Synthesis of enantiopure 7-substituted azepane-2-carboxylic acids as templates for conformationally constrained peptidomimetics

Article abstract of DOI:10.1002/ejoc.201101387

The introduction of a cyclic amino acid in a peptide is one of the best methods to rigidify a strand. A general approach towards a new class of seven-membered ring amino acids is described starting from (S)-tribenzyl glutamic acid γ-aldehyde, which reacts with β-keto phosphonates to generate the Horner-Wadsworth-Emmons product. In the presence of H₂ and a Pd catalyst, a four-step process occurs involving double-bond hydrogenation, hydrogenolysis of three benzyl protecting groups, imine formation, and reductive amination to produce the 7-substituted azepane carboxylic acid in good overall yield and with good to excellent diastereomeric ratios. An amino function can be introduced in the 7-position as an additional orthogonal chemical handle for readily generating diversity on the cyclic amino acid scaffold by using a β-keto phosphonate derived from amino acids. A cyclic RGD (Arg-Gly-Asp) pentapeptide analogue containing this new class of noncoded amino acids was also prepared by microwave-assisted cyclization, showing a promising activity as α_vβ₃ integrin inhibitor.

Full text of DOI:10.1002/ejoc.201101387

FULL PAPER
DOI: 10.1002/ejoc.201101387
Synthesis of Enantiopure 7-Substituted Azepane-2-carboxylic Acids as
Templates for Conformationally Constrained Peptidomimetics
Elena Cini,[a] Giuseppe Bifulco,[b] Gloria Menchi,[c] Manuela Rodriquez,*[b] and
Maurizio Taddei*[a]
Keywords: Amino acids / Peptidomimetics / Azepanes / Cyclization / Synthetic methods
The introduction of a cyclic amino acid in a peptide is one of
the best methods to rigidify a strand. A general approach
towards a new class of seven-membered ring amino acids is
described starting from (S)-tribenzyl glutamic acid γ-alde-
hyde, which reacts with β-keto phosphonates to generate the
Horner–Wadsworth–Emmons product. In the presence of H2
and a Pd catalyst, a four-step process occurs involving
double-bond hydrogenation, hydrogenolysis of three benzyl
protecting groups, imine formation, and reductive amination
to produce the 7-substituted azepane carboxylic acid in good
overall yield and with good to excellent diastereomeric ra-
tios. An amino function can be introduced in the 7-position
as an additional orthogonal chemical handle for readily gen-
erating diversity on the cyclic amino acid scaffold by using a
β-keto phosphonate derived from amino acids. A cyclic RGD
(Arg-Gly-Asp) pentapeptide analogue containing this new
class of noncoded amino acids was also prepared by micro-
wave-assisted cyclization, showing a promising activity as
αvβ3 integrin inhibitor.
intended to introduce a constraint into the backbone that
forces the system into a predefined secondary structure,
corresponding roughly to α-, β-, or γ-turns or β-strands,
which are structural motifs frequently found in the biolo-
gically active conformations of peptides. Although the in-
troduction of cyclic constraints into peptides has proven to
be advantageous to organize the backbone, such cycliza-
tions are generally not able to preferentially orient the
amino acid side chains. Because these side chains contribute
significantly to specificity and affinity, controlling their spa-
tial orientation is an important aspect in the design of
peptidomimetics.[4] One of the best methods to constrain
a peptide, with simultaneous projection of the side chain
orientations, is to introduce a cyclic amino acid into the
strand. Besides proline, other kinds of mono-, bi-, and even
tricyclic amino acids have been designed and synthesized to
serve as useful templates for turn mimetics.[5]
Introduction
The development of new small molecules with high affin-
ity and selectivity towards therapeutically relevant targets is
one of the main efforts of contemporary bioorganic and
medicinal chemists.[1] Peptides serve as leads in this discov-
ery process; although flexible, they work in the so-called
biologically active conformation adopted to bind proteins.
A conformationally constrained peptide analogue is ex-
pected to have higher affinity for the target because the pre-
organized molecule should pay a lower entropic cost for
complexation, assuming that the flexible and the con-
strained molecules interact in the same way with the solvent
and the receptor.[2] Moreover, conformationally constrained
molecules often show better selectivity profiles. Evidence of
candidates with reduced flexibility showing improved high
oral bioavailability have been also demonstrated.[3]
One of the possible options to rigidify a structure in-
serted into a flexible peptide is to prepare a cyclic analogue
of the natural linear peptide. Such a cyclization is primarily
Results and Discussion
[a] Dipartimento Farmaco Chimico Tecnologico,
Università degli Studi di Siena,
Azepanes are heterocyclic rings that are well represented
in nature and in pharmaceutically relevant structures such
as Balanol and Tuberostemonine (Scheme 1),[6] but no
enantiopure substituted azepane amino acid has been syn-
thesized until now, with the exception of simple azepane 2-
carboxylic acid.[7]
Considering our experience in the application of glut-
amic acid as a synthon to access bioactive molecules,[8] we
figured out a possible rapid approach to a collection of en-
antiomerically pure azepane carboxylic acids based on the
Via A. Moro 2, 53100 Siena, Italy
Fax: +39-0577-234280
E-mail: taddei.m@unisi.it
[b] Dipartimento di Scienze Farmaceutiche e Biomediche,
Università degli Studi di Salerno,
Via Ponte don Melillo, 84084 Fisciano, Salerno, Italy
[c] Dipartimento di Chimica “Ugo Schiff”,
Università degli Studi di Firenze,
Polo Scientifico di Sesto Fiorentino,
50019 Sesto Fiorentino, Firenze, Italy
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201101387.
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Table 1. Synthesis of benzyl 2-dibenzylamino-7-oxooct-5-enoates
13–22.
Scheme 1. Azepane-containing bioactive molecules.
reductive cyclization of 2-amino-7-ketocarboxylates. For
their preparation, a HWE (Horner–Wadsworth–Emmons)
reaction between the γ-aldehyde derived l-Glu and a phos-
phonate (derivable form the corresponding ester) was pro-
posed (see the scheme in Table 1).
Aldehyde 1, obtained in two steps from l-glutamic
acid,[9] was treated with phosphonates 3–12. With the ex-
ception of commercially available phosphonates 3–5, com-
pounds 6–12 were prepared by reaction of lithium methyl
phosphonate 2 (generated in situ from dimethyl methyl-
phosphonate with BuLi in THF at –78 °C) with different
esters.[8a] β-Ketophosphonates 6–12 were obtained in good
to acceptable yields, although some of them were unstable
at room temperature and were prepared just before their
use. An additional HWE reaction was carried out by em-
ploying DIPEA in the presence of dry LiCl in MeCN to
[a] Yield of isolated and fully characterized product.
Table 2. Optimization of the cyclization step.
Entry
Catalyst
Conditions
Product (% Yield)
1
2
3
4
5
6
7
8
Pd/C
Pd/C
Pd/C
Pd(OH)2/C
Pd(OH)2/C
Pd(OH)2/C
Pd(OH)2/C
Pd(OH)2/C
HCOONH4, 2-propanol, MW, 150 °C, 2 min
H2 (3 atm), MeOH, 150 °C, 60 min
H2 (6 atm), MeOH, 150 °C, 4 h
H2 (6 atm), MeOH, r.t., 4 h
H2 (6 atm), MeOH, 150 °C, 4 h
H2 (6 atm), MeOH, MW, 150 °C, 10 min
H2 (6 atm), MeOH/AcOH (10:1), r.t., 12 h
H2 (6 atm), MeOH/AcOH (10:1), MW, 100 °C, 20 min
23 (87)[a]
23 (48), 24 (32), 25 (8)[b]
23 (6), 24 (27), 25 (41)[b]
24 (81)[a]
24 (85)[a]
24 (88)[a]
26 (79)[a] 95:5dr[c]
26 (91)[a] 70:30dr[c]
[a] Yield of isolated and fully characterized product. [b] Yield determined by HPLC analysis by using p-aminobenzoic acid as an internal
standard. [c] Ratio determined by HPLC analysis of the corresponding N-Cbz derivative prepared directly from the crude reaction mixture.
