β-methyl substitution of cyclohexylalanine in Dmt-Tic-Cha-Phe peptides results in highly potent δ opioid antagonists

Article abstract of DOI:10.1021/jm060721u

The opioid peptide TIPP (H-Tyr-Tic-Phe-Phe-OH, Tic: 1,2,3,4- tetrahydroisoquinoline-3-carboxylic acid) was substituted with Dmt (2′,6′-dimethyltyrosine) and a new unnatural amino acid, β-MeCha (β-methylcyclohexylalanine). This double substitution led to a

Full text of DOI:10.1021/jm060721u

328
J. Med. Chem. 2007, 50, 328-333
â-Methyl Substitution of Cyclohexylalanine in Dmt-Tic-Cha-Phe Peptides Results in Highly
Potent δ Opioid Antagonists
Ge´za To´th,*,†,‡ Enik_o Ioja,† Csaba To¨mbo¨ly,†,‡ Steven Ballet,‡ Dirk Tourwe´,‡ Antal Pe´ter,‡,§ Tama´s Martinek,| Nga N. Chung,
Peter W. Schiller, Sa´ndor Benyhe,† and Anna Borsodi†
Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Post Office Box 521 H-6701 Szeged, Hungary,
Department of Organic Chemistry, Vrije UniVersiteit Brussel, B-1050 Brussels, Belgium, Department of Inorganic and Analytical Chemistry,
UniVersity of Szeged, Post Office Box 440, Szeged, Hungary, Institute of Pharmaceutical Chemistry, UniVersity of Szeged, Post Office Box 121
H-6701 Szeged, Hungary, and Clinical Research Institute of Montreal, Montreal, Quebec, H2W 1R7 Canada
ReceiVed June 15, 2006
The opioid peptide TIPP (H-Tyr-Tic-Phe-Phe-OH, Tic:1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) was
substituted with Dmt (2′,6′-dimethyltyrosine) and a new unnatural amino acid, â-MeCha (â-methyl-
cyclohexylalanine). This double substitution led to a new series of opioid peptides displaying subnanomolar
δ antagonist activity and µ agonist or antagonist properties depending on the configuration of the â-MeCha
residue. The most promising analog, H-Dmt-Tic-(2S,3S)-â-MeCha-Phe-OH was a very selective δ antagonist
both in the mouse vas deferens (MVD) assay (Ke ) 0.241 ( 0.05 nM) and in radioligand binding assay (Kiδ
) 0.48 ( 0.05 nM, Kiµ/Kiδ ) 2800). The epimeric peptide H-Dmt-Tic-(2S,3R)-â-MeCha-Phe-OH and the
corresponding peptide amide turned out to be mixed partial µ agonist/δ antagonists in the guinea pig ileum
and MVD assays. Our results constitute further examples of the influence of Dmt and â-methyl substitution
as well as C-terminal amidation on the potency, selectivity, and signal transduction properties of TIPP
related peptides. Some of these compounds represent valuable pharmacological tools for opioid research.
Introduction
potency.8 TIPP and its C-terminal amide analog were systemati-
cally modified topologically by substitution of each amino acid
residue with all stereoisomers of the corresponding â-methyl
amino acid. The TIPP analogs containing (2S,3R)-â-MeTic in
position 2 or (2S,3R)-â-MePhe in position 3 displayed extra-
ordinarily high δ antagonist potency and δ selectivity, while
substitution of (2S,3S)-â-MePhe for Phe3 in TIPP resulted in a
peptide with mixed µ agonist/δ antagonist properties. These
results suggest a profound influence of â-methyl substitution
on the potency, selectivity, and signal transduction properties
of TIPP peptides.9
The tetrapeptide H-Tyr-Tic-Phe-Phe-OH (TIPP)1 represents
the prototype of a class of potent and selective δ opioid
antagonists that contain a 1,2,3,4-tetrahydroisoquinoline-3-
carboxylic acid (Tic) in the position 2 of the peptide sequence.2
In an effort to further improve the δ opioid antagonist potency
and δ receptor selectivity of TIPP, numerous analogs have been
prepared and pharmacologically characterized in Vitro. First of
all, the parent peptide, TIPP, was shown to undergo slow
spontaneous Tyr-Tic diketopiperazine formation with cleavage
of the Tic-Phe peptide bond. To prevent this degradation, a
pseudo peptide containing a reduced peptide bond between the
Tic and Phe residues (Tyr-Tic-ψ[CH2-NH]-Phe-Phe-OH, TIPPψ)
was synthesized. This peptide analog showed considerable
higher stability against chemical and enzymatic degradation3
and proved to be a more potent and more selective δ opioid
antagonist compared to the parent peptide. Replacement of Tyr1
with 2′,6′-dimethyltyrosine (Dmt) in TIPP resulted in a signifi-
cant enhancement of δ antagonist potency, but it was ac-
companied by a slight decrease in δ opioid selectivity.4 This
observation confirms that the replacement of Tyr with Dmt in
general results in an increase both in µ- and in δ-receptor affinity
in a wide variety of opioid peptides.5-7 Numerous substitutions
with natural and unnatural amino acids were performed in
position 3, and some Phe3-substituted TIPP analogs exhibited
very potent δ opioid antagonist properties.8 Most interestingly,
saturation of the Phe3 aromatic ring in TIPP led to H-Tyr-Tic-
Cha-Phe-OH [TICP] with substantially increased δ antagonist
The first known compound with a mixed µ agonist/δ
antagonist profile was TIPP-NH2.2 Substitution of Dmt for the
Tyr1 in the TIPP-NH2 resulted in the compound H-Dmt-Tic-
Phe-Phe-NH2 (DIPP-NH2) with increased µ agonist potency in
the guinea pig ileum (GPIa) assay and unchanged high δ
antagonist activity in the mouse vas deferens (MVD) assay.10
Reduction of the peptide bond between Tic2 and Phe3 of DIPP-
NH2 led to the compound DIPPψ-NH2, which displayed further
enhanced µ agonist potency and still high δ antagonist activity.
