Expanding the scope of oligo-pyrrolinone-pyrrolidines as protein-protein interface mimics

Article abstract of DOI:10.1021/jo400323k

Oligo-pyrrolinone-pyrrolidines (generic structure 1) have the potential to interfere with protein-protein interactions (PPIs), but to reduce this to practice it is necessary to be able to synthesize these structures with a variety of different side chains

Full text of DOI:10.1021/jo400323k

Article
pubs.acs.org/joc
Expanding the Scope of Oligo-pyrrolinone−Pyrrolidines
as Protein−Protein Interface Mimics
Arjun Raghuraman,† Dongyue Xin,† Lisa M. Perez,‡ and Kevin Burgess*,†
‡
†Department of Chemistry and Laboratory for Molecular Simulation, Texas A&M University, Box 30012, College Station,
Texas 77841, United States
S
* Supporting Information
ABSTRACT: Oligo-pyrrolinone−pyrrolidines (generic structure 1) have the
potential to interfere with protein−protein interactions (PPIs), but to reduce
this to practice it is necessary to be able to synthesize these structures with a variety
of different side chains corresponding to genetically encoded proteins. This paper
describes expansion of the synthetic scope of 1, the difficulties encountered in this
process, particularly issues with epimerization and slow coupling rates, and
methods to overcome them. Finally, spectroscopic and physicochemical properties
as well as proteolytic stabilities of molecules in this series were measured; these
data highlight the suitability of oligo-pyrrolinone−pyrrolidines for the development
of pharmacological probes or pharmaceutical leads.
INTRODUCTION
■
Minimalist mimics of secondary structures is a term we used to
describe organic scaffolds that present amino acid side chains
(usually three) in restricted conformations relative to peptides
A. Typical examples of these include Hamilton’s helical systems
beginning with terphenyls and now encompassing a range of
variations from his group1−5 and others6−12 and Hirschmann−
Smith turn and sheet mimics.13−17 Some specific examples
of recent minimalist mimics include Arora’s oxopiperazines B,
Smith−Hirschmann pyrrolinones C,17 selected Bartlett’s
aza-@tide structures D,18,19 and our own oligo-pyrrolinone−
pyrrolidines 1.20
Several attributes shared by the generalized structures B−D
and 1 enhance their value as minimalist mimics. First, they all
must occupy more narrowly defined regions of conformational
space than the peptides A because their backbones have fewer
significant degrees of freedom (red arrows). Second, they are
not composed of repeating secondary amide units, enhancing
the likelihood that their derivatives will be proteolytically stable,
cell permeable, and orally bioavailable,21 though each of these
parameters is partly determined by the side chains. Their
relative conformational rigidities,22 and the fact that they will
not form polycationic ammonium species under physiological
conditions, are also desirable features with respect to retarding
proteolysis and promoting absorption. Third, water solubilities
of compounds having these structures will tend to be promoted
by their heteroatoms and heterocycles, relative to structures
featuring mostly benzenoid rings. Fourth, each of these
minimalist mimic scaffolds have side chains arranged in 1, 4,
7, 10 ..... relationships (we colloquially refer to this feature of
some minimalist mimics as side-chain periodicities). The fact that
this is the same side-chain periodicity as peptides and proteins
is almost certainly advantageous when attempting to mimic
consecutive amino acid side chains. Finally, and related to this,
all these structures presumably could be made from amino
acids, and this may facilitate preparations of systems with more
Received: February 14, 2013
Published: May 8, 2013
© 2013 American Chemical Society
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functionalized, less synthetically accessible, side chains like
those found in Arg, His, Trp, and Cys.
this “Meldrum’s acid route” described above also has drawbacks.
In our hands, the condensation procedure does not proceed
to completion for some amino acids (e.g., Phe) unless excess
carboxylate activating agents are used (e.g., 2.0−2.5 equiv of
EDCI, even though some procedures have reported using less
EDCI).27 Consequently, a procedure was devised wherein an
aqueous work up was used to remove excess EDCI and DMAP
salts after first step of the process, and the crude material was
used in the next transformation without further purification.
The procedures described above were based on work by
Tønder and co-workers24−27 who made these products but
exclusively using hydrophobic amino acids for which the
purified products could be obtained via precipitation from
ether. The current studies showed this approach was not
universally applicable to functionalized side chains, depending
on the scale, the side-chain, and residual TFA levels in the
crude material. Thus, isolation of some tetramic acids (e.g.,
that from Trp) required chromatograpy, but several others
(Met/Phe/Thr and Leu) were crystallized directly after
rigorous removal of TFA. Overall, we find this “Meldrum’s
acid route” to be the most useful entry point into tetramic
acids with NH N-termini; it tends to be practical because many
of the requisite BOC-protected amino acids starting materials
are commercially available.
In practice, the fact that structures like B−D could be made
from functionalized amino acids is very different to actually
demonstrating the methods and conditions by which they can
be prepared with these side chains. Just as protocols in peptide
synthesis have taken decades to develop and refine, syntheses of
any of these systems with different combinations of side chains
corresponding to a diverse set of amino acids are difficult.
Consequently, one key objective of this article is to describe
syntheses of compounds 1 incorporating a variety of amino acid
side chains. We have not prepared a set of systems that shows
all 20 naturally encoded amino acids can be incorporated, but
have made enough to support an immediate need for further
validating the concept of exploring key orientations (EKO).23
This technique features data mining to match preferred
conformations of a minimalist mimic with PPI interface regions.
RESULTS AND DISCUSSION
■
Preparation of Tetramic Acid Derivatives 2. The first of
two routes to the requisite tetramic acid synthons involved BOC-
protected amino acids as starting materials in condensation/
cyclization processes involving Meldrum’s acid as shown in
Scheme 1a.24−27 This strategy has the advantage that many
Scheme 1b shows an alternative route to tetramic acid
derivatives based on cumulative literature, largely originating
from Schobert’s laboratory,29−33 regarding additions of
(triphenylphosphoranylidine)ketene (the Bestmann ylide) to
N-alkyl α-amino esters. These routes are conceptually different
from those based on Meldrum’s acid insofar as the starting
materials are C- rather than N-protected. Removal of Ph3PO
after the cyclization in method b can be problematic, particularly
for >5 g scale. When trying to overcome this difficulty, we found
the N-PMB-protected products tend to be ether soluble, thus
facilitating precipitation of Ph3PO from concentrated ether
solutions of the crude material at −20 °C. Samples obtained in
this way could be subjected to column chromatography, even
on >5 g scales. This approach is more practical, if less elegant,
than ones based on supported variants of Bestmann’s ylide33
that involve inconveniently big amounts of solid-phase reagent
for large-scale reactions.
Scheme 1. Two Methods for Preparing Tetramic Acid
Derivatives: (a) Meldrum’s Acid Approach; (b) Bestmann’s
Ylide28−30 Route
Overall, the Bestmann’s ylide approach in Scheme 1b is
preferred if N-capped tetramic acid derivatives are required.
N-PMB tetramic derivatives are useful in the syntheses
described below, and the starting materials can be prepared
conveniently from amino acids via reductive amination with
4-methoxybenzaldehyde.
A possible complication for preparation of tetramic acids
from aspartic acid derivatives is competing cyclization to give
the six-membered ring product in competition with the desired
material 2d′. To explore this possibility, the di-tert-butyl ester
of aspartic acid was transformed as indicated in reaction 1, but
only the five-membered ring compound 2d′ was observed. The
corresponding reaction with glutamic acid was not attempted,
but in that case the competitive processes would be between
formation of a 5-membered ring and a 7-membered ring; the
latter is less likely, so side-chain complications are predicted to
be even less of a concern.
Table 1 shows all of the tetramic acids isolated in the course
of this work and indicates how the one-letter codes for side chains
in this paper correspond to those of the parent amino acids.
Preparation of Pyrrolinone−pyrrolidine Nucleophiles 2.
Our preparation of the scaffold systems 1 begin with a Merck
BOC-protected amino acids with appropriate side-chain
protection are commercially available and inexpensive relative
to other protected amino acid derivatives. Moreover, the
condensation/cyclization process is experimentally convenient
because strictly anhydrous conditions are not required. However,
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procedure to decarboxylate trans-4-hydroxyproline that can afford
more than 50 g of crystalline (R)-3-hydroxypyrrolidine without
Scheme 2. (a) Syntheses of the Target Systems 1. (b) One-
Pot Modification of the Key Coupling Step
a
Table 1. Preparation of Tetramic Acid Derivatives
a
All tetramic acids were synthesized from the corresponding L-amino
acids except w, which was made from the D-amino acid.
chromatography.34 N-Protection of (R)-3-hydroxypyrrolidine
followed by nucleophilic displacement of its triflate derivative
(under conditions optimized to avoid elimination)35 gave the
amino esters 4 (Scheme 2). Variable amounts of epimerization
occurred in these reactions, but this is not a serious problem
because all of the esters 4 featured in this paper were obtained as
single diastereomers via one crystallization from either ethanol or
acetonitrile. The communication that precedes this work reported
an X-ray analysis of 4f·HCl (after crystallization from ethanol) that
proved it formed via a single inversion, i.e., without a neighboring
group effect.20 Here, 4i·HCl (after crystallization from acetonitrile)
was similarly shown to be the product of one inversion.
was made by reacting the Cbz-pyrrolidine 3-triflate precursor
with (rac)-tert-butyl phenylalanine. Second, racemic Cbz-
pyrrolidine 3-triflate was reacted with optically pure tert-
butylphenylalanine to form the isomers that would be
formed from “ring epimerization”, i.e., (S,S)-4f + (R,S)-4f and
(S,R)-4f + (R,R)-4f. After removal of the Cbz group, 13C NMR
spectroscopy of these mixtures allowed (S,S)-4f and (R,S)-4f to
be differentiated [and, of course, the enantiomers (R,R)-4f from
(S,R)-4f]. Comparison of that 13C NMR data with analytical
A series of experiments were undertaken to elucidate which
of the two chiral centers in structure 4 epimerized in the
synthesis of this material. First a mixture that would be formed
from “acyclic epimerization”, (S,S)-4f + (R,S)-4f (see below),
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tendencies for compounds 5 to epimerize are suppressed when
the side-chain and/or the O-substituent are large, probably
because this sterically disfavors formation of planar, extended
enolates.