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Azepane-2-carboxylic Acids as Templates for Peptidomimetics
give α,β-unsaturated ketones 13–22 in good yields (Table 1). Table 3. Synthesis of different 7-substituted-azepane 2-carboxylic
Careful inspection of the 1H NMR (400 MHz) spectra
acids.
showed that exclusively the E isomer was always formed.
Compounds 13–22 were then submitted to double-bond
hydrogenation/hydrogenolysis of the benzyl groups to ob-
tain directly the cyclic azepane. The amine was first formed
under reductive conditions and further cyclized to generate
the imine, which was promptly reduced by hydrogen em-
ployed in the reaction. First attempts were carried out on
compound 13, used as a model, by transfer hydrogenolysis
with HCOONH4 and Pd/C as catalyst under microwave
(MW) dielectric heating[10] (Table 2, Entry 1). Unfortu-
nately, only reduction of the double bond occurred (com-
pound 23; Table 2, Entry 1). Also, the use of H2 at a dif-
ferent pressure and temperature did not lead to removal of
the benzyl groups (Table 2, Entries 2 and 3). Moving from
Pd/C to Pd(OH)2/C (Pearlman’s catalyst), monobenzyl de-
rivative 24 was obtained (Table 2, Entry 4). However, every
attempt to remove the last benzyl group failed, even when
working at a higher temperature or by using a MW appara-
tus equipped to operate under gas pressure[11] (Table 2, En-
tries 4–6). Compound 26 was finally obtained in 79% yield
by using Pd(OH)2/C in MeOH/AcOH (10:1) under 6 atm of
hydrogen at room temperature for 12 h (Table 2, Entry 7).
7-Methylazepane-2-carboxylic acid 26 was obtained as a
mixture of diastereomers in 95:5 ratio (HPLC and NMR
analysis). Under the optimized conditions, once the imine
is formed, it is rapidly reduced avoiding racemization of the
stereocenter at C2. The enantiomeric purity of compound
26 was proved by comparison of the NMR spectroscopic
data and HPLC data of the (+)-Mosher acid amide with
those obtained for the analogous racemic compound pre-
pared in the same way starting from racemic glutamic acid
(see the Supporting Information). To speed up the step, hy-
drogenation under MW dielectric heating was attempted
under the same reaction conditions. Product 26 was ob-
tained in very good yields but with lower diastereoselectiv-
ity (Table 2, Entry 8). The optimized reaction conditions
were applied to ketones 14–22 and the corresponding
azepane carboxylic acids 27–35 were obtained in very good
yields after 12 h (see Table 3).
The reaction proceeded with a high level of stereoselecti-
vity except in the case of compounds 32 and 34. However,
the isomer formed in higher quantity was always isolated
by column chromatography on silica gel. The 1H NMR
spectra of the cycles show large and sharp signals relative
to different parts of the molecule. Preliminary analysis of
the ROESY spectra for assessing the relative configuration
of the azepane sustituents in the 2- and 7-positions sug-
[a] Yield of isolated and fully characterized product. [b] Ratio de-
termined by HPLC analysis of the corresponding N-Cbz derivative
prepared directly from the crude reaction mixture. [c] Dia-
stereomeric ratio determined by HPLC analysis.
gested their cis arrangement on the basis of a ROE peak
between H-2 and H-7. This attribution was confirmed by a
NOESY spectrum carry out on compound 26 showing a
cross-peak between H-2 and H-7.
For securing this stereochemical assignment, we calcu-
lated, at the DFT MPW1PW91 level using the 6-31G(d,p) pared to the experimental values.[12] As shown in Table 4, a
basis set, the 13C NMR chemical shifts for the most signifi- mean absolute error (MAE = Σ [|(δexp. – δcalcd.)|]/n) of
cant conformers of the trans and cis stereoisomers, and 2.3 ppm for the cis stereoisomer vs. 4.6 ppm observed for
their Boltzmann-weighted averaged shieldings were com- the trans stereoisomer strongly suggested the cis configura-
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E. Cini, G. Bifulco, G. Menchi, M. Rodriquez, M. Taddei
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tion for the azepane ring. As the original stereocenter was oxylic groups, 7-alkylaminoazepane 2-carboxylic acids re-
derived from (S)-lactic acid, we could assign the absolute tain the same topology of a dipeptide. Moreover, by intro-
configuration (S) to the carbon atom in the 7-position of ducing an alkylamino group derived from an amino acid,
azepanes 26–28 and the (R) configuration to azepanes 29– all the analogues of the coded amino acids can be poten-
35, thus resulting in a cis relative configuration (for the tially prepared.
most abundant isomer). This spatial arrangement is com-
With the aim of proving that these azepane carboxylic
patible with the mode of formation of the imine and attack acids could be successfully employed in the synthesis of
of the H2 adsorbed on the Pd surface form the less-hindered conformationally constrained peptides, the preparation of a
side (opposite to the substituent in the 2-position). The in- cyclic RGD (Arg-Gly-Asp) cyclic pseudopeptide, incorpo-
crease in the hindrance around the C=N gave an increase rating our seven-membered ring template, was attempted.
in the selectivity, whereas the presence of two substituents To carry out a solid-phase synthesis of the cyclic peptide,
of comparable size at the adjacent stereocenter (as in 32 the endocyclic nitrogen of 31 was protected as Cbz and the
and 34) reduced the stereoselection.
Boc protection of the exocyclic nitrogen was replaced with a
Fmoc group through acid deprotection followed by reaction
with Fmoc-OSu (Scheme 3).
Table 4. 13C NMR calculated [MPW1PW91/6-31G(d,p) level]
chemical shifts for the cis and trans forms of 30 and the corre-
sponding 13C NMR chemical shift values (δ, ppm).
Atom
Calculated 13C NMR chemical shifts
cis
trans
30 (Exp.)
2
3
4
5
6
7
9
59.0
29.8
26.1
24.3
32.1
64.9
71.6
21.4
2.3
60.0
31.2
28.0
30.2
35.8
58.9
72.7
20.5
4.6
60.2
27.6
23.9
24.1
26.3
65.1
68.8
17.5
Scheme 3. Preparation of Fmoc-Aca(Cbz)-OH.
10
Compound 36 [stated as Fmoc-Aca(Cbz)-OH] was iso-
lated in 65% yield. The preparation of cyclic peptide 42 was
carried out on solid phase starting with the acid-labile PS-
DVB-2-ClTrt-Gly-NH2 resin (Scheme 4). N-Fmoc-Arg-
(Mtr)OH was loaded onto the resin by using HBTU/
HOBT. Then the synthesis proceeded through a cycle of
Fmoc deprotection (10% piperidine in DMF) and further
coupling with Fmoc-Aca(Cbz)-OH (36) first and N-Fmoc-
Asp(OtBu)-OH after, until linear pseudopeptide 40 was ob-
tained after removal from the resin by using AcOH/TFE/
DCM (2:2:6) for 2 h. This product, obtained in good overall
yields (84%), was cyclized in a 7ϫ10–4 m solution in
CH2Cl2 under microwave dielectric heating at 75 °C,
25 min, 25 W, using HATU/DIPEA as the coupling
agent.[13] Product 41 (protected at the amino acid side
chains) was obtained in exceptionally high yield (79%). Fi-
MAE[a]
[a] Mean absolute error (MAE) found for 13C NMR chemical shifts
of cis and trans vs. 30 13C NMR experimental values: MAE = Σ
[|(δexp. – δcalcd.)|]/n.
