Oral Bioavailability of a New Class of μ-Opioid Receptor Agonists Containing 3,6-Bis[Dmt-NH(CH2)n]-2(1H)-pyrazinone with Central-Mediated Analgesia

Article abstract of DOI:10.1021/jm0304616

The inability of opioid peptides to be transported through epithelial membranes in the gastrointestinal tract and pass the blood-brain barrier limits their effectiveness for oral application in an antinociceptive treatment regime. To overcome this limitat

Full text of DOI:10.1021/jm0304616

J . Med. Chem. 2004, 47, 2599-2610
2599
Or a l Bioa va ila bility of a New Cla ss of µ-Op ioid Recep tor Agon ists Con ta in in g
3,6-Bis[Dm t-NH(CH2)n ]-2(1H)-p yr a zin on e w ith Cen tr a l-Med ia ted An a lgesia
Yunden J insmaa, Anna Miyazaki,† Yoshio Fujita,† Tingyou Li,‡ Yutaka Fujisawa,† Kimitaka Shiotani,†
Yuko Tsuda,‡,† Toshio Yokoi,‡,† Akihiro Ambo,§ Yusuke Sasaki,§ Sharon D. Bryant, Lawrence H. Lazarus,*, and
Yoshio Okada*,†,‡
Medicinal Chemistry Group, LCBRA, National Institutes of Environmental Health Sciences,
Research Triangle Park, North Carolina 27709, Faculty of Pharmaceutical Sciences,
Department of Medicinal Chemistry, and High Technology Research Center, Kobe Gakuin University,
Nishi-ku, Kobe 651-2180, J apan, and Department of Biochemistry, Tohoku Pharmaceutical University,
4-1, Komatsushima 4-chome, Aoba-ku, Sendai 981-8558, J apan
Received September 16, 2003
The inability of opioid peptides to be transported through epithelial membranes in the
gastrointestinal tract and pass the blood-brain barrier limits their effectiveness for oral
application in an antinociceptive treatment regime. To overcome this limitation, we enhanced
the hydrophobicity while maintaining the aqueous solubility properties in a class of opioid-
mimetic substances by inclusion of two identical N-termini consisting of Dmt (2′,6′-dimethyl-
L-tyrosine) coupled to a pyrazinone ring platform by means of alkyl chains to yield the class of
3,6-bis[Dmt-NH-(CH2)n]-2(1H)-pyrazinones. These compounds displayed high µ-opioid receptor
affinity (Kiµ ) 0.042-0.115 nM) and selectivity (Kiδ/Kiµ ) 204-307) and functional µ-opioid
receptor agonism (guinea-pig ileum, IC50 ) 1.3-1.9 nM) with little or undetectable bioactivity
toward δ-opioid receptors (mouse vas deferens) and produced analgesia in mice in a naloxone
reversible manner when administered centrally (intracerebroventricular, icv) or systemically
(subcutaneously and orally). Furthermore, the most potent compound, 3,6-bis(3’-Dmt-amino-
propyl)-5-methyl-2(1H)-pyrazinone (7′), lacked functional δ-opioid receptor bioactivity and was
50-63-fold and 18-21-fold more active than morphine by icv administration as measured
analgesia using tail-flick (spinal involvement) and hot-plate (supraspinal effect) tests,
respectively; the compound ranged from 16 to 63% as potent upon systemic injection. These
analgesic effects are many times greater than unmodified opioid peptides. The data open new
possibilities for the rational design of potential opioid-mimetic drugs that pass through the
epithelium of the gastrointestinal tract and the blood-brain barrier to target brain receptors.
In tr od u ction
onslaught of a broad spectrum of proteolytic enzymes2
and specific peptidases, termed the “blood-brain enzy-
matic barrier” that acts as another active deterrent in
the cerebral microvasculature.5
A major concern in the application of opioid peptides
as pharmacological drugs for clinical or therapeutic
situations, such as the amelioration of pain associated
with postsurgery procedures, cancer, or birth, involves
their bioavailability.1 The oral bioavailability of bioac-
tive peptides requires not only a means to overcome
physiological and metabolic barriers, but also to be able
to transit the physical barriers encompassed in the
epithelial membrane of the gastrointestinal tract2 and
at the microcapillary-brain junction, known as the
blood-brain barrier (BBB).3,4 In general, this involves
the exclusionary properties of membrane tight junctions
associated with cellular and tissue components.2 Fur-
thermore, passage through these barriers requires
overcoming the physicochemical features of peptides,
which includes their metabolic liability, charge at physi-
ological pH, lipophilicity, molecular weight, and struc-
ture.2,4 In regard to stability, peptides must survive the
In addition to the necessity for enzymic stability, the
passage of peptides through membranes involves tissue
and cellular transport mechanisms that also depend on
the specific peptide in question.3 Of the known transport
systems by which peptides enter and exit membranes,3
the most common include passive diffusion,2 endocytotic
mechanisms,2 such as pinocytosis and transcytosis,6 and
carrier-mediated, energy dependent active transport
systems3 that involve peptide transporters,7 particle
transporters,7 and lipid-absorption pathways.2 Further-
more, the ability of a peptide to pass through the BBB
appears to be mediated by an uptake mechanism that
is correlated with its lipid solubility.2,4 One character-
istic of cerebral endothelium is that it acts as an
effective barrier to penetration by hydrophilic mol-
ecules,3 which is one rationale for the extensive covalent
modification of opioid peptides in order to enhance their
overall hydrophobic properities,8-16 although intramo-
lecular hydrogen bonding was also suggested to explain
membrane transit by nonsaturable transmembrane
diffusion.17
* To whom correspondence should be addressed. E-mail:
okada@pharm.kobegakuin.ac.jp or lazarus@niehs.nih.gov.
