Parallel synthesis of peptide-like macrocycles containing imidazole-4,5-dicarboxylic acid

Article abstract of DOI:10.3390/molecules17055346

We prepared a series of peptide-like 14-membered macrocycles containing an imidazole-4,5-dicarboxylic acid scaffold by using known coupling reagents and protecting group strategies. Yields of the purified macrocycles were poor on average, yet seemingly independent of amino acid substitution or stereochemistry. The macrocycles retain some level of conformational variability as observed by both molecular modeling and X-ray crystallography. These macrocycles represent a new class of structures for further development and for future application in high-throughput screening against a variety of biological targets.

Full text of DOI:10.3390/molecules17055346

Molecules 2012, 17, 5346-5362; doi:10.3390/molecules17055346
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Parallel Synthesis of Peptide-Like Macrocycles Containing
Imidazole-4,5-dicarboxylic Acid
Zhigang Xu 1, Kraig A. Wheeler 2 and Paul W. Baures 3,*
1
Department of Pharmacology and Toxicology, The University of Arizona, Tucson, AZ 85724,
USA; E-Mail: zxu@pharmacy.arizona.edu
2
Department of Chemistry, Eastern Illinois University, Charlestown, IL 61920, USA;
E-Mail: kawheeler@eiu.edu
3
Department of Chemistry, Keene State College, Keene, NH 03435, USA
* Author to whom correspondence should be addressed; E-Mail: pbaures@keene.edu;
Tel.: +1-603-358-2769; Fax: +1-603-358-2525.
Received: 5 April 2012; in revised form: 26 April 2012 / Accepted: 3 May 2012 /
Published: 8 May 2012
Abstract: We prepared a series of peptide-like 14-membered macrocycles containing an
imidazole-4,5-dicarboxylic acid scaffold by using known coupling reagents and protecting
group strategies. Yields of the purified macrocycles were poor on average, yet seemingly
independent of amino acid substitution or stereochemistry. The macrocycles retain some
level of conformational variability as observed by both molecular modeling and X-ray
crystallography. These macrocycles represent a new class of structures for further development
and for future application in high-throughput screening against a variety of biological targets.
Keywords: macrocycle; scaffold; library synthesis; imidazole-4,5-dicarboxylic acid
1. Introduction
Natural products contain a wide variety of conformationally-constrained, macrocyclic, peptide-based
rings that present both backbone and side chain pharmacophoric groups for interacting with biological
targets [1]. Such biologically-active compounds are generally used by plants and animals in chemical
warfare in order to gain an advantage against competitors or assaults [2]. Peptide based macrocycles
from marine sources continue to be investigated for their biological activity [2,3], as are similar
compounds from plants [4].

Molecules 2012, 17
5347
Medicinal chemists have long used macrocyclization as a tool in drug discovery. A classic illustration
of this technique was the discovery of potent cyclic peptide somatostatin mimics three decades ago [5,6].
The use of cyclization has since developed into a general paradigm in peptidomimetic drug design that
has been widely used in the discovery of biologically-active compounds for studying the cell [7].
Indeed, there are both natural product and synthetic macrocyclic peptides with known anticancer use or
potential [8], thereby illustrating their functional similarity.
Oxazoles and thiazoles, as well as oxazoline and thiazoline rings, are commonly found in natural
product macrocyclic peptides [9,10]. Several research groups have reported on the synthesis of
macrocyclic peptides containing heterocyclic amino acids [11–16]. Other heterocyclic amino acids that
have been incorporated into cyclic peptides include pyridine [17] and 1,4-triazole [18].
In addition to the potency and selectivity gained by macrocyclization of peptides, cyclic peptides
also resist proteolytic degradation and other metabolism, plus they are more cell permeable than the
corresponding linear peptides [19–27]. These are important considerations for compounds to be
valuable as chemical tools for studying the cell.
We now report the synthesis and characterization of a peptide-like macrocyclic library that contains
imidazole-4,5-dicarboxylic acid, thereby yielding a unique compound class out of inspiration from
macrocyclic natural products and known paradigms in drug discovery. The final compounds are
14-membered macrocycles with variations in stereochemistry and amino acid side chains. The side
chain variations are limited in comparison with those possible, but nonetheless illustrate the ability to
form the macrocycles regardless of stereochemistry or larger side chains. Indeed, macrocycles are
underrepresented in screening libraries due in part to their synthetic challenge [28,29], and this work is
an important first step in the development of imidazole-4,5-dicarboxylic acid-based macrocycles for
screening efforts. The value of the imidazole-4,5-dicarboxylic acid scaffold in drug discovery has been
previously reported [30], as have other libraries for screening efforts that were built around this
scaffold [31–33].
The library members obey Lipinski’s rule-of-five (RO5) for drug-like compounds [34–36], and also
have limited conformational flexibility that is favorable for pharmacokinetic properties [37]. Regardless,
tool-like compounds that are outside the RO5 in molecular weight, hydrogen bond donors or acceptors,
and Log P values are still valuable for use in the early screening and chemical genomic research [38].
It is important to note that these macrocycles represent a new compound class and are different
from the small compound libraries previously reported that used the imidazole-4,5-dicarboxylic acid
scaffold [31–33]. Their features mimic those of known bioactive natural products, and we anticipate
that such compounds would have reasonable pharmacokinetic properties. Thus, we reason the
macrocycles will be valuable for screening efforts in a variety of bioassays and these results will be
reported in time.
2. Results and Discussion
2.1. Synthesis
The building blocks for the synthesis of the macrocycles are shown in Chart 1.

