Analogues of both Leu- and Met-enkephalin containing a constrained dipeptide isostere prepared from a Baylis-Hillman adduct

Article abstract of DOI:10.1007/s00726-009-0314-z

An efficient route was developed for the synthesis of the Fmoc-protected dipeptide 4, isostere of Gly-Gly containing an α-methylene β-amino acid; the conformationally restricted analogues of Leu-enkephalin, 3a, and Met-enkephalin, 3b, respectively, were prepared by changing 4 for Gly ²-Gly³ in the native compounds 3a and 3b whose biological activities were significantly lower than the parent compounds.

Full text of DOI:10.1007/s00726-009-0314-z

Amino Acids (2010) 38:1057–1065
DOI 10.1007/s00726-009-0314-z
ORIGINAL ARTICLE
Analogues of both Leu- and Met-enkephalin containing
a constrained dipeptide isostere prepared from a Baylis-Hillman
adduct
Roberta Galeazzi Æ Gianluca Martelli Æ
Eleonora Marcucci Æ Mario Orena Æ Samuele Rinaldi Æ
Roberta Lattanzi Æ Lucia Negri
Received: 4 March 2009 / Accepted: 8 June 2009 / Published online: 8 July 2009
Ó Springer-Verlag 2009
Abstract An efficient route was developed for the
synthesis of the Fmoc-protected dipeptide 4, isostere of
Gly-Gly containing an a-methylene b-amino acid; the
conformationally restricted analogues of Leu-enkephalin,
3a, and Met-enkephalin, 3b, respectively, were prepared by
changing 4 for Gly2-Gly3 in the native compounds 3a and
3b whose biological activities were significantly lower than
the parent compounds.
biological activity in an iterative process that ultimately
provides insights concerning the bound conformation of the
peptide. Thus, there is a need for peptide mimics that pre-
organize the peptide backbone and the side chains in the
biologically active conformation of the native peptide
(Gillespie et al. 1997; Hruby et al. 1997; Hruby and Balse
2000; Hruby 2005).
Within an ongoing program directed to identify appro-
priate peptide replacements that can be useful to elucidate
the biologically active conformation of pharmacologically
relevant oligopeptides (Galeazzi et al. 2003, 2005a, b,
2007, 2008), we considered Leu-enkephalin 1 and Met-
enkephalin 2, endogeneous peptide ligands towards the
d-opioid receptor isolated from brain tissue (Hughes et al.
1975), that are involved in a lot of physiological processes,
their roles in behavior, neuroendocrinology and pain
transmission being well documented (Kieffer and Evans
2009; Monnier and Bride 1995; Acosta and Lopez 1999).
Keywords Isostere ꢀ Dipeptide ꢀ
Conformational restriction ꢀ Baylis–Hillman ꢀ Activity
Introduction
A significant goal of drug discovery is to design small
molecules having the key structural and functional elements
of biologically active peptides, whereas a major challenge in
this area is to define their biologically active conformation,
or the bound structure. The strategies aimed at elucidating
this structure typically involve introducing conformational
restraints at specific sites of the peptide (Olson et al. 1993;
Giannis and Kolter 1993; Gante 1994; Hanessian et al. 1997;
Goodman and Zhang 1997; Hanessian and Auzzas 2008)
and the solution structures of the constrained analogues
can then be determined and correlated with binding and
H-Tyr1-Gly2-Gly3-Phe4-Leu5-OH 1
H-Tyr1-Gly2-Gly3-Phe4-Met5-OH 2
As for other bioactive endogenous peptides, there are
several major considerations that limit the clinical appli-
cations of naturally occurring enkephalins including
selectivity for receptor subtypes, modest absorption, and
relatively low central bioavailability (Chen et al. 2001).
Moreover, both enkephalins undergo rapid degradation
´
under physiological conditions (Fernandez et al. 2002;
R. Galeazzi ꢀ G. Martelli ꢀ E. Marcucci ꢀ M. Orena (&) ꢀ
Carrera et al. 2005). Since cleavage performed by pepti-
dases can be prevented by the incorporation in the peptide
chains of non-natural amino acids, every change at these
positions could increase the lifetime of the peptides in vivo,
thus increasing the biological activity (Shinada et al. 2007).
A single crystal X-ray diffraction study of Leu-enkeph-
alin and Met-enkephalin was reported (Smith and Griffin
S. Rinaldi
Dipartimento I.S.A.C., Universita Politecnica delle Marche,
`
Via Brecce Bianche, 60131 Ancona, Italy
e-mail: m.orena@univpm.it
R. Lattanzi ꢀ L. Negri
Dipartimento di Fisiologia e Farmacologia ‘‘Vittorio Erspamer’’,
`
Universita di Roma, La Sapienza, 00815 Rome, Italy
123

1058
R. Galeazzi et al.
1978) and the molecule in the solid state was shown to
display a Gly2-Gly3 b-turn stabilized by two intramolecular
hydrogen bonds between the CO and NH groups of Tyr1 and
the NH and CO groups of Phe4. In addition, although
enkephalin itself has a random structure in water, a b-turn
conformation at the Gly2-Gly3 positions seems to be
important for its biological activity (Hersh 1982; Currie
et al. 1993; Martin et al. 1998; Maricic et al. 2002; Eguchi
et al. 1999, 2002; Rew and Goodman 2002; Gu et al. 2004)
and conformational calculations of enkephalins were car-
ried out in order to assign their three-dimensional structures
(Meirovitch et al. 1994; Koca and Carlsen 1995). Deter-
mining the three-dimensional structures of enkephalins, 1
and 2, bound to the l- and d-opioid receptors was the focus
of numerous investigations.(Belleney et al. 1989; Wu et al.
