N -aminoimidazolidin-2-one peptidomimetics

Article abstract of DOI:10.1021/ol500739k

The synthesis of N-aminoimidazolidin-2-one (Aid) peptidomimetics has been achieved by alkylation of the urea nitrogen of a semicarbazone residue using ethylene bromide. The Aid scaffold combines electronic and structural constraints to rigidify the peptide backbone in the equivalent of an aza variant of a Freidinger-Veber lactam. The syntheses and isolation of 25 Aid peptides, including eight GHRP-6 analogues, are reported to demonstrate the utility of this method for controlling conformation.

Full text of DOI:10.1021/ol500739k

Letter
pubs.acs.org/OrgLett
N‑Aminoimidazolidin-2-one Peptidomimetics
Ngoc-Duc Doan, Robert Hopewell, and William D. Lubell*
́ ́ ́ ́ ́
Departement de Chimie, Universite de Montreal, C.P. 6128, Succursale Centre-Ville, Montreal, Quebec Canada H3C 3J7
S
* Supporting Information
ABSTRACT: The synthesis of N-aminoimidazolidin-2-one
(Aid) peptidomimetics has been achieved by alkylation of
the urea nitrogen of a semicarbazone residue using ethylene
bromide. The Aid scaffold combines electronic and structural
constraints to rigidify the peptide backbone in the equivalent of
an aza variant of a Freidinger−Veber lactam. The syntheses
and isolation of 25 Aid peptides, including eight GHRP-6
analogues, are reported to demonstrate the utility of this
method for controlling conformation.
onstrained analogues are useful for characterizing bio-
amino lactam and azapeptide strategies for peptide mimicry has
been achieved by the synthesis of N-aminoimidazolin-2-one
(Nai) peptide mimics.12 Designed to favor β-turn secondary
structures, these dipeptide surrogates employ both electronic
and structural constraints to hinder rotation about the
backbone ϕ-, ψ- and ω-dihedral angles. X-ray crystallographic
and NMR spectroscopic analyses have shown that the Nai
residue prefers to sit in the central position of γ- and type II′ β-
turns.12,13 To study the importance of unsaturation for
conformational control as well as to simplify the synthesis of
such aza-variants of Freidinger−Veber lactams, we have
pursued a method to produce the saturated version of the
Nai residue. The N-aminoimidazolidin-2-one (Aid) residue has
now been synthesized by double alkylation of a semicarbazone-
protected azaglycinamide residue using 1,2-dibromoethane.
A solution-phase approach was first employed to synthesize
Aid dipeptides. Benzhydrylidene azaglycinylphenylalanine tert-
butyl ester14 (1, 1 equiv) was treated with 1,2-dibromoethane
(2.5 equiv) using different bases (5 equiv) in THF to alkylate
first the acidic semicarbazone nitrogen followed by the urea
nitrogen.15,16 Tetraethylammonium hydroxide (TEAH, 53%
yield) gave better results than its tetrabutylammonium
counterpart and the nonionic phosphazene base tert-
butyliminotri(pyrrolidino)phosphorane (BTPP, Table 1). No
cyclic product was isolated using sodium hydride and potassium
tert-butoxide, possibly due to β-elimination. Similarly, 1,2-
diiodo- and 1,2-dichloroethanes failed to give Aid products.
Although racemization (up to 30%) was detected using tert-
butyl ester 1, no loss of configuration was observed after similar
alkylation of its isopropyl amide counterpart 3 (55% yield,
Table 1, Supporting Information). In agreement with the
alkylation of semicarbazones on solid phase,15 benzylidene 6
gave a higher yield (87%) of Aid dipeptide than its
benzhydrylidene counterpart 3. Removal of the benzylidene
group from 6 was effectively achieved using 1 N HCl in THF
C
logically active peptide conformers. Optimal restraint can
enhance affinity by preorganizing geometry that favors receptor
binding surmounting the required loss of entropy for correct
folding.1 Moreover, rigid peptide analogues may avoid
undesirable conformers prone to degradation and off-target
interactions and may thus exhibit improved pharmacological
properties, such as enhanced stability and bioavailability.2
Among approaches for rigidifying peptides, the utilization of
α-amino-γ-lactam (Agl, so-called “Freidinger−Veber lactam”,
Figure 1) residues as backbone constraints has proven
Figure 1. Peptide and Agl, Aza, Nai, and Aid mimics.