Pyrrolidines 5 tend to be protonated by adventitious protons
upon standing. One attempt to crystallize the free base 5f using
untreated glassware deposited crystals of the corresponding
ammonium chloride (X-ray analysis). It is important to avoid
such protonation events because the salts of 5 do not couple
with tetramic acids to give the desired vinylogous ureas 6. For
this reason we routinely purified the free-bases 5 immediately
before the next coupling step via column chromatography using
an eluent containing 1% Et3N. Epimerization may occur if the
fractions are directly concentrated in that basic medium, but
azeotropic removal with toluene prevents epimerization in the
rotary evaporation process, because, we hypothesize, the mol
fraction of Et3N is not increased under these conditions.
It is to be anticipated that C-terminal tetramate-OMe units
will be preferred when dealing with those PPI targets where the
corresponding tert-butyl esters are too bulky and lipophilic
to be accommodated. This consideration led us to investigate
the methyl tetramate forms of 5; however, these were found to
epimerize faster than the corresponding tert-butyl compounds.
Consequently, a “one-pot” procedure was developed to
transform intermediate E directly into the products 9 (methyl
forms) and 6 (tert-butyl). In the event, the modification that
was developed (Scheme 2b) facilitated these reactions without
epimerization and made it easier to remove triphenylphosphine
oxide from the products. Moreover, selective hydrogenolysis
of the Cbz group in the presence of O-benzyl side-chain
protection occurred in this process; that is an advantage in
situations where downstream steps require side-chain O-benzyl
protection.
traces from a chiral HPLC column established that (R,S)-4f
could be chromatographically distinguished from the other three
stereoisomers that eluted at almost the same time under these
conditions. Comparison of all this data with 13C NMR and
chiral HPLC analyses of the partially epimerized mixture
(step 1, Scheme 1) was sufficient to confirm that ring epimeriza-
tion was the dominant process and acyclic epimerization did
not occur to within the limits of detection in the 13C spectra that
were accumulated (see the Supporting Information).
Epimerization in the first step of Scheme 1 was found to be
dependent upon the side chains involved and the scale of the
reaction. Loss of stereochemical integrity in this reaction could
occur via minor pathways involving SN1 mechanisms and/or
a neighboring group effect from the carbamate. It was con-
spicuous in this study because the nucleophile is homochiral.
Several papers in the literature feature reactions of N-carbamate-
protected 3-hydroxypyrrolidine derivatives with achiral nucleo-
philes;36−43 partial racemization in these reactions would have
been less noticeable.
Pyrrolinone−Pyrrolidine−Pyrrolinone−Pyrrolidine−
Pyrrolinones 1. Optimized conditions for coupling of the
pyrrolidine 5 with the tetramic acids 2 as described above
worked well for several substrates, but coupling of the same
pyrrolidines 5 with the larger electrophiles derived from
deprotection of vinylogous carbamates 6 was significantly
slower. We therefore investigated reaction of the nucleophiles
with mesylates derived from the tetramic acid, with or without
Once crystallized, the hydrochlorides 4 are stable on the bench
for at least several weeks. It is convenient that the products
are hydrochlorides because this provides the 1 equiv of acid
that is required to activate Bestmann’s ylide28−30 in the next
step: cyclization to Cbz-protected forms of the pyrrolidines 5.
Intermediates E are labeled with a letter and not a number
because they were subjected to hydrogenolysis “as is”. Epimeriza-
tion can occur in cyclizations using Bestmann’s ylide, but we
found it is negated in THF using relatively short reaction times
(3 h). Dimerization of the ketene formed from Bestmann’s ylide
after protonation competes with the desired cyclizations in these
processes. To minimize this complication, the solid ylid was
added portionwise every 15 min until completion of the reac-
tion (monitored via 1H NMR spectroscopy; see the Supporting
Information).
Condensation of the free pyrrolidine-NH of 5 with 5-
substituted 2,4-pyrrolidinediones (tetramic acids) 2 gave the
pyrrolinone−pyrrolidine−pyrrolinone systems 6. Unfortunately,
the free pyrrolidines 5 seem to be basic enough to mediate self-
epimerization, at rates dependent on the side chains and the
C-terminal cap but, in the worst cases, even during storage at
−20 °C. For instance, the tert-butyl ester 5a (Me side-chain,
OtBu C-terminus) was isolated and characterized as one
stereoisomer and then used immediately in the next step
without compromising its stereochemical purity, whereas the
corresponding methyl ester derivatives were far less stable (see
below). Similarly, to illustrate dependence of epimerization
on side chains, storage of 5a at −20 °C for 4 d resulted in
epimerization, but none was detected for the derivative from
Ile (5i) under the same conditions. Overall, we conclude the
44
i
catalytic Lewis acids like Yb(OTf)3 and PrOH/AcOH/
molecular sieves,45 but this did not improve the product yield.
Qualitatively, we observed that reaction of the pyrrolidines 5
was faster in alcohol solvents, but MeOH caused methanolysis
of the vinylogous urea bonds leading to byproducts, so a more
hindered alcohol medium, iPrOH, was used. Trimethyl
orthoformate was added to scavenge water in this reaction; it
was more effective than molecular sieves or azeotropic removal.
Better product yields were obtained with 1.5 equiv of
trimethylorthoformate, though use of this as a cosolvent was
not helpful. No epimerization was observed when products 1 or
6 were formed under the conditions specified in Scheme 2a
and, most particularly, using a slight excess of the C-terminal
tetramate electrophile, whereas it was problematic in test
reactions in which the pyrrolidine nucleophile was used in
excess. Fortunately, protecting group requirements for function-
alized nucleophiles 5 and electrophiles 2 do not place severe
constraints on the reaction; for instance, the unprotected indole
side-chain in 2w tolerated coupling with the nucleophiles 5.
However, when the R2 substituent is large, e.g., for Thr(OBn) or
Ile, the desired reaction is relatively slow, and the product yield
is diminished.
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Properties of the Pyrroline−Pyrrolidine Oligomers.
Stabilities of the oligomers containing pyrroline-pyrrolidine
were challenged under commonly encountered conditions to
determine synthetic limitations and constraints for in vitro
assays and to reveal how the featured compounds might be
changed in vivo. In the first instance, influence of aqueous acid
was examined by HPLC for 6t′l in pH 7.4 phosphate buffer
(100 mM) and at pH 4.5 (100 mM acetate buffer) and 25 °C.
At pH 7.4 there was no change to 6t′l over 15 h (after which
time the experiment was stopped, see the Supporting
Information), but at pH 4.5 this compound hydrolyzed with
a half-life of ∼40 h−1 (Figure 1a). Similarly, 6ll was completely
stable in pH 7.8 10 mM Tris buffer at 55 °C for 60 h (after
which time the experiment was stopped).
Proteolytic stabilities were assessed by treating 1fla and 6ll
with 63 μM pronase (a mixture of proteases from Streptomyces
griseus that is routinely used to hydrolyze peptides)46 at pH 7.8
in 10 mM Tris buffer containing 5 mM CaCl2, at 37 °C. No
decomposition of either compound was observed by HPLC over a
period of 60 h (after which time the experiment was stopped).
Under the same conditions a control tetrapeptide GATV-DAP
(GATV fluorescently labeled on the N-terminus with 4-
dimethyolaminophthalimide) was completely hydrolyzed to smaller
fragments within 2 h. Collectively, these data indicate the com-
pounds 1 and 6 tend to be stable under neutral aqueous conditions
at 37 °C, even in the presence of proteases, but they would be
hydrolyzed at decreased pH values, such as in the stomach.
Figure 1 shows these compounds have a maximal UV
absorbance at around 295 nm: it has an extinction coefficient
of 3.88 × 104 M−1 cm−1. The products with an alkylated
C-terminus have no significant fluorescence, but the deprotected
forms like the C-deprotected form of 6fl (i.e., 6fl-OH; Φ =
0.006 in 100 mM acetate buffer, pH 4.5) fluoresce with a low
quantum yield, but which might influence assays set to observe
around 400 nm.
Two molecular features govern the bioavailability of
structures 1, specifically, parameters of the (i) scaffold unit
and (ii) the side chains. Jorgenssen’s QikProp program47,48
was used to simulate rates of permeation into Caco-2 cells
(Table 2) in comparison with corresponding peptides
Table 2. Rates of Permeation into Caco-2 Cells Simulated
Using QikProp
Figure 1. (a) Compound 6t′l in pH 4.5 100 mM acetate buffer has a
half-life of ca. 40 h−1 at 25 °C. (b) UV absorbance spectrum of 1fla in
100 mM acetate buffer pH 4.5. (c) Fluorescence spectrum of the
C-deprotected form of 6fl.
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solvents under anhydrous conditions. Glassware for anhydrous
reactions were dried in an oven at 140 °C for minimum 6 h prior
to use. Dry solvents were obtained by passing the previously degassed
solvents through activated alumina columns. Yields refer to chromato-
graphically and spectroscopically (1H NMR) homogeneous materials,
unless otherwise stated. Reagents were purchased at a high commercial
quality (typically 97% or higher) and used without further purification,
unless otherwise stated. Analytical thin layer chromatography (TLC)
was carried out on Merck silica gel plates with QF-254 indicator and
visualized by UV, ceric ammonium molybdate, and/or potassium
permanganate stains. Flash column chromatography was performed
using silica gel 60 (Silicycle, 230−400 mesh) as per the Still protocol.
1H and 13C spectra were recorded on a 300 MHz spectrometer and
were calibrated using residual nondeuterated solvent as an internal
Table 3. Rates of Permeation into Cells Simulated via
QikProp
1
reference (CDCl3: H NMR = 7.26, 13C NMR = 77.16, DMSO-d6:
1
13C NMR = 39.52, CD3OD: H NMR = 3.31, 13C NMR = 49.00).
(Table 3) to begin to understand these properties. Rates of
>20 nm/s−1 are widely considered to be a good indicator of
favorable oral bioavailability; standards for permeation into cells
would be somewhat less than this.
The following abbreviations or combinations thereof were used to
explain the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet,
m = multiplet, p = pentet, br = broad, dd = doublet of doublet, app =
apparent. IR spectra were recorded using NaCl plates. Melting points
were recorded on an automated melting point apparatus and are
uncorrected. Optical rotations were obtained on a polarimeter at the
D line of sodium.
All of the HPLC analyses were carried out with UV detection
monitored at 254 nm. Analytical reversed-phase HPLC analyses were
performed with a 150 × 4.6 mm C-18 column using gradient
conditions (10−90% acetonitrile in water, flow rate = 0.75 mL/min).
Chiralpak AD (250 × 4.6 mm ID) column was utilized for the chiral
HPLC analysis (hexanes: isopropyl alcohol 85:15, flow rate = 1 mL/min).
Table 2 lists simulated rates of permeation into Caco-2 cells
for compounds based on the generic structures 1 and 6, and
Table 3 gives the data for peptides with the corresponding side
chains. This data covers six compounds 1 that were prepared
in this work and six more that have not been prepared yet.
