Role of 2′,6′-dimethyl-l-tyrosine (Dmt) in some opioid lead compounds

Article abstract of DOI:10.1016/j.bmc.2010.06.073

Here we evaluated how the interchange of the amino acids 2′,6′-dimethyl-l-tyrosine (Dmt), 2′,6′-difluoro-l- tyrosine (Dft), and tyrosine in position 1 can affect the pharmacological characterization of some reference opioid peptides and pseudopeptides. Generally, Dft and Tyr provide analogues with a similar pharmacological profile, despite different pK_a values. Dmt/Tyr(Dft) replacement gives activity changes depending on the reference opioid in which the modification was made. Whereas, H-Dmt-Tic-Asp-Bid is a potent and selective δ agonist (MVD, IC₅₀ = 0.12 nM); H-Dft-Tic-Asp-Bid and H-Tyr-Tic-Asp-Bid are potent and selective δ antagonists (pA₂ = 8.95 and 8.85, respectively). When these amino acids are employed in the synthesis of deltorphin B and its Dmt¹ and Dft¹ analogues, the three compounds maintain a very similar δ agonism (MVD, IC₅₀ 0.32-0.53 nM) with a decrease in selectivity relative to the Dmt¹ analogue. In the less selective H-Dmt-Tic-Gly-Bid the replacement of Dmt with Dft and Tyr retains the δ agonism but with a decrease in potency. Antagonists containing the Dmt-Tic pharmacophore do not support the exchange of Dmt with Dft or Tyr.

Full text of DOI:10.1016/j.bmc.2010.06.073

Bioorganic & Medicinal Chemistry 18 (2010) 6024–6030
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Role of 20,60-dimethyl-L-tyrosine (Dmt) in some opioid lead compounds
b
c
c
d
Gianfranco Balboni a, , Erika Marzola , Yusuke Sasaki , Akihiro Ambo , Ewa D. Marczak ,
*
Lawrence H. Lazarus d, Severo Salvadori b,
a Department of Toxicology, University of Cagliari, I-09124 Cagliari, Italy
*
b Department of Pharmaceutical Sciences and Biotechnology Center, University of Ferrara, I-44100 Ferrara, Italy
c Department of Pharmacology, Tohoku Pharmaceutical University, 4-1, Komatsushima 4-chome, Aoba-Ku, Sendai 981-8558, Japan
d Medicinal Chemistry Group, Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA
a r t i c l e i n f o
a b s t r a c t
Here we evaluated how the interchange of the amino acids 20,60-dimethyl- -tyrosine (Dmt), 20,60-difluoro-
L
Article history:
Received 17 May 2010
Revised 16 June 2010
Accepted 20 June 2010
Available online 25 June 2010
L
-tyrosine (Dft), and tyrosine in position 1 can affect the pharmacological characterization of some refer-
ence opioid peptides and pseudopeptides. Generally, Dft and Tyr provide analogues with a similar phar-
macological profile, despite different pKa values. Dmt/Tyr(Dft) replacement gives activity changes
depending on the reference opioid in which the modification was made. Whereas, H-Dmt-Tic-Asp -Bid
*
*
*
is a potent and selective d agonist (MVD, IC50 = 0.12 nM); H-Dft-Tic-Asp -Bid and H-Tyr-Tic-Asp -Bid
are potent and selective d antagonists (pA2 = 8.95 and 8.85, respectively). When these amino acids are
employed in the synthesis of deltorphin B and its Dmt1 and Dft1 analogues, the three compounds main-
tain a very similar d agonism (MVD, IC50 0.32–0.53 nM) with a decrease in selectivity relative to the Dmt1
analogue. In the less selective H-Dmt-Tic-Gly*-Bid the replacement of Dmt with Dft and Tyr retains the d
agonism but with a decrease in potency. Antagonists containing the Dmt-Tic pharmacophore do not sup-
port the exchange of Dmt with Dft or Tyr.
Keywords:
Dmt-Tic pharmacophore
Opioid peptides
Opioid receptors
d-Opioid agonists
UFP-512
d-Opioid antagonists
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
bution nearly all research groups working in the opioid peptide field
used this amino acid in the synthesis of new and interesting opioids.
20,60-Dimethyl-
L
-tyrosine (Dmt) has become one of the most
The importance of Dmt is further documented by different synthetic
methods published or patented until now in order to obtain the
chiral amino acid in high yield avoiding the use of very expensive
chiral catalysts.4–7 Generally, structure–activity studies on a variety
of opioid ligands containing Dmt revealed important alterations in
their activities through the elevation of affinity (especially for the
widely used amino acids in the synthesis of opioid peptides and
pseudopeptides.1,2 To the best of our knowledge, Dmt was first intro-
duced in opioid peptides by Hansen et al.3 After this seminal contri-
Abbreviations: AcOEt, ethyl acetate; AcOH, acetic acid; Asp*, –NH–CH(CH2–
COOH)–; Bid, 1H-benzimidazol-2-yl; Boc, tert-butyloxycarbonyl; Bzl, benzyl;
l
receptor), modification of receptor selectivity, and change in the
spectrum of their bioactivity profile.1,2 Once the importance of the
Dmt substitution in lieu of Tyr1 was established, other Tyr/Dmt sur-
rogates were also considered in further structure–activity studies.8,9
In some cases the new analogues yielded interesting results,9 but the
use of Dmt in opioids still remains unsurpassed.
DAMGO, [
Tyr- -Ala-Phe-Glu-Val-Val-Gly-NH2; Deltorphin C or Deltorphin-II, H-Tyr-
Phe-Asp-Val-Val-Gly-NH2; Dft, 20,60-difluoro-
-tyrosine; DMF, N,N-dimethylform-
amide; DMSO-d6, hexadeuteriodimethyl sulfoxide; Dmt, 20,60-dimethyl-
-tyrosine;
DPDPE,
-Pen2, -Pen5)-enkephalin; Endomorphin-2, H-Tyr-Pro-Phe-Phe-NH2;
D
-Ala2,N-Me-Phe4,Gly-ol5]enkephalin; Deltorphin B or Deltorphin I, H-
D
D-Ala-
L
L
(D
D
Et2O, diethyl ether; Gly*, –NH–CH2–; GPI, guinea-pig ileum; HOBt, 1-hydroxyben-
zotriazole; HPLC, high performance liquid chromatography; IBCF, isobutyl chloro-
formate; MALDI-TOF, matrix assisted laser desorption ionization time-of-flight;
MVD, mouse vas deferens; NMM, 4-methylmorpholine; OBzl, benzyl ester; pA2,
negative log of the molar concentration required to double the agonist concentra-
tion to achieve the original response; PE, petroleum ether; PL017, H-Tyr-Pro-(N-
Starting from this point it is quite difficult to suggest new Tyr/
Dmt analogues able to maintain or improve the activity when in-
serted in opioid peptides or pseudopeptides. Nonetheless, we fo-
cused our attention on a new Tyr/Dmt analogue in which the 20
and 60 methyl groups are substituted by fluorine atoms (20,60-di-
Me)Phe-D-Pro-NH2; TFA, trifluoroacetic acid; Tic, 1,2,3,4-tetrahydroisoquinoline-3-
carboxylic acid; TLC, thin-layer chromatography; WSC, 1-ethyl-3-[30-dimethyl)ami-
nopropyl]-carbodiimide hydrochloride; Z, benzyloxycarbonyl.
