCI (6.90 g, 36.0 mmol) and HOBt (4.87 g, 36.0 mmol). The
mixture was then stirred at 0 °C for 1 h, whereupon the hydrochloride
salt in DMF (30 mL) was added. The mixture was warmed to rt and
stirred for 16 h. The mixture was concentrated under reduced pressure
to give the crude product which was purified via flash chromatography
(silica gel, petroleum ether/EtOAc (83:17 to 50:50)) leading to
tripeptide 17 (9.0 g, 14 mmol, 58%): 1H NMR (600 MHz, CD3OD) δ
7.62 (d, J = 1.7 Hz, 1H), 7.49 (d, J = 7.3 Hz, 2H), 7.36 (t, J = 7.6 Hz,
2H), 7.29 (t, J = 7.3 Hz, 1H), 7.17−7.12 (m, 1H), 6.91 (d, J = 8.4 Hz,
1H), 5.12 (s, 2H), 4.58 (dd, J = 8.0, 6.1 Hz, 1H), 4.37−4.30 (m, 1H),
4.11−4.01 (m, 1H), 3.67 (s, 3H), 3.05 (dd, J = 14.0, 6.1 Hz, 1H), 2.92
(dd, J = 14.0, 8.0 Hz, 1H), 1.43 (s, 9H), 1.31 (d, J = 7.1 Hz, 3H), 1.27
(d, J = 7.1 Hz, 3H); 13C NMR (150 MHz, CD3OD) δ 175.4, 174.7,
173.0, 157.7, 141.2, 138.2, 132.4, 131.5, 129.5, 128.8, 128.2, 113.8,
87.1, 80.6, 71.8, 55.2, 52.7, 51.4, 50.1, 36.9, 28.7, 18.3; HRMS (ESI)
calcd for C28H37IN3O7 654.1671; found, 654.1680.
CsF (1 M in H2O, 1.5 mL, 1.5 mmol) in degassed dioxane (62.5 mL).
The flask was sealed and heated to 90 °C for 18 h. The reaction
mixture was diluted with EtOAc (150 mL) and washed with water
(2 × 25 mL) and brine (2 × 25 mL). The organic layer was dried
(Na2SO4) and concentrated in vacuo, the residue was purified by an
automated system (KP-C-18-HS 12 g column; H2O/MeCN 100:0 to
H2O/MeCN 0:100) leading to cyclic peptide 20 (91 mg, 0.129 mmol,
1
51%): H NMR (600 MHz, CD3CN) δ 7.60 (s, 1H), 7.41−7.35 (m,
4H), 7.35−7.29 (m, 3H), 7.16 (s, 1H), 7.10−7.01 (m, 3H), 6.78 (d, J =
3.8 Hz, 2H), 5.52 (d, J = 6.1 Hz, 1H), 5.08 (d, J = 11.6 Hz, 1H), 5.05
(d, J = 11.6 Hz, 1H), 4.96−4.88 (m, 1H), 4.34−4.28 (m, 1H), 4.25−
4.18 (m, 1H), 4.15−4.08 (m, 1H), 3.72 (s, 3H), 3.19−3.09 (m, 2H),
3.03−2.92 (m, 2H), 1.46 (s, 9H), 1.23 (d, J = 7.2 Hz, 3H), 1.18 (d, J =
7.0 Hz, 3H); 13C NMR (150 MHz, CD3CN) δ 173.0, 172.6, 171.3,
155.7, 155.1, 140.8, 139.8, 138.0, 133.1, 132.2, 131.1, 130.6, 129.9,
129.6, 129.3, 129.2, 129.1, 128.7, 128.5, 128.3, 113.8, 80.0, 70.9, 55.3,
52.8, 52.7, 50.0, 49.6, 38.9, 36.9, 28.5, 18.0, 17.5; HRMS (ESI) calcd
for C37H44ClN4O8 707.2842; found, 707.2833.
Ac-(Cyclo-m,m)-[(3-Cl)FAAY]-CO2Me (21). (m,m)-Cyclo Boc-
F(3-Cl)AAY-CO2Me (20) (194 mg, 0.274 mmol) was dissolved in 4
M HCl in dioxane (4 mL, 16.0 mmol) and stirred at rt for 3 h. The
suspension was concentrated in vacuo. The hydrochloride salt of the
tetrapeptide was suspended in DMF (5 mL). To this suspension,
DIPEA (0.479 mL, 2.74 mmol) and Ac2O (0.259 mL, 2.74 mmol)
were added and the reaction mixture was stirred at rt for 12 h. The
reaction mixture was diluted with EtOAc (100 mL), and washed with
1 M aqueous HCl (2 × 20 mL), saturated aqueous NaHCO3 (2 × 20
mL), and brine (20 mL). The organic layer was dried (Na2SO4) and
concentrated in vacuo and the residue was purified by an automated
system (KP-Sil 10 g column; CH2Cl2/MeOH 97:3 to CH2Cl2/MeOH
1
90:10) leading to cyclic peptide 21 (111 mg, 0.171 mmol, 62%). H
NMR (600 MHz, CD3OD) δ 7.51 (s, 1H), 7.43 (d, J = 2.0 Hz, 1H),
7.41 (s, 1H), 7.32−7.26 (m, 4H), 7.25−7.21 (m, 1H), 7.13 (s, 1H),
7.04 (dd, J = 8.4, 2.0 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 5.02 (d, J =
11.9 Hz, 1H), 4.99 (d, J = 11.9 Hz, 1H), 4.91 (dd, J = 9.1, 3.8 Hz, 1H),
4.61 (dd, J = 8.5, 2.5 Hz, 1H), 4.17 (q, J = 7.3 Hz, 1H), 4.10 (q, J = 7.0
Hz, 1H), 3.74 (s, 3H), 3.20−3.12 (m, 2H), 3.02−2.92 (m, 2H), 1.99
(s, 3H), 1.24 (d, J = 7.3 Hz, 3H), 1.22 (d, J = 7.0 Hz, 3H); 13C NMR
(150 MHz, CD3OD) δ 174.4, 174.0, 173.1, 172.7, 172.4, 155.8, 141.5,
139.7, 138.5, 133.8, 132.8, 131.1, 130.9, 130.7, 130.4, 129.8, 129.4,
129.2, 128.7, 128.3, 114.5, 71.6, 55.3, 53.6, 52.8, 50.6, 50.4, 39.2, 37.2,
22.5, 17.8, 17.6; HRMS (ESI) calcd for C34H38ClN4O7 649.2423;
found, 649.2435.
Ac-(Cyclo-m,m)-[FAAY]-NH2 (22). To a suspension of palladium
hydroxide on carbon (19.4 mg, 20 wt %, 0.027 mmol) in MeOH (2.7
mL), (m,m)-cyclo Ac-F(3-Cl)AAY-CO2Me (21) (88 mg, 0.136 mmol)
and NH4OH (30% in H2O, 0.352 mL, 2.71 mmol) were added. The
reaction mixture was flushed with hydrogen gas and the reaction
mixture was stirred under hydrogen for 12 h at 40 °C. The crude
reaction mixture was filtered through a short plug of Celite (MeOH)
and concentrated in vacuo. Carrying this material forward without
further purification, the newly formed intermediate was dissolved in
THF (1.3 mL), MeOH (0.26 mL), and H2O (0.13 mL). LiOH (40
mg, 1.68 mmol) was added and the reaction mixture was stirred at rt
for 5 h. The reaction mixture was acidified with 1 M aqueous HCl to
pH ∼ 2. The aqueous layer was extracted with EtOAc (5 × 15 mL).
The combined organic layers were dried (Na2SO4), filtered, and
concentrated in vacuo. Carrying this material forward without further
purification, the newly formed intermediate was dissolved in DMF (0.7
mL) and CH2Cl2 (3.5 mL). To this solution, PyBOP (66 mg, 0.126
mmol) was added. After ammonia gas was bubbled through the
solution for 5 min, the reaction mixture was stirred at rt for 5 h. The
reaction mixture was concentrated in vacuo and the residue was
purified by an automated system (KP-Sil 10 g column; CH2Cl2/
MeOH 95:5 to CH2Cl2/MeOH 80:20) leading to cyclic peptide 22
(28 mg, 0.08 mmol, 40%). Note concerning the NMR data of the
following compound: Due to a mixture of conformers, the proton
assignment of 1H NMR data was carried out for the two major
compounds (1:0.4 ratio) in this mixture. The 13C NMR data represents a
mixture of all conformers. 1H NMR (600 MHz, d6-DMSO) δ 9.31 (s, 1H),
Boc-(Cyclo-m,m)-[(3-Cl)FAAY]-CO2Me (20). (6S,9S,12S)-Methyl
12-(4-(benzyloxy)-3-iodobenzyl)-2,2,6,9-tetramethyl-4,7,10-trioxo-3-
oxa-5,8,11-triazatridecan-13-oate (17) (163 mg, 0.250 mmol) was
dissolved in 4 M HCl in dioxane (8 mL, 32.0 mmol) and stirred at rt
for 5 h. The suspension was concentrated in vacuo. The hydrochloride
salt (18) of the tripeptide was suspended in DMF (12.0 mL). To this
suspension, DIPEA (0.131 mL, 0.75 mmol), PyBOP (169 mg, 0.325
mmol), and (S)-2-((tert-butoxycarbonyl)amino)-3-(3-chloro-5-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propanoic acid
(8) (138 mg, 0.325 mmol) were added and the reaction mixture
was stirred at rt for 12 h. The reaction mixture was diluted with EtOAc
(80 mL) and washed with 1 M aqueous HCl (2 × 30 mL), saturated
aqueous NaHCO3 (25 mL), and brine (40 mL). The organic layer was
dried (Na2SO4) and concentrated in vacuo to give tetrapeptide 19.