Compounds 31–33 (derived from encoded amino acids
and carrying a protected exocyclic NH2) can be considered
as potential cyclic analogues of dipeptides (Scheme 2). With
the same 6-atom-distance between the amino and the carb-
Scheme 2. Similarities between azepane carboxylic acids and di-
peptides.
Scheme 4. Synthesis of a pentapeptide RGD analogue containing the Aca moiety.
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Azepane-2-carboxylic Acids as Templates for Peptidomimetics
multiple resonances, br. s = broad singlet), coupling constant (J)
in Hz and integration. Carbon nuclear magnetic resonance spectra
(13C NMR) were recorded at 27 °C. Carbon chemical shifts are
expressed in parts per million (ppm, δ scale) and are referenced to
the carbon resonances of the NMR solvent (CDCl3, δ = 77.0 ppm;
CD3OD, δ = 49.1 ppm, [D6]DMSO, δ = 39.5 ppm). Mass spectrom-
etry data of the products were collected with LC-MS ESI mass
spectrometers. LC/MS conditions: ES ionisation after passage
through a C-18, 35ϫ5 mm, 3 μ column, elution: mixture A (99.9%
water, 0.1% HCOOH); mixture B (99.9% acetonitrile, 0.1%
nal removal of all the protections with TFA/thioanisole/
H2O (90:5:5) for 3 h at room temperature gave compound
42, which was purified by reverse-phase chromatography on
a C-18 cartridge followed by precipitation in diethyl ether.
Product 42 was submitted to solid-phase binding competi-
tion studies[14] against ST1646,[15] a well-known integrin li-
gand, showing IC50 = 1.8Ϯ0.4 μm towards αvβ3 and IC50
= 2.9Ϯ0.8 μm towards αvβ5 receptors. Conformational
analysis carried out on compound 42 suggested the possible
existence of a turn consistent with the binding data (Fig- HCOOH): 0–6.0 min, 95% mixture A; 6.0–9.0 min 95–0% mixture
A; 9.0–15 min 0–95% mixture A, flow 1.0 mL/min, T = 40 °C. Re-
actions carried out under microwave dielectric heating were per-
formed with a microwave oven (Discover from CEM) under mono-
mode irradiation in a 10 mL sealed vial. The internal temperature
was monitored through an internal IR sensor and the maximum
internal pressure monitored and maintained under the value of
200 psi.
ure 1). In particular, 42 showed a preferred cyclopeptide
conformation very similar to the X-ray-binding conforma-
tion of EMD121974.[15]
Synthesis of (S,E)-Benzyl 2-(Dibenzylamino)-7-oxooct-5-enoate (13)
as a Representative Procedure: To a solution of phosphonate 3
(1.00 g, 1.3 mmol) in dry MeCN (40 mL) was added dry LiCl
(254 mg, 6.0 mmol) followed by freshly distilled DIPEA (626 mg,
830 μL, 4.9 mmol) under an atmosphere of N2. After stirring for
2 h at room temperature, aldehyde 1 (1.86 g, 4.6 mmol) in MeCN
(25 mL) was added, and the mixture was stirred at room tempera-
ture for 12 h. A saturated solution of NaCl was used for quenching,
and the organic layer was separated. The aqueous phase was ex-
tracted with EtOAc, and all the organic fractions were collected
and dried with Na2SO4. The solvent was removed in vacuo. The
crude mixture was purified by column chromatography (petroleum
ether/EtOAc, 8:2) to give compound 13 as a colorless oil (1.56 g,
Figure 1. (a) Minimum energy conformation of 42 sampled during
the 10-ns Monte Carlo/stochastic dynamics simulation after energy
minimization.
(b) X-ray
conformation
of
ανβ3-bound
EMD121974.[16]
1
78% yield). H NMR (400 MHz, CDCl3): δ = 7.44–7.22 (m, 15 H,
ArH), 6.58 (dt, J = 15.6, 7.8 Hz, 1 H, CH), 5.90 (d, J = 15.6 Hz,
1 H, CH), 5.22 (AB system, 2 H, CH2), 3.61 (AB system, 4 H, 2
CH2), 3.37 (t, J = 7.0 Hz, 1 H, CH), 2.58–2.24 (m, 2 H, CH2), 2.23
(s, 3 H, CH3) 2.09–1.91 (m, 2 H, CH2) ppm. 13C NMR (50 MHz,
CDCl3): δ = 197.7, 171.9, 146.6, 139.0, 135.8, 131.5, 128.7 (4 C),
128.4 (3 C), 128.3 (2 C), 128.1 (4 C), 128.0, 126.9 (2 C), 65.9, 59.9,
54.3 (2 C), 28.5, 27.7, 26.4 ppm. MS (ESI+, C29H31NO3): m/z =
464 [M + Na]+. HRMS (ESI+): calcd. for C29H31NNaO3 464.2202;
found 464.2205.
Conclusions
In conclusion we have described the synthesis of a new
class of enantiopure cyclic amino acid based on the azepane
ring in four simple steps starting from readily available (l)-
glutamic acid. These structures can be used as a template
for the introduction of constraints into a peptide as a turn
inducer. A cyclic RGD analogous cyclic pseudopeptide that
showed micromolar binding affinity towards αvβ3 and αvβ5
receptors has also been prepared.
(S,E)-Benzyl 2-(Dibenzylamino)-7-oxonon-5-enoate (14): 1H NMR
(400 MHz, CDCl3): δ = 7.44–7.20 (m, 15 H, ArH), 6.58 (dt, J =
15.6, 7.8 Hz, 1 H, CH), 5.90 (d, J = 15.8 Hz, 1 H, CH), 5.22 (AB
system, 2 H, CH2), 3.49 (AB system, 4 H, 2 CH2), 3.37 (t, J =
7.0 Hz, 1 H, CH), 2.41 (q, J = 7.6 Hz, 2 H, CH2), 2.27–2.11 (m, 2
H, CH2), 1.96–1.84 (m, 2 H, CH2), 1.04 (t, J = 7.6 Hz, 3 H, CH3)
ppm. 13C NMR (50 MHz, CDCl3): δ = 200.9, 171.9, 145.2, 138.9
(2 C), 135.8, 130.4, 128.7 (4 C), 128.4 (2 C), 128.3, 128.2 (2 C),
128.1 (4 C), 126.8 (2 C), 65.9, 60.0, 54.3 (2 C), 32.7, 28.5, 21.4,
7.6 ppm. MS (ESI+): m/z = 456 [M + H]+. HRMS (ESI+): calcd.
for C30H34NO3 456.2539; found 456.2537.
Experimental Section
General Methods: All reagents and solvents were used after purifi-
cation: THF distilled from Na with benzophenone, CH2Cl2 dis-
tilled from dry CaCl2, MeOH distilled from Na, DMF and MeCN
dryed with molecular sieves (4 Å). The reactions were carried out
in oven-dried or flamed vessels (vials) and performed under nitro-
gen. Flash column chromatography was performed with Merck sil-
ica gel 60 (0.040–0.063 mm, 230–400 mesh). Merck aluminum- (S,E)-Benzyl 2-(Dibenzylamino)-7-oxododec-5-enoate (15): 1H
backed plates pre-coated with silica gel 60 (UV254) were used for
TLC and were visualized by staining with a solution of KMnO4.