Molecules 2012, 17
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Chart 1. Building blocks for the macrocycles.
The synthesis of the macrocycles begins with commercially available N,N'-dialkylalkanediamines,
1{1-2}, as shown in Scheme 1. These are monoprotected with benzyloxycarbonyl chloride by using an
excess of the diamine to yield 2{1-2} following workup and purification as similarly reported [39].
However, this method worked poorly for 1{1} and resulted in significant amounts of diprotected
diamine. An alternative synthesis was therefore used that employed N-Boc protection first [40]
followed by benzyloxycarbonyl chloride protection to give the orthogonally protected diamine.
Subsequent removal of the N-Boc protecting group with CF3CO2H in CH2Cl2 [31] yielded 2{1}. These
intermediates were coupled to the Boc-amino acid building blocks, 3{1-4}, with EDC and DMAP in
CH2Cl2 [29], providing chemset 4. Hydrogenolysis of chemset 4 in MeOH [31] yielded chemset 5
following workup. The pyrazine building blocks, 6{1-4}, were prepared as previously described [33],
and reacted with chemset 5 in CH2Cl2 to yield acyclic chemset 7 following purification by column
chromatography. The stoichiometry of the reaction between chemset 5 and chemset 6 is 2:1,
respectively, and yields 2 equivalents of chemset 7. The tert-butyl ester and N-Boc protecting
groups were removed by using CF3CO2H in CH2Cl2 [31]. The trifluoroacetate was exchanged for a
chloride by adding dilute HCl, freezing the solution, and lyophilizing to give chemset 8.
Macrocyclic ring closure to chemset 9 was affected by dissolving chemset 8 in CH2Cl2 and adding
EDC with DMAP [31]. The final macrocycles were also purified by column chromatography and
characterized by both LC-MS and 1H-NMR spectroscopy.

Molecules 2012, 17
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Scheme 1. Synthetic strategy for the macrocycle synthesis.
O
R2
O
O
Ph
O
O
Ph
O
H
N
OH
O
N
N
N
N
H
R1
R1
3{1-4}
Ph
O
Cl
R1
R1
R1
R1
O
EDC, DMAP, CH2Cl2
CH2Cl2, -78 C
N
H
N
H
H
N
O
O
1{1-2}
2{1-2}
R2
O
H2, Pd/C
CH3OH
4{1-2,1-4}
H
N
R1
O
R2
R1
O
N
O
N
H
H
N
O
R3
N
O
O
H
N
N
R1
O
O
N
H
O
R2
O
O
R3
N
5{1-2,1-4}
O
N
O
O
O
H
N
H
CH2Cl2
R3
O
N
R1
O
N
N
N
O
6{1-4}
7{1-2,1-4,1-4}
1. CF3CO2H, CH2Cl2
2. aq HCl, lyophilization
R2
R2
O
R1
H
O
N
N
Cl H3N
O
R3
N
R1 = CH3, CH2CH2
R1
O
R3
H
HO
EDC, DMAP
CH2Cl2
N
R2 = H, S-CH3, R-CH3, S-CH2CH(CH3)2
R3 = H, S-CH3, R-CH3, S-CH2CH(CH3)2
N
O
N
R1
H
N
O
O
H
O
R1
N
N
N
N
H
8{1-2,1-4,1-4}
9{1-2,1-4,1-4}
No extensive effort was made to optimize the synthesis of these macrocycles, and the yields to
purified macrocycles are poor across the series, averaging 12 ± 11% yield, with the best yield
being 37% for both 9{1,1,4} and 9{1,4,3}. A single reaction trying to form the macrocycle
9{1,1,2} with HBTU and Et3N in DMF was attempted [41], but no product was observed by
LC-MS analysis. Indeed, no final product was identified for nine attempted macrocyclization
reactions, and the starting material was not isolated for an additional four macrocycles, leaving 19 of 32
planned macrocycles being synthesized in the study. The yields to the chromatographically-pure
macrocycles are given in Table 1. Importantly, we did not observe a clear trend relating
stereochemistry or size of the side chain in preventing the formation of the macrocycle, so we
reason it would be possible to expand this library in the future to include greater diversity in these
areas. This result is nonetheless an important and critical first step in the goal of targeting
screening libraries of these and closely related compounds, especially given the general challenge
in the synthesis of macrocycles [28,29].
The final macrocycles all contain three hydrogen bond donors and five hydrogen bond acceptors,
thereby conforming to the principles of fewer than five hydrogen bond donors and 10 hydrogen bond
acceptors as outlined in Lipinski’s rule-of-five (RO5) for drug-like compounds [34–36]. The final
macrocycles molecular weights and cLogP values are provided in Table 2. The molecular weights are
all less than 500 g/mol, with cLogP values ranging from −1.70 to 3.31, clearly below the desired limit of 5.