Because of the instability of this material upon purification, the crude
product was submitted to the next reaction. In a 100 mL flask were the
tetrapeptide 19, Pd(dppf)Cl2·CH2Cl2 (10.2 mg, 0.013 mmol), and
3108
dx.doi.org/10.1021/jo202105v | J. Org. Chem. 2012, 77, 3099−3114

The Journal of Organic Chemistry
Article
9.24 (s, 0.4H), 8.55 (d, J = 7.9 Hz, 1H), 8.22 (d, J = 9.5 Hz, 1H), 8.10
(t, J = 7.4 Hz, 0.8H), 7.98 (d, J = 8.6 Hz, 0.4H), 7.64 (d, J = 7.6 Hz,
1H), 7.50−7.45 (m, 2.4H), 7.38 (d, J = 6.9 Hz, 1H), 7.32−7.21
(m, 2.2H), 7.16−7.09 (m, 3.8H), 7.08−7.03 (m, 0.8H), 6.97−6.93 (m,
1.4H), 6.92−6.88 (m, 1H), 6.82 (d, J = 8.3 Hz, 0.4H), 6.80 (d, J = 8.2
Hz, 1H), 4.90−4.83 (m, 1H), 4.65 (dt, J = 7.5, 1.8 Hz, 1H), 4.49−4.33
(m, 0.8H), 4.24−4.10 (m, 2.8H), 3.15−3.11 (m, 1H), 3.05 (dd, J =
13.1, 3.2 Hz, 0.4H), 2.98−2.89 (m, 2.4H), 2.85−2.77 (m, 1.4H), 2.73
(dd, J = 13.6, 10.6 Hz, 0.4H), 1.92 (s, 3H), 1.91 (s, 1.2H), 1.19 (d, J =
7.5 Hz, 3H), 1.14 (d, J = 6.8 Hz, 3H), 1.05 (d, J = 6.8 Hz, 1.2H), 0.95
(d, J = 6.9 Hz, 1.2H); 13C NMR (150 MHz, d6-DMSO) δ 173.1, 171.1,
170.7, 169.7, 169.4, 168.9, 168.8, 154.2, 152.4, 138.6, 138.4, 136.2,
136.1, 130.9, 130.1, 129.7, 129.4, 129.0, 128.9, 128.5, 128.0, 127.7,
127.6, 127.5, 127.5, 127.4, 127.2, 127.1, 126.7, 115.8, 115.7, 115.7,
62.7, 54.9, 54.1, 52.8, 50.5, 48.5, 48.4, 47.7, 47.5, 47.4, 38.4, 37.9, 36.9,
36.5, 22.6, 22.4, 18.8, 18.7, 18.2, 18.0; HRMS (ESI) calcd for
C26H32N5O6 510.2347; found, 510.2350.
H2O/MeCN 0:100) leading to cyclic peptide 26 (66 mg, 0.129 mmol,
34%): H NMR (600 MHz, CDCl3) δ 7.72 (d, J = 7.2 Hz, 1H), 7.66
1
(s, 1H), 7.38−7.31 (m, 5H), 7.31−7.27 (m, 2H), 7.17 (dd, J = 8.4, 1.9
Hz, 1H), 7.15−7.10 (m, 2H), 6.95 (s, 1H), 6.93 (d, J = 8.4 Hz, 1H),
6.84 (br s, 1H), 5.59 (br s, 1H), 5.10 (d, J = 12.0 Hz, 1H), 5.05 (d, J =
12.0 Hz, 1H), 4.64 (t, J = 9.6 Hz, 1H), 4.47−4.39 (m, 1H), 4.26−4.19
(m, 1H), 4.17−4.11 (m, 1H), 3.98−3.94 (m, 1H), 3.71 (s, 3H), 3.19
(d, J = 12.9 Hz, 1H), 3.09−3.03 (m, 1H), 3.02−2.83 (m, 2H), 1.48 (s,
9H), 1.44 (d, J = 7.3 Hz, 3H), 1.38 (d, J = 7.1 Hz, 3H), 1.09 (d, J = 7.2
Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 173.3, 173.1, 172.6, 172.3,
172.0, 156.2, 154.1, 139.3, 136.8, 136.2, 133.0, 132.1, 131.0, 130.1,
129.9, 129.0, 128.4, 127.7, 126.9, 126.8, 113.1, 81.6, 70.3, 58.4, 54.0,
52.4, 51.1, 49.0, 37.9, 37.2, 29.6, 28.2, 24.8, 17.5, 16.9; HRMS (ESI)
calcd for C40H49ClN5O9 778.3213; found, 778.3212.
Ac-(Cyclo-m,m)-[(3-Cl)FAAAY]-CO2Me (27). (m,m)-Cyclo Boc-
F(3-Cl)AAAY-CO2Me (26) (120 mg, 0.154 mmol) was dissolved in 4
M HCl in dioxane (4 mL, 16.0 mmol) and stirred at rt for 3 h. The
suspension was concentrated in vacuo. The hydrochloride salt of the
pentapeptide was suspended in DMF (5 mL). To this suspension,
DIPEA (0.269 mL, 1.54 mmol) and Ac2O (0.145 mL, 1.54 mmol)
were added and the reaction mixture was stirred at rt for 12 h. The
reaction mixture was diluted with EtOAc (100 mL), and washed with
1 M aqueous HCl (2 × 20 mL), saturated aqueous NaHCO3 (2 × 20
mL), and brine (20 mL). The organic layer was dried (Na2SO4) and
concentrated in vacuo and the residue was purified by an automated
system (KP-Sil 10 g column; CH2Cl2/MeOH 97:3 to CH2Cl2/MeOH
(6S,9S,12S,15S)-Methyl 15-(4-(Benzyloxy)-3-iodobenzyl)-
2,2,6,9,12-pentamethyl-4,7,10,13-tetraoxo-3-oxa-5,8,11,14-
tetraazahexadecan-16-oate (23). (6S,9S,12S)-Methyl 12-(4-(benz-
yloxy)-3-iodobenzyl)-2,2,6,9-tetramethyl-4,7,10-trioxo-3-oxa-5,8,11-tri-
azatridecan-13-oate (17) (9.0 g, 14 mmol) in ethyl acetate (35 mL) at
0 °C was treated with 4 M HCl in ethyl acetate (35 mL, 140 mmol)
and stirred at rt for 5 h. The suspension was concentrated in vacuo to
yield the hydrochloride salt 18 of the tripeptide. To a solution of Boc-
Ala-OH (3.10 g, 16.4 mmol) and DIPEA (7.2 mL, 41.4 mmol) in
DMF (70 mL) at 0 °C was added EDCI (3.96 g, 20.7 mmol) and
HOBt (2.79 g, 21.5 mmol). The mixture was then stirred at 0 °C for
1 h, whereupon the hydrochloride salt in DMF (30 mL) was added.
The mixture was warmed to rt and stirred for 16 h. The mixture was
concentrated under reduced pressure to give the crude product which
was purified via preparatory HPLC (250 × 50 mm, 10 μm column,
mobile phase H2O/CH3CN (65:35 to 35:65) containing 0.1%
ammonia, flow rate 80 mL/min, UV detection at 220 nm) leading
1
80:20) leading to cyclic peptide 27 (72 mg, 0.100 mmol, 65%): H
NMR (600 MHz, CDCl3) δ 8.07 (d, J = 5.9 Hz, 1H), 7.82 (br s, 1H),
7.71 (br s, 1H), 7.55 (s, 1H), 7.47 (d, J = 8.7 Hz, 1H), 7.28−7.23 (m,
5H), 7.22−7.18 (m, 2H), 7.08−7.04 (m, 2H), 6.87 (s, 1H), 6.84 (d,
J = 8.5 Hz, 1H), 5.02 (d, J = 12.2 Hz, 1H), 4.95 (d, J = 12.2 Hz, 1H),
4.47 (t, J = 10.1 Hz, 1H), 4.25−4.18 (m, 1H), 4.16−4.10 (m, 1H),
4.10−4.05 (m, 1H), 3.86 (d, J = 10.9 Hz, 1H), 3.59 (s, 3H), 3.11 (d,
J = 13.3 Hz, 1H), 3.03−2.95 (m, 2H), 2.95−2.88 (m, 1H), 1.95 (s, 3H),
1.37 (d, J = 7.3 Hz, 3H), 1.35 (d, J = 7.2 Hz, 3H), 1.00 (d, J = 7.2 Hz,
3H),; 13C NMR (150 MHz, CDCl3) δ 174.4, 174.1, 173.8, 173.6,
172.7, 172.0, 154.2, 138.7, 137.1, 136.7, 132.8, 132.1, 130.7, 129.8,
129.8, 129.4, 128.5, 128.4, 127.6, 127.1, 126.8, 113.1, 70.2, 58.4, 54.4,
52.7, 52.5, 51.3, 49.8, 37.4, 37.0, 29.6, 22.8, 16.8, 16.7; HRMS (ESI)
calcd for C37H43ClN5O8 720.2795; found, 720.2812.
1
to tetrapeptide 23 (5.5 g, 7.6 mmol, 55%): H NMR (600 MHz, d6-
DMSO) δ 8.21 (d, J = 7.3 Hz, 1H), 7.90 (d, J = 7.5 Hz, 1H), 7.84 (d, J =
7.3 Hz, 1H), 7.63 (d, J = 1.9 Hz, 1H), 7.48 (d, J = 7.4 Hz, 2H), 7.40
(t, J = 7.6 Hz, 2H), 7.32 (t, J = 7.3 Hz, 1H), 7.19 (dd, J = 8.4, 1.9 Hz,
1H), 6.98 (dd, J = 7.7, 4.7 Hz, 2H), 5.15 (s, 2H), 4.42−4−38 (m, 1H),
4.31−4.21 (m, 2H), 3.98−3.89 (m, 1H), 3.57 (s, 3H), 2.94 (dd, J =
13.9, 5.7 Hz, 1H), 2.85 (dd, J = 13.9, 8.8 Hz, 1H), 1.37 (s, 9H), 1.17−
1.14 (m, 9H); 13C NMR (150 MHz, d6-DMSO) δ 172.3, 172.1, 171.5,
171.5, 155.4, 155.0, 139.3, 136.6, 131.4, 130.2, 128.3, 127.6, 127.0,
112.6, 86.4, 77.9, 69.9, 53.5, 51.7, 49.5, 47.7, 47.7, 34.9, 28.1, 18.2,
18.1, 17.9; HRMS (ESI) calcd for C31H42IN4O8 725.2042; found,
725.2022.
