A new conformationally restricted mimetic of dipeptide EG - Synthesis of an analogue of FEG

Article abstract of DOI:10.1002/ejoc.200700300

Starting from the chiral pyrrolidin-2-one 2, the carboxy group at C-4 underwent homologation, and subsequent removal of the 1-(4-methoxyphenyl)ethyl group gave lactam 6. Alkylation of N-1 with benzyl bromoacetate led to 7, a new conformationally restricte

Full text of DOI:10.1002/ejoc.200700300

FULL PAPER
DOI: 10.1002/ejoc.200700300
A New Conformationally Restricted Mimetic of Dipeptide EG – Synthesis of
an Analogue of FEG
Roberta Galeazzi,[a] Gianluca Martelli,[a] Eleonora Marcucci,[a] Giovanna Mobbili,[a]
Desiré Natali,[b] Mario Orena,*[a] and Samuele Rinaldi[a]
Keywords: Peptides / Mimetics / Conformational restriction / Molecular modeling / Active conformation / Conformation
analysis
Starting from the chiral pyrrolidin-2-one 2, the carboxy group
at C-4 underwent homologation, and subsequent removal of
the 1-(4-methoxyphenyl)ethyl group gave lactam 6. Alky-
lation of N-1 with benzyl bromoacetate led to 7, a new con-
formationally restricted analogue of the dipeptide EG (Glu-
Gly). The usefulness of 7 was demonstrated by its eventual
conversion into 8, an orthogonally protected analogue of
bioactive tripeptide FEG. In order to provide the biological
activity of the new mimetic 9, available from 8 after the re-
moval of the protecting groups, the conformational prefer-
ence of 9 was ascertained by a detailed conformational
analysis and a comparison with that of FEG (1).
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2007)
properties in allergic airways inflammation in Brown–
Norway rats,[10] reduces endotoxin-provoked perturbation
of intestinal motility and inflammation[11,12] and regulates
leukocyte adhesion to the heart.[13,14]
Introduction
The incorporation of conformational constraints into
biologically active peptides can provide them with very use-
ful chemical and biological properties including a well-de-
fined backbone conformation, specific topographical prop-
erties, high potency and selectivity at biological receptors,
together with increased stability against enzymatic degrada-
tion. Thus, the synthesis of conformationally restricted
amino acids and dipeptides and their application to both
the general study of peptide behaviour and the preparation
of drugs has received widespread attention.[1] In addition,
recent developments in the field of protein engineering by
site-directed mutagenesis[2] and total chemical synthesis[3]
insure an important and continued role for peptide ana-
logues in protein science and in drug discovery.
Within an ongoing research program aimed at preparing
conformationally restricted analogues of amino acids or oli-
gopeptides,[4] we hypothesized that mimetics of the salivary
gland tripeptide FEG (Phe-Glu-Gly) (1)[5,6] and its enanti-
omer feG (ent-1),[5,7,8] could be of interest owing to the bio-
logical activity of the parent peptides. In fact, FEG was
reported to display anti-hypotensive properties against ana-
phylactic shock,[5] together with a potent inhibition of intes-
tinal anaphylaxis and inhibitory effects on inflammatory re-
actions.[9] On the other hand, feG has anti-inflammatory
Thus, at first we devised that compound 2, having a
(3R,4S) configuration, which was recently synthesized in
our laboratory,[15] could be an attractive starting material
for the preparation of a conformationally restricted dipep-
tide EG (Glu-Gly) suitable for the insertion in FEG, in or-
der to change or improve its biological activity. In fact it is
well known that insertion of a γ-lactam moiety into a pep-
tide chain leading to severe conformational constrictions
can give rise to significant changes in both the potency and
biological activity.[16]
In addition, the diastereomer of 2 with the (3S,4R) con-
figuration is also available,[15] so that the same synthetic
route could be directed to obtain a mimetic of tripeptide
feG ent-1 which in turn displays interesting biological ac-
tivity somewhat different from FEG.[10–14]
Results and Discussion
The ester 2[15] was smoothly converted into the corre-
sponding acid 3 in quantitative yield, which by reaction
with methyl chloroformate gave the corresponding mixed
anhydride which was directly treated with an ethereal solu-
tion of diazomethane to afford the diazo ketone 4 in moder-
ate yield.
The homologation pathway was carried out by using an
Arndt–Eistert rearrangement,[17] and the ester 5 was ob-
tained in moderate yield with total retention at C-4, a sole
diastereomer being recovered from the reaction mixture.