* Corresponding authors. Tel.: +39 70 675 8625; fax: +39 70 675 8612 (G.B.); tel.:
+39 532 455 918; fax: +39 532 455 953 (S.S).
fluoro-L-tyrosine; Dft). This amino acid was selected because is
commercially available at the same costs of Dmt (RSP Amino
Acids), fluorine atoms maintain the same positions in the aromatic
ring, and finally as generally asserted by Thayer; ‘Fluorine can be
highly advantageous in pharmaceutical and agrochemical com-
pounds. One or just a few atoms in an organic molecule can
E-mail addresses: gbalboni@unica.it (G. Balboni), sal@unife.it (S. Salvadori).
Abbreviations are taken from the IUPAC-IUB Commission on Biochemical
Nomenclature (J. Biol. Chem. 1985, 260, 14).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.073

G. Balboni et al. / Bioorg. Med. Chem. 18 (2010) 6024–6030
6025
dramatically alter its chemical and biological nature, including its
stability, lipophilicity, and bioavailability’.10,11 Furthermore, in
the light of the relatively small dimensions of fluorine atoms, we
also revaluated the possibility of using Tyr in place of Dmt. The bio-
logical effects induced by Dft and Tyr were assessed by their inser-
the condensation of Z-Lys(Boc)-OH with benzylamine via WSC/
HOBt. The Z-protected intermediate was treated with catalytic
hydrogenation (10%, Pd/C) and condensed with Z-Tic-OH via
WSC/HOBt. After Tic deprotection by catalytic hydrogenation
(10%, Pd/C), the dipeptide was subsequently condensed with Boc-
Dft-OH or Boc-Tyr-OH. In final compounds (11 and 12), Boc pro-
tecting groups were simultaneously removed by TFA treatment.
N-terminal dimethylated dipeptides were obtained as reported
in Schemes 3 and 4 (Supporting Information) by condensation via
WSC/HOBt of the commercially available H-Tic-OtBu with Boc-Dft-
OH (14) or Boc-Tyr-OH (15); or H-Tic-NH2 with Boc-Dft-OH (17) or
Boc-Tyr-OH (18), respectively. After N-terminal deprotection,
dipeptides were N-dimethylated by aqueous formaldehyde and
NaBH3CN treatment in acetonitrile solution at room temperature.15
Deltorphin B (9) and its analogues (7 and 8) were prepared by
solid phase peptide synthesis using a Fmoc strategy as detailed
in Section 5.
tion in lieu of Dmt1 in some of our opioid lead compounds
12
*
containing the Dmt-Tic pharmacophore: H-Dmt-Tic-Asp -Bid
and H-Dmt-Tic-Gly*-Bid13 (d agonists); H-Dmt-Tic-Lys-NH-Bzl (
l
antagonist/weak d antagonist);14 N,N(Me)2-Dmt-Tic-OH15 (d antag-
15,16
onist) and N,N(Me)2-Dmt-Tic-NH2
(d inverse agonist). Deltor-
phin B was also modified in light of the fact that its Dmt1
analogue was previously synthesized and pharmacologically
evaluated.17
2. Chemistry
All new peptides and pseudopeptides (2, 3, 5, 6, 11, 12, 14, 15,
17 and 18) (except deltorphin B and its analogues) were prepared
in solution by the same methods we previously used for the corre-
sponding lead compounds (1, 4, 9, 10, 13 and16). Briefly, mixed car-
bonic anhydride coupling of Boc-Asp(OMe)-OH or Boc-Gly-OH
with o-phenylendiamine gave the corresponding crude intermedi-
ate monoamides, which were converted without purification to the
desired 1H-benzimidazol-2-yl (Bid) derivatives by cyclization and
dehydration in acetic acid (AcOH) as outlined in Scheme 1 (Sup-
porting Information) for analogues 2 and 3. Each Boc-protected
intermediate was treated with trifluoroacetic acid (TFA) and con-
densed with Boc-Tic-OH via WSC/HOBt. After Tic deprotection by
TFA, the pseudodipeptides were subsequently condensed with
3. Results and discussion
3.1. Receptor affinity analysis
Receptor binding data for d- and l-opioid receptors and d-selec-
l
tivity (Ki /Kid) are reported in Table 1. Interestingly, analogues of
the more highly selective d agonist reference compounds 1 and 7
maintained the same affinity for d receptors despite the replace-
ment of Dmt by Dft or Tyr. In the less selective reference d agonist
(4), the same substitutions caused a ca. 30-fold drop in d affinity.
On the other hand, Dft and Tyr when introduced in place of Dmt
Boc-Dft-OH (Dft = 20,60-difluoro-
L
-tyrosine) or Boc-Tyr-OH. In final
in non-selective (weak d antagonist/l antagonist; 10), selective d
compounds (2,3, 5 and 6), methyl esters and Boc protecting groups
were removed by basic hydrolysis (1 N NaOH) and TFA treatment,
respectively.12,13
antagonist (13) or selective d inverse agonist (16), decreased d
affinity at least of 2 orders of magnitude. The substitution of Dmt
in lieu of Tyr is generally thought to be linked with an increase
Compounds (11 and 12) were prepared essentially (Scheme 2,
in
l affinity and consequently to a loss of d selectivity. Whilst this
Supporting Information) as reported previously14 starting from
observation holds for the references d agonists (1, 4 and 7) in com-
Table 1
Receptor binding affinities and functional bioactivities of compounds 1–14
Structure
Receptor affinitya (nM)
Selectivity
Functional bioactivity
l
l
c
b
c
b
Kid
Ki
Ki /Kid
MVD pA2
MVD IC50 (nM)
GPI pA2
GPI IC50 (nM)
1
2
3
4
H-Dmt-Tic-Asp*-Bidd
H-Dft-Tic-Asp*-Bid
0.44
53.9
122
1390
3864
14
0.12
1724
0.162 0.02 (5)
0.177 0.009 (4)
0.035
225.1 36 (4)
683.9 91 (6)
0.50
8.95
8.85
H-Tyr-Tic-Asp*-Bid
e
*
H-Dmt-Tic-Gly -Bid
0.13
26.92
*
5
6
7
8
H-Dft-Tic-Gly -Bid
1.01 0.05 (3)
0.913 0.03 (3)
0.16 0.03 (4)
0.241 0.03 (3)
0.12
54.5 4.4 (5)
66.9 8.8 (6)
1.68 0.14 (3)
154.3 19 (3)
638
54
73
10
640
5317
8
7
26
20,636
20
35
1655
3
7
67.3 6.8
50.1 1.67
0.32 0.02
0.40 0.08
0.53 0.02
>10,000
*
H-Tyr-Tic-Gly -Bid
4216 544
48.2 1.76
3559 566
>10,000
[Dmt1]Deltorphin Bf
[Dft1]Deltorphin B
9
Deltorphin Bf
10
11
12
13
14
15
16
17
18
H-Dmt-Tic-Lys-NH-Bzlg
H-Dft-Tic-Lys-NH-Bzl
H-Tyr-Tic-Lys-NH-Bzl
N,N(Me)2-Dmt-Tic-OHh
N,N(Me)2-Dft-Tic-OH
N,N(Me)2-Tyr-Tic-OHh
N,N(Me)2-Dmt-Tic-NH2
N,N(Me)2-Dft-Tic-NH2
N,N(Me)2-Tyr-Tic-NH2
0.50
4.05
6.01
9.4
7.96
5.8
153.4 21 (6)
63.9 7.6 (3)
0.118
29.2 3.8 (5)
34.8 1.7 (4)
0.309
1029 83 (4)
1649 231 (6)
2435
573.3 69 (4)
1234 48.5 (3)
511.4
h
9.9
6.3
176.4 7.5 (5)
201.3 14 (4)
596.4 58 (4)
1451 25 (5)
a
The Ki values (nM) were determined according to Cheng and Prusoff.33 Means SEM with n repetitions in parenthesis is based on independent duplicate binding assays
conducted with five to eight peptide doses using several different synaptosomal preparations.