particularly effective in the design of ligands for therapeutic
targets.3−6 The Agl residue restrains rotation about the ψ- and
ω-dihedral angles, forces the C-terminal amide to adopt a trans-
conformation,3 and predisposes the peptide backbone to mimic
β-turn secondary structures,7 which are commonly involved in
molecular recognition events of biologically active peptides.8,9
In contrast to the structural restraints of Agl residues,
azapeptides, in which the CαH group is substituted for a N
atom (Figure 1), utilize electronic forces to stabilize β-turn
conformations in peptides.10 As demonstrated by computa-
tional and X-ray analyses, the semicarbazide structure of the
azapeptide rigidifies the ψ-dihedral angle, due to the planarity of
the urea moiety, and the ϕ-dihedral angle, because of hydrazine
N−N lone-pair repulsion.11 Recently, the confluence of the
Received: March 10, 2014
Published: April 4, 2014
© 2014 American Chemical Society
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Organic Letters
Letter
(1:2 v/v) at 40 °C to provide the corresponding semi-
carbazide,17 which was acylated using 4-methoxybenzoyl
chloride and DIEA, as well as with the symmetric anhydride
prepared from Fmoc-Ala and DIC to give,15 respectively, Aid
peptides 7a and 7b in 89% and 68% overall yields (Scheme 1).
showed formation of multiple unidentifiable products. Using
tetraethylammonium hydroxide as base, the benzyl ester side
chain of aspartate 8e was hydrolyzed during alkylation leading
to undesired side products. Similar conditions were not fruitful
using Wang ester resin, likely due to competitive hydrolytic
resin cleavage. To avoid hydrolysis, BTPP was employed and
provided respectively 10e and 10j in 63% and 82% conversion,
as determined by HPLC analysis. Acid 10j was cleaved from
Wang resin 9j using TFA/phenol (95:5), and isolated by
preparative HPLC in 21% overall yield. Analysis of the cleavage
product from an aliquot of resin 9d by HPLC indicated two
closely eluting peaks with the same mass and may be due to
ring-opening of succinimide formed during alkylation to
produce a mix of aspartamide and asparagine analogues.18
Table 1. Aid Dipeptide Solution-Phase Synthesis
a
substrate
R
X
product
base
yield (%)
(S)-1
Ph
OtBu
(S)-2
TBAH
TEAH
BTPP
KOtBu
NaH
15 (71)
53 (41)
20 (37)
trace (0)
trace (54)
62 (15)
55 (31)
trace (78)
51 (18)
trace (0)
52 (41)
87 (−)
Table 2. Solid-Phase Aid Dipeptide Synthesis
(R)-1
(S)-3
Ph
Ph
OtBu
(R)-2
(S)-4
TEAH
TEAH
KOtBu
TEAH
TEAH
BTPP
TEAH
NHiPr
(R)-3
Ph
H
NHiPr
OtBu
(R)-4
5
5c
R
6
H
NHiPr
6c
a
a
8
10
conv (%)
% recovered starting material is indicated in parentheses.
CH2Ph (8a)
CH2Ph (10a)
quant
quant
b
(CH2)4NHBoc (8b)
CH2STrt (8c)
CH2CO2tBu (8d)
CH2CO2Bn (8e)
CH2CONHTrt (8f)
H (8g)
(CH2)4NH2 (10b)
CH2SH (10c)
Scheme 1. Aid Peptide Solution-Phase Synthesis
CH2CO2H (10d)
CH2CO2Bn (10e)
CH2CONH2 (10f)
H (10g)
quant
c
63
quant
quant
quant
quant
(CH2)2SCH3 (8h)
CH2OtBu (8i)
CH2Ph (8j)
(CH2)2SCH3 (10h)
CH2OH (10i)
CH2Ph (10j)
c,d
82
a
Conversion determined by HPLC of cleaved product (λ = 214 nm).
