Studies on the structure-activity relationship of 2′,6′- dimethyl-l-tyrosine (Dmt) derivatives: Bioactivity profile of H-Dmt-NH-CH 3

Article abstract of DOI:10.1016/j.bmcl.2004.11.040

The 2′,6′-dimethyl-l-tyrosine (Dmt) enhances receptor affinity, functional bioactivity and in vivo analgesia of opioid peptides. To further investigate its direct influence on these opioid parameters, we developed a series of compounds (H-Dmt-NH-X). Among them, H-Dmt-NH-CH₃ showed the highest affinity (K_iμ = 7.45 nM) equal to that of morphine, partial μ-opioid agonism (E_max = 66.6%) in vitro and a moderate antinociception in mice.

Full text of DOI:10.1016/j.bmcl.2004.11.040

Bioorganic & Medicinal Chemistry Letters 15 (2005) 599–602
Studies on the structure–activity relationship of
20,60-dimethyl-L-tyrosine (Dmt) derivatives: bioactivity profile
of H–Dmt–NH–CH3
Yoshio Fujita,a Yuko Tsuda,a,b,c Takashi Motoyama,b Tingyou Li,a Anna Miyazaki,b
Toshio Yokoi,a,b,c Yusuke Sasaki,d Akihiro Ambo,d Hideko Niizuma,d Yunden Jinsmaa,e
Sharon D. Bryant,e Lawrence H. Lazaruse and Yoshio Okadaa,b,c,
*
aThe Graduate School of Food and Medicinal Sciences, Kobe Gakuin University, Nishi-ku, Kobe 651-2180, Japan
bDepartment of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Nishi-ku, Kobe 651-2180, Japan
cHigh Technology Research Center, Kobe Gakuin University, Nishi-ku, Kobe 651-2180, Japan
dDepartment of Biochemistry, Tohoku Pharmaceutical University, Aoba-ku, Sendai 981-8558, Japan
eMedicinal Chemistry Group, LCBRA, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA
Received 11 October 2004; revised 16 November 2004; accepted 17 November 2004
Available online 2 December 2004
Abstract—The 20,60-dimethyl-L-tyrosine (Dmt) enhances receptor affinity, functional bioactivity and in vivo analgesia of opioid pep-
tides. To further investigate its direct influence on these opioid parameters, we developed a series of compounds (H–Dmt–NH–X).
Among them, H–Dmt–NH–CH3 showed the highest affinity (Kil = 7.45 nM) equal to that of morphine, partial l-opioid agonism
(Emax = 66.6%) in vitro and a moderate antinociception in mice.
ꢀ 2004 Elsevier Ltd. All rights reserved.
The common N-terminal amino acid of endogenous opi-
oid peptides (enkephalins,1 endorphins,2 dynorphins,3
endomorphins,4 deltorphins,5 dermorphins,6 and so
on) is Tyr, except Phe in nociceptin,7 and is considered
an important pharmacophore to interact with opioid
receptors according to the message–address concept.8
Recent studies verified that Dmt (20,60-dimethyl-L-tyro-
sine)9 can readily replace Tyr in opioid substances and
dramatically enhances receptor affinity, in vitro func-
tional bioactivity and in vivo analgesic action.10 For
example, affinity increased by several hundredfold for
enkephalin derivatives, such as [Leu5]enkephalin11 and
DPDPE (cyclo[D-Pen2,5]enkephalin),12 the dermorphin
analogue DALDA (H-Tyr-D-Arg-Phe-Lys-NH2),13 delt-
orphin,14 and endomorphin-2 (EM-2)15 as well as the
Tic-containing di- and tripeptide DOR (d-opioid recep-
tor) antagonists.10,16 A study on [Dmt1]EM-2 deletion
analogues also revealed that H–Dmt–Pro–NH2 exhibi-
ted moderate affinity (Kil = 41.7 nM) in contrast to
the inactive Tyr derivative.15 Furthermore, the design
and synthesis of the novel opioid ligands, H–Dmt–
NH–X–NH–Dmt–H, in which the Dmt pharmaco-
phores were linked by either diaminoalkane or 3,6-
bis-(aminoalkyl)-5-methyl-2(1H)pyrazinone, produced
analogues that had higher MOR (l-opioid receptor)
affinities and greater functional bioactivities than those
containing single or multiple Tyr residues.17 This report
presents comprehensive data on the development of an
opioid compound library using Dmt as the single core
amino acid (Scheme 1).