Boc-(Cyclo-m,m)-[(3-Cl)FAAAY]-CO2Me (26). (6S,9S,12S,15S)-
Methyl 15-(4-(benzyloxy)-3-iodobenzyl)-2,2,6,9,12-pentamethyl-
4,7,10,13-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-oate (23)
(181 mg, 0.250 mmol) was dissolved in 4 M HCl in dioxane (4
mL, 16.0 mmol) and stirred at rt for 5 h. The suspension was
concentrated in vacuo. The hydrochloride salt (24) of the tetrapeptide
was suspended in DMF (12.0 mL). To this suspension, DIPEA (0.131
mL, 0.75 mmol), PyBOP (169 mg, 0.325 mmol), and (S)-2-((tert-
butoxycarbonyl)amino)-3-(3-chloro-5-(4,4,5,5-tetramethyl-1,3,2-dioxa-
borolan-2-yl)phenyl)propanoic acid (8) (138 mg, 0.325 mmol) were
added and the reaction mixture was stirred at rt for 12 h. The reaction
mixture was diluted with EtOAc (80 mL), and washed with 1 M
aqueous HCl (2 × 30 mL), saturated aqueous NaHCO3 (25 mL), and
brine (40 mL). The organic layer was dried (Na2SO4) and
concentrated in vacuo to give tetrapeptide (25). Because of the
instability of this material upon purification, the crude product was
submitted to the next reaction. In a 100 mL flask were the
pentapeptide (25), Pd(dppf)Cl2·CH2Cl2 (10.2 mg, 0.013 mmol),
and CsF (1 M in H2O, 1.5 mL, 1.5 mmol) in degassed dioxane (62.5
mL). The flask was sealed and heated to 90 °C for 18 h. The reaction
mixture was diluted with EtOAc (150 mL) and washed with water
(2 × 25 mL) and brine (2 × 25 mL). The organic layer was dried
(Na2SO4) and concentrated in vacuo, the residue was purified by an
automated system (KP-C-18-HS 12 g column; H2O/MeCN 0:100 to
Ac-(Cyclo-m,m)-[FAAAY]-NH2 (28). To a suspension of palla-
dium hydroxide on carbon (11.1 mg, 20 wt %, 0.016 mmol) in MeOH
(2.9 mL), (m,m)-cyclo Ac-F(3-Cl)AAAY-CO2Me (27) (57 mg, 0.079
mmol) and NH4OH (30% in H2O, 0.205 mL, 1.58 mmol) were added.
The reaction mixture was flushed with hydrogen gas and the reaction
mixture was stirred under hydrogen for 12 h at 40 °C. The crude
reaction mixture was filtered through a short plug of Celite (MeOH)
and concentrated in vacuo. Carrying this material forward without
further purification, the newly formed intermediate was dissolved in
THF (1.2 mL), MeOH (0.24 mL) and H2O (0.12 mL). LiOH (38 mg,
1.58 mmol) was added and the reaction mixture was stirred at rt for
5 h. The reaction mixture was acidified with 1 M aqueous HCl to
pH ∼ 2. The aqueous layer was extracted with EtOAc (5 × 15 mL).
The combined organic layers were dried (Na2SO4), filtered, and
concentrated in vacuo. Carrying this material forward without further
purification, the newly formed intermediate was dissolved in DMF
(0.7 mL) and CH2Cl2 (3.3 mL). To this solution, PyBOP (62 mg,
0.119 mmol) was added. After ammonia gas was bubbled through the
solution for 5 min, the reaction mixture was stirred at rt for 5 h. The
reaction mixture was concentrated in vacuo and the residue was
purified by an automated system (KP-Sil 10 g column; CH2Cl2/
MeOH 95:5 to CH2Cl2/MeOH 70:30) leading to cyclic peptide 28
(25 mg, 0.043 mmol, 55%). Note concerning the NMR data of the
following compound: Due to a mixture of conformers, the proton
assignment of 1H NMR data was carried out for the two major
compounds (1:0.2 ratio) in this mixture. The 13C NMR data represents a
1
mixture of all conformers. H NMR (600 MHz, d6-DMSO) δ 9.32 (s,
1H), 9.29 (s, 0.2H), 8.55 (d, J = 7.6 Hz, 1H), 8.50 (d, J = 7.8 Hz,
0.2H), 8.48 (d, J = 7.7 Hz, 1H), 8.36 (d, J = 7.9 Hz, 0.2H), 8.30 (d, J =
7.3 Hz, 0.2H), 8.24 (d, J = 6.5 Hz, 1H), 7.98 (d, J = 8.0 Hz, 0.2H),
3109
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7.85 (d, J = 8.2 Hz, 1H), 7.62 (d, J = 6.7 Hz, 0.2H), 7.58 (s, 1H), 7.49
(d, J = 7.7 Hz, 1H), 7.42 (d, J = 7.8 Hz, 0.2H), 7.41−7.35 (m, 1.4H),
7.31−7.26 (m, 2H), 7.26−7.20 (m, 1.2H), 7.20−7.17 (m, 1.2H), 7.15
(d, J = 7.7 Hz, 0.2H), 7.07 (s, 1.2H), 7.00−6.95 (m, 1.2H), 6.85−6.79
(m, J = 8.2 Hz, 1.2H), 4.42−4.36 (m, 1.2H), 4.27−4.16 (m, 4.4H),
4.16−4.08 (m, 1.4H), 3.18−3.04 (m, 1.2H), 2.93−2.85 (m, 1.6H),
2.85−2.79 (m, 2H), 1.87 (s, 3H), 1.85 (s, 0.6H), 1.22 (d, J = 7.4 Hz,
3H), 1.22−1.13 (m, 8.4H); 13C NMR (150 MHz, d6-DMSO) δ 172.9,
172.8, 172.4, 172.2, 171.9, 171.7, 171.5, 171.5, 170.9, 169.2, 169.1,
152.8, 138.6, 138.4, 131.6, 131.0, 130.2, 130.0, 129.3, 129.2, 128.4,
127.5, 127.5, 127.3, 127.2, 126.8, 126.7, 126.6, 125.5, 115.5, 54.5, 54.2,
54.2, 49.1, 48.5, 48.3, 48.2, 47.7, 47.4, 36.9, 36.5, 22.4, 18.8, 18.0, 17.3,
17.0, 16.9; HRMS (ESI) calcd for C29H37N6O7 581.2781; found,
581.2708.
7.18 (s, 1H), 4.66−4.60 (m, 1H), 4.60−4.55 (m, 1H), 4.14−4.07 (m,
2H), 3.23−3.19 (m, 1H), 3.11 (d, J = 13.7 Hz, 1H), 3.02−2.95 (m,
1H), 2.78−2.70 (m, 1H), 1.91−1.86 (m, 3H), 1.41 (s, 3H), 1.25 (s,
3H), 1.04 (d, J = 6.2 Hz, 3H), 0.96 (d, J = 6.3 Hz, 3H); HRMS (ESI)
calcd for C30H37ClN6O6 613.2536; found, 613.2532.
Ac-(Cyclo-m,m)-[(3-Cl)F(Aib)AAF]-NH2 (34). The pentapeptide
macrocyclization precursor was synthesized on solid support using
general procedure A. It was subjected to Suzuki−Miyaura macro-
cyclization general procedure C1. The macrocyclic product was
cleaved from the resin using general procedure D and acetylated using
general procedure F1. The resultant peptide was isolated as two
isomers, which were separated and purified by HPLC using general
procedure G1 to yield the solid 34a (3.0 mg, 2%) as a single peak
(purity >98%; see Supporting Information for pdf of HPLC trace): 1H
NMR (700 MHz, d6-DMSO) δ 8.41 (d, J = 9.5 Hz, 1H), 8.16 (d, J =
7.6 Hz, 1H), 7.74 (d, J = 7.8 Hz, 1H), 7.68−7.66 (m, 2H), 7.64 (s,
1H), 7.54−7.50 (m, 3H), 7.32 (d, J = 7.9 Hz, 2H), 7.28 (s, 1H), 7.19
(s, 1H), 7.17 (d, J = 7.5 Hz, 1H), 4.62−4.55 (m, 1H), 4.40−4.34 (m,
1H), 4.34−4.28 (m, 1H), 4.13−4.06 (m, 1H), 3.09 (dd, J = 13.7, 2.9
Hz, 1H), 3.07−3.02 (m, 1H), 2.96 (dd, J = 13.7, 9.3 Hz, 1H), 2.73
(t, J = 12.9 Hz, 1H), 1.92 (s, 3H), 1.47 (s, 3H), 1.16 (d, J = 6.8 Hz, 3H),
1.11 (d, J = 7.3 Hz, 3H), 1.05 (s, 3H); HRMS (ESI) calcd for
C30H37ClN6O6 613.2536; found, 613.2532; and the solid 34b (6.0 mg,
4%) as a single peak (purity >98%; see Supporting Information for pdf
of HPLC trace): 1H NMR (700 MHz, d6-DMSO) δ 8.52 (s, 1H), 8.15
(d, J = 7.2 Hz, 1H), 8.13 (d, J = 6.9 Hz, 1H), 8.12−8.05 (m, 1H),
7.93−7.89 (m, 1H), 7.86 (s, 1H), 7.74−7.68 (m, 1H), 7.56−7.50 (m,
3H), 7.35−7.30 (m, 2H), 7.23 (s, 1H), 7.19 (s, 1H), 4.50−4.44 (m,
2H), 4.36 (t, J = 7.2 Hz, 1H), 4.18−4.14 (m, 1H), 3.16−3.10 (m, 2H),
3.02−2.96 (m, 1H), 2.85 (t, J = 12.3 Hz, 1H), 1.95−1.92 (m, 3H),
1.51 (s, 3H), 1.33 (d, 3H), 1.28 (d, J = 6.4 Hz, 3H), 0.90−0.86 (m,
3H); HRMS (ESI) calcd for C30H37ClN6O6 613.2536; found,
613.2536.