Similarly, that same table covers four compounds 6 that were
made and four others that were not. None of the peptides are
predicted to be bioavailable (PCaco <11 nm/s). However, most
of the PCaco data is above the 20 nm/s threshold for the
smaller mimics 6 that have about the same molecular masses as
the tripeptides. The only exceptions were for the most polar
analogues, e.g., ones containing a Lys side-chain. Predicted
Caco-2 data for the larger compounds 1 was found to be less, as
expected from their larger sizes. Nevertheless, the least polar
of these structures had pCaco values in an acceptable region
(15 − 25 nm/s). Overall, we conclude that molecules based on
1 and 6 are reasonable candidates for experimental determi-
nations of bioavailabilities, and they are probably much more
cell-permeable than closely related peptides.
QikProp 3.5 from Schrodinger (2012) was used to evaluate
̈
pharmaceutically relevant properties for compounds 1 and 6 and
tripeptides with various side chains.
General Procedure for the Synthesis of N-PMB α-Amino
Ester Hydrochlorides. To a solution of the α-amino ester hydro-
chloride (40.0 mmol) in methanol (50 mL) at 0 °C was added
triethylamine (5.6 mL, 40.0 mmol, 1.0 equiv) dropwise. p-Methox-
ybenzaldehyde (5.82 mL, 48.0 mmol, 1.2 equiv) was added, and the
reaction was allowed to proceed at 25 °C for 2 h. Sodium borohydride
(3.02 g, 80.0 mmol, 2.0 equiv) was carefully added portionwise at
0 °C over 60 min. The reaction was stirred for 30 min at 25 °C and
concentrated. The residue was partitioned between Et2O (250 mL) and
saturated NaHCO3 (150 mL). The aqueous layer was extracted twice
with Et2O (75 mL), and the combined organic layers were washed with
brine (75 mL), dried over MgSO4, and filtered. The filtrate was cooled
to 0 °C and treated with a 4 M solution of HCl in dioxane (10 mL,
dropwise addition) to precipitate the product, which was collected by
filtration.
(S)-tert-Butyl 2-((4-methoxybenzyl)amino)propanoate
(N-PMB Ala tert-butyl ester·HCl): white solid, 10.4 g, 86%;
1H NMR (300 MHz, CDCl3) δ 7.47 (d, J = 8.4 Hz, 2H), 6.82 (d,
J = 8.4 Hz, 2H), 4.05 (m, 2H), 3.69 (s, 3H), 3.45 (m, 1H), 1.59 (d, J =
7.2 Hz, 3H), 1.50 (s, 9H) 13C NMR (75 MHz, CDCl3) δ 168.2, 160.2,
132.1, 122.4, 114.3, 83.9, 55.2, 53.8, 48.6, 27.9, 15.7; IR (film, cm−1)
1735, 1613, 1562, 1421, 1390, 1257, 1090; MS (ESI-TOF) m/z calcd
for C15H23NO3 (M + H)+ 266.18, found 266.19
CONCLUSIONS
■
Several issues emerged when attempting to expand the scope
of the procedures to obtain interface mimics 1. First,
preparation of the requisite tetramic acids with several different
side chains is relatively easy. The first real problem encountered
in the syntheses was “ring-epimerization” in the C-protected
N-nucleophile syntheses corresponding to fragments 4. This
issue was address by crystallizing those nucleophiles; this
proved to be possible for every different side chain R2 or R3
examined here. The second issue relates to slow couplings of
the nucleophiles 5 with electrophiles like 6; this was an issue
that was improved, and steric constraints regarding the side
chains were elucidated. Furthermore, a one-pot procedure was
developed for the first key coupling step in the syntheses of 6.
The target molecules 1 are stable at neutral pH levels
around physiological, but have a half-life of about 40 h−1 at
pH 4.5. They are proteolytically stable, thermally resilient to
55 °C, and potentially cell permeable based on QikProp
calculations. The molecular scaffolds 1 and 6 explored in this
work have potentially much better cell permeability than the
corresponding peptides.
(2S,3S)-tert-Butyl 2-((4-methoxybenzyl)amino)-3-methyl-
pentanoate (N-PMB Ile tert-butyl ester·HCl): white solid, 8.1 g,
1
79%; [α]20 +15.5 (c 1.0, MeOH); H NMR (300 MHz, CDCl3) δ
10.52 (br s, 1H), 9.60 (br s, 1H), 7.58 (d, J = 8.7 Hz, 2H), 6.90 (d, J =
8.7 Hz, 2H), 4.32 (d, J = 13.2 Hz, 1H), 4.15 (d, J = 13.5 Hz, 1H), 3.77
(s, 3H), 3.38 (br s, 1H), 2.45−2.32 (m, 1H), 1.50 (s, 10H), 1.45−1.27
(m, 1H), 1.14 (d, J = 6.9 Hz, 3H), 0.87 (t, J = 7.4 Hz, 3H); 13C NMR
(75 MHz, CDCl3) δ 165.9, 160.4, 132.5, 121.7, 114.4, 84.3, 61.7, 55.2,
49.7, 36.1, 28.1, 26.8, 15.0, 11.6; IR (film, cm−1) 1732, 1614, 1557,
1421, 1393, 1257, 1092, 1031; MS (ESI-TOF) m/z calcd for C18H30NO3
(M + H)+ 308.22, found 308.29
(S)-Di-tert-butyl 2-((4-methoxybenzyl)amino)succinate
(N-PMB Asp(OtBu) tert-butyl ester·HCl): light yellow solid, 8.1 g,
74%; mp = 112.0−113.3 °C; 1H NMR (300 MHz, CDCl3) δ 7.27 (dd,
J1 = 2.1 Hz, J2 = 6.6 Hz, 2H), 6.87 (dd, J1 = 2.1 Hz, J2 = 6.6 Hz, 2H),
EXPERIMENTAL SECTION
General Experimental Methods. All reactions were carried out
under an inert atmosphere (nitrogen or argon where stated) with dry
■
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3.87−3.82 (m, 4H), 3.67 (d, J = 12.6 Hz, 1H), 3.51 (dd, J1 = 5.7 Hz,
J2 = 6.9 Hz, 1H), 2.63 (dd, J1 = 5.7 Hz, J2 = 15.6 Hz, 1H), 2.54 (dd,
J1 = 6.9 Hz, J2 = 15.6 Hz, 1H), 1.92 (b, 1H), 1.50 (s, 9H), 1.47 (s,
9H); 13C NMR (75 MHz, CDCl3) δ 173.0, 170.3, 158.7, 132.0, 129.5,
113.7, 81.3, 80.8, 57.8, 55.3, 51.4, 39.5, 28.1; IR (film, cm−1) 3420(br),
2981, 1735, 1614, 1517, 1371, 1255, 1154; HRMS (ESI-TOF) m/z
calcd for C20H31NO5 (M + H)+ 366.2275, found 366.2274 (0.3 ppm)
General Procedure for the Cyclization of N-PMB α-Amino
Ester Hydrochlorides. To a stirred suspension of the PMB-protected
α-amino ester hydrochloride (5.0 mmol) in anhydrous dioxane (0.25 M)
under Argon was added Bestmann’s ylide (11.0 mmol, 2.2 equiv) in
one portion at 25 °C. The reaction was heated at 100 °C under Argon
for 12 h. Upon cooling, the reaction mixture was concentrated and
resuspended in 50 mL of ether. The suspension was stirred vigorously
for 30 min, and the solids were removed by filtration. The filtrate was
concentrated to ∼10 mL and placed at −20 °C for 14 h following which
the crystallized Ph3PO byproduct was removed by filtration. The filtrate
was concentrated to obtain an oil which was purified by flash
chromatography.
EDCI (1.2 g, 7.2 mmol, 2.4 equiv) was added in one portion, and the
reaction mixture was stirred at 25 °C for 14 h. The yellow reaction
mixture was transferred to a separatory funnel, diluted with EtOAc
(80 mL), and washed with cold 5% KHSO4 (3 × 100 mL) and brine
(75 mL). The organic layer was dried over MgSO4 and filtered. The
filtrate was refluxed for 30 min under N2. Upon concentration, the
residue was dissolved in dichloromethane (5 mL) and cooled to 0 °C.
TFA (5 mL) was added, and the reaction was stirred for 30 min.
Toluene (25 mL) was added, and the solution was concentrated.
Residual TFA was azeotroped two times with toluene (25 mL each),
and then 5 mL ether was added. After the mixture was stirred for
10 min, the solid product was collected by filtration.
(S)-5-((1H-Indol-3-yl)methyl)pyrrolidine-2,4-dione (2w): white
1
solid, 407.3 mg, 58%; H NMR (300 MHz, CDCl3) δ 8.41 (br s,
1H), 7.56 (d, J = 7.8 Hz, 1H), 7.38−7.32 (m, 1H), 7.26−7.18 (m,
1H), 7.17−7.10 (m, 1H), 7.25 (br s, 1H), 6.93 (d, J = 2.4 Hz, 1H),
4.23 (dd, J = 7.5, 3.3 Hz, 1H), 3.01 (dd, J = 14.9, 7.9 Hz, 1H), 2.89 (d,
J = 22.2 Hz, 1H), 2.68 (dd, J = 22.2, 1.5 Hz, 1H); 13C NMR (75 MHz,
CDCl3) δ 207.3, 171.4, 136.2, 126.9, 123.5, 122.5, 119.9, 118.6, 111.5,
109.2, 64.7, 40.9, 28.1; IR (film, cm−1) 3400 (br), 2918, 1767, 1684,
1361, 1236, 1093, 745; HRMS (ESI-TOF) m/z calcd for C13H12N2O2
(M + Li)+ 235.1020, found 235.1028 (3.4 ppm).
(S)-5-((S)-sec-Butyl)pyrrolidine-2,4-dione (2i): white solid, 316.4
mg, 68%; 1H NMR (300 MHz, CDCl3) δ 8.06 (br s, 1H), 3.90 (d, J =
3.6 Hz, 1H), 2.95 (m, 2H), 1.91−1.84 (m, 1H), 1.38−1.27 (m, 2H),
0.99 (d, J = 6.9 Hz, 3H), 0.89 (t, J = 7.2 Hz, 3H); 13C NMR (75 MHz,
CDCl3) δ 207.7, 172.3, 69.1, 41.7, 37.7, 24.3, 15.3, 11.6; IR (film,
cm−1) 3200 (br), 2957, 1766, 1697, 1471, 1363, 1291; HRMS (ESI-
TOF) m/z calcd for C8H13NO2 (M + H)+ 156.1019; found 156.1018
(0.6 ppm).