[a] Dipartimento di Scienze e Tecnologie Chimiche, Università
Politecnica delle Marche,
Via Brecce Bianche, 60131 Ancona, Italy
Fax: +39-071-2204714
E-mail: m.orena@univpm.it
[b] Dipartimento di Patologia Molecolare e Terapie Innovative,
Università Politecnica delle Marche,
Via Tenna 30, 60132 Ancona, Italy
4402
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 4402–4407

A New Conformationally Restricted Mimetic of Dipeptide EG
FULL PAPER
Removal of the chiral 1-(p-methoxyphenyl)ethyl group was (1), which could be of interest owing to the biological ac-
easily accomplished by a reaction with CAN (cerium am- tivity of the parent peptide. At first, the tBoc protecting
monium nitrate), to give the corresponding γ-lactam 6 in group in compound 7 was carefully removed by using TFA.
good yield. Once the required product 6 was in hand, it was Then, the corresponding trifluoroacetate salt underwent di-
alkylated with benzyl bromoacetate in dry THF, using NaH rectly a reaction with Et3N, followed by condensation with
as the base, to give in good yield the chiral pyrrolidin-2-one tBoc-Phe in the presence of N-[3-(dimethylamino)propyl]-
7, a conformationally restricted analogue of dipeptide EG NЈ-ethylcarbodiimide hydrochloride (EDCl), to afford in
(Glu-Gly) (Scheme 1).
moderate yield 8, which was eventually deprotected to give
9, a constrained analogue of tripeptide FEG (1) (Scheme 2).
Scheme 2. Structures of FEG (1) and of its constrained analogue
9.
In order to ascertain the conformational preference of
the fully deprotected analogue 9 with respect to FEG (1,
Scheme 2), and to correlate the biological activity with the
conformational behaviour, a detailed conformational analy-
sis was performed. The MC search protocol was used to-
gether with the AMBER* force field[19] taking into account
the dipolar interaction of the charged groups simulating the
aqueous polar environment (GB/SA H2O model).[20,21] In
addition, a full exploration of the conformational potential
energy surface (PES) of FEG (1) was carried out using the
same protocol in order to locate all the possible stable ori-
entations of the functional groups. In fact, the conforma-
tional behaviour of 7 was previously studied, and the most
probable bioactive conformation for this peptide displays a
strong interaction occurring between the glutamyl carboxyl
group and the N-terminus of the peptide, together with an
interaction between the aromatic ring and the terminal car-
boxyl group.[6,8]
Scheme 1. (a) 1 m NaOH, then 1 m HCl; (b) ClCOOCH3, Et3N,
DCM, 0 °C, then CH2N2, –10 °C; (c) PhCOOAg, Et3N, MeOH,
0 °C; (d) CAN, CH3CN/H2O, room temp.; (e) NaH, THF, 0 °C,
then BrCH2COOBn, THF, 0 °C to room temp.; (f) TFA, DCM,
room temp., then Et3N, tBoc-Phe, EDCl, DCM, room temp.; (g)
2 m NaOH, followed by 1 m HCl; (h) TFA, then Dowex 50, 1 m
NH4OH.
Our results are in total agreement with these findings and
prompted us to compare the conformational preferences of
the two compounds identifying the most probable bioactive
conformation of 1 having the following φ and ψ values
(conformer number 1 at lowest energy): φ1 = –174.0°, ψ1 =
The dipeptide EG is rather ubiquitous in peptide chains 128.8°; φ2 = –137.3°, ψ2 = 126.8°; φ3 = –179.7°, ψ3 = –0.40°.
and in bioactive synthetic peptides[18] and the mimetic 7
Thus, from the cluster analysis of FEG conformers, we
could be useful for substitution in order to improve or could divide the structures into two clusters by varying the
change biological activity.
central φ2 and ψ2 values (cluster value 0.4) although we
Thus, with the aim to test its usefulness, we prepared 9, noticed a very high flexibility of the lateral chains, in par-
a conformationally restricted analogue of tripeptide FEG ticular for the phenyl group of Phe (Figure 1).
Eur. J. Org. Chem. 2007, 4402–4407
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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M. Orena et al.
FULL PAPER
Figure 2. Cluster analysis of mimetic 9. Cluster no. 1 (top) and no.
2 (bottom), with a cluster value of 0.2 for superimposing φ2 and
ψ2.
Figure 1. Cluster analysis of FEG (1). Cluster no. 1 (top) and no.
