Synthesis of 3,6-bis[H-Tyr/H-Dmt-NH(CH2)m,n]-2(1H)pyrazinone derivatives: Function of alkyl chain length on opioid activity

Article abstract of DOI:10.1016/j.bmcl.2006.08.079

Dimeric opioid analogues linked to a pyrazinone platform, 3-[Tyr/Dmt-NH(CH₂)_m]-6-[Tyr/Dmt-NH(CH₂)_n]-2(1H)-pyrazinone (m, n = 3 or 4), were synthesized. The Tyr-containing compound (m = 4, n = 3) exhibited μ-rece

Full text of DOI:10.1016/j.bmcl.2006.08.079

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5793–5796
Synthesis of 3,6-bis[H-Tyr/H-Dmt-NH(CH2)m,n]-2(1H)pyrazinone
derivatives: Function of alkyl chain length on opioid activity
Kimitaka Shiotani,a Tingyou Li,a Anna Miyazaki,b Yuko Tsuda,a,b
Sharon D. Bryant,c Akihiro Ambo,d Yusuke Sasaki,d
Lawrence H. Lazarusc 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
cMedicinal Chemistry Group, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences,
Research Triangle Park, NC 27709, USA
dDepartment of Biochemistry, Tohoku Pharmaceutical University, Aoba-ku, Sendai 981-8558, Japan
Received 11 May 2006; revised 22 July 2006; accepted 17 August 2006
Available online 1 September 2006
Abstract—Dimeric opioid analogues linked to a pyrazinone platform, 3-[Tyr/Dmt-NH(CH2)m]-6-[Tyr/Dmt-NH(CH2)n]-2(1H)-pyr-
azinone (m, n = 3 or 4), were synthesized. The Tyr-containing compound (m = 4, n = 3) exhibited l-receptor affinity (Kil; 7.58 nM)
comparable to that of morphine, while the Dmt derivatives exhibited considerably higher affinity (Kil; 0.021–0.051 nM) with
corresponding agonism (IC50 = 1.79–4.93 nM). Interestingly one compound (m = 4, n = 3) revealed modest d-opioid agonism; the
converse analogue (m = 3, n = 4), however, was inactive in MVD assay.
ꢀ 2006 Elsevier Ltd. All rights reserved.
The N-terminal Tyr residue is essential for the opioid
receptor interactions in peptides,1–6 except nociceptin,7
which contains Phe in lieu of Tyr, and is an integral
component of the ‘message domain’.8 While several ami-
no acids were found to replace Tyr in opioids,9 20,60-di-
methyl-L-tyrosine (Dmt) dramatically enhanced
receptor affinity and functional bioactivity, and consis-
tently altered receptor selectivity. In fact, H-Dmt-NH-
not only high l- and d-opioid affinities, but also unique
in vitro functional biological activity profiles (GPI and
MVD bioassays), and potent in vivo antinociceptive
activity after intracerebroventricular (icv), subctaneous
(sc), and oral (po) administration.16 Thus, we directed
our studies to the further development of 3,6-bis[Tyr-
or Dmt-aminoalkyl]-pyrazinone derivatives for their
application as possible therapeutics. Previously,16 we
synthesized eight kind of 3,6-bis[Tyr- or Dmt-amin-
oalkyl]-pyrazinone derivatives (Fig. 1, m = n: 1–4) and
revealed that 3,6-bis[Tyr or Dmt-aminoalkyl]-pyrazi-
none derivatives (Fig. 1, m = n: 3) exhibited the highest
10
CH3 was found to specifically interact with the l-opi-
oid receptor (Kil = 7.45 nM), although H-Tyr-NH-CH3
interacted with neither l- nor d-opioid receptors
(Kil = 23,000 nM); however, H-Dmt-NH-CH3 could
not trigger a biological reaction. Incorporation of
Dmt into opioid ligands greatly increased biological
activity as seen with Dmt-Tic pharmacophore,11 endo-
HO
R
morphins,12,13 [Dmt1,Leu5]-enkephalin,
bis[Dmt-
14
O
NH]alkane,15 and the symmetrically substituted
3,6-bis[Dmt-aminoalkyl]-pyazinone derivatives.16 The
3,6-bis[Dmt-aminoalkyl]-pyazinone analogues exhibited
(
)m
N
NH HCl
R
N
H
H
R
N
(
)n
N
H
O
HCl HN
O
R
OH
5 m=3, n=4, R=H
6 m=3, n=4, R=CH3
7 m=4, n=3, R=H
8 m=4, n=3, R=CH3
Keywords: 20,60-Dimethyl-L-tyrosine; Pyrazinone; l-Selective opioid
ligand.
*
Corresponding author. Tel.: +81 78 974 1551; fax: +81 78 974
5689; e-mail: okada@pharm.kobegakuin.ac.jp
Figure 1. Structure of compounds 5–8.
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.079