National Institutes of Environmental Health Sciences.
† Faculty of Pharmaceutical Sciences, Department of Medicinal
Chemistry, Kobe Gakuin University.
‡ High Technology Research Center, Kobe Gakuin University.
§ Tohoku Pharmaceutical University.
10.1021/jm0304616 CCC: $27.50 © 2004 American Chemical Society
Published on Web 03/30/2004

2600 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
J insmaa et al.
Ra tion a le
The inclusion of Dmt into a variety of opioid peptides
in lieu of Tyr as the N-terminal residue brought about
marked changes in their physical and biological proper-
ties.18 Dmt was responsible for the change in peptide
conformation, as indicated by low-energy conformational
searching paradigms and supported by the X-ray dif-
fraction analysis of the crystalline structures of three
analogues with distinct bioactivities.19 The passage of
the bis[Dmt-NH]-alkyl compounds through the BBB
appears to involve the increased hydrophobicity at-
tributed to the two methyl groups in Dmt since the Tyr
analogues were inactive,20 and that apparently differs
from effects observed with H-Dmt-Tic-OH.21 An anal-
gesic endpoint is crucial in identifying the biological
effect elicited by opioid peptides to provide inferential
and intuitive evidence on the composition of the com-
pounds that enable them to transit the BBB. In this
communication, we report the substitution of Dmt for
Tyr in the series of symmetric 3,6-bis[Tyr-NH-(CH2)n]-
2(1H)-pyrazinone substances (Figure 1) that not only
increased receptor affinity and selectivity to the µ-opioid
receptor by several orders of magnitude but also pro-
duced antinociception following a regime of central,
systemic or oral administration.
Ch em istr y
Enantiomerically pure 2′,6′-dimethyl-L-tyrosine (Dmt)
was prepared according to Dygos et al.22 Enantiomeric
purity (>98%) was ascertained both by HPLC using a
chiral column [CROWNPAC CR(+)] and by the reaction
with D- and L-amino acid oxidases followed by amino
acid analysis. Boc-Dmt-OH and Boc-Dmt-NHNH2 were
prepared as described.23 The 3,6-bis(aminoalkyl)-5-
methyl-2(1H)-pyrazinones were synthesized by pub-
lished procedures.23 The Boc-X(Z)-OH intermediates
were coupled with H-X(Z)-CH2Cl by a mixed anhydride
method to produce Boc-X(Z)-X(Z)-CH2Cl [2a : X ) Dap
(2,3-diaminopropionic acid);24 2b: X ) Dab (2,4-diami-
nobutyric acid);24 2c: X ) Orn; 2d : X ) Lys]. After
removal of Boc groups by HCl in dioxane, the corre-
sponding amine hydrochloride in MeOH or THF was
heated at a reflux for 1 h to produce the protected
pyrazinone derivatives, which were then converted to
the corresponding amine23 by catalytic hydrogenation
or by HBr/AcOH (Scheme 1). The resulting amines were
coupled with Boc-Tyr-NHNH2 or Boc-Dmt-NHNH2 by
use of azide,25 or with Boc-Tyr-OH or Boc-Dmt-OH using
Bop reagent,26 to produce protected 1′-8′ (5a -d and
6a -d ). The products were treated with TFA to give
crude 1′-8′ (Scheme 2). All final products were purified
by semipreparative reverse-phase HPLC; each com-
pound exhibited a single peak on analytical HPLC with
a unique retention time.20 Analysis by MALDI-TOF
mass spectrometry (MS), 1H and 13C NMR, and by
HPLC revealed that they were the desired compounds
with greater than 98% purity. These data are sum-
marized in Supporting Information (Table 1).
F igu r e 1. (a) Structure of 3,6-bis[Tyr-NH(CH2)n]-2(1H)-
pyrazinone or 3,6-bis[Dmt-NH(CH2)n]-2(1H)-pyrazinone where
n ) 1-4 and R ) H (Tyr) or CH3 in Dmt. (b) A low energy
representation of 3,6-bis[Dmt-NH-(CH2)3]-2(1H)-pyrazinone
(7′). Carbon atoms are green, nitrogens blue and oxygens red;
hydrogen atoms are not displayed. Only one possible confor-
mation is shown since many low energy structures exist. In
fact, due to the large number of number rotatable alkyl (C-
C) bonds, the necessity to access g+, g-, and trans orientations
associated with the alkyl groups, as well as to take into account
the side chains of Dmt, selecting several low energy structures
to serve as starting structures would be extremely difficult if
not imprecise. Each of those structures in turn can generate
thousands of conformations, such that both the starting and
final structures will be based on assumptions about the best
orientations of the alkyl bonds and not necessarily depict the
conformation bound to the receptor.
3,6-bis[Z-NH(CH2)n]-2(1H)-pyrazinones, the Z groups
were removed by catalytic hydrogenation over a Pd cat-
alyst (Scheme 1: 4c and 4d ;23 n ) 3 and 4, respectively).