Molecules 2012, 17
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Table 1. Purified yields to the final macrocycles, chemset 9.
R1 = Me
R2 = (S)-
R2 = H
R2 = (S)-CH3
R2 = (R)-CH3
CH2CH(CH3)2
9{1,4,1}; —
9{1,4,2}; 14%
9{1,4,3}; 37%
9{1,4,4}; ‡
R3 = H
R3 = (S)-CH3
9{1,1,1}; 7%
9{1,1,2}; 7%
9{1,1,3}; 17%
9{1,1,4}; 37%
9{1,2,1}; —
9{1,2,2}; 32%
9{1,2,3}; —
9{1,2,4}; 12%
R1 = Et
9{1,3,1}; —
9{1,3,2}; 22%
9{1,3,3}; —
R3 = (R)-CH3
R3 = (S)-CH2CH(CH3)2
9{1,3,4}; 11%
R2 = (S)-
CH2CH(CH3)2
9{2,4,1}; 8%
9{2,4,2}; 11%
9{2,4,3}; —
9{2,4,4}; —
R2 = H
R2 = (S)-CH3
R2 = (R)-CH3
R3 = H
R3 = (S)-CH3
9{2,1,1}; —
9{2,1,2}; 9%
9{2,1,3}; 8%
9{2,1,4}; ‡
9{2,2,1}; —
9{2,2,2}; 7%
9{2,2,3}; 8%
9{2,2,4}; ‡
9{2,3,1}; 3%
9{2,3,2}; 10%
9{2,3,3}; 9%
9{2,3,4}; ‡
R3 = (R)-CH3
R3 = (S)-CH2CH(CH3)2
‡: No starting material was available; —: No product was identified.
Table 2. Molecular weights and cLogP values for the macrocycles.
cmpd
MW (g/mol)
322.32
336.35
336.35
378.43
336.35
350.37
350.37
392.45
336.35
350.37
350.37
392.45
378.43
392.45
434.53
434.53
cLogP
−1.70
−1.36
−1.36
0.10
cmpd
MW (g/mol)
cLogP
−0.64
−0.12
−0.12
1.33
9{1,1,1}
9{1,1,2}
9{1,1,3}
9{1,1,4}
9{1,2,1}
9{1,2,2}
9{1,2,3}
9{1,2,4}
9{1,3,1}
9{1,3,2}
9{1,3,3}
9{1,3,4}
9{1,4,1}
9{1,4,2}
9{1,4,3}
9{1,4,4}
9{2,1,1}
9{2,1,2}
9{2,1,3}
9{2,1,4}
9{2,2,1}
9{2,2,2}
9{2,2,3}
9{2,2,4}
9{2,3,1}
9{2,3,2}
9{2,3,3}
9{2,3,4}
9{2,4,1}
9{2,4,2}
9{2,4,3}
9{2,4,4}
350.37
364.40
364.40
406.40
364.40
378.43
378.43
420.51
364.40
378.43
378.43
420.51
406.48
420.51
420.51
462.59
−1.36
−0.84
−0.84
0.62
−0.12
0.40
0.40
1.85
−1.36
−0.84
−0.84
1.85
−0.12
0.40
0.40
1.85
0.10
1.33
0.62
1.85
0.62
1.85
2.08
3.31
2.2. Characterization
X-ray crystallography was done on 9{2,4,1}, 9{2,4,3}, and 9{2,1,3} in order to gain insights on the
conformation of the macrocyclic ring system and select crystal data is provided in Table 3.
Interestingly, four different conformers were observed in the asymmetric unit for 9{2,4,1} and these
are labeled conformer A, conformer B, conformer C, and conformer D in the crystal structure. There
are also four conformers (labeled conformers A–D) in the X-ray crystal structure of 9{2,4,3} and two
conformers (labeled conformers A and B) in the X-ray crystal structure of 9{2,1,3}.

Molecules 2012, 17
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Table 3. Selected crystal data collection and refinement data.
Crystal data
formula
9{2,4,1}
C19H30N6O4 + 0.16O
409.03
9{2,4,3}
C20H32N6O4
420.51
9{2,1,3}
C16H24N6O4 + H2O
382.43
weight (g mol−1)
crystal size (mm)
crystal system
space group
a (Å)
0.45  0.38  0.28
monoclinic
P21
0.45  0.13  0.07
monoclinic
P21
0.35  0.07  0.05
orthorhombic
P212121
13.7441(2)
14.4322(2)
21.3948(3)
94.192(1)
4,232.5(1)
8
14.0826(7)
13.9531(7)
22.4144(12)
96.766(3)
4,373.7(4)
8
8.6170(1)
17.9394(3)
24.4406(4)
-
b (Å)
c (Å)
 (°)
cell volume (Å3)
Z
3,778.1(1)
8
temp (K)
S
100(2)
100(2)
100(2)
1.017
1.093
0.994
A labeling diagram for the macrocycles, averaged torsion angles with deviations for the four
conformers A–D of both 9{2,4,1} and 9{2,4,3}, the averaged torsion angles with deviations for the two
conformers A and B in 9{2,1,3}, an illustration of conformer A in 9{2,4,1} shown in a ball-and-stick
representation, as well as the comparable torsion angles observed in a Spartan’06 [42,43]
energy-minimized structure of 9{2,4,1} are all given in Table 4.
Table 4. Crystallographic details for 9{2,4,1}, 9{2,4,3}, and 9{2,1,3}.
R1 = CH2CH(CH3)2 or H
R1
O
H
6
7
N 5
8N
O
4
9
10
3
R2 = H or CH3
R2
11N
N 2
1
12
H
14 13
O
H
O
N
N
Compound
9{2,4,3} ‡
Torsion Angle
1-2-3-4
9{2,4,1} ‡
9{2,1,3} † Minimized 9{2,4,1} §
131.2 ± 4.3°
138.8 ± 6.4°
−66.3 ± 3.0°
165.5 ± 5.2°
−109.0 ± 6.8°
111.1 ± 9.1°
177.1 ± 2.3°
100.8 ± 5.4°
−77.0 ± 4.2°
99.4 ± 2.9°
175.4 ± 3.0°
145.9 ± 3.1°
−3.8 ± 1.9°
134.1 ±4.0°
−77.4 ± 0.3°
165.0 ± 1.6°
−95.2 ± 1.7°
112.0 ± 5.7°
174.7 ± 1.8°
98.5 ± 3.9°
−71.9 ± 0.9°
103.6 ± 3.1°
173.3 ± 0.2°
137.3 ± 1.6°
−1.1 ± 4.1°
109.6°
−82.8°
156.0°
−93.7°
129.1°
−176.5°
95.5°
2-3-4-5
−60.3 ± 13.8°
3-4-5-6
164.2 ± 4.5°
4-5-6-7
−108.4 ± 12.5°
106.1 ± 7.6°
175.4 ± 3.2°
102.2 ± 1.7°
−75.6 ± 4.2°
96.3 ± 0.9°
5-6-7-8
6-7-8-9
7-8-9-10
8-9-10-11
9-10-11-12
10-11-12-13
11-12-13-14
12-13-14-1
−89.4°
90.5°
176.1 ± 1.0°
147.2 ± 3.9°
−2.2 ± 1.2°
−175.0°
149.8°
−2.6°

