Design and synthesis of opioidmimetics containing 2′,6′-dimethyl-l-tyrosine and a pyrazinone-ring platform

Article abstract of DOI:10.1016/j.bmcl.2007.08.058

Twelve 2′,6′-dimethyl-l-tyrosine (Dmt) analogues linked to a pyrazinone platform were synthesized as 3- or 6-[H-Dmt-NH(CH₂)_n],3- or 6-R-2(1H)-pyrazinone (n = 1-4). 3-[H-Dmt-NH-(CH₂)₄]-6-β-phenethyl-5-methyl-2(1H)-pyrazinone 11 bound to μ-opioid receptors with high affinity (K_iμ = 0.13 nM; K_iδ/K_iμ = 447) with μ-agonism (GPI IC₅₀ = 15.9 nM) and weak δ-antagonism (MVD pA₂ = 6.35). Key factors affecting opioid affinity and functional bioactivity are the length of the aminoalkyl chain linked to Dmt and the nature of the R residue. These data present a simplified method for the formation of pyrazinone opioidmimetics and new lead compounds.

Full text of DOI:10.1016/j.bmcl.2007.08.058

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5768–5771
Design and synthesis of opioidmimetics containing
20,60-dimethyl-L-tyrosine and a pyrazinone-ring platform
Kimitaka Shiotani,a Tingyou Li,a Anna Miyazaki,b Yuko Tsuda,a,b Toshio Yokoi,a,b
Akihiro Ambo,c Yusuke Sasaki,c Sharon D. Bryant,d
Lawrence H. Lazarusd and Yoshio Okadaa,b,
*
aThe Graduate School of Food and Medicinal Sciences, Kobe Gakuin University, Nishi-ku, Kobe 651-2180, Japan
bFaculty of Pharmaceutical Sciences, Kobe Gakuin University, Nishi-ku, Kobe 651-2180, Japan
cDepartment of Biochemistry, Tohoku Pharmaceutical University, Aoba-ku, Sendai 981-8558, Japan
dMedicinal Chemistry Group, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences,
Research Triangle Park, NC 27709, USA
Received 19 June 2007; revised 16 August 2007; accepted 24 August 2007
Available online 28 August 2007
Abstract—Twelve 20,60-dimethyl-L-tyrosine (Dmt) analogues linked to a pyrazinone platform were synthesized as 3- or 6-[H-Dmt-
NH(CH2)n],3- or 6-R-2(1H)-pyrazinone (n = 1–4). 3-[H-Dmt-NH-(CH2)4]-6-b-phenethyl-5-methyl-2(1H)-pyrazinone 11 bound to
l-opioid receptors with high affinity (Kil = 0.13 nM; Kid/Kil = 447) with l-agonism (GPI IC50 = 15.9 nM) and weak d-antagonism
(MVD pA2 = 6.35). Key factors affecting opioid affinity and functional bioactivity are the length of the aminoalkyl chain linked to
Dmt and the nature of the R residue. These data present a simplified method for the formation of pyrazinone opioidmimetics and
new lead compounds.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The N-terminal Tyr residue is essential for the opioid
receptor interactions in opioid peptides,1–6 except noci-
ceptin,7 which contains Phe instead of Tyr. It is well
known that replacement of Tyr in opioid peptides and
opioidmimetics with 20,60-dimethyl-L-tyrosine (Dmt)
dramatically enhances receptor affinity and functional
bioactivity.8 While H-Dmt-NHCH3 specifically interacts
with l-opioid receptor (Kil = 7.45 nM), indicating that
Dmt itself could act as a part of message domain, it
was insufficient to trigger a biological reaction.9 Re-
cently, the dimerization of the Dmt pharmacophore
through diaminoalkane10 or diaminoalkyl-pyrazinone,11
exhibited high l-affinity and potent in vitro and in vivo
functional bioactivity. It could be assumed that one
Dmt residue interacts within message-binding domain,
while another might lie in the address domain of the
receptor.