Studies on the hydrogenation of Z-protected pyrazinone
derivatives revealed that deamination occurred at posi-
tion 6 of compound 3a [Scheme 1: 3,6-bis(Z-NHCH2)-
2(1H)-pyrazinone] due to the benzylic or allylic proper-
ties of the CN bond at position 6.27 Because of the
similarity in the CN bond at position 6 of 3b [Scheme
1: 3,6-bis(Z-NHC2H4)-2(1H)-pyrazinone], which also has
homobenzylic or homoallylic properties, catalytic hy-
drogenation was avoided and 25% HBr/AcOH was
employed to remove Z groups. Furthermore, Boc-Tyr-
N2H3 or Boc-Dmt-N2H3 was coupled with 3,6-bis[NH2-
(CH2)n]-2(1H)-pyrazinones (Scheme 2: 4a and 4b, n )
1 and 2, respectively) by an azide coupling procedure,25
because 4a and 4b have a tendency to form pyrazinol-
type rather than pyrazinone-type rings (data not shown);
on the other hand, Boc-Tyr-OH or Boc-Dmt-OH was
In the synthesis of the (1H)-pyrazinone derivatives,
it was necessary to take specific precautions in the
removal of the Z protecting groups. For example, to
produce 3,6-bis[NH2(CH2)n]-2(1H)-pyrazinones (Scheme
1: 4a and 4b: n ) 1 and 2, respectively), the Z groups
were removed by 25% HBr/AcOH; however, with the

Oral Bioavailability of µ-Opioid Receptor Agonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2601
Sch em e 1
Sch em e 2
coupled with 4c and 4d 23 by using Bop reagent.26 These
procedures provided high quality products: each of the
compounds (1′-8′) exhibited single peak on reverse
phase HPLC with greater than 98% purity.
Ta ble 1. Opioid Receptor Interactions of
3,6-Bis[Tyr-NH(CH2)n]-2(1H)-pyrazinone (1′-4′) and
3,6-Bis[Dmt-NH(CH2)n]-2(1H)-pyrazinone (5′-8′)a
no. (CH2)n
Kiµ (nM)
n
Kiδ (nM)
n
Kiδ/Kiµ
Resu lts
1′
2′
3′
4′
5′
6′
7′
8′
1
2
3
4
1
2
3
4
460 ( 14
309 ( 11.2
25.7 ( 3.0
3
3
5
3
3
3
3
3
1830 ( 520
2900 ( 590
435 ( 84
2190 ( 130
15.7 ( 2.1
7.26 ( 1.2
13.2 ( 1.7
23.2 ( 2.5
3
4
4
3
3
4
3
3
4.0
9.4
17
31
13.5
63
Op ioid Recep tor Affin ity (Ki). The replacement of
Tyr (1′-4′) by Dmt (5′-8′) to yield 3,6-bis[Dmt-NH-
(CH2)n]-2(1H)-pyrazinone compounds dramatically in-
creased the affinity to both δ- and µ-opioid receptors,
but predominately the latter by several orders of
magnitude (6′-8′, Kiµ ) 0.042-0.115 nM) and elevated
the selectivity for µ-opioid receptors (7′ and 8′, δ/µ )
307 and 204, respectively) (Table 1). Compound 7′ [3,6-
bis(3′-Dmt-aminopropyl)-5-methyl-2(1H)-pyrazinone] ex-
hibited the highest affinity (Kiµ ) 0.042 nM) and was
ca. 3-fold greater than that of either 6′ [3,6-bis(2′-Dmt-
aminoethyl)-5-methyl-2(1H)-pyrazinone] or 8′ [3,6-bis-
(4′-Dmt-aminobutyl)-5-methyl-2(1H)-pyrazinone] and
nearly 30 times that of 5′ [3,6-bis(Dmt-aminomethyl)-
5-methyl-2(1H)-pyrazinone]. Thus, the length of the
interposing alkyl chain determines efficacy of receptor
binding: propyl > ethyl, butyl . methyl.
70.2 ( 4.8
1.16 ( 0.18
0.115 ( 0.009
0.042 ( 0.003
0.114 ( 0.008
307
204
a
Binding affinities are given as Ki values determined using
[3H]DPDPE for δ-opioid receptors and [3H]DAGO for µ-opioid
receptors (Experimental Section). the mean ( SE is based on three
to five independent assays (n) conducted in duplicate using five
to seven dosages of each compound. as seen in Figure 1a, (CH2)n
represents the n number of methylene units in the linkage between
the pyrazinone ring and the N-terminal Dmt residues.
pig ileum (GPI) and mouse vas deferens (MVD) char-
acterizes the action of a ligand as having agonist or
antagonist activity. The data verify that the bis-Dmt-
derivatives (5′-8′) are biologically active (Table 2) and
generally reflect the values obtained for the affinity
constants (Table 1). The weak Ki values of the Tyr
F u n ction a l Bioa ctivity in Vitr o. The in vitro
functional bioactivity with isolated tissues from guinea-

2602 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
J insmaa et al.
OH21 suggested this might involve the formation of a
lipid-soluble diketopiperazine29 analogous to TRH in the
formation of the His-Pro diketopiperazine.3 Owing to the
high opioid receptor affinity and bioactivity of 7′ (Tables
1-3), it is equally plausible that only a small proportion
of the administered compound might have crossed the
luminal membrane and the BBB,3 or they might be
relatively more stable in vivo than natural opioid
peptides, which have relatively short half-lives (in the
range of a few minutes).30,31 However, since a dif-
ferential analgesic sensitivity exists in mice strains,32
it may be also possible that bioactivity expressed by 7′
might exhibit similar differences. However, neither the
mode of transit across membrane barriers nor its
stability is addressed in this report.
Ta ble 2. Functional Bioactivity of
3,6-Bis[Dmt-NH(CH2)n]-2(1H)-pyrazinone Compounds 5′-8′a
IC50, nM (mean ( SE)
no. (CH2)n
GPI (µ)
MVD (δ)
antagonism pA2 (δ)
5′
6′
7′
8′
1
2
3
4
1695 ( 365 >10000
12.9 ( 2.4 >10000
1.33 ( 0.2 >10000
6.47
6.56
none
none
1.90 ( 0.67
41.5 ( 10.4
a
In vitro bioactivity was determined as given in the Experi-
mental Section. Repetitions are 5-7 for each bioassay. Agonists
inhibited the electrically evoked twitch (IC50); deltorphin C and
dermorphin were used as the δ- and µ-opioid receptor agonist
standard peptides, Respectively. the pA2 defines antagonism and
is the negative log(M concentration) required to double the
concentration of a δ-opioid receptor agonist (deltorphin C) to
achieve the original response. From Figure 1a, (CH2)n is the
number of n methylene units in the sequence.