2 (bottom), with a cluster value of 0.4 for superimposing φ2 and
ψ2.
On the contrary, a restriction of the conformational free-
dom was observed for the constrained mimetic 9, leading
to a smaller number of stable conformations (66 for 9 ver-
sus 155 for 1 within 6.0 kcal/mol). However, in all these
conformations the required strong interaction between the
+
N-terminal NH3 group and the glutamyl carboxy group
takes place, whereas the two remaining groups (Phe and the
terminal carboxyl group) are instead kept at a distance ow-
ing to the constrained pyrrolidin-2-one ring mimicking Glu.
In addition, from the cluster analysis we can divide all the
conformations into two main clusters, which differ in the φ2
and ψ2 values (cluster value 0.2, cluster no. 1: φ2 = –175.6°,
ψ2 = –138°; cluster no. 2: φ2 = –112.7°, ψ2 = –137.9°, Fig-
ure 2).
Figure 3. Superimposition of the FEG lowest-energy conformer
and the most representative structure in cluster no. 2 of analogue
Particularly relevant is cluster no. 2 since by superimpos- 9.
ing the representative structure of this cluster with the FEG
lowest-energy conformer we could observe a high corre-
spondence (Figure 3) since the N-terminus interacts with
At present the role of each group in displaying the bio-
the carboxyl group of Glu through a hydrogen bond, logical activity has not been well ascertained but it is only
whereas the phenyl ring interacts with the C=O group of deduced from conformational analogies between FEG and
the constrained mimetic ring instead of the terminal car- some of its active (feG) and non-active analogues (weG).[5,6]
boxy group.
Thus, the ability of compound 9 to inhibit rat intestinal
4404
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Eur. J. Org. Chem. 2007, 4402–4407

A New Conformationally Restricted Mimetic of Dipeptide EG
FULL PAPER
(3R,4S)-{4-(tert-Butoxycarbonylamino)-1-[(1S)-1-(4-methoxy-
phenyl)ethyl]-5-oxopyrrolidin-3-yl}carboxylic Acid (3): The ester 2[15]
(1.57 g; 4.0 mmol) was dissolved in methanol (5 mL) and then
aqueous NaOH (1 m, 1.5 equiv, 6.0 mL) was added. After stirring
the mixture at room temp. for 4 h, it was extracted with AcOEt
(2ϫ20 mL), the aqueous phase cooled to 0 °C, and 1 m HCl was
added until the pH = 2. After extraction with AcOEt (2ϫ20 mL),
the solution was dried and the solvent eventually removed under
reduced pressure, to give the acid 3 (1.47 g, 97% yield) as a white
anaphylaxis was evaluated in comparison with that of FEG
(1), according to the literature methods (Table 1).[5,6]
Table 1. Inhibition of intestinal anaphylaxis by FEG (1) and mi-
metic 9.
Entry
Compound
A/BC[a,b]
N[c]
1
2
3
control
FEG (1)
9
0.29Ϯ0.06
0.13Ϯ0.02
0.22Ϯ0.05
4
4
4
1
solid. M.p. 72–74 °C. H NMR (200 MHz, CDCl3): δ = 1.43 (s, 9
H, tBu), 1.54 (d, J = 7.0 Hz, 3 H, CHCH3), 3.09–3.35 (m, 2 H, 3-
CH + 2-CH-pro-R), 3.41–3.51 (m, 1 H, 2-CH-pro-S), 3.80 (s, 3 H,
OCH3), 4.52 (dd, J = 6.1, J = 8.4 Hz, 1 H, 1 H, 4-CH), 5.46 (q, J
= 7.0 Hz, 1 H, CHCH3), 5.79 (d, J = 6.1 Hz, 1 H, NH), 6.88 (d, J
= 8.6 Hz, 2 ArH), 7.25 (d, J = 8.6 Hz, 2 ArH), 11.54 (br. s, 1 H,
COOH) ppm. 13C NMR (50 MHz, CDCl3): δ = 16.1, 28.1, 42.1,
43.9, 46.4, 50.2, 50.3, 55.3, 81.6, 114.1, 128.3, 130.1, 155.1, 159.3,
163.9, 174.6 ppm. [α]2D0 = –140.0 (c = 0.5, CHCl3). MS-ESI: m/z =
379.4 [M + H]+, 401.4 [M + Na]+. C19H26N2O6 (378.42): calcd. C
60.30, H 6.93, N 7.40; found C 60.25, H 6.89, N 7.44.