In the rat tail-flick test, DIPPψ-NH2 produced a potent analgesic
effect, about three times higher than morphine. This peptide
produced less-acute tolerance upon continuous infusion, and
no physical dependence was observed after chronic admin-
istration.10
In the present article, we describe further structural modifica-
tions of the TIPP peptide, which resulted not only in highly
potent δ opioid antagonists with very high δ selectivity, but
* To whom correspondence should be addressed. Phone: 36-62-599647.
Fax: 36-62-433-506. E-mail: geza@nucleus.szbk.u-szeged.hu.
† Hungarian Academy of Sciences.
a Abbreviations: Boc, tert-butyloxycarbonyl; DAMGO, H-Tyr-D-Ala-
Gly-NMe-Phe-Gly-ol; DPDPE, H-Tyr-c[D-Pen-Gly-Phe-D-Pen]-OH; FDAA,
NR-(2,4-dinitro-5-fluorophenyl)-l-alaninamide; GITC, 2,3,4,6-tetra-O-acetyl-
â-D-glucopyranosyl isothiocyanate; GPI, guinea pig ileum; MVD, mouse
vas deferens; RP-HPLC, reversed-phase high-performance liquid chroma-
tography; TAPP, H-Tyr-D-Ala-Phe-Phe-NH2; TFA, trifluoroacetic acid; TLC,
thin-layer chromatography.
‡ Vrije Universiteit Brussel.
§ Department of Inorganic and Analytical Chemistry, University of
Szeged.
| Institute of Pharmaceutical Chemistry, University of Szeged.
Clinical Research Institute of Montreal.
10.1021/jm060721u CCC: $37.00 © 2007 American Chemical Society
Published on Web 12/21/2006

â-Methyl Substitution on Cha Residue in Peptides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 329
Scheme 1. Synthesis of Dmt-Tic-â-MeCha-Phe Peptidesa
a Reagents and conditions: (i) 50 psi H2, 50 °C, PtO2, AcOH-H2O (2:1); (ii) (Boc)2O, pH 9; (iii) Boc solid-phase peptide synthesis on Phe-Merrifield
or Phe-MBHA resin
Table 1. Analytical Properties of TIPP and TIPP-NH2 Analogs
TLCa
HPLCb
k′
ESI-MS
no.
peptides
Rf(A)
Rf(B)
Rf(C)
[M + H]+
1
2
3
4
5
6
7
8
H-Dmt-Tic-(2S,3S)-â-MeCha-Phe-OH
H-Dmt-Tic-(2R,3R)-â-MeCha-Phe-OH
H-Dmt-Tic-(2S,3R)-â-MeCha-Phe-OH
H-Dmt-Tic-(2R,3S)-â-MeCha-Phe-OH
H-Dmt-Tic-(2S,3S)-â-MeCha-Phe-NH2
H-Dmt-Tic-(2R,3R)-â-MeCha-Phe-NH2
H-Dmt-Tic-(2S,3R)-â-MeCha-Phe-NH2
H-Dmt-Tic-(2R,3S)-â-MeCha-Phe-NH2
0.65
0.65
0.66
0.64
0.64
0.60
0.63
0.60
0.56
0.55
0.60
0.50
0.58
0.56
0.62
0.60
0.48
0.53
0.52
0.46
0.64
0.49
0.63
0.45
8.94
11.17
9.44
10.94
8.31
10.23
8.76
9.82
683
683
683
683
682
682
682
682
a Retention factor on silica gel 60 F254 precoated glass plates. Solvent systems: (A) butanol/acetic acid/water (4:1:1), (B) acetonitrile/methanol/water
(4:1:1), (C) ethyl acetate/pyridine/acetic acid/water (60:20:6:11). b Capacity factors for Vydac 218TP54 (25 × 0.46 cm, dp ) 5 µm) column with gradient
of 0.75%/min acetonitrile (0.08% (v/v) TFA) in water (0.1% TFA) over 40 min starting from 25% acetonitrile. Flow rate: 1 mL/min, t0 ) 3.1 min, λ )
216 nm.
