Synthesis and activity of endomorphin-2 and morphiceptin analogues with proline surrogates in position 2

Article abstract of DOI:10.1016/j.ejmech.2010.07.022

The opioid agonists endomorphins (Tyr-Pro-Trp-Phe-NH₂; EM1 and Tyr-Pro-Phe-Phe-NH₂; EM2) and morphiceptin (Tyr-Pro-Phe-Pro-NH ₂) exhibit an extremely high selectivity for μ-opioid receptor. Here a series of novel EM2 and morphiceptin analogues containing in place of the proline at position 2 the S and R residues of β-homologues of proline (HPro), of 2-pyrrolidinemethanesulphonic acid (HPrs) and of 3- pyrrolidinesulphonic acid (βPrs) have been synthesized and their binding affinity and functional activity have been investigated. The highest μ-receptor affinity is shown by [(S)βPrs²]EM2 analogue (6e) which represents the first example of a β-sulphonamido analogue in the field of opioid peptides.

Full text of DOI:10.1016/j.ejmech.2010.07.022

European Journal of Medicinal Chemistry 45 (2010) 4594e4600
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and activity of endomorphin-2 and morphiceptin analogues
with proline surrogates in position 2
,
a
a
a
b
c
Cesare Giordano a , Anna Sansone , Annalisa Masi , Gino Lucente , Pasqualina Punzi , Adriano Mollica ,
Francesco Pinnen c, Federica Feliciani c, Ivana Cacciatore c, Peg Davis d, Josephine Lai d, Shou-Wu Ma d,
Frank Porreca d, Victor Hrubye
*
a Istituto di Chimica Biomolecolare del CNR, c/o Dipartimento di Chimica e Tecnologie del Farmaco, “Sapienza” Università di Roma, P.le A. Moro 5, 00185 Roma, Italy
b Dipartimento di Chimica, “Sapienza” Università di Roma, P.le A. Moro 5, 00185 Roma, Italy
c Dipartimento di Scienze del Farmaco, Università di Chieti-Pescara “G. D’Annunzio”, Via dei Vestini 31, 66100 Chieti, Italy
d Departments of Pharmacology, University of Arizona, Tucson, AZ 85719, USA
e Departments of Chemistry, University of Arizona, Tucson, AZ 85721, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The opioid agonists endomorphins (TyreProeTrpePheeNH2; EM1 and TyreProePheePheeNH2; EM2)
Received 16 March 2010
Received in revised form
13 July 2010
Accepted 13 July 2010
Available online 21 July 2010
and morphiceptin (TyreProePheeProeNH2) exhibit an extremely high selectivity for
Here a series of novel EM2 and morphiceptin analogues containing in place of the proline at position 2
the S and R residues of -homologues of proline (HPro), of 2-pyrrolidinemethanesulphonic acid (HPrs)
and of 3-pyrrolidinesulphonic acid ( Prs) have been synthesized and their binding affinity and functional
activity have been investigated. The highest -receptor affinity is shown by [(S)
Prs2]EM2 analogue (6e)
which represents the first example of a
m-opioid receptor.
b
b
m
b
b
-sulphonamido analogue in the field of opioid peptides.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Endomorphins
Opioid peptides
Peptide synthesis
b-Sulphonamido peptides
Unusual amino acids
1. Introduction
(TyreProePheeProeNH2) isolated from an enzymatic digest of
bovine -casein [1e3] and by the structurally related opioid ligands
endomorphin-1 (EM1: TyreProeTrpePheeNH2) and endomor-
b
Due to their role in the modulation and perception of pain the
opioid receptors continue to be an extremely important target in
phin-2 (EM2: TyreProePheePheeNH2) which exhibit high
m-
medicinal chemistry. Three major subtypes (
protein coupled family of receptors have been defined and it is well
established that the group represents the major target of the
analgesics. Although several endogenous peptide ligands of opioid
receptors are known most of them, including enkephalins, does not
m
,
k
,
d
) of this G-
opioid receptor selectivity and agonist potency [4].
As it is well established [5,6] and clearly discussed by Goodman
and Schiller [7], in the structure of natural opioid peptides two
biological relevant fragments can be identified, namely the N-
terminal message sequence, containing the two pharmacophoric
aromatic residues of Tyr and Phe, and the remaining C-terminal
fragment which represents the address sequence. EM2 and mor-
phiceptin have a different C-terminal address sequence [3] which
is ePheeNH2 for EM2 and eProeNH2 for morphiceptin, but an
identical TyreProePhee N-terminal message sequence. This
feature, common to EMs and morphiceptin, is different from that
found in enkephalins and other endogenous opioid peptides such
as endorphins and dynorphins. These latter ligands possess the
tetrapeptidic fragment TyreGlyeGlyePhee as a characteristic N-
terminal message sequence. Thus, a topochemical characteristic
which differentiates the N-terminal sequences present in the two
m
show significant
m-selective agonistic activity. A notable exception
is represented by morphiceptin,
a
tetrapeptide amide
Abbreviations: HPreOH, homoproline (2-pyrrolidineacetic acid); HPrs-OH,
2-pyrrolidinemethanesulphonic acid; Pro-OH, -proline (3-pyrrolidinecarboxylic
acid); Prs-OH, 3-pyrrolidinesulphonic acid; DMSO, dimethylsulfoxide; Cbz, ben-
b
b
b
zyloxycarbonyl; Boc, tert-butyloxycarbonyl; DCM, dichloromethane; HOBt,
hydroxybenzotriazole; TEA, triethylamine; EDC, N-(3-dimethylaminopropyl)-N0-
ethylcarbodiimide hydrochloride.
* Corresponding author. Tel.: þ39 06 49913237; fax: þ39 06 491491.
E-mail address: cesare.giordano@uniroma1.it (C. Giordano).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.07.022

C. Giordano et al. / European Journal of Medicinal Chemistry 45 (2010) 4594e4600
4595
classes of peptides is the type of the spacer group separating the Tyr
and Phe aromatic residues. It thus can be inferred that different
spacers, namely the Pro residue at position 2 in morphiceptin [8,9],
and in EMs [10e12], as well as the GlyeGly dipeptide fragment in
enkephalins, fulfill the stereochemical requirements needed for
a correct interaction of the N-terminal phaarmacophore aromatic
side chains with the involved receptor area.
On the basis of the above considerations and in order to obtain
additional information on the biochemical consequences of the
structural modifications performed at the level of the Tyr1/Phe3
spacer residue we synthesized and biologically evaluated a new
group of analogues of EM2 and morphiceptin reported in Fig. 1.