The antinociceptive activity of one of Dmt-pyrazinone
dimers, 3-[40-(H-Dmt)-aminobutyl]-6-[30-(H-Dmt)-ami-
nopropyl]-5-methyl-2(1H)-pyrazinone was 65–71 times
greater than with morphine after icv administration in
mice based on the tail-flick and hot-plate tests.12 On
the other hand, after sc administration it was equivalent
to morphine in tail-flick test and 89% as effective by the
hot-plate test; however, after oral administration it
exhibited only 65% and 16% the activity of morphine
on these tests, respectively.12 The results indicated a de-
gree of enzymatic stability and the ability to transit epi-
thelial membranes through the gastrointestinal tract and
blood–brain barrier.12–14 Thus, the pyrazinone-ring may
be an ideal platform on which to develop stable opioids
with sufficient lipophilicity suitable for clinical and ther-
apeutic applications.
To further examine the role of the pyrazinone-ring in
opioidmimetics, we prepared compounds (1–12) as
shown in Figure 1 and examined their receptor affinities
and in vitro functional bioactivities.
Keywords: 20,60-Dimethyl-L-tyrosine; Pyrazinone; l-Selective opioid
ligand; l-Agonist/d-antagonist; l-Antagonist/d-antagonist.
Corresponding author. Tel.: +81 78 974 1551; fax: +81 78 974
5689; e-mail: okada@pharm.kobegakuin.ac.jp
*
Compounds (1–12) were synthesized according to
Scheme 1 starting from dipeptidyl chloromethyl ketones
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.08.058

K. Shiotani et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5768–5771
5769
N
R2
O
ring. Compound 3 exhibited the highest l-affinity, indi-
cating that the H-Dmt-NH-(CH2)4-moiety is the most
suitable derivative among compounds 3,5–7. Compound
1 (R1 = H) exhibited subnanomolar l-affinity, indicating
that the pyrazinone-ring played a role to enhance affin-
ity. Although the Kil of 9 (R1 = benzyl) is only 1.5-fold
higher than that of 12 (R1 = 20,60-dimethylbenzyl), the
increase in hydrophobicity introduced by dimethylation
on phenyl-ring of benzyl moiety at position 6 did not
substantially or significantly affect l-affinity. Compound
11 (R1 = b-phenethyl) exhibited the highest l-affinity
(Kil = 0.125 nM) of the substances in Table 1. The phe-
nyl moiety at R1 contributed to increased l-affinity.
( )n
Dmt
N
H
N
N
H
( )
R1
N
H
O
nN
H
Dmt
2 n = 4, R2 = H
1 n = 4, R1 = H
4 n = 4, R2 = CH3
3 n = 4, R1 = CH3
5 n = 3, R1 = CH3
6 n = 2, R1 = CH3
7 n = 1, R1 = CH3
8 n = 3, R1 = benzyl
9 n = 4, R1 = benzyl
10 n = 3, R1 = β-phenethyl
11 n = 4, R1 = β-phenethyl
12 n = 4, R1 = 2',6'-dimethylbenzyl
Figure 1. The structure of Dmt-pyrazinone derivatives (1–12).
The GPI (guinea-pig ileum) and MVD (mouse vas def-
erens) functional bioactivity assays were performed as
described previously.19 The results are summarized in
Table 1. All the compounds exhibited a relatively weak
d-antagonism (pA2 = 5.5 to 6.61) with very low agonist
potency (less than 34% inhibition at a dose of 10 lM).
(1a–l).15 After removal of the Boc group from the dipep-
tidyl chloromethyl ketones (1a–l), the resulting amine
hydrochlorides were treated in methanol at 60 ꢁC to give
Z-protected pyrazinone derivatives (2a–l), in which the
different and desired moieties are covalently bound to
positions 3 and 6. Z-Protection was removed by HBr/
CH3COOH to release the amine group, which was then
coupled with Boc-Dmt-OH8b,16 using PyBop to produce
Boc-protected Dmt analogues containing a pyrazinone-
ring (3a–l). The Boc group was removed by 7 M HCl/
dioxane to give the crude final compounds as hydrochlo-
ride salt (1–12), which were purified by semi-preparative
HPLC [column: Cosmosil ODS (20 · 250 mm) in an ini-
tial 0.05% TFA acetonitrile/0.05% TFA water gradient
(10:90) to (90:10) for 80 min, flow rate: 10 mL/min].