Table 2. Equilibrium Competition Binding Data for [â-MeCha3]TIPP
also led to peptides with mixed partial µ agonist/δ antagonist
Tetrapeptides in Rat Brain Membranesa
properties. For this purpose, we substituted â-methylcyclohexy-
peptides
Kiµ (nM)
Kiδ (nM)
Kiµ/Kiδ
lalanine (â-MeCha) for Phe in position 3, and the effects of the
different configurations of the isomers of this amino acid on
the biological properties of TIPP and TIPP-NH2 were explored.
H-Tyr-Tic-Phe-Phe-OHb
1720 ( 50
3600
1.22 ( 0.07
0.61
1410
5890
2800
290
140
37
82
22
41
29
H-Tyr-Tic-Cha-Phe-OHb
1
2
3
4
5
6
7
8
1400 ( 149
580 ( 80
280 ( 5
0.48 ( 0.05
2.0 ( 0.6
2.0 ( 0.3
1.5 ( 0.1
4.0 ( 1.4
13 ( 2.6
4.1 ( 0.7
7.0 ( 1.3
Results and Discussion
Chemistry. The racemic erythro-(2S,3S and 2R,3R) and
threo-(2S,3R and 2R,3S)-â-MeCha diastereoisomers were ob-
tained from the corresponding erythro- or threo-â-MePhe9,11,12
by hydrogenation at 60 psi H2 and 50 °C using a PtO2 catalyst
in aqueous acetic acid solution, as was previously reported for
the preparation of cyclohexylalanine from phenylalanine.13-17
Boc-protection of erythro- or threo-â-MeCha and peptide
synthesis on a Merrifield or MBHA resin were carried out by
known, published methods (Scheme 1).9 The relative configura-
tions were confirmed indirectly by NMR analysis of the Boc-
â-MeCha diastereomers. Similarly to the â-MePhe and â-MeNal
isomers, the coupling constant between CR-H and Câ-H of the
Boc-erythro-â-MeCha was larger (J ) 8.5 Hz) than in the Boc-
threo-â-MeCha (J ) 5.0 Hz).9,11,12,18 Furthermore, the relative
NOE intensity between the NH and â-methyl protons of the
Boc-threo-â-MeCha was found to be significantly higher than
that of the erythro isomers, as published for the â-MePhe
isomers.19 The configuration of the R-carbon was determined
directly by enzymatic digestion of the â-MeCha isomers with
L-amino acid oxidase,20 followed by RP-HPLC separation of
the chiral derivatized â-MeCha isomers or by chiral TLC
separation of the amino acid isomers. The GITC derivatization21
was more useful, as it made it possible to separate all four
â-MeCha isomers and, thus, the 2R and 2S diastereomers were
unambiguously identified (see Supporting Information). In
contrast, the resolution of the FDAA derivatives of the 2S and
2R diastereomers was very low. Racemic erythro- and threo-
â-MeCha was digested with L-amino acid oxidase in 250 mg
scale as well to obtain (2R,3R)- and (2R,3S)-â-MeCha as
standards.
55 ( 1
330 ( 106
290 ( 99
170 ( 11
200 ( 37
a Receptor binding data are presented as the means ( SEM of two or
three independent assays. [3H]DAMGO as µ ligand and [3H]Ile5,6-deltorphin-
2 as δ ligand were used. b See ref 5.
cal properties of the new TIPP and TIPP-NH2 analogs are
summarized in Table 1. The configuration of the â-MeCha
residue in the new TIPP analogs was determined by chiral TLC
analysis of the peptide hydrolysates and by analytical HPLC
analyses of the GITC-derivatized hydrolysates.20,21
Biological Evaluation. The opioid receptor binding
assays22 were performed by displacement of [3H]DAMGO and
[3H]Ile5,6-deltorphin-223 as µ and δ opioid receptor selective
radioligands by the novel TIPP analogs in rat brain membrane
preparations. The opioid receptor binding affinities (Ki values)
of the eight novel peptides (four peptide acids and four
C-terminal amidated analogs) are presented in Table 2. Com-
pound 1, [Dmt,1 (2S,3S)-â-MeCha3]TIPP, exhibited 2.5-fold
higher δ opioid receptor affinity (Kiδ ) 0.48 ( 0.05 nM) and
2-fold higher selectivity for the δ opioid receptor (Kiµ/Kiδ
)
2800) than the parent TIPP. The other analogs had lower δ
binding affinity than TIPP and were less δ opioid receptor
selective. Comparing the peptide acid and amide analogs of
TIPP, it turned out that the TIPP amides in general had higher
affinities for the µ opioid receptor, with the exception of
compound 8, and slightly decreased δ affinities.