b
Agonist activity is expressed as IC50 obtained from dose–response curves. These values represent means SE for at least four tissue samples. DPDPE and PL017 were the
internal standards for MVD (d-opioid receptor bioactivity) and GPI (
l
-opioid receptor bioactivity) tissue preparation, respectively.
c
The pA2 values of opioid antagonists against the d and
l agonists (deltorphin-II and endomorphin-2, respectively) were determined by the method of Kosterlitz and
Watt.36
Data taken from Balboni et al.12
Data taken from Balboni et al.13
Affinity data taken from Guerrini et al.17
Data taken from Balboni et al.14
Data taken from Salvadori et al.15
d
e
f
g
h

6026
G. Balboni et al. / Bioorg. Med. Chem. 18 (2010) 6024–6030
parison with their corresponding analogues (2, 3; 5, 6; 8, 9), antag-
onists and the inverse agonist revealed a different behaviour. In
fact, whereas reference (10) and its analogues exhibited only a
minimal variation in d selectivity, references (13, 16) and their ana-
logues (14, 15; 17, 18) yielded the opposite receptor binding
behaviour; namely, the compounds containing Dmt were far more
d selective than the analogues containing either Dft or Tyr. This
remarkable differential profile was essentially due to a loss in d
affinity by more than 2 orders of magnitude.
antagonists (2, MVD, pA2 = 8.95; 3, MVD, pA2 = 8.85, respectively);
the same modification made with the less selective d agonist (4)
carries to less potent d agonists. Deltorphin B (9) supports the same
degree of d agonism in spite of the amino acid introduced in position
1 (Dmt, Dft or Tyr) the only variation concerns the increase of laffin-
ity and functional bioactivity induced by Dmt in comparison to Dft
or Tyr.
Antagonists (11, 12, 14, 15, 17 and 18) listed in Table 1 do not
tolerate such a change, but in other compounds it is well tolerated;
such as the pair formed by the d antagonist H-Tyr-Tic-Phe-Phe-
NH2 and the
l
agonist/d antagonist H-Dmt-Tic-Phe-Phe-NH2.26
3.2. Functional bioactivity
Furthermore, H-Tyr-Tic-Phe-Phe-OH, H-Dmt-Tic-Phe-Phe-OH, H-
Dbcp-Tic-Phe-Phe-OH; H-Cdp-Tic-Phe-Phe-OH have d antagonist
activities with Ke values ranging from 0.196 to 4.80 nM, whereas
H-Bcp-Tic-Phe-Phe-OH is a d agonist (IC50 = 3.42 nM).9 In conclu-
sion, we should recall that the history of Tyr/Dmt substitution
started from the couple of d selective antagonist dipeptides H-
All newly synthesized compounds were evaluated in the electri-
cal stimulated MVD and GPI pharmacological assays for intrinsic
functional bioactivity (Table 1). Interestingly and totally unexpect-
*
edly, in the d-opioid agonist H-Dmt-Tic-Asp -Bid (1; coded UFP-
512) the substitution of Dmt1 with Dft or Tyr reverted its activity
from a potent and selective d agonism (IC50 = 0.12 nM) into potent
and selective d antagonism (pA2 = 8.95 and 8.85, respectively). The
27
28
Tyr-Tic-OH/NH2 and H-Dmt-Tic-OH/NH2 initially reported by
us and later by Schiller and co-workers,9 in which the difference
in activity induced by Dmt aroused great interest.
*
same substitution in the less selective d agonist H-Dmt-Tic-Gly -
In summary, especially working in the field of d selective opioid
peptide/pseudopeptide ligands, besides to consider the insertion of
expensive or difficult to prepare Tyr/Phe analogues in position 1, it
could also be helpful to prepare the corresponding analogue con-
taining the inexpensive Tyr in position 1, it could raise interesting
surprises.
Bid (MVD IC50 = 0.13 nM; GPI IC50 = 26.92 nM) yielded less active
d agonists [MVD (5) IC50 = 67.3 nM; (6) IC50 = 50.1 nM]. Further-
more, analogues (7 and 8) of the d selective agonist deltorphin B
(9) maintained the same degree of
d agonism [MVD (7)
IC50 = 0.32 nM; (8) IC50 = 0.40 nM; (9) IC50 = 0.53 nM], but with a
loss of d selectivity relative to the Dmt1 analogue (7). Derivatives
(11, 12), (14, 15) and (17, 18) of the corresponding reference antag-
onists (10), (13) and (16), respectively, on the basis of their poor
5. Experimental
5.1. Chemistry
affinities for d and
l receptors were not assessed for functional
bioactivities.
5.1.1. General methods
4. Conclusion
Crude peptides and pseudopeptides were purified by prepara-
tive reversed-phase HPLC [Waters Delta Prep 4000 system with
Waters Prep LC 40 mm Assembly column C18 (30 cm ꢀ 4 cm,
To shed more light in the structure–activity relationship of opi-
oid peptides and pseudopeptides, we synthesized analogues in
which 20,60-dimethyl-
L
-tyrosine in position 1 was substituted by
-tyrosine (Dft) or -tyrosine. From this study some
15 lm particle)] and eluted at a flow rate of 20 mL/min with mo-
20,60-difluoro-
L
L
bile phase solvent A (10% acetonitrile + 0.1% TFA in H2O, v/v), and
a linear gradient from 10% to 60% B (60%, acetonitrile + 0.1% TFA
in H2O, v/v) in 25 min. Analytical HPLC analyses were performed
with a Beckman System Gold (Beckman ultrasphere ODS column,
interesting observations can be drawn: Tyr and Dft seem to show
a very similar behaviour when inserted in position 1 of opioid pep-
tides and pseudopeptides despite their very different pKa values
(Dft has a pKa of 8.1, about 2 U lower than Tyr).18 To give more com-
plexity, if possible, to the SAR related to the d agonist containing the
Dmt-Tic pharmacophore (1 and 4), here we complete the demon-
stration that all parts of the compounds can be altered resulting in
different activities. For example, alkylation of 1H-benzimidazol-2-
yl (Bid) at N1 yields only d antagonists19–21; substitution of Bid with
other aromatic nuclei (benzoxazol-2-y; benzothiazol-2-yl; 1H-in-
dol-2-yl; 4-phenyl-1H-imidazol-2-yl) generally furnish d antago-
nists, but occasionally d agonism can be maintained (1H-imidazol-
250 mm ꢀ 4.6 mm, 5
lm particle). Analytical determinations and
capacity factor (K0) of the products used HPLC in solvents A and B
programmed at flow rate of 1 mL/min with linear gradients from
0% to 100% B in 25 min. Analogues had less than 5% impurities at
220 and 254 nm.