Proton nuclear magnetic resonance (1H NMR) spectra were re-
corded at 27 °C. Proton chemical shifts are expressed in parts per
million (ppm, δ scale) and are referred to the residual hydrogen
NMR (200 MHz, CDCl3): δ = 7.44–7.20 (m, 15 H, ArH), 6.60 (dt,
J = 15.0, 7.6 Hz, 1 H, CH), 5.93 (d, J = 15.0 Hz, 1 H, CH), 5.22
(AB system, 2 H, CH2), 3.74 (AB system, 4 H, 2 CH2), 3.37 (t, J
= 7.0 Hz, 1 H, CH), 2.55 (t, J = 7.4 Hz, 2 H, CH2), 2.46–2.15 (m,
2 H, CH2), 2.11–1.91 (m, 2 H, CH2), 1.60–1-50 (m, 2 H, CH2),
in the solvent (CHCl3, δ = 7.27 ppm; CD2HOD, δ = 3.31 ppm; 1.54–1.28 (m, 4 H, 2 CH2), 0.98 (t, J = 7.2 Hz, 3 H, CH2) ppm.
CHD2SOCD3, δ = 2.50 ppm). Data are represented as follows:
chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q
= quartet, sext = sextuplet, sept = septuplet m = multiplet and/or
13C NMR (50 MHz, CDCl3): δ = 200.4, 172.2, 145.5, 139.2 (2 C),
135.9, 130.7, 128.8 (4 C), 128.5 (2 C), 128.4, 128.3 (2 C), 128.2 (4
C), 127.0, 66.1, 60.2, 59.9, 54.5 (2 C), 39.8, 31.4, 28.7, 27.9, 23.8,
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22.4, 13.8 ppm. MS (ESI+): m/z = [M + Na]+. HRMS (ESI+):
calcd. for C33H39NNaO3 520.2828; found 520.2830.
65.8, 63.9, 60.2, 54.6 (2 C), 29.2, 28.3 (3 C), 27.9, 25.2 ppm. MS
(ESI+): m/z = 649 [M + Na]+. HRMS (ESI+): calcd. for
C38H46N2NaO6 649.3254; found 649.3257.
(S,E)-Benzyl
2-(Dibenzylamino)-8,8-dimethyl-7-oxonon-5-enoate
1
(16): H NMR (400 MHz, CDCl3): δ = 7.44–7.20 (m, 15 H, ArH),
6.80 (dt, J = 15.2, 7.8 Hz, 1 H, CH), 6.34 (d, J = 15.2 Hz, 1 H,
CH), 5.22 (AB system, 2 H, CH2), 3.71 (AB system, 4 H, 2 CH2),
3.37 (t, J = 7.0 Hz, 1 H, CH), 2.44–2.01 (m, 2 H, CH2), 1.94–1.83
(m, 2 H, CH2), 1.09 (s, 9 H, 3 CH3) ppm. 13C NMR (50 MHz,
CDCl3): δ = 203.7, 172.3, 146.1, 139.4 (2 C), 136.1, 128.8 (4 C),
128.6, 128.5 (2 C), 128.4, 128.3 (2 C), 127.1 (4 C), 124.5 (2 C), 66.1,
60.5, 54.7 (2 C), 42.7, 29.1, 28.1, 26.2 (3 C) ppm. MS (ESI+): m/z =
506 [M + Na]+. HRMS (ESI+): calcd. for C32H37NNaO3 506.2671;
found 506.2671.
(S,E)-Benzyl 2-(Dibenzylamino)-7-[(R)-2,2-dimethyl-1,3-dioxolan-4-
yl]-7-oxohept-5-enoate (21): 1H NMR (400 MHz, CDCl3): δ = 7.44–
7.20 (m, 15 H, ArH), 6.83 (dt, J = 15.6, 7.6 Hz, 1 H, CH), 6.34 (d,
J = 15.6 Hz, 1 H, CH), 5.22 (AB system, 2 H, CH2), 4.49 (dd, J =
7.2, 6.8 Hz, 1 H, CH), 4.03–3.80 (m, 2 H, CH2), 3.73 (AB system,
4 H, 2 CH2), 3.37 (t, J = 6.8 Hz, 1 H, CH), 2.47–2.06 (m, 2 H,
CH2), 1.96–1.84 (m, 2 H, CH2), 1.47 (s, 3 H, CH3), 1.41 (s, 3 H,
CH3) ppm. 13C NMR (50 MHz, CDCl3): δ = 197.3, 172.1, 148.6,
139.2 (2 C), 135.9, 128.8 (4 C), 128.6 (2 C), 128.5 (2 C), 128.3,
128.2 (4 C), 127.1 (2 C), 125.4, 110.8, 79.3, 66.4, 66.1, 60.2, 54.6 (2
C), 29.2, 27.8, 25.9, 25.3 ppm. MS (ESI+): m/z = 528 [M + H]+.
HRMS (ESI+): calcd. for C33H37NNaO5 550.2569; found
550.2569.
(2S,8S,E)-Benzyl 8-(tert-Butyldimethylsilyloxy)-2-(dibenzylamino)-
7-oxonon-5-enoate (17): 1H NMR (400 MHz, CDCl3): δ = 7.43–
7.17 (m, 15 H, ArH), 6.87 (dt, J = 15.6, 6.8 Hz, 1 H, CH), 6.48 (d,
J = 15.8 Hz, 1 H, CH), 5.21 (AB system, 2 H, CH2), 4.21 (q, J =
6.8 Hz, 1 H, CH), 3.73 (AB system, 4 H, 2 CH2), 3.38 (t, J =
7.0 Hz, 1 H, CH), 2.45–1.95 (m, 2 H, CH2), 1.91–1.83 (m, 2 H,
CH2), 1.25 (d, J = 6.8 Hz, 3 H, CH3), 0.92 (s, 9 H, 3 CH3), 0.11
(s, 6 H, 2 CH3) ppm. 13C NMR (50 MHz, CDCl3): δ = 201.3, 172.1,
147.5, 139.1 (2 C), 135.8, 128.7 (4 C), 128.5, 128.2 (2 C), 128.1,
126.9 (2 C), 126.8 (4 C), 124.3 (2 C), 74.1, 66.0, 60.3, 54.4 (2 C),
29.2, 27.8, 25.6 (3 C), 20.9, 18.0, –4.9, –5.1 ppm. MS (ESI+): m/z
= 608 [M + Na]+. HRMS (ESI+) calcd. for C36H47NNaO4Si
608.3172; 608.3173.
(S,E)-Benzyl 2-(Dibenzylamino)-7-oxo-9-phenylnon-5-enoate (22):
1H NMR (400 MHz, CDCl3): δ = 7.44–7.20 (m, 20 H, ArH), 6.09
(dt, J = 15.8, 7.6 Hz, 1 H, CH), 5.94 (d, J = 15.8 Hz, 1 H, CH),
5.22 (AB system, 2 H, CH2), 3.71 (AB system, 4 H, 2 CH2), 3.37
(t, J = 7.0 Hz, 1 H, CH), 2.94–2.65 (m, 4 H, 2 CH2), 2.44–1.61 (m,
4 H, 2 CH2) ppm. 13C NMR (50 MHz, CDCl3): δ = 199.2, 172.3,
146.1, 141.3, 139.3 (2 C), 135.9, 130.1 (4 C), 128.9 (2 C), 128.7 (2
C), 128.6 (4 C), 128.5 (2 C), 128.4 (4 C), 127.2 (2 C), 126.1 (1 C),
66.2, 60.0, 54.6 (2 C), 41.5, 30.0, 28.7, 28.0 ppm. MS (ESI+): m/z =
554 [M + Na]+. HRMS (ESI+): calcd. for C36H37NNaO3 554.2671;
found 554.2674.