Ac-(Cyclo-m,m)-[(α-Me)FAAAF]-NH2 (35). The pentapeptide
macrocyclization precursor was synthesized on solid support using
general procedure A. It was subjected to Suzuki−Miyaura macro-
cyclization general procedure C1. The macrocyclic product was
cleaved from the resin using general procedure D and acetylated using
general procedure F1. The resultant peptide was purified by HPLC
using general procedure G3 to yield the solid 35 (1.5 mg, 2%) as a
single peak (purity >98%; see Supporting Information for pdf of
HPLC trace): 1H NMR (700 MHz, d6-DMSO) δ 9.20 (s, 1H), 8.84 (s,
1H), 8.25−8.15 (m, 2H), 8.00−7.95 (m, 1H), 7.57−7.50 (m, 2H),
7.45 (d, J = 12.4 Hz, 1H), 7.42−7.36 (m, 2H), 7.33 (t, J = 7.5 Hz, 1H),
7.25 (d, J = 7.3 Hz, 1H), 7.17 (d, J = 6.2 Hz, 1H), 6.98 (s, 1H), 6.58−
6.50 (m, 1H), 4.21−4.17 (m, 1H), 4.00−3.93 (m, 2H), 3.93−3.86 (m,
1H), 3.23 (d, J = 12.1 Hz, 1H), 3.18−3.12 (m, 1H), 3.08−3.01 (m,
1H), 2.96 (d, J = 13.4 Hz, 1H), 1.92 (s, 3H), 1.45 (d, J = 7.1 Hz, 3H)
1.35 (d, J = 7.0 Hz, 3H), 0.95 (s, 3H), 0.89−0.85 (m, 3H); HRMS
(ESI) calcd for C30H38N6O6Na 601.2745; found, 601.2749.
(2R,4R)-Benzyl 4-Methyl-5-oxo-2-phenyloxazolidine-3-car-
boxylate (38). To a solution of (R)-2-(((benzyloxy)carbonyl)-
amino)propanoic acid (37) (10 g, 44.8 mmol) and (dimethoxymethyl)
benzene (6.82 g, 44.8 mmol) in THF (75 mL) at 0 °C was added
SOCl2 (3.27 mL, 44.8 mmol). After stirring the reaction mixture for 5
min, ZnCl2 (6.11 g, 44.8 mmol) was added and the reaction mixture
was stirred for 3 h at 0 °C. At his stage, another portion of SOCl2
(0.654 mL, 8.96 mmol) and ZnCl2 (1.22 g, 8.96 mmol) was added,
and the reaction mixture was stirred for an additional 1 h. The reaction
mixture was quenched by dropwise addition of water so that the
reaction temperature did not exceed 10 °C. It was extracted with Et2O
(200 mL). The organic phase was washed with water until almost
neutral, with saturated aqueous NaHCO3 (2 × 40 mL) and water (40
mL). The organic layer was dried (Na2SO4) and concentrated in
vacuo, and the residue was purified by an automated system (FLASH
65i column; hexanes/EtOAc 92:8 to hexanes/EtOAc 83:17) leading to
oxazolidine 38 (8.8 g, 28.3 mmol, 63%): 1H NMR (600 MHz, CDCl3)
δ 7.52−7.12 (m, 10H), δ 6.64 (br s, 1H), 5.23−5.12 (m, 2H), 4.52−
4.46 (m, 1H), 1.59 (d, J = 4.9 Hz, 3H); 13C NMR (150 MHz, CDCl3)
δ 172.3, 136.8, 135.2, 129.6, 128.7, 128.6, 128.5, 128.3, 127.9, 126.4,
Ac-(Cyclo-m,m)-[(3-Cl)FAAAF]-NH2 (31). The pentapeptide
macrocyclization precursor was synthesized on solid support using
general procedure A. It was subjected to Suzuki−Miyaura macro-
cyclization general procedure C1. The macrocyclic product was
cleaved from the resin using general procedure D and acetylated using
general procedure F1. The resultant peptide was purified by HPLC
using general procedure G4 to yield the solid 31 (15.0 mg, 12.5%
yield) as a single peak (purity >98%; see Supporting Information for
1
pdf of HPLC trace): H NMR (700 MHz, d6-DMSO) δ 8.59 (d, J =
8.0 Hz, 1H), 8.50 (d, J = 8.0 Hz, 1H), 8.30 (d, J = 6.4 Hz, 1H), 7.93
(d, J = 8.0 Hz, 1H), 7.88 (s, 1H), 7.70 (s, 1H), 7.58−7.54 (m, 2H),
7.49−7.44 (m, 2H), 7.34 (t, J = 7.7 Hz, 1H), 7.29 (s, 1H), 7.20 (d, J =
7.5 Hz, 1H), 7.15 (s, 1H), 4.47−4.42 (m, 2H), 4.31−4.24 (m, 2H),
4.17−4.12 (m, 1H), 3.06 (d, J = 15.0 Hz, 1H), 3.04−3.00 (m, 1H),
2.97−2.93 (m, 1H), 2.89 (dd, J = 14.7, 10.0 Hz, 1H), 1.87 (s, 3H),
1.22 (d, J = 7.5 Hz, 3H), 1.20 (d, J = 6.9 Hz, 3H), 1.17 (d, J = 7.3 Hz,
3H); HRMS (ESI) calcd for C29H35ClN6O6 599.2379; found,
599.2392.
Ac-(Cyclo-m,m)-[(3-Cl)FAA(Aib)F]-NH2 (32). The pentapeptide
macrocyclization precursor was synthesized on solid support using
general procedure A. It was subjected to Suzuki−Miyaura macro-
cyclization general procedure C1. The macrocyclic product was
cleaved from the resin using general procedure D and acetylated using
general procedure F1. The resultant peptide was purified by HPLC
using general procedure G2 to yield the solid 32 (9.2 mg, 6%) as a
single peak (purity >98%; see Supporting Information for pdf of
HPLC trace): 1H NMR (700 MHz, d6-DMSO) δ 8.47−8.44 (m, 1H),
8.02−7.97 (m, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.63−7.57 (m, 1H), 7.54
(d, J = 6.2 Hz, 1H), 7.52 (s, 1H), 7.44 (d, J = 8.0 Hz, 2H), 7.40 (s,
1H), 7.29 (d, J = 7.3 Hz, 2H), 7.25 (s, 1H), 7.18 (s, 2H), 4.61−4.56
(m, 1H), 4.56−4.52 (m, 1H), 4.20−4.15 (m, 1H), 3.88−3.82 (m, 1H),
3.15 (d, J = 13.7 Hz, 1H), 3.06 (d, J = 12.8 Hz, 1H), 2.93−2.84 (m,
2H), 1.85 (s, 3H), 1.42 (s, 3H), 1.28 (s, 3H), 1.11 (d, J = 6.7 Hz, 3H),
0.88 (d, J = 6.8 Hz, 3H); HRMS (ESI) calcd for C30H37ClN6O6
613.2536; found, 613.2557.
Ac-(Cyclo-m,m)-[(3-Cl)FA(Aib)AF]-NH2 (33). The pentapeptide
macrocyclization precursor was synthesized on solid support using
general procedure A. It was subjected to Suzuki−Miyaura macro-
cyclization general procedure C1. The macrocyclic product was
cleaved from the resin using general procedure D and acetylated using
general procedure F1. The resultant peptide was isolated as two
isomers, which were separated and purified by HPLC using general
procedure G1 to yield the solid 33a (5.0 mg, 4%) as a single peak
(purity >98%; see Supporting Information for pdf of HPLC trace): 1H
NMR (700 MHz, d6-DMSO) δ 8.30 (d, J = 9.1 Hz, 1H), 8.10 (d, J =
7.7 Hz, 1H), 8.04 (s, 1H), 7.70 (s, 1H), 7.64 (d, J = 5.3 Hz, 1H), 7.56
(s, 1H), 7.49−7.44 (m, 3H), 7.30 (d, J = 7.7 Hz, 2H), 7.22−7.16 (m,
3H), 4.56−4.53 (m, 1H), 4.45−4.40 (m, 1H), 4.30−4.22 (m, 1H),
4.16−4.10 (m, 1H), 3.09 (d, J = 12.9 Hz, 1H), 2.96−2.88 (m, 2H),
2.78 (t, J = 12.6 Hz, 1H), 1.89 (s, 3H), 1.35 (s, 3H), 1.18−1.13 (m,
6H), 0.91 (d, J = 6.7 Hz, 3H); HRMS (ESI) calcd for C30H37ClN6O6
613.2536; found, 613.2532; and the solid 33b (3 mg, 2%) as a single
peak (purity >98%; see Supporting Information for pdf of HPLC
1
trace): H NMR (700 MHz, d6-DMSO) δ 8.30 (s, 1H), 8.25 (d, J =
9.0 Hz, 1H), 8.15−8.09 (m, 1H), 7.74 (s, 1H), 7.68−7.63 (m, 1H),
7.51 (s, 1H), 7.49−7.44 (m, 2H), 7.32−7.25 (m, 4H), 7.23 (s, 1H),
3110
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126.1, 88.9, 67.8, 52.0; HRMS (ESI) calcd for C18H18NO4 312.1230;
found, 312.1228.
135.8, 129.8, 128.9, 94.1, 61.1, 52.7, 40.1, 23.9, 23.3; HRMS (ESI)
calcd for C13H17INO3 362.0248; found, 362.0250.