Interestingly, hydrophobic analogues of the Dmt-Tic
pharmacophore, which should be readily transmitted
through the BBB, selectively inhibited the action of the
membrane-bound hMDR-1 P-glycoprotein in vitro.33
cognates precluded any determination of their bioactiv-
ity. Not only was 7′ the most biologically active (GPI,
IC50 ) 1.33 nM) and also more potent than the bis[Dmt-
NH]-alkyl compounds (GPI, IC50 ) 3.08-5.33 nM),20 but
also it was a µ-opioid receptor agonist without measur-
able δ-opioid receptor bioactivity (Table 2). However,
compound 8′, which only had 30% less µ-opioid receptor
agonism than 7′, exhibited moderate δ-opioid receptor
agonism (IC50 ) 41.5 nM), while 5′ and 6′ exerted weak
δ-opioid receptor antagonism (pA2 ) 6.56 and 6.47,
respectively) similar to that of the bis[Dmt-NH]-alkyl
analogues (pA2 ) 6.4-5.5).20
An tin ocicep tion in Mice. Compound 7′ produced
analgesia in vivo following icv administration in mice
as well as by subcutaneous (sc) injection and per oral
(po) lavage (Figures 2-4). The effect was 50-fold and
ca. 20-fold more potent than morphine in the tail-flick
(Figure 2a,b) and hot-plate tests (Figure 3a,b), respec-
tively, following icv administration. Naloxone (2 mg/kg)
injected sc 30 min before 7′ suppressed the effect in both
tests (Figure 2c,d and Figure 3c,d), suggesting the effect
is mediated by opioid receptors. Injection of 7′ by sc and
po routes exhibited a dose dependent analgesia in both
tests (Figure 4). The bioactivity data are summarized
in Table 3 relative to the action of morphine, which
served as a positive control.
P yr a zin on e-Con ta in in g Op ioid -Mim etic Com -
p ou n d s. The 3,6-bis[Tyr-NH-[(CH2)n]-2(1H)-pyrazinone
compounds weakly interact with opioid receptors,24,34
while the bis[Dmt-NH]-alkyl compounds provided evi-
dence that a substance with two Dmt residues at the
N-termini exhibited high µ-opioid receptor affinity and
agonism.20 In comparison to the 3,6-bis[Tyr-NH-(CH2)n]-
2(1H)-pyrazinone derivatives (1′-4′) (Table 1), the
inclusion of Dmt (5′-8′) enhanced affinity to µ-opioid
receptors by factors of 400-, 2,700-, 612- and 616-fold
relative to the Tyr cognates (1′-4′), respectively. The
Dmt-containing compounds were µ-opioid receptor ago-
nists with minimal δ-opioid activity except 8′, which had
modest δ-opioid receptor agonism. Comparable to the
data on the bis[Dmt-NH]-alkyl compounds,20 the length
of the alkyl linker, in this case between the pyrazinone
ring and the aromatic residue, markedly affects receptor
interaction. Furthermore, the difference of a single
methylene group between these opioid-mimetic com-
pounds substantially changes their bioactivity profile,
which is similar to the observations with the H-Dmt-
Tic-NHCH(R)-R′ substances.35
Bifu n ction a l Op ioid -Mim etic P ep tid es. Earlier
studies on opioid peptides with two identical N-termini
were derived by tail-to-tail dimerization of enkephalin36
and dermorphin;37 in both cases, they exhibited in-
creased activity toward opioid receptors. Generally, the
increase in affinity and biological activity of bis-der-
morphin37 as well as bis[D-Ala2,Leu5]-enkephalin,36 bis-
[D-Ala2-des-Leu5]-enkephalin,36 and a cystamine-en-
kephalin dimer38 was slightly more potent for both µ-
and δ-opioid receptors. The enhanced activity might be
accounted for by their interaction with two opioid
receptors in close proximity or they induced receptor
aggregation due to their increased length.36 On the other
hand, the relatively small dimensions of 7′ or the bis-
[Dmt-NH]-alkyl compounds20 suggest that they bind
within a single µ-opioid receptor pocket (unpublished
observations).
Discu ssion
System ic a n d Or a l Bioa va ila bility of Op ioid -
Mim etic Com p ou n d s. Our results unequivocally dem-
onstrated that an unmodified opioid-mimetic compound
(7′) administered systemically and orally produced
analgesia through a naloxone reversible mechanism
(Figures 2-4). This implied that without chemical
modification to enhance its physicochemical prop-
erties,8-16 compound 7′ crossed epithelial membrane
barriers in both the intestine and mirocapillaries in
mouse brain. Furthermore, absorption on nanoparticles
coated with polysorbate 807 was also unwarranted.