Only in the case of the synthesis of the sulphonamido
junction couplings were performed by using sulfonyl chlorides.
The N-protected pseudoamino acids 1a-f were coupled with
HePheeOMe.HCl and the resulting dipeptides 2a-f were subjected
to catalytic hydrogenation. Successive coupling with BoceTyreOH
gave the tripeptides 3a-f which were O-deprotected by alkaline
hydrolysis and successively coupled with HePheeNH2 or
HeProeNH2 leading to the tetrapeptides 4a-f and 5a,b, respec-
tively. Treatment with TFA 95% afforded the required final free
peptides 6a-f and 7a,b as trifluoroacetate salts (final products were
characterized by mass spectra analysis, see Table 1).
The N-Cbz (S) and (R) homoprolines 1a,b, necessary for the
synthesis of peptides 6a,b and 7a,b, were prepared according to
literature [27] through the diazomethyl ketone route, starting from
the appropriate N-Cbz derivative.
The(S)and (R)pyrrolidinesulfonylchlorides 1c-f, requiredfor the
preparation of peptides 6c-f, were synthesized starting from the
corresponding amino alcohol according to Scheme 2 [28e30].
In the first group of analogues (6a,b and 7a,b) the (S) and (R) b-
homologues of proline, namely (S)-homoproline [(S)-HProeOH]
and (R)-homoproline [(R)-HProeOH], respectively, replace the
native Pro residue at position 2. In the remaining four EM2
analogues (6c,d and 6e,f) two different types of Pro b-homologues
have been inserted as spacer groups and in both of them the
eSO2eNHe sulphonamido group replaces the eCOeNHe peptide
bond. This bioisosteric replacement, initially introduced in 1989 by
Lucente and coworkers [13,14] and then extensively investigated
[15e18], generates a metabolically stable junction associated with
significant changes in backbone conformational properties, polarity
and H-bonding capacity [19]. Although the potentiality of sulfo-
namidopeptides is now well established [20,21] and applied to
different classes of bioactive peptides [22e24] and enzyme inhib-
itors [25], analogues of EMs containing the sulfonamide junction
have not been described. The here reported compounds 6c and 6d
contain the (R) and (S) residues of the 2-pyrrolidinemethanesul-
3. Biological evaluation and conclusions
The binding affinities for opioid receptors and functional
bioactivities, exhibited by the EM2 and the morphiceptin analogues
6a-f and 7a,b are summarized in Table 2.
All compounds show very low k- and
(micromolar range) and high selectivity. Results concerning the
four EM2 derivatives containing -homologues of proline, both of
carboxylic and -sulfonic type (namely 6a,b and 6c,d respectively),
d-opioid receptor affinities
d/m
b
b-
b
confirm the strong influence of the stereochemistry at position 2 on
the binding affinity. In this group of four analogues only the [(S)
phonic acid [(S)-HPrseOH and (R)-HPrseOH] and are then
phonamido analogues of 6a and 6b, respectively. To a second and
new type of -sulphonamido peptides belong the analogues 6e and
6f. These are characterized by the presence of the 3-pyrrolidine-
sulphonic acid [(S) or (R) PrseOH] residue. This cyclic -amino
sulfonic acid, synthesized for the first time for the present research,
is the sulfonyl analogue of -proline (3-pyrrolidinecarboxylic acid;
-ProeOH), a well known -amino carboxylic acid, previously
incorporated into the EM1 molecule to give the highly active Tyre
(R)- -ProeTrpePheeNH2 tetrapeptide [26].
b-sul-
HPro2]-EM2 (6a), containing the (S)-
b-homoproline residue, main-
tains good
m
-opioid receptor affinity (Kmi ¼ 58 nM) whereas the
b
corresponding [(R)HPro2]-EM2 (6b) is sensibly less potent
(Kmi ¼ 290 nM). The same trend is found in the case of the two
b
b
derivatives containing the b-homo-sulfonyl residue HPrs where the
[(R)HPrs2]-EM2 (6d) is 5-fold less active then [(S)HPrs2]-EM2 (6c).
These results are in agreement with those previously reported for
b
b
b
b
-
amino carboxylic acid containing EM1 analogues [33] where the [(S)
HPro2]-EM1 has been found to be about 33-fold more potent than
the [(R)HPro2]-EM1. Although with an opposite trend, a high influ-
ence on the affinity is also shown by the stereochemistry at position
2 of the two morphiceptin analogues 7a and 7b containing enan-
b
2. Chemistry
The synthesis of peptides 6a-f and 7a,b (Scheme 1) was per-
formed in solution using the carbodiimide method for coupling
steps.
tiomeric b
-homologues of proline. Here it has been found that the Kim
values for [(S)HPro2]-morphiceptin (7a) and [(R)HPro2]-morphi-
ceptin (7b) are Kmi ¼ 6100 nM and Kmi ¼ 30 nM, respectively. Thus,
Fig. 1. Schematic representation of the reported EM2 (6aef) and morphiceptin (7a,b) analogues.

4596
C. Giordano et al. / European Journal of Medicinal Chemistry 45 (2010) 4594e4600
Scheme 1. Synthesis of pseudopeptides 6aef and 7a,b. Reagents: (i) for 2a,b: PheeOMe.HCl, EDC, HOBt, TEA, DCM; for 2cef: PheeOMe.HCl, TEA, DCM; (ii) a: H2, Pd/C, MeOH, TFA;
b: BoceTyreOH, EDC, HOBt, TEA, DCM; (iii) a: 1 M NaOH, MeOH; b: Phe-NH2HCl, EDC, HOBt, TEA, DCM; (iv) a: 1 M NaOH, MeOH; b: Pro-NH2HCl, EDC, HOBt, TEA, DCM; (v) TFA/H2O
95:5. Structures of Xaa residues are reported in Fig. 1.
when these data are compared to those shown by the above cited
affinities of 6a and 6b, it can be seen that the absolute configuration
of the HPro residue at position 2 exerts an opposite effect on binding
of the morphiceptin and EM2 analogues and leads to the [(R)HPro2]-
morphiceptin (7b) endowed with the highest potency in the GPI
[(R)-b
Prs2]-EM2 (6f), in which the stereochemistry of the residue at
position2 has beenchanged, shows a sensible decrease of the binding
affinity (Kmi ¼ 63nM). Thus, aspreviously foundfor theEM1 analogues
containing the bPro residue (i.e. the carboxylic analogue of bPrs) [26],
the absolute configuration at position 2 highly influences the binding.