(S)-Methyl 2-Acetamido-2-methyl-3-(3-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)phenyl)propanoate (42). In a 100 mL
flask was (S)-methyl 2-acetamido-3-(3-iodophenyl)-2-methylpropa-
noate (41) (1.8 g, 4.98 mmol), Pd(dppf)Cl2·CH2Cl2 (182 mg, 0.249
mmol) B2pin2 (2.53 g, 9.97 mmol) and KOAc (1.96 g, 19.9 mmol) in
degassed DMSO (36 mL). The flask was sealed and heated to 85 °C
for 6 h. The reaction mixture was poured into brine/water (1:1, 40
mL) and extracted with EtOAc (2 × 80 mL). The combined organic
layers were washed with brine (3 × 40 mL), dried (Na2SO4), and
concentrated in vacuo, the residue was purified by an automated
system (Flash 40+M column; hexanes/EtOAc 65:35 to hexanes/
EtOAc 30:70) yielding boronic ester 42 (1.63 g, 4.51 mmol, 90%): 1H
NMR (500 MHz, CDCl3) δ 7.66 (d, J = 7.4 Hz, 1H), 7.48 (s, 1H),
7.26 (t, J = 7.4 Hz, 1H), 7.13 (d, J = 7.4 Hz, 1H), 6.00 (br s, 1H), 3.77
(s, 3H), 3.53 (d, J = 13.5 Hz, 1H), 3.19 (d, J = 13.5 Hz, 1H), 1.97 (s,
3H), 1.65 (s, 3H), 1.32 (s, 12H); 13C NMR (125 MHz, CDCl3) δ
174.3, 169.6, 136.2, 135.6, 133.1, 132.6, 127.6, 83.7, 61.1, 52.5, 40.7,
24.9, 24.8, 23.9, 23.1; HRMS (ESI) calcd for C19H29BNO5 362.2133;
found, 362.2138.
(S)-2-Acetamido-3-(3-boronophenyl)-2-methylpropanoic
Acid (36). To a stirred solution of (S)-methyl 2-acetamido-2-methyl-
3-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propanoate
(42) (361 mg, 1.0 mmol) in MeOH (4 mL) was added LiOH·H2O
(210 mg, 5.0 mmol) in H2O (4 mL) at rt. The mixture was stirred at
the same temperature for 12 h. The reaction mixture was acidified with
1 M aqueous HCl to pH ∼ 2. The aqueous layer was extracted with
EtOAc (4 × 50 mL). The combined organic layers were dried
(Na2SO4), filtered, and concentrated in vacuo providing crude acid 36.
Because of the instability of this material, it was carried forward
without further purification.
(2R,4S)-Benzyl 4-(3-Iodobenzyl)-4-methyl-5-oxo-2-phenyl-
oxazolidine-3-carboxylate (39). A solution of (2R,4R)-benzyl 4-
methyl-5-oxo-2-phenyloxazolidine-3-carboxylate (38) (6.5 g, 20.9
mmol) and 3-iodo-benzyl bromide (6.2 g, 20.88) in THF (42 mL)
was added dropwise at −30 °C to a solution of LiHMDS (1 M in
THF, 22.1 mL, 22.1 mmol) diluted in THF (167 mL). The reaction
mixture was stirred at this temperature for 1 h and then allowed to
warm to rt and stirred for 3 h. Saturated aqueous NaHCO3 (100 mL)
was added and the mixture was extracted with Et2O (2 × 200 mL).
The organic layer was dried (Na2SO4) and concentrated in vacuo, and
the residue was purified by an automated system (FLASH 65i column;
hexanes/EtOAc 95:5 to hexanes/EtOAc 81:19) leading to oxazolidine
39 (7.9 g, 14.98 mmol, 72%). Note concerning the NMR data of the
following compound: Due to a mixture of rotamers, the proton assignment
1
of H NMR data was carried out for the two compounds in this mixture
(3:1 ratio). The 13C NMR data represents a mixture of the two rotamers.
1H NMR (600 MHz, CDCl3) δ 7.64 (d, J = 7.8 Hz, 1H), 7.61 (s, 1H),
7.49 (d, J = 7.1 Hz, 0.6H), 7.44 (t, J = 7.2 Hz, 0.6H), 7.42−7.33 (m,
2.6H), 7.33−7.27 (m, 2.9H), 7.27−7.24 (m, 0.9H), 7.21 (t, J = 7.3 Hz,
2H), 7.17 (d, J = 7.2 Hz, 2H), 7.12 (d, J = 7.6 Hz, 1H), 7.00−6.97 (t, J =
7.8 Hz, 1.3H), 6.89−6.84 (m, 2.3H), 5.52 (s, 0.3H), 5.38 (d, J = 12.0
Hz, 0.3H), 5.36 (s, 1H), 5.13 (d, J = 12.0 Hz, 0.3H), 5.07 (d, J = 12.2
Hz, 1H), 5.00 (d, J = 12.2 Hz, 1H), 3.72 (d, J = 13.5 Hz, 1H), 3.33 (d,
J = 13.7 Hz, 0.3H), 3.07 (d, J = 13.5 Hz, 1H), 3.02 (d, J = 13.7 Hz,
0.3H), 1.95 (s, 3H), 1.87 (s, 1H); 13C NMR (150 MHz, CDCl3) δ
174.0, 173.8, 152.2, 151.9, 138.6, 138.3, 137.5, 136.9, 136.7, 136.6,
136.5, 136.0, 134.9, 134.9, 130.4, 129.8, 129.7, 128.9, 128.9, 128.8,
128.6, 128.5, 128.4, 128.2, 128.1, 127.9, 126.7, 126.7, 94.6, 94.5, 89.4,
89.2, 68.0, 67.5, 64.5, 64.0, 41.9, 40.2, 24.9, 23.9; HRMS (ESI) calcd
for C25H23INO4 528.0666; found, 528.0675.
Ac-(Cyclo-m,o)-[(3-Cl)FAF]-NH2 (43). The tripeptide macro-
cyclization precursor was synthesized on solid support using general
procedure A. It was subjected to Suzuki−Miyaura macrocyclization
general procedure C1. The macrocyclic product was cleaved from the
resin using general procedure D and acetylated using general
procedure F1. The resultant peptide was purified by HPLC using
general procedure G2 to yield the solid 43 (23.0 mg, 25%) as a single
peak (purity >98%; see Supporting Information for pdf of HPLC
(S)-2-Amino-3-(3-iodophenyl)-2-methylpropanoic Acid (40).
A mixture of (2R,4S)-benzyl 4-(3-iodobenzyl)-4-methyl-5-oxo-2-
phenyloxazolidine-3-carboxylate (39) (1.35 g, 2.56 mmol) and
KOSiMe3 (90% pure, 1.10 g, 7.68 mmol) was suspended in THF
(45 mL) and heated to 75 °C for 2.5 h. MeOH (75 mL) was added
and the reaction mixture was concentrated in vacuo. The residue was
redissolved in MeOH and applied to a 20 g SCX-2 ion exchange
cartridge (0.59 mmol/g loading) eluting with MeOH and then with
Et3N (0.2 M in MeOH). The Et3N/MeOH fraction was concentrated
in vacuo leading to amino acid 40 (0.72 g, 2.36 mmol, 92%): 1H NMR
(600 MHz, CD3OD) δ 7.69 (s, 1H), 7.65 (d, J = 7.7 Hz, 1H), 7.29 (d,
J = 7.7 Hz, 1H), 7.10 (t, J = 7.7 Hz, 1H), 3.23 (d, J = 14.1 Hz, 1H),
2.86 (d, J = 14.1 Hz, 1H), 1.49 (s, 3H); 13C NMR (150 MHz,
CD3OD) δ 175.6, 140.3, 138.7, 137.8, 131.5, 130.7, 95.3, 62.8, 43.7,
23.6; HRMS (ESI) calcd for C10H13INO2 305.9986; found, 305.9984.
(S)-Methyl 2-Acetamido-3-(3-iodophenyl)-2-methylpropa-
noate (41). To MeOH (46 mL), SOCl2 (1.91 mL, 26.2 mmol) was
added dropwise at 0 °C. (S)-2-amino-3-(3-iodophenyl)-2-methylpro-
panoic acid (40) (0.72 g, 2.36 mmol) in MeOH (46 mL) was added,
and after stirring for 30 min at 0 °C, the reaction mixture was allowed
to warm to rt After 2 h, the reaction mixture was concentrated in
vacuo. Carrying this material forward without further purification, the
newly formed intermediate was suspended in CH2Cl2 (131 mL). To
this suspension, DIPEA (2.86 mL, 16.4 mmol), Ac2O (1.24 mL, 13.1
mmol), and DMAP (16 mg, 0.13 mmol) were added at 0 °C. After
stirring for 12 h at rt, the reaction mixture was concentrated in vacuo.
The residue was redissolved in MeONa (0.2 M in MeOH, 100 mL,
20.0 mmol) and heated to reflux for 3 h. The reaction mixture was
concentrated in vacuo and the residue was taken up in EtOAc (150
mL) and washed with water/brine (1:1, 2 × 80 mL). The organic layer
was dried (Na2SO4) and concentrated in vacuo and the residue was
purified by an automated system (Flash 40+M column; hexanes/
EtOAc 90:10 to hexanes/EtOAc 40:60) yielding amino acid 41 (1.84
1
trace): H NMR (700 MHz, d6-DMSO) δ 8.18 (d, J = 7.1 Hz, 1H),
7.60 (s, 1H), 7.49−7.35 (m, 3H), 7.32 (t, J = 7.2 Hz, 1H), 7.27 (t, J =
7.4 Hz, 1H), 7.25 (s, 1H), 7.23 (s, 1H), 7.16−7.12 (m, 2H), 6.98 (br s,
1H), 4.61−4.44 (m, 1H), 4.38−4.31 (m, 1H), 4.31−4.26 (m, 1H),
3.28−3.16 (m, 1H), 3.06−2.90 (m, 2H), 2.80 (br s, 1H), 1.88 (s, 3H),
0.99 (d, J = 7.1 Hz, 3H). HRMS (ESI) calcd for C23H25ClN4O4
457.1637; found, 457.1646.