Passage of opioids through the BBB by a nonsaturable
(passive diffusion) mechanism of the metabolic stable
µ-opioid receptor agonist [D-Arg2,Lys4]dermorphin ana-
logue (DALDA)4 or NR-1-iminoethyl-Tyr-D-MetO-Phe-
MeâAla-OH16 correlated with their lipid solubility,
whereas the δ-opioid receptor agonist DPDPE appeared
to be transmitted through the BBB by “an energy-
dependent transcytotic mechanism”.28 The appearance
of analgesia by systemically administered H-Dmt-Tic-
Con clu sion s
Changes in the number of methylene units (n ) 1-4)
on either side of the pyrazinone ring between the Dmt
N-termini produced opioid-mimetic substances with

Oral Bioavailability of µ-Opioid Receptor Agonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2603
F igu r e 2. Effect of intracerebroventricular administrated 3,6-bis(3′-Dmt-aminopropyl)-5-methyl-2(1H)-pyrazinone (7′) and
morphine on tail flick (spinal analgesia) latency in mice. Compounds dissolved in physiological saline (pH 7) were injected icv
into male Swiss-Webster mice (20-25 g). Morphine (morph.) was used as a positive control; the antagonist naloxone was applied
to counter the effect and to determine if analgesia involved opioid receptors.20 The area under the curve (AUC) is derived from
data based on the response (mean ( SE) of five to seven mice per time point. The dose response curve is seen in (a) [( ) saline;
O ) 2.04 ng 7′; 9 ) 6.8 ng 7′; b ) 20.4 ng 7′; 4 ) morphine (0.5 µg)] with the AUC (b). Asterisks (*) denote values that are
significantly different (ANOVA) from the control mice (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Dose-response curves with or
without naloxone are in (c) [( ) saline; b ) 20.4 ng 7′; O ) 7′+ 2 mg naloxone injected sc 30 min before 7′; 4 ) morphine (0.5 µg);
2 ) morphine + naloxone] and with the AUC (d). The symbol ### denotes the statistical differences between the effect of 7′ or
morphine treatment relative to naloxone-treated mice (### p < 0.001).
F igu r e 3. Effect of intracerebroventricularly injected 3,6-bis(3′-Dmt-aminopropyl)-5-methyl-2(1H)-pyrazinone (7′) and morphine
on hot-plate (supraspinal analgesia) latency. Complete details are given in the legend to Figure 2. Statistical significances: (b)
**, p < 0.01; (d) ###, p < 0.001.
distinct binding affinities to µ- and δ-opioid receptors,
different bioactivity profiles, and analgesia following icv,
sc, and po administration. Compound 7′ traversed the
epithelial tissue in the gastrointestinal tract, trans-
ported by the blood plasma to the brain to where it
passed through the BBB to interact with brain µ-opioid

2604 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
J insmaa et al.
F igu r e 4. Effect of subcutaneously injected and orally administrated 3,6-bis(3′-Dmt-aminopropyl)-5-methyl-2(1H)-pyrazinone
(7′) and morphine in tail-flick and hot-plate tests. The dose-response curves for sc injection determined in the tail-flick test is in
(a) [( ) saline; O ) 1 mg/kg 7′; 4 ) 3 mg/kg 7′; 9 ) 10 mg/kg 7′; b ) 3 mg/kg morphine] and with the AUC (b). Per oral dose-
response curves (c) [( ) saline; 2 ) 10 mg/kg 7′; O ) 30 mg/kg 7′; 0 ) 100 mg/kg 7′; 9 ) 10 mg/kg morphine] and AUC (d) (***,
p < 0.001). The analgesia represented by the hot-plate tests is in parts e and f. Complete details are given in the legend to Figure
2. Statistical significance: (b, d, and f) *, p < 0.05, **, p < 0.01, ***, p < 0.001.
Ta ble 3. Biological Potency of
8′) greatly affected opioid receptor binding affinities and
in vitro bioactivity profiles, which might provide clues
on the design of new opioid agonists. In light of the oral
3,6-Bis(3′-Dmt-aminopropyl)-5-methyl-2(1H)-pyrazinone (7′)
Relative to Morphine in Micea
mode of delivery
tail flicka
hot platea
bioavailability of 7′, further studies are underway to
evaluate its therapeutic advantages and differences
relative to morphine in terms of tolerance required to
determine its potential use in clinical approaches in the
treatment of postoperative or cancer pain in humans,
pain associated with birthing, or as a possible veterinary
drug. Our results open the way for the rational design
of a new class of synthetic opioid-mimetic substances
that target brain opioid receptors.39
intracerebroventricular
subcutaneous
per oral
50-63
0.63
0.42
18-21
0.55
0.16-0.24
a
These values were derived from concentrations of 7′ that
produced an equivalent analgesia as morphine.
receptors. Interestingly, 7′ was systemically 5- to 6-fold
more potent than the bis[Dmt-NH]-alkyl class of opioid
compounds,20 which suggests that the pyrazinone ring
might confer enhanced structural and chemical proper-
ties permitting greater absorption through tissues. We
can only conjecture at this point whether the binding
of the pyrazinone derivatives occurs in an analogous
manner to the bis[Dmt-NH]-alkyl compounds20 or other
opioid substances for that matter since the presence of
the pyrazinone ring in the molecule introduces an
unknown factor. Nonetheless, we observed that the
presence of Dmt and different length alkyl chains (5′-
Exp er im en ta l Section
Melting points were determined on a Yanagimoto micro-
melting point apparatus and are uncorrected. Optical rotations
were measured with an automatic polarimeter, model DIP-
1000 (J apan Spectroscopic Co.). Mass spectra were measured
1
with a KRATOS-MALDI TOF-MS. H (400 MHz or 500 MHz)
and 13C (100 MHz or 125 MHz) NMR spectra were recorded
on a Bruker DPX-400 or ARX-500 spectrometers, respectively.