Compounds were synthesized as shown in Scheme 1.
The NH2–CH2–NH–Boc was prepared from Boc–Gly–
NH2 by Hofmann rearrangement using bis-(trifluoro-
acetoxy)-iodobenzene.18 It was reported that although
diaminomethane is unstable, mono-protected diami-
nomethane is comparatively stable.19 The NH2–CH2–
NH–Boc obtained here was identified by TLC, NMR,
and MS.20 The NH2–C2H4–NH–Boc was prepared from
commercially available NH2–C2H4–NH221 and the other
amino components were purchased from commercial
sources. Dmt was prepared according to the DygosÕ
method.9
Keywords: Dmt; l-Opioid agonist; Structure–activity relationship.
*
Corresponding author. Tel.: +81 789741551; fax: +81 789745689;
e-mail: okada@pharm.kobegakuin.ac.jp
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.11.040

600
Y. Fujita et al. / Bioorg. Med. Chem. Lett. 15 (2005) 599–602
i, ii, iii
iv
v
HCl . H-Dmt-NH2 (2)
Me2-Dmt-NH2 (20)
H-Dmt-OH (1)
Boc-Dmt-NH2
ii
vi or vii
iv
Boc-Dmt-OH
Boc-Dmt-NH-Y
HCl . H-Dmt-NH-X (3-19)
(9 and 10)
CH2CH2OH (14)
X =
CH2CH2OH
OCH3
CH3 (3)
CH2CH3 (4)
CH
CH2CH3
Y =
3
OCH3 (15)
(11)
CH2COO-t-Bu
CH2CH2CH3 (5)
CH(CH3)2 (6)
CH2COOH (16)
CH2CH2COOH (17)
CH2CH2CH3
CH(CH3)2
CH2CH2COO-t-Bu
CH2NH-Boc
C(CH3)3 (7)
(8)
CH2NH2 (18)
C(CH3)3
(12)
CH2CH2NH-Boc
CH2CH2NH2 (19)
OH
OH (13)
Scheme 1. Synthetic scheme for the Dmt compound library. Reagents and conditions: (i) SOCl2/CH3OH; (ii) (Boc)2O and Et3N; (iii) NH3/CH3OH;
(iv) HCl/dioxane; (v) HCHO and NaBH3CN; (vi) PyBop, N,N-diisopropylethylamine and NH2–Y; (vii) isobutyl chloroformate, Et3N, and NH2–Y.
The competitive displacement assay15 was performed
using [3H]DAMGO (H-Tyr-D-Ala-Gly-NaMePhe-Gly-
ol) and [3H]DPDPE for MOR and DOR, respectively
(Table 1). The data for Tyr derivatives are not shown
due to negligible affinities (Ki = 3.4–56 lM). Although
the free amino acid H–Dmt–OH (1) was almost inactive
toward both opioid receptors, amidation of the carboxyl
group (2) greatly enhanced receptor affinity, particularly
toward MOR. Since the C-terminal substituent influ-
ences the degree of binding affinity and selectivity be-
tween MOR and DOR,10 the carboxyl moiety was
further modified.
(Ki = 7.45 nM), nearly equivalent to morphine22 and
approximately one-tenth of EM-2,4 however, increasing
the size of the alkyl group decreased MOR affinity in the
series of CH3 (3) > C2H5 (4) > n-propyl (5) > iso-propyl
(6) > tert-butyl (7). Moreover, the introduction of bulky
hydrophobic residues, such as cyclohexyl (8) and ada-
mantyl (9, 10), and aromatic residues, such as phenyl
(11) and naphthyl (12), decreased MOR affinity and in-
creased DOR affinity, such that adamantyl (9, 10), phe-
nyl (11), and naphthyl (12) exhibited DOR selectivity.