Ac-(Cyclo-m,o)-[(3-Cl)FAAF]-OH (44). The tetrapeptide macro-
cyclization precursor was synthesized on solid support using general
procedure A. It was subjected to Suzuki−Miyaura macrocyclization
general procedure C1. The macrocyclic product was cleaved from the
resin using general procedure D and acetylated using general
procedure F1. The resultant peptide was purified by HPLC using
general procedure G3 to yield the solid 44 (2.0 mg, 2%) as a single
peak (purity >98%; see Supporting Information for pdf of HPLC
1
trace): H NMR (700 MHz, d6-DMSO) δ 8.35 (d, J = 6.1 Hz, 1H),
8.15 (s, 1H), 7.66 (d, J = 6.8 Hz, 1H), 7.33−7.29 (m, 3H), 7.28−7.24
(m, 2H), 7.21 (d, J = 7.1 Hz, 1H), 7.18 (s, 1H), 7.15−7.11 (m, 1H),
7.07 (s, 1H), 7.05 (s, 1H), 4.80−4.76 (m, 1H), 4.38−4.33 (m, 1H),
4.01−3.96 (m, 2H), 3.18−3.12 (m, 1H), 3.06−3.01 (m, 1H), 2.98 (d, J =
12.3 Hz, 1H), 2.96−2.91 (m, 1H), 1.92 (s, 3H), 1.22 (d, J = 7.4 Hz,
3H), 0.97 (d, J = 7.1 Hz, 3H); HRMS (ESI) calcd for C26H29ClN4O6
529.1854 ; found, 529.1866.
Ac-(Cyclo-m,o)-[(3-Cl)FAAAF]-NH2 (45). The pentapeptide
macrocyclization precursor was synthesized on solid support using
general procedure A. It was subjected to Suzuki−Miyaura macro-
cyclization general procedure C1. The macrocyclic product was
cleaved from the resin using general procedure D and acetylated using
general procedure F1. The resultant peptide was purified by HPLC
using general procedure G1 to yield the solid 45 (1.5 mg, 1%) as a
1
g, 5.09 mmol, 78%): H NMR (600 MHz, CDCl3) δ 7.57−7.53 (m,
1H), 7.40 (s, 1H), 7.02−6.96 (m, 2H), 6.08 (br s, 1H), 3.79 (s, 3H),
3.53 (d, J = 13.5 Hz, 1H), 3.13 (d, J = 13.5 Hz, 1H), 1.98 (s, 3H), 1.64
(s, 3H); 13C NMR (150 MHz, CDCl3) δ 174.1, 169.6, 138.9, 138.8,
3111
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single peak (purity >98%; see Supporting Information for pdf of
HPLC trace): 1H NMR (700 MHz, d6-DMSO) δ 8.36 (d, J = 7.7 Hz,
1H), 8.20−8.13 (m, 2H), 8.11−8.06 (m, 1H), 7.44 (d, J = 8.3 Hz,
1H), 7.32 (s, 1H), 7.30 (d, J = 9.5 Hz, 1H), 7.28−7.26 (m, 2H), 7.25
(s, 1H), 7.19 (s, 1H), 7.17 (d, J = 7.3 Hz, 1H), 7.15 (s, 1H), 7.08−7.04
(m, 1H), 4.46 (d, J = 6.9 Hz, 1H), 4.28 (d, J = 8.6 Hz, 1H), 4.11 (t, J =
6.6 Hz, 1H), 4.03 (dd, J = 11.3, 3.8 Hz, 1H), 3.97 (t, J = 6.5 Hz, 1H),
3.09−3.03 (m, 1H), 3.01−2.96 (m, 1H), 2.96−2.92 (m, 2H), 1.87 (s,
3H), 1.13 (d, J = 6.6 Hz, 3H), 1.12−1.10 (m, 3H), 1.08 (d, J = 6.6 Hz,
3H); HRMS (ESI) calcd for C29H35ClN6O6 599.2379; found,
599.2380.
101.5, 101.4, 101.4, 89.3, 68.0, 67.3, 64.0, 63.6, 45.8, 44.5, 25.4, 25.4,
24.4; HRMS (ESI) calcd for C25H22INO4 528.0666; found, 528.0669.
(S)-2-Amino-3-(2-iodophenyl)-2-methylpropanoic Acid (52).
A mixture of (2R,4S)-benzyl 4-(2-iodobenzyl)-4-methyl-5-oxo-2-
phenyloxazolidine-3-carboxylate (51) (2.0 g, 3.79 mmol) and
KOSiMe3 (90% pure, 1.62 g, 11.4 mmol) was suspended in THF
(63 mL) and heated to 75 °C for 2.5 h. MeOH (100 mL) was added
and the reaction mixture was concentrated in vacuo. The residue was
redissolved in MeOH and applied to a 40 g SCX-2 ion exchange
cartridge (0.59 mmol/g loading) eluting with MeOH and then with
Et3N (0.2 M in MeOH). The Et3N/MeOH fraction was concentrated
in vacuo leading to amino acid 52 (1.13 g, 3.7 mmol, 98%): 1H NMR
(600 MHz, CD3OD) δ 7.90 (d, J = 7.6 Hz, 1H), 7.43 (d, J = 7.6 Hz,
1H), 7.34 (t, J = 7.6 Hz, 1H), 6.99 (t, J = 7.6 Hz, 1H), 3.40 (d, J = 14.5
Hz, 1H), 3.36 (d, J = 14.5 Hz, 1H), 1.52 (s, 3H); 13C NMR (150
MHz, CD3OD) δ 176.0, 141.3, 139.7, 132.2, 130.2, 129.7, 103.3, 63.5,
47.5, 23.2; HRMS (ESI) calcd for C10H13INO2 305.9986; found,
305.9987.
(S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-io-
dophenyl)-2-methylpropanoic Acid (50). A mixture of (S)-2-
amino-3-(2-iodophenyl)-2-methylpropanoic acid (52) (575 mg, 1.89
mmol) and TMSCl (0.48 mL, 3.77 mmol) was suspended in CH2Cl2
(20 mL) and heated to reflux for 6 h. DIPEA (0.69 mL, 3.96 mmol)
and FmocCl (0.54 g, 2.07 mmol) were added to the reaction mixture
at 0 °C. The reaction mixture was allowed to warm to rt and stirred for
30 h. The reaction mixture was concentrated in vacuo and residue was
redissolved in EtOAc (100 mL). The organic layer was washed with 1
M HCl (2 × 30 mL) and brine (30 mL). The organic layer was dried
(Na2SO4) and concentrated in vacuo, and the residue was purified by
an automated system (KP-Sil 25 g column; CH2Cl2/MeOH 95:5 to
CH2Cl2/MeOH 80:20) leading to Fmoc-carbamate 50 (570 mg, 1.08
mmol, 57%): 1H NMR (600 MHz, CDCl3) δ 7.84 (d, J = 7.1 Hz, 1H),
7.77 (d, J = 6.8 Hz, 2H), 7.60 (t, J = 7.7 Hz, 2H), 7.40 (t, J = 7.0 Hz,
2H), 7.31 (t, J = 7.1 Hz, 2H), 7.19 (t, J = 6.4 Hz, 1H), 7.04 (br s, 1H),
6.91 (t, J = 6.3 Hz, 1H), 5.29 (s, 1H), 4.58−4.48 (m, 1H), 4.48−4.36
(m, 1H), 4.23 (t, J = 6.3 Hz, 1H), 3.62−3.43 (m, 2H), 1.57 (s, 3H);
13C NMR (150 MHz, CDCl3) δ 177.8, 155.1, 143.7, 141.3, 139.9,
Ac-(Cyclo-m,o)-[(3-Cl)FAAA(α-Me)F]-NH2 (48). The pentapep-
tide macrocyclization precursor was synthesized on solid support using
general procedure B. It was subjected to Suzuki−Miyaura macro-
cyclization general procedure C2. The macrocyclic product was
cleaved from the resin using general procedure E and acetylated using
general procedure F2. The resultant peptide was purified by HPLC
using general procedure G6 to yield the solid 48 (2 mg, 2%) as a single
peak (purity >98%; see Supporting Information for pdf of HPLC
1
trace): H NMR (700 MHz, d6-DMSO) δ 8.50−8.44 (m, 1H), 8.29−
8.23 (m, 1H), 8.22−8.14 (m, 1H), 8.01 (d, J = 6.9 Hz, 1H), 7.63 (s,
1H), 7.40−7.34 (m, 1H), 7.30−7.27 (m, 1H), 7.26−7.20 (m, 3H),
7.20−7.16 (m, 1H), 7.13 (s, 1H), 7.05 (d, J = 7.3 Hz, 2H), 4.51−4.46
(m, 1H), 4.28−4.21 (m, 2H), 4.08−4.03 (m, 1H), 3.31 (d, J = 13.8
Hz, 1H), 3.26−3.20 (m, 1H), 3.01 (d, J = 13.4 Hz, 1H), 2.71 (t, J =
13.0 Hz, 1H), 1.75 (s, 3H), 1.33 (s, 3H), 1.25−1.17 (m, 9H); HRMS
(ESI) calcd for C30H38ClN6O6 613.2536; found, 613.2531.
Ac-(Cyclo-m,o)-[(3-Cl)F(Aib)AAF]-NH2 (49). The pentapeptide
macrocyclization precursor was synthesized on solid support using
general procedure B. It was subjected to Suzuki−Miyaura macro-
cyclization general procedure C2. The macrocyclic product was
cleaved from the resin using general procedure E and acetylated using
general procedure F2. The resultant peptide was purified by HPLC
using general procedure G3 to yield the solid 49 (5 mg, 4%) as a single
peak (purity >98%; see Supporting Information for pdf of HPLC
1
trace): H NMR (700 MHz, d6-DMSO) δ 8.84 (s, 1H), 8.68 (s, 1H),
7.53 (d, J = 7.6 Hz, 1H), 7.44 (d, J = 7.1 Hz, 1H), 7.37 (d, J = 7.8 Hz,
1H), 7.32 (t, J = 7.5 Hz, 1H), 7.26 (t, J = 7.4 Hz, 1H), 7.22 (s, 1H),
7.20−7.17 (m, 3H), 7.13 (s, 1H), 7.10 (s, 1H), 6.92 (s, 1H), 4.58−
4.51 (m, 1H), 4.32−4.26 (m, 1H), 4.03 (p, J = 7.2 Hz, 1H), 3.92 (p,
J = 7.3 Hz, 1H), 3.27 (dd, J = 13.6, 6.3 Hz, 1H), 3.04 (dd, J = 14.9, 6.3
Hz, 1H), 2.92 (dd, J = 13.5, 10.6 Hz, 1H), 2.59 (dd, J = 14.8, 8.5 Hz,
1H), 1.98 (s, 3H), 1.27 (s, 3H), 1.24 (d, J = 7.4 Hz, 3H), 1.08 (s, 3H),
0.95 (d, J = 7.1 Hz, 3H); HRMS (ESI) calcd for C30H38ClN6O6
613.2536; found, 613.2538.