Multiple products observed. BTPP as base. from Wang resin.
b
c
d
Chemoselective benzylidene removal and liberation of
With a solution-phase method for the synthesis of N-
aminoimidazolidin-2-one peptide mimics in hand, we turned
our attention toward a solid-phase approach. On Rink amide
resin, a set of benzylidene azaglycinyl dipeptides 8 were
prepared using a diverse array of C-terminal amino acid
residues (Table 2).15 In order to verify the compatibility of this
synthetic strategy, benzylidene azaglycinyl-D-phenylalanine 8j
was also synthesized on Wang resin. Semicarbazone alkylation
was performed on resin 8 swollen in THF by treatment with
TEAH (5 equiv) for 30 min, followed by 1,2-dibromoethane
(2.5 equiv) and agitation overnight, after which time LC−MS
analysis was performed on an aliquot of cleaved resin. For the
most part, bis-alkylation of azaglycinyl dipeptide 8 linked to
Rink amide resin was effectively achieved and provided a new
peak with a molecular ion corresponding to the mass of the
desired Aid dipeptide in high conversion (Table 2). For
example, after cleavage using a freshly made solution of TFA/
H2O/TES (95:2.5:2.5, v/v/v), benzylidene N-aminoimidazoli-
dinones 10a and 10b were isolated by preparative HPLC in
41% and 47% overall yields, respectively. On the other hand,
multiple attempts failed to synthesize imidazolidinone from
alkylation of azaglycinylcysteine 10c; instead, HPLC analysis
semicarbazide 9 was effectively accomplished employing
NH2OH·HCl in pyridine at 60 °C for 12 h.15 Acylation was
subsequently performed with phenylacetyl chloride (3 equiv)
and DIEA (5 equiv) in DCM or by coupling N-protected
amino acids (Boc-Gly, Boc-Met, Fmoc-Cys(Trt), Boc-Ala, Boc-
Tyr(OtBu), Fmoc-Pro and Boc-Pro) activated by way of their
symmetric anhydrides, which were freshly prepared using DIC.
Resin cleavage using TFA/H2O/TES provided Aid peptides 12
in 17−47% overall yields after purification by preparative
HPLC (Figure 2). Multiple attempts to couple different
protected amino acids (Boc-Tyr(OtBu), Fmoc-Pro, and Boc-
Ala) to the semicarbazide derived from aspartate resin 9f were,
however, unsuccessful using symmetric anhydrides.
An Aid scan of a peptide sequence was next performed.
Growth hormone-releasing peptide-6 (GHRP-6, His-D-Trp-
Ala-Trp-D-Phe-Lys-NH2) was selected for this purpose because
of its interesting biological activity and challenging chemical
structure. This synthetic hexapeptide exhibited affinity for the
ghrelin receptor (GHS-R1a) and the cluster of differentiation
36 (CD36) scavenger receptor.19,20 Activation of GHS-R1a
stimulates growth hormone secretion, appetite, food intake,
weight gain, and gastric emptying.21−23 Recently, GHS-R1a
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Letter
THF gave imidazolidinone 14 in quantitative conversion as
indicated by LC−MS analysis of a cleaved resin aliquot.