TLC was performed on precoated plates of silica gel F254
(Merck, Darmstadt, Germany): (A) 1-butanol/AcOH/H2O (3:1:1, v/
v/v); (B) CH2Cl2/toluene/methanol (17:1:2). Ninhydrin (1% ethanol,
Merck), fluorescamine (Hoffman-La Roche) and chlorine spray re-
agents. Melting points were determined on a Kofler apparatus
and are uncorrected. Optical rotations were assessed at 10 mg/
mL in methanol with a Perkin-Elmer 241 polarimeter in a 10 cm
water-jacketed cell. Molecular weights of the compounds were
determined by a MALDI-TOF analysis (Hewlett Packard G2025A
2-yl).22 The portion –Gly -Bid can be replaced by –Gly-NH-Ph or
*
–Gly-NH-Bzl resulting in a dual
l agonist/d agonist or a l agonist/
d antagonist, respectively.13 Again, if we consider the replacement
of the benzyl group with surrogates or the introduction of substitu-
ents in the para position, its pharmacological profile further
changes.23 Furthermore, the portion –Asp*-Bid can be replaced by
–Asp-NH-Ph or –Asp-NH-Bzl giving d antagonists in both cases.20
The side chain next to Bid can be altered to obtain a variety of differ-
ent activities, for example, Gly, Ala and Asp side-chains result in d
agonism; Phe side chain results in d antagonism, and the side chains
LD-TOF system mass spectrometer) and
a-cyano-4-hydroxycin-
namic acid as a matrix. 1H NMR (d) spectra were measured, when
not specified, in DMSO-d6 solution using a Bruker AC-200 spec-
trometer, and peak positions are given in parts per million down-
field from tetramethylsilane as internal standard. The purity of
tested compounds was determined by combustion elemental anal-
yses conducted by the Microanalytical Laboratory of the Chemistry
Department, University of Ferrara, with a Yanagimoto MT-5 CHN
recorder elemental analyser. All tested compounds possess a purity
of at least 95% of the theoretical values.
of the other more representing amino acids yield
stitution of Tic2 by 4-amino-1,2,4,5-tetrahydrobenzo[c]-azepin-3-
one25 or agonism.23 Finally,
-Tic yields compounds endowed with
in the highly selective agonist (1) (MVD, IC50 = 0.12 nM),
replacement of Dmt1 by Dft or Tyr provides potent and selective d
l
agonism.24 Sub-
D
l
d

G. Balboni et al. / Bioorg. Med. Chem. 18 (2010) 6024–6030
6027
5.2. Synthesis
NMM (0.06 mL, 0.54 mmol), HOBt (0.05 g, 0.30 mmol), and WSC
(0.06 g, 0.30 mmol) were added. The reaction mixture was stirred
for 3 h at 0 °C and 24 h at room temperature. After DMF was evap-
orated, the residue was dissolved in AcOEt and washed with NaH-
CO3 (5% in H2O), and brine. The organic phase was dried (Na2SO4)
and evaporated to dryness. The residue was precipitated from
Et2O/PE (1:9, v/v): yield 0.16 g (88%); Rf (B) 0.91; HPLC K0 8.94;
5.2.1. tert-Butyl (S)-2-(methoxycarbonyl)-1-(1H-
benzo[d]imidazol-2-yl)ethylcarbamate [Boc-Asp(OMe)*-Bid]
(Asterisk represents an abbreviation as suggested by Maekawa
and Ohtani.29) A solution of Boc-Asp(OMe)-OH (1 g, 4.05 mmol)
and NMM (0.4 mL, 4.05 mmol) in DMF (10 mL) was treated at
ꢁ20 °C with IBCF (0.5 mL, 4.05 mmol). After 10 min at ꢁ20 °C, o-
phenylendiamine (0.44 g, 4.05 mmol) was added. The reaction mix-
ture was allowed to stir whilst slowly warming to room temperature
(1 h) and was then stirred for 3 h. The solvent was evaporated, and
the residue was partitioned between AcOEt and H2O. The AcOEt
layer was washed with NaHCO3 (5% in H2O) and brine and dried over
Na2SO4. The solution was filtered, the solvent was evaporated, and
the residual solid was dissolved in glacial acetic acid (10 mL). The
solution was heated at 60 °C for 1 h. After the solvent was evapo-
rated and the residue was precipitated from Et2O/PE (1:9, v/v): yield
mp 162–164 °C; ½a D20
ꢂ
+6.3; m/z 679 (M+H)+; 1H NMR (DMSO-d6)
d 1.38–1.42 (d, 9H), 2.67–3.17 (m, 6H), 3.67 (s, 3H), 4.41–4.51
(m, 2H), 4.92–5.51 (m, 3H), 6.16 (s, 2H), 6.96–7.70 (m, 8H).
5.2.7. Boc-Dft-Tic-Asp*-Bid
*
To a solution of Boc-Dft-Tic-Asp(OMe) -Bid (0.13 g, 0.19 mmol)
in methanol (10 mL) at room temperature, 1 N NaOH (0.38 mL,
0.38 mmol) was added. The reaction mixture was stirred for 3 h
at room temperature. After methanol was evaporated, the residue
was dissolved in solvent B (60% acetonitrile + 0.1% TFA in H2O, v/v)
and directly lyophilized. A small amount were purified by prepara-
tive HPLC for characterization: yield 0.12 g (93%); Rf (B) 0.84; HPLC
1.07 g (83%); Rf (B) 0.45; HPLC K0 6.52; mp 129–131 °C; ½a 2D0
ꢂ
+16.4;
m/z 320 (M+H)+; 1H NMR (DMSO-d6) d 1.40 (s, 9H), 2.67–2.92 (m,
2H), 3.67 (s, 3H), 5.48–5.53 (m, 1H), 7.26–7.70 (m, 4H).
K0 8.54; mp 168–170 °C; ½a 2D0
ꢂ
+7.4; m/z 665 (M+H)+; 1H NMR
(DMSO-d6) d 1.38–1.42 (d, 9H), 2.67–3.17 (m, 6H), 4.41–4.51 (m,
*
5.2.2. 2TFAꢃH-Asp(OMe) -Bid
2H), 4.92–5.20 (m, 3H), 6.16 (s, 2H), 6.96–7.70 (m, 8H).
Boc-Asp(OMe)*-Bid (1.02 g, 3.2 mmol) was treated with TFA
(2 mL) for 0.5 h at room temperature. Et2O/PE (1:1, v/v) were
added to the solution until the product precipitated: yield 1.33 g
*
5.2.8. 2TFAꢃH-Dft-Tic-Asp -Bid (2)
Boc-Dft-Tic-Asp*-Bid (0.09 g, 0.14 mmol) was treated with TFA
(1 mL) for 0.5 h at room temperature. Et2O/PE (1:1, v/v) were
added to the solution until the product precipitated: yield 0.10 g
(93%); Rf (A) 0.46; HPLC K0 5.20; mp 137–139 °C; ½a 2D0
ꢂ
+18.1; m/z
220 (M+H)+; 1H NMR (DMSO-d6) d 2.67–2.92 (m, 2H), 3.67 (s,
3H), 4.51–4.56 (m, 1H), 7.26–7.70 (m, 4H).
(94%); Rf (A) 0.63; HPLC K0 7.12; mp 172–154 °C; ½a 2D0
ꢂ
+8.6; m/z
565 (M+H)+; 1H NMR (DMSO-d6) d 2.65–3.17 (m, 6H), 3.91–3.99
(m, 1H), 4.41–4.51 (m, 2H), 4.92–5.20 (m, 2H), 6.16 (s, 2H), 6.96–
7.70 (m, 8H). Anal. Calcd for C33H29F8N5O9: C; H; N.