2007) A number of conformationally constrained enkeph-
alin derivatives were prepared that display high potency and
selectivity (Lee et al. 2007) thus suggesting that a 5 ? 2 b-
turn might be an important structural motif in the biologi-
cally active conformation of these important neuropeptides
(Blomberg et al. 2006). On the basis of modeling studies,
we reasoned that replacing the Gly2-Gly3 subunit with 4
might stabilize such a reverse turn via the geometric con-
straints due to the methylene group. Consequently, the
pseudopeptides 3a and 3b, analogues of Leu- and Met-
enkephalin, respectively, were selected as the primary tar-
gets for synthesis (Scheme 1).
Fig. 1 Geometries of the two lowest energy conformers for pseudo-
peptide 3a [Left lowest energy minimum. E = 0.0 kcal/mol
(-262.12 kcal/mol): /1 = -54.4, w1 = -177.3, /2 = 132.7,
w2 = 179.3, /3 = -110.2, h3 = 27.6, w3 = -174.7, /4 = 12.7,
w4 = -179.7, /5 = -146.6, w5 = 135.8. Right Conformer c2.
E = 0.53 kcal/mol (-261.59 kcal/mol)]
Scheme 2
H
O
O
FmocN
N
H
OH
In order to confirm the relevance of the structural con-
straints introduced by the methylene insertion in inducing a
peculiar secondary structure rearrangement for the designed
pseudopeptides, i.e. the predicted b-turn arrangement, a
molecular modeling investigation was carried out. Thus, the
conformational space of compound 3a in its zwitterionic
form was undertaken by means of the standard quenched
molecular dynamics (QMD) protocol, which have already
shown to predict reliable distribution of conformers
(O’Connor et al. 1992) (Carstens et al. 2005) using AMBER
force field (Weiner et al. 1986) together with implicit sol-
vation model GA/SA in water (Still et al. 1990), to take
account of the biological environment.
4
arrangement, and stabilized by the classical H-bond
between the C = O of Gly2 and the NH of the Leu5
forming a 11-membered ring (not a 10 because of the
presence of the b-amino acid W-Gly3) (Fig. 1). Further-
more this secondary structure is stabilized strongly also by
the presence of other H-bonding interactions formed
directly between the charged terminal carboxylate and
´
ammonium groups (Sanbonmatsu and Garcıa 2002).
Thus, with the aim to identify appropriate changes, we
evaluated the W-dipeptide 4 that was chosen for insertion
in both Leu- and Met-enkephalin as novel mimic of the
Gly-Gly unit, also considering that replacement of Gly3 for
dehydroalanine strongly enhances the biological activity
of the enkephalins (Shimoigashi and Stammer 1982;
Shimoigashi et al. 1982) (Scheme 2).
All the stable conformations showed common structural
features which correspond indeed to a real b-turn
HO
H
N
O
O
H
N
COOH
H3N
N
H
N
H
Synthesis of the enkephalin analogues 3a and 3b
O
O
R
CF3COO
3a,b
a. R = CH CH(CH ) ; b. R = CH CH SCH
3
A significant challenge associated with the synthesis of
peptidomimetics 3a and 3b was the design of a convenient
entry to the dipeptide isostere 4 originating from non-
amino acid precursors. To address this problem, we
2
3 2
2
2
Scheme 1
123

Analogues of Leu- and Met-enkephalin
1059
Scheme 3
O
N
C O
Cl
6
O
O
O
+
DABCO (20%)
DCM
Cl
O
Ot-Bu
N
H
O
rt
DCM, 0 °C
47%
quantitative
Ot-Bu
HO
7
5
O
O
O
O
Zn, NH Cl sat.
FmocOSu, Na2CO3
4
NaN , DMSO, rt
3
N3
Cl
Ot-Bu
Ot-Bu
N
H
N
quantitative
THF
70%
H
8
9
H
O
O
H
O
O
TFA, DCM, rt
quantitative
FmocN
FmocN
Ot-Bu
N
H
N
H
OH
10
4
developed a straightforward approach to 4 starting from the
alcohol 5 (Byun et al. 1994) that was treated with an
equimolar amount of chloroacetyl isocyanate, 6 (Speziale
and Smith 1963) to give the corresponding acyl carbamate
7 in very good yield (Ciclosi et al. 2002) (Scheme 3).
Reaction of 7 with DABCO in DCM gave the amide 8
Biological assays
The opioid activities of the compound 3a and 3b were
evaluated in vitro using mouse vas deferens (MVD, rich
in d-opioid receptors) and guinea pig ileum (GPI, rich in
l-opioid receptors) in comparison with the activity of
deltorphin I, a full agonist highly selective for d-opioid
receptors, and dermorphin, a full agonist highly selective
for l-opioid receptors. Comparison was also carried
out against Leu-enkephalin, 1, and Met-enkephalin, 2
(Table 1). Compounds 3a and 3b behaved as partial
agonists both on MVD and on GPI. Whereas 3b displayed
higher potency on MVD than on GPI, indicating a pref-
erential affinity for d-receptors than for l-receptors, 3a
displayed comparable low potency on both MVD and
GPI.
0
through two subsequent SN reactions whereas substitution
of the halogen, performed with sodium azide in DMSO,
gave the azide 9 in good yield.
The elaboration of the N-terminus of 9 involved
reduction of the azido group with Zn in NH4Cl aqueous
solution, which was performed in the presence of Fmoc–
OSu, with the aim to directly obtain the corresponding
N-Fmoc derivative 10. Eventual cleavage of the t-butyl ester,
carried out with TFA in DCM, afforded the key acid 4.
