6-Hydroxy-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid mimics active conformation of tyrosine in opioid peptides

Article abstract of DOI:10.1016/j.bmcl.2005.03.075

6-Hydroxy-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid (6Htc) has been proposed as a rigid mimic of tyrosine conformation in opioid ligand-receptor complex. The significant receptor binding to mu and delta opioid receptors of respective analogues of

Full text of DOI:10.1016/j.bmcl.2005.03.075

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2467–2469
6-Hydroxy-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic
acid mimics active conformation of tyrosine in opioid peptides
Emanuela Sperlinga,a Piotr Kosson,b Zofia Urbanczyk-Lipkowska,d
Giuseppe Ronsisvalle,a Daniel B. Carrc and Andrzej W. Lipkowskia,b,c,
*
aDepartment of Pharmaceutical Sciences, University of Catania, Catania 95125, Italy
bMedical Research Centre, Polish Academy of Sciences, Warsaw 02106, Poland
cTufts-New England Medical Center, Boston MA 02111, USA
dInstitute of Organic Chemistry, Polish Academy of Sciences, Warsaw 01224, Poland
Received 3 March 2004; revised 10 March 2005; accepted 17 March 2005
Available online 12 April 2005
Abstract—6-Hydroxy-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid (6Htc) has been proposed as a rigid mimic of tyrosine con-
formation in opioid ligand–receptor complex. The significant receptor binding to mu and delta opioid receptors of respective ana-
logues of deltorphin, dermorphin, and endomorphin with D,L-6Htc prove initial prediction.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
peptides.2 Trimethyltyrosine (TMT), which substituted
Tyr(1) resulted in biologically potent analogues, possess-
ing rotamers that well overlap with alkaloid tyramine.3
Substitution of tyrosine with rigid tyrosine analogue,
7-hydroxy-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic
acid (Htc) (Fig. 1), resulted in dramatic loss of affinity to
mu opioid receptor types but with preservation of affin-
ity to delta opioid receptors.4
Tyramine moiety is a common element of opioid phar-
macophore of both, alkaloids and peptides. In rigid opi-
oid alkaloids, tyramine has a ÔfreezedÕ conformation.
This conformation is not related to any of those that
have been found for tyrosine residue in peptides or pro-
teins. It has been postulated that opioid peptides inter-
act with their receptors in
a multistep ÔzipperÕ
mechanism of complementary groups fitting to receptor
binding sites.1 Adaptation of N-terminal tyrosine, espe-
cially tyramine moiety, is one of the critical steps of this
mechanism. The energetically unfavored transformation
of tyrosine conformation probably needs additional sup-
porting groups in the formed peptide–receptor complex.
Therefore in opioid peptides, aromatic phenylalanine
has been defined as a second determinant of affinity to
opioid receptors. The possible common conformation
of tyramine moiety of benzomorphan alkaloids and opi-
oid peptides in ligand–receptors complex has been evi-
denced indirectly through hybridization of rigid
benzomorphan alkaloids with peptide C-terminal (Ôad-
dressÕ) fragment of various selective opioid peptides that
resulted in modulation of receptor selectivity of the final
hybride compounds in the same manner as the parent
To get additional data to support hypothesis of confor-
mational similarity of tyramine in peptides and alka-
loids, search for constrained amino acid analogues
that may simulate benzomorphan tyramine has been
performed.
The molecular modeling studies indicated that 6-hydroxy-
1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid (6Htc)
(Fig. 1) better than Htc simulates tyrosine in
OH
HO
HO
OH
OH
OH
H2N
Tyr
N
H
N
H
O
O
O
6Htc
Htc
*
Corresponding author. Tel./fax: +48 22 6685388; e-mail:
andrzej@lipkowski.org
Figure 1. Molecular formulas of Tyr, Htc, and 6Htc.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.075