Purified compounds (30 mg) were dissolved in 1.0 mL pyridine-

Oral Bioavailability of µ-Opioid Receptor Agonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2605
d5 (99.9% isotopic purity). Chemical shift values are expressed
as ppm downfield from tetramethylsilane, used as an internal
standard (δ-value), and the J values are given in hertz. The
13C signals were assigned with the aid of distortionless
enhancement by polarization transfer (DEPT) and two-
dimensional experiments, and multiplicities are indicated by
p (primary), s (secondary), t (tertiary), or q (quaternary).
Reversed phase HPLC analysis used Waters model 600E (A)
or Waters model delta 600 (B) with a COSMOSIL C18-AR-II
(4.6 × 250 mm) column in the following solvent systems: A,
0.05% TFA in water; B, 0.05% TFA in CH3CN. The retention
time was reported as tR (min). On TLC (Kieselgel G 60, Merck),
Rf values refer to the following systems: (A) AcOEt-hexane
(1:1), (B) CHCl3-MeOH-AcOH (90:8:2), (C) CHCl3-AcOEt-
EtOH (6:4:1), (D) n-BuOH-AcOH-pyridine-water (4:1:1:2),
(E) n-BuOH-AcOH-pyridine-water (1:1:1:1), and (F) CHCl3-
MeOH-water (8:3:1). Open column chromatography was run
on silica gel 60 (70-230 mesh, YMC).
Syn th esis of Boc-X(Z)-CH2Cl (1a -c, X: a ) Da p , b )
Da b, or c ) Or n ). To a solution of mixed anhydride [prepared
from Boc-X(Z)-OH (13.6 mmol), isobutyl chloroformate (16.4
mmol) and Et3N (16.4 mmol)] in THF (50 mL) was added
diazomethane in ether (60 mL) [prepared from p-toluensulfo-
nyl-N-methyl-N-nitrosoamide (40.9 mmol)]. The reaction mix-
ture was stirred at 4 °C overnight and 6.9 N HCl/dioxane (34.1
mmol) added to the solution under cooling with an ice-salt
solution. The reaction mixture was stirred at -15 °C for 15
min before water and ice were added to the reaction mixture,
and the product was extracted with AcOEt. The extract was
washed with water, 5% NaHCO3, and saturated sodium
chloride solution, dried over Na2SO4, and evaporated. The
residue was crystallized from petroleum ether, and the crystals
were collected by filtration. Yield, mp, [R]25D, Rf and elemental
analyses of 1a -c are summarized in Supporting Information
(Table 2).
Boc-Da p (Z)-CH2Cl (1a ). 1H NMR (CDCl3) δ 1.44 (s, 9H,
tert-butyl), 3.59 (m, 1H, â-CH2), 3.70 (br, 1H, â-CH2), 4.30 and
4.42 (AB-q, J ) 15.6 Hz, 2H, COCH2Cl), 4.59 (br, 1H, R-CH),
5.07 (s, 2H, benzyl), 5.22 (br, 1H, â-NH), 5.69 (d, J ) 6.0 Hz,
1H, R-NH), 7.31-7.37 (m, 5H, ArH); 13C NMR (CDCl3) δ 28.3
(p, tert-butyl), 41.9 (s, â-CH2), 46.5 (s, COCH2Cl), 58.2 (t,
R-CH), 67.3 (s, benzyl), 80.7 (q, tert-butyl), 128.1, 128.3 and
128.6 (t, aryl C), 136.9 (q, aryl C), 155.5 and 157.1 (q × 2),
202.3 (q, COCH2Cl).
Boc-Da b(Z)-CH2Cl (1b). 1H NMR (CDCl3) δ 1.44 (s, 9H,
tert-butyl), 1.67 (br, 1H, â-CH2), 2.09 (m, 1H, â-CH2), 3.11 (br,
1H, γ-CH2), 3.47 (br, 1H, γ-CH2), 4.23 (s, 2H, COCH2Cl), 4.57
(br, 1H, R-CH), 5.10 (s, 2H, benzyl), 5.45 (br, 2H, γ-NH and
R-NH), 7.31-7.36 (m, 5H, ArH); 13C NMR (CDCl3) δ 28.4 (p,
tert-butyl), 31.7 (s, â-CH2), 37.1 (s, γ-CH2), 46.5 (s, COCH2Cl),
55.0 (t, R-CH), 66.9 (s, benzyl), 80.7 (q, tert-butyl), 128.3-128.6
(t, aryl C), 136.5 (q, aryl C), 157.7 and 159.1 (q × 2), 202.1 (q,
COCH2Cl).
residue was crystallized from ether, and the crystals were
collected by filtration. The crude product was purified by
column chromatography. Yield, mp, [R]25D, Rf, and elemental
analyses of 2a -c are summarized in Supporting Information
(Table 2).
1
Boc-Da p (Z)-Da p (Z)-CH2Cl (2a ). H NMR (DMSO-d6 con-
taining 1% pyridine-d5) δ 1.38 (s, 9H, tert-butyl), 3.24-3.58
(m, 4H, â-CH2), 4.03 (br, 1H, R-CH), 4.40 (br, 1H, R-CH), 4.53
and 4.60 (AB-q, 2H, J ) 16.8 Hz, COCH2Cl), 5.02 (s, 2H,
benzyl), 5.03 (s, 2H, benzyl), 6.90 (d, J ) 6.7 Hz, 1H, R-NH),
7.14 (br, 1H, â-NH), 7.23 (br, 1H, â-NH), 7.29-7.37 (m, 10H,
ArH), 8.54 (d, J ) 7.1 Hz, 1H, R-NH); 13C NMR (CDCl3) δ 28.0
(p, tert-butyl), 40.1 (s, â-CH2), 41.6 (s, â-CH2), 47.9 (s, COCH2-
Cl), 54.9 (t, R-CH), 56.7 (t, R-CH), 65.5 (s, benzyl), 78.6 (q, tert-
butyl), 127.6-128.3 (t, aryl C), 136.8 (q, aryl C), 136.9 (q, aryl
C), 155.3, 156.3 and 170.9 (q × 3), 199.2 (q, COCH2Cl).