The hydrochlorides of dipeptides Phe-Leu-OMe, 11a,
and Phe-MetOMe, 11b, underwent N-acylation with 4 under
standard peptide coupling conditions (EDCl in DCM), to
give in good yield 12a or 12b, respectively (Scheme 4).
However, at the outset of these investigations removal
of the Fmoc protecting group in both 12a and 12b under
usual conditions (Carpino, 1987; Flinn et al. 1995) resulted
difficult and heating with 1 equiv DABCO in dry DCM was
required in order to prepare derivatives 13a and 13b
bearing the free amino group. Again, by using standard
peptide coupling (EDCl in DCM) and starting from com-
mercial t-Bu,t-Boc-tyrosine, the full protected analogues
14a and 14b were obtained in good yield. Eventually, by
cleavage of the methyl ester moiety and of both the t-butyl
and t-Boc protecting groups, the corresponding trifluoro-
acetates 3a and 3b were obtained.
Conclusions
Two conformationally constrained analogues of Leu-
enkephalin and Met-enkephalin that contain the novel
dipeptide mimic 4 were prepared. A concise and efficient
protocol was developed for the synthesis of this dipeptide
isostere, which may be applied to the preparation of ana-
logues of other peptides. Our investigation indicated that
the incorporation of compound 4 had little effect con-
cerning the activity of derivatives 3a, b, and highlighted
that insertion of a methylene group, with respect to dehy-
droalanine (Shimoigashi et al. 1982) leads to a dramatic
loss of activity.
123

1060
R. Galeazzi et al.
Scheme 4
a. Phe-Leu-OMe HCl, 11a, or
b. Phe-Met-OMe HCl, 11b
H
O
O
H
EDCl, TEA, DCM, 0 °C to rt
FmocN
N
COOCH3
4
N
H
N
H
60%
R
O
12a,b
t-Boc(t-Bu)-Tyr, EDCl,
TEA, DCM, 0 °C to rt
O
O
H
DABCO, DCM
quantitative yield
N
COOCH3
H2N
60%
N
H
N
H
O
R
13a,b
t-BuO
1. NaOH, H2O, MeOH
quantitative yield
H
O
O
H
N
N
COOCH3
t-BocN
N
N
2. TFA, DCM
quantitative yield
H
O
H
H
O
R
14a,b
HO
H
O
O
H
N
N
COOH
H3N
N
N
CF3COO
O
H
H
O
R
3a,b
a. R = CH CH(CH )
b. R = CH CH SCH
2 2 3
2
3 2
Experimental
Table 1 Functional biological activity of compounds 3a and 3b
MVD (d) IC50 (nM)
15
GPI (l) IC50 (nM)
Melting points were measured on an Electrothermal IA
9,000 apparatus and are uncorrected. Ir spectra were
recorded in CHCl3 on a Nicolet Fourier Transform Infrared
Dermorphine
Deltorphine
Met-enkephalin, 1
Leu-enkephalin, 2
3a
2
1.24 0.2
1,300 150
90.1 8.7
0.4 0.03
17.3 1.9
1
20-SX spectrophotometer. H and 13C NMR spectra were
12.1 1.5
504.2 43
recorded at 200 and 50 MHz, respectively, on a Varian
Gemini 200 spectrometer, using CDCl3 as a solvent unless
otherwise stated. Chemical shifts (d) are reported in ppm
relative to TMS and coupling constants (J) in Hz.
Assignments were aided by decoupling and homonuclear
two-dimensional experiments. Optical rotations were
measured on a Perkin Elmer 341 polarimeter. The samples
were analyzed with a liquid chromatography Agilent
Technologies HP1100 equipped with a Zorbax Eclipse
XDB-C8 Agilent and Technologies column (flow rate
0.5 mL/min) and equipped with a diode-array UV detector
(220 and 254 nm). Acetonitrile and methanol for HPLC
were purchased from a commercial supplier. All the sam-
ples were prepared by diluting 1 mg in 5 mL of a 1:1
3,000.0 445
202.0 37.5
2,200.0 355
990.0 105
3b
mixture of H2O and acetonitrile in pure acetonitrile or in
pure methanol. The MSD1100 mass detector was utilized
under the following conditions: mass range 100–
2,500 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
4,500 V. Column chromatography was performed with
silica gel 60 (230–400 mesh). Compound 5 was synthe-
sized according (Byun et al. 1994); L-Phe-L-Leu-OMe
123

Analogues of Leu- and Met-enkephalin
1061
hydrochloride 11a was purchased from Chem-Impex
International, Wood Dale, IL, USA, and L-Phe-L-Met-OMe
hydrochloride 11b from Astatech, Princeton, MA, USA.
3.99 (s, 2H), 4.10 (d, J = 6.2 Hz, 2H), 5.73 (s, 1H), 6.19
(s, 1H), 6.79 (br s, 1H, NH); 13C NMR (50 MHz, CDCl3):
d 27.9, 40.5, 52.7, 81.5, 126.4, 137.4, 165.2, 166.3. ESI–
MS: m/z 241.2 [MH]?, 263.1 [M?Na]?. Anal. calcd for
C10H16N4O3: C, 49.99; H, 6.71; N, 23.32. Found: C, 49.95;
H, 6.68; N, 23.36.