The identification and purity of the final compounds
Compound 11 (n = 4, R1 = b-phenethyl) exhibited
mixed l-agonism/d-antagonism. The phenyl moiety at
R1 of 11 might be able to bind to the l-opioid receptor
as part of the address domain of the ligand. While 10
(n = 3, R1 = b-phenethyl) and 12 (n = 4, R1 = 20,60-dim-
ethylbenzyl) had very weak l-agonism (IC50 = 5547 and
6273 nM, respectively), 1-9 exhibited essentially no l-
agonism with inhibition of less than 47% at a dose of
10 lM. Interestingly, only compounds 2–4, 10 and 12
behaved as weak antagonists toward the l-opioid recep-
tor as well, which were not previously observed with
other Dmt-pyrazinone analogues.10–12
1
were assessed using MS, H and 13C NMR, analytical
HPLC,17 and elemental analysis.18 The compounds
exhibited greater than 98% purity.
In the series of opioidmimetics which contains a single
Dmt residue, l-affinity is consistently lower than that
of the opioidmimetic analogues with two Dmt resi-
dues.10–12 In the case of Dmt dimers, both Dmt residues
might participate in binding to the l-opioid receptor as
constituents of the message and address domains, while
in the Dmt monomeric analogues, the Dmt residue
could act by anchoring the compound only within l-opi-
oid receptors. The distance between the two Dmt phar-
macophores in the dimerized ligands might be better
accommodated in the l-opioid receptor. On the other
hand, the distance between Dmt and another aromatic
The radioreceptor assay19 used [3H]DAMGO (H-Tyr-D-
Ala-Gly-N-MePhe-Gly-ol) and [3H]deltorphin II for l-
and d-opioid receptors, respectively. The affinities of
compounds 1–12 are summarized in Table 1. Com-
pounds 1, 9, 10, 11 and 12 exhibited subnanomolar affin-
ities (Kil). All the compounds exhibited l-selectivity
(Kid/Kil = 1.2–447). Compounds 1 and 3 exhibited 92-
fold and 3.6-fold higher l-affinity relative to 2 and 4,
respectively, indicating that the Dmt residue at position
3 is preferable relative to position 6 of the pyrazinone-
H H O H H O
N
R3
Boc N C C N C C CH2Cl
i, ii
R3
R4
R4
N
H
O
1a-l
2a-l
R3, R4 = H, CH3, benzyl, β-phenethyl,
2',6'-dimethylbenzyl or Z-NH-(CH2)1-4
iii, iv
N
R5
O
R5, R6 = H, CH3, benzyl, β-phenethyl,
2',6'-dimethylbenzyl or Boc-Dmt-NH-(CH2)1-4
R6
i
N
H
R2
O
N
3a-l
R1, R2 = H, CH3, benzyl, β-phenethyl,
2',6'-dimethylbenzyl or H-Dmt-NH-(CH2)1-4
R1
N
H
1-12
Scheme 1. Synthetic method for pyrazinone-ring containing opioidmimetics (1–12). Reagents and condition: (i) 4MHCl/dioxane; (ii) CH3OH, at
60 ꢁC; (iii) 25% HBr/CH3COOH; (iv) Boc-Dmt-OH, PyBop, DIPEA.