However, an opposite spatial orientation of the acylating group at
position 2 characterizes the most active analogues in the two cases. In
assays(IC50 ¼17) as well as high
d/m binding selectivity. This result is
not in disagreement with previous studies which highlight the role
of the spacer residue structure at position 2 in giving correct spatial
orientation to the aromatic residues of the ligand [6,34] and
confirms the observation that each class of opioid peptides shows
distinct chiral requirements for the spacers between the biologically
important Tyr and Phe residues [7].
fact, when the
b
-carboxylic residue is involved, the [(R)-
b
Pro2]-EM1
Prs2]-
[26] was found to be more active than the corresponding [(S)-
b
EM1 (Kmi ¼ 0.33 nM versus Kmi ¼ 10.4 nM) [26].
Table 2 summarizes, in addition to the affinity data, the func-
tional activity on
m- and
d-opioid receptors of the here studied
In addition to the analogues 6aed and 7a,b containing the
homoproline residues HPro and HPrs, the EM2 analogues 6e,f
contain at position 2 the enantiomers of the -proline sulfonyl
residue Prs. These are characterized by relevant structural differ-
ences as compared with the -homoproline (HPro and HPrs) con-
b
-
analogues. The highest potency as m
-agonist is that of the analogue
7b which, in the GPI functional assay, shows a value (IC50 ¼ 17)
practical equal to that of the EM2 (IC50 ¼ 15) although with
a sensibly lower selectivity as indicated by the MVD/GPI IC50 ratio
which is 34 versus 5.7, respectively. Notable is in this case the
strong influence of the absolute configuration of the residue at
position 2 as shown by the binding and activity values (see Table 2)
found for 7b, the epimeric analogue of 7a. Although to a lower
degree, a strong effect of the chirality at position 2 is here observed
in the case of 6a and 6b and was previously reported for the couple
of EM1 analogues containing (R) and (S) HPro residues [33]. The
two groups of peptides 6c,d and 6e,f contain a sulphonamido
junction replacing the native Pro2-Phe3 peptide bond. In this case,
structurally characterized by a CH2 group inserted between the
b
b
b
taining residues 6aed and 7a,b. In fact, in addition to the presence
of the SO2eNH junction, replacing the usual COeNH peptide bond,
in the
bPrs residues the acylating group is directly bound to
a carbon atom of the 5-membered ring. Conversely, in 6aed and
7a,b a eCH2e bridge is inserted between the pyrrolidine ring and
the acylating group. This feature may greatly change the confor-
mational preferences of the entire molecule, leading to systems
with higher flexibility and conformational freedom. As shown in
Table 2 the
peptide amide 6e, containing the (S)-
m
-opioid receptor affinity (Kmi ¼ 19 nM) of the tetra-
b
Prs sulfonyl residue, is the
SO2eNH bond and the pyrrolidine ring, a clear
m-receptor prefer-
highest among the here studied ligands. The epimeric analogue
ence is shown by the analogue 6c, possessing the (S)
b
Prs residue at
position 2 (IC50, GPI ¼ 730 and 2700 nM, for 6c and 6d, respec-
tively). This behavior is analogous to that exhibited by 6a and 6b
containing the (S)HPro and (R)HPro residue, respectively. In the
models 6e and 6f the SO2eNH junction is directly bound to the five-
membered ring and this strongly limits the backbone flexibility as
Table 1
Sequence and mass spectra analysis of the reported analogues 6aef and 7a,b.
Peptide
Sequencea
M(Hþ) obsd (MW calcd)
compared with 6c and 6d. In this case both the epimers [(S)
EM2 6e and [(R)
Prs2]EM2 6f show unexpectedly low GPI and MVD
potencies while it is still retained significant receptor affinity for
both - and -opioid receptor types. This suggests that this couple
b
Prs2]
6a
6b
6c
6d
6e
6f
HeTyre(S)-HProePheePheeNH2
HeTyre(R)-HProePheePheeNH2
HeTyre(S)-HPrsePheePheeNH2
HeTyre(R)-HPrsePheePheeNH2
HeTyre(S)-
HeTyre(R)-
586.3792 (585.2951)
586.2837 (585.2951)
622.3729 (621.2621)
622.4567 (621.2621)
608.5681 (607.7216)
608.6743 (607.7216)
536.4761 (535.2795)
536.3954 (535.2795)
b
m
d
b
PrsePheePheeNH2
PrsePheePheeNH2
of analogues may have a mixed agonist/antagonist property for
both receptor types.
b
7a
7b
HeTyre(S)-HProePheeProeNH2
HeTyre(R)-HProePheeProeNH2
To the best of our knowledge the here reported
bPrs containing
a
Modification are shown in bold letters.
tetrapeptides 6e,f are the first examples of analogues containing

C. Giordano et al. / European Journal of Medicinal Chemistry 45 (2010) 4594e4600
4597
Scheme 2. Synthesis of sulfonyl chlorides 1cef. Reagents: i) a: MsCl, TEA, DCM; b: HCl, dioxane; ii) a: Na2SO3, H2O; b: CbzeCl, aq. NaOH; c: COCl2/toluene, DMF, DCM.
a
b
-sulphonamido replacement in the field of opioid peptides. The
4.1.1. Preparation of sulfonic acids. General procedure
high -receptor affinity shown by the tetrapeptide [(S)-
(6e) suggests further study of this type of derivatives and
underlines, at the same time, the highly different interactions
which
m
b
Prs2]EM2
An HCl saturated solution of the Boc-mesylates (1.00 mmol) in
dioxane (30 mL) was allowed to stand at room temperature for
30 min. The precipitate was filtered and crystallized from ethanol-
ethyl ether. A solution of the recovered precipitate and Na2SO3
(2.6 mmol) in water (5 mL) was stirred for 24 h at room tempera-
ture, then passed firstly on Amberlite IR-120 (Hþ form) and then on
Dowex 11 (acetate form) columns. Evaporation of the eluate under
b-sulphonamido pseudopeptides, as compared with the
b
-carboxyamido counterparts, may establish with the receptors.
vacuum gave
watereethanol.
a crude residue which was crystallized from
4. Experimental protocols
4.1. General
4.1.1.1. (S)-3-pyrrolidinesulfonic acid. From N-Boc-(S)-3-pyrrolidi-
nemethansulfonate [30] (4.90 g, 18.48 mmol). White solid (78%). 1H
IR spectra were recorded in 1% CHCl3 solution employing a Per-
kin-Elmer FT-IR Spectrum 1000 spectrophotometer. [
NMR (D2O)
d: 2.12 (m, 2H, Pyr
gCH2), 3.06e3.28 (two m, 3H, Pyr
a
]
was
D
b
CH and CH2), 4.02 (m, 2H, Pyr
d
a
CH2). Anal. Calcd for C4H9NO3S: C
measured at 20 ꢀC with a SchmidteHaensch polarimeter at a 1%
concentration in CHCl3 (unless otherwise specified) with a 1 cm cell.