t-Butyl 3-(chloroacetylaminocarbonyloxy)-2-
methylenepropanoate (7)
A solution containing compound 5 (Byun et al. 1994)
(2.2 g; 13 mmol) and chloroacetyl isocyanate 6 (Speziale
and Smith 1963) (15 mmol) in DCM (20 mL) was stirred
for 3 h at rt. After removal of the solvent, the residue was
purified by silica gel chromatography (cyclohexane:ethyl
acetate 50:50) to give the N-acyl carbamate 7 (3.2 g; 89%
yield) as a colorless oil. 1H NMR (200 MHz, CDCl3):
d 1.50 (s, 9H), 4.49 (s, 2H), 4.88 (s, 2H), 5.83 (s, 1H), 6.33
(s, 1H), 7.97(s, 1H, NH). 13C NMR (50 MHz, CDCl3): d 28.0,
43.6, 64.8, 81.8, 127.9, 135.8, 150.7, 164.0, 166.6. ESI–MS:
m/z 278.1 [MH]?, 281.1 [MH?2]?, 300.1 [M?Na]?, 302.1
[M?2?Na]?. Anal. calcd for C11H16ClNO5: C, 47.58; H,
5.81; N, 5.04. Found: C, 47.61; H, 5.75; N, 5.01.
t-Butyl 3-(Fmoc-glicylamino)-2-methylenepropanoate (10)
To a solution containing the azide 9 (1.32 g; 5.5 mmol)
and FmocOSu (5.56 g; 16.5 mmol) in THF (25 mL) a
saturated NH4Cl aqueous solution (5.5 mL) was added at
rt, followed by Zn (1.07 g; 16.5 mmol) under vigorous
stirring. After 10 min a saturated Na2CO3 aqueous solution
was added until pH = 9 and then the mixture was stirred
for 12 h. After addition of H2O (30 mL) and ethyl acetate
(50 mL), the mixture was extracted with ethyl acetate
(2 9 50 mL), the organic layer wad dried (Na2SO4) and
solvents removed under reduced pressure. The residue was
purified by silica gel chromatography (cyclohexane:ethyl
acetate 90:10 as eluent) to give the product 10 (1.68 g;
1
t-Butyl 3-(chloroacetylamino)-2-methylenepropanoate (8)
70% yield) as a white amorphous solid. Mp: 49–51°C. H
NMR (200 MHz, CDCl3):
d
1.49 (s, 9H), 3.86
To a solution containing the N-acyl carbamate 7 (1.4 g;
5 mmol) in DCM (20 mL), DABCO (0.11 g; 1.0 mmol)
was added and the misture was stirred for 10 min at rt.
After dilution with ethyl acetate (100 mL), the organic
layer was washed with 1 M HCl (30 ml) and then with
brine (50 mL). After drying (Na2SO4), the solvent was
removed under reduced pressure and the residue was
purified by silica gel chromatography (cyclohexane:ethyl
acetate 80:20), to give compound 8 (0.91 g; 78% yield) as
(d, J = 5.6 Hz, 2H), 4.08 (d, J = 6.2 Hz, 2H), 4.22
(t, J = 6.9 Hz, 1H), 4.43 (d, J = 6.9 Hz, 2H), 5.46
(t, J = 6.7 Hz, 1H, NH), 5.70 (s, 1H), 6.16 (s, 1H), 6.42
(t, J = 6.7 Hz, 1H, NH), 7.22–7.81 (m, 8 ArH); 13C NMR
(50 MHz, CDCl3): d 28.0, 40.5, 44.6, 47.1, 67.2, 81.4,
120.0, 125.0, 126.0, 127.0, 127.7, 128.4, 128.6, 131.9,
132.1, 137.6, 141.2, 143.7, 156.5, 165.3, 168.6. ESI–MS:
m/z 437.4 [MH]?, 459.3 [M?Na]?. Anal. Calcd for
C25H28N2O5: C, 68.79; H, 6.47; N, 6.42. Found: C, 68.75;
H, 6.44; N, 6.45.
1
white solid. Mp: 58–60°C. H NMR (200 MHz, CDCl3):
d 1.51 (s, 9H), 4.06 (s, 2H), 4.11 (d, J = 6.6 Hz, 2H), 5.73
(s, 1H), 6.20 (s, 1H), 7.08 (br s, 1H, NH); 13C NMR
(50 MHz, CDCl3): d 28.0, 40.8, 42.6, 81.6, 126.5, 137.3,
165.0, 165.6. ESI–MS: m/z 234.1 [MH]?, 236.1 [MH?2]?,
256.1 [M?Na]?, 258.1 [M?2?Na]?. Anal. calcd for
C10H16ClNO3: C, 51.40; H, 6.90; N, 5.99. Found: C, 51.37;
H, 6.86; N, 6.01.