5794
K. Shiotani et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5793–5796
l-opioid affinity (Kil = 25.7 and 0.042 nM for Tyr and
Dmt derivative, respectively); and the compound
(m = n: 4), followed the above compounds (Kil = 70.2
and 0.114 nM for Tyr and Dmt derivative, respectively).
To determine the effect of different chain lengths of the
aminoalkyl linkers of pyrazinone derivatives on opioid
properties and to develop more potent opioidmimetic
analgesics, the unsymmetrical aminoalkyl linkers (m,
n = 3 or 4) were covalently bound at the positions 3
and 6 of pyrazinone ring platform (Fig. 1, 5–8), which
could provide a further basis for uncovering the discrete
and subtle differences in opioidmimetic substances to
produce biological effect.
(Kid/Kil = 1519). Similarly, the affinity of 5 increased
537-fold upon replacement of Tyr by Dmt (6), compara-
ble to published data;16 however, in the case of 1,4-bis-
[Dmt-NH]butane, the relative activity rose to 7500,15
while in the case of endomorphin-2, RA value was only
4.6.12 These results further support the observations that
Dmt in opioidmimetics plays an important role to
anchoring the compounds within opioid receptors to
elicit a higher degree of binding12–16 and suggest that
an unsymmetrical alkyl chain at position 3 or 6 of the
pyrazinone ring produced a more favorable conforma-
tion for affinity and selectivity to the receptor. The
d-opioid receptor affinity of 5 and 7 is essentially non-ex-
istent, whereas the Dmt derivatives (6 and 8) exhibited
Kid = 18.8 and 31.9 nM, respectively, with relative
activity of 147- and 29-fold, respectively. Interestingly,
although Dmt derivatives increased affinities compared
with those of the corresponding Tyr derivatives for both
l- and d-opioid receptors, they are still l-selective opioid
ligands.
Compounds were synthesized as shown in Scheme 1
starting from dipeptidyl chloromethyl ketones.17 After
removal of Boc group of dipeptidyl chloromethyl ke-
tone, the resulting hydrochlorides were treated in aceto-
nitrile at 60 ꢁC to afford Z-protected pyrazinone
derivative, in which the different and desired aminoalkyl
moieties are covalently bound to positions 3 and 6.
Z-Protection of compounds was removed by HBr/
AcOH to release the amine groups, which were then
coupled with Boc-Tyr-OH or Boc-Dmt-OH18 using
PyBop reagent to produce asymmetrical Boc-protected
3,6-bis(Tyr- or Dmt-aminoalkyl)-pyrazinone derivatives
(1–4). The Boc group was removed by 7 N HCl/dioxane
to give the 3,6-bis(H-Tyr- or H-Dmt-aminoalkyl)-pyraz-
inoneÆ2HCl (5–8). The crude final products were purified
by semi-preparative HPLC [column: YMC pack R&D
ODS (4.6 · 250 mm) in an initial acetonitrile/water
gradient (10:90) for 20 min to 50:50 for 40 min and
finally 90:10 for 5 min]. The identification and purity
of the final compounds were assessed using MS, 1H
and 13C NMR, analytical HPLC, and elemental analy-
sis.19 The compounds exhibited greater than 98% purity.
In the functional bioactivity12 (Table 1), 6 and 8 exhib-
ited l-agonism and they were either inactive (6) or
exhibited weak agonism (8); however, even at this level,
the maximum inhibition of muscle contraction was only
60% in the MVD assay. Other Dmt opioid deriva-
tives12,15,16 were reported to exhibit full l-agonist activ-
ity, but [Dmt1]-endomorphin-2 was a full l and d
agonist.12 While the differences in the opioid receptor
affinity between 6 and 8 are not significant, the function-
al bioactivity revealed a major difference in the effect of
the length of the aminoalkyl chain and its position on
the pyrazinone ring; namely, relative to 6, the agonism
elicited by 8 for l- and d-opioid receptors more than
doubled and increased by over 40-fold, respectively.
Thus, from the data on asymmetrical pyrazinone
derivatives (5–8), differences in not only chain lengths,
but also the position (3 or 6) on the pyrazinone ring
to which aminoalkyl chains are bound, affected bio-
logical activity and allowed the opioidmimetics to dif-
ferentially interact with the ligand-binding domains of
l- and d-opioid receptors. These data also opened fur-
ther possibility for rational design of potential opioid
mimetic drugs that are more l-selective. It is also
emphasized that our rapid and facile synthetic proce-
dure of pyrazinone ring from dipeptidyl chloromethyl
ketones20 could provide the asymmetric pyrazinone
derivatives.
The competitive displacement assay12 was performed
using [3H]DAMGO (H-Tyr-D-Ala-Gly-Na-MePhe-Gly-
ol) and [3H]deltorphin II for l- and d-opioid receptors,
respectively. The affinities are summarized in Table 1
with the relative activity (RA, the difference between
Tyr and Dmt). Of the Tyr dimers, 7 (m = 4, n = 3) exhib-
ited a higher l-receptor affinity (Kil 7.38 nM), similar to
that of morphine with a higher receptor selectivity
(Kid/Kil = 220) compared with
a previous report
(Kil = 25.7–460 nM and Kid/Kil = 4.0–31).16 Substitu-
tion by Dmt (8) enhanced l-opioid receptor affinity
361-fold (Kil = 0.021 nM) with higher selectivity
O
C
O
C
(
)m
N
H
C
H
Boc
N
N
O
O
C
H
Boc-Orn(Z)-Lys(Z)-CH2Cl
H
H
N
(
)m
iii, iv
i
i, ii
Boc
C
N
CH
CH2
N
NHZ
5, 6, 7, 8
CH2
or
(
)n
N
H
O
ZHN
R
R
Boc-Lys(Z)-Orn(Z-CH2Cl
(
)n
N
H
O
R
R
1
2
3
4
:R=H, m=3, n=4
:R=CH3 , m=3, n=4
:R=H, m=4, n=3
:R=CH3 , m=4, n=3
OH
OH
Scheme 1. Synthesis of bis-Tyr-pyrazinone (5 and 7) or bis-Dmt-pyrazinone opioidmimetics (6 and 8). Reagents and conditions: (i) 7 N HCl/dioxane;
(ii) CH3CN, 60 ꢁC; (iii) 25% HBr/AcOH; (iv) Boc-Tyr-OH or Boc-20,60-dimethyl-L-tyrosine (Boc-Dmt-OH), PyBop, DIEA, DMF;
Z = benzyloxycarbonyl.