The introduction of a polar functional group such as
an OH group (13, 14) decreased MOR affinity in com-
parison to substances 2 and slightly decreased compared
with compound 4, respectively. Furthermore, com-
pound 15, in which the OH group was masked as
OCH3, showed higher receptor affinity than that of 13.
Alkylation of the carboxylamide group of H–Dmt–NH2
further increased the affinity toward both opioid recep-
tors: H–Dmt–NH–CH3 (3) exhibited high MOR affinity
Table 1. Competitive receptor affinities of opioid ligands with the single amino acid Dmt
Compounds
Receptor affinity Kil (nM)a
Receptor affinity Kid (nM)a
Kid/Kil
1
H–Dmt–OH
H–Dmt–NH–X
X = H
145,300 28,800
46,500 11,800
1.55
2
112 6.2
7.45 0.4
15.6 2.7
16.9 0.56
22.4 0.92
36 2.5
1,470 123
460 30
13
3
CH3
C2H5
n-Propyl
61.7
41.6
9.82
22
4
649 119
166 32
492 32
5
6
iso-Propyl
tert-Butyl
Cyclohexyl
1-Adamantyl
2-Adamantyl
Phenyl
7
531 7.0
197 2.0
208 38
14.8
1.71
0.27
0.23
0.069
0.054
5.58
28.1
26.3
0.43
1
8
115 3.8
767 26
746 50
9
10
11
12
13
14
15
16
17
18
19
20
175 29
120 18
1,730 98
966 87
Naphthyl
OH
C2H4OH
52.8 6.5
9,490 2,650
455 38
1,700 246
16.2 3.0
174 15
OCH3
4,580 1,310
1,150 490
328 93
CH2COOH
C2H4COOH
CH2NH2
2,700 214
329 2.45
180 55
2,410 640
3,650 724
1,450 270
13.4
338
C2H4NH2
N,N-(CH3)2–Dmt–NH2
10.8 1.85
216 17
6.71
Morphine22
EM-24
H–Dmt–Tic–OH16b
6.55
0.69
3220
217
9230
0.022
33.1
13400
6.6 · 10ꢀ7
a Values are means of four to seven experiments, Ki values as the mean SE.

Y. Fujita et al. / Bioorg. Med. Chem. Lett. 15 (2005) 599–602
601
Table 2. Analgesia of Dmt compounds relative to morphine in mice
Compounds
MED HP test
(nmol) (%)
TF test
(%)
Antago-
nism (%)
2
3
H–Dmt–NH2
H–Dmt–NH–CH3
200
30
0.26 0.48–0.55 100
0.64–0.85 1.3–1.7 73
The analgesia was determined by hot-plate (HP) and tail-flick (TF)
tests after icv administration. Antagonism was evaluated using
naloxone.
data (Table 1). The maximum effect (Emax) value of
inhibiting electrical induced twitches in GPI assay by
both H–Dmt–Pro–NH2 and H–Dmt–NH2 (2) were
below 50%. H–Dmt–NH–CH3 (3), however, produced
a significant activity with Emax of 66.6%, although its
potency was three orders of magnitude lower than that
of EM-2 and [Dmt1]EM-2. From these results, we can
deduce that these compounds might be weak MOR
antagonists at high doses. Neither H–Dmt–NH2 (2)
nor H–Dmt–NH–CH3 (3) exhibited agonism in the
MVD assay (IC50 > 10,000 nM) and therefore the data
are not shown.
Figure 1. Dose–response curves in the GPI assay. Substances: Ç EM-2
(Emax = 88.0%, IC50 = 5.79 nM);
[Dmt1]EM-2 (Emax = 85.7%,
IC50 = 0.071 nM); d H–Dmt–Pro-NH2 (Emax = 36.9%); m H–Dmt–
NH2 (Emax = 18.2%); H–Dmt–NH–Me (Emax = 66.6%,
IC50 = 6568 nM).