[a] Data are the meanϮS.D. for six independent experiments. [b]
Albumin (A) induced contractile response relative to bethanechol
chloride (BC) induced contractile response. [c] Number of rats.
A significant decrease in activity was observed for 9 com-
pared to that of 1, and this result suggests that the orienta-
tion of the functionalities observed in FEG by using MD
simulations plays an important role in the bioactivity of the
natural product, so that an effective mimetic of 1 must pre-
serve both structural and conformational features of the
parent tripeptide.
(3R,4S)-{4-(tert-Butoxycarbonylamino)-1-[(1S)-1-(4-methoxy-
phenyl)ethyl]-5-oxopyrrolidin-3-yl} Diazomethyl Ketone (4): Methyl
chloroformate (0.49 mL, 3.0 mmol) and Et3N (0.28 mL, 3.0 mmol)
were added at 0 °C to a solution of 3 (0.76 g, 2.0 mmol) in dry
THF (15 mL). The reaction mixture was stirred at 0 °C for 40 min
and then at room temp. for 30 min. The reaction mixture was co-
oled again to 0 °C, and an ethereal solution of CH2N2 (0.2 m,
20 mL) was added. After 14 h, excess CH2N2 was carefully de-
stroyed by the dropwise addition of TFA/AcOEt (50:50) until N2
evolution had ceased. The solvents were removed under reduced
pressure, and the residue was purified by silica gel chromatography
(cyclohexane/AcOEt, 50:50) to give the title product 4 (0.45 g, 56%
Conclusions
Starting from the chiral pyrrolidin-2-one 2, the con-
strained analogue of EG (Glu-Gly) (7), was obtained,
which was eventually converted into compound 9, a mi-
metic of bioactive tripeptide FEG. The biological activity
of 9 was significantly lower than that of the natural tripep-
tide owing to a different conformational behaviour. Thus,
we are currently focusing on novel analogues of both FEG
and its enantiomer, feG, with the aim to improve the thera-
peutic potential, and the results will be reported in due
course.
yield) as a yellow oil. FTIR (CHCl ): ν = 2207, 1667, 1650 cm–1.
˜
3
1H NMR (200 MHz, CDCl3): δ = 1.42 (s, 9 H, tBu), 1.49 (d, J =
7.1 Hz, 3 H, CHCH3), 2.96–3.12 (m, 2 H, 3-CH and 2-CH-pro-R),
3.36–3.51 (m, 1 H, 2-CH-pro-S), 3.77 (s, 3 H, OCH3), 4.36 (dd, J
= 6.6, J = 8.7 Hz, 1 H, 4-CH), 5.32 (d, J = 6.6 Hz, 1 H, NH), 5.41
(q, J = 7.1 Hz, 1 H, CHCH3), 5.55 (br. s, 1 H, CHN2), 6.84 (d, J
= 8.7 Hz, 2 ArH), 7.22 (d, J = 8.7 Hz, 2 ArH) ppm. 13C NMR
(50 MHz, CDCl3): δ = 16.2, 28.2, 41.3, 49.2, 49.4, 55.3, 55.7, 56.4,
Experimental Section
80.4, 114.0, 128.3, 131.2, 155.6, 159.1, 169.3, 192.1 ppm. [α]2D0
=
Methods: Melting points were measured with an Electrothermal IA
9000 apparatus and are uncorrected. IR spectra were recorded in
CHCl3 with a Nicolet Fourier Transform Infrared 20-SX spectro-
–89.0 (c = 0.8, CHCl3). MS-ESI: m/z = 403.4 [M + H]+, 425.3 [M
+ Na]+. C20H26N4O5 (402.45): calcd. C 59.69, H 6.51, N 13.92;
found C 59.63, H 6.44, N 13.98.
1
photometer. H and 13C NMR spectra were recorded at 200 MHz
and 50 MHz, respectively, with a Varian Gemini 200 spectrometer,
using CDCl3 as a solvent unless otherwise stated. Chemical shifts
(δ) are reported in ppm relative to TMS, and coupling constants
(J) are reported in Hz. Assignments were aided by decoupling and
homonuclear two-dimensional experiments. Optical rotations were
measured with a Perkin–Elmer 341 polarimeter. The samples were
analyzed with a liquid chromatography Agilent Technologies
HP1100 equipped with a Zorbax Eclipse XDB-C8 Agilent Technol-
ogies column (flow rate 0.5 mL/min) and equipped with a diode-
array UV detector (220 nm and 254 nm). Acetonitrile and meth-
anol for HPLC were purchased from a commercial supplier. All
the samples were prepared by diluting 1 mg in H2O/acetonitrile
(1:1, 5 mL) in pure acetonitrile or in pure methanol. The MSD1100
mass detector was utilized under the following conditions: mass
range 100–2500 uma, positive scanning, energy of fragmentor 50 V,
drying gas flow (nitrogen) 10.0 mL/min, nebulizer pressure 45 psig,
drying gas temperature 350 °C, capillary voltage 4500 V. Column
chromatography was performed with silica gel 60 (230–400 mesh).