In Vitro agonist and antagonist activities of the novel TIPP
compounds were determined in the GPI and MVD assays. GPI
preparations contain mainly µ opioid receptors, whereas δ opioid
The solid-phase peptide synthesis resulted in diastereomeric
peptides that were separated by preparative RP-HPLC. Analyti-

330 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
To´th et al.
Table 3. In Vitro Opioid Activity (GPI and MVD Assays) of
[â-MeCha3]TIPP Analogs
In the functional assays, compounds 3 and 7 were mixed
partial µ agonist/δ antagonists. The other compounds 1, 2, 4,
5, 6, and 8 behaved as µ and δ antagonists. The configuration
of the â-MeCha residue has a profound influence on the µ
affinity and µ agonist properties, similar to our earlier results
obtained with [â-MePhe3]TIPP analogs.9,25 On the other
hand, all compounds displayed high δ antagonist activity,
indicating that the configuration or the conformation of the
â-MeCha3 in the TIPP analogs does not influence the antagonist
properties. This further confirms that the aromatic ring in
position 3 in TIPP or TIPP-NH2 is not required for δ antagonist
properties.
We also tested the G-protein activation of these analogs in
[35S]GTPγS binding assays using rat brain membranes or
Chinese hamster ovary (CHO) cells stably expressing µ or δ
opioid receptor.26 In rat brain membranes, none of the com-
pounds activated [35S]GTPγS binding. Similarly, in δhCHO cell
membranes, no increase in [35S]GTPγS binding was found in
the presence of these new analogs. In contrast, in µhCHO cell
membranes, all amidated analogs displayed weak-to-moderate
partial agonist properties. These results are in agreement
with the data obtained in the GPI and MVD assays presented
here.
K
e (nM)
GPIa
peptides
GPI
>10 000
1700 ( 220
MVDb
H-Tyr-Tic-Phe-Phe-OHc
H-Tyr-Tic-Phe-Phe-NH2
4.8 ( 0.2
18.0 ( 2.2
0.20
c
H-Dmt-Tic-Phe-Phe-OHc
H-Tyr-Tic-Cha-Phe-OHc
0.44
c
H-Dmt-Tic-Phe-Phe-NH2 18.0 ( 1.8
0.21 ( 0.04
0.24 ( 0.05
0.55 ( 0.02
0.81 ( 0.81
1
2
970 ( 31
290 ( 53
3
4
5
6
7
8
150 ( 28 (IC30)
19.0 ( 2.6 0.11 ( 0.03
340 ( 50
670 ( 50
0.77 ( 0.09
1.70 ( 0.33
0.68 ( 0.04
1.94 ( 0.02
110 ( 17 (IC40)
120 ( 12
a Determined against H-Tyr-D-Ala-Phe-Phe-NH2 (TAPP). b Determined
against DPDPE. c See ref 5 and references therein.
receptors are predominantly present in MVD tissues. δ Opioid
antagonist potencies were determined against the δ agonist
DPDPE, while µ opioid antagonist potencies were determined
against the µ agonist Tyr-D-Ala-Phe-Phe-NH2 (TAPP). The
results are presented in Table 3. All compounds were found to
be δ opioid antagonists with high potencies in the MVD assay.
In conclusion, we prepared new â-MeCha3-containing TIPP
analogs that show very potent δ antagonist properties. The
configuration of the â-methyl-substituted carbon has a large
influence on the affinity and activity profile of the peptide
analogs. H-Dmt-Tic-(2S,3S)-â-MeCha-Phe-OH (1) is a δ opioid
antagonist with increased potency and higher δ selectivity as
compared to the parent TIPP. Compounds 3 and 7 are mixed
partial µ agonist/δ antagonists.
µ
δ
Compound 1 had the highest δ opioid selectivity (Ke /Ke
)
4042) in these functional assays. In the GPI assays, compounds
3 and 7 behave as partial µ opioid agonists, whereas compounds
1, 2, 5, 6, and 8 displayed moderate µ antagonist properties. In
this assay, compound 4 had quite high µ antagonist potency
(Ke ) 19.0 ( 2.6 nM).