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Table 4. Cont.
Compound
9{2,4,3} ‡
Torsion Angle
13-14-1-2
9{2,4,1} ‡
15.7 ± 4.5°
9{2,1,3} † Minimized 9{2,4,1} §
13.6 ± 4.3°
−166.9 ± 2.7°
16.3 ± 0.9°
39.9°
14-1-2-3
−167.9 ± 2.1°
−166.4 ± 0.3°
−163.5°
‡ Four conformers were observed in the X-ray crystal structure and the data presented is the
average with the observed deviation in these torsion angles; † Two conformers were observed in
the X-ray crystal structure and the data presented is the average with the observed deviation in
these torsion angles; § The emboldened torsion angles are outside the range observed in conformers
A–D for the crystal structure of 9{2,4,1}; The ball-and-stick illustration is Conformer A from the
X-ray crystal structure of 9{2,4,1}.
The  torsion angle (N2-C3-C4-N5; −60.3 ± 13.8°) of the glycine residue in conformers A–D of
9{2,4,1} is a principal site of observed variability between the conformers found in the crystal
structure, as evidenced by the value of the deviation in this angle compared to others in the
macrocycle. This is not particularly surprising as glycine is quite flexible relative to the other natural
amino acids. Yet, it was surprising to find considerable variability in both the  (C4-N5-C6-C7;
−108.4 ± 12.5°) and  (N5-C6-C7-N8; 106.1 ± 7.6°) torsion angles of the leucine, along with striking
similarity in the torsion angles of the N,N'-diethylethylenediamine linker. We would have expected the
diamine torsion angles to be the observed site of differences more so than the amino acid residues. In
hindsight, we reason this has some relation to the strong intramolecular 7-membered hydrogen
bond [44,45], which is seen in each of the conformers of the asymmetric units across the crystal
structures, although crystal packing influences cannot be ruled out. The observed NH…O distances of
this hydrogen bond across conformers A–D in the X-ray crystal structures of both 9{2,4,1} and
9{2,4,3}, as well as for conformers A and B in 9{2,1,3} are provided in Table 5. The comparable
values observed in the energy-minimized structure of 9{2,4,1} is also given in Table 5.
Table 5. Intramolecular hydrogen bonding details for 9{2,4,1}, 9{2,4,3}, and 9{2,1,3}.
Molecule in Asymmetric Unit
Measurement
9{2,4,1}
9{2,4,3}
9{2,1,3}
Minimized 9{2,4,1}
N2…O(=C12)
N2-H…O(=C12)
C12=O…H(-N2)
2.69 ± 0.02 Å 2.72 ± 0.02 Å 2.78 ± 0.01 Å 2.74 Å
166.8 ± 3.5°
106.2 ± 0.8°
160.9 ± 2.6°
106.4 ± 1.3°
161.7 ± 5.9°
103.4 ± 2.1°
151.4°
114.4°
The , torsion angles of the leucine (−109.0 ± 6.8°, 111.1 ± 9.1°) and D-alanine (138.8 ± 6.4°,
−66.3 ± 3.0°) in 9{2,4,3} are also principal sites of variability in the conformers A–D observed in this
X-ray crystal structure, as again evidenced by the larger deviations in these angles as compared with
other torsion angles. Smaller deviations for the respective , torsion angles of the glycine (−95.2 ± 1.7°,
112.0 ± 5.7°) and D-alanine (134.1 ± 4.0°, −77.4 ± 0.3°) residues are observed in the two conformers
(A and B) of 9{2,1,3}, although the greater of these deviations are still significant when compared the
other torsion angles of the macrocycle.
As indicated, the intramolecular hydrogen bond is observed in all of the crystal structures, just as
previously noted across a wide variety of imidazole-4,5-dicarboxylic acid derivatives [44,45]. The