(S)-4-tert-Butoxy-1-(4-methoxybenzyl)-5-methyl-1H-pyrrol-
1
2(5H)-one: colorless oil, 1.30 g, 90%; H NMR (300 MHz, CDCl3) δ
7.15 (d, J = 8.4 Hz, 2H), 6.81 (d, J = 9.0 Hz, 2H), 5.03−4.96 (m, 2H),
3.96 (d, J = 15.0 Hz, 1H), 3.76 (s, 3H), 3.66 (q, J = 6.7 Hz, 1H), 1.39
(s, 9H), 1.18 (d, J = 6.9 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ
172.2, 172.0, 158.8, 130.0, 129.2, 113.9, 94.9, 81.4, 56.6, 55.2, 42.3,
27.4, 16.0; MS (ESI-TOF) m/z calcd for C17H24NO3 (M + H)+
290.17, found 290.10.
(S)-4-tert-Butoxy-5-((S)-sec-butyl)-1-(4-methoxybenzyl)-1H-pyr-
rol-2(5H)-one: colorless oil, 1.23 g, 74%; [α]20 +11.8 (c 1.0, MeOH);
1H NMR (300 MHz, CDCl3) δ 7.18 (d, J = 8.7 Hz, 2H), 6.87 (d, J =
9.0 Hz, 2H), 5.18−5.10 (m, 2H), 3.88 (d, J = 15.3 Hz, 1H), 3.82 (s,
3H), 3.72 (d, J = 3.0 Hz, 1H), 1.96−1.82 (m, 1H), 1.45 (s, 9H), 1.55−
1.33 (m, 2H), 0.89 (t, J = 7.4 Hz, 3H), 0.77 (d, J = 6.9 Hz, 3H); 13C
NMR (75 MHz, CDCl3) δ 172.9, 170.8, 158.8, 129.9, 129.3, 113.9,
96.5, 81.6, 63.4, 55.2, 42.3, 34.9, 27.5, 25.7, 12.9, 12.6; IR (film, cm−1)
1682, 1514, 1372, 1329, 1246, 1171; MS (ESI-TOF) m/z calcd for
C20H30NO3 (M + H)+ 332.22, found 332.23
General Procedure for the Syntheses of 4. To a solution of
Cbz-protected pyrrolidin-3-ol (2.57 g, 11.6 mmol) in dry dichloro-
methane (15 mL) at −78 °C was added diisopropylethylamine
(2.2 mL, 12.8 mmol, 1.1 equiv) dropwise. Freshly distilled (P2O5)
triflic anyhydride (2.0 mL, 12.1 mmol, 1.05 equiv) was added using a
syringe pump at rate of 4 mL/h ensuring that the bath temperature did
not exceed −70 °C. The reaction mixture turned pink. On complete
addition of triflic anyhydride, the reaction was stirred for 10 min.
A solution of amino acid tert-butyl ester (17.4 mmol, 1.5 equiv)
in dichloromethane (15 mL) was then added at a rate of 30 mL/h.
The reaction was stirred for 10 min at −78 °C and allowed to warm to
25 °C. After 18 h, the reaction mixture was transferred to a separatory
funnel and diluted with dichloromethane (125 mL). The organic layer
was extracted with saturated sodium bicarbonate (2 × 150 mL) and
brine (1 × 100 mL). The organic layer was dried over MgSO4, filtered,
and concentrated. The residue was purified by column chromatog-
raphy (SiO2, 1:9 ethyl acetate/dichloromethane; ceric ammonium
molybdate stain and UV for visualization) to afford the product as a
mixture of diastereomers. To a solution of the mixture in dry ether
(70 mL) at 0 °C was added HCl in dioxane (4 M, 1.1 equiv) dropwise.
Removal of the ether by filtration afforded a hydroscopic white solid.
The solid was dissolved in dichloromethane (10 mL) and transferred
to a 50 mL erlenmeyer flask. Hexanes (35 mL) was slowly added to
form a second layer. The flask was placed at −20 °C for 12 h to afford
a white, nonhydroscopic solid. The resulting solid was collected by
filtration and to afford the required pure single diastereomer.
(S)-Di-tert-butyl 2-(((S)-1-((benzyloxy)carbonyl)pyrrolidin-3-yl)-
amino)pentanedioate (4e′): white crystals, crystallized from hot
MeCN (∼12 mL/g), 1.62 g after crystallization, 28%; mp = 171−
(S)-tert-Butyl 2-(3-tert-butoxy-1-(4-methoxybenzyl)-5-oxo-2,5-
1
dihydro-1H-pyrrol-2-yl)acetate (2d′): colorless oil, 1.54 g, 75%, H
NMR (300 MHz, CDCl3) δ 7.22 (dd, J1 = 2.1 Hz, J2 = 6.6 Hz, 2H),
6.86 (dd, J1 = 2.1 Hz, J2 = 6.6 Hz, 2H), 5.10 (s, 1H), 5.05 (d, J = 15.3
Hz), 4.07 (t, J = 5.1 Hz), 4.03 (d, J = 15.3 Hz), 3.81 (s, 3H), 2.62 (dd,
J1 = 5.1 Hz, J2 = 15.3 Hz, 1H), 2.51 (dd, J1 = 5.1 Hz, J2 = 15.3 Hz, 1H),
1.45 (s, 18H); 13C NMR (75 MHz, CDCl3) δ 172.5, 170.2, 169.0,
158.9, 129.7, 129.3, 114.0, 95.7, 82.1, 81.3, 57.8, 55.2, 42.8, 36.6; IR
(film, cm−1) 2979, 2361, 1731, 1615, 1513, 1368, 1248, 1147; HRMS
(ESI-TOF) m/z calcd for C22H31NO5 (M + Na)+ 412.2094, found
412.2101 (1.7 ppm).
Procedure for the Syntheses of 3i. (S)-4-tert-Butoxy-5-((S)-sec-
butyl)-1-(4-methoxybenzyl)-1H-pyrrol-2(5H)-one (1 mmol) was dis-
solved in dichloromethane (5 mL) and cooled to 0 °C. TFA (5 mL)
was added, and the reaction was allowed to warm to room temperature.
After being stirred for 2 h, toluene (25 mL) was added and the solution
was concentrated. Residual TFA was azeotroped two times with
toluene (25 mL × 2), and the residue was purified by flash chromato-
graphy (50% ethyl acetate in dichloromethane) to afford (S)-5-((S)-sec-
butyl)-1-(4-methoxybenzyl)pyrrolidine-2,4-dione (3i) in 78% yield:
1
colorless oil, 215.4 mg, 78%; H NMR (300 MHz, CDCl3) δ 7.12 (d,
J = 8.4 Hz, 2H), 6.80 (d, J = 9.0 Hz, 2H), 5.22 (d, J = 14.7 Hz, 1H),
3.82 (d, J = 14.7 Hz, 1H), 3.74 (s, 3H), 3.61 (d, J = 3.3 Hz, 1H), 2.92
(s, 2H), 1.90−1.78 (m, 1H), 1.49−1.33 (m, 2H), 0.86−0.73 (m, 6H);
13C NMR (75 MHz, CDCl3) δ 205.5, 170.4, 159.5, 129.8, 126.6, 114.3,
68.6, 55.2, 43.1, 42.4, 35.1, 25.1, 13.4, 12.0; IR (film, cm−1) 1770, 1514,
1418, 1248, 1034; MS (ESI-TOF) m/z calcd for C16H22NO3 (M + H)+
276.15, found 276.19
General Procedure for the Syntheses of Tetramic Acids 2.
A modified literature procedure was used. To a stirred solution of
meldrum’s acid (476 mg, 3.3 mmol, 1.1 equiv) and DMAP (550 mg,
4.5 mmol, 1.5 equiv) at 0 °C in dichloromethane (30 mL) was
added N-Boc-amino acid (3.0 mmol, 1.0 equiv) in one portion.
1
172 °C; H NMR (300 MHz, CDCl3) δ 7.36−7.26 (m, 5H), 5.16−
5.02 (m, 2H), 3.98−3.86 (m, 1H), 3.85−3.62 (m, 4H), 3.44−3.24 (m,
1H), 2.72−2.42 (m, 4H), 2.39−2.22 (m, 2H), 1.50 (s, 9H), 1.40 (s,
9H); 13C NMR (75 MHz, CDCl3) δ 170.8, 166.4, 154.4, 136.4, 128.5,
128.2, 128.09, 128.03, 127.9, 85.2, 81.1, 67.2 and 67.1, 59.2, 55.7
and 54.9, 47.5 and 47.1, 44.3 and 44.0, 30.7, 28.9, 28.1, 28.0, 25.4,
25.2. Note: Carbon spectra show more than the expected number of
peaks due to restricted rotation about the NH−CO bond. This was
confirmed by hydrogenolysis of the product (see the Supporting
Information); IR (film, cm−1) 2978, 2628, 1714, 1697, 1417, 1153;
4829
dx.doi.org/10.1021/jo400323k | J. Org. Chem. 2013, 78, 4823−4833

The Journal of Organic Chemistry
Article
HRMS (ESI-TOF) m/z calcd for C25H39N2O6 (M + H)+ 463.2808,
found 463.2817 (1.9 ppm)
4.14 (d, J = 9.6 Hz, 1H), 4.07−3.88 (m, 1H), 3.86 (d, J = 2.7 Hz, 1H),
3.83−3.69 (m, 1H), 3.46−3.15 (m, 3H), 2.74−2.52 (m, 1H), 1.87−
1.47 (m, 5H), 1.42 (s, 9H), 1.40−1.35 (m, 1H), 1.0−0.89 (m, 6H),
0.75 (d, J = 6.9 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 176.2, 173.3,
170.4, 166.7, 97.4, 88.3, 82.0, 65.8, 55.7, 52.6, 49.6, 47.4, 42.2, 36.4,
27.4, 26.0, 25.9, 23.7, 21.4, 12.6, 12.4; HRMS (ESI-TOF) m/z calcd
for C24H40N3O3 (M + H)+ 418.3070, found 418.3083 (3.1 ppm).