Compound 1 was synthesized according to ref.[15]
Methyl (3R,4S)-{4-(tert-butoxycarbonylamino)-1-[(1S)-1-(4-meth-
oxyphenyl)ethyl]-5-oxopyrrolidin-3-yl}acetate (5): The diazo ketone
4 (0.4 g, 1.0 mmol) was dissolved in a mixture of dry THF (20 mL)
and MeOH (20 mL) and at 0 °C a solution of silver benzoate
(30 mg, 0.1 mmol) in Et3N (0.5 mL) was slowly added, and after
0.5 h the mixture was stirred at room temp. for 3 h. Water (10 mL)
was then added, and the mixture was extracted with AcOEt
(2ϫ50 mL). After drying (Na2SO4) of the organic layer and re-
moval of the solvent under reduced pressure, the residue was puri-
fied by silica gel chromatography (cyclohexane/AcOEt, 4:6) to give
the product 5 (0.27 g, 66% yield) as a colourless viscous oil. FTIR
(CHCl ): ν = 3345, 1742, 1685, 1665 cm–1. 1H NMR (200 MHz,
˜
3
CDCl3): δ = 1.43 (s, 9 H, tBu), 1.48 (d, J = 7.1 Hz, 3 H, CHCH3),
2.25–2.52 (m, 2 H, CH2COOMe), 2.90–2.98 (m, 1 H, 3-CH), 2.93
(dd, J = 9.2, J = 9.8 Hz, 1 H, 2-CH-pro-R), 3.21 (dd, J = 7.9, J =
9.8 Hz, 1 H, 2-CH-pro-S), 3.63 (s, 3 H, OCH3), 3.79 (s, 3 H,
OCH3), 5.08 (dd, J = 7.0, J = 9.9 Hz, 1 H, 4-CH), 5.06 (d, J =
7.0 Hz, 1 H, NH), 5.41 (q, J = 7.1 Hz, 1 H, CHCH3), 6.85 (d, J =
8.8 Hz, 2 ArH), 7.19 (d, J = 8.8 Hz, 2 ArH) ppm. 13C NMR
Eur. J. Org. Chem. 2007, 4402–4407
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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M. Orena et al.
FULL PAPER
(50 MHz, CDCl3): δ = 16.2, 28.1, 35.9, 38.3, 44.2, 48.9, 51.5, 55.0,
57.3, 79.7, 113.8, 127.9, 131.3, 156.0, 158.8, 170.6, 172.0 ppm.
[α]2D0 = –101.8 (c = 1.83, CHCl3). MS-ESI: m/z = 407.4 [M + H]+,
residue was purified by silica gel chromatography (AcOEt), to give
8 (199 mg, 70% yield) as a colourless viscous oil. FTIR (CHCl3):
1
ν = 3344, 1741, 1687, 1668 cm–1. H NMR (200 MHz, CDCl ): δ
˜
3
430.2 [M + Na]+. C21H30N2O6 (406.47): calcd. C 62.05, H 7.44, N = 1.38 (s, 9 H, tBu), 2.47 (dd, J = 9.7, J = 15.5 Hz, 1 H,
6.89; found C 61.98, H 7.38, N 6.85.