(2S,8S,E)-Benzyl 8-(tert-Butoxycarbonylamino)-2-(dibenzylamino)-
7-oxonon-5-enoate (18): 1H NMR (400 MHz, CDCl3): δ = 7.43–
7.17 (m, 15 H, ArH), 6.87 (dt, J = 15.8, 6.8 Hz, 1 H, CH), 6.07 (d,
J = 15.8 Hz, 1 H, CH), 5.48–5.40 (m, 1 H, NH) 5.25 (AB system,
2 H, CH2), 4.61–4.48 (m, 1 H, CH), 3.72 (AB system, 4 H, 2 CH2),
3.38 (t, J = 7.4 Hz, 1 H, CH), 2.55–2.05 (m, 2 H, CH2), 2.01–1.83
(m, 2 H, CH2), 1.60 (s, 9 H, 3 CH3), 1.25 (d, J = 6.8 Hz, 3 H, CH3)
ppm. 13C NMR (50 MHz, CDCl3): δ = 197.8, 172.1, 155.0, 139.3,
139.1 (2 C), 136.0, 128.7 (4 C), 128.5, 128.4 (2 C), 128.3, 128.2 (2
C), 128.1 (2 C), 127.1 (2 C), 126.9 (2 C), 78.2, 65.9, 60.0, 59.8, 54.5
(2 C), 28.9, 28.2 (3 C), 27.7, 18.5 ppm. MS (ESI+): m/z = 593 [M
+ Na]+. HRMS (ESI+): calcd. for C35H42N2NaO5 593.2991; found
593.2990.
Synthesis of (2S,7S)-7-Methylazepane-2-carboxylic Acid (26) as a
Representative Procedure: A solution of 13 (900 mg, 2.04 mmol) in
MeOH (9 mL) was introduced into a bottle connected with a Parr
apparatus. Pd(OH)2/C (20%) (115 mg, 0.153 mmol) and acetic acid
(61 mg, 58 μL, 1.02 mmol) were also added. The bottle was filled
with H2 (6 atm) and shaked at room temperature for 8 h. The bottle
was degassed, the catalyst filtered (ATTENTION: the Pd residue
may be pyrophoric) and washed several times with MeOH. The
MeOH fractions were collected, and the solvent was removed under
vacuum to give compound 26 as a yellow oil (310 mg, 79% yield,
95:5dr). A portion of the crude (2 mg) was dissolved in THF
(0.5 mL) and CbzCl (2 mg) was added followed by 10% NaOH
(0.2 mL). The mixture was stirred in a vial for 4 h, and the product
was analyzed at HPLC on a column EC 125/4.6 NUCLEODUR
100–5 C18 ec using the following eluent: 32% B (A: H2O + 0.1%
HCOOH, B: CH3CN) at flow rate of 0.8 mL/min. The retention
time of the minor isomer was 41.78 min and for the major isomer
it was 44.39 min. The crude was then submitted to column
chromatography on silica gel (EtOAc/MeOH, 95:5) to isolate the
major isomer. 1H NMR (300 MHz, CD3OD): δ = 3.66 (dd, J =
3.2, 8.4 Hz, 1 H, CH), 3.40–3.37 (m, 1 H, CH), 2.24–2.19 (m, 1 H,
CHH), 2.09–2.00 (m, 1 H, CHH), 1.91–1.88 (m, 1 H, CHH), 1.77–
1.55 (m, 5 H, 2 CH2, CHH), 1.37 (d, J = 6.4 Hz, 3 H, CH3) ppm.
13C NMR (75 MHz, CD3OD): δ = 173.6, 61.5, 54.8, 33.9, 28.9,
24.9, 24.7, 20.1 ppm. MS (ESI+): m/z = 158 [M + H]+. HRMS
(ESI+): calcd. for C8H16NO2 158.1181; found 158.1183. The dia-
stereomeric ratio was determined by analytical LC–MS on the
NCbz derivative.
(2S,8S,E)-Benzyl 8-(tert-Butoxycarbonylamino)-2-(dibenzylamino)-
9-methyl-7-oxodec-5-enoate (19): 1H NMR (400 MHz, CDCl3): δ =
7.44–7.20 (m, 15 H, ArH), 6.82 (dt, J = 15.8, 7.6 Hz, 1 H, CH),
6.04 (d, J = 15.8 Hz, 1 H, CH), 5.22 (AB system, 2 H, CH2), 4.47–
4.39 (m, 1 H, CH), 3.73 (AB system, 4 H, 2 CH2), 3.37 (t, J =
7.0 Hz, 1 H, CH), 2.44–2.01 (m, 3 H, CH, CH3), 1.94–1.83 (m, 2
H, CH2), 1.41 (s, 9 H, 3 CH3), 0.94 (d, J = 7.4 Hz, 3 H, CH3), 0.75
(d, J = 7.4 Hz, 3 H, CH3) ppm. 13C NMR (50 MHz, CDCl3): δ =
197.8, 172.0, 155.7, 147.7, 139.1 (2 C), 135.8, 128.7 (4 C), 128.5 (2
C), 128.4 (2 C), 128.3 (2 C), 128.2 (4 C), 127.0 (2 C), 79.2, 66.0,
60.1, 54.5, 30.5, 29.0, 28.2 (3 C), 27.7, 19.7 (2v) ppm. MS (ESI+):
m/z = 621 [M + Na]+. HRMS (ESI+): calcd. for C37H46N2NaO5
631.3304; found 631.3302.
(S)-tert-Butyl 4-[(S,E)-7-(Benzyloxy)-6-(dibenzylamino)-7-oxohept-
2-enoyl]-2,2-dimethyloxazolidine-3-carboxylate (20): 1H NMR
(400 MHz, CDCl3): δ = 7.44–7.20 (m, 15 H, ArH), 6.75 (dt, J =
15.2, = 7.8 Hz, 1 H, CH), 6.20 (d, J = 15.2 Hz, 1 H, CH), 5.23 (AB (2S,7S)-7-Ethylazepane-2-carboxylic Acid (27): The diastereomeric
system, 2 H, CH2), 4.60–4.47 (m, 1 H, CH), 4.12–4.01 (m, 2 H, ratio was determined by analytical LC–MS on the NCbz derivative
CH2), 3.69 (AB system, 4 H, 2 CH2), 3.37 (t, J = 6.8 Hz, 1 H, CH), (EC 125/4.6 NUCLEODUR 100–5 C18 ec) using the following elu-
2.47–2.06 (m, 2 H, CH2), 1.98–1.84 (m, 2 H, CH2), 1.62–0.89 (m,
15 H, 5 CH3) ppm. 13C NMR (50 MHz, CDCl3): δ = 198.1, 172.0,
148.0, 139.2 (2 C), 135.9, 128.8 (4 C), 128.7 (2 C), 128.5 (2 C),
128.4 (2 C), 128.3 (4 C), 127.2 (2 C), 81.4, 80.7, 67.1, 66.2 (2 C),
ent: 40% B (A: H2O + 0.1% HCOOH, B: CH3CN) at flow rate of
0.8 mL/min. The retention time for the minor isomer was 43.78 min
and for the major isomer it was 46.23 min. The crude was submit-
ted to column chromatography on silica gel (EtOAc/MeOH, 95:5)
2138
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© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 2133–2141

Azepane-2-carboxylic Acids as Templates for Peptidomimetics
to isolate the major isomer. 1H NMR (400 MHz, [D6]DMSO): δ =
3.40 (dd, J = 4.0, 8.0 Hz, 1 H, CH), 2.98–2.96 (m, 1 H, CH), 2.03–
1.38 (m, 10 H, 5 CH2), 0.79 (t, J = 7.6 Hz, 3 H, CH3) ppm. 13C
NMR (100 MHz, [D6]DMSO): δ = 172.5, 60.4, 58.4, 30.3, 27.8,
26.8, 24.9, 24.4, 10.0 ppm. MS (ESI+): m/z = 194 [M + Na]+.