K. Shiotani et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5793–5796
5795
Table 1. The competitive opioid receptor affinities and functional bioactivities of compounds 5–8
R
N
(
)
m
H
N
H
N
(
)n
N
O
R
H
Compounds
Receptor affinity
Ki (nM)
Receptor
selectivity
Functional bioactivity
IC50 SE (nM)
l
(n)
RA
d
(n)
RA
Ki d/Ki l
GPI assaya
MVD assaya
5 R = Tyr, m = 3, n = 4
6 R = Dmt, m = 3, n = 4
7 R = Tyr, m = 4, n = 3
8 R = Dmt, m = 4, n = 3
27.4 5.2
(5)
(5)
(5)
(5)
1
537
1
2770 446
18.8 2.9
924.4 125
31.9 2.9
(5)
(3)
(4)
(5)
1
147
1
65
369
0.051 0.009
7.58 0.21
0.021 0.003
4.93 1.73
1.79 0.57
>10,000b
220
1519
361
29
25.8 4.3c
Displacement of [3H]DAMGO (l-selective) and [3H]deltorphin II (d-selective) from rat synaptosomes (P2 fraction).16 Values are means SE with (n)
the number of independent repetitions. (RA: relative activity of Dmt derivative compared with the corresponding Tyr derivatives.)
a The data are means of over five independent repetitions that used different isolated tissue preparations.
b Partial agonist (max inhibition 44%).
c Partial agonist (max inhibition 60%).
Y.; Bryant, S. D.; Lazarus, L. H.; Okada, Y. Bioorg. Med.
Chem. Lett. 2005, 15, 599.
Acknowledgments
11. 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.
12. 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.
13. Jinsmaa, Y.; Marczak, E.; Fujita, Y.; Shiotani, K.;
Miyazaki, A.; Li, T.; Tsuda, Y.; Ambo, A.; Sasaki, Y.;
Bryant, S. D.; Okada, Y.; Lazarus, L. H. Pharmaco.
Biochem. Behavior 2006, in press.
14. Sasaki, Y.; Suto, T.; Ambo, A.; Ouchi, H.; Yamamoto, Y.
Chem. Pharm. Bull. 1999, 47, 1506.
15. 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.
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16. Jinsmaa, Y.; Miyazaki, A.; Fujita, Y.; Li, T.; Fujisawa, Y.;
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Bryant, S. D.; Lazarus, L. H.; Okada, Y. J. Med. Chem.
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17. Okada, Y.; Fukumizu, A.; Takahashi, M.; Yamazaki, J.;
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18. Dygos, J. H.; Yonan, E. E.; Scaros, M. D.; Goodmomson,
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19. Physicochemical data of compounds 5–8. 6-[40-(H-Tyr)-
aminobutyl]-3-[30-(H-Tyr)-amino-propyl]-5-methyl-2(1H)-
This work was supported in part by a grant from Kobe
Gakuin University to K.S. and the Intramural Research
Program of the NIH, and NIEHS.
References and notes
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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,
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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.;
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U.S.A. 1989, 86, 5188; (d) Lazarus, L. H.; Bryant, S. D.;
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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. Li, T.; Fujita, Y.; Tsuda, Y.; Miyazaki, A.; Ambo, A.;
Sasaki, Y.; Jinsmaa, Y.; Bryanat, S. D.; Lazarus, L. H.;
Okada, Y. J. Med. Chem. 2005, 48, 586.
20:2
pyrazinone hydrochloride (5): Yield 5.0 mg (65.0%), ½aꢀD
+39.9ꢁ (c 0.1, MeOH), Rf (n-BuOH/AcOH/pyridine/
H2O = 4:1:1:2) 0.32, tR 20.7 (min), TOF-MS m/z 565.50
(M+1)+. Calcd 564.68. HPLC conditions: column, YMC
pack R&D ODS (4.6 · 250 mm); detection: 220 nm, purity
(99.7%). Anal. Calcd for C30H40N6O5Æ2HClÆ5.6ÆH2O: C,
50.4; H, 6.96; N, 11.0. Found: C, 50.3; H, 6.64; N, 10.9. 6-
[40-(H-Dmt)-aminobutyl]-3-[30-(H-Dmt)-aminopropyl]-5-
methyl-2(1H)-pyrazinone hydrochloride (6): Yield 9.0 mg
22:1
(59.5%), ½aꢀD +32.4ꢁ (c 0.1, MeOH), Rf (n-BuOH/AcOH/
pyridine/H2O = 4:1:1:2) 0.64, tR 31.9 (min), TOF-MS m/z:
622.05 (M+1)+. Calcd 621.78. HPLC conditions: column,
YMC pack R&D ODS (4.6 · 250 mm); detection: 220 nm,
purity (99.1%). Anal. Calcd for C34H48N6O5Æ2HClÆ5.5-
H2O: C, 47.2; H, 6.42; N, 9.71. Found. C, 47.0; H, 6.23; N,
9.99.
3-[40-(H-Tyr)-aminobutyl]-6-[30-(H-Tyr)-amino-propyl]-5-
methyl-2(1H)-pyrazinone hydrochloride (7): Yield 5.0 mg
10. Fujita, Y.; Tsuda, Y.; Motoyama, T.; Li, T.; Miyazaki, A.;
Yokoi, T.; Sasaki, Y.; Ambo, A.; Niizuma, H.; Jinsmaa,