Semicarbazone 14 was then treated with hydroxylamine
hydrochloride in pyridine, and the resulting semicarbazide 15
was coupled to Fmoc-Ala by way of its symmetric anhydride,
which was prepared using DIC. Removal of the Fmoc
protection from the resulting tetrapeptide 16, elongation of
the sequence by standard Fmoc-based solid-phase peptide
synthesis,37 and resin cleavage using a cocktail of TFA/H2O/
TES gave [Aid4]-GHRP-6 (17) in 44% crude purity (Table 3,
entry 4). After purification by preparative HPLC, 99% pure Aid
peptide 17 was obtained in 16% overall yield. For comparison,
mimic 17 was also prepared from building block 2c by a route
involving tert-butyl ester removal and coupling of the resulting
Aid dipeptide to Lys(Boc)-Rink amide resin using HBTU and
DIEA. After benzylidene removal, peptide elongation, resin
cleavage and HPLC purification, [Aid4]-GHRP-6 (17) was
obtained in 13% yield and exhibited identical retention time as
material prepared by alkylation on resin. Employing the solid-
phase method, four additional [Aid]-GHRP-6 analogues were
successfully prepared in 6−13% yields and >99% purity (Table
3, entries 1−3 and 5). Notably, in the syntheses of [Aid1]- and
[Aid2]-GHRP-6, the alkylation step was incomplete, con-
sequently complicating HPLC purification of the final Aid
peptides. Shorter [Aid]-GHRP-6 peptides were also prepared in
11−14% overall yield using this method (Table 3, entries 6−8).
Figure 2. Aid peptide solid-phase synthesis.
activation has been recognized to regulate glucose-induced
insulin secretion.24−26 Consequently, GHS-R1a has been
targeted to treat obesity and type 2 diabetes.27 The CD36
receptor exhibited pleiotropic effects on immunity, metabolism,
behavior, and angiogenesis.28 Implicated in the clearance of
subretinal deposits,29 the CD36 receptor plays roles in
facilitating fatty acid transport to the heart30 and reversing
cholesterol transport.31−33 Selective CD36 receptor ligands are
thus being pursued as potential treatments for age-related
macular degeneration29 and cardiovascular diseases.34 Interest
in receptor selective GHRP-6 analogues inspired earlier studies
employing aza-amino acid residues, as well as α- and β-amino γ-
lactams to control conformation.7,16,35 Receptor selectivity was
shown to be contingent on peptide conformation. For example,
[aza-Tyr4]GHRP-6 exhibited significantly reduced affinity
toward the GHS-R1a yet retained affinity for the CD36
receptor.36 Similarly, [S-Agl3]GHRP-6 had CD36 receptor
selectivity.35
Scheme 2. Solid-Phase Synthesis of [Aid4]GHRP-6
Targeting [Aid4]GHRP-6 initially, a general solid-phase
protocol was developed for introducing N-aminoimidazolidin-
2-one residues into the GHRP-6 sequence (Scheme 2).
Azaglycine resin 13 was prepared by acylation of D-Phe-
Lys(Boc)-Rink amide resin with an activated carbazate, which
was formed in situ from benzaldehyde hydrazone and p-
nitrophenyl chloroformate.15 Alkylation of semicarbazone 13
with 1,2-dibromoethane (2.5 equiv) and TEAH (5 equiv) in
In summary, we have developed an effective method for
systematically introducing the N-aminoimidazolidin-2-one
(Aid) scaffold into peptide sequences by alkylation of
semicarbazones with 1,2-dibromoethane. Employing both
electronic and structural constraints to induce conformational
rigidity, the Aid residue offers interesting potential for exploring
structure−activity relationships to elucidate active conformers
of biologically relevant peptides. The Aid method was used to
Table 3. Yields and Purities of Aid-Containing Peptides
HRMS
entry
peptides
crude purity (%)
purity (%)
isolated yield (%)
m/z (cal)
m/z (obs)
1
2
3
4
5
6
7
8
Aid-D-Trp-Ala-Trp-D-Phe-Lys-NH2
His-Aid-Ala-Trp-D-Phe-Lys-NH2
His-D-Trp-Aid-Trp-D-Phe-Lys-NH2
His-D-Trp-Ala-Aid-D-Phe-Lys-NH2 (17)
His-D-Trp-Ala-Trp-Aid-Lys-NH2
Ala-Aid-D-Phe-Lys-NH2
35
51
51
44
44
72
68
67
>99
>99
>99
>99
>99
>99
>99
>99
6.8
5.8
820.4259
793.3872
908.4318
793.3867
832.3995
448.2685
758.3521
495.2727
820.4253
793.3869
908.4291
793.3869
832.3978
448.2667
758.3538
495.2714
13.2
15.8
9.7
12.8
14.0
11.1
His-D-Trp-Aid-Trp-D-Phe-NH2
PhAc-Aid-D-Phe-Lys-NH2
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Letter
(24) Sun, Y. X.; Wang, P.; Zheng, H.; Smith, R. G. Proc. Nat. Acad.