1H NMR spectra were determined in CDCl3 solution with a Bruker
AM 400 spectrometerand chemical shifts wereindirectly referred to
TMS. The mass spectra were performed on a Q-TOFMICRO spec-
trometer (Micromass, now Waters, Manchester, UK) equipped with
an ESI source, in the positive ion mode and datawere analyzed using
the MassLynx software (Waters). Thin-layer and preparative layer
chromatographies were performed on silica gel Merck 60 F254
plates. The drying agent was sodium sulfate. Elemental analyses for
C, H and N (where necessary a sample was further purified by
preparative TLC) were performed in the laboratories of the Servizio
Microanalisi del CNR, Area della Ricerca di Roma, Montelibretti, Italy,
and were within 0.4% of the theoretical values.
31.78, H 6.00, N 9.26; found C 31.85, H 6.23, N 9.21.
4.1.1.2. (R)-3-pyrrolidinesulfonic acid. From N-Boc-(R)-3-pyrrolidi-
nemethansulfonate [30] (3.83 g, 14.43 mmol). White solid (86%). 1H
NMR (D2O)
d: 2.12 (m, 2H, Pyr
gCH2), 3.06e3.28 (two m, 3H, Pyr
b
CH and CH2), 4.02 (m, 2H, Pyr
d
a
CH2). Anal. Calcd for C4H9NO3S: C
31.78, H 6.00, N 9.26; found C 31.91, H 6.20, N 9.33.
4.1.2. Preparation of sulfonyl chlorides. General procedure
To a solution of the appropriate sulfonic acid (1.00 mmol) in H2O
(10 mL), adjusted to pH 8.5 with NaOH, Cbz-chloride (1.1 mmol)
was added in five portions at room temperature and under vigorous
stirring, maintaining the pH at 8e8.5 by small amounts of 1 N
Table 2
Binding affinity and in vitro activity for compounds 6 aef and 7 a,b.
,
Compound
Receptor affinitya b (nM)
Selectivity
Kdi /Kim
Bioassayb (nM)
MVD (IC50
510 ꢁ 35d
Kid
Kim
Kik
)
GPI (IC50)
EM2c
8360 ꢁ 1314
> 10000
8.23 ꢁ 0.48
e
e
1016
> 74.1
85
> 34
86
15 ꢁ 2d
Morphyceptinc
135 ꢁ 18.4
58 ꢁ 5
e
e
6a
6b
6c
6d
6e
6f
4900 ꢁ 540
990 ꢁ 12
1300 ꢁ 300
1900 ꢁ 230
1100 ꢁ 260
260 ꢁ 32
420 ꢁ 91
730 ꢁ 64
2700 ꢁ 220
260 ꢁ 56
220 ꢁ 41
1100 ꢁ 115
17 ꢁ 3
> 10000
293 ꢁ 27
100 ꢁ 8
510 ꢁ 48
19 ꢁ 3
10000 ꢁ 1200
4800 ꢁ 367
6300 ꢁ 540
3400 ꢁ 330
7100 ꢁ 628
4400 ꢁ 356
10000 ꢁ 982
8600 ꢁ 940
7800 ꢁ 850
> 10000
15.3
36% at 1 mM
> 532.9
113.1
> 6
530 ꢁ 91
390 ꢁ 56
33.7% at 1
96 ꢁ 12
7100 ꢁ 782
63 ꢁ 5
7a
7b
> 10000
6100 ꢁ 553
mM
> 10000
30 ꢁ 4
> 333
Displacement of [3H]DAMGO (
m
-selective) and [3H]DPDPE (
k
-selective) from rat brain membrane binding sites; displacement of [3H]U69593 (
k
-selective) from guinea pig
a
brain membrane binding sites.
b
ꢁ S.E.M.
c
Data from reference [31].
d
Data from reference [32].

4598
C. Giordano et al. / European Journal of Medicinal Chemistry 45 (2010) 4594e4600
NaOH. After additional 2 h from the last addition, water was added
(10 mL) and the aqueous phase was washed with Et2O (2 ꢂ 10 mL),
evaporated under reduced pressure and coevaporated with
toluene. The crude sodium sulfonate salt, white solid, was dried
overnight under high vacuum on P2O5. To a suspension of the dried
residue in DCM (30 mL) a solution of phosgene in toluene (20% p/p,
4.0 mL) and DMF (0.6 mL) in DCM (30 mL) was added under N2. If
the reaction was not complete (TLC) after 1 h, an additional amount
(1e2 mL) of the latter solution was added. After stirring for 2 h at
room temperature, concentration of the mixture and purification
on silica gel column (CH2Cl2) gave the products which were stored
under argon and used as such.
5.10 (s, 1H, Phe NH), 7.01e7.23 (m, 10H, Ar). Anal. Calcd for
22H26N2O6S: C 59.18, H 5.87, N 6.27; found C 59.33, H 6.07, N 6.12.
C
4.1.3.3. Cbze(R)-
bPrsePheeOMe (2f). From 1f (0.975 g, 3.21 mmol).
Colourless oil (52%); [
a
]
þ 2ꢀ (1, H2O); IR
n:3691, 3020, 1687,
D
1432 cmꢃ1
;
1H NMR
Prs CH and
d: 1.98 (m, 2H, bPrs gCH2), 2.93e3.12 (m, 5H,
Phe
Prs
b
a
CH2,
b
b
dCH2), 3.87 (s, 3H, OCH3), 4.26 (br d, 2H,
b
CH2), 4.92e5.15 (m, 3H, Phe
a
CH and Cbz CH2), 5.11 (s,1H, Phe
NH), 7.08e7.21 (m, 10H, Ar). Anal. Calcd for C22H26N2O6S: C 59.18, H
5.87, N 6.27; found C 59.14, H 6.02, N 6.19.
4.1.4. Carbodiimide coupling. General procedure
To an ice-cooled mixture containing the C-protected amino acid
or peptide salt (1.0 mmol), the required N-protected amino acid
(1.0 mmol), HOBt (1.2 mmol) and TEA (2.2 mmol) in anhydrous
DCM (6.0 mL), EDC (1.2 mmol) was added and the reaction mixture
was allowed to warm slowly to room temperature overnight. The
mixture was then diluted with DCM (20 mL) and washed with 1 M
KHSO4 (2 ꢂ 15 mL), saturated aqueous NaHCO3 (2 ꢂ 15 mL) and
brine (15 mL). The organic phase was dried and evaporated under
reduced pressure.