.
j
Antinociception of intracerebroventricularly (icv)
administered H–Dmt–NH2 (2) and H–Dmt–NH–CH3
(3) was measured by the tail-flick (TF) and hot-plate
(HP) tests in mice as described previously.17b While
H–Dmt–NH–CH3 (3) exhibited a 6-fold stronger re-
sponse than that of H–Dmt–NH2 (2), it was only
1.3–1.7% and 0.64–0.85% as effective as morphine in
TF and HP tests, respectively; in both cases naloxone
blocked the response (Table 2), which verifies that the
compound interacted with opioid receptors.17b These
data are consistent with the data of functional bioassays
in GPI suggesting that the 2 and 3 recognizes similar
opioid receptors in in vitro and in vivo described above
to the same degree.
The incorporation of COOH (16, 17), which introduces
a negative charge, also clearly demonstrated a decrease
in MOR affinity since it might repel ionic site of
MOR;23 although both compounds had weak affinities,
they were either slightly DOR selective (16) or nonselec-
tive (17). On the other hand, the incorporation of NH2
to the C-terminus of H–Dmt–NH–C2H5 (4) to produce
H–Dmt–NH–C2H4–NH2 (19), which enhanced MOR
affinity and decreased DOR affinity, resulting in the
highest MOR selective ligand (Kid/Kil = 338), so far
examined. In summary, these data shown in Table 1
confirmed the principle that opioid ligands containing
a C-terminal hydrophobic, aromatic, or negatively
charged group are selective for DOR.10,13,17,23–25 The
alkylation of the amino group, N,N-dimethylation
(20), had a negative effect on opioid receptor interaction,
and produced a compound with a very weak DOR affin-
ity even though Na-alkylation is known to enhance
receptor affinity to DOR sites.23,26 The C-terminal step-
wise deletion of amino acids from [Dmt1]EM-2 de-
creased MOR affinity and the final dipeptide (H–Dmt–
In summary, our data demonstrate for the first time that
a single C-terminally hydrophobically modified amino
acid selectively interacts with MOR. These results sub-
stantiate the flood of data that supports the observation
that Dmt is the key residue responsible for the enhance-
ment of affinity to MOR in opioid ligands which bear
hydrophobic C-terminal substituents.10,16b,17,23–25 In
addition these results provided a clue to develop MOR
antagonists. The presence of Dmt was responsible for
the transmission of opioid mimetic substances through
the blood–brain barrier16b,17 and the further application
of Dmt in the synthesis of opioid peptides will contrib-
ute to the development of additional compounds that
can readily pass through membrane barriers.
Pro–NH2)
exhibited
moderate
MOR
affinity
(Ki = 41.7 nM) that was, however, two orders of magni-
tude weaker than that of the parent analogue.15 Interest-
ingly, although the molecular dimension of H–Dmt–
NH–CH3 (3) is somewhat smaller than that of H–
Dmt–Pro–NH2, it exhibited a 5.6-fold higher MOR
affinity suggesting that the Pro residue interfered with
alignment of the ligand in the receptor pocket.
References and notes
Preparation of GPI (guinea-pig ileum) and MVD
(mouse vas deferens) tissues and stimulation tests were
performed as described previously.15 The results of func-
tional bioactivity in vitro are illustrated in Figure 1. The
agonist potency was ranked in the order of H–Dmt–
NH–CH3 (3) > H–Dmt–Pro-NH215 > H–Dmt–NH2 (2)
and a similar trend was noted with the receptor binding
1. Scientific articles: (a) Cox, B. M.; Goldstein, A.; Li, C. H.
Proc. Natl. Acad. Sci. U.S.A. 1976, 73, 1821–1823; (b)
Ling, N.; Burgus, R.; Guillemin, R. Proc. Natl. Acad. Sci.
U.S.A. 1976, 73, 3042.
2. Goldstein, A.; Tachibana, S.; Lowney, L. H.; Hunkapiller,
M.; Hood, L. Proc. Natl. Acad. Sci. U.S.A. 1979, 77,
6666.