5770
K. Shiotani et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5768–5771
Table 1. Opioid receptor binding affinity and functional bioactivity of compounds (1–12)
b
c
b
MVD IC50 (nM)
c
Compound
DAMGOd
Kil (nM) (n)a
Kid (nM) (n)a
Kid/Kil GPI IC50 (nM)
pA2
pA2
2.29
130
0.24 0.06 (6)
57
1135e
11.5
—
—
—
76
0.14 0.06
>10,000
—
—
Deltorphin IIe 272 50 (11)
420 95
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
5547 741
15.9 2.8
6273 895
1
0.62 0.068 (6)
56.9 5.30 (3)
4.23 0.65 (3)
15.4 1.36 (3)
8.88 0.77 (3)
76.5 9.20 (7)
73.80 6.40 (5) 119
343.7 26.0 (5)
77.7 6.1 (4)
(47.6%)
(25.7%) <5.5
(28.1%) <5.5
(26.2%) 5.60
2
6
18
(37.1%) <6.0 >10,000
(35.1%) 6.70 >10,000
(30.1%) <6.0 >10,000
3
4
208.5 23.0 (6) 14
269.4 29.0 (3) 30
93.10 16.0 (5) 1.2
(0.0%)
5.63
5
(22.7%)
(45.0%)
(20.6%)
(36.7%)
(39.5%)
—
—
—
—
—
>10,000
>10,000
>10,000
>10,000
>10,000
(31.1%) <5.5
(25.2%) 5.87
(28.1%) 5.83
(34.6%) 5.91
(19.9%) 6.23
6
7
129.1 19.00 (3) 698.1 30.0 (5) 5.4
8
1.00 0.02 (3)
0.42 0.01 (3)
0.55 0.09 (3)
59.60 5.30 (4) 60
9
8.60 0.70 (3)
38.2 2.60 (3)
20
69
447
8
10
11
12
<6.0 >10,000
>10,000
<6.0 >10,000
(4.1%)
(12.8%) 6.35
(6.4%) 6.61
5.87
0.125 0.002 (3) 55.9 8.30 (3)
0.66 0.05 (5) 5.3 0.8 (5)
—
—, denotes no antagonism.
a Repetitions (n) are 5–7 times for each bioassay.
b Agonists inhibited the electrically evoked twitch (IC50). Values in parentheses indicate maximal inhibition of the tissue contraction at the con-
centration of 10,000 nM. Endomorphin 2 and deltorphin II were used as the l- and d-opioid receptor agonist standard peptides, respectively.
c The pA2 (antagonism) is the negative logarithm (M = concentration) required to double the concentration of a d-opioid receptor agonist (deltorphin
II) in MVD assay or of a l-opioid receptor agonist (endomorphin 2) in GPI assay to achieve the original response.
d DAMGO is the standard l-selective opioid agonist (data from Ref. 21).
e Deltorphin II is a d-selective (Kil/Kid = 1135) amphibian opioid agonist (data from Ref. 22).
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residue or the residue itself might not be appropriate for
binding to the l-opioid receptor. While l-antagonists
are important pharmacological tools, not only to delin-
eate critical biochemical, pharmacological, and physio-
logical roles played by these receptors, but also to
serve as clinically and therapeutically relevant agents,20
compounds 10 and 12 may be lead compounds for
the further development of new l-opioid receptor
antagonists.
Acknowledgments
This work was supported in part by a grant from Kobe
Gakuin University to K.S., in part by the Intramural
Research Program of the NIH, and NIEHS, and in part
by Grant-in-Aid for Scientific Research and by ‘Aca-
demic Frontier’ Project for Private Universities: match-
ing fund subsidy from the Japanese Ministry of
Education, Culture, Sports, Science and Technology,
2006–2010.
References and notes
10. Okada, Y.; Tsuda, Y.; Fujita, Y.; Yokoi, T.; Sasaki, Y.;
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11. Jinsmaa, Y.; Miyazaki, A.; Fujita, Y.; Li, T.; Fujisawa, Y.;
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17. The analytical HPLC condition for Compounds 1–12.
HPLC conditions: column, Cosmosil ODS (4.6 ·
250 mm); solvents program, A: 0.05%TFA (trifluoroacetic
acid)/water, B: 0.05%TFA/acetonitrile, A:B (90:10) to A:B
(10:90) in 40 min; flow rate: 1 mL/min; detection: UV
220 nm.