4.1.2.1. Cbze(R)-2-pyrrolidinemethanesulfonyl chloride [Cbze(R)-
HPrs-Cl] (1d). From (R)-2-pyrrolidinemethanesulfonic acid [29]
(2.05 g, 12.42 mmol). Colourless oil (56%). IR
1701, 1414 cmꢃ1; 1H NMR
: 1.81e2.34 (m, 4H, Pyr
(m,2H, CH2SO2), 3.25 (m,1H, Pyr CH), 3.96e4.25 (m, 2H, Pyr
n
: 3020, 2957, 2873,
and CH2),3.16
CH2),
d
b
g
a
d
5.21 (s, 2H, Cbz CH2), 7.21e7.53 (m, 5H, Ar). Anal. Calcd for
C13H16ClNO4S: C 49.13, H 5.07, N 4.41; found C 49.01, H 5.18, N 4.56.
4.1.2.2. Cbz-(S)-3-pyrrolidinesulfonyl chloride [Cbze(S)-
(1e). From (S)-3-pyrrolidinesulfonic acid (1.93 g, 12.77 mmol).
Colourless oil (62%). IR
: 3018, 2955, 2871, 1769, 1414 cmꢃ1 1H
NMR : 1.96e2.43 (m, 4H, Pyr and CH2), 3.02 (m, 1H, Pyr CH),
3.4 (m, 2H, Pyr CH2), 5.19 (s, 2H, Cbz CH2), 7.20e7.54 (m, 5H, Ar).
bPrs-Cl]
4.1.4.1. Cbze(R)-HProePheeOMe (2b). From 1b [27] (0.650 g,
2.47 mmol) and HePheeOMe. HCl (0.532 g, 2.47 mmol). Pale yellow
n
;
oil (81%); [
a
]
þ 10ꢀ; IR
n ;
:3689, 3425, 3029, 3009,1740,1676 cmꢃ1
D
d
g
d
b
1H NMR
d: 2.06e2.10 (m, 4H, HPro
b
and
g
CH2), 2.90e2.97 (dd, 2H,
CH2), 3.18 (m, 2H,
CH2), 3.74 (s, 3H, OCH3), 4.13 (br, 1H,
CH), 5.10 (m, 2H, Cbz CH2), 5.89 (br,
a
A and B of an ABX, J ¼ 8.0, 5.5 and 13.5 Hz, Phe
CH2CO), 3.55 (m, 2H, HPro
HPro CH), 4.92 (m, 1H, Phe
b
Anal. Calcd for C12H14ClNO4S: C 47.45, H 4.65, N 4.61; found C 47.38,
H 4.72, N 4.57.
d
a
a
1H, Phe NH), 6.98e7.57 (m, 10H, Ar). Anal. Calcd for C24H28N2O5: C
67.91, H 6.65, N, 6.60; found: C 67.82, H 6.58, N 6.49.
4.1.2.3. Cbz-(R)-3-pyrrolidinesulfonyl chloride [Cbze(R)-
(1f). From (R)-3-pyrrolidinesulfonic acid (1.45 g, 9.60 mmol). Col-
ourless oil (48%). IR
: 3018, 2955, 2871,1769, 1414 cmꢃ1; 1H NMR
1.96e2.43 (m, 4H, Pyr and CH2), 3.02 (m, 1H, Pyr CH), 3.4 (m,
2H, Pyr CH2), 5.19 (s, 2H, Cbz CH2), 7.20e7.54 (m, 5H, Ar). Anal.
bPrs-Cl]
n
d:
4.1.4.2. BoceTyre(S)-HProePheeOMe (3a). Hydrogenolysis of 2a
[28] (1.37 g, 3.23 mmol) in MeOH on 10% Pd/C in the presence of
TFA afforded the corresponding trifluoroacetate salt (white foam,
92%) which was coupled with BoceTyreOH (0.838 g, 2.98 mmol).
g
d
b
a
Calcd for C12H14ClNO4S: C 47.45, H 4.65, N 4.61; found C 47.29, H
4.86, N 4.45.
Purified on SiO2 (DCM/EtOAc 1:1). White solid (63%); [
:3689, 3432, 3032, 1743, 1670, 1629 cmꢃ1; 1H NMR
(CH3)3], 1.20e1.54 (m, 4H, HPro and
Tyr CH2, Phe CH2, HPro CH2 and CH2CO), 3.73 (s, 3H, OCH3), 4.20
(br s, 1H, HPro CH), 4.51 (m, 1H, Phe CH), 4.93 (m, 1H, Tyr CH),
a
]
þ 46ꢀ; IR
D
n
d: 1.30 [s, 9H, C
4.1.3. Preparation of peptidosulfonamides. General procedure
To an ice-cooled mixture containing the appropriate Cbzesul-
fonyl chloride (1.0 mmol) and PheeOMe.HCl (2.0 mmol) in anhy-
drous DCM (10 mL) a solution of TEA (2.0 mmol) in DCM (4.0 mL)
was added dropwise under stirring. The reaction was stirred
overnight at room temperature, then was diluted with DCM
(25 mL) and consecutively washed with 1 N HCl (2 ꢂ 20 mL), sat.
NaHCO3 (2 ꢂ 20 mL) and brine (20 mL). The organic phase was
dried and evaporated. The crude products were purified on silica
gel flash chromatography (CHCl3) and were obtained as pale yellow
oil which solidified on standing.
b
gCH2), 2.39e3.45 (four m, 8H,
b
b
d
a
a
a
5.23 (m, 1H, Tyr NH), 6.80e7.20 (m, 10H, Ar), 7.54 (s, 1H, Phe NH),
9.83 (m, 1H, OH). Anal. Calcd for C30H39N3O7: C 65.08, H 7.10, N 7.59;
found: C 65.22, H 6.97, N 7.71.