HRMS (ESI+) calcd. for C9H17NNaO2 194.1157; found 194.1158.
25.9, 25.0 (2 C), 16.3 ppm. MS (ESI+): m/z = 287 [M + H]+.
HRMS (ESI+): calcd. for C14H27N2O4 287.1971; found 287.1970.
(2S,7R)-7-[(S)-1-(tert-Butoxycarbonylamino)-2-methylpropyl]azep-
ane-2-carboxylic Acid (32): The diastereomeric ratio was deter-
mined by analytical LC–MS (EC 125/4.6 NUCLEODUR 100–5
C18 ec) using the following eluent: 75 % B (A: H2O + 0.1 %
HCOOH, B: CH3CN) at flow rate of 0.8 mL/min. The retention
time for the major isomer was 10.37 min and for the minor isomer
it was 14.52 min. The crude was submitted to column chromatog-
raphy on silica gel (EtOAc followed by EtOAc/MeOH, 95:5) to
(2S,7S)-7-Pentylazepane-2-carboxylic Acid (28): The diastereomeric
ratio was determined by analytical LC–MS on the NCbz derivative
(EC 125/4.6 NUCLEODUR 100–5 C18 ec) using the following elu-
ent: 45% B (A: H2O + 0.1% HCOOH, B: CH3CN) at flow rate of
0.8 mL/min. The retention time for the major isomer was
50.37 min; the minor was not detected. 1H NMR (400 MHz,
CD3OD): δ = 3.67 (dd, J = 3.2, 8.4 Hz, 1 H, CH), 3.21–3.19 (m, 1
H, CH), 2.25–2.20 (m, 1 H, CHH), 2.06–1.94 (m, 3 H, CHH, CH2),
1.77–1.26 (m, 12 H, 6 CH2), 0.90 (t, J = 6.8 Hz, 3 H, CH3) ppm.
13C NMR (75 MHz, CD3OD): δ = 173.6, 61.6, 58.9, 34.5, 31.7,
31.1, 28.6, 25.4, 25.1, 24.6, 22.5, 13.2 ppm. MS (ESI+): m/z = 236
[M + Na]+. HRMS (ESI+): calcd. for C12H23NNaO2 236.1626;
found 236.1626.
1
isolate the major isomer. H NMR (400 MHz, CD3OD): δ = 6.95
(d, J = 9.2 Hz, 1 H, NH), 3.83 (dd, J = 3.2, 6.8 Hz, 1 H, CH),
3.62–3.57 (m, 1 H, CH), 3.46 (d, J = 8.8 Hz, 1 H, CH), 2.25–2.01
(m, 2 H, CH2), 1.90–1.33 (m, 16 H, CH, 3 CH2, 3 CH3), 0.98 (d,
J = 6.4 Hz, 6 H) ppm. 13C NMR (100 MHz, CD3OD): δ = 172.9,
157.8, 79.1, 60.5, 59.5, 59.2, 29.4, 27.0 (3 C), 26.6, 25.1, 24.7, 24.4,
18.5, 18.1 ppm. MS (ESI+): m/z = 315 [M + H]+. HRMS (ESI+):
calcd. for C16H31N2O4 315.2284; found 315.2287.
(2S,7R)-7-[(S)-3-(tert-Butoxycarbonyl)-2,2-dimethyloxazolidin-4-yl]-
azepane-2-carboxylic Acid (33): The diastereomeric ratio was deter-
mined by analytical LC–MS (EC 125/4.6 NUCLEODUR 100–5
C18 ec) using the following eluent: 38 % B (A: H2O + 0.1 %
HCOOH, B: CH3CN) at flow rate of 0.8 mL/min. The retention
time for the major isomer was 21.26 min and for the minor isomer
it was 23.57 min. The crude was submitted to column chromatog-
raphy on silica gel (EtOAc/MeOH, 95:5) to isolate the major iso-
(2S,7R)-7-tert-Butylazepane-2-carboxylic Acid (29): The dia-
stereomeric ratio was determined by analytical LC–MS on the
NCbz derivative (EC 125/4.6 NUCLEODUR 100–5 C18 ec) using
the following gradient: from 50% to 80%B (A: H2O + 0.1%
HCOOH, B: CH3CN) at flow rate of 0.8 mL/min. The retention
time for the minor isomer was 7.18 min and for the major isomer
it was 7.91 min. The crude was submitted to column chromatog-
raphy on silica gel (EtOAc/MeOH, 95:5) to isolate the major iso-
1
mer. H NMR (400 MHz, CD3OD): δ = 4.24–4.20 (m, 2 H, CH2),
1
mer. H NMR (300 MHz, CD3OD): δ = 3.89–3.75 (m, 1 H, CH),
3.97–3.92 (m, 1 H, CH), 3.77 (dd, J = 3.6, 8.0 Hz, 1 H, CH), 3.55–
3.52 (m, 1 H, CH), 2.17–2.02 (m, 2 H, CH2), 1.85–1.75 (m, 2 H),
1.65–1.43 (m, 19 H, 2 CH2, 5 CH3) ppm. 13C NMR (100 MHz,
CD3OD): δ = 173.2, 154.2, 94.9, 81.8, 65.1, 61.3, 59.7, 40.2, 27.7,
27.2 (3 C), 25.5, 25.1, 22.6 (2 C), 22.1 ppm. MS (ESI+): m/z = 365
[M + Na]+. HRMS (ESI+): calcd. for C17H30N2NaO5 365.2052;
found 365.2053.
3.09–3.01 (m, 1 H, CH), 2.36–1.27 (m, 8 H, 4 CH2), 1.09 (s, 9 H,
3 CH3) ppm. 13C NMR (75 MHz, CD3OD): δ = 172.8, 65.3, 62.8,
34.2, 27.5, 27.2, 25.3 (3 C), 25.1, 24.8 ppm. MS (ESI+): m/z = 200
[M + H]+. HRMS (ESI+): calcd. for C11H22NO2 200.1651; found
200.1652.
(2S,7R)-7-[(S)-1-(tert-Butyldimethylsilyloxy)ethyl]azepane-2-carb-
oxylic Acid (30): The diastereomeric ratio was determined by ana-
lytical LC–MS (EC 125/4.6 NUCLEODUR 100–5 C18 ec) using
the following gradient: from 20% B to 75% B (A: H2O + 0.1%
HCOOH, B: CH3CN) over 90 min, at flow rate of 0.8 mL/min. The
retention time for the minor isomer was 29.79 min and for the
major isomer it was 32.00 min. The crude was submitted to column
chromatography on silica gel (EtOAc/MeOH, 90:10) to isolate the
(2S,7R)-7-[(R)-2,2-Dimethyl-1,3-dioxolan-4-yl]azepane-2-carboxylic
Acid (34): The diastereomeric ratio was determined by analytical
LC–MS on the NCbz derivative (EC 125/4.6 NUCLEODUR 100–
5 C18 ec) using the following eluent: 30 % B (A: H2O + 0.1 %
HCOOH, B: CH3CN) at flow rate of 0.8 mL/min. The retention
time for the major isomer was 42.32 min and for the minor isomer
it was 45.63 min. The crude was submitted to column chromatog-
raphy on silica gel (EtOAc followed by EtOAc/MeOH, 95:5) to
isolate the major isomer. 1H NMR (600 MHz, CD3OD): δ = 4.21–
4.08 (m, 2 H, CH2), 3.91–3.85 (m, 1 H, CH), 3.71–3.66 (m, 1 H,
CH), 3.56–3.49 (m, 1 H, CH), 2.24–1.97 (m, 4 H, 2CH2), 1.81–1.72
(m, 2 H, CH2), 1.67–1.54 (m, 8 H, CH2, 2 CH3) ppm. 13C NMR
(150 MHz, CD3OD): δ = 178.6, 91.5, 81.9, 66.0, 59.7, 48.5, 27.2,
26.2, 24.5, 22.6, 20.1, 19.0 ppm. MS (ESI+): m/z = 244 [M + H]+.