*
5.2.3. Boc-Tic-Asp(OMe) -Bid
To a solution of Boc-Tic-OH (0.79 g, 2.86 mmol) and 2TFAꢃH-
*
Asp(OMe) -Bid (1.28 g, 2.86 mmol) in DMF (10 mL) at 0 °C, NMM
(0.62 mL, 5.72 mmol), HOBt (0.48 g, 3.15 mmol), and WSC (0.60 g,
3.15 mmol) were added. The reaction mixture was stirred for 3 h
at 0 °C and 24 h at room temperature. After DMF was evaporated,
the residue was dissolved in AcOEt and washed with NaHCO3 (5%
in H2O), and brine. The organic phase was dried (Na2SO4) and evap-
orated to dryness. The residue was precipitated from Et2O/PE (1:9,
v/v): yield 1.18 g (86%); Rf (B) 0.88; HPLC K0 8.14; mp 149–151 °C;
5.2.9. Boc-Tyr-Tic-Asp(OMe)*-Bid
This intermediate was obtained by condensation of Boc-Tyr-OH
with 2TFAꢃH-Tic-Asp(OMe)*-Bid via WSC/HOBt as reported for
*
Boc-Dft-Tic-Asp(OMe) -Bid: yield 0.21 g (86%); Rf (B) 0.64; HPLC
K0 6.28; mp 143–145 °C; ½a 2D0
ꢂ
+5.8; m/z 643 (M+H)+; 1H NMR
(DMSO-d6) d 1.38–1.42 (d, 9H), 2.67–3.17 (m, 6H), 3.67 (s, 3H),
4.41–4.51 (m, 2H), 4.92–5.51 (m, 3H), 6.68–7.70 (m, 12H).
½
a 2D0 +12.2; m/z 480 (M+H)+; 1H NMR (DMSO-d6) d 1.38–1.42 (d,
ꢂ
9H), 2.67–3.17 (m, 4H), 3.67 (s, 3H), 4.17–4.27 (m, 2H), 4.92–
5.2.10. Boc-Tyr-Tic-Asp*-Bid
*
5.51 (m, 2H), 6.96–7.70 (m, 8H).
Boc-Tyr-Tic-Asp(OMe) -Bid was treated with 1 N NaOH as re-
ported for Boc-Dft-Tic-Asp*-Bid: yield 0.13 g (94%); Rf (B) 0.58;
*
5.2.4. 2TFAꢃH-Tic-Asp(OMe) -Bid
HPLC K0 5.85; mp 150–152 °C; ½a 2D0
ꢂ
+6.2; m/z 629 (M+H)+; 1H
NMR (DMSO-d6) d 1.38–1.42 (d, 9H), 2.67–3.17 (m, 6H), 4.41–
Boc-Tic-Asp(OMe)*-Bid (1.13 g, 2.36 mmol) was treated with
TFA (1.5 mL) for 0.5 h at room temperature. Et2O/PE (1:1, v/v) were
added to the solution until the product precipitated: yield 1.29 g
4.51 (m, 2H), 4.92–5.20 (m, 3H), 6.68–7.70 (m, 12H).
(90%); Rf (A) 0.57; HPLC K0 6.48; mp 145–147 °C; ½a 2D0
ꢂ
+13.1; m/z
5.2.11. 2TFAꢃH-Tyr-Tic-Asp -Bid (3)
*
379 (M+H)+; 1H NMR (DMSO-d6) d 2.67–3.17 (m, 4H), 3.67–3.95
Boc-Tyr-Tic-Asp -Bid was treated with TFA as reported for
*
(m, 6H), 5.47–5.52 (m, 1H), 6.96–7.70 (m, 8H).
2TFAꢃH-Dft-Tic-Asp*-Bid: yield 0.09 g (94%); Rf (A) 0.35; HPLC K0
4.01; mp 165–167 °C; ½a D20
ꢂ
+7.5; m/z 529 (M+H)+; 1H NMR
(DMSO-d6) d 2.65–3.17 (m, 6H), 3.91–3.99 (m, 1H), 4.41–4.51 (m,
5.2.5. Boc-Dft-OH
To a solution of H-Dft-OH (0.5 g, 2.3 mmol) in tBuOH/H2O (2:1,
15 mL), 1 N NaOH (2.3 mL, 2.3 mmol) and di-tert-butyl dicarbonate
(0.55 g, 2.53 mmol) at 0 °C, were added. The reaction mixture was
stirred for 3 h at 0 °C and 24 h at room temperature. H2O (20 mL)
and solid citric acid (5 g) were added. The product was extracted
with AcOEt (100 mL), dried (Na2SO4), and evaporated to dryness.
The residue was precipitated from Et2O/PE (1:9, v/v): yield 0.66 g
2H), 4.92–5.20 (m, 2H), 6.68–7.70 (m, 12H). Anal. Calcd for
C33H31F6N5O9: C; H; N.
5.2.12. Boc-Dft-Tic-Gly*-Bid
This intermediate was obtained by condensation of Boc-Dft-OH
with 2TFAꢃH-Tic-Gly*-Bid13 via WSC/HOBt as reported for Boc-Dft-
0
*
Tic-Asp(OMe) -Bid: yield 0.15 g (85%); Rf (B) 0.65; HPLC K 6.34;
(91%); Rf (B) 0.27; HPLC K0 6.76; mp 148–150 °C; ½a 2D0
ꢂ
ꢁ5.02; m/z
mp 149–151 °C; ½a D20
ꢂ
+5.1; m/z 607 (M+H)+; 1H NMR (DMSO-d6)
d 1.38–1.42 (d, 9H), 2.92–3.17 (m, 4H), 4.41–4.51 (m, 4H), 4.85–
317 (M+H)+; 1H NMR (DMSO-d6) d 1.40 (s, 9H), 2.91–3.16 (dd,
2H), 4.72–4.93 (m, 1H), 6.16 (s, 2H).
4.96 (m, 2H), 6.16 (s, 2H), 6.96–7.70 (m, 8H).
5.2.6. Boc-Dft-Tic-Asp(OMe)*-Bid
5.2.13. 2TFAꢃH-Dft-Tic-Gly*-Bid (5)
*
To a solution of Boc-Dft-OH (0.09 g, 0.27 mmol) and 2TFAꢃH-
Boc-Dft-Tic-Gly -Bid was treated with TFA as reported for
Tic-Asp(OMe)*-Bid (0.16 g, 0.27 mmol) in DMF (10 mL) at 0 °C,
2TFAꢃH-Dft-Tic-Asp*-Bid: yield 0.11 g (94%); Rf (A) 0.37; HPLC K0

6028
G. Balboni et al. / Bioorg. Med. Chem. 18 (2010) 6024–6030
4.19; mp 158–160 °C; ½a D20
ꢂ
ꢁ4.3; m/z 507 (M+H)+; 1H NMR (DMSO-
5.2.20. Boc-Dft-Tic-Lys(Boc)-NH-Bzl
d6) d 2.92–3.17 (m, 4H), 3.91–3.99 (m, 1H), 4.41–4.92 (m, 5H), 6.16
To a solution of Boc-Dft-OH (0.1 g, 0.32 mmol) and H-Tic-Lys-
(Boc)-NH-Bzl (0.16 g, 0.32 mmol) in DMF (10 mL) at 0 °C, HOBt
(0.05 g, 0.35 mmol), and WSC (0.07 g, 0.35 mmol) were added.
The reaction mixture was stirred for 3 h at 0 °C and 24 h at room
temperature. After DMF was evaporated, the residue was dissolved
in AcOEt and washed with citric acid (10% in H2O), NaHCO3 (5% in
H2O), and brine. The organic phase was dried (Na2SO4) and evapo-
rated to dryness. The residue was precipitated from Et2O/PE (1:9,
v/v): yield 0.23 g (90%); Rf (B) 0.93; HPLC K0 8.76; mp 138–
(s, 2H), 6.96–7.70 (m, 8H). Anal. Calcd for C31H27F8N5O7: C; H; N.
*
5.2.14. Boc-Tyr-Tic-Gly -Bid
This intermediate was obtained by condensation of Boc-Tyr-OH
with 2TFAꢃH-Tic-Gly -Bid13 via WSC/HOBt as reported for Boc-Dft-
*
Tic-Asp(OMe)*-Bid: yield 0.28 g (87%); Rf (B) 0.61; HPLC K0 5.98;
mp 142–144 °C; ½a D20
ꢂ
+6.2; m/z 571 (M+H)+; 1H NMR (DMSO-d6)
d 1.38–1.42 (d, 9H), 2.92–3.17 (m, 4H), 4.41–4.51 (m, 4H), 4.85–
4.96 (m, 2H), 6.68–7.70 (m, 12H).