Molecules 2012, 17
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amino acid from chemset 6{1-4} provides the donor NH (N2) in this intramolecular hydrogen bond,
with the acceptor (C12=O) coming from the scaffold itself. The macrocycles then form intermolecular
dimers in the solid-state, with the imidazole NH and other amide carbonyl from the scaffold (C1=O)
forming NH…O hydrogen bonds to C7=O and N5-H, respectively, from the amino acid chemset 3{1-4}
in the synthetic scheme. These interactions can be observed in the asymmetric units from the X-ray
crystal structures as illustrated in Figure 1.
Figure 1. Asymmetric units in the crystal structures of (a) 9{2,4,1}, (b) 9{2,4,3}, and (c) 9{2,1,3}.
Molecular modeling with Spartan’06 was done on 9{2,4,1} in order to compare the torsion angles
observed in the energy-minimized structure with the averaged torsion angles from the four conformers
(A–D) in the X-ray crystal structure (Table 4). Interestingly, a majority of the torsion angles in the
energy-minimized model deviate from the observed ranges seen across conformers A–D of 9{2,4,1},
and only two torsion angles connecting the intramolecular hydrogen bond acceptor (C12=O) with the
imidazole ring fall into the observed range. Yet, the parameters for the intramolecular hydrogen bond
in the energy-minimized structure do not compare with the ranges of the four conformers (A–D) in the
crystal structure (Table 5). While the source of these differences is not clear at this time, the variability
in the torsion angles is considered advantageous in the future screening applications of the macrocycle
libraries. The thinking is that the macrocycle will provide overall preorganization of the scaffold, but
retain enough torsion angle variability about the amino acid residues to help optimize the binding
interactions of side chain pharmacophores at targets.

Molecules 2012, 17
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3. Experimental
3.1. General
All apparatus was oven-dried and cooled in a desiccator. Reagents were purchased from
commercial suppliers and used without further purification except reagent grade CH2Cl2 that was
distilled from CaH2 before use. Thin layer chromatography (TLC) was performed on 250 µm glass-backed
silica gel plates and visualized using UV. Column chromatography was performed on silica gel
(Merck, grade 9385, 230–400 mesh, 60 Å). The columns were prepared in plastic 20 or 30 mL syringe
bodies and filled to approximately 2/3 of their capacity with silica gel.
3.2. LC-MS Analysis
Characterization of the purity and identity of the library members and intermediates was carried out
by liquid chromatography-mass spectrometry (LC-MS) using a Varian 500-MS LC Ion Trap mass
spectrometer. Solutions of the compounds were prepared at an approximate concentration of 1 mg/mL
by first adding <10% by volume of CH2Cl2 to dissolve the sample and then diluting to the final volume
with HPLC grade methanol. Five microliters of the sample was injected onto a Polaris 5 mm C18-A
(50  2.0 mm) HPLC column and eluted with a gradient of CH3CN/H2O containing 0.1% CH3CO2H at
a 0.2 mL/min flow rate. Compounds were detected at 214 nm. The gradient was as follows: 0 min., 4:6
CH3CN/H2O  1 min., 4:6 CH3CN/H2O  6 min., 9:1 CH3CN/H2O  8 min., 9:1 CH3CN/H2O 
9 min., 4:6 CH3CN/H2O  10 min., 4:6 CH3CN/H2O. The mass spectrum was recorded for the entire
elution time by using ESI detection from 50–800 (m/z) with the following parameters: capillary
voltage at 60.0, RF loading at 100%, drying gas at 250 °C, spray chamber at 50 °C, nebulizer gas at
50.0 psi, drying gas at 25 psi, and damping gas at 0.8 mL/min.
3.3. NMR Spectroscopy
13
1H-NMR and C-NMR spectra were recorded for the intermediates at 400 MHz and 100.5 MHz,
1
respectively, in CDCl3 with CHCl3 as the internal reference for H ( 7.24) and CDCl3 as the internal
13
1
reference for C ( 77.00) or DMSO-d6 with DMSO as the internal reference for H ( 2.49) and
13
1
DMSO-d6 as the internal reference for C ( 39.50). The H-NMR spectra of the final macrocycles
were determined at 270 MHz in MeOH-d4 with MeOH as the internal reference for H ( 3.34). The
1
spectra for the final macrocycles are provided in the ESI. Coupling constants are expressed in Hertz (Hz).
Data are reported in this sequence: (1) chemical shifts (ppm); (2) spin multiplicities given as,
s (singlet), bs (broad singlet), d (doublet), t (triplet), q (quartet), quintet, sextet and m (multiplet);
(3) coupling constant J (Hz); and (4) integration.
3.4. X-ray Crystallography
Samples for X-ray diffraction were obtained from the slow evaporation of a wet methanolic
solution. Data using CuK radiation were collected with a Bruker APEX II CCD and integrated with
SAINT and XPREP in order to provide the observable reflections [Fo > 4sig(Fo)]. Crystal stability
was monitored by measuring three standard reflections after every 97 reflections with no significant