139.0, 131.1, 128.7, 128.1, 127.7, 127.0, 125.0, 119.9, 102.7, 66.6, 60.2,
47.2, 44.3, 23.2; HRMS (ESI) calcd for C25H23INO4 528.0676; found,
528.0666.
Ac-(Cyclo-o,m)-[FAAAF]-NH2 (55). The pentapeptide macro-
cyclization precursor was synthesized on solid support using general
procedure B. It was subjected to Suzuki−Miyaura macrocyclization
general procedure C2. The macrocyclic product was cleaved from the
resin using general procedure E and acetylated using general procedure
F2. The resultant peptide was purified by HPLC using general
procedure G3 to yield the solid 55 (22 mg, 8%) as a single peak
(purity >98%; see Supporting Information for pdf of HPLC trace): 1H
NMR (700 MHz, d6-DMSO) δ 8.37 (d, J = 6.4 Hz, 1H), 8.31 (d, J =
8.6 Hz, 1H), 7.77 (br s, 1H), 7.67 (br s, 1H), 7.41 (d, J = 7.6 Hz, 1H),
7.38−7.23 (m, 4H), 7.23−7.18 (m, 2H), 7.18−7.08 (m, 3H), 4.67−
4.63 (m, 1H), 4.32 (br s, 1H), 4.05 (p, J = 6.9 Hz, 1H), 3.99 (p, J = 6.5
Hz, 1H), 3.89−3.83 (m, 1H), 3.18 (dd, J = 14.7, 4.0 Hz, 1H), 3.13−
3.05 (m, 2H), 2.78−2.73 (m, 1H), 1.85 (s, 3H), 1.25 (d, J = 7.4 Hz,
3H), 1.19 (d, J = 7.1 Hz, 3H), 1.13 (d, J = 7.0 Hz, 3H); HRMS (ESI)
calcd for C29H36N6O6 565.2769; found, 565.2773.
Ac-(Cyclo-o,m)-[F(Aib)AAF]-NH2 (56). The pentapeptide macro-
cyclization precursor was synthesized on solid support using general
procedure B. It was subjected to Suzuki−Miyaura macrocyclization
general procedure C2. The macrocyclic product was cleaved from the
resin using general procedure E and acetylated using general procedure
F2. The resultant peptide was purified by HPLC using general
procedure G3 to yield the solid 56 (7.6 mg, 7%) as a single peak
(purity >98%; see Supporting Information for pdf of HPLC trace): 1H
NMR (700 MHz, d6-DMSO) δ 8.52 (s, 1H), 8.32 (s, 1H), 7.83 (d, J =
6.8 Hz, 1H), 7.59 (d, J = 7.1 Hz, 1H), 7.51 (d, J = 8.2 Hz, 1H), 7.42
(d, J = 7.5 Hz, 1H), 7.33−7.21 (m, 5H), 7.14−7.08 (m, 4H), 4.52 (s,
1H), 4.32−4.24 (m, 1H), 4.03 (t, J = 7.2 Hz, 1H), 3.88 (t, J = 7.0 Hz,
1H), 3.17 (dd, J = 14.5, 4.0 Hz, 1H), 3.13 (dd, J = 14.3, 3.9 Hz, 1H), 3.09−
3.02 (m, 1H), 2.92−2.85 (m, 1H), 1.83 (s, 3H), 1.29 (d, J = 3.8 Hz, 6H),
(2R,4S)-Benzyl 4-(2-Iodobenzyl)-4-methyl-5-oxo-2-phenyl-
oxazolidine-3-carboxylate (51). A solution of (2R,4R)-benzyl 4-
methyl-5-oxo-2-phenyloxazolidine-3-carboxylate (38) (8.82 g, 28.3
mmol) and 2-iodo-benzyl bromide (8.41 g, 28.3 mmol) in THF (38
mL) was added dropwise at −30 °C to a solution of LiHMDS (1 M in
THF, 31.2 mL, 31.2 mmol) diluted in THF (151 mL). The reaction
mixture was stirred at this temperature for 1 h and then allowed to
warm to rt and stirred for 3 h. Saturated aqueous NaHCO3 (100 mL)
was added and the mixture was extracted with Et2O (2 × 200 mL).
The organic layer was dried (Na2SO4) and concentrated in vacuo, and
the residue was purified by an automated system (KP-Sil 340 g
column; hexanes/EtOAc 95:5 to hexanes/EtOAc 82:18) leading to
oxazolidine 51 (11.38 g, 21.58 mmol, 76%). Note concerning the NMR
data of the following compound: Due to a mixture of rotamers, the proton
assignment of 1H NMR data was carried out for the two compounds in this
mixture (2:1 ratio). The 13C NMR data represents a mixture of the two
rotamers. 1H NMR (600 MHz, CDCl3) δ 7.90−7.87 (m, 1.5H), 7.41−
7.28 (m, 8H), 7.28−7.12 (m, 7.5H), 7.09−7.03 (m, 0.5H), 6.98−6.91
(m, 1.5H), 6.84−6.78 (m, 2H), 5.89 (br s, 0.5H), 5.72 (s, 1H), 5.27−
5.14 (m, 1H), 4.98 (d, J = 12.2 Hz, 1H), 4.95 (d, J = 12.2 Hz, 1H),
3.90−3.80 (m, 1H), 3.63 (d, J = 13.4 Hz, 0.5H), 3.43−3.38 (m, 1.5H),
2.04 (s, 3H), 1.92 (s, 1.5H); 13C NMR (150 MHz, CDCl3) δ 173.2,
173.2, 173.1, 173.1, 173.0, 152.3, 152.2, 151.6, 140.5, 138.4, 138.0,
137.9, 136.8, 136.2, 136.2, 135.1, 130.5, 130.1, 129.8, 129.2, 129.2,
129.1, 128.6, 128.4, 128.3, 128.2, 128.2, 128.1, 127.9, 126.7, 101.5,
3112
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1.20 (d, J = 7.1 Hz, 3H), 1.03 (d, J = 7.3 Hz, 3H); HRMS (ESI) calcd
for C30H38N6O6 579.2925; found, 579.2923.
The resultant peptide (59) was subjected to hydrogenation using 10
mol % Pd/C in DMF for 24 h to yield the crude product, which was
purified by HPLC using general procedure G5 to yield the solid 63
(79 mg, 50%) as a single peak (purity >98%; see Supporting
Information for pdf of HPLC trace): 1H NMR (700 MHz, d6-DMSO)
δ 9.08−8.40 (m, 3H), 8.01−7.38 (m, 4H), 7.38−6.92 (m, 9H), 4.39−
3.50 (m, obscured by H2O peak, baseline correction shows 5H), 3.01−
2.79 (m, 2H), 2.79−2.65 (m, 1H), 2.65−2.53 (m, 1H), 2.48−2.40 (m,
1H), 2.25−2.17 (m, 1H), 2.12−1.93 (m, 1H), 1.62−1.48 (m, 3H), 1.48−
1.37 (m, 1H), 1.36−1.20 (m, 2H), 1.08−0.85 (m, 8H), 0.84−0.63
(m, 3H); HRMS (ESI) calcd for C33H45N7O6 636.3504; found, 636.3501.
(S)-Methyl 3-(2-Bromophenyl)-2-((tert-butoxycarbonyl)-
amino)propanoate (66). To a suspension of (S)-2-((tert-
butoxycarbonyl)amino)-3-(2-bromophenyl)propanoic acid (65) (4.0 g,
11.6 mmol) and NaHCO3 (1.95 g, 23.2 mmol) in DMF (39 mL),
methyl iodide (3.63 mL, 58.1 mmol) was added and stirred at room
temperature for 12 h. The reaction mixture was poured into water
(100 mL) and extracted with EtOAc (2 × 150 mL). The combined
organic phases were washed with brine (100 mL), dried (Na2SO4),
and concentrated in vacuo. The residue was purified by an automated
system (Flash 40+M column; hexanes/EtOAc 100:0 to hexanes/
Ac-(Cyclo-o,m)-[FAA(Aib)F]-NH2 (57). The pentapeptide macro-
cyclization precursor was synthesized on solid support using general
procedure B. It was subjected to Suzuki−Miyaura macrocyclization
general procedure C2. The macrocyclic product was cleaved from the
resin using general procedure E and acetylated using general procedure
F2. The resultant peptide was purified by HPLC using general
procedure G3 to yield the solid 57 (7.8 mg, 7%) as a single peak
(purity >98%; see Supporting Information for pdf of HPLC trace): 1H
NMR (700 MHz, d6-DMSO) δ 8.58 (s, 1H), 8.46 (s, 1H), 8.21 (s,
1H), 8.02 (s, 1H), 7.44−7.35 (m, 1H), 7.34−7.31 (m, 1H), 7.31−7.27
(m, 2H), 7.27−7.23 (m, 2H), 7.22−7.17 (m, 1H), 7.09−7.05 (m, 1H),
7.04−6.99 (m, 1H), 6.92 (s, 1H), 6.85−6.79 (m, 1H), 4.75−4.68 (m,
1H), 4.29−4.10 (m, 1H), 4.03−3.95 (m, 1H), 3.73 (s, 1H), 3.32−3.21
(m, 2H), 3.21−3.15 (m, 1H), 3.10−2.97 (m, 1H), 1.84−1.74 (m, 3H),
1.38−1.31 (m, 3H), 1.27−1.20 (m, 6H), 1.17−1.12 (m, 3H); HRMS
(ESI) calcd for C30H38N6O6 579.2925; found, 579.2924.
Ac-(Cyclo-o,m)-[FAF]-NH2 (60). The tripeptide macrocyclization
precursor was synthesized on solid support using general procedure B.