140 °C; ½a 2D0
ꢂ
ꢁ19.1; m/z 795 (M+H)+; 1H NMR (DMSO-d6) d 1.29–
1.79 (m, 24H), 2.92–3.17 (m, 6H), 4.41–4.53 (m, 5H), 4.88–4.95
(m, 2H), 6.16 (s, 2H), 6.96–7.14 (m, 9H).
*
5.2.15. 2TFAꢃH-Tyr-Tic-Gly -Bid (6)
Boc-Tyr-Tic-Gly*-Bid was treated with TFA as reported for
2TFAꢃH-Dft-Tic-Asp*-Bid: yield 0.18 g (95%); Rf (A) 0.32; HPLC K0
5.2.21. 2TFAꢃH-Dft-Tic-Lys-NH-Bzl (11)
3.57; mp 151–153 °C; ½a D20
ꢂ
ꢁ6.1; m/z 471 (M+H)+; 1H NMR
Boc-Dft-Tic-Lys(Boc)-NH-Bzl (0.18 g, 0.23 mmol) was treated
with TFA (1 mL) for 0.5 h at room temperature. Et2O/PE (1:1, v/v)
were added to the solution until the product precipitated: yield
(DMSO-d6) d 2.92–3.17 (m, 4H), 3.91–3.99 (m, 1H), 4.41–4.92 (m,
5H), 6.68–7.70 (m, 12H). Anal. Calcd for C31H29F6N5O7: C; H; N.
0.17 g (92%); Rf (A) 0.41; HPLC K0 5.19; mp 152–154 °C; ½a 2D0
ꢂ
5.2.16. Z-Lys(Boc)-NH-Bzl
ꢁ17.1; m/z 595 (M+H)+; 1H NMR (DMSO-d6) d 1.29–1.79 (m, 6H),
2.65–3.17 (m, 6H), 3.95–4.53 (m, 6H), 4.90–4.94 (m, 1H), 6.16 (s,
2H), 6.96–7.14 (m, 9H). Anal. Calcd for C36H39F8N5O8: C; H; N.
To a solution of Z-Lys(Boc)-OH (1 g, 2.63 mmol) and benzyl-
amine (0.29 mL, 2.63 mmol) in DMF (10 mL) at 0 °C, HOBt
(0.44 g, 2.89 mmol), and WSC (0.56 g, 2.89 mmol) were added.
The reaction mixture was stirred for 3 h at 0 °C and 24 h at room
temperature. After DMF was evaporated, the residue was dissolved
in AcOEt and washed with citric acid (10% in H2O), NaHCO3 (5% in
H2O), and brine. The organic phase was dried (Na2SO4) and evapo-
rated to dryness. The residue was precipitated from Et2O/PE (1:9,
v/v): yield 1.09 g (88%); Rf (B) 0.89; HPLC K0 7.99; mp 104–
5.2.22. Boc-Tyr-Tic-Lys(Boc)-NH-Bzl
This intermediate was obtained by condensation of Boc-Tyr-OH
with H-Tic-Lys(Boc)-NH-Bzl via WSC/HOBt as reported for Boc-Dft-
Tic-Lys(Boc)-NH-Bzl: yield 0.25 g (88%); Rf (B) 0.87; HPLC K0 8.32;
mp 132–134 °C; ½a D20
ꢂ
ꢁ20.3; m/z 759 (M+H)+; 1H NMR (DMSO-
d6) d 1.29–1.79 (m, 24H), 2.92–3.17 (m, 6H), 4.41–4.53 (m, 5H),
106 °C; ½a 2D0
ꢂ
ꢁ11.8; m/z 471 (M+H)+; 1H NMR (DMSO-d6) d 1.29–
4.88–4.95 (m, 2H), 6.68–7.14 (m, 13H).
1.79 (m, 15H), 2.92–2.99 (t, 2H), 4.46–4.53 (m, 3H), 5.34 (s, 2H),
7.06–7.19 (m, 10H).
5.2.23. 2TFAꢃH-Tyr-Tic-Lys-NH-Bzl (12)
Boc-Tyr-Tic-Lys(Boc)-NH-Bzl was treated with TFA as reported
for 2TFAꢃH-Dft-Tic-Lys-NH-Bzl: yield 0.19 g (92%); Rf (A) 0.35;
5.2.17. H-Lys(Boc)-NH-Bzl
To a solution of Z-Lys(Boc)-NH-Bzl (1.04 g, 2.21 mmol) in meth-
anol (30 mL) was added Pd/C (10%, 0.1 g), and H2 was bubbled for
1 h at room temperature. After filtration, the solution was evapo-
rated to dryness. The residue was precipitated from Et2O/PE (1:9,
v/v): yield 0.62 g (84%); Rf (A) 0.64; HPLC K0 5.56; mp 104–
HPLC K0 4.36; mp 147–149 °C; ½a 2D0
ꢂ
ꢁ18.9; m/z 559 (M+H)+; 1H
NMR (DMSO-d6) d 1.29–1.79 (m, 6H), 2.65–3.17 (m, 6H), 3.95–
4.53 (m, 6H), 4.90–4.94 (m, 1H), 6.68–7.14 (m, 13H). Anal. Calcd
for C36H41F6N5O8: C; H; N.
106 °C; ½a 2D0
ꢂ
ꢁ13.2; m/z 336 (M+H)+; 1H NMR (DMSO-d6) d 1.29–
5.2.24. Boc-Dft-Tic-OtBu
1.79 (m, 15H), 2.92–2.99 (t, 2H), 3.56–4.46 (m, 3H), 7.06–7.14
(m, 5H).
To a solution of Boc-Dft-OH (0.11 g, 0.34 mmol) and H-Tic-OtBu
(0.08 g, 0.34 mmol) in DMF (10 mL) at 0 °C, HOBt (0.06 g,
0.37 mmol), and WSC (0.07 g, 0.37 mmol) were added. The reaction
mixture was stirred for 3 h at 0 °C and 24 h at room temperature.
After DMF was evaporated, the residue was dissolved in AcOEt
and washed with citric acid (10% in H2O), NaHCO3 (5% in H2O),
and brine. The organic phase was dried (Na2SO4) and evaporated
to dryness. The residue was precipitated from Et2O/PE (1:9, v/v):
yield 0.15 g (84%); Rf (B) 0.76; HPLC K0 8.12; mp 112–115 °C;
5.2.18. Z-Tic-Lys(Boc)-NH-Bzl
To a solution of Z-Tic-OH (0.53 g, 1.70 mmol) and H-Lys(Boc)-
NH-Bzl (0.57 g, 1.70 mmol) in DMF (10 mL) at 0 °C, HOBt (0.29 g,
1.87 mmol), and WSC (0.36 g, 1.87 mmol) were added. The reac-
tion mixture was stirred for 3 h at 0 °C and 24 h at room tempera-
ture. After DMF was evaporated, the residue was dissolved in
AcOEt and washed with citric acid (10% in H2O), NaHCO3 (5% in
H2O), and brine. The organic phase was dried (Na2SO4) and evapo-
rated to dryness. The residue was precipitated from Et2O/PE (1:9,
v/v): yield 0.93 g (87%); Rf (B) 0.92; HPLC K0 8.56; mp 110–
½
a 2D0 +11.3; m/z 534 (M+H)+; 1H NMR (DMSO-d6) d 1.29 (s, 9H),
1.38 (s, 9H), 2.92–3.29 (m, 4H), 4.41–4.51 (m, 2H), 4.81–4.92 (m,
ꢂ
2H), 6.16 (s, 2H), 6.96–7.02 (m, 4H).