1
Boc-Da b(Z)-Da b(Z)-CH2Cl (2b). H NMR (CDCl3) δ 1.42
(s, 9H, tert-butyl), 1.77-2.21 (m, 4H, â-CH2), 3.11 (br, 1H,
γ-CH2), 3.28 (m, 1H, γ-CH2), 3.52 (br, 1H, γ-CH2), 3.63 (d, J )
10.8 Hz, 1H, COCH2Cl), 3.73 (m, 1H, γ-CH2), 4.09-4.30 (m,
2H, COCH2Cl and R-CH), 4.73 (br, 1H, R-CH), 5.08-5.16 (m,
4H, 2 × benzyl), 5.40 (br, 2H, γ-NH and R-NH), 6.89 (br, 1H,
R-NH), 7.29-7.37 (m, 10H, ArH); 13C NMR (CDCl3) δ 27.9 (s,
â-CH2), 28.3 (p, tert-butyl), 33.9 (s, â-CH2), 37.9 (s, γ-CH2), 44.8
(s, COCH2Cl), 45.5 (s, γ-CH2), 52.2 (t, R-CH), 52.6 (t, R-CH),
67.1 (s, benzyl), 67.3 (s, benzyl), 80.3 (q, tert-butyl), 127.8-
128.7 (t, aryl C), 136.0 (q, aryl C), 136.3 (q, aryl C), 154.5, 156.9
and 172.0, (q × 3), 199.0 (q, COCH2Cl).
Boc-Or n (Z)-Or n (Z)-CH2Cl (2c). 1H NMR (CDCl3) δ 1.42
(s, 9H, tert-butyl), 1.55 (br, 6H, â-CH2 and 2 × γ-CH2), 1.78-
1.85 (m, 2H, â-CH2), 3.17 (br, 3H, δ-CH2), 3.33 (br, 1H, δ-CH2),
4.21 (s, 2H, COCH2Cl), 4.27 (br, 1H, R-CH), 4.69 (br, 1H,
R-CH), 5.06 (s, 4H, 2 × benzyl), 5.22 (br, 2H, 2 × δ-NH), 5.30
(br, 1H, R-NH), 7.29-7.32 (m, 11H, ArH and R-NH); 13C NMR
(CDCl3) δ 26.2 (s, 2 × γ-CH2), 28.3 (s, â-CH2), 28.4 (p, tert-
butyl), 29.8 (s, â-CH2), 39.7 (s, δ-CH2), 40.2 (s, δ-CH2), 46.6 (s,
COCH2Cl), 53.1 (t, R-CH), 56.0 (t, R-CH), 66.8 (s, 2 × benzyl),
80.1 (q, tert-butyl), 127.8-128.5 (t, aryl C), 136.5 (q × 2, aryl
C), 155.9, 156.8, 157.1 and 172.9 (q × 4), 200.8 (q, COCH2Cl).
Syn th esis of 3,6-Bis[Z-NH(CH2)n]-5-m eth yl-2(1H)-pyr azi-
n on e (3a : n ) 1 or 3c: n ) 3). A solution of HCl‚H-X(Z)-
X(Z)-CH2Cl [prepared from Boc-X(Z)-X(Z)-CH2Cl (1.35 mmol)
and 7.2 N HCl/dioxane (27.1 mmol)] in MeOH (10 mL) was
heated at a reflux for 1 h. After removal of the solvent, the
residue was dissolved in CHCl3, washed with water, 10% citric
acid and water, dried over Na2SO4, and evaporated. The
residue was crystallized from ether, and the crystals were
collected by filtration and recrystallized from MeOH.
3,6-Bis(b e n zyloxyca r b on yla m in om e t h yl)-5-m e t h yl-
2(1H)-p yr a zin on e (3a ). Yield 52.6%, mp 183-187 °C, Rf 0.58
(B). Anal. Calcd for C23H24N4O5‚0.5H2O: C, 62.0; H, 5.66; N,
12.6. Found: C, 62.3; H, 5.47; N,12.7. 1H NMR (pyridine-d5) δ
2.42 (s, 3H, 5-CH3), 4.56 (d, J ) 4.9 Hz, 2H, CH2NH-Z), 4.93
(d, J ) 4.8 Hz, 2H, CH2NH-Z), 5.32 and 5.34 (2s, 4H, benzyl),
7.23-7.48 (m, 10H, ArH), 8.03 (br, 1H, NH-Z), 8.49 (br, 1H,
NH-Z); 13C NMR (pyridine-d5) δ 19.1 (p, 5-CH3), 42.5 and 42.8
(s × 2, 2 × CH2NH-Z), 66.5 (s, benzyl), 66.7 (s, benzyl), 128.1-
128.8 (t, aryl C), 137.8, 138.1, 156.1, and 157.4 (q × 4).
Boc-Or n (Z)-CH2Cl (1c). 1H NMR (CDCl3) δ 1.44 (s, 9H,
tert-butyl), 1.48-1.62 (m, 3H, γ-CH2 and â-CH2), 1.87 (br, 1H,
â-CH2), 3.23 (m, 2H, δ-CH2), 4.24 (s, 2H, COCH2Cl), 4.51 (br,
1H, R-CH), 4.94 (br, 1H, δ-NH), 5.09 (s, 2H, benzyl), 5.18 (br,
1H, R-NH), 7.30-7.36 (m, 5H, ArH); 13C NMR (CDCl3) δ 26.1
(s, γ-CH2), 28.3 (p, tert-butyl), 28.5 (s, â-CH2), 40.5 (s, δ-CH2),
46.5 (s, COCH2Cl), 57.0 (t, R-CH), 66.8 (s, benzyl), 80.5 (q, tert-
butyl), 128.1-128.6 (t, aryl C), 136.5 (q, aryl C), 156.6 and
156.6 (q × 2), 201.6 (q, COCH2Cl).
