Synthesis of hexa- and pentasubstituted diketopiperazines from sterically hindered amino acids

Article abstract of DOI:10.1021/ol100048g

Chemical equation presented Steric hindrance assists In the formation of hindered diketopiperazines using acyl-transfer coupling. In acyl-transfer coupling, the carboxylate of an unprotected N-alkylamino acid attacks an active ester to form a transient anhydride that undergoes an O,N acyl transfer to form a tertiary amide. If the active ester Is part of an N-alkylamino acid It will form a diketopiperazine. It is demonstrated here that acyl-transfer coupling can assemble highly functionalized bis-peptides bearing a functional group on every monomer.

Full text of DOI:10.1021/ol100048g

ORGANIC
LETTERS
2010
Vol. 12, No. 7
1436-1439
Synthesis of Hexa- and Pentasubstituted
Diketopiperazines from Sterically
Hindered Amino Acids
Zachary Z. Brown† and Christian E. Schafmeister*,‡
Chemistry Department, UniVersity of Pittsburgh, Pittsburgh, PennsylVania 15260, and
Chemistry Department, Temple UniVersity, Philadelphia, PennsylVania 19122
meister@temple.edu
Received January 12, 2010
ABSTRACT
Steric hindrance assists in the formation of hindered diketopiperazines using acyl-transfer coupling. In acyl-transfer coupling, the carboxylate
of an unprotected N-alkylamino acid attacks an active ester to form a transient anhydride that undergoes an O,N acyl transfer to form a
tertiary amide. If the active ester is part of an N-alkylamino acid it will form a diketopiperazine. It is demonstrated here that acyl-transfer
coupling can assemble highly functionalized bis-peptides bearing a functional group on every monomer.
While exploring new approaches to forming asymmetric
hexa- and pentasubstituted diketopiperazines, we recently
developed a new reaction for forming extremely hindered
tertiary amides.1-3 In this reaction, the carboxylic acid of
an unprotected N-alkylamino acid acts as a nucleophile to
attack an active ester to form a transient anhydride. This
anhydride spontaneously undergoes an O,N acyl transfer to
form a tertiary amide bond. This O,N acyl transfer is similar
to the driving step of the Ugi reaction,4 the acyl transfer of
native chemical ligation,5 and others.6-8 We have continued
to develop this reaction and demonstrate here that it can be
used to synthesize asymmetric hexa- and pentasubstituted
diketopiperazines under mild conditions with excellent yields.
Diketopiperazines are considered privileged structures in
medicinal chemistry and are valuable motifs for the discovery
of new lead compounds by combinatorial chemistry and the
rational development of new therapeutic agents.9-11 We also
demonstrate that this reaction can be applied sequentially to
† University of Pittsburgh.
‡ Temple University.
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10.1021/ol100048g  2010 American Chemical Society
Published on Web 03/10/2010