3-(Fmoc-glicylamino)-2-methylenepropanoic acid (4)
To a solution of the ester 10 (0.5 g; 1.14 mmol) in dry
DCM (2.5 mL), trifluoroacetic acid (1.31 mL; 17.1 mmol)
was added at rt. After 10 min volatiles were removed under
reduced pressure and the residue was washed with dry
ethyl ether to give the product 4 as a white solid (0.44 g;
quantitative yield). Mp: 67–69°C. 1H NMR (200 MHz,
CDCl3): d 0.87 (d, J = 5.9 Hz, 3H), 0.88 (d, J = 5.9 Hz,
3H), 1.41–1.55 (m, 3H), 3.11 (d, J = 7.0 Hz, 2H), 3.68
(s, 3H), 3.77–3.89 (m, 2H), 4.00 (dd, J = 5.8 Hz,
J = 15.4 Hz, 1H), 4.14–4.27 (m, 2H), 4.43 (d, J = 7.0 Hz,
2H), 4.46–4.71 (m, 2H), 5.57 (s, 1H), 5.64 (bt, J = 6.9 Hz,
1H, NH), 5.77 (s, 1H), 6.38 (d, J = 8.3, 1H, NH), 6.63
(t, J = 6.5 Hz, 1H, NH), 6.90 (d, J = 7.2 Hz, 1H, NH), 7.12–
7.42 (m, 9 ArH), 7.58 (d, J = 7.0 Hz, 2 ArH), 7.77 (d,
J = 6.6 Hz, 2 ArH) 13C NMR (50 MHz, CDCl3): d 41.3,
44.9, 47.4, 68.3, 120.6, 125.4, 127.6, 128.4, 130.9, 135.3,
141.8, 143.8, 157.9, 170.3, 171.6. ESI–MS: m/z 381.1
t-Butyl 3-(azidoacetylamino)-2-methylenepropanoate (9)
To a solution of compound 8 (1.2 g; 5 mmol) in DMSO
(5 mL), NaN3 (0.65 g; 10 mmol) was added and the sus-
pension was stirred for 20 h at rt. Then H2O (20 mL) was
added and the mixture was extracted with ethyl acetate
(2 9 100 mL). After drying (Na2SO4) the solvent was
removed under reduced pressure and the residue was
purified by silica gel chromatography (cyclohexane:ethyl
acetate 70:30), to give the azide 9 (1.2 g; quantitative
yield) as colorless oil. IR (CHCl3): 3302, 2106, 1704,
1
1667 cm-1. H NMR (200 MHz, CDCl3): d 1.51 (s, 9H),
123

1062
R. Galeazzi et al.
[MH]?, 403.2 [M?Na]?. Anal. Calcd for C21H20N2O5: C,
21.9, 22.7, 24.7, 37.5, 40.4, 41.3, 44.1, 50.9, 52.2, 54.8,
123.6, 126.8, 128.5, 129.1, 129.2, 136.8, 139.9, 166.5,
170.8, 173.0, 173.1. [a]D -22.8 (c 0.5, CHCl3). ESI–MS:
m/z 433.2 [MH]?, 455.2 [M?Na]?. Anal. Calcd for
C22H32N4O5: C, 61.09; H, 7.46; N, 12.95. Found: C, 61.02;
H, 7.51; N, 12.88.
66.31; H, 5.30; N, 7.36. Found: C, 66.26; H, 5.25; N, 7.41.
[3-(Fmoc-glicylamino)-2-methylenepropanoyl]-L-
phenylalaninyl-L-leucine methyl ester (12a)
To a solution containing the acid 4 (1.0 g; 2.6 mmol), the
L-Phe-L-Leu-OMe hydrochloride 11a (1.06 g; 2.6 mmol)
and EDCl (0.55 g; 2.86 mmol) in dry DCM (15 mL), Et3N
(361 lL; 2.6 mmol) was added and the mixture was stirred
for 3 h at rt. Then water (20 mL) was added and the
mixture was extracted with ethyl acetate (3 9 50 mL).
After drying (Na2SO4) and removal of the solvent under
(reduced pressure, the residue was purified by silica gel
chromatography (ethyl acetate as eluent) to give the com-
pound 12a (1.14 g; 66% yield) as a white solid. Mp: 65–
67°C. 1H NMR (200 MHz, CDCl3): d 0.87 (d, J = 5.9 Hz,
3H), 0.88 (d, J = 5.9 Hz, 3H), 1.48–1.74 (m, 3H), 3.11
(d, J = 7.0 Hz, 2H), 3.68 (s, 3H), 3.84 (dd, J = 6.2 Hz,
J = 8.0 Hz, 2H), 4.00 (dd, J = 5.8 Hz, J = 15.4 Hz, 1H),
4.13–4.28 (m, 2H), 4.43 (d, J = 7.0 Hz, 1H), 4.45–4.55
(m, 1H), 4.64–4.71 (m, 1H), 5.57 (s, 1H), 5.63 (br s, 1H,
NH), 5.77 (s, 1H), 6.38 (d, J = 8.3 Hz, 1H, NH), 6.27
(t, J = 6.2 Hz, 1H, NH), 6.90 (d, J = 7.2 Hz, 1H, NH),
7.13–7.45 (m, 9 ArH), 7.58 (d, J = 7.0 Hz, 2 ArH) 7.67
(d, J = 6.6 Hz, 2 ArH); 13C NMR (50 MHz, CDCl3): d
21.8, 22.6, 24.7, 37.9, 40.8, 41.1, 47.0, 50.8, 52.2, 54.6,
60.3, 67.1, 119.9, 122.4, 125.0, 126.9, 127.0, 127.7, 128.4,
128.6, 129.0, 129.2, 129.8,13 6.5, 140.0, 141.2, 143.7,
156.6, 166.8, 169.7, 170.8, 173.2; [a]D -31.6 (c 0.5,
CHCl3). ESI–MS: m/z 655.2 [MH]?, 677.2 [M?Na]?.
Anal. Calcd for C37H42N4O7: C, 67.87; H, 6.47; N, 8.56.
Found: C, 67.81; H, 6.42; N, 8.61.