4.1.4.3. BoceTyre(R)-HProePheeOMe (3b). Hydrogenolysis of 2b
(0.806 g, 1.90 mmol) in MeOH on 10% Pd/C in the presence of TFA
afforded the corresponding trifluoroacetate salt (white foam, 88%)
which was coupled with BoceTyreOH (0.470 g, 1.67 mmol). Puri-
fied on SiO2 (DCM/EtOAc 1:1). Colourless oil (76%); [
3639, 3429, 3012, 2835, 1742, 1697, 1631 cmꢃ1 1H NMR
9H, C(CH3)3], 1.51e1.74 (m, 4H, HPro and
m, 8H, Tyr CH2, Phe CH2, HPro CH2 and CH2CO), 3.70 (s, 3H,
OCH3), 4.22 (br s, 1H, HPro CH), 4.50 (br s, 1H, Phe CH), 4.91 (m,
1H, Tyr CH), 5.24 (m, 1H, Tyr NH), 6.81e7.23 (m, 10H, Ar), 7.52 (m,
a
]
D
þ 2ꢀ; IR
n:
4.1.3.1. Cbze(R)-HPrsePheeOMe
5.50 mmol). Pale yellow solid (48%); [
3008, 1747, 1668, 1430 cmꢃ1 1H NMR
and
and 13.5 Hz, Phe
CH2SO2), 3.76 (s, 3H, OCH3), 4.18 (m,1H, HPrs
CH), 5.21 (s, 2H, Cbz CH2), 5.84 (br d, 1H, Phe NH), 7.04e7.12 (m,
(2d). From 1d (1.750 g,
;
d: 1.32 [s,
a
]
þ 7ꢀ (1, H2O); IR
n: 3430,
d: 1.87e2.40 (m, 4H, HPrs
b
gCH2), 2.40e3.42 (four
D
;
b
b
d
b
g
CH2), 2.94 and 3.12 (dd, 2H, A and B of an ABX, J ¼ 7.9, 5.5
CH2), 3.26 (m, 2H, HPrs CH2), 3.54 (m, 2H,
CH), 4.22 (m,1H, Phe
a
a
b
d
a
a
1H, Phe NH), 9.80 (s, 1H, OH). Anal. Calcd for C30H39N3O7: C 65.08, H
7.10, N 7.59; found: C 65.14, H 7.02, N 7.67.
a
10H, Ar). Anal. Calcd for C23H28N2O6S: C 59.98, H 6.13, N 6.08; found
C 60.12, H 6.27, N 5.96.
4.1.4.4. BoceTyre(S)-HPrsePheeOMe (3c). Hydrogenolysis of 2c
[28] (0.161 g, 0.35 mmol) in MeOH on 10% Pd/C in the presence of
TFA afforded the corresponding trifluoroacetate salt (white foam,
78%) which was coupled with BoceTyreOH (0.075 g, 0.27 mmol).
4.1.3.2. Cbze(S)-
2.72 mmol). Pale yellow solid (46%); [
3012, 1699, 1426 cmꢃ1 1H NMR
: 2.02 (m, 2H,
2.90e3.05 (m, 5H, Phe CH2, Prs CH and CH2), 3.80 (s, 3H, OCH3),
4.31 (br d, 2H, Prs CH2), 4.90e5.12 (m, 3H, Phe CH and Cbz CH2),
bPrsePheeOMe
(2e). From
1e
(0.827
g,
a
]
- 3ꢀ (1, H2O); IR
n:3689,
D
;
d
bPrs gCH2),
Purified on SiO2 (DCM/MeOH 99:1). Pale yellow solid (90%); [
5ꢀ; IR : 3433, 3027, 1704, 1671 cmꢃ1 1H NMR
: 1.34 [s, 9H, C
(CH3)3], 1.81 (m, 2H, HPrs CH2), 2.10e2.81 (two m, 4H, Tyr CH2
a] -
D
b
b
b
d
n
;
d
b
a
a
g
b

C. Giordano et al. / European Journal of Medicinal Chemistry 45 (2010) 4594e4600
4599
and HPrs
CH2SO2), 3.72 (s, 3H, OCH3), 4.27 (br d, 1H, HPrs
Phe CH), 4.78 (m, 1H, Tyr CH), 5.45 (m, 1H, Tyr NH), 6.32 (m, 1H,
b
CH2), 2.84e3.33 (m, 6H, Phe
b
CH2, HPrs
d
CH2 and
(m, 1H, Tyr NH), 6.92e7.29 (m, 14H, Ar), 8.09e8.25 (m, 4H, NH2 and
two Phe NH), 9.26 (m, 1H, OH). Anal. Calcd for C38H47N5O7: C 66.55,
H 6.91, N 10.21; found: C 66.33, H 6.76, N 10.29.
aCH), 4.47 (m, 1H,
a
a
Phe NH), 6.89e7.21 (m, 10H, Ar), 9.01 (s, 1H, OH). Anal. Calcd for
C29H39N3O8S: C 59.07, H 6.67, N 7.13; found: C 59.25, H 6.58, N 7.01.
4.1.4.10. BoceTyre(S)-HPrsePheePheeNH2 (4c). Alkaline hydro-
lysis of 3c (0.271 g, 0.46 mmol) in MeOH with aq. NaOH 2.0 N
afforded the corresponding acid (white solid, 84%) which was
coupled with HePheeNH.2 HCl (0.077 g, 0.38 mmol). Triturated
4.1.4.5. BoceTyre(R)-HPrsePheeOMe (3d). Hydrogenolysis of 2d
(0.850 g, 1.84 mmol) in MeOH on 10% Pd/C in the presence of TFA
afforded the corresponding trifluoroacetate salt (white foam, 90%)
which was coupled with BoceTyreOH (0.467 g, 1.66 mmol). Puri-
with hexane. White solid (58%); [
2931, 1685 cmꢃ1 1H NMR (d6-DMSO)
1.80e2.00 (m, 4H, HPrs and CH2), 2.70e3.49 (four m, 8H, Tyr
CH2, two Phe CH2 and HPrs CH2), 3.96 (m, 2H, CH2SO2),
3.91e4.32 (four m, 4H, two Phe, HPrs and Tyr CH), 5.22 (br s, 1H,
a]
- 36ꢀ; IR
n
:3691, 3372, 3012,
D
;
d: 1.42 [s, 9H, C(CH3)3],
fied on SiO2 (DCM/MeOH 99:1). Pale yellow solid (70%); [
IR
: 3432, 3032, 3028, 1702, 1671 cmꢃ1 1H NMR
(CH3)3], 1.83 (m, 2H, HPrs CH2), 2.12e2.80 (two m, 4H, Tyr
HPrs CH2), 2.85e3.37 (m, 6H, Phe CH2, HPrs CH2 and CH2SO2),
3.75 (s, 3H, OCH3), 4.29 (br s, 1H, HPrs CH), 4.54 (m, 1H, Phe CH),
4.80 (m, 1H, Tyr CH), 5.48 (m, 1H, Tyr NH), 6.38 (m, 1H, Phe NH),
a
]
þ 6ꢀ;
b
g
D
n
;
d
: 1.34 [s, 9H, C
b
b
d
g
bCH2,
a
b
b
d
Tyr NH), 5.90e6.22 (m, 2H, two Phe NH), 6.72e7.64 (m, 14H, Ar),
9.79 (m, 1H, OH). Anal. Calcd for C37H47N5O8S: C 61.56, H 6.56, N
9.70; found: C 61.37, H 6.76, N 9.98.
a
a
a
6.90e7.24 (m, 10H, Ar), 9.07 (s, 1H, OH). Anal. Calcd for
C29H39N3O8S: C 59.07, H 6.67, N 7.13; found: C 59.13, H 6.42, N 6.98.