112 °C; ½a 2D0
ꢂ
ꢁ17.9; m/z 630 (M+H)+; 1H NMR (DMSO-d6) d 1.29–
5.2.25. TFAꢃH-Dft-Tic-OH
1.79 (m, 15H), 2.92–3.17 (m, 4H), 4.17–4.53 (m, 5H), 4.92–5.34
(m, 3H), 7.06–7.19 (m, 14H).
Boc-Dft-Tic-OtBu (0.12 g, 0.23 mmol) was treated with TFA
(1 mL) for 0.5 h at room temperature. Et2O/PE (1:1, v/v) were
added to the solution until the product precipitated: yield 0.1 g
5.2.19. H-Tic-Lys(Boc)-NH-Bzl
(93%); Rf (A) 0.57; HPLC K0 3.13; mp 141–143 °C; ½a 2D0
ꢂ
+16.3; m/z
To a solution of Z-Tic-Lys(Boc)-NH-Bzl (0.88 g, 1.40 mmol) in
methanol (30 mL) was added Pd/C (10%, 0.09 g), and H2 was bub-
bled for 1 h at room temperature. After filtration, the solution
was evaporated to dryness. The residue was precipitated from
Et2O/PE (1:9, v/v): yield 0.59 g (85%); Rf (A) 0.68; HPLC K0 6.72;
377 (M+H)+; 1H NMR (DMSO-d6) d 2.91–3.17 (m, 4H), 3.92–3.98
(m, 1H), 4.41–4.85 (m, 3H), 6.16 (s, 2H), 6.96–7.02 (m, 4H).
5.2.26. TFAꢃN,N(Me)2-Dft-Tic-OH (14)
To a stirred solution of TFAꢃH-Dft-Tic-OH (0.07 g, 0.14 mmol) in
acetonitrile/H2O (1:1, v/v; 5 mL) at room temperature, NMM
(0.03 mL, 0.28 mmol), 37% aqueous formaldehyde (0.1 mL,
1.4 mmol), and NaBH3CN (0.03 g, 0.42 mmol) were added. Acetic
mp 123–125 °C; ½a D20
ꢂ
ꢁ18.5; m/z 496 (M+H)+; 1H NMR (DMSO-
d6) d 1.29–1.79 (m, 15H), 2.92–3.17 (m, 4H), 3.76–3.95 (m, 3H),
4.46–4.53 (m, 3H), 6.96–7.14 (m, 9H).

G. Balboni et al. / Bioorg. Med. Chem. 18 (2010) 6024–6030
6029
acid (0.04 mL) was added over 10 min and the reaction was stirred
for 15 min. The reaction mixture was acidified with TFA (0.1 mL)
and directly purified by reverse phase preparative HPLC: yield
Deltorphin B (9) and its analogues (7 and 8) were prepared
by solid phase peptide synthesis. Fmoc Rink Amide resin
(0.69 mmol/g, 0.2 g) was treated with 40% piperidine in DMF and
linked with Fmoc-Gly-OH using DIPCI/HOBt as coupling reagent.
The following protected amino acids were sequentially coupled
to the growing peptide chain: Fmoc-Val-OH, Fmoc-Val-OH, Fmoc-
0.07 g (94%); Rf (A) 0.59; HPLC K0 3.23; mp 149–151 °C; ½a 2D0
ꢂ
+24.3; m/z 405 (M+H)+; 1H NMR (DMSO-d6) d 2.27 (s, 6H), 2.77–
3.16 (m, 4H), 3.95–4.51 (m, 3H), 4.82–4.88 (m, 1H), 6.16 (s, 2H),
6.96–7.02 (m, 4H). Anal. Calcd for C23H23F5N2O6: C; H; N.
Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-D-Ala-OH and Boc-Dmt-OH
or Boc-Dft-OH or Boc-Tyr-OH. Protected amino acids were reacted
in a fourfold excess using DIPCI/HOBt (fourfold excess) for 1 h of
coupling. To improve the purity of the final crude peptides, capping
with acetic anhydride (0.5 M/DMF) in the presence of NMM
(0.25 M/DMF) (3:1 v/v; 2 mL/0.2 g resin) was performed at any
step. Fmoc protecting group was removed at every step by 40%
piperidine/DMF treatment for 25 min. Each protected peptide
linked to the resin, after washing with methanol and drying in va-
cuo, was fully deprotected and removed from the resin by treat-
ment with a mixture containing TFA/triethylsilane/H2O (10 mL;
9:0.5:0.5) for 1.5 h at room temperature. The resin was filtered
and the solution was evaporated in vacuo to yield an oil that was
triturated with diethyl ether and collected by centrifugation. Each
crude peptide was purified by reverse phase preparative HPLC.
5.2.27. Boc-Dft-Tic-NH2
To a solution of Boc-Dft-OH (0.1 g, 0.32 mmol) and TFAꢃH-Tic-
NH2 (0.09 g, 0.32 mmol) in DMF (10 mL) at 0 °C, NMM (0.03 mL,
0.32 mmol), HOBt (0.05 g, 0.35 mmol), and WSC (0.07 g,
0.35 mmol) were added. The reaction mixture was stirred for 3 h
at 0 °C and 24 h at room temperature. After DMF was evaporated,
the residue was dissolved in AcOEt and washed with citric acid
(10% in H2O), NaHCO3 (5% in H2O), and brine. The organic phase
was dried (Na2SO4) and evaporated to dryness. The residue was
precipitated from Et2O/PE (1:9, v/v): yield 0.14 g (89%); Rf (B)
0.63; HPLC K0 7.54; mp 121–123 °C; ½a 2D0
ꢂ
+15.4; m/z 476 (M+H)+;
1H NMR (DMSO-d6) d 1.38 (s, 9H), 2.92–3.17 (m, 4H), 4.41–4.51
(m, 2H), 4.85–4.94 (m, 2H), 6.16 (s, 2H), 6.96–7.02 (m, 4H).
5.2.28. TFAꢃH-Dft-Tic-NH2
5.2.33. TFAꢃ[Dft1]deltorphin B (8)
Boc-Dft-Tic-NH2 (0.11 g, 0.23 mmol) was treated with TFA
(1 mL) for 0.5 h at room temperature. Et2O/Pe (1:1, v/v) were
added to the solution until the product precipitated: yield 0.08 g
Estimated yield 0.05 g (39%); Rf (A) 0.67; HPLC K0 4.39; mp
176–178 °C; ½a 2D0
ꢂ
ꢁ22.5; m/z 820 (M+H)+; 1H NMR (DMSO-d6) d
1.01–1.48 (m, 15H), 2.06–2.23 (m, 4H), 2.68–3.17 (m, 6H), 3.95–
4.09 (m, 3H), 4.52–4.92 (m, 5H), 6.16 (s, 2H), 7.08–7.21 (m, 5H).
Anal. Calcd for C40H53F5N8O12: C; H; N.
(94%); Rf (A) 0.65; HPLC K0 3.42; mp 137–139 °C; ½a 2D0
ꢂ
+15.8; m/z
376 (M+H)+; 1H NMR (DMSO-d6) d 2.92–3.17 (m, 4H), 3.92–3.98
(m, 1H), 4.41–4.92 (m, 3H), 6.16 (s, 2H), 6.96–7.02 (m, 4H).