assemble bis-peptides that display a functional group on
every monomer.
The approach we chose toward functionalizing bis-peptides
is to combine N-alkyl-R,R-disubstituted bis-amino acids such
as 3a-g through diketopiperazine linkages (Scheme 1).
Toward the rational design of preorganized arrays of
functional groups that can be designed to selectively bind
protein surfaces we have developed bis-peptides.12 Bis-
peptides are spiro-cyclic oligomers assembled from stere-
ochemically pure, cyclic bis-amino acids. Bis-amino acids
display two R-amino acid groups mounted on a cyclic core.
In the assembly of bis-peptides, diketopiperazine rings are
formed sequentially between adjacent monomers to create
spiro-ladder oligomers with well-defined three-dimensional
structures. We have demonstrated the synthesis of many bis-
amino acids and their assembly into a wide variety of
oligomers with different shapes.12 Modeling suggests that
properly designed functionalized bis-peptides could mimic
the display of side chains on R-helical peptides. In recent
years, many groups have described non-natural oligomers
that adopt helical structures.13,14 ꢀ-Peptides and R/ꢀ-peptide
hybrids adopt a variety of helical structures, some of which
are able to mimic the presentation of R-helical peptides and
disrupt helix-protein interactions.15-18 Other oligomers have
been demonstrated to fold into helical structures that present
side chains including peptoids,19-21 N,N′-linked oligo ure-
as,22 aromatic amino acid oligomers,23 quinoline oligoam-
ides,24 and pyridine dicarboxamides.25 Another approach to
mimicking R-helices is to construct scaffolds that present
functional groups with spacing and orientation that is as
similar to R-helical side-chain presentation as possible. This
approach is demonstrated by the trisubstituted terphenyl
derivatives26 and the benzoylurea oligomers27 of Hamilton
and co-workers.
Scheme 1
.
Synthesis of N-Functionalized, Activated Amino
Esters
These amino acids are very sterically hindered and have a
history as poor substrates for amide bond formation using
traditional coupling agents.28 In terms of forming diketopi-
perazines, even tetrasubstituted diketopiperazines are difficult
to form from linear dipeptide precursors and traditionally
require forcing conditions.11,29,30 While steric hindrance has
traditionally been regarded as an obstacle to the coupling of
hindered amino acids, we demonstrate here that it can
actually assist in the formation of tertiary amides and hexa-
and pentasubstituted diketopiperazines.
Compounds 2 and ent-2 are synthesized in five and seven
steps, respectively, from trans-L-4-hydroxyproline 1 in 49%
and 18% overall yield using previously described proce-
dures.12 The synthesis of each amino acid involves only one
chromatographic step. The amino acids 2 and ent-2 were
functionalized on the amine using reductive alkylation with
the aldehydes a-g shown in Table 1 to form 3a-g with
quantitative yield. The aldehydes a-g were chosen because
they demonstrate that a variety of proteogenic and nonpro-
teogenic functional groups can be incorporated using acyl-
transfer coupling. The unprotected amino acids 3a-g were
then combined with 6 equiv of 1-hydroxy-7-azabenzotriazole
(HOAt) followed by 1 equiv of diisopropylcarbodiimide
(DIC). The excess HOAt is used to trap the O-acylisourea
to form 4a-g in near quantitative yield. When less than 6
equiv of HOAt was used, we observed the formation of
symmetric hexasubstituted diketopiperazines formed from
two molecules of 3. The activated esters 4a-g do not
spontaneously self-react in DMF/CH2Cl2 solutions for several
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Org. Lett., Vol. 12, No. 7, 2010
1437

observed come about from the reaction of ent-3g with 5 to
form the anhydride 6 followed by acyl transfer to form 12.
Each of the amide products 7 and 9 proceed to the same
diketopiperazine product 11 by spontaneous dehydration or
assisted by the addition of a dehydrating agent such as DIC.
Diketopiperazine closure requires the first amide 7 and/or 9
to adopt a cis conformation; this is greatly assisted in these
cases by the sterically crowded nature of the tertiary amides.
To determine if we could use this approach to sequentially
form pentasubstituted diketopiperazines and assemble func-
tionalized bis-peptides we synthesized compounds 16 and
17 (Scheme 3). To synthesize the functionalized bis-peptide
Table 1. Variety of Aldehydes Used to Functionalize Bis-amino
Acids
Scheme 3. Synthesis of Oligomers 16 and 17
hours at room temperature presumably because of the
extremely hindered nature of the otherwise unprotected
secondary amine.
To test if the N-alkylamino-OAt esters 4a-g could couple
with hindered N-alkylamino acids through an acyl-transfer
mechanism we combined 110 µmol of ent-4d with 110 µmol
of ent-3g in 2 mL of 1:2 DMF/CH2Cl2 and stirred at room
temperature for 1.5 h (Scheme 2). High-performance liquid
Scheme 2. Proposed Mechanism for the Synthesis of
Hexasubstituted Diketopiperazines
16, we started from compound 13, a protected bis-amino acid
that we have previously described.12 The Boc group was
removed with 50% trifluoroacetic acid (TFA/CH2Cl2) and
the solution was concentrated under reduced pressure. The
resulting amino acid was combined with active ester 4a and
allowed to stir at room temperature overnight. By C18 RP-
HPLC-MS we observed two products, one had a mass
consistent with the formation of a single amide bond between
the two amino acids and the other had a mass consistent
with the desired diketopiperazine product in a ratio of 27:
73. We then added 1 equiv of DIC directly to the reaction
mixture and stirred for 2 h at room temperature. At this point,
the diketopiperazine was the only product observed by RP-
HPLC-MS. The diketopiperazine was isolated and treated
with HBr in acetic acid to form the amino acid 14. We
isolated compound 14 by C18 RP-HPLC (68% yield relative
to 13). We then repeated the process with a new monomer,
chromatography with mass spectrometry (RP-HPLC-MS)
analysis of the reaction mixture showed a ratio of ent-3g:
7/9:11:8/10:12 of 20:35:35:8:2. We then added 1 equiv of
DIC to the reaction mixture, allowed it to stir for 1 h, and
observed the ratio of ent-3g:11:12 of 4:81:15. Thus, the yield
of 11 was 81% relative to ent-3g. In the initial reaction of
ent-3g with ent-4d, the only competent nucleophile is the
carboxylic acid of ent-3g which we propose attacks the -OAt
ester of ent-4d to form the transient anhydride 5 which
spontaneously rearranges one of two ways to form 7 and/or
9. We propose that the traces of 8, 10, and 12 that are
1438
Org. Lett., Vol. 12, No. 7, 2010