[3-(t-BuO,t-Boc-L-tyrosylglicylamino)-2-
methylenepropanoyl]-L-phenylala-ninyl-L-leucine
methyl ester (14a)
To a solution containing the amino derivative 13a
(310 mg; 0.71 mmol) and t-Boc,t-Bu-tyrosine (241 mg;
0.71 mmol) in DCM (8.0 mL), EDCl (164 mg;
0.85 mmol) was added and the mixture was stirred for 3 h
at rt. Then water (10 mL) was added and the mixture was
extracted with ethyl acetate (3 9 50 mL). After drying
(Na2SO4) and removal of the solvents under reduced
pressure, the residue was purified by silica gel chroma-
tography (ethyl acetate as eluent), to give the title product
14a (0.34 g; 63% yield) as a white foam. 1H NMR
(200 MHz, CDCl3): d 0.89 (d, J = 6.0 Hz, 6H), 1.32
(s, 9H), 1.36 (s, 9H), 1.42–1.64 (m, 3H), 2.92–3.20 (m,
4 H), 3.68 (s, 3H), 3.74 (dd, J = 5.4 Hz, J = 15.1 Hz, 1H),
3.92 (dd, J = 6.2 Hz, J = 15.1 Hz, 1H), 4.02–4.10
(m, 2H), 4.16–4.27 (m, 1H), 4.45–4.69 (m, 2H), 4.98
(d, J = 6.5 Hz, 1H, NH), 5.52 (s, 1H), 5.82 (s, 1H), 6.54
(d, J = 6.9 Hz, 1H, NH), 6.93 (d, J = 8.5 Hz, 2 ArH),
7.11 (d, J = 8.5 Hz, 2 ArH), 7.15–7.42 (m, 8H, 5 ArH ? 2
NH); 13C NMR (50 MHz, CDCl3): d 21.9, 22.7, 24.7, 28.2,
28.8, 37.7, 40.9, 41.2, 42.9, 50.9, 52.2, 54.9, 56.5, 78.2,
80.5, 122.7, 124.3, 126.8, 128.5, 129.2, 129.6, 131.1,
136.7, 1400, 154.4, 166.9, 169.4, 171.0, 172.2, 173.2; [a]D
-12.6 (c 0.5, CHCl3). ESI–MS: m/z 752.4 [MH]?, 774.3
[M?Na]?. Anal. Calcd for C40H57N5O9: C 63.90; H 7.64;
N 9.31. Found: C, 63.81; H, 7.69; N, 9.41.
(3-Glicylamino-2-methylenepropanoyl)-L-phenyl-
alaninyl-L-leucine methyl ester (13a).
To a solution containing the compound 12a (505 mg;
0.77 mmol) in dry DCM (10 mL), DABCO (87 mg;
0.77 mmol) was added and the clear solution was refluxed
for 6 h and stirred for further 12 h at rt. After removal of
the solvent under reduced pressure, the residue was purified
by silica gel chromatography (ethyl acetate:methanol 90:10
as eluent) to give 13a (0.32 g; 94% yield) as a white solid.
Mp: 47–48°C. 1H NMR (200 MHz, CDCl3): d 0.89
(d, J = 6.1 Hz, 6H), 1.34–1.63 (m, 3H), 2.81 (br s, 2H, NH2),
3.05–3.21 (m, 2H), 3.20–3.41 (m, 2H), 3.69 (s, 3H), 4.04
(dd, J = 4.4 Hz, J = 15.2 Hz, 1H), 4.15 (dd, J = 6.0 Hz,
(3-L-Tyrosylglicylamino-2-methylenepropanoyl)-L-
phenylalaninyl-L-leucine trifluoroacetate (3a)
The ester 14a (0.24 g; 0.31 mmol) was dissolved in metha-
nol (3 mL) and then 1 M NaOH aqueous solution was added
(1.55 equiv; 210 lL). After stirring for 4 h at rt, the mixture
was extracted with ethyl acetate (2 9 10 mL), the aqueous
phase cooled to 0°C and 1 M HCl was added until pH = 2.
After extraction with ethyl acetate (2 9 10 mL), the solvent
was dried and eventually removed under reduced pressure, to
give a residue which crystallized on standing To a solution of
the this solid in DCM (3 mL), TFA (0.513 g; 4.5 mmol) was
added. After stirring for 7 h at rt, the solvent was removed
under reduced pressure and the residue was washed with dry
ethyl ether (2 9 5 mL), to give the trifluoroacetate 3a
(0.22 g; quantitative yield) as a white solid. Mp: 44–45°C.
J = 15.2 Hz,
1 H), 4.46–4.60 (m, 1H), 4.72 (dt,
J = 6.6 Hz, J = 7.6 Hz, 1H), 5.55 (s, 1H), 5.94 (s, 1H),
6.59 (d, J = 7.6 Hz, 1H, NH), 7.15–7.35 (m, 5 ArH), 7.48
(d, J = 8.0 Hz, 1H, NH), 7.51 (dd, J = 4.4 Hz,
J = 6.0 Hz, 1 H, NH); 13C NMR (50 MHz, CDCl3): d
123

Analogues of Leu- and Met-enkephalin
1063
1
1H NMR (200 MHz, DMSO-d6): d 0.87 (d, J = 6.0 Hz, 3H),
0.93 (d, J = 6.0 Hz, 3H), 1.47–1.77 (m, 3H), 2.78 (dd,
J = 8.2 Hz, J = 15.0 Hz, 1H), 2.91–3.15 (m, 3H), 3.22–
3.33 (m, 1H), 3.34 (bs, 3H, NH3?), 3.68–4.07 (m, 4H), 4.19–
4.32 (m, 1H), 4.51–4.68 (m, 1H), 5.34 (s, 1H), 5.76 (s, 1H),
6.72 (d, J = 8.3 Hz, 2 ArH), 7.07 (d, J = 8.3 Hz, 2 ArH),
7.16–7.35 (m 5 ArH), 8.07–8.18 (m, 2H, NH), 8.31
(d, J = 7.5 Hz, 1H, NH), 9.38 (s, 1H, OH); 13C NMR
(50 MHz, DMSO-d6): d 21.4, 22.8, 24.3, 36.2, 38.1, 40.2,
40.6, 41.8, 50.3, 53.7, 54.9, 115.3, 124.7, 126.2, 128.0, 129.1,
10.4, 138.2, 140.4, 156.5, 166.2, 168.1, 168.5, 171.3, 173.9;
[a]D -11.9 (c 0.5, CHCl3). ESI–MS: m/z 582.3 [MH]?,
604.3 [M?Na]?. Anal. Calcd for C32H40F3N5O9: C, 55.34;
H, 5.79; N, 10.07. Found: C, 55.25; H, 5.82; N, 10.01.