(S)-1-((S)-1-((R)-2-((R)-1-(Benzyloxy)ethyl)-5-oxo-2,5-dihydro-1H-
pyrrol-3-yl)pyrrolidin-3-yl)-4-tert-butoxy-5-methyl-1H-pyrrol-2(5H)-
one (6t′a): white solid, 212.6 mg, 67%; 1H NMR (300 MHz, CDCl3)
δ 7.37−7.19 (m, 5H), 6.29 (br s, 1H), 4.94 (s, 1H), 4.62−4.54 (m,
2H), 4.43 (d, J = 11.7 Hz, 1H), 4.39−4.26 (m, 1H), 4.24 (d, J =
1.5 Hz, 1H), 3.87−3.76 (m, 1H), 3.69−3.58 (m, 1H), 3.38−3.24 (m,
3H), 3.23−3.09 (m, 1H), 2.30−2.04 (m, 2H), 1.42 (s, 9H), 1.23 (d,
J = 6.6 Hz, 3H), 1.17 (d, J = 6.3 Hz, 3H); 13C NMR (75 MHz,
CDCl3) δ 176.7, 172.7, 172.0, 163.8, 138.1, 128.3, 127.8, 127.7, 95.2,
90.6, 81.8, 74.2, 71.1, 61.2, 57.3, 51.0, 50.9, 48.1, 29.0, 27.4, 18.2, 15.3;
IR (film, cm−1) 3250 (br), 2978, 2872, 1597, 1398, 1375, 1257, 1213,
1167, 1096, 735; MS (ESI-TOF) m/z calcd for C26H36N3O4 (M + H)+
454.27, found 454.26
(S)-Benzyl 3-(((2S,3R)-3-(benzyloxy)-1-methoxy-1-oxobutan-2-
yl)amino)pyrrolidine-1-carboxylate (4t′): white crystals, crystallized
from hot MeCN (∼ 14 mL/g), 1.85 g after crystallization, 40%; mp =
1
164−166 °C; H NMR (300 MHz, CDCl3) δ 7.41−7.21 (m, 10 H),
5.16−4.98 (m, 2H), 4.72−4.56 (m, 2H), 4.50−4.39 (m, 1H), 4.01−
3.90 (m, 1H), 3.88−3.78 (m, 2H), 3.78 (m, 3H), 3.70−3.59 (m, 1H),
3.34−3.19 (m, 1H), 2.63−2.40 (m, 1H), 2.40−2.27 (m, 1H), 1.38 (d,
J = 6.3 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 166.4, 154.4, 137.3,
136.5, 128.51, 128.48, 128.28, 128.12, 128.05, 127.9, 73.2, 71.5, 67.0,
63.3, 57.3 and 56.7, 53.4, 48.1 and 47.6, 44.2 and 43.9, 28.5 and 27.7,
16.7; IR (film, cm−1) 2953, 2886, 2636, 1747, 1213, 1123, 741; HRMS
(ESI-TOF) m/z calcd for C24H31N2O5 (M + H)+ 427.2233, found
427.2219 (3.3 ppm)
General Procedure for the Syntheses of 5. The recrystallized
hydrochloride salt 4 (0.97 mmol) was suspended in dry THF (10 mL),
and Bestmann’s ylide (recrystallized from PhMe, 352 mg, 1.2 equiv)
was added in one portion. The reaction mixture was heated to
75 °C under an argon atmosphere. After 30 min, a second portion of
Bestmann’s ylide (59 mg, 0.2 equiv) was added, and this process was
repeated four additional times at 15 min intervals to complete the
addition of 2.2 equiv of ylide. The reaction was monitored by NMR
spectroscopy. After completion of reaction (∼ 3 h), the solvent was
evaporated. Upon cooling, the THF was removed in vacuo, and the
residue was loaded onto a short SiO2 column. Elution with 5%
EtOAc/CH2Cl2 (to remove traces of unreacted starting material)
followed by 50% % EtOAc/CH2Cl2 afforded a mixture of the cyclized
product and triphenylphosphine oxide. The mixture was directly
utilized in the next step.
tert-Butyl 3-((S)-1-((S)-1-((S)-2-benzyl-5-oxo-2,5-dihydro-1H-pyr-
rol-3-yl)pyrrolidin-3-yl)-3-tert-butoxy-5-oxo-2,5-dihydro-1H-pyrrol-
2-yl)propanoate (6fe′): colorless oil that turned into a solid under
1
vacuum, 134.0 mg, 64%; H NMR (300 MHz, CDCl3) δ 7.35−7.13
(m, 5H), 5.23 (br s, 1H), 5.03 (s, 1H), 4.52 (s, 1H), 4.32 (dd, J = 9.9,
3.0 Hz, 1H), 0.3.04−4.14 (m, 1H), 4.04−3.95 (m, 1H), 3.82−3.66 (m,
1H), 3.65−3.53 (m, 1H), 3.52−3.30 (m, 2H), 3.25 (dd, J = 13.5, 3.0
Hz, 1H), 2.66−2.41 (m, 2H), 2.30−2.16 (m, 1H), 2.15−1.95 (m, 4H),
1.44 (s, 9H), 1.43 (s, 9H); 13C NMR (75 MHz, CDCl3) δ 175.6,
173.3, 172.3, 170.1, 165.5, 136.8, 129.1, 128.8, 128.6, 127.1, 97.0, 88.8,
82.3, 80.9, 60.7, 58.4, 52.1, 49.9, 47.7, 39.4, 28.1, 27.4, 24.8; IR (film,
cm−1) 3250 (br), 2978, 2931, 2872, 1724, 1667, 1601, 1395, 1371,
1339, 1371, 1339, 1258, 1165, 1151, 702; HRMS (ESI-TOF) m/z
calcd for C30H42N3O5 (M + H)+ 524.3124, found 524.3115 (1.8 ppm).
(S)-1-((S)-1-((S)-2-((1H-Indol-3-yl)methyl)-5-oxo-2,5-dihydro-1H-
pyrrol-3-yl)pyrrolidin-3-yl)-4-tert-butoxy-5-isobutyl-1H-pyrrol-
General Procedure for the Hydrogenolysis of the N-Cbz
Group. To a solution of the substrate in methanol (0.1 M) under
nitrogen was added 10 wt % of Pd/C (0.1 equiv Pd). The reaction was
placed under an atmosphere of hydrogen (1 atm, balloon) for 12 h and
purged with N2. The reaction mixture was filtered over a Celite pad
and concentrated to afford the crude product. The crude product was
purified by flash chromatography (SiO2, 3% MeOH/CH2Cl2 → 3%
MeOH/CH2Cl2 containing 1% Et3N) to afford the product.
tert-Butyl 3-((S)-3-tert-butoxy-5-oxo-1-((S)-pyrrolidin-3-yl)-2,5-di-
hydro-1H-pyrrol-2-yl)propanoate (5e′): colorless oil, 225.4 mg, 64%
1
2(5H)-one (6wl): pale yellow solid, 127.4 mg, 52%; H NMR (300
MHz, CDCl3) δ 9.79 (s, 1H), 7.47 (d, J = 7.5 Hz, 1H), 7.36 (d, J = 7.8
Hz, 1H), 7.14 (t, J = 7.8 Hz, 1H), 7.06 (t, J = 7.2 Hz, 1H), 6.95 (d, J =
1.5 Hz, 1H), 5.35 (br s, 1H), 5.00 (s, 1H), 4.56 (s, 1H), 4.38−4.28 (m,
1H), 4.26−4.12 (m, 1H), 3.94−3.76 (m, 2H), 3.76−3.55 (m, 2H),
3.49−3.31 (m, 2H), 2.69 (dd, J = 14.7, 9.6 Hz, 1H), 2.59−2.41 (m,
1H), 2.28−2.13 (m, 1H), 1.91−1.74 (m, 1H), 1.69−1.56 (m, 2H),
1.44 (s, 9H), 0.94 (d, J = 0.9 Hz, 3H), 0.92 (d, J = 0.9 Hz, 3H); 13C
NMR (75 MHz, CDCl3) δ 176.4, 173.4, 171.9, 166.2, 136.6, 126.9,
123.4, 121.9, 119.3, 118.0, 111.9, 110.4, 96.2, 88.9, 82.1, 61.4, 57.5,
52.4, 50.9, 47.8, 39.6, 30.2, 28.8, 27.4, 24.1, 24.0, 23.1; IR (film, cm−1)
3414, 3246 (br), 2976, 2928, 2868, 1647, 1597, 1422, 1341, 1167, 908,
735; HRMS (ESI-TOF) m/z calcd for C29H39N4O3 (M + H)+
491.3022, found 491.3035 (2.6 ppm)
1
over two steps; H NMR (300 MHz, CDCl3) δ 5.23 (s, 1H), 4.22−
4.10 (m, 1H), 3.95−3.89 (m, 1H), 3.63−3.51 (m, 3H), 3.50−3.36 (m,
1H), 2.50−2.29 (m, 1H), 2.12−1.94 (m, 5H), 1.44 (s, 9H), 1.41 (s,
9H) (Note: Complete removal of residual solvent (e.g., Et3N, CH2Cl2)
used in chromatography was not done to avoid epimerization. After
characterization, the sample was immediately carried to the next step.);
13C NMR (75 MHz, CDCl3) δ 174.2, 171.9, 171.1, 96.7, 83.1, 81.0,
61.8, 51.9, 49.5, 45.8, 45.2, 30.1, 28.1, 27.4, 23.4; IR (film, cm−1) 2980,
2935, 2870, 1728, 1682, 1614, 1369, 1339, 1258, 1167, 844, 808;
HRMS (ESI-TOF) m/z calcd for C19H33N2O4 (M + H)+ 353.2440,
found 353.2431 (2.6 ppm)
General Procedure for the One-Pot Syntheses of 6. To a
solution of the tetramic acid substrate (1.3 mmol) and N-Cbz
protected dimer (1.0 mmol) in ethanol (0.1 M) under nitrogen
was added trimethylorthoformate (1.5 equiv) and 10 wt % of Pd/C
(0.2 equiv Pd). The reaction was stirred under an atmosphere of H2
(1 atm, balloon) for 12 h. The reaction was purged with N2 for a few
minutes and filtered over a pad of Celite. The filtrate was concentrated
to obtain the crude product, which was purified by flash chromato-
graphy (4−5% MeOH/CH2Cl2) to afford the trimers as a white solid.