5.3. Pharmacology
5.2.29. TFAꢃN,N(Me)2-Dft-Tic-NH2 (17)
To a stirred solution of TFAꢃH-Dft-Tic-NH2 (0.06 g, 0.12 mmol)
in acetonitrile/H2O (1:1, v/v; 5 mL) at room temperature, NMM
(0.01 mL, 0.12 mmol), 37% aqueous formaldehyde (0.09 mL,
1.2 mmol), and NaBH3CN (0.02 g, 0.42 mmol) were added. Acetic
acid (0.04 mL) was added over 10 min and the reaction was stirred
for 15 min. The reaction mixture was acidified with TFA (0.1 mL)
and directly purified by reverse phase preparative HPLC: yield
5.3.1. Competitive binding assays
Opioid receptor affinities were determined under equilibrium
conditions [2.5 h room temperature (23 °C)] in competition assays
using brain P2 synaptosomal membranes prepared from Sprague–
Dawley rats.30,31 Synaptosomes were preincubated to remove
endogenous opioid peptides and stored at ꢁ80 °C in buffered 20%
glycerol.30,32 Each analogue was analysed in duplicate assays using
five to eight dosages and conducting three to five independent rep-
etitions with different synaptosomal preparations (n values are
listed in Table 1 in parenthesis and results are means SE). Unla-
0.06 g (95%); Rf (A) 0.69; HPLC K0 3.58; mp 144–146 °C; ½a 2D0
ꢂ
+18.6; m/z 404 (M+H)+; 1H NMR (DMSO-d6) d 2.27 (s, 6H), 2.77–
3.17 (m, 4H), 3.95–4.51 (m, 3H), 4.89–4.95 (m, 1H), 6.16 (s, 2H),
6.96–7.02 (m, 4H). Anal. Calcd for C23H23F5N3O5: C; H; N.
belled peptide (2 lM) was used to determine non-specific binding
in the presence of 1.9 nM [3H]deltorphin-II (45.0 Ci/mmol, Perkin-
Elmer, Boston, MA; KD = 1.4 nM) for d-opioid receptors and 3.5 nM
[3H]DAMGO (50.0 Ci/mmol), Amersham Bioscience, Buckingham-
5.2.30. Boc-Tyr-Tic-NH2
This intermediate was obtained by condensation of Boc-Tyr-OH
with TFAꢃH-Tic-NH2 via WSC/HOBt as reported for Boc-Dft-Tic-
NH2: yield 0.23 g (87%); Rf (B) 0.58; HPLC K0 6.84; mp 129–
shire, UK; KD = 1.5 nM) for
l-opioid receptors. Glass fibre filters
(Whatman GFC) were soaked in 0.1% polyethylenimine in order
to enhance the signal-to-noise ratio of the bound radiolabeled-syn-
aptosome complex, and the filters were washed thrice in ice-cold
buffered 0.1% BSA,30 and the affinity constants (Ki) were calculated
according to Cheng and Prusoff.33
131 °C; ½a 2D0
ꢂ
+16.3; m/z 441 (M+H)+; 1H NMR (DMSO-d6) d 1.38
(s, 9H), 2.92–3.17 (m, 4H), 4.41–4.51 (m, 2H), 4.85–4.94 (m, 2H),
6.68–7.02 (m, 8H).
5.2.31. TFAꢃH-Tyr-Tic-NH2
Boc-Tyr-Tic-NH2 was treated with TFA as reported for TFAꢃH-
5.3.2. Biological activity in isolate tissue preparations
The myenteric plexus longitudinal muscle preparations (2–3 cm
segments) from the small intestine of male Hartley strain of guinea
Dft-Tic-NH2: yield 0.15 g (94%); Rf (A) 0.62; HPLC K0 3.31; mp
132–134 °C; ½a 2D0
ꢂ
+19.5; m/z 340 (M+H)+; 1H NMR (DMSO-d6) d
2.92–3.17 (m, 4H), 3.92–3.98 (m, 1H), 4.41–4.92 (m, 3H), 6.68–
7.02 (m, 8H).
pigs (GPI) measured l-opioid receptor agonism, and a single
mouse vas deferens (MVD) was used to determine d-opioid recep-
tor agonism as described previously.34 The isolated tissues were
suspended in organ baths containing balanced salt solutions in a
physiological buffer, pH 7.5. Agonists were tested for the inhibition
of electrically evoked contraction and expressed as IC50 (nM) ob-
tained from the dose–response curves. The IC50 values represent
means SE of five or six separate assays, and the d-antagonist
potencies in the MVD assay were determined against the d agonist
5.2.32. TFAꢃN,N(Me)2-Tyr-Tic-NH2 (18)
This compound was obtained by N,N-dimethylation of TFAꢃH-
Tyr-Tic-NH2 as reported for TFA.N,N(Me)2-Dft-Tic-NH2: yield
0.09 g (93%); Rf (A) 0.61; HPLC K0 3.39; mp 138–140 °C; ½a 2D0
ꢂ
+20.2; m/z 368 (M+H)+; 1H NMR (DMSO-d6) d 2.27 (s, 6H), 2.77–
3.17 (m, 4H), 3.95–4.51 (m, 3H), 4.89–4.95 (m, 1H), 6.68–7.02
(m, 8H). Anal. Calcd for C23H26F3N3O5: C; H; N.
deltorphin-II, whilst
l antagonism (GPI assay) used the l agonist

6030
G. Balboni et al. / Bioorg. Med. Chem. 18 (2010) 6024–6030
14. Balboni, G.; Onnis, V.; Congiu, C.; Zotti, M.; Sasaki, Y.; Ambo, A.; Bryant, S. D.;
Jinsmaa, Y.; Lazarus, L. H.; Trapella, C. J. Med. Chem. 2006, 49, 5610.
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endomorphin-2. Antagonism is expressed as pA2 determined using
the Schild Plot.35
16. Tryoen-Tóth, P.; Décaillot, F. M.; Filliol, D.; Befort, K.; Lazarus, L. H.; Schiller, P.
W.; Schmidhammer, H.; Kieffer, B. L. J. Pharmacol. Exp. Ther. 2005, 313,
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Acknowledgements
This work was supported in part by the University of Cagliari (to
G.B.), the University of Ferrara (S.S.), and in part by the Intramural
Research Program of the NIH and NIEHS (to L.H.L.).
20. Balboni, G.; Fiorini, S.; Baldissserotto, A.; Trapella, C.; Sasaki, Y.; Ambo, A.;
Marczak, E. D.; Lazarus, L. H.; Salvadori, S. J. Med. Chem. 2008, 51, 5109.
21. Lopez, A.; Balboni, G.; Martinez, H.; Giuvelis, D.; Negus, S. S.; Salvadori, S.;
Bilsky, E. J. FASEB J. 2009, 23, 742.6 [Meeting Abstract].
22. Salvadori, S.; Fiorini, S.; Trapella, C.; Porreca, F.; Davis, P.; Sasaki, Y.; Ambo, A.;
Marczak, E. D.; Lazarus, L. H.; Balboni, G. Bioorg. Med. Chem. 2008, 16, 3032.
23. Balboni, G.; Salvadori, S.; Trapella, C.; Knapp, B. I.; Bidlack, J. M.; Lazarus, L. H.;
Peng, X.; Neumeyer, J. L. ACS Chem. Neurosci. 2010, 1, 155.
24. Balboni, G.; Trapella, C.; Sasaki, Y.; Ambo, A.; Marczak, E. D.; Lazarus, L. H.;
Salvadori, S. J. Med. Chem. 2009, 52, 5556.
Supplementary data
Supplementary data (synthetic Schemes 1–4) associated with
this article can be found, in the online version, at doi:10.1016/
j.bmc.2010.06.073.
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