Molecules 2012, 17
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decay in observed intensities. A-2 scanning technique was used for peak collection with Lorenz and
polarization corrections applied. Hydrogen atom positions were located from difference Fourier maps,
and a riding model with fixed thermal parameters [uij = 1.2Uij(eq)] for the atom to which they are
bonded] was used for subsequent refinements. The SHELXL-93 package [46] was used for data
reduction, structure solution, and refinement.
CCDC 874142-874144 contain the supplementary crystallographic data for this paper. These data
can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12
Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033; e-mail: deposit@ccdc.cam.ac.uk).
3.5. Molecular Modeling
Spartan’06 [42,43] was used to calculate a low-energy conformation for 9{2,4,1}. A 100-membered
conformer library was generated and the 10 lowest energy conformers were minimized with PM3
semi-empirical methods. The lowest energy conformer from the molecular modeling was then
compared with the four conformers (A–D) observed in the solid-state by X-ray crystallography.
3.6. Synthetic Procedures
Procedures are provided for the protection of the N,N'-dialkylalkanediamines, along with general
methods for the remaining intermediates and final macrocycles. A specific example for each
intermediate and for one macrocycle is also provided here. The remaining synthetic details and
analytical data are provided in the ESI.
2{1}. Alternative procedure: To an ice-cooled solution of N,N'-dimethylethylenediamine (7.4 mL,
68.8 mmol) in dry THF (60 mL) was added a solution of Boc2O (5.0 g, 22.9 mmol) in dry THF (40 mL).
The reaction mixture was stirred at 0 °C for 30 min. then warmed to room temperature (rt) and stirred
another 19.5 h. The reaction mixture was filtered and the filtrate concentrated under vacuum. The
residue was dissolved in EtOAc, washed with brine (3 × 50 mL), dried over MgSO4, and concentrated
under vacuum to yield 3.83 g (85%) of a colorless oil used without further purification. The residue
from above was dissolved in 40 mL of dry THF and cooled to 0 °C before concurrently adding Et3N
(2.84 mL, 20.4 mmol) and benzyl chloroformate (2.86 mL, 20.4 mmol) dropwise over 20 min. The
reaction was warmed to rt and stirred another 3 h. The solvent was removed under vacuum. The
residue was dissolved in EtOAc (100 mL), washed with brine (3 × 30 mL), dried over MgSO4, and
concentrated under vacuum to yield 6.35 g (97%) of a colorless oil. TLC Rf (EtOAc/Hex 1:1) = 0.54;
1H-NMR (CDCl3) δ 7.32 (m, 5H), 5.11 (s, 2H), 3.30 (s, 4H), 2.58 (s, 4H), 1.70 (m, 2H), 1.48 (s, 1H),
1.06 (m, 6H); LC-MS (Method A) 5.25 min; ESI MS m/z 345 [M+Na]+.
To a solution of the above intermediate (6.35 g, 19.7 mmol) in 20 mL of acetone was added 36%
aqueous HCl (20 mL) dropwise at rt. The reaction mixture was stirred for 16 h before removing the
solvent under vacuum. The residue was dissolved in EtOAc (100 mL), washed with sat. NaHCO3,
brine, dried over MgSO4, and concentrated under vacuum to yield 1.20 g (27%) of a pale yellow oil.
LC-MS (Method A) 1.19 min; ESI MS m/z 223 [M+H]+.

Molecules 2012, 17
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2{2}. To a dry roundbottom flask was added 40 mL of CH2Cl2 and N,N'-diethylethylenediamine (10.1 mL,
70.4 mmol). To this stirred solution at −78 °C was added a solution of benzyl chloroformate (1.0 mL,
7.04 mmol) in 40 mL of CH2Cl2 over 1 h. The reaction was stirred for 1.5 h at −78 °C before warming
to rt and stirring for another 18 h. The reaction was filtered and the filtrate washed with brine (3 × 50 mL),
dried over MgSO4, and concentrated under vacuum. The product was purified by column chromatography
using a gradient of EtOAc/MeOH (19:1  3:2), yielding 1.40 g (80%) of a brown oil. TLC
Rf (EtOAc) = 0.05; 1H-NMR (CDCl3) δ 7.27–7.33 (m, 5H), 5.11 (s, 2H), 3.32 (m, 4H), 2.59–2.76 (m,
4H), 1.00–1.12 (m, 6H); LC-MS (Method A) 1.45 min; ESI MS m/z 251 [M+H]+.
4{1-2,1-4}. General Method. To a solution of the N,N'-dialkylalkanediamine in CH2Cl2 was added the
Boc-amino acid, EDC.HCl, and DMAP at rt. The reaction was stirred for a period of time before
removing the solvent under vacuum. The product was purified by column chromatography on silica gel.
4{1,1}. Example. As described using 2{1} (2.00 g, 9.01 mmol) in 100 mL of CH2Cl2 with 3{1} (1.58 g,
9.01 mmol), EDC.HCl (3.79 g, 19.82 mmol), and DMAP (0.22 g, 1.80 mmol) at rt. The reaction was
stirred for 16 h and the product was purified by column chromatography on silica gel using
EtOAc/hexanes (1:3  1:0), as the gradient, yielding 2.94 g (86%) of a colorless oil. TLC Rf
(EtOAc/Hex 1:1) = 0.37; 1H-NMR (CDCl3) δ 7.26–7.33 (m, 5H), 5.38 (s, 1H), 5.08 (d, 2H), 3.73–3.81
(m, 2H), 3.41–3.54 (m, 4H), 2.77–2.97 (m, 6H), 1.43 (s, 9H); LC-MS (Method A) 3.49 min; ESI MS
m/z 380 [M+H]+.
5{1-2,1-4}. General Method. A suspension of one intermediate, 4{1-4,1-4}, and 5% Pd/C in CH3OH
was stirred at rt under 1 atm of H2. The reaction was stirred until complete, filtered to remove the
Pd/C, and concentrated under vacuum. The product was used without further purification.
5{1,1}. Example. As described using 4{1,1} (2.90 g, 7.65 mmol) and 5% Pd/C (0.40 g) in 120 mL of
CH3OH at rt. The reaction was stirred for 11 h and workup yielded 1.80 g (96%) of a colorless oil.
1H-NMR (CDCl3) δ 5.49 (s, 1H), 3.92 (q, J = 8.0 Hz, 2H), 3.49 (t, J = 6.4 Hz, 1H), 3.30 (t, J = 6.4 Hz,
1H), 2.94 (d, J = 2.8 Hz, 3H), 2.72 (m, 2H), 2.41 (d, J = 3.6 Hz, 3H), 1.42 (s, 9H), 1.28 (s, 1H);
LC-MS (Method A) 1.20 min; ESI MS m/z 246 [M+H]+.
6{1-4}. Synthesis was done as previously described [33]. Briefly, to a dry round-bottom flask under
argon was added dry CH2Cl2. To this stirred solvent at −78 °C were added, in order, the pyrazine
diacid chloride (1 equiv.), the amino acid ester hydrochloride salt (2 equiv.), and N,N-diethylaniline
(4 equiv.). The resulting solution was held at −78 °C for 30 min before stirring at room temperature.
The reaction was washed against water (3×) and the organic fraction was dried over MgSO4, filtered
and concentrated. The residue was suspended in boiling EtOAc, stirred for 15 min and then cooled at
0 °C. The solid product 6{1-4} was collected by vacuum filtration and washed with EtOAc. The final
product was characterized by 1H-NMR spectroscopy.
1
6{1}. Example. H-NMR (CDCl3) δ 8.64 (s, 2H), 8.59 (t, J = 4.8 Hz, 2H), 4.17 (d, J = 4.8 Hz, 2H),
1.49 (s, 18H).