HRMS (ESI+): calcd. for C12H22NO4 244.1549; found 244.1550.
1
major isomer. H NMR (400 MHz, CD3OD): δ = 4.19–4.15 (m, 1
H, CH), 3.79–3.75 (m, 1 H, CH), 3.28–3.25 (m, 1 H, CH), 2.21–
2.17 (m, 1 H, CHH), 2.04–1.99 (m, 1 H, CHH), 1.87–1.83 (m, 2
H, CH2), 1.59–1.50 (m, 4 H, 2 CH2), 1.24 (d, J = 6.0 Hz, 3 H,
CH3), 0.96 (s, 9 H, 3 CH3), 0.17 (s, 6 H, 2 CH3) ppm. 13C NMR
(100 MHz, CD3OD): δ = 175.9, 72.4, 67.6, 64.8, 30.6 (2 C), 28.4 (3
C), 28.2, 28.0, 27.6, 20.8, –2.1, –2.7 ppm. MS (ESI+): m/z = 302
[M + H]+. HRMS (ESI+): calcd. for C15H32NO3Si 302.2151; found
302.2149.
(2S,7R)-7-[(S)-1-(tert-Butoxycarbonylamino)ethyl]azepane-2-carb-
oxylic Acid (31): The diastereomeric ratio was determined by ana-
lytical LC–MS (EC 125/4.6 NUCLEODUR 100–5 C18 ec) using
(2S,7R)-7-Phenethylazepane-2-carboxylic Acid (35): The dia-
stereomeric ratio was determined by analytical LC–MS on the
NCbz derivative (EC 125/4.6 NUCLEODUR 100–5 C18 ec) using
the following eluent: 82% B (A: H2O + 0.1% HCOOH, B: CH3CN) the following eluent: 65% B (A: H2O + 0.1% HCOOH, B: CH3CN)
at flow rate of 0.8 mL/min. The retention time for the major isomer
was 4.57 min and for the minor isomer it was 5.54 min. The crude
was submitted to column chromatography on silica gel (EtOAc/
at flow rate of 0.8 mL/min. The retention time for the major isomer
was 11.04 min and for the minor isomer it was 12.32 min. The
crude was submitted to column chromatography on silica gel
MeOH, 90:10) to isolate the major isomer. 1H NMR (300 MHz, (EtOAc/MeOH, 90:10) to isolate the major isomer. 1H NMR
CD3OD): δ = 4.11–3.96 (m, 1 H, CH), 3.91–3.80 (m, 1 H, CH), (600 MHz, CD3OD): δ = 7.28–7.23 (m, 5 H, ArH), 3.69–3.62 (m,
3.40–3.27 (m, 1 H, CH), 2.24–1.36 (m, 8 H, 4 CH2), 1.33 (s, 9 H, 1 H, CH), 3.30–3.14 (m, 1 H, CH), 2.79–2.64 (m, 2 H, CH2), 2.27–
3 CH3), 1.16 (d, J = 6.8 Hz, 3 H, CH3) ppm. 13C NMR (75 MHz, 1.51 (m, 10 H, 5 CH2) ppm. 13C NMR (150 MHz, CD3OD): δ =
CD3OD): δ = 172.9, 155.8, 80.1, 63.1, 61.5, 49.6, 27.8 (3 C), 26.3,
173.3, 141.9, 126.7 (2 C), 126.4 (2 C), 125.1, 61.2, 57.8, 37.4, 35.8,
Eur. J. Org. Chem. 2012, 2133–2141
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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2139

E. Cini, G. Bifulco, G. Menchi, M. Rodriquez, M. Taddei
FULL PAPER
31.7, 26.3, 24.6, 23.9 ppm. MS (ESI+): m/z = 248 [M + H]+.
HRMS (ESI+): calcd. for C15H22NO2 248.1651; found 248.1654.
(850 mg, 1.4 mmol), HOBt (190 mg, 1.4 mmol), HBTU (530 mg,
1.4 mmol), and DIEA (226 mg, 300 μL, 1.75 mmol). After incorpo-
ration of Fmoc-Aca-OH, the Fmoc protecting group was removed
according to general procedure b. Third coupling: Fmoc-As-
p(OtBu)-OH (850 mg, 1.4 mmol), HOBt (190 mg, 1.4 mmol),
HBTU (530 mg, 1.4 mmol), and DIEA (226 mg, 300 μL,
1.75 mmol). After incorporation of Fmoc-Asp(OtBu)-OH, the
Fmoc protecting group was removed according to general pro-
cedure b.
(2S,7R)-7-[(S)-1-{[(9H-Fluoren-9-yl)methoxy]carbonylamino}ethyl]-
1-(benzyloxycarbonyl)azepane-2-carboxylic Acid (36): To a solution
of 31 (286 mg, 1.0 mmol) in THF (4 mL) was added 10% NaOH
(2.7 mL). The mixture was cooled to –5 °C, and then CbzCl
(205 mg, 168 μL, 1.2 mmol) was added. The mixture was warmed
up to room temperature and stirred for 1 h. A saturated aqueous
solution of NH4Cl was added until the solution reached pH 4, and
the organic layer was separated. The aqueous phase was extracted
with EtOAc, and all the organic fractions were collected and dried
with Na2SO4. The solvent was removed in vacuo. The crude mix-
ture was purified by column chromatography (CHCl3/MeOH, 9:1)
to give the Cbz derivative as a colorless oil (286 mg, 68% yield).
1H NMR (200 MHz, CDCl3): δ = 7.51–7.22 (m, 5 H, ArH), 5.25
(s, 2 H, CH2), 4.71–3.93 (m, 3 H, 3 CH), 2.33–1.95 (m, 2 H, CH2),
1.93–0.98 (m, 18 H, 3 CH2, 4 CH3) ppm. 13C NMR (100 MHz,
CDCl3): δ = 177.8, 157.1, 137.3, 129.0 (2 C), 128.4 (2 C), 127.8,
127.7, 80.1, 70.1, 68.0, 64.5, 50.7, 31.4, 29.9, 28.0 (3 C), 25.9, 24.7,
19.2 ppm. MS (ESI+): m/z = 442 [M + Na]+. HRMS (ESI+): calcd.
for C22H32N2NaO6 443.2158; found 443.2158. To a solution of the
NCbz derived previously prepared (130 mg, 0.31 mmol) in CH2Cl2
(1.2 mL) was added Et3SiH (150 mg, 207 μL, 1.30 mmol) and TFA
(1.705 g, 1.305 mL, 17.05 mmol), and the mixture was stirred at
room temperature for 3 h. The solvent was removed in vacuo. To
the residue was added H2O (4 mL), Et3N (84 mg, 116 μL,
0.83 mmol), and FmocOSu (136 mg, 0.40 mmol, in 4 mL of
CH3CN), and the mixture was stirred for 1 h. HCl (1 n) was added
until pH 4, and the organic layer was separated. The aqueous phase
was extracted with EtOAc, and all the organic fractions were col-
lected and dried with Na2SO4. The solvent was removed in vacuo.