a colorless oil. H NMR (200 MHz, CDCl3): d 1.83–2.19
(m, 2H), 2.04 (s, 3H), 2.38–2.46 (m, 2H), 2.88 (br s, 2H,
NH2), 2.95–3.21 (m, 2H), 3.25–3.39 (m, 2H), 3.70 (s, 3H),
3.98 (dd, J = 4.2 Hz, J = 15.1 Hz, 1H), 4.20 (dd,
J = 6.1 Hz, J = 15.1 Hz, 1H), 4.55–4.66(m, 1H),4.66–4.78
(m, 1H), 5.55 (s, 1H), 5.97 (s, 1H), 6.97 (d, J = 7.7 Hz, 1H,
NH), 7.18–7.35 (m, 5 ArH), 7.65 (d, J = 7.8 Hz, 1H, NH),
7.81 (dd, J = 4.2 Hz, J = 6.1 Hz, 1H, NH); 13C NMR
(50 MHz, CDCl3): d 15.3, 29.7, 31.4, 37.3, 40.3, 44.2, 51.5,
52.4, 55.1, 124.3, 126.8, 128.5, 129.2, 136.8, 139.8, 166.3,
170.7, 172.0, 173.3; [a]D -13.4 (c 0.5, CHCl3). ESI–MS:
m/z 451.2 [MH]?, 473.2 [M?Na]?. Anal. Calcd for
C21H30N4O5S: C, 55.98; H, 6.71; N, 12.44. Found: C, 56.06;
H, 6.63; N, 12.37.
[3-(Fmoc-glicylamino)-2-methylenepropanoyl]-L-
[3-(t-BuO,t-Boc-L-tyrosylglicylamino)-2-
methylenepropanoyl]-L-phenylalaninyl-L-methionine
methyl ester (14b)
phenylalaninyl-L-methionine methyl ester (12b)
To a solution containing the acid 4 (0.88 g; 2.3 mmol), the
hydrochloride 11b (0.95 g; 2.3 mmol) and EDCl (0.49 g;
2.5 mmol) in dry DCM (15 mL), Et3N (320 lL; 2.3 mmol)
was added and the mixture was stirred for 3 h at rt. Then
water (20 mL) was added and the mixture was extracted with
ethyl acetate (3 9 50 mL). After drying (Na2SO4) and
removal of the solvent under reduced pressure, the residue
was purified by silica gel chromatography (ethyl acetate as
eluent) to give the compound 12b (0.97 g; 64% yield) as a
white solid. Mp: 68–70°C. 1H NMR (200 MHz, CDCl3): d
1.88–2.17 (m, 2H), 2.01 (s, 3H), 2.32–2.43 (m, 2H), 3.07 (dd,
J = 7.6 Hz, J = 14.7 Hz, 1H), 3.15 (dd, J = 6.5 Hz,
J = 14.7 Hz, 1H), 3.68 (s, 3H), 3.73–4.00 (m, 3H), 4.17–
4.28 (m, 2H), 4.43 (d, J = 7.3 Hz, 2H), 4.57–4.71 (m, 2H),
5.56 (s, 1H), 5.72 (t, J = 6.7 Hz, 1H, NH), 5.82 (s, 1H), 6.68
(bt, J = 7.2 Hz, 1H, NH), 6.74 (d, J = 7.7 Hz, 1H, NH),
7.03 (d, J = 7.6 Hz, 1H, NH), 7.16–7.44 (m, 9 ArH), 7.58
(d, J = 7.0 Hz, 2 ArH), 7.76(d, J = 7.4 Hz, 2ArH);13CNMR
(50 MHz, CDCl3): d 15.3, 29.7, 31.3, 37.6, 41.0, 44.6, 47.1,
51.5, 52.5, 54.9, 67.2, 120.0, 123.3, 125.0, 127.1, 127.8,
128.7, 129.2, 136.4, 139.8, 141.3, 143.7, 166.6, 169.9, 170.7,
172.2; [a]D -16.4 (c 0.5, CHCl3). ESI–MS: m/z 673.2
[MH]?, 695.1 [M?Na]?. Anal. Calcd for C36H40N4O7S: C,
64.27; H, 5.99; N, 8.33. Found: C, 64.18; H, 6.09; N, 8.24.
To a solution containing the amino derivative 13b (0.55 g;
1.25 mmol) and t-Boc,t-Bu-tyrosine (0.42 g; 1.25 mmol) in
DCM (15 mL), EDCl (288 mg; 1.5 mmol) was added and
the mixture was stirred for 2 h at rt. Then water (10 mL)
was added and the mixture was extracted with ethyl acetate
(3 9 50 mL). After drying (Na2SO4) and removal of the
solvents under reduced pressure, the residue was purified by
silica gel chromatography (ethyl acetate as eluent), to give
the title product 14b (0.58 g; 51% yield) as a white foam.