Molecules 2012, 17
5357
7{1-2,1-4,1-4}. General Method. To a suspension of one amino acid ester substituted pyrazine, 6{1-4}
in CH2Cl2 was added one intermediate, 5{1-2,1-4} at rt. The reaction was stirred until complete, the
solvent removed under vacuum, and the product purified by column chromatography on silica gel.
7{1,1,1}. Example. As described using 6{1} (0.20 g, 0.40 mmol) and 5{1,1} (0.25 g, 1.00 mmol) in 20 mL
of CH2Cl2 at rt. The reaction was stirred for 19.5 h. Column chromatography using a gradient of
EtOAc/Hex (1:1  1:0) followed by EtOAc/MeOH (1:19  4:1) yielded 0.25 g (63%) of a white
1
solid. TLC Rf (EtOAc/MeOH 9:1) = 0.25; H-NMR (CDCl3) δ 12.26 (m, 1H), 10.50–10.93 (m, 1H),
7.58 (d, J = 5.6 Hz, 1H), 5.56 (t, J = 11.2 Hz, 1H), 3.64–4.19 (m, 8H), 2.85–3.42 (m, 6H), 1.47 (s, 9H),
1.39 (s, 9H); 13C-NMR (CDCl3) δ 169.0, 168.9, 168.4, 168.3, 168.2, 168.1, 165.9, 165.8, 165.4, 159.7,
159.5, 158.6, 155.8, 136.5, 135.4, 134.8, 134.6, 134.5, 134.4, 133.8, 130.7, 129.7, 129.3, 129.2, 129.0,
81.9, 79.5, 79.3, 52.9, 49.5, 48.6, 47.9, 47.2, 46.7, 46.6, 45.9, 45.4, 42.6, 42.4, 42.3, 41.9, 41.6, 39.0,
38.6, 35.9, 35.6, 35.0, 34.5, 34.2, 34.0, 28.3, 28.0; LC-MS (Method A) 1.67 min; ESI MS m/z 497 [M+H]+.
8{1-2,1-4,1-4}. General Method. To a solution of one intermediate, 7{1-2,1-4,1-4} in CH2Cl2 was
added trifluoroacetic acid at rt. The reaction was stirred until complete and the solvent removed under
vacuum. The residue was taken up in aqueous HCl and lyophilized to dryness, yielding a material that
was used without further purification.
8{1,1,1}. Example. As described using 7{1,1,1} (210 mg, 0.423 mmol), 5 mL of CH2Cl2 and 1 mL of
TFA. The reaction was stirred for 13 h. LC-MS (Method A) 1.14 min; ESI MS m/z 341 [M+H]+.
9{1-2,1-4,1-4}. General Method. To 20 mL of CH2Cl2 in a dry roundbottom flask was added dropwise
over 1 h a solution of one intermediate, 7{1-32} (1 equiv.) in 20 mL of CH2Cl2 concurrent with the
dropwise addition of a solution containing EDC.HCl (2.2 equiv.) and DMAP (0.2 equiv.) in 20 mL of
CH2Cl2. The resulting solution was stirred for a period of time before removing the solvent under
vacuum and purifying the macrocycle by column chromatography on silica gel. The chemset numbers
for the final products and their associated names are provided in Table 6.
9{1,1,1}. Example. As described using 8{1,1,1} (72 mg, 0.210 mmol), EDC.HCl (89 mg, 0.466 mmol),
and DMAP (5 mg, 0.042 mmol). The reaction was stirred for 10.5 h. Column chromatography using a
gradient of EtOAc/Hex (1:1  1:0) followed by EtOAc/MeOH (1:19  3:2) yielded 4.9 mg (7%) of a
white solid. TLC Rf (EtOAc/MeOH 1:1) = 0.25; LC-MS (Method A) 1.21 min; ESI MS m/z 323 [M+H]+.
Table 6. Final product numbers and nomenclature.
compound
Name
9{1,1,1} cyclo(N,N'dimethylethylenediamine-glycyl-glycyl-4,5-dicarbonyl)-1H-imidazole
9{1,1,2} cyclo(N,N'dimethylethylenediamine-glycyl-S-alanyl-4,5-dicarbonyl)-1H-imidazole
9{1,1,3} cyclo(N,N'dimethylethylenediamine-glycyl-R-alanyl-4,5-dicarbonyl)-1H-imidazole
9{1,1,4} cyclo(N,N'dimethylethylenediamine-glycyl-S-leucyl-4,5-dicarbonyl)-1H-imidazole
9{1,2,1} cyclo(N,N'dimethylethylenediamine-S-alanyl-glycyl-4,5-dicarbonyl)-1H-imidazole
9{1,2,2} cyclo(N,N'dimethylethylenediamine-S-alanyl-S-alanyl-4,5-dicarbonyl)-1H-imidazole
9{1,2,3} cyclo(N,N'dimethylethylenediamine-S-alanyl-R-alanyl-4,5-dicarbonyl)-1H-imidazole
9{1,2,4} cyclo(N,N'dimethylethylenediamine-S-alanyl-S-leucyl-4,5-dicarbonyl)-1H-imidazole