The crude mixture was purified by column chromatography
(CHCl3/MeOH, 9:1) to give compound 36 as colorless oil (112 mg,
67% yield). 1H NMR (200 MHz, CDCl3): δ = 9.02, (br. s, 1 H,
OH), 7.77–7.63 (m, 2 H, ArH), 7.61–7.49 (m, 2 H, ArH), 7.47–7.21
(m, 9 H, ArH), 5.20 (s, 2 H, CH2), 4.52–3.89 (m, 6 H, CH2, 4 CH),
2.31–1.06 (m, 11 H, 4 CH2, CH3) ppm. 13C NMR (50 MHz,
CDCl3): δ = 174.1, 155.4, 154.8, 143.4, 143.1 (2 C), 135.1 (2 C),
128.9 (2 C), 128.6 (2 C), 128.0, 127.7 (4 C), 127.8 (2 C), 126.9 (2
C), 65.9, 64.7, 56.0, 50.8, 47.1, 37.6, 39.4, 28.9, 25.6, 24.1,
19.2 ppm. MS (ESI+): m/z = 565 [M + Na]+. HRMS (ESI+): calcd.
for C32H34N2NaO6 565.2315; found 565.2315.
(c) Resin Cleavage (2-ClTrt-Gly-NH2): The dried peptide resin was
treated for 2 h, whilst stirring, with AcOH/TFE/DCM (2:2:6; 10 μL
ϫ1 mg of resin) cleavage mixture. Then the resin was filtered off
and washed with neat cleavage mixture (3 mL, 3ϫ1.5 min). After
addition of hexane (15 times volume) to remove acetic acid as an
azeotrope, the filtrate was concentrated and lyophilized to give
270 mg of the crude linear peptide 40 (84 % overall yield). MS
(ESI+): m/z = 917 [M + H]+.
(d) Cyclization: The cyclization step was performed on 65 mg of the
crude linear peptide dissolved in dry CH2Cl2 (7ϫ10–4 m) and to
this solution, cooled to 0 °C, HATU (1.5 equiv.) and DIEA
(2 equiv.) were added. The vial was inserted in the cavity of a Dis-
cover synthesizer (CEM Corporation) and heated at 75 °C (25 W
power, max internal pressure 5 atm) for two cycles of 10 min each
(with a no irradiation interval of 2 min). The mixture was concen-
trated in vacuo and washed with 1 n HCl and purified by crystalli-
zation from ether to give 49 mg of crude protected cyclopeptide 41
(79% yield). MS (ESI+): m/z = 899 [M + H]+. HRMS: calcd. for
C43H63N8O11S+ 899.4332; found 899.4329.
(e) Protections Cleavage: Side chain deprotection was obtained by
treatment with TFA/thioanisole/H2O (90:5:5) for 3 h whilst stirring.
The solvent was removed in vacuo to give the crude cyclopeptide
that was purified by crystallization from ether (17 mg, 61%). The
product was analyzed by analytical LC–MS (EC 125/4.6 NUCLE-
ODUR 100–5 C18 ec) using the following gradient: from 0% B to
15% B over 15 min, at flow rate of 1 mL/min. The binary solvent
system (A/B) was as follows: water (A), MeOH (B). The ab-
sorbance was detected at 214 nm. The HPLC analysis showed one
main peak (tR = 2.573 min) that was identified as pure 42 on the
basis of MS (ESI). The product was found to be over 95% pure by
HPLC analysis. MS (ESI+): m/z = 417 [M + H]+.
(2S,7S)-7-Methyl-1-[(R)-3,3,3-trifluoro-2-methoxy-2-phenylprop-
anoyl]azepane-2-carboxylic Acid: To a solution of 26 (10 mg,
0.064 mmol) in CH2Cl2 (1 mL) was added (S)-(+)-α-methoxy-α-
(trifluoromethyl)phenylacetyl chloride (19 mg, 0.077 mmol). The
mixture was cooled to –5 °C, and then Et3N (10 mg, 13 μL,
0.096 mmol) was added. The mixture was warmed up to room tem-
perature and stirred for 1 h. HCl (1 n) was added, the organic layer
was separated, and the aqueous phase was extracted with CH2Cl2.
All the organic fractions were collected and dried with Na2SO4.
The solvent was removed in vacuo. The crude mixture was purified
by column chromatography (CHCl3/MeOH, 9:1) to give the chiral
Solid-Phase Synthesis of the Linear Precursor and Cyclization of
cyclo[-L-Gly-L-Asp-2-(S)-ACA-L-Arg-] (42): The 2-ClTrt-gly-NH2
resin was placed in a 25 mL polypropylene ISOSOLUTE syringe
on a VAC MASTER system, swollen in DMF (3 mL) for 1 h, and
then washed with 2ϫ3 mL of DCM.
(a) Peptide Coupling Conditions: A mixture of Fmoc-amino acid
(3–4 equiv.), HOBt (3–4 equiv.), HBTU (3–4 equiv.), and NMM or
DIEA (4–5 equiv.) was stirred under an atmosphere of N2 in DMF
(2.5 mL) for 2 h. After each coupling, washings were carried out
with DMF (3 mL, 3ϫ1.5 min), and DCM (3 mL, 3ϫ1.5 min), and
the Kaiser test was used to assess coupling efficiency.
1
amide as a colorless oil (21 mg, 51% yield). H NMR (400 MHz,
[D6]benzene): δ = 7.78 (d, J = 7.6 Hz, 1 H CH), 7.15–7.02 (m, 4
H, ArH), 4.05–4.00 (m, 1 H, CH), 3.87 (s, 3 H, CH3), 3.21 (d, J =
10.4 Hz, 1 H, CH), 2.52–2.40 (m, 2 H, CH2), 1.61–1.11 (m, 6 H,
3CH2), 0.99 (d, J = 6.0 Hz, 3 H, CH3) ppm. MS (ESI+): m/z = 396
[M + Na]+.
(b) Fmoc Deprotection: Removal of the Fmoc protecting group was
carried out using 20% piperidine in DMF (3 mL, 1ϫ1.5 min), 20%
piperidine in DMF (3 mL, 1ϫ10 min or 1ϫ5 min); washings in
DMF 2ϫ3 mL, DCM 2ϫ3 mL, DMF 2ϫ3 mL, (1.5 min each).
First coupling: Fmoc-Arg(Mtr)-OH (850 mg, 1.4 mmol), HOBt
(190 mg, 1.4 mmol), HBTU (530 mg, 1.4 mmol), and DIEA
Computational Details: To allow a full exploration of the conforma-
tional space, stochastic dynamics (10 ns) calculations at 300 K were
(226 mg, 300 μL, 1.75 mmol). After incorporation of Fmoc- performed with a Pentium-4 at 2.8 GHz processor using the
Arg(Mtr)-OH, the Fmoc protecting group was removed according
to general procedure b. Second coupling: Fmoc-Aca(Cbz)-OH
AMBER force field included in the MacroModel package. All the
structures so obtained (in number of 100) were minimized by using
2140
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© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 2133–2141

Azepane-2-carboxylic Acids as Templates for Peptidomimetics
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the Polak-Ribier Conjugate Gradient algorithm (PRCG, 1000
steps, maximum derivative less than 0.05 kcal/mol). This led to the
selection of the lowest-energy minimum conformer for 42 (Fig-
ure 1a). The initial geometries of the minimum energy conformers
for 30 were optimized at the hybrid DFT MPW1PW91 level using
the 6-31G(d) basis set (Gaussian 03 Software Package). GIAO 13
C
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the 6-31G(d,p) basis set, using as input the geometry previously
optimized at MPW1PW91/6-31G(d) level.
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Supporting Information (see footnote on the first page of this arti-
1
cle): Copies of the H NMR and 13C NMR spectra.
Acknowledgments
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Published Online: February 28, 2012
Eur. J. Org. Chem. 2012, 2133–2141
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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