Experimental Section
Discussion
Earlier structural modifications of TIPP peptides resulted in
analogs with interesting pharmacological profiles. For example,
Dmt-Tic-Phe-Phe-NH2 showed high affinity for δ opioid
receptors and µ opioid receptors with moderate δ receptor
selectivity in radioligand binding assays. This compound
behaved as µ agonist and δ antagonist in the in Vitro GPI and
MVD assays.10,24 Saturation of the Phe3 aromatic ring in TIPP
led to a compound (Tyr-Tic-Cha-Phe-OH, TICP) with substan-
tially increased δ antagonist potency and higher δ selectivity
than the parent peptide.8 Methylation of the â-carbon in the
Phe3 in TIPP resulted in different activity profiles depending
on the configuration of the â-MePhe in the TIPP and TIPP-
NH2.9 It demonstrated that â-methyl substitution of the Phe3
residue in TIPP can have important effects on receptor selectivity
and on agonist/antagonist properties. On the basis of these earlier
results, we decided to prepare new TIPP and TIPP-NH2 analogs
containing L-Dmt,1 and the four different â-MeCha3 isomers.
Consequently, we have prepared erythro- and threo-â-MeCha
by saturation of the aromatic ring of â-MePhe using catalytic
hydrogenation in the presence of PtO2. The racemic Boc-
erythro- and Boc-threo-â-MeCha isomers were introduced into
the TIPP or TIPP-NH2 sequences by solid-phase peptide
synthesis. The resulting diasteromeric peptides were separated,
and the configuration of the â-MeCha residue was assigned by
chiral HPLC or TLC methods.
General Methods. TLC was performed on 2.5 × 10 cm plates
precoated with silica gel 60 F254 from Merck (Darmstadt, Germany)
or on chiral TLC plates (Macherey Nagel, Du¨rer, Germany), using
the following solvent systems: (A) 1-butanol/acetic acid/water
(4:1:1), (B) acetonitrile/methanol/water (4:1:1), and (C) ethyl
acetate/pyridine/acetic acid/water (60:20:6:11). TLC spots were
visualized under UV light or with ninhydrin reagent. Melting
points were measured with a Bu¨chi 510 apparatus. Enhanced
NMR spectra were obtained on a Bruker AC 250-P 250 MHz and
on a Bruker Avance DRX 400 MHz spectrometer. The assignments
were based on 1H, 13C, DEPT, HMQC, and HMBC NMR
experiments.
RP-HPLC was performed on a Merck-Hitachi instrument equipped
with a Vydac 218TP54 column for analytical purposes or with a
Vydac 218TP1010 column for semipreparative separations. UV
detection was carried out at 216 nm or 275 nm. Mass spectra were
recorded on a VG Quattro II spectrometer (ES ionization; cone
voltage, 70 V; source temperature, 80 °C).
Specific optical rotation was determined on an Optical Activity
AA-5 automatic polarimeter at 589.4 nm.
Protected amino acids (except â-MeCha) and Merrifield and
4-methylbenzhydrylamine resins were purchased from Sigma
Aldrich Kft. (Budapest, Hungary) or from BACHEM Feinchemi-
calen AG (Bubendorf, Switzerland). Boc-L-Dmt was obtained from
RSP Amino Acids LLC (Sherley, MA). PtO2 catalyst was purchased
from Acros Chimica N.V. (Geel, Belgium).
erythro-â-MePhe and threo-â-MePhe were prepared in racemic
form in our laboratory by fractional crystallization12 using the
method of Kataoka et al.11
Radioligands were prepared in our laboratory using the precursor
peptides [Dit1]DAMGO, [Dit1]Ile5,6-deltorphin-2 and tritium gas
in the presence of PdO/BaSO4 catalyst of Merck (Darmstadt,
Germany).27
The affinity for the µ or δ opioid receptor was determined in
rat brain membranes. As expected, all compounds show high
affinity for the δ receptor and are less potent at the µ receptor.
The δ selectivity was higher for the C-terminal carboxylic acid
analogs than for the C-terminal amides; Dmt-Tic-(2S,3S)-â-
MeCha-Phe-OH (1) displaying the highest δ selectivity.

â-Methyl Substitution on Cha Residue in Peptides
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 331
erythro-â-Methylcyclohexylalanine. An amount equal to 1.9 g
of erythro-(2S,3S and 2R,3R)-â-MePhe hydrochloride (8.8 mmol)
was dissolved in 100 mL of acetic acid/water (2:1). PtO2 (250 mg)
was added to the solution, and it was shaken under 50 psi of
hydrogen at 50 °C in a Parr-hydrogenator for 20 h. The reaction
mixture was cooled to room temperature, the catalyst was filtered
over a layer of Celite, and the filtrate was evaporated in vacuum.