18. Elemental analysis data of compounds 1–12. 3-[40-(H-
Dmt)-aminobutyl]-5-methyl-2(1H)-pyrazinone hydrochlo-
ride (1): Calcd for C20H28N4O3 Æ HCl Æ 2.5 H2O: C, 47.2;
H, 6.85; N, 10.0. Found:C, 46.9; H, 6.93; N, 10.3. 6-[40-(H-
Dmt)-aminobutyl]-5-methyl-2(1H)-pyrazinone hydrochlo-
ride (2): Calcd for C20H28N4O3 Æ HCl Æ H2O: C, 46.9; H,
6.93; N, 11.0. Found:C, 46.5 H, 6.88, N, 10.7. 3-[40-(H-
Dmt)-aminobutyl]-5,6-dimethyl-2(1H)-pyrazinone hydro-
chloride (3): Calcd for C21H30N4O3 Æ HCl Æ 2.5H2O: C,
50.6; H, 6.93; N, 10.3. Found:C, 50.3; H, 7.13, N, 10.7.
6-[40-(H-Dmt)-aminobutyl]-3,5-dimethyl-2(1H)-pyrazi-
none hydrochloride (4): Calcd for C21H30N4O3 Æ HCl Æ 4-
H2O: C, 48.3; H, 6.87; N, 9.79. Found:C, 48.5 H, 6.82, N,
10.0. 3-[30-(H-Dmt)-aminopropyl]-5,6-dimethy-2(1H)-pyr-
azinone hydrochloride (5): Calcd for C20H28N4O3 Æ HCl Æ
H2O: C, 48.9; H, 6.53; N, 10.5. Found:C, 49.0; H, 6.38; N,
10.4. 3-[20-(H-Dmt)-aminoethyl]-5,6-dimethy-2(1H) pyr-
azinone hydrochloride (6): Calcd for C19H26N4O3 Æ H-
Cl Æ 3H2O: C, 47.9; H, 6.32; N, 10.7. Found:C, 47.9; H,
6.42; N, 10.6. 3-[(H-Dmt)-aminomethyl]-5,6-dimethy-
2(1H)-pyrazinone
hydrochloride
(7):
Calcd
for
C18H24N4O4 Æ HCl Æ 2H2O: C, 51.9; H, 7.01; N, 13.4.
Found:C, 51.7 H, 6.93, N, 13.5. 6-Benzyl-3-[30-(H-Dmt)-
aminopropyl]-5-methyl-2(1H)-pyrazinone hydrochloride
(8): Calcd for C26H32N4O3 Æ HCl Æ 3H2O: C, 54.9; H,
6.37; N, 9.09. Found:C, 54.5; H, 6.31, N, 8.83. 6-Benzyl-
3-[40-(H-Dmt)-aminobutyl]-5-methyl-2(1H)-pyrazinone
hydrochloride (9): Calcd for C27H34N4O3 Æ HCl Æ H2O: C,
62.7; H, 7.21; N, 10.8. Found:C, 62.9; H, 7.23; N, 11.2. 6-
b-phenethyl-3-[30-(H-Dmt)-aminopropyl]-5-methyl-2(1H)-
pyrazinone hydrochloride (10): Calcd for C27H34N4O3 Æ
HCl Æ 2.5H2O: C, 59.6; H, 7.41; N, 10.3. Found:C, 59.6; H,
6.99; N, 10.7. 6-b-phenethyl-3-[40-(H-Dmt)-aminobutyl]-5-
methyl-2(1H)-pyrazinone hydrochloride (11): Calcd for
C28H36N4O3 Æ HCl Æ 2H2O: C, 61.3; H, 7.53; N, 10.2.
Found: C, 61.5; H, 7.66; N, 9.81. 3-[40-(H-Dmt)-amin-
obutyl]-6-[20,60-dimethylbenzyl]-5-methyl-2(1H)-pyrazi-
none hydrochloride (12): Calcd for C29H38N4O3Æ
HCl Æ 1.3H2O: C, 60.2; H, 6.88; N, 9.22. Found: C, 60.2;
H, 6.95; N, 9.25.
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