(S)-4-tert-Butoxy-5-isobutyl-1-((S)-1-((S)-2-isobutyl-5-oxo-2,5-di-
hydro-1H-pyrrol-3-yl)pyrrolidin-3-yl)-1H-pyrrol-2(5H)-one (6ll):
General Procedure for the Syntheses of 6. These compounds
were prepared via the general procedure described previously.49
(S)-4-tert-Butoxy-5-methyl-1-((S)-1-((S)-2-(2-(methylthio)ethyl)-5-
oxo-2,5-dihydro-1H-pyrrol-3-yl)pyrrolidin-3-yl)-1H-pyrrol-2(5H)-one
(6ma): pale yellow oil, 231.9 mg, 59%; 1H NMR (300 MHz, CDCl3) δ
6.37 (br s, 1H), 5.00 (app s, 1H), 4.56 (d, J = 1.2 Hz, 1H), 4.37−4.25
(m, 2H), 3.94−3.82 (m, 1H), 3.64−3.52 (m, 1H), 3.53−3.38 (m, 2H),
3.38−3.30 (m, 1H), 2.57 (t, J = 7.3 Hz, 2H), 2.53−2.40 (m, 1H),
2.28−2.13 (m, 2H), 2.11 (s, 3H), 1.90−1.75 (m, 1H), 1.45 (s, 9H),
1.35 (d, J = 6.6 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 176.2, 172.7,
172.1, 165.4, 95.5, 89.2, 81.9, 77.3, 57.9, 56.3, 51.7, 50.1, 47.6, 31.9,
29.9, 27.4, 18.0, 15.7; IR (film, cm−1) 3214 (br), 2958, 2873, 1653,
1599, 1480, 1456, 1399, 1373, 1339, 1302, 1259, 1214, 1168, 1096,
880, 840, 781, 757; HRMS (ESI-TOF) m/z calcd for (M + H)+
C20H32N3O3S 394.2164, found 394.2175 (2.7 ppm).
1
white solid, 157.7 mg, 54%; H NMR (300 MHz, CDCl3) δ 5.78
(br s, 1H), 4.97 (s, 1H), 4.51 (d, J = 1.5 Hz, 1H), 4.21−3.97 (m, 2H),
3.87 (dd, J = 6.3, 3.8 Hz, 1H), 3.79−3.64 (m, 1H), 3.46−3.33 (m,
2H), 3.32−3.14 (m, 1H), 2.65−2.43 (m, 1H), 2.23−2.06 (m, 2H),
1.87−1.67 (m, 2H), 1.67−1.53 (m, 3H), 1.42 (s, 9H), 0.99−0.83 (m,
12H); 13C NMR (75 MHz, CDCl3) δ 176.3, 173.3, 171.8, 166.8, 96.1,
88.3, 82.0, 61.3, 55.8, 52.5, 49.8, 47.4, 42.1, 39.7, 27.4, 25.8, 24.1, 23.9,
23.7, 23.0, 21.3; IR (film, cm−1) 3211 (br), 2955, 1661, 1651, 1601,
(S)-4-tert-Butoxy-5-((S)-sec-butyl)-1-((S)-1-((S)-2-isobutyl-5-oxo-
2,5-dihydro-1H-pyrrol-3-yl)pyrrolidin-3-yl)-1H-pyrrol-2(5H)-one
(6li): pale yellow solid, 115.1 mg, 55%; 1H NMR (300 MHz,
CDCl3) δ 5.70 (br s, 1H), 4.99 (s, 1H), 4.52 (d, J = 1.5 Hz, 1H),
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1472, 1371, 1341, 1268, 1167, 731; HRMS (ESI-TOF) m/z calcd for
C24H40N3O3 (M + H)+ 418.3070, found 418.3067 (0.6 ppm).
(S)-4-tert-Butoxy-5-isobutyl-1-((S)-1-((S)-2-methyl-5-oxo-2,5-di-
hydro-1H-pyrrol-3-yl)pyrrolidin-3-yl)-1H-pyrrol-2(5H)-one (6al):
tert-Butyl 3-((S)-1-((S)-1-((S)-2-benzyl-1-((S)-1-((S)-2-methyl-5-
oxo-2,5-dihydro-1H-pyrrol-3-yl)pyrrolidin-3-yl)-5-oxo-2,5-dihydro-
1H-pyrrol-3-yl)pyrrolidin-3-yl)-3-tert-butoxy-5-oxo-2,5-dihydro-1H-
pyrrol-2-yl)propanoate (1afe′): pale yellow solid, 86.6 mg, 42%;
1H NMR (300 MHz, CDCl3) δ 7.25−7.01 (m, 5H), 5.72 (br s, 1H),
5.00 (s, 1H), 4.41 (s, 1H), 4.38 (s, 1H), 4.32 (t, J = 4.5 Hz, 1H), 4.25−
4.16 (s, 1H), 4.15−4.05 (s, 1H), 4.01−3.82 (m, 2H), 3.71−3.42 (m,
3H), 3.42−3.17 (m, 4H), 3.17−3.05 (m, 2H), 2.91 (dd, J = 14.3, 5.3
Hz, 1H), 2.55−2.29 (m, 2H), 2.25−2.13 (m, 1H), 2.12−1.95 (m, 5H),
1.42 (s, 9H), 1.40 (s, 9H), 1.31 (d, J = 6.3 Hz, 3H); 13C NMR (75
MHz, CDCl3) δ 176.2, 174.3, 173.3, 172.3, 170.1, 167.3, 163.5, 135.1,
129.3, 128.3, 127.1, 96.9, 91.1, 87.4, 82.4, 80.8, 61.2, 60.6, 53.4, 52.9,
52.0, 50.1, 50.0, 47.6, 47.4, 37.6, 28.1, 27.4, 24.7, 19.0; IR (film, cm−1)
3123 (br), 2976, 2871, 1724, 1670, 1600, 1395, 1369, 1341, 1258,
1167, 922, 887, 847, 731; HRMS (MALDI-TOF) m/z calcd for
C39H54N5O6 (M + H)+ 688.4067; found 688.4045 (3.2 ppm).
General Procedure for the Deprotection of tert-Butyl Group.
(3′S,5S)-5-((S)-sec-Butyl)-1′-((S)-1-((S)-1-((S)-2-Isobutyl-5-oxo-2,5-di-
hydro-1H-pyrrol-3-yl)pyrrolidin-3-yl)-2-methyl-5-oxo-2,5-dihydro-
1H-pyrrol-3-yl)-[1,3′-bipyrrolidine]-2,4-dione (8lai): off-white solid,
84.2 mg, 80%; 1H NMR (300 MHz, CDCl3) δ 6.01 (br s, 1H),
4.65−4.48 (m, 2H), 4.27−4.02 (m, 4H), 3.93 (d, J = 3.0 Hz, 1H), 3.77
(t, J = 9.5 Hz, 1H), 3.68−3.51 (m, 3H), 3.50−3.37 (m, 3H), 3.37−
3.22 (m, 1H), 2.98 (s, 2H), 2.69−2.42 (m, 2H), 2.36−2.11 (m, 2H),
1.93−1.78 (m, 1H), 1.77−1.48 (m, 2H), 1.47−1.32 (m, 3H), 1.03−
0.92 (m, 12 H), 0.89 (d, J = 6.6 Hz, 3H); 13C NMR (75 MHz, CDCl3)
δ 205.1, 176.2, 173.2, 169.7, 167.1, 165.0, 89.7, 87.8, 71.4, 56.2, 55.9,
53.2, 52.2, 51.8, 50.1, 49.7, 47.4, 47.3, 43.3, 41.9, 37.5, 27.8, 25.8,
25.3, 23.7, 21.4, 18.6, 13.3, 12.1; HRMS (MALDI-TOF) m/z calcd for
C29H44N5O4 (M + H)+ 526.3393, found 526.3381 (2.3 ppm)
General Procedure for the pH Stability Assay. Procedure:
Stock solutions of trimer 6t′l (200 μM) in 100 mM acetate buffer
pH 4.5 (containing less than 5% DMSO) and 100 mM phosphate
buffer pH 7.4 (containing less than 5% DMSO) were prepared and
stored at 25 °C. Samples (25 μL) were injected into a RP-HPLC
system (see general methods) at regular intervals.
General Procedure for the Heat Stability Assay. Procedure:
A stock solution of 1fla in the protease assay buffer (pH 7.8) was
prepared as described above and incubated at 55 °C. Samples (200 μL)
were aliquoted, periodically diluted with 200 μL of MeCN and 600 μL
of protease assay buffer II, and analyzed by RP-HPLC (see the General
Experimental Methods).
Protease Assay. Procedure: Two buffers were used in the protease
stability assay. Buffer I consisted of 50 mM Tris, 10 mM CaCl2, pH 7.8
in 50% (v/v) glycerol. Buffer II consisted of 50 mM Tris, 10 mM
CaCl2, pH 7.8. Pronase (3.2 mg) from Streptomyces griseus (commercial
supplier) was dissolved in buffer I (1 mL) to obtain a stock solution of
160 μM (assuming an average molecular weight of 20 kDa). Pentamer
1fla or trimer 6ll (2 mg) was dissolved in 50 μL of DMSO and diluted
with 500 μL of buffer I and then buffer II to make up 2 mL of solution
to obtain the sample stock solutions (1.6 mM 1fla, 2.4 mM 6ll).
The stock solutions were filtered through a 0.2 μL membrane filter to
remove particulate matter for HPLC analysis. To the respective sample
stock solutions, 70 μL of Pronase stock solution was added and gently
mixed to obtain the reaction mixture, which was incubated at 37 °C.
1
white solid, 180.1 mg, 48%; H NMR (300 MHz, CDCl3) δ 5.72
(br s, 1H), 4.97 (s, 1H), 4.48 (d, J = 1.2 Hz, 1H), 4.20 (q, J = 6.7 Hz,
1H), 4.14−3.99 (m, 1H), 3.87 (dd, J = 6.4, 3.8 Hz, 1H), 3.77−3.62
(m, 1H), 3.51−3.33 (m, 2H), 3.33−3.16 (m, 1H), 2.62−2.43 (m, 1H),
2.19−2.06 (m, 1H), 1.87−1.71 (m, 1H), 1.65−1.53 (m, 2H), 1.42 (s,
9H), 1.35 (d, J = 6.6 Hz, 3H), 0.90 (d, J = 6.3 Hz, 6H); 13C NMR (75
MHz, CDCl3) δ 176.2, 173.3, 171.9, 167.3, 96.1, 87.7, 82.0, 61.2, 52.9,
52.5, 49.8, 47.5, 39.7, 27.7, 27.4, 24.1, 23.9, 23.0, 19.0; IR (film, cm−1)
3304 (br), 2976, 2871, 1653, 1601, 1397, 1339, 1258, 1167, 731;
HRMS (ESI-TOF) m/z calcd for C21H34N3O3 (M + H)+ 376.2600,
found 376.2610 (2.6 ppm).