Molecules 2012, 17
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Table 6. Cont.
compound
Name
9{1,3,1} cyclo(N,N'dimethylethylenediamine-R-alanyl-glycyl-4,5-dicarbonyl)-1H-imidazole
9{1,3,2} cyclo(N,N'dimethylethylenediamine-R-alanyl-S-alanyl-4,5-dicarbonyl)-1H-imidazole
9{1,3,3} cyclo(N,N'dimethylethylenediamine-R-alanyl-R-alanyl-4,5-dicarbonyl)-1H-imidazole
9{1,3,4} cyclo(N,N'dimethylethylenediamine-R-alanyl-S-leucyl-4,5-dicarbonyl)-1H-imidazole
9{1,4,1} cyclo(N,N'dimethylethylenediamine-S-leucyl-glycyl-4,5-dicarbonyl)-1H-imidazole
9{1,4,2} cyclo(N,N'dimethylethylenediamine-S-leucyl-S-alanyl-4,5-dicarbonyl)-1H-imidazole
9{1,4,3} cyclo(N,N'dimethylethylenediamine-S-leucyl-R-alanyl-4,5-dicarbonyl)-1H-imidazole
9{1,4,4} cyclo(N,N'dimethylethylenediamine-S-leucyl-S-leucyl-4,5-dicarbonyl)-1H-imidazole
9{2,1,1} cyclo(N,N'diethylethylenediamine-glycyl-glycyl-4,5-dicarbonyl)-1H-imidazole
9{2,1,2} cyclo(N,N'diethylethylenediamine-glycyl-S-alanyl-4,5-dicarbonyl)-1H-imidazole
9{2,1,3} cyclo(N,N'diethylethylenediamine)-glycyl-R-alanyl-4,5-dicarbonyl)-1H-imidazole
9{2,1,4} cyclo(N,N'diethylethylenediamine-glycyl-S-leucyl-4,5-dicarbonyl)-1H-imidazole
9{2,2,1} cyclo(N,N'diethylethylenediamine-S-alanyl-glycyl-4,5-dicarbonyl)-1H-imidazole
9{2,2,2} cyclo(N,N'diethylethylenediamine-S-alanyl-S-alanyl-4,5-dicarbonyl)-1H-imidazole
9{2,2,3} cyclo(N,N'diethylethylenediamine-S-alanyl-R-alanyl-4,5-dicarbonyl)-1H-imidazole
9{2,2,4} cyclo(N,N'diethylethylenediamine-S-alanyl-S-leucyl-4,5-dicarbonyl)-1H-imidazole
9{2,3,1} cyclo(N,N'diethylethylenediamine-R-alanyl-glycyl-4,5-dicarbonyl)-1H-imidazole
9{2,3,2} cyclo(N,N'diethylethylenediamine-R-alanyl-S-alanyl-4,5-dicarbonyl)-1H-imidazole
9{2,3,3} cyclo(N,N'diethylethylenediamine-R-alanyl-R-alanyl-4,5-dicarbonyl)-1H-imidazole
9{2,3,4} cyclo(N,N'diethylethylenediamine-R-alanyl-S-leucyl-4,5-dicarbonyl)-1H-imidazole
9{2,4,1} cyclo(N,N'diethylethylenediamine-S-leucyl-glycyl-4,5-dicarbonyl)-1H-imidazole
9{2,4,2} cyclo(N,N'diethylethylenediamine-S-leucyl-S-alanyl-4,5-dicarbonyl)-1H-imidazole
9{2,4,3} cyclo(N,N'diethylethylenediamine-S-leucyl-R-alanyl-4,5-dicarbonyl)-1H-imidazole
9{2,4,4} cyclo(N,N'diethylethylenediamine-S-leucyl-S-leucyl-4,5-dicarbonyl)-1H-imidazole
4. Conclusions
Macrocyclic peptides are useful tools in the drug discovery, owing in part to the conformational
preorganization of their pharmacophores, reduced proteolytic degradation and metabolism, as well as
increased bioavailability compared to acyclic peptides. Imidazole-4,5-dicarboxylic acid is a useful
scaffold for drug discovery that is readily diversified with amines to yield imidazole-4,5-dicarboxamides.
We now illustrate the ability of such derivatives to be further modified into 14-membered peptide-like
macrocyclic rings. These final products are natural product-like and the synthesis accepts modifications
in stereochemistry and amino acid side chains. The yields to the chromatographically pure final
products are poor at this time, but efforts to optimize the macrocyclization and extend the types of
substituents presented by the macrocyclic ring will be undertaken. The products may also be valuable
in screening collections looking for hits against a wide variety of drug targets, and these efforts too
will be reported on in due time.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/17/5/5346/s1.

Molecules 2012, 17
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Acknowledgments
The authors thank the NIH-NIGMS for funding this research (P41 GM79589-02). K.A.W. and
P.W.B. thank the NSF for support of the EIU X-ray facility (0722547).
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