Sci. U.S.A. 2004, 101, 4679−4684.
prepare short peptides and to scan the hexapeptide GHRP-6.
The conformations and biological activities of these compounds
are currently under evaluation and will be reported in due time.
(25) Reimer, M. K.; Pacini, G.; Ahren, B. Endocrinology 2003, 144,
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ASSOCIATED CONTENT
■
S
* Supporting Information
(27) Maletinska, L.; Matyskova, R.; Maixnerova, J.; Sykora, D.;
Pychova, M.; Spolcova, A.; Blechova, M.; Drapalova, J.; Lacinova, Z.;
Haluzik, M.; Zelezna, B. Mol. Cell. Endocrinol. 2011, 343, 55−62.
(28) Silverstein, R. L.; Febbraio, M. Sci. Signal. 2009, 2, re3.
(29) Picard, E.; Houssier, M.; Bujold, K.; Sapieha, P.; Lubell, W.;
Dorfman, A.; Racine, J.; Hardy, P.; Febbraio, M.; Lachapelle, P.; Ong,
H.; Sennlaub, F.; Chemtob, S. Aging (Albany NY) 2010, 2, 981−989.
(30) Bessi, V. L.; Labbe, S. M.; Huynh, D. N.; Menard, L.; Jossart, C.;
Febbraio, M.; Guerin, B.; Bentourkia, M.; Lecomte, R.; Carpentier, A.
C.; Ong, H.; Marleau, S. Cardiovasc. Res. 2012, 96, 99−108.
(31) Bujold, K.; Mellal, K.; Zoccal, K. F.; Rhainds, D.; Brissette, L.;
Febbraio, M.; Marleau, S.; Ong, H. Atherosclerosis 2013, 229, 408−414.
(32) Bujold, K.; Rhainds, D.; Jossart, C.; Febbraio, M.; Marleau, S.;
Ong, H. Cardiovasc. Res. 2009, 83, 457−464.
(33) Rubic, T.; Lorenz, R. L. Cardiovasc. Res. 2006, 69, 527−535.
(34) Bodart, V.; Febbraio, M.; Demers, A.; McNicoll, N.; Pohankova,
P.; Perreault, A.; Sejlitz, T.; Escher, E.; Silverstein, R. L.; Lamontagne,
D.; Ong, H. Circ. Res. 2002, 90, 844−849.
(35) Boutard, N.; Jamieson, A. G.; Ong, H.; Lubell, W. D. Chem. Biol.
Drug. Des. 2009, 75, 40−50.
Experimental procedures, characterization data, NMR spectra,
and analytical HPLC profiles of representative peptides. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: william.lubell@umontreal.ca.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This research was supported by the Natural Sciences and
Engineering Research Council of Canada (NSERC), the
■
Minister
de l’exportation du Quebec (#878-2012, Traitement de la
degenerescence maculaire), and Amorchem.
̀ ́ ́
e du developpement economique de l’innovation et
́
́
(36) Proulx, C.; Picard, E.; Boeglin, D.; Pohankova, P.; Chemtob, S.;
Ong, H.; Lubell, W. D. J. Med. Chem. 2012, 55, 6502−6511.
(37) Lubell, W. D.; Blankenship, J. W.; Fridkin, G.; Kaul, R. In Science
of Synthesis 21.11, Chemistry of Amides.; Thieme: Stuttgart, 2005; pp
713−809.
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