(5S)-1-((3S)-1-(2-((R)-1-(Benzyloxy)ethyl)-5-oxo-2,5-dihydro-1H-
pyrrol-3-yl)pyrrolidin-3-yl)-4-tert-butoxy-5-isobutyl-1H-pyrrol-
1
2(5H)-one (6t′l): white solid, 131.2 mg, 53%; H NMR (300 MHz,
CDCl3) δ 7.36−7.21 (m, 5H), 5.78 (br s, 1H), 4.98 (s, 1H), 4.64−4.55
(m, 2H), 4.44 (d, J = 11.7 Hz, 1H), 4.21 (d, J = 1.5 Hz, 1H),
4.19−4.07 (m, 1H), 3.83−3.74 (m, 1H), 3.52−3.27 (m, 3H), 3.24−
3.11 (m, 1H), 2.41−2.25 (m, 1H), 2.14−2.05 (m, 1H), 1.99−1.86 (m,
1H), 1.82−1.69 (m, 1H), 1.63−1.51 (m, 2H), 1.44 (s, 9H), 1.19 (d,
J = 6.3 Hz, 3H), 0.89 (d, J = 6.3 Hz, 6H); 13C NMR (75 MHz,
CDCl3) δ 176.6, 173.3, 171.8, 163.8, 138.2, 128.3, 127.8, 127.6, 96.0,
90.3, 81.9, 73.8, 71.0, 61.3, 60.8, 51.9, 50.8, 48.1, 39.7, 28.1, 27.4,
24.0, 23.1, 15.7; IR (film, cm−1) 3227 (br), 2976, 2868, 1599, 1339,
1256, 1167, 1098, 737; HRMS (ESI-TOF) m/z calcd for C29H42N3O4
(M + H)+ 496.3175, found 496.3171 (0.9 ppm).
tert-Butyl 3-((S)-3-tert-butoxy-1-((S)-1-((S)-2-isobutyl-5-oxo-2,5-
dihydro-1H-pyrrol-3-yl)pyrrolidin-3-yl)-5-oxo-2,5-dihydro-1H-pyr-
rol-2-yl)propanoate (6le′): white solid, 119.9 mg, 49%; 1H NMR
(300 MHz, CDCl3) δ 5.71 (br s, 1H), 5.02 (s, 1H), 4.51 (s, 1H),
4.29−4.10 (m, 2H), 4.01−3.94 (m, 1H), 3.67−3.54 (m, 1H), 3.48−
3.32 (m, 2H), 3.32−3.19 (m, 1H), 2.57−2.37 (m, 2H), 2.26−1.96
(m, 5H), 1.80−1.67 (m, 1H), 1.66−1.53 (m, 1H), 1.43 (s, 9H), 1.41
(s, 9H), 0.95 (d, J = 6.6 Hz, 3H), 0.92 (d, J = 6.6 Hz, 3H); 13C NMR
(75 MHz, CDCl3) δ 176.2, 173.2, 172.2, 170.0, 166.7, 97.0, 88.4, 82.3,
80.8, 60.7, 55.7, 52.0, 49.7, 47.4, 42.1, 28.1, 27.4, 25.8, 24.7, 23.7, 21.4;
IR (film, cm−1) 3123 (br), 2976, 2871, 1724, 1670, 1600, 1395, 1369,
1341, 1258, 1167, 922, 887, 847, 731; HRMS (ESI-TOF) m/z calcd
for C27H44N3O5 (M + H)+ 490.3281; found 490.3270 (2.2 ppm).
(S)-5-((R)-1-(Benzyloxy)ethyl)-4-methoxy-1-((S)-1-((S)-2-methyl-5-
oxo-2,5-dihydro-1H-pyrrol-3-yl)pyrrolidin-3-yl)-1H-pyrrol-2(5H)-one
1
(9at′): white solid, 104.1 mg, 51%; H NMR (300 MHz, CDCl3) δ
7.35−7.24 (m, 5H), 5.68 (br s, 1H), 5.04 (s, 1H), 4.70 (d, J = 12.0 Hz,
1H), 4.50−4.41 (m, 2H), 4.11−3.96 (m, 3H), 3.78 (s, 3H), 3.78−3.70
(m, 1H), 3.67−3.55 (m, 1H), 3.33−3.21 (m, 2H), 3.21−3.08 (m, 1H),
2.42−2.25 (m, 1H), 2.01−1.88 (m, 1H), 1.30 (d, J = 6.6 Hz, 3H), 1.18
(d, J = 6.0 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 176.1, 175.0,
172.7, 167.2, 137.7, 128.6, 128.0, 127.7, 95.8, 87.7, 74.0, 71.1, 64.5,
58.3, 53.6, 52.8, 49.7, 47.3, 27.2, 19.0, 14.8; IR (film, cm−1) 3260 (br),
2928, 2868, 1670, 1595, 1396, 1361, 1238, 1099, 995, 733, 696;
HRMS (ESI-TOF) m/z calcd for C23H30N3O4 (M + H)+ 412.2236,
found 412.2277 (3.7 ppm)
General Procedure for the Syntheses of 1. These compounds
were prepared via the general procedure described previously.49
(S)-1-((S)-1-((S)-2-Benzyl-5-oxo-2,5-dihydro-1H-pyrrol-3-yl)-
pyrrolidin-3-yl)-4-((S)-3-((S)-3-tert-butoxy-2-methyl-5-oxo-2,5-dihy-
dro-1H-pyrrol-1-yl)pyrrolidin-1-yl)-5-isobutyl-1H-pyrrol-2(5H)-one
1
(1fla): white solid, 99.7 mg, 54%; H NMR (300 MHz, CDCl3) δ
7.32−7.14 (m, 5H), 5.11 (br s, 1H), 4.96 (s, 1H), 4.58 (s, 1H), 4.50
(s, 1H), 4.33−4.23 (m, 2H), 4.24−4.08 (m, 2H), 3.86 (q, J = 6.6 Hz,
1H), 3.63−3.20 (m, 8H), 2.66−2.38 (m, 4H), 2.26−2.08 (m, 2H),
1.77−1.59 (m, 3H), 1.42 (s, 9H), 1.32 (d, J = 6.6 Hz, 3H), 0.91 (d, J =
6.3 Hz, 3H), 0.86 (d, J = 6.0 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ
175.5, 174.2, 172.7, 172.1, 165.5, 163.5, 137.0, 129.1, 128.8, 127.1,
95.5, 90.6, 88.6, 82.0, 60.2, 58.5, 57.8, 52.9, 51.7, 50.3, 49.9, 47.6, 47.5,
39.4, 38.2, 27.4, 24.1, 23.9, 23.1, 18.0; HRMS (MALDI-TOF) m/z
calcd for C36H50N5O4 (M + H)+ 616.3857; found 616.3865 (1.3 ppm).
The positive control was prepared was follows. Pronase stock
solution (70 μM) was added to a solution of the N-labeled tetrapeptide
GATV−OH (3.8 mM, 2 mL), which was prepared in a manner similar
to that for the sample stock solution above. The reaction mixture was
incubated at 37 °C.
Samples (200 μL) were aliquoted periodically, quenched with
200 μL of MeCN to halt the reaction, diluted with 600 μL of buffer II,
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and analyzed by RP-HPLC (see the General Experimental Methods).
Under the described conditions, the control peptide completely
decomposed in 2 h.
General Procedure for X-ray Structure Determination. A micro-
scope was used to identify a suitable colorless multifaceted crystal
with very well-defined faces with dimensions (max, intermediate,
and min) 0.05 mm × 0.03 mm × 0.01 mm from a representative
sample of crystals of the same habit. The crystal mounted on a nylon
loop was then placed in a cold nitrogen stream maintained at
110 K.
A X-ray diffractometer was employed for crystal screening, unit
cell determination, and data collection. The goniometer was con-
trolled using the FRAMBO software suite.50 The sample was optically
centered with the aid of a video camera such that no translations were
observed as the crystal was rotated through all positions. The detector
was set at 6.0 cm from the crystal sample (MWPC Hi-Star Detector,
512 × 512 pixel). The X-ray radiation employed was generated
from a Cu sealed X-ray tube (Kα = 1.54184 Å with a potential of
40 kV and a current of 40 mA) fitted with a graphite monochromator
in the parallel mode (175 mm collimator with 0.5 mm monocapillary
optics).
The rotation exposure indicated acceptable crystal quality and the
unit cell determination was undertaken. 2100 data frames were taken
at widths of 0.5° with an exposure time of 10 s. Over 6000 reflections
were centered and their positions were determined. These reflections
were used in the autoindexing procedure to determine the unit cell.
A suitable cell was found and refined by nonlinear least-squares and
Bravais lattice procedures and reported in Table 1. No supercell or
erroneous reflections were observed.
After careful examination of the unit cell, a standard data collection
procedure was initiated. This procedure consists of collection of one
hemisphere of data collected using ω scans involving the collection
0.5° frames at fixed angles for ϕ, 2θ, and χ (2θ = −28°, χ = 54.73°,
2θ = −90°, χ = 54.73°), while varying ω. Addition data frames were
collected to complete the data set. Each frame was exposed for 10 s.
The total data collection was performed for duration of approximately
24 h at 110 K. No significant intensity fluctuations of equivalent reflections
were observed.
Data Reduction, Structure Solution, and Refinement.
Integrated intensity information for each reflection was obtained
by reduction of the data frames with the program SAINT.51 The
integration method employed a three-dimensional profiling algorithm
and all data were corrected for Lorentz and polarization factors,
as well as for crystal decay effects. Finally the data was merged and
scaled to produce a suitable data set. The absorption correction
program SADABS52 was employed to correct the data for absorption
effects.
Systematic reflection conditions and statistical tests for the data
suggested the space group P21. A solution was obtained readily using
SHELXTL (SHELXS).53 All non-hydrogen atoms were refined with
anisotropic thermal parameters. The Hydrogen atoms bound to
carbon were placed in idealized positions [C−H = 0.96 Å, Uiso(H) =
1.2Uiso(C)]. The structure was refined (weighted least-squares refine-
ment on F2) to convergence. X-seed was employed for the final data
presentation and structure plots.54
AUTHOR INFORMATION
Corresponding Author
*E-mail: burgess@tamu.edu.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the National Institutes of Health (GM087981) and
the Robert A. Welch Foundation (A-1121) for financial support.
REFERENCES
■
(1) Yin, H.; Lee, G.; Sedey, K. A.; Kutzki, O.; Park, H. S.; Orner, B.
P.; Ernst, J. T.; Wang, H.-G.; Sebti, S. M.; Hamilton, A. D. J. Am.
Chem. Soc. 2005, 127, 10191.
(2) Davis, J. M.; Truong, A.; Hamilton, A. D. Org. Lett. 2005, 7, 5405.
(3) Ernst, J. T.; Becerril, J.; Park, H.; Yin, H.; Hamilton, A. D. Angew.
Chem., Int. Ed. 2003, 42, 535.
(4) Davis, J. M.; Tsou, L. K.; Hamilton, A. D. Chem. Soc. Rev. 2007,
36, 326.