The remaining solid was dissolved in hot water and kept in a
refrigerator overnight. The precipitate was filtered, and a yield of
1.77 g (81%) was obtained; mp 258 °C; Rf(A) 0.48; Rf(B) 0.56
stored in a refrigerator overnight, after which it solidified. Yield
3.0 g (94%); mp 86.7-88.7 °C; Rf(A) 0.9; ESI-MS 286 [M + H]+;
1H NMR (DMSO-d6) δ 6.95 (d, 1H, J ) 8.5 Hz, NH), 3.85 (t, 1H,
J ) 8.5 Hz, R-H), 1.67-0.80 (m, 11H, chx), 1.65 (br s, 1H, â-H),
1.38 (s, 9H, Boc-CH3), 0.75 (d, 3H, J ) 7.0 Hz, â-CH3); 13C NMR
(DMSO-d6) δ 174.2 (COOH), 155.8 (Boc-CO), 78.3 (C(CH3)3),
56.6 (C-R), 39.7 (C-â), 38.0 (chx-CH), 31.4 (chx-CH2), 28.6
(C(CH3)), [27.1, 26.8, 26.5] (chx-CH2), 12.4 (â-CH3); Anal. (C15H27-
NO4) C, H, N.
NR-Boc-threo-â-methylcyclohexylalanine. An amount equal to
2.0 g of threo-â-MeCha‚HOAc was dissolved in 50 mL of dioxane
and 25 mL of water. The pH was adjusted to 9 with 2 N NaOH,
and 2.2 g of (Boc)2O was added to the solution. The reaction
mixture was stirred for 3 h at 0 °C and then at room temperature
overnight. The work up was as described above. Yield 2.45 g (99%);
1
and 0.36 on chiral plate; ESI-MS 186 [M + H]+; H NMR (DCl/
DMSO-d6) δ 8.60 (m, NH3+), 3.82 (d, 1H, J ) 4.2 Hz, R-H), 1.79-
0.78 (m, 12H, â-H, chx), 0.85 (d, 3H, J ) 7.0 Hz, â-CH3); 13C
NMR (DCl/DMSO-d6) δ 170.2 (COOH), 54.2 (C-R), 39.7 (C-â),
38.4 (chx-CH), [31.0, 29.2, 26.2, 26.1, 25.9] (chx-CH2), 12.0 (â-
CH3); Anal. (C10H19NO2) C, H, N.
1
mp 132.2 °C; TLC Rf(A) 0.9; ESI-MS 286 [M + H]+; H NMR
threo-â-Methylcyclohexylalanine. An amount equal to 0.85 g
of threo-(2S,3R and 2R,3S)-â-MePhe was dissolved in 40 mL of
acetic acid/water (2:1) and, in the presence of 100 mg of PtO2
catalyst, was hydrogenated under 55 psi of hydrogen at 50 °C for
23 h, followed by the above work up. Yield was 0.79 g (88%); mp
260.1 °C; Rf(A) 0.48; Rf(B) 0.54 and 0.45 on chiral plate; ESI-MS
186 [M + H]+; 1H NMR (DCl/DMSO-d6) δ 8.42 (m, NH3+), 3.82
(d, 1H, J ) 4.0 Hz, R-H), 1.79 (1H, m, â-H), 1.63-0.77 (m, 10H,
(DMSO-d6) δ 6.77 (d, 1H, J ) 9.2 Hz, NH), 4.15 (dd, 1H, J ) 5.0
Hz, 9.2 Hz, R-H), 1.67-0.84 (m, 10H, chx), 1.70 (br s, 1H, â-H),
1.38 (s, 9H, Boc-CH3), 1.10 (m, 1H, chx-CH), 0.79 (d, 3H, J )
7.0 Hz, â-CH3); 13C NMR (DMSO-d6) δ 174.6 (COOH), 156.2
(Boc-CO), 78.3 (C(CH3)3), 55.8 (C-R), 39.7 (C-â), 39.3 (chx-CH),
[30.9, 29.5] (chx-CH2), 28.6 (C(CH3)), 26.4 (chx-CH2), 12.3 (â-
CH3); Anal. (C15H27NO4) C, H, N.