4.1.4.11. BoceTyre(R)-HPrsePheePheeNH2 (4d). Alkaline hydro-
lysis of 3d (0.200 g, 0.34 mmol) in MeOH with aq. NaOH 2.0 N
afforded the corresponding acid (white solid, 87%) which was
coupled with HePheeNH.2 HCl (0.059 g, 0.29 mmol). Triturated
4.1.4.6. BoceTyre(S)-bPrsePheeOMe (3e). Hydrogenolysis of 2e
(0.161 g, 0.36 mmol) in MeOH on 10% Pd/C in the presence of TFA
afforded the corresponding trifluoroacetate salt (white foam, 85%)
which was coupled with BoceTyreOH (0.086 g, 0.31 mmol). Puri-
with hexane. White solid (64%); [
2930, 1676 cmꢃ1 1H NMR (d6-DMSO)
1.84e2.03 (m, 4H, HPrs and CH2), 2.67e3.45 (four m, 8H, Tyr
CH2, two Phe CH2 and HPrs CH2), 3.89 (m, 2H, CH2SO2),
3.89e4.28 (four m, 4H, two Phe, HPrs and Tyr CH), 5.20 (br s, 1H,
a]
- 38ꢀ; IR
n
:3689, 3367, 3021,
D
;
d: 1.42 [s, 9H, C(CH3)3],
fied on SiO2 (DCM/MeOH 95:5). Colourless oil (80%); [
:3689, 3432, 3032, 3011, 1744 cmꢃ1 1H NMR
: 1.49 [s, 9H, C
(CH3)3], 2.47 (m, 2H, Prs CH2), 2.70e3.51 (m, 6H, Phe CH2, Prs
and CH2), 3.81 (s, 3H, OCH3), 4.31 (br d, 1H, Prs CH), 4.41e4.72
(m, 2H, Phe and Tyr CH), 5.22 (s, 1H, Phe NH), 5.52 (m, 1H, Tyr NH),
a
]
D
- 5ꢀ; IR
b
g
n
;
d
b
b
d
b
g
b
b
a
a
d
b
b
Tyr NH), 5.87e6.19 (m, 2H, two Phe NH), 6.78e7.59 (m, 14H, Ar),
9.84 (m, 1H, OH). Anal. Calcd for C37H47N5O8S: C 61.56, H 6.56, N
9.70; found: C 61.28, H 6.34, N 9.56.
a
7.02e7.24 (m, 10H, Ar), 9.01 (s, 1H, OH). Anal. Calcd for
C28H37N3O8S: C 58.42, H 6.48, N 7.30; found: C 58.63, H 6.39, N 7.57.
4.1.4.12. BoceTyre(S)-bPrsePheePheeNH2 (4e). Alkaline hydro-
4.1.4.7. BoceTyre(R)-
b
PrsePheeOMe (3f). Hydrogenolysis of 2f
lysis of 3e (0.109 g, 0.19 mmol) in MeOH with aq. NaOH 2.0 N
afforded the corresponding acid (pale yellow solid, 89%) which was
coupled with HePheeNH.2 HCl (0.034 g, 0.17 mmol). Triturated
(0.270 g, 0.60 mmol) in MeOH on 10% Pd/C in the presence of TFA
afforded the corresponding trifluoroacetate salt (white foam, 92%)
which was coupled with BoceTyreOH (0.156 g, 0.55 mmol). Puri-
with hexane. White solid (66%); [
1683 cmꢃ1; 1H NMR (d6-DMSO)
: 1.36 [s, 9H, C(CH3)3], 2.20e2.79
(m, 8H, Tyr CH2, two Phe CH2 and Prs CH2), 3.10e3.29 (m, 4H,
CH2), 3.51e4.72 (four m, 4H, Prs CH, two Phe and Tyr
CH), 6.32e6.54 (m, 2H, Tyr NH and NHSO2), 6.73e7.68 (m, 14H,
a] n:3689, 3032, 2337,
- 10ꢀ; IR
D
fied on SiO2 (DCM/MeOH 95:5). Colourless oil (78%); [
:3689, 3432, 3033, 3011, 1744 cmꢃ1 1H NMR
: 1.53 [s, 9H, C
(CH3)3], 2.49 (m, 2H, Prs CH2), 2.68e3.52 (m, 6H, Phe CH2, Prs
and CH2), 3.79 (s, 3H, OCH3), 4.33 (br d, 1H, Prs CH), 4.40e4.68
(m, 2H, Phe and Tyr CH), 5.20 (s, 1H, Phe NH), 5.53 (m, 1H, Tyr NH),
a
]
D
- 5ꢀ; IR
d
n
;
d
b
b
b
g
b
g
b
b
bPrs
a
and
d
b
b
a
d
b
b
a
a
Ar), 7.51e8.00 (m, 3H, Phe NH and NH2), 8.90 (br m, 1H, OH). Anal.
Calcd for C36H45N5O8S: C 61.09, H 6.41, N 9.89; found: C 61.17, H
6.29, N 9.48.
7.00e7.23 (m, 10H, Ar), 9.02 (s, 1H, OH). Anal. Calcd for
C28H37N3O8S: C 58.42, H 6.48, N 7.30; found: C 58.59, H 6.72, N 7.47.