602
Y. Fujita et al. / Bioorg. Med. Chem. Lett. 15 (2005) 599–602
3. Hughes, J.; Smith, T. W.; Kosterlitz, H. W.; Forthergill, L.
A.; Morgan, B. A.; Morris, H. R. Nature 1975, 258,
577.
4. Zadina, J. E.; Hackler, L.; Ge, L. J.; Kastin, A. J. Nature
1997, 386, 499.
15. (a) Okada, Y.; Fujita, Y.; Motoyama, T.; Tsuda, Y.;
Yokoi, T.; Li, T.; Sasaki, Y.; Ambo, A.; Jinsmaa, Y.;
Bryant, S. D.; Lazarus, L. H. Bioorg. Med. Chem. 2003,
11, 1983; (b) Fujita, Y.; Tsuda, Y.; Li, T.; Motoyama, T.;
Takahashi, M.; Shimizu, Y.; Yokoi, T.; Sasaki, Y.; Ambo,
A.; Kita, Atsuko; Jinsmaa, Y.; Bryant, S. D.; Lazarus, L.
H.; Okada, Y. J. Med. Chem. 2004, 47, 3591.
16. (a) Salvadori, S.; Attila, M.; Balboni, G.; Bianchi, C.;
Bryant, S. D.; Crescenzi, O.; Guerrini, R.; Picone, D.;
Tancredi, T.; Temussi, P. A.; Lazarus, L. H. Mol. Med.
1995, 1, 678; (b) Capasso, A.; Guerrini, R.; Balboni, G.;
Sorrentino, L.; Temussi, P.; Lazarus, L. H.; Bryant, S. D.;
Salvadori, S. Life Sci. 1996, 59, PL93.
17. (a) Okada, Y.; Tsuda, Y.; Fujita, Y.; Yokoi, T.; Sasaki,
Y.; Ambo, A.; Konishi, R.; Nagata, M.; Salvadori, S.;
Jinsmaa, Y.; Bryant, S. D.; Lazarus, L. H. J. Med. Chem.
2003, 46, 3201; (b) Jinsmaa, Y.; Miyazaki, A.; Fujita, Y.;
Li, T.; Fujisawa, Y.; Shiotani, K.; Tsuda, Y.; Yokoi, T.;
Ambo, A.; Sasaki, Y.; Bryant, S. D.; Lazarus, L. H.;
Okada, Y. J. Med. Chem. 2004, 47, 2599.
18. Almquist, R. G.; Kadambi, S. R.; Yasuda, D. M.; Weitl,
F. L.; Polgar, W. E.; Toll, L. R. Int. J. Pept. Res. 1989, 34,
455.
5. (a) Erspamer, V.; Melchiorri, P. Trends Pharmacol.
Sci. 1980, 1, 391; (b) Broccardo, M.; Erspamer, V.;
Falconieri; Erspamer, G.; Improta, G.; Linari, G.;
Melchiorri, P.; Montecucchi, P. C. Br. J. Pharmacol.
1981, 73, 625; (c) Montecucchi, P. C.; de Castiglione, R.;
Piani, S.; Gozzini, L.; Erspamer, V. Int. J. Pept. Protein
Res. 1981, 17, 275; (d) Montecucchi, P. C.; de Castiglione,
R.; Erspamer, V. Int. J. Pept. Protein Res. 1981, 17, 316;
(e) Melchiorri, P.; Negri, L. Gen. Pharmacol. 1996, 27,
1099.
6. (a) Kreil, G.; Barra, D.; Simmaco, M.; Erspamer-Falconi-
eri, G.; Melchiorri, P.; Negri, L.; Severini, C.; Corsi, R.
Eur. J. Pharmacol. 1989, 162, 123; (b) Lazarus, L. H.; de
Castiglione, R.; Guglietta, A.; Wilson, W. E. J. Biol.
Chem. 1989, 264, 3047; (c) Erspamer, V.; Melchiorri, P.;
Erspamer-Falconieri, G.; Negri, L.; Corsi, R.; Severini, C.;
Barra, D.; Simmaco, M.; Kreil, G. Proc. Natl. Acad. Sci.