CH2COOMe), 2.56–2.66 (m, 1 H, CH2COOMe), 2.83–3.16 (m, 3
H, 3-CH + l-Phe β-CH2), 3.24 (dd, J = 8.9, J = 8.9 Hz, 1 H, 2-
CH-pro-R), 3.61 (dd, J = 8.1, J = 8.9 Hz, 1 H, 2-CH-pro-S), 3.66
(s, 3 H, OCH3), 4.16–4.27 (m, 1 H, l-Phe α-CH), 4.21 (ABq, J =
17.7 Hz, 2 H, CH2COOBn), 4.37 (dd, J = 5.9, J = 7.1 Hz, 1 H, 4-
CH), 5.03 (d, J = 6.9 Hz, 1 H, NH), 5.15 (s, 2 H, COOCH2Ph),
6.57 (d, J = 5.9 Hz, 1 H, NH), 7.15–7.35 (m, 10 ArH) ppm. 13C
NMR (50 MHz, CDCl3): δ = 28.2, 36.0, 37.7, 38.4, 44.5, 50.1, 51.7,
55.4, 55.5, 55.7, 67.2, 80.1, 80.2, 126.9, 128.4, 128.6, 128.7, 129.4,
Methyl (3R,4S)-[4-(tert-Butoxycarbonylamino)-5-oxopyrrolidin-3-
yl]acetate (6): A solution of 5 (0.41 g, 1.0 mmol) in CH3CN (5 mL)
was treated at room temperature with CAN (1.1 g, 2.0 mmol) dis-
solved in H2O (5 mL), and the reaction mixture was stirred for
1 h. The aqueous layer was extracted with AcOEt (3ϫ25 mL), the
organic layers were combined, washed with brine and dried
(Na2SO4). Removal of the solvent under reduced pressure gave a
crude residue, which was purified by silica gel chromatography (cy-
clohexane/AcOEt, 3:7) to give 6 (0.25 g, 90% yield) as a low-melt-
135.1, 136.5, 155.2, 155.3, 168.0, 171.4, 172.0, 172.3 ppm. [α]2D0
=
–21.5 (c = 0.1, CHCl3). MS-ESI: m/z = 568.2 [M + H]+, 590.2 [M
+ Na]+. C30H37N3O8 (567.26): calcd. C 63.48, H 6.57, N 7.40;
found C 63.55, H 6.51, N 7.35.
ing solid. M.p. 36–38 °C. FTIR (CHCl ): ν = 3350, 1743, 1684,
˜
3
1665 cm–1. 1H NMR (200 MHz, CDCl3): δ = 1.44 (s, 9 H, tBu),
2.48 (dd, J = 9.2, J = 15.8 Hz, 1 H, CH2COOMe), 2.58–2.73 (m,
1 H, CH2COOMe), 2.75–2.88 (m, 1 H, 3-CH), 3.04 (dd, J = 9.1, J (3R,4S)-[1-(Carboxymethyl)-4-(L-phenylalanylamino)-5-oxopyrrol-
= 9.5 Hz, 1 H, 2-CH-pro-R), 3.54–3.63 (m, 1 H, 2-CH-pro-S), 3.68
(s, 3 H, OCH3), 4.02 (dd, J = 6.7, J = 9.9 Hz, 1 H, 4-CH), 5.04 (d,
idin-3-yl]acetic Acid (9): Compound 8 (0.57 g, 1.0 mmol) dissolved
in MeOH (1 mL) was added to NaOH (2 m, 3 mL), and the mixture
J = 6.7 Hz, 1 H, NH), 6.35 (br. s, 1 H, NH) ppm. 13C NMR was stirred at 0 °C for 4 h. The clear solution was extracted with
(50 MHz, CDCl3): δ = 28.2, 35.9, 39.7, 44.7, 51.7, 56.3, 80.1, 156.3,
172.1, 172.2 ppm. [α]2D0 = –23.7 (c = 1.0, CHCl3). MS-ESI: m/z =
273.2 [M + H]+, 296.2 [M + Na]+. C12H20N2O5 (272.30): calcd. C
52.93, H 7.40, N 10.29; found C 52.87, H 7.36, N 10.33.
AcOEt (2ϫ10 mL), HCl (1 m, 3 mL) was added, and the mixture
was extracted with AcOEt (3ϫ10 mL). After drying (Na2SO4) of
the organic layer and removal of the solvent, the residue was
treated with TFA (2 mL), and the solution was stirred at room
temp. for 24 h. Removal of the volatiles under reduced pressure
gave an oil, which was dissolved in H2O and subjected to an ion-
exchange column (Dowex 50, elution with 1 m NH4OH) to give the
title product 9 as a white solid (0.24 g; 66% yield). M.p. 200–204 °C
(dec). 1H NMR (200 MHz, D2O): δ = 2.47 (dd, J = 9.7, J =
15.5 Hz, 1 H, CH2COOH), 2.56–2.66 (m, 1 H, CH2COOH), 2.83–
3.16 (m, 3 H, 3-CH + l-Phe β-CH2), 3.24 (dd, J = 8.9, J = 8.9 Hz,
1 H, 2-CH-pro-R), 3.61 (dd, J = 8.1, J = 8.9 Hz, 1 H, 2-CH-pro-
S), 4.16–4.27 (m, 1 H, l-Phe α-CH), 4.18 (ABq, J = 17.4 Hz, 2 H,
NCH2COOH), 4.41 (d, J = 5.7 Hz, 1 H, 4-CH), 4.97 (br. s, 5 H,
NH and COOH), 7.24–7.49 (m, 5 ArH) ppm. 13C NMR (50 MHz,
D2O): δ = 28.2, 36.0, 37.7, 38.4, 44.5, 50.1, 51.7, 55.4, 55.5, 55.7,
67.2, 126.9, 128.4, 128.6, 128.7, 129.4, 135.1, 136.5, 155.2, 155.3,
166.2, 169.3, 171.0, 171.4 ppm. [α]2D0 = –14.6 (c = 0.2, H2O). MS-
ESI: m/z = 364.1 [M + H]+, 386.2 [M + Na]+. C17H21N3O6
(363.14): calcd. C 56.19, H 5.83, N 11.56; found C 56.13, H 5.89,
N 11.49.