1H NMR (200 MHz, CDCl3): d 1.32 (s, 9H), 1.35 (s, 9H),
1.88–2.18 (m 2H), 2.04 (s, 3H), 2.31–2.49 (m, 2H), 2.92
(dd, J = 7.8 Hz, J = 14.7 Hz, 1H), 3.01 (dd, J = 6.2 Hz,
J = 15.2 Hz, 1H), 3.09 (dd, J = 8.4 Hz, J = 15.2 Hz, 1H),
3.17 (dd, J = 5.9 Hz, J = 14.7 Hz, 1H), 3.68 (s, 3H), 3.86
(d, J = 5.9 Hz, 2H), 4.01–4.10 (m, 2H), 4.19–4.31 (m, 1H),
4.54–4.72 (m, 2H), 5.04 (d, J = 5.2 Hz, 1H, NH), 5.54
(s, 1H), 5.88 (s, 1H), 6.84–6.94 (m, 3H, 2 ArH ? 1 NH), 7.08
(d, J = 8.6 Hz, 2 ArH), 7.15–7.33 (m, 6H, 5 ArH ? 1
NH), 7.52 (d, J = 5.7 Hz, 1H, NH); 13C NMR (50 MHz,
CDCl3): d 15.3, 28.3, 28.8, 29.7, 31.2, 37.2, 37.3 (50%),
37.4 (50%), 41.0, 42.9, 51.5, 52.5, 55.3, 56.7, 78.5, 80.8,
124.1, 124.4, 126.9, 128.6, 129.2, 129.6, 129.7, 130.9,
136.7, 139.6, 154.5, 166.5, 169.8, 171.1, 172.0, 172.3; [a]D
-22.4 (c 0.5, CHCl3). ESI–MS: m/z 770.4 [MH]?, 792.3
[M?Na]?. Anal. Calcd for C39H55N5O9S: C, 60.84; H,
7.20; N, 9.10. Found: C, 60.75; H, 7.27; N, 9.01.
(3-Glicylamino-2-methylenepropanoyl)-L-
phenylalaninyl-L-methionine methyl ester (13b)
To a solution containing the compound 12b (513 mg;
0.77 mmol) in dry DCM (10 mL), DABCO (87 mg;
0.77 mmol) was added and the clear solution was refluxed
for 7 h and stirred for further 12 h at rt. After removal of
the solvent under reduced pressure, the residue was purified
by silica gel chromatography (ethyl acetate:methanol 80:20
as eluent) to give the compound 13b (0.32 g; 94% yield) as
(3-L-Tyrosylglicylamino-2-methylenepropanoyl)-L-
phenylalaninyl-L-methionine trifluoroacetate (3b)
The ester 14b (0.39 g; 0.51 mmol) was dissolved in
methanol (5 mL) and then 1 M NaOH aqueous solution
(0.8 mL; 0.8 mmol) was added. After stirring for 5 h at rt,
the mixture was extracted with ethyl acetate (2 9 10 mL),
123

1064
R. Galeazzi et al.
the aqueous phase cooled to 0°C and 1 M HCl was added
until pH = 2. After extraction with ethyl acetate
(2 9 10 mL), the solvent was dried and eventually
removed under reduced pressure, to give a residue which
crystallized on standing. The solid was dissolved in DCM
(5 mL) and TFA (0.85 g; 7.4 mmol) was added. After
stirring for 7 h at rt, the solvent was removed under
reduced pressure and the residue was washed with dry
ethyl ether (2 9 5 mL), to give the trifluoroacetate 3b
(0.36 g; quantitative yield) as a white solid. 1H NMR
(200 MHz, D2O): d 1.83–2.27 (m, 2H), 2.08 (s, 3H), 2.41–
2.61 (m, 2H), 2.82–3.21 (m, 4H), 3.22–3.33 (m, 1H), 3.88
- 4.09 (m, 5H), 4.58 (dd, J = 4.8 Hz, J = 9.2 Hz, 1H),
4.71 (dd, J = 4.8 Hz, J = 9.6 Hz, 1H), 4.91 (br s, 9H,
NH?OH), 5.51 (s, 1H), 5.76 (s, 1H), 6.78 (d, J = 8.1 Hz, 2
ArH), 7.11 (d, J = 8.1 Hz, 2 ArH), 7.18–7.32 (m, 5 ArH);
13C NMR (50 MHz, CD3OD): d 15.2, 31.1, 32.2, 37.7,
38.5, 41.4, 43.3, 50.3, 52.7, 56.1, 109.8, 116.9, 121.7,
126.0, 127.8, 129.5, 129.6, 130.2, 130.3, 131.5, 138.5,
141.5, 158.3, 169.2, 170.3, 171.2, 173.8, 174.8; [a]D -16.8
(c 0.5, CHCl3). ESI–MS: m/z 600.2 [MH]?, 622.2
[M?Na]?. Anal. Calcd for C31H38F3N5O9S: C, 52.17; H,
5.37; N, 9.81. Found: C, 52.06; H, 5.29; N, 9.88.
In vitro bioactivity assays
Preparations of the myenteric plexus-longitudinal muscle
obtained from male guinea pig ileum (GPI, rich in l-opioid
receptors) and preparations of mouse vas deferens (MVD,
rich in d-opioid receptors) were used for field stimulation
with bipolar rectangular pulses of supramaximal voltage.
Agonists were evaluated for their ability to inhibit the
electrically evoked twitch. The results are expressed as the
IC50 values obtained from concentration–response curves
(Prism). IC50 values represent the mean of not less than five
tissue samples SEM.
Acknowledgments We wish to acknowledge Nanodream s.r.l. (Iesi,
Italy) for funding this research, and Mr. Andrea Garelli, University of
Bologna, for mass spectral analyses.
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