2468
E. Sperlinga et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2467–2469
O3
C5
C4
C6
C3
C7
C2
C1
O2
C8
C9
C10
O1
N1
C11
O4
Figure 2. Comparison of overlaped conformations of oxymorphone
(A), R-6Htc (B), and S-6Htc (C).
Figure 3. ORTEP diagram of D,L-6Htc hydrochloride monohydrate
showing thermal ellipsoids at 30% probability level.
Ôbenzomorphan related conformationÕ (Fig. 2). Carboxyl
group of 6Htc is responsible for link of N-terminal Ôtyr-
amine likeÕ element with the other part of opioid peptides.
Flexible peptide chains express high degree of structure
modification and adaptation on a stage of interactions
with receptors. In the molecular simulation level of struc-
ture–activity studies of analogs with 6Htc, it was hard to
predict, which stereoisomer should be introduced to pep-
tide analogues. Therefore, in the preliminary stage of
study, the analogues of opioid selective peptides with
racemic 6Htc have been synthesized and their affinities
to opioid mu and delta receptors have been tested.
ysis of 15 reflections in h range 23.1–41.3°: a = 8.5303(4),
˚
b = 6.6333(3), c = 20.0341(9) (A) b = 90.136(3) (°);
3
˚
V = 1133.61(9) (A ) monoclinic space group P21/n,
Z = 4, calculated density 1.445 (mg mꢀ3). Final R indices
for 2075 data and 170 refined parameters with I > 2sig-
ma(I): R1 = 0.0473, wR2 = 0.1344. The structure (Fig.
3) was solved with SHELXS-97 and refined with SHELXL
97.6 Hydrogen atoms were located from Dq maps (water,
N, and O hydrogens) or placed in geometrically idealized
positions and constrained to ride on their parent atoms,
with Uiso(H) = 1.2Ueq(C).7
2. Synthesis
4. Receptor affinity measurement
The Picted-Spengler reaction of formaldehyde and D,L-
meta-tyrosine performed overnight at room temperature
in water resulted in precipitation of D,L-6Htc in high
(89%) yield and purity, and has been used in next steps
without additional purification. The used reaction con-
ditions were much milder than previously used.5 D,L-
6Htc has been transformed into Boc-D,L-6Htc using
standard procedure with commercial (Boc)2O. Peptide
analogues 3 and 6 have been synthesized in solid phase
using Fmoc-strategy with Rink-amide resin. 2-(1H-ben-
zotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoro-
borate (TBTU) in presence of N-hydroxybenzotriazole
(HOBt) and N-diisopropylethyl amine (DIPEA) has
been used for coupling reaction. Piperidine has been
used for Fmoc-deprotection. Peptides have been
liberated from the resin with 95% trifluoroacetic acid.
Peptides 8 and 10 have been synthesized in solution
step-by-step procedure using commercial Boc-amino
acids and phenylalanine amide. For coupling reactions,
N,N0-dicyclohexylcarbodiimide (DCC) in presence of
HOBt and DIPEA has been used. Hydrochloride in ace-
tic acid has been used for Boc-group deprotection.
Crude peptide analogues have been purified using gel fil-
tration on Sephadex LH-20 in methanol.
Radioreceptor binding assays have been performed as
described previously8 after obtaining permission from
the Animal Rights Commission of Medical Research
Centre of Polish Academy of Sciences. The binding as-
says were performed on adult male Wistar rats brain
homogenates. [3H]Deltorphin II, and [3H]naloxone syn-
thesized by Dr. G. Toth,9 Biological Institute, Hungar-
ian Academy of Sciences, Szeged, Hungary have been
used as a radioligands of delta and mu receptors, respec-
tively. Naltrexone hydrochloride was used to define non-
specific tissue binding. The data were analyzed by a
nonlinear least-squares regression analysis computer
program Prism Graph Pad. The obtained result are
summarized in Table 1.
5. Results and discussion
Molecular modeling search for amino acid that might
mimic tyramine moiety of benzomorphan opioid alkalo-
ides resulted in selecting of 6-hydroxy-1,2,3,4-tetrahy-
dro-isoquinoline-3-carboxylic acid (6Htc). The X-ray
structure of synthetic D,L-6Htc confirmed predicted
model structure. Phenol and amino groups of 6Htc and
benzomorphan overlapped well. Small, but potentially
significant difference in distance between phenol and
amino group has been observed. It was not possible to
choose between amino acid stereoisomers on the basis
of available data. Therefore, for preliminary studies,
the synthesis of peptide analogues with racemic 6Htc
has been decided. The opioid peptide analogues with
3. Crystal structure determination
The 6Htc has been transformed into hydrochloride salt
and crystallized from water. The X-ray experiment was
performed using Nonius BV MACH3 diffractometer.
Unit cell parameters were obtained by least-squares anal-

E. Sperlinga et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2467–2469
Table 1. Opioid receptor binding of opioid peptide analogs containing D,L-6Htc
2469
Amino acid sequence
Ki (nM)
Mu
Delta
295
1. Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2
2. Htc-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2
3. D,L-6Htc-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2
4. Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2
5. Htc-D-Ala-Phe-Asp-Val-Val-Gly-NH2
6. D,L-6Htc-D-Ala-Phe-Asp-Val-Val-Gly-NH2
7. Tyr-Pro-Phe-Phe-NH2
2.4
1615a
253
>10,000a
>10,000
0.73
1700
>10,000a
>10,000
3.7
304a
10.4
2300
8. D,L-6Htc-Pro-Phe-Phe-NH2
9. Tyr-D-Ala-Phe-Phe-NH2
10. D,L-6Htc-D-Ala-Phe-Phe-NH2
216.0
5.1
57.8
>10,000
1820
>10,000
a Taken from Ref. 4.
tyrosine stereoisomers could express opposite functional
properties (agonist vs antagonist). Nevertheless, their
potential cross interferences should not be visible in
binding assay and their values should be the sum of affin-
ities of both enantiomers. Dermorphin (1), deltorphin II
(4), endomorphin (7), and D-Ala2-endomorphin (9) have
been used as a parent sequences of synthetic analogues.
The obtained results of binding to opioid receptors
showed that 6Htc indeed is able to simulate active con-
formation of tyrosine. Substitution of N-terminal tyro-
sine with 6Htc in delta selective peptide deltorphin II,
resulted in delta selective and potent analogue 6. Substi-
tution of tyrosine in dermorphin, endomorphin, and D-
Ala2-endomorphin, three mu selective peptides resulted
in 3, 8, and 10 analogues that preserved high selectivity
to mu receptor types. Nevertheless, the affinity levels
are lower than for delta selective analogue. This, with
previous data of analogues with Htc,4 may strongly indi-
cate the difference in tyramine conformation require-
ments of delta and mu receptors. The obtained
interesting preliminary results rationalize further studies
of analogues with individual isomers of 6Htc.
References and notes
1. (a) Misicka, A.; Lipkowski, A. W.; Fang, L.; Knapp, R. J.;
Davis, P.; Kramer, T.; Burks, T. F.; Yamamura, H. I.;
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Horvat, R.; Davis, P.; Kramer, T. H.; Yamamura, H. I.;
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7. X-ray parameters for crystal structure of D,L-6Htc have
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Acknowledgments
8. Olma, A.; Łachwa, M.; Lipkowski, A. W. J. Pept. Res.
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A. Life Sci. 1992, 50, PL75–PL78.
Financial supports from the Italian MIUR (GR) and
Maria Curie 5FP Grant (AWL) are gratefully
acknowledged.