Methyl (3R,4S)-[1-(Benzyloxycarbonylmethyl)-3-(tert-butoxycar-
bonylamino)-5-oxopyrrolidin-3-yl]acetate (7): To a solution contain-
ing 6 (0.27 g, 1.0 mmol) in dry THF (5 mL) under argon, NaH
(50 mg of a 50% dispersion in oil, 1.01 mmol) was added, and the
solution was stirred at 0 °C for 50 min. Benzyl bromoacetate
(0.17 mL, 1.01 mmol) dissolved in dry THF (3 mL) was then added
at 0 °C, and the reaction mixture was subsequently stirred at room
temp. for 4 h. Water (5 mL) and AcOEt (40 mL) were added, and
the mixture was extracted with AcOEt (2ϫ50 mL). After drying
(Na2SO4) of the organic layer and removal of the solvent under
reduced pressure, the residue was purified by silica gel chromatog-
raphy (cyclohexane/AcOEt, 3:7) to give 7 (0.23 g, 54% yield) as a
colourless oil. 1H NMR (200 MHz, CDCl3): δ = 1.43 (s, 9 H, tBu),
2.47 (dd, J = 9.8, J = 15.8 Hz, 1 H, CH2COOMe), 2.56–2.67 (m,
1 H, CH2COOMe), 2.91–3.01 (m, 1 H, 3-CH), 3.21 (dd, J = 9.2, J
= 9.4 Hz, 1 H, 2-CH-pro-R), 3.58 (dd, J = 7.9, J = 9.4 Hz, 1 H, 2-
CH-pro-S), 3.66 (s, 3 H, OCH3), 4.08 (dd, J = 6.7, J = 9.9 Hz, 1
H, 1 H, 4-CH), 4.10 (ABq, J = 17.6 Hz, 2 H, CH2COOBn), 5.08
Computational Methods: All calculations were carried out with SGI
(d, J = 6.7 Hz, 1 H, NH) 5.14 (s, 2 H, COOCH2Ph), 7.34 (m, 5 Octane2 IRIX 6.5 workstations. Molecular mechanics calculations
ArH) ppm. 13C NMR (50 MHz, CDCl3): δ = 28.2, 36.1, 38.5, 44.5,
were performed using the implementation of the AMBER force
49.8, 51.7, 56.6, 67.2, 80.1, 128.3, 128.5, 128.6, 135.0, 168.0, 172.1 field (AMBER*)[19] within the framework of Macromodel version
ppm. [α]2D0 = –14.4 (c = 0.9, CHCl3). MS-ESI: m/z = 421.4 [M +
H]+, 444.1 [M + Na]+. C21H28N2O7 (420.46): calcd. C 59.99, H
6.71, N 6.66; found C 59.92, H 6.65, N 6.71.