Solid-Phase Peptide Synthesis and Peptide Purification. The
TIPP peptide analogs were prepared by manual solid-phase peptide
synthesis using Boc chemistry on Merrifield resin for the C-terminal
carboxylic acid analogs and 4-methylbenzhydrylamine resin for the
C-terminal amides. Boc-Phe was attached to the Merrifield resin
by the Gisin method.28 Diisopropylcarbodiimide and 1-hydroxy-
benzotriazole were used as coupling reagents. The coupling steps
were monitored by ninhydrin test29 or chloranil test. Removal of
the Boc protecting group was performed by washing with a TFA/
DCM/anisole (50:48:2) solution for 2 and 20 min, followed by
DCM washes (4), a neutralization with 20% DIEA in DCM (2 ×
2 min), and DCM washing (3×) again. The peptides were cleaved
from the resin with anhydrous HF (5 mL/g resin) in the presence
of anisole as scavenger for 1 h at 0 °C. The peptide was separated
from the resin by dissolving in 30% acetic acid solution. The
solution was filtered and lyophilized. The purification of crude
peptide was performed by semipreparative RP-HPLC using a Vydac
218TP1010 column with a linear gradient of 0.1% TFA in
water and 0.08% TFA in acetonitrile. Each peptide was at least
98% pure as assessed by TLC and analytical RP-HPLC. The
molecular weights of the peptides were confirmed by ESI-MS (see
Table 1).
Identification of Enantiomers of â-Methylcyclohexylalanine
in Peptides. The acidic hydrolysate (6 N HCl, 24 h, 110 °C) of 1
mg of peptide was separated by analytical HPLC to isolate
â-MeCha. The configuration of the pure amino acid was determined
by chiral TLC in acetonitrile/methanol/water (4:1:1). The Rf value
was compared with that of standard optical pure â-MeCha isomers.20
GITC derivatization of the amino acid mixture obtained after
hydrolysis was also used as an alternative method.21 The derivatized
amino acid mixture was separated by analytical HPLC on a Vydac
218TP54 column, and the peak of the derivatized â-MeCha was
compared with those of the prepared standard â-MeCha isomers
(see Supporting Information, p. S5).
Radioligand Binding Assays. Rat brain membranes were
prepared from Wistar rat brain according to Simon et al.22 The
binding experiments were performed in 50 mM Tris‚HCl buffer,
pH 7.4, at a final volume of 1 mL containing 200-300 µg protein.
In competition experiments the following conditions were used for
incubations: [3H]DAMGO (35 °C, 45 min), [3H]Ile5,6-deltorphin-
2 (35 °C, 45 min).23 Incubations were started by the addition of
the membrane suspension and terminated by rapid filtration
through Whatman GF/C glass fiber filters using Brandel M24R
Cell Harvester. The filters were washed three times with ice cold
Tris‚HCl buffer and dried for 3 h at 37 °C, and the radioactivity
was measured in UltimaGOLD scintillation cocktail using a Packard
TriCarb 2300 TR counter. Affinities of competing ligands were
determined by co-incubation with 10-12-10-5 M freshly prepared
chx), 1.32 (m, 1H, chx-CH), 0.82 (d, 3H, J ) 7.0 Hz, â-CH3); 13
C
NMR (DCl/DMSO-d6) δ 171.2 (COOH), 54.5 (C-R), 39.3 (C-â),
37.6 (chx-CH), [31.1, 29.3, 26.1(2C), 25.9] (chx-CH2), 12.0 (â-
CH3); Anal. (C10H19NO2) C, H, N.
Identification of Enantiomers of â-Methylcyclohexylalanine.
For the preparation of the (2R)-â-MeCha isomers, 75 mg (16.5
units) of L-amino acid oxidase (Sigma-Aldrich Kft, Budapest) was
added to the solution of 250 mg of erythro- or threo-â-MeCha in
200 mL of water at pH 7.3. The solution was stirred at room
temperature overnight. After gel filtration on Sephadex G-10, the
(2R,3R)- or (2R,3S)-â-MeCha was obtained. On analytical scale,
250 µL of a 10 mM solution of racemic erythro- or threo-â-MeCha
in 100 mM Tris buffer pH 7.2 was digested with 0.05 units of
L-amino acid oxidase at room temperature overnight.20 The
chiral TLC spots and the HPLC peaks of the GITC derivatives
were identified with these (2R)-diastereomers (see Supporting
Information).
Optically pure (2S,3S)- and (2R,3R)-â-MeCha were prepared
from resolved benzyloxycarbonyl erythro-â-MePhe. The optical
resolution of the erythro-â-MePhe was attained by fractional
crystallization of the quinine salt of the benzyloxycarbonyl deriva-
tive.11 Hydrogenation of the resolved erythro-â-MePhe resulted in
the removal of the benzyloxycarbonyl group and the saturation of
the aromatic ring (see Supporting Information, p. S2).
(2S,3S)-â-MeCha: [R]25D ) +25.3 (c ) 0.75; 25% (v/v) AcOH),
Rf(B) 0.56 on chiral plate, tR(GITC derivative) ) 19.7 min. (2R,3R)-
â-MeCha: [R]25 ) -27.3 (c ) 0.75; 25% (v/v) AcOH), Rf(B)
D
0.36 on chiral plate, tR(GITC derivative) ) 16.7 min. (2S,3R)-â-
MeCha: Rf(B) 0.54 on chiral plate, tR(GITC derivative) ) 18.8
min. (2R,3S)-â-MeCha: [R]25 ) -7.0 (c ) 0.75; 25% (v/v)
D
AcOH), Rf(B) 0.45 on chiral plate, tR(GITC derivative) ) 15.7 min.