combining 14 and the activated, 2-methylenenaphthylene-
functionalized bis-amino acid 4c which again formed a
mixture of amide and diketopiperazine products to which
we added 1 equiv of DIC and then treated with HBr/AcOH
to form 15 (77% yield from 14). The process was repeated
a third time, combining 15 with the activated, carboxyben-
zoyl-aminopropyl-functionalized bis-amino acid 4e. The
single amide and diketopiperazine products of this were
treated with DIC and then HBr/AcOH to form 16 (59% yield
from 15). Oligomer 17 was synthesized using a different
sequence of functionalized monomers with the indicated
stereochemistry with similar yields.
The composition of 16 and 17 was verified by high-
resolution mass spectrometry, and the structures were
validated using two-dimensional nuclear magnetic reso-
nance experiments. ROESY correlations were consistent
with minimum energy conformations of 16 and 17 identified
using the Amber94 force field.31 In the structure of 16 the
stereochemistry of the component monomers arranges the
three functional groups in a left-handed helical arrangement,
and they superimpose well on the side chains of residues i,
i + 3, and i + 6 of a model R-helical peptide (Figure 1).
The distance between each successive pairs of functionalized
amide nitrogens is 5.6 Å. In the structure of 17 the functional
groups are in a right-handed helical arrangement that
superimpose well on the side-chains of residues i, i + 4,
and i + 8 of a model R-helix with a distance of 5 Å between
each successive pair of functionalized amide nitrogens.
We have demonstrated that acyl-transfer coupling allows
the synthesis of hexa- and pentasubstituted diketopiperazines
and the sequential solution phase synthesis of short bis-
peptide oligomers that carry a different functional group on
each monomer. The three-dimensional display of the func-
Figure 1. Models of 16 and 17. Pyrrolidine rings with (S,S)
stereochemistry are shaded blue and those with (R,R) stereochem-
istry are shaded red. In 16, the side chains project in a left-handed
helical sense, and in 17, they project in a right-handed helical sense.
tional groups is controlled by the sequence and stereochem-
istry of the monomers and a wide assortment of proteogenic
and nonproteogenic functional groups can be incorporated.
Acknowledgment. This work was supported by the NIH/
NIGMS (GM067866) and the Defense Threat Reduction
Agency (DOD-DTRA) (HDTRA1-09-1-0009).
Supporting Information Available: Synthesis and char-
acterization of the functionalized bis peptides and coupling
experiments. This material is available free of charge via
the Internet at http://pubs.acs.org.
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