It was subjected to Suzuki−Miyaura macrocyclization general
procedure C2. The macrocyclic product was cleaved from the resin
using general procedure E and acetylated using general procedure F2.
The resultant peptide was purified by HPLC using general procedure
G1 to yield the solid 60 (3.6 mg, 4%) as a single peak (purity >98%;
1
EtOAc 85:15) to give ester 66 (4.06 g, 11.33 mmol, 98% yield): H
NMR (500 MHz, CDCl3) δ 7.54 (d, J = 7.9 Hz, 1H), 7.26−7.18 (m,
2H), 7.10 (t, J = 6.8 Hz, 1H), 5.07 (d, J = 6.7 Hz, 1H), 4.64 (dd, J =
13.7, 7.3 Hz, 1H), 3.71 (s, 3H), 3.30 (dd, J = 13.6, 5.8 Hz, 1H), 3.10
(dd, J = 13.6, 8.4 Hz, 1H), 1.37 (s, 9H); 13C NMR (125 MHz, CDCl3)
δ 172.3, 154.9, 136.0, 132.8, 131.2, 128.5, 127.4, 125.0, 79.8, 53.5, 52.3,
38.6, 28.2; HRMS (ESI) calcd for C15H21BrNO4 358.0654; found,
358.0649.
1
see Supporting Information for pdf of HPLC trace): H NMR (500
MHz, d6-DMSO) δ 8.11 (br s, 1H, NH), 7.71 (br s, 1H, NH), 7.52 (s,
1H, NH), 7.46−7.43 (m, 1H, Ar−H), 7.41−7.26 (m, 5H, Ar−H), 7.25
(s, 2H, NH2), 7.14 (dd, J = 7.5 Hz, 1.5, 1H, Ar−H), 7.08 (dt, J = 7.5,
1.5 Hz, 1H, Ar−H), 4.62 (td, J = 9.0, 4.4 Hz, 1H, CHN), 4.36−4.30
(m, 2H, CHN), 3.28 (d, J = 14.8 Hz, 1H, CH2Ar), 3.20−3.13 (m, 1H,
CH2Ar), 2.98−2.89 (m, 1H, CH2Ar), 2.84 (dd, J = 14.7, 6.9 Hz, 1H,
CH2Ar), 1.95 (s, 3H, Ac), 1.09 (d, J = 6.8 Hz, 3H, CHCH3). HRMS
(ESI) calcd for C23H27N4O4 423.2027; found, 423.2035.
Ac-(Cyclo-o,m)-[FAAF]-NH2 (61). The tetrapeptide macrocycliza-
tion precursor was synthesized on solid support using general
procedure B. It was subjected to Suzuki−Miyaura macrocyclization
general procedure C2. The macrocyclic product was cleaved from the
resin using general procedure E and acetylated using general procedure
F2. The resultant peptide was purified by HPLC using general
procedure G1 to yield the solid 61 (3.9 mg, 4%) as a single peak
(purity >98%; see Supporting Information for pdf of HPLC trace): 1H
NMR (500 MHz d6-DMSO) δ 8.58 (d, J = 7.4 Hz, 1H, NH), 8.34 (s,
2H, NH2), 8.25 (d, J = 9.5 Hz, 1H, NH), 7.98 (d, J = 8.3 Hz, 1H, NH),
7.47 (s, 1H, NH), 7.28−7.15 (m, 4H, Ar−H), 7.10−6.99 (m, 4H, Ar−
H), 4.93 (m, 1H, CHN), 4.68 (m, 1H, CHN), 3.98−3.87 (m, 2H,
CHN), 3.12 (d, J = 13.2 Hz, 2H, CH2Ar), 2.75 (dd, J = 15.2 Hz, 11.9,
1H, CH2Ar), 2.61−2.54 (m, 1H, CH2Ar), 1.68 (s, 3H, Ac), 1.14 (d, J =
7.4 Hz, 3H, CHCH3), 1.02 (d, J = 6.6 Hz, 3H, CHCH3). HRMS (ESI)
calcd for C26H32N5O5 494.2398; found, 494.2400.
(S)-Methyl 2-((tert-Butoxycarbonyl)amino)-3-(2-(4,4,5,5-tet-
ramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propanoate (67). In
a 250 mL flask was (S)-methyl 3-(2-bromophenyl)-2-((tert-
butoxycarbonyl)amino)propanoate (65) (4.06 g, 11.33 mmol),
Pd(dppf)Cl2·CH2Cl2 (415 mg, 0.567 mmol), B2pin2 (4.32 g, 17.0
mmol), and KOAc (4.45 g, 45.3 mmol) in degassed dioxane (113 mL).
The flask was sealed and heated to 85 °C for 3 h. The reaction mixture
was poured into brine/water (1:1, 80 mL) and extracted with EtOAc
(2 × 100 mL). The combined organic layers were washed with brine
(80 mL), dried (Na2SO4), and concentrated in vacuo; the residue was
purified by a Biotage system (Flash 40+M column; hexanes/EtOAc
91:9 to hexanes/EtOAc 80:20) yielding boronic ester 67 and Boc-Phe-
CO2Me (2.84 g). This mixture was submitted to HPLC purification
1
yielding boronic ester 67 (1.62 g, 4.0 mmol, 35%): H NMR (600
MHz, CDCl3) δ 7.81 (d, J = 7.2 Hz, 1H), 7.40 (dt, J = 7.6, 1.2 Hz,
1H), 7.29−7.21 (m, 3H), 5.95 (d, J = 8.1 Hz, 1H), 4.37 (ddd, J = 10.7,
8.1, 4.2 Hz, 1H), 3.75 (s, 3H), 3.29−3.23 (m, 1H), 3.20 (dd, J = 13.3,
4.2 Hz, 1H), 1.39 (s, 6H), 1.38 (s, 6H), 1.32 (s, 9H); 13C NMR (150
MHz, CDCl3) δ 173.3, 155.5, 143.5, 136.1, 131.4, 130.0, 126.1, 84.0,
79.2, 56.2, 52.0, 37.1, 28.2, 24.9, 24.6; HRMS (ESI) calcd for
C21H33BNO6 406.2395; found, 406.2402.
(S)-2-((tert-Butoxycarbonyl)amino)-3-(2-(4,4,5,5-tetrameth-
yl-1,3,2-dioxaborolan-2-yl)phenyl)propanoic Acid (64). To a
stirred solution of (S)-methyl 2-((tert-butoxycarbonyl)amino)-3-(2-
(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propanoate (67)
(405 mg, 1.0 mmol) in MeOH (4 mL) was added LiOH·H2O (121
mg, 3.0 mmol) in H2O (4 mL) at rt. The mixture was stirred at the
same temperature for 50 min. The reaction mixture was acidified with
1 M aqueous HCl to pH ∼ 2. The aqueous layer was extracted with
EtOAc (3 × 50 mL). The combined organic layers were dried
(Na2SO4), filtered, and concentrated in vacuo providing crude acid 64.
Because of the instability of this material, it was carried forward
without further purification.
Ac-(Cyclo-o,m)-[FAKAF]-NH2 (62). The pentapeptide macro-
cyclization precursor was synthesized on solid support using general
procedure B. It was subjected to Suzuki−Miyaura macrocyclization
general procedure C2. The macrocyclic product was cleaved from the
resin using general procedure E and acetylated using general procedure F2.
The resultant peptide (58) was subjected to hydrogenation using
10 mol % Pd/C in DMF for 24 h to yield the crude product, which
was purified by HPLC using general procedure G5 to yield the solid
62 (75 mg, 48%) as a single peak (purity >98%; see Supporting
Information for pdf of HPLC trace): 1H NMR (700 MHz, d6-DMSO)
δ 8.58−7.25 (m, 7H), 7.25−6.73 (m, 10H), 4.39−3.50 (m, obscured
by H2O peak, baseline correction shows 5H), 2.97−2.90 (m, 2H), 2.77
(q, J = 7.8 Hz, 2H), 2.33−2.20 (m, 1H), 1.62−1.48 (m, 3H), 1.45−
1.22 (m, 4H), 1.16−0.74 (m, 9H); HRMS (ESI) calcd for
C32H43N7O6 622.3347; found, 622.3342.
ASSOCIATED CONTENT
■
S
* Supporting Information
Ac-(Cyclo-o,m)-[F(Aib)KAF]-NH2 (63). The pentapeptide macro-
cyclization precursor was synthesized on solid support using general
procedure B. It was subjected to Suzuki−Miyaura macrocyclization gen-
eral procedure C2. The macrocyclic product was cleaved from the resin
using general procedure E and acetylated using general procedure F2.
1H and 13C NMR data for compounds 7−28, 38−42, 50−52,
1
and 66, and H NMR and HPLC data for compounds 31−35,
43−49 and 55−63. This material is available free of charge via
the Internet at http://pubs.acs.org.
3113
dx.doi.org/10.1021/jo202105v | J. Org. Chem. 2012, 77, 3099−3114

The Journal of Organic Chemistry
Article
(15) (a) Bois-Choussy, M.; Cristau, P.; Zhu, J. Angew. Chem., Int. Ed.
2003, 42, 4238. (b) Lepine, R.; Zhu, J. Org. Lett. 2005, 7, 2981.
́
AUTHOR INFORMATION
Corresponding Author
*Email: kjames@scripps.edu
Notes
■
(c) Roberts, T. C.; Smith, P. A.; Cirz, R. T.; Romesberg, F. E. J. Am.
Chem. Soc. 2007, 129, 15830. (d) Dufour, J.; Luc Neuville, L.; Zhu, J.
Synlett 2008, 15, 2355. (e) Dufour, J.; Luc Neuville, L.; Zhu, J.
Chem.Eur. J. 2010, 16, 105238. (f) Wang, Z.; Bois-Choussy, M.; Jia,
Y.; Zhu, J. Angew. Chem., Int. Ed. 2010, 49, 2018.
(16) Meyer, F. −M.; Liras, S.; Bian, J.; Perreault, C.; Guzman-Perez,
A.; James, K. Org. Lett. 2010, 12, 3870.
The authors declare no competing financial interest.
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