This information was used for the identification of the
configuration of the â-MeCha in the diastereomeric peptides (see
below).
NR-Boc-erythro-â-methylcyclohexylalanine. A solution of 2.74
g of erythro-â-MeCha‚HOAc salt (11.2 mmol) in 50 mL of dioxane
and 25 mL of water was cooled in an ice bath. The pH was adjusted
to 9 with 2 N NaOH, and then 3.09 g of (Boc)2O was added to the
solution with stirring for 3 h, followed by overnight stirring at room
temperature. If the pH changed it was adjusted to 9. After
evaporation of dioxane, the pH of water solution was adjusted to
2-3 with a saturated KHSO4 solution under ice cooling. The
aqueous layer was extracted three times with 50 mL of ethyl acetate.
The combined organic layers were washed with water, brine, and
again with water. The organic phase was dried over Na2SO4 and
evaporated in vacuum. Because precipitation of the resulting oil
from an ethyl acetate/petroleum ether mixture failed, the oil was

332 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
To´th et al.
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effect, no physical dependence, and less tolerance than morphine in
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The synthesis and the optical resolutions of â-methylphenylalanine
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Topographically designed analogues of [D-Pen2, D-Pen5]enkephalin.
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solution of the unlabeled peptides with 0.5-1 nM tritiated ligand.
Nonspecific binding was defined as a radioactivity bound in the
presence of 10 µM naloxone. All assays were performed in duplicate
and repeated several times. Experimental data were analyzed by
GraphPad Prism 2.01 software.30
GPI and MVD In Vitro Bioassays. The bioassays of the
peptides were based on electrically induced smooth muscle
contractions of MVD and GPI myenteric plexus-longitudinal
muscle strips, as reported in detail elsewere.31,32 The dose-response
curve was determined with Leu-enkephalin as standard for each
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tested were normalized according to a published procedure.33 Ke
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values obtained with δ agonist, DPDPE in MVD assay in the
presence and absence of a fixed antagonist concentration (5 nM).
Ke values of compounds for µ antagonist properties were determined
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Acknowledgment. This work was supported by operating
grants from bilateral governmental Flemish-Hungarian Scientific
and Development Grant (B-03/04, D.T. and A.P.), NKTH RET
08/2004 DNT from National Bureau of Research and Technol-
ogy (A.B., S.B., and G.T.), OTKA TS 049817 (G.T.), T464334
(A.B.), and Ja´nos Bolyai fellowship (Cs.T.).
(14) Wakamiya, T.; Ueki, Y.; Shiba, T.; Kido, Y.; Motoki, Y. Structural
determination of ancovenin, a peptide inhibitor of angiotensin I
converting enzyme. Bull. Chem. Soc. Jpn. 1990, 63, 1032-1038.
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K. T.; King, C. H. R. Large-scale synthesis of anticoagulant
decapeptide MDL 28050. Tetrahedron 1992, 48, 307-318.
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Van de Vusse, J. Unique and efficient synthesis of [2S-(2R*,3S*,4R*)]-
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Supporting Information Available: 1H NMR spectra of Boc-
erythro- and threo-â-MeCha, 1D NOESY slices taken at the
chemical shifts of HN for Boc-erythro and threo-â-MeCha, GITC
derivatization, RP-HPLC analysis of â-MeCha, and elemental
analysis data. This material is available free of charge via the
Internet at http://pubs.acs.org.
(17) Ishida, K.; Matsuda, H.; Murakami, M. Micropeptins 88-A to 88-F,
chymotrypsin inhibitors from the cyanobacterium Microcystis aerugi-
nosa (NIES-88). Tetrahedron 1998, 54, 5545-5556.
(18) EÄ rchegyi, J.; Penke, B.; Simon, L.; Michaelson, S.; Wenger, S.;
Waser, B.; Cescato, R.; Schaer, J. C.; Reubi, J. C.; Rivier, J. Novel
sst4-selective somatostatin (SRIF) agonists. 2. Analogues with
â-methyl-3-(2-naphthyl)alanine substitutions at position 8. J. Med.
Chem. 2003, 46, 5587-5596.
(19) Nogle, L. M.; Mann, C. W.; Watts, W. L., Jr.; Zhang, Y. Preparative
separation and identification of derivatized â-methylphenylalanine
enantiomers by chiral SFC, HPLC and NMR for development of
new peptide ligand mimetics in drug discovery. J. Pharm. Biomed.
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