4.1.4.8. BoceTyre(S)-HProePheePheeNH2 (4a). Alkaline hydro-
lysis of 3a (0.372 g, 0.69 mmol) in MeOH with aq. NaOH 2.0 N
afforded the corresponding acid (white solid, 72%) which was
coupled with HePheeNH.2 HCl (0.100 g, 0.50 mmol). Triturated
4.1.4.13. BoceTyre(R)-bPrsePheePheeNH2 (4f). Alkaline hydro-
lysis of 3f (0.196 g, 0.34 mmol) in MeOH with aq. NaOH 2.0 N
afforded the corresponding acid (pale yellow solid, 89%) which was
coupled with HePheeNH.2 HCl (0.061 g, 0.30 mmol). Purified on
with hexane. White solid (64%); [
2975, 1643 cmꢃ1 1H NMR (d6-DMSO)
1.75e1.80 (m, 4H, HPro and CH2), 2.61e2.95 (m, 8H, Tyr
two Phe CH2 and CH2CO), 3.12 (br s, 2H, HPro CH2), 3.73 (br s, 1H,
HPro CH), 3.91 (m, 2H, two Phe CH), 4.28 (m, 1H, Tyr CH), 6.71
a
]
- 12ꢀ; IR
n
:3684, 3295, 3029,
: 1.37 [s, 9H, C(CH3)3],
CH2,
SiO2 (DCM/MeOH 95:5). White foam (48%); [
3029, 2330, 1687 cmꢃ1; 1H NMR
: 1.34 [s, 9H, C(CH3)3], 2.23e2.84
(m, 8H, Tyr CH2, two Phe CH2 and Prs CH2), 3.12e3.33 (m, 4H,
CH2), 3.49e4.76 (four m, 4H, two Phe and Tyr CH, Prs
CH), 6.30e6.48 (m, 2H, Tyr NH and NHSO2), 6.70e7.66 (m, 14H,
a] n:3691,
- 10ꢀ; IR
D
D
;
d
d
b
g
b
b
b
b
g
b
d
bPrs
a
and
d
a
b
a
a
a
b
(m, 1H, Tyr NH), 6.90e7.22 (m, 14H, Ar), 8.01e8.22 (m, 4H, NH2 and
two Phe NH), 9.21 (m, 1H, OH). Anal. Calcd for C38H47N5O7: C 66.55,
H 6.91, N 10.21; found: C 66.27, H 7.03, N 10.35.
Ar), 7.48e7.95 (m, 3H, Phe NH and NH2), 8.92 (br m, 1H, OH). Anal.
Calcd for C36H45N5O8S: C 61.09, H 6.41, N 9.89; found: C 61.25, H
6.18, N 9.67.
4.1.4.9. BoceTyre(R)-HProePheePheeNH2 (4b). Alkaline hydro-
lysis of 3b (0.448 g, 0.81 mmol) in MeOH with aq. NaOH 2.0 N
afforded the corresponding acid (white solid, 87%) which was
coupled with HePheeNH.2 HCl (0.141 g, 0.70 mmol). Triturated
4.1.4.14. BoceTyre(S)-HProePheeProeNH2 (5a). Alkaline hydro-
lysis of 3a (0.112 g, 0.19 mmol) in MeOH with aq. NaOH 2.0 N
afforded the corresponding acid (pale yellow solid, 89%) which was
coupled with HeProeNH.2 HCl (0.026 g, 0.17 mmol). Purified on
with hexane. White solid (66%); [
2989, 1682 cmꢃ1 1H NMR (d6-DMSO)
1.72e1.81 (m, 4H, HPro and CH2), 2.59e2.91 (m, 8H, Tyr
two Phe CH2 and CH2CO), 3.14 (br s, 2H, HPro CH2), 3.70 (br s, 1H,
HPro CH), 3.89 (m, 2H, two Phe CH), 4.33 (m, 1H, Tyr CH), 6.68
a]
- 9ꢀ; IR
n
:3690, 3336, 3034,
: 1.39 [s, 9H, C(CH3)3],
CH2,
SiO2 (DCM/MeOH 95:5). White solid (56%);
n
[
a
]
D
-
12ꢀ; IR
D
;
d
:3687,3295, 2975, 2345, 1643 cmꢃ1; 1H NMR (d6-DMSO)
d: 1.32 [s,
b
g
b
9H, C(CH3)3], 1.52e2.00 (m, 4H, HPro and Pro
g
CH2), 2.70e3.02 (m,
6H, Tyr and Phe CH2),
4.12e4.81 (four m, 4H, Phe, HPro, Pro and Tyr aCH), 6.63 (m, 1H, Tyr
b
d
bCH2, CH2CO), 3.11 (m, 4H, HPro and Pro d
a
a
a

4600
C. Giordano et al. / European Journal of Medicinal Chemistry 45 (2010) 4594e4600
NH), 6.65e7.28 (m, 11H, Ar, Phe NH and ProeNH2), 9.25 (br m, 1H,
OH). Anal. Calcd for C34H45N5O7: C 64.23, H 7.13, N 11.02; found: C
64.17, H 6.98, N 11.23.
Acknowledgments
This research was supported in part by grants from the U.S.
Public Health Service, National Institutes of Health, DR 06784 and
DA 13449 (VJH).
4.1.4.15. BoceTyre(R)-HProePheeProeNH2 (5b). Alkaline hydro-
lysis of 3a (0.134 g, 0.26 mmol) in MeOH with aq. NaOH 2.0 N
afforded the corresponding acid (pale yellow solid, 83%) which was
coupled with HeProeNH.2 HCl (0.032 g, 0.21 mmol). Purified on
References
SiO2 (DCM/MeOH 95:5). White solid (51%); [
3336, 2989, 2345, 1682 cmꢃ1; 1H NMR (d6-DMSO)
(CH3)3], 1.49e1.98 (m, 4H, HPro and Pro CH2), 2.75e3.10 (m, 6H,
Tyr and Phe CH2, CH2CO), 3.09 (m, 4H, HPro and Pro CH2),
4.10e4.75 (four m, 4H, Phe, HPro, Pro and Tyr CH), 6.59 (m, 1H, Tyr
a
]
D
- 9ꢀ; IR
n
:3690,
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9706e9712.
d: 1.30 [s, 9H, C
g
b
d
a
[4] J.E. Zadina, L. Hackler, L.J. Ge, A.J. Kastin, Nature 386 (1997) 499e502.
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[7] T. Yamazaki, S. Ro, M. Goodman, N.N. Chung, P.W. Schiller, J. Med. Chem. 36
(1993) 708e719.
NH), 6.73e7.32 (m, 11H, Ar, Phe NH and ProeNH2), 9.28 (br m, 1H,
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mM bes-
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m
m
m
methylsulfonyl-fluoride (PMSF). Radioligand competition analysis
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lines that expressed either
(1.6 nM) was used to label the
k-, d- or m
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k
-opioid receptors, 1 nM [3H]DAMGO
was used to label the
used to label the
m
d
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-opioid receptors in the respective cell
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