U.S.A. 1989, 86, 5188; (d) Lazarus, L. H.; Bryant, S. D.;
Cooper, P. S.; Salvadori, S. Prog. Neurobiol. 1999, 57,
377.
19. DeBons, F. E.; Loudon, G. M. J. Org. Chem. 1980, 45,
1704.
7. Meunier, J.-C.; Mollereau, C.; Toll, L.; Suaudeau, C.;
Moisand, C.; Alvinerie, P.; Butour, J.-L.; Guillemot, J.-C.;
Ferrara, P.; Monsarrat, B.; Mazarguil, H.; Vassart, G.;
Parmentier, M.; Costentin, J. Nature 1995, 377, 532.
8. (a) Schwyzer, R. Biochemistry 1986, 25, 6335; (b) Por-
toghese, P. S. Trends. Pharmacol. Sci. 1989, 10, 230.
9. Dygos, J. H.; Yonan, E. E.; Scaros, M. G.; Goodmonson,
O. J.; Getman, D. P.; Periana, R. A.; Beck, G. R.
Synthesis 1992, 741.
10. Bryant, S. D.; Jinsmaa, Y.; Salvadori, S.; Okada, Y.;
Lazarus, L. H. Biopolymers (Pept. Sci.) 2003, 71, 86.
11. Sasaki, Y.; Suto, T.; Ambo, A.; Ouchi, H.; Yamamoto, Y.
Chem. Pharm. Bull. 1999, 47, 1506.
20. Rf = 0.49 (CHCl3–CH3OH–AcOH = 8:3:1), Rf = 0.60 (n-
BuOH–AcOH–H2O = 4:1:5), MNa+ 169.1, MK+ 185.0;
1H NMR (CDCl3): d 5.10 (1H, br, NH), 3.61–3.79 (2H, m,
CH2), 3.11 (2H, t, J = 5.7 Hz, NH2), 1.45 (9H, s,
C(CH3)3).
21. Saari, W. S.; Schwering, J. E.; Lyle, P. A.; Smith, S. J.;
Engelhardt, E. L. J. Med. Chem. 1990, 33, 97.
¨
22. Spetea, M.; Toth, F.; Schutz, J.; Otvo¨s, F.; Toth, G.;
¨
´
´
Benyhe, S.; Borsodi, A.; Schmidhammer, H. Eur. J.
Neurosci. 2003, 18, 290.
23. Salvadori, S.; Balboni, G.; Guerrini, R.; Tomatis, R.;
Bianchi, C.; Bryant, S. D.; Cooper, P. S.; Lazarus, L. H. J.
Med. Chem. 1997, 40, 3100.
12. Hansen, D. W., Jr.; Stapelfeld, A.; Savage, M. A.;
Reichman, M.; Hammond, D. L.; Haaseth, R. C.;
Mosberg, H. I. J. Med. Chem. 1992, 35, 684.
24. Balboni, G.; Guerrini, R.; Salvadori, S.; Bianchi, C.; Rizzi,
D.; Bryant, S. D.; Lazarus, L. H. J. Med. Chem. 2002, 45,
713.
13. Schiller, P. W.; Nguyen, T. M.-D.; Berezowska, I.;
Dupuis, S.; Weltrowska, G.; Chung, N. N.; Lemieux, C.
Eur. J. Med. Chem. 2000, 35, 895.
14. Guerrini, R.; Capasso, A.; Sorrentino, L.; Anacardio, R.;
Bryant, S. D.; Lazarus, L. H.; Attila, M.; Salvadori, S.
Eur. J. Pharmacol. 1996, 302, 37.
25. Balboni, G.; Salvadori, S.; Guerrini, R.; Negri, L.;
Gionnini, E.; Jinsmaa, Y.; Bryant, S. D.; Lazarus, L. H.
J. Med. Chem. 2002, 45, 5556.
26. Summers, M. C.; Hayes, R. J. The interaction of Na-
alkylenkephalins with opiate receptors. J. Biol. Chem.
1981, 256, 4951.