5.5.[19b] The torsional space of each molecule was randomly varied
with the usage-directed Monte Carlo conformational search.[19c]
For each search, at least 1000 starting structures for each variable
torsion angle was generated and minimized until the gradient was
less than 0.05 kJ/Åmol. Duplicate conformations and those with
an energy in excess of 6.0 kcal/mol above the global minimum were
discarded. The solvent effect was included by using the implicit
water GB/SA solvation method,[20] to take into account polar sol-
vent effects. The cluster analysis was performed within the Macro-
model package using Xcluster.[21,22]
Methyl (3R,4S)-{1-(Benzyloxycarbonylmethyl)-4-[N-(tert-butoxy-
carbonyl)-L-phenylalanylamino]-5-oxopyrrolidin-3-yl}acetate (8): To
a solution of 7 (210 mg, 0.5 mmol) in DCM (7 mL), TFA (0.6 mL)
was added, and the clear solution was stirred at room temp. for
2 h. The volatiles were then removed under reduced pressure, and
the residue was washed twice with diethyl ether. The raw trifluo-
roacetate salt obtained in a quantitative yield (217 mg, 0.5 mmol)
was dissolved in DCM (10 mL) and then tBoc-l-Phe (131 mg,
In Vitro Experiments: Male Sprague–Dawley (Harlan, Italy) rats
0.5 mmol), TEA (70 μL, 0.6 mmol) and EDCl (113 mg, 0.6 mmol) weighing 170–180 g were sensitized to albumin from chicken egg
were subsequently added, and the solution was stirred at room
temp. for 12 h. Water (5 mL) was added, and the mixture was ex-
tracted with DCM (3ϫ10 mL). After drying (Na2SO4) of the or-
ganic layer and removal of the solvent under reduced pressure, the
white (A, 1 mg) and to pertussis toxin (50 ng, Sigma). Six to eight
weeks after sensitisation, 1.5 cm sections were obtained from the
terminal ileum and were mounted in 10 mL of organ baths contain-
ing Krebs–Henseleit buffer solution (118.9 mm NaCl, 4.6 mm KCl,
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© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 4402–4407

A New Conformationally Restricted Mimetic of Dipeptide EG
FULL PAPER
[9] F. Turesin, A. Sadr, J. S. Davison, R. Mathison, BMC Physiol.
2002, 2, 13 (http://www.biomedcentral.com/1472–6793/2/13).
[10] R. E. Dery, M. Ulanova, L. Puttagunta, G. R. Stenton, D.
James, S. Merani, R. Mathison, J. Davison, A. D. Befus, Eur.
J. Immunol. 2004, 34, 3315–3325.
[11] R. D. Mathison, J. S. Davison, A. D. Befus, Proc. West. Pharm-
acol. Soc. 2001, 44, 157–158.
[12] R. Mathison, P. Lo, D. Tan, B. Scott, J. S. Davison, Neurogas-
troenterol. Motil. 2001, 13, 599–603.
[13] a) R. Mathison, R. Woodman, J. S. Davison, Can. J. Physiol.
Pharmacol. 2001, 79, 785–792; b) F. Bao, S. M. John, Y. Chen,
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[14] a) R. Mathison, A. D. Befus, J. S. Davison, R. C. Woodman,
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1471–2172/4/3); b) R. Mathison, Curr. Immunol. Rev. 2006, 2,
247–259; c) R. D. Mathison, J. S. Davison, J. Inflammation
2006, 3, 9 (http://www.journal-inflammation.com/content/3/1/
9).
1.2 mm KH2PO4, 2.5 mm CaCl2, 25.0 mm NaHCO3, 1.2 mm
MgSO4·7H2O, 10.1 mm glucose) under 1.0 g of tension. The con-
tractions generated by albumin and [(RS)-2-carbamoyloxypropyl]-
trimethylammonium chloride (bethanechol chloride, BC) were
measured isometrically (Basile Mod. 7080, Italy) and recorded with
a two-channel recorder (Basile Mod. 7070, Italy). The tissues were
washed several times in Krebs–Henseleit solution and allowed to
equilibrate for 15 min. Anti-anaphylactic properties of FEG (1) and
its analogue 9 were determined by adding 10 μg of product to a
bath and incubating for 10 min. After washing, the segments were
treated with 1 mg of the A antigen and the corresponding contrac-
tile response was measured at peak contraction. The segments were
washed again and peak contractile response was obtained by add-
ing 10–5 m BC. Eventually, the mass of the muscle was measured,
the tension was calculated in gram force per gram wet tissue, and
the results are given by the ratio of A-induced contractile response
relative to BC-induced contractile response (Table 1).
[15] R. Galeazzi, G. Martelli, M. Orena, S. Rinaldi, P. Sabatino,
Tetrahedron 2005, 61, 5465–5473.
Acknowledgments
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We would like to thank M.I.U.R (Roma, Italy) for funding within
the framework PRIN 2004 and Mr. Andrea Garelli (Università di
Bologna) for recording mass spectra.
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Received: April 4, 2007
Published Online: July 6, 2007
Eur. J. Org. Chem. 2007, 4402–4407
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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