(5) Rodriguez, J. M.; Hamilton, A. D. Angew. Chem., Int. Ed. 2007, 46,
8614.
(6) Ahn, J.-M.; Han, S.-Y. Tetrahedron Lett. 2007, 48, 3543.
(7) Shaginian, A.; Whitby, L. R.; Hong, S.; Hwang, I.; Farooqi, B.;
Searcey, M.; Chen, J.; Vogt, P. K.; Boger, D. L. J. Am. Chem. Soc. 2009,
131, 5564.
(8) Moisan, L.; Odermatt, S.; Gombosuren, N.; Carella, A.; Rebek, J.,
Jr. Eur. J. Org. Chem. 2008, 1673.
(9) Maity, P.; Koenig, B. Org. Lett. 2008, 10, 1473.
(10) Marimganti, S.; Cheemala, M. N.; Ahn, J.-M. Org. Lett. 2009, 11,
4418.
(11) Tosovska, P.; Arora, P. S. Org. Lett. 2010, 12, 1588.
(12) Lee, Ji, H.; Zhang, Q.; Jo, S.; Chai Sergio, C.; Oh, M.; Im, W.;
Lu, H.; Lim, H.-S. J. Am. Chem. Soc. 2011, 133, 676.
(13) Hirschmann, R.; Nicolaou, K. C.; Pietranico, S.; Salvino, J.;
Leahy, E. M.; Sprengeler, P. A.; Furst, G.; Smith, A. B., III. J. Am.
Chem. Soc. 1992, 114, 9217.
(14) Hirschmann, R.; Nicolaou, K. C.; Pietranico, S.; Leahy, E. M.;
Salvino, J.; Arison, B.; Cichy, M. A.; Spoors, P. G.; Shakespeare, W. C.;
Sprengeler, P. A.; Hamley, P.; Smith, A. B., III; Reisine, T.; Raynor, K.;
Maechler, L.; Donaldson, C.; Vale, W.; Freidinger, R. M.; Cascieri, M.
R.; Strader, C. D. J. Am. Chem. Soc. 1993, 115, 12550.
(15) Hirschmann, R.; Sprengeler, P. A.; Kawasaki, T.; Leahy, J. W.;
Shakespeare, W. C.; Amos B. Smith, I. J. Am. Chem. Soc. 1992, 114,
9699.
(16) Mowery, B. P.; Prasad, V.; Kenesky, C. S.; Angeles, A. R.;
Taylor, L. L.; Feng, J.-J.; Chen, W.-L.; Lin, A.; Cheng, F.-C.; Smith, A.
B., III; Hirschmann, R. Org. Lett. 2006, 8, 4397.
(17) Smith, A. B., III; Charnley, A. K.; Hirschmann, R. Acc. Chem.
Res. 2011, 44, 180.
(18) Hammond, M. C.; Bartlett, P. A. J. Org. Chem. 2007, 72, 3104.
(19) Hammond, M. C.; Harris, B. Z.; Lim, W. A.; Bartlett, P. A.
Chem. Biol. 2006, 13, 1247.
(20) Raghuraman, A.; Ko, E.; Burgess, K. J. Am. Chem. Soc. 2011,
133, 12350.
(21) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv.
Drug Delivery Rev. 1997, 23, 3.
(22) Veber, D. F.; Johnson, S. R.; Cheng, H.-Y.; Smith, B. R.; Ward,
K. W.; Kopple, K. D. J. Med. Chem. 2002, 45, 2615.
(23) Ko, E.; Perez, L. M.; Ioerger, T. R.; Burgess, K. Chem. Commun.
2013, submitted.
(24) Jouin, P.; Castro, B. J. Chem. Soc., Perkin Trans. 1 1987, 1177.
(25) Courcambeck, J.; Bihel, F.; De Michelis, C.; Quelever, G.; Kraus,
J. L. J. Chem. Soc., Perkin Trans. 1 2001, 1421.
(26) Ma, D.; Ma, J.; Ding, W.; Dai, L. Tetrahedron Asymmetry 1996,
7, 2365.
(27) Hosseini, M.; Kringelum, H.; Murray, A.; Tonder, J. E. Org. Lett.
2006, 8, 2103.
ASSOCIATED CONTENT
■
S
* Supporting Information
1
Copies of H and 13C NMR spectra for all new compounds.
X-ray crystallographic data (CIF) for compound 4i·HCl. HPLC
chromatograms for stability assays. Photophysical properties
and quantum yields of tetramic acids. HPLC and NMR study
of epimerization in the syntheses of 4. All Qikprop predicted
properties of compounds 1 and 6 and tripeptides.This material
is available free of charge via the Internet at http://pubs.acs.org.
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(28) Bestmann, H. J. Angew. Chem., Int. Ed. Engl. 1977, 16, 349.
(29) Schobert, R.; Dietrich, M.; Mullen, G.; Urbina-Gonzalez, J.-M.
Synthesis 2006, 3902.
(30) Schobert, R.; Boeckman, R. K., Jr.; Pero, J. E. Org. Synth. 2005,
82, 140.
(31) Schobert, R.; Jagusch, C. Tetrahedron 2005, 61, 2301.
(32) Schobert, R.; Schlenk, A. Bioorg. Med. Chem. 2008, 16, 4203.
(33) Schobert, R.; Jagusch, C.; Melanophy, C.; Mullen, G. Org.
Biomol. Chem. 2004, 2, 3524.
(34) Houghton, P. G.; Humphrey, G. R.; Kennedy, D. J.; Roberts, D.
C.; Wright, S. H. B. J. Chem. Soc., Perkin Trans. 1 1993, 1421.
(35) Corruble, A.; Davoust, D.; Desjardins, S.; Fressigne, C.;
Giessner-Prettre, C.; Harrison-Marchand, A.; Houte, H.; Lasne, M.-
C.; Maddaluno, J.; Oulyadi, H.; Valnot, J.-Y. J. Am. Chem. Soc. 2002,
124, 15267.
(36) Sanchez, J. P.; Domagala, J. M.; Heifetz, C. L.; Priebe, S. R.;
Sesnie, J. A.; Trehan, A. K. J. Med. Chem. 1992, 35, 1764.
(37) Zhang, H.; Mitsumori, S.; Utsumi, N.; Imai, M.; Garcia-Delgado,
N.; Mifsud, M.; Albertshofer, K.; Cheong, P. H.-Y.; Houk, K. N.;
Tanaka, F.; Barbas, C. F., III. J. Am. Chem. Soc. 2008, 130, 875.
(38) Bell, I. M.; Gallicchio, S. N.; Abrams, M.; Beshore, D. C.; Buser,
C. A.; Culberson, J. C.; Davide, J.; Ellis-Hutchings, M.; Fernandes, C.;
Gibbs, J. B.; Graham, S. L.; Hartman, G. D.; Heimbrook, D. C.;
Homnick, C. F.; Huff, J. R.; Kassahun, K.; Koblan, K. S.; Kohl, N. E.;
Lobell, R. B.; Lynch, J. J.; Miller, P. A.; Omer, C. A.; Rodrigues, A. D.;
Walsh, E. S.; Williams, T. M. J. Med. Chem. 2001, 44, 2933.
(39) Hoehne, M.; Robins, K.; Bornscheuer, U. T. Adv. Synth. Catal.
2008, 350, 807.
(40) Rejman, D.; Kovackova, S.; Pohl, R.; Dracinsky, M.; Fiedler, P.;
Rosenberg, I. Tetrahedron 2009, 65, 8513.
(41) Pouliquen, M.; Blanchet, J.; De Paolis, M.; Rema Devi, B.;
Rouden, J.; Lasne, M.-C.; Maddaluno, J. Tetrahedron: Asymmetry 2010,
21, 1511.
(42) Tice, C. M.; Zhao, W.; Krosky, P. M.; Kruk, B. A.; Berbaum, J.;
Johnson, J. A.; Bukhtiyarov, Y.; Panemangalore, R.; Scott, B. B.; Zhao,
Y.; Bruno, J. G.; Howard, L.; Togias, J.; Ye, Y.-J.; Singh, S. B.;
McKeever, B. M.; Lindblom, P. R.; Guo, J.; Guo, R.; Nar, H.; Schuler-
Metz, A.; Gregg, R. E.; Leftheris, K.; Harrison, R. K.; McGeehan, G.
M.; Zhuang, L.; Claremon, D. A. Bioorg. Med. Chem. Lett. 2010, 20,
6725.
(43) Macdonald, S. J. F.; Clarke, G. D. E.; Dowle, M. D.; Harrison, L.
A.; Hodgson, S. T.; Inglis, G. G. A.; Johnson, M. R.; Shah, P.; Upton,
R. J.; Walls, S. B. J. Org. Chem. 1999, 64, 5166.
(44) Phillips, S. T.; Rezac, M.; Abel, U.; Kossenjans, M.; Bartlett, P.
A. J. Am. Chem. Soc. 2002, 124, 58.
(45) Hosseini, M.; Grau Jakob, S.; Sorensen Kasper, K.; Sotofte, I.;
Tanner, D.; Murray, A.; Tonder Janne, E. Org. Biomol. Chem. 2007, 5,
2207.
(46) Jurasek, L.; Johnson, P.; Olafson, R. W.; Smillie, L. B. Can. J.
Biochem. 1971, 49, 1195.
(47) Jorgensen, W. L.; Duffy, E. M. Bioorg. Med. Chem. Let 2000, 10,
1155.
(48) Duffy, E. M.; Jorgensen, W. L. J. Am. Chem. Soc. 2000, 122,
2878.
(49) Raghuraman, A.; Ko, E.; Perez, L. M.; Ioerger, T. R.; Burgess, K.
J. Am. Chem. Soc. 2011, 133, 12350.
(50) APEX2 “Program for Data Collection on Area Detectors”,
BRUKER AXS Inc., 5465 East Cheryl Parkway, Madison, WI.
(51) SAINT, “Program for Data Reduction from Area Detectors”,
BRUKER AXS Inc., 5465 East Cheryl Parkway, Madison, WI.
(52) SADABS: Sheldrick, G. M. “Program for Absorption Correction of
Area Detector Frames”, BRUKER AXS Inc., 5465 East Cheryl Parkway,
Madison, WI.
(53) SHELXTL: Sheldrick, G. M. Acta Crystallogr. 2008, A64, 112.
(54) X-Seed - A software tool for supramolecular crystallography.
Barbour, L. J. J. Supramol. Chem. 2001, 1, 189.
4833
dx.doi.org/10.1021/jo400323k | J. Org. Chem. 2013, 78, 4823−4833