5796
K. Shiotani et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5793–5796
21:2
25
(6.021%), ½aꢀD +3.58ꢁ (c 0.1, MeOH), Rf (n-BuOH/
AcOH/pyridine/H2O = 4:1:1:2) 0.42, tR: 19.1 (min),
TOF-MS m/z: 565.50 (M+1)+. Calcd 564.68. HPLC
7.0 mg (48.2%), ½aꢀD +76.3ꢁ (c 0.1, MeOH), Rf (n-BuOH/
AcOH/pyridine/H2O = 4:1:1:2) 0.48, tR: 25.7 (min), TOF-
MS m/z: 621.88 (M+1)+. Calcd 621.78. HPLC conditions:
column, YMC pack R&D ODS (4.6 · 250 mm); detection:
220 nm, purity (98.3%). Anal. Calcd for C34H48N6O5Æ2H-
ClÆ6H2O: C, 50.9; H, 7.49; N, 10.5. Found: C, 50.7; H,
7.23; N, 10.1.
conditions: column, YMC pack R&
D
ODS
(4.6 · 250 mm); detection: 220 nm, purity (99.7%). Anal.
Calcd for C30H40N6O5Æ2HClÆ5.5 H2O: C, 50.4; H, 6.96; N,
11.0. Found: C, 50.4; H, 6.74; N, 11.4.
3-[40-(H-Dmt)-aminobutyl]-6-[30-(H-Dmt)-aminopropyl]-
5-methyl-2(1H)-pyrazinone hydrochloride (8): Yield
20. Okada, Y.; Taguchi, H.; Nishiyama, Y.; Yokoi, T.
Tetrahedron Lett. 1994, 35, 1231.