3,6-Bis(2′-b en zyloxyca r b on yla m in oet h yl)-5-m et h yl-
2(1H)-p yr a zin on e (3b). A solution of 2b (198 mg, 0.32 mmol)
and 3 N HCl (4 mL) in THF (2 mL) was heated at a reflux for
1 h. After removal of THF, the residue was dissolved in CHCl3,
washed with water, 5% NaHCO3, and saturated sodium
chloride solution, dried over Na2SO4, and evaporated. The
residue was crystallized from ether, and the crystals were
collected by filtration, and recrystallized from EtOH. Yield
Syn th esis of Boc-X(Z)-X(Z)-CH2Cl (2a -c, X: a ) Da p ,
b ) Da b, or c ) Or n ). To a solution of Boc-X(Z)-OH (3.37
mmol) in THF (30 mL) were added Et3N (3.37 mmol) and
isobutyl chloroformate (3.37 mmol) under cooling to -15 °C.
After 10 min, a cold solution of HCl‚H-X(Z)-CH2Cl [prepared
from Boc-X(Z)-CH2Cl (4.04 mmol) and 7.2 N HCl/dioxane (12.1
mmol)] in DMF (30 mL) containing Et3N (4.04 mmol) was
added. The reaction mixture was stirred for 1 h at 0 °C and
overnight at room temperature. After removal of the solvent,
the residue was dissolved in AcOEt, which was washed with
water, 10% citric acid, 5% NaHCO3, and saturated sodium
chloride solution, dried over Na2SO4, and evaporated. The
54.8%, mp 183-185 °C, Rf 0.28 (C). Anal. Calcd for C25H28
-
N4O5: C, 64.6; H, 6.08; N, 12.1. Found: C, 64.4; H, 5.87; N,
1
12.0. H NMR (pyridine-d5) δ 2.33 (s, 3H, 5-CH3), 2.92 (t, J )
6.9 Hz, 2H, CH2CH2NH-Z), 3.32 (t, J ) 6.9 Hz, 2H, CH2CH2-
NH-Z), 3.72 (q, J ) 6.5 Hz, 2H, CH2CH2NH-Z), 3.99 (q, J )
6.5 Hz, 2H, CH2CH2NH-Z), 5.29 and 5.32 (2s, 4H, benzyl),
7.24-7.45 (m, 10H, ArH), 8.04 (br, 1H, NH-Z), 8.34 (br, 1H,
NH-Z); 13C NMR (pyridine-d5) δ 19.0 (p, 5-CH3), 32.5 and 33.6

2606 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
J insmaa et al.
(s × 2, 2 × CH2CH2NH-Z), 39.5 and 40.2 (s × 2, 2 × CH2CH2-
NH-Z), 66.2 and 66.3 (s × 2, 2 × benzyl), 128.1-128.8 (t, aryl
C), 138.0, 138.2, 157.3, 157.3, and 157.2 (q × 5).
(m, 2H, â-CH2), 3.75 (bq, J ) 5.1 Hz, 2H, CH2CH2NH), 3.96
(bm, 1H, CH2CH2NH), 4.05 (bm, 1H, CH2CH2NH), 4.95-4.88
(m, 2H, 2 × R-CH), 7.32-7.05 (m, 8H, 2 × ArH), 7.86 (d, J )
8.4 Hz, 1H, NH of Tyr), 7.99 (d, J ) 8.4 Hz, 1H, NH of Tyr),
8.78 (br, 1H, C2H4NH), 9.08 (br, 1H, C2H4NH); 13C NMR
(pyridine-d5) δ 18.8 (p, 5-CH3), 28.2 (p, tert-butyl), 31.8 and
32.8 (s × 2, 2 × CH2CH2NH), 37.3 (s, CH2CH2NH), 38.3 (s,
CH2CH2NH), 38.6 (s, 2 × â-CH2), 57.0 (t, 2 × R-CH), 78.4 and
78.5 (q × 2, 2 × tert-butyl), 115.9 (t, aryl C), 128.4 and 128.6
(q × 2), 130.8 (t, aryl C), 156.1, 156.8, 157.3, 157.4, 172.1,
172.6, and 173.3 (q × 7).
3,6-Bis(3′-ben zyloxyca r b on yla m in op r op yl)-5-m et h yl-
2(1H)-p yr a zin on e (3c). Yield 52.3%, mp 133-135 °C, Rf 0.52
(B). Anal. Calcd for C27H32N4O5‚0.3H2O: C, 65.1; H, 6.55; N,
11.3. Found: C, 65.2; H, 6.59; N, 11.3. 1H NMR (CDCl3) δ 1.79
(br, 4H, CH2CH2CH2NH-Z), 2.25 (s, 3H, 5-CH3), 2.53 (t, 2H, J
) 7.0 Hz, CH2CH2CH2NH-Z), 2.78 (t, J ) 7.0 Hz, 2H, CH2-
CH2CH2NH-Z), 3.14-3.20 (m, 4H, 2 × CH2CH2CH2NH-Z), 5.06
and 5.08 (2s, 4H, benzyl), 5.57 (br, 1H, NH-Z), 6.11 (br, 1H,
NH-Z), 7.28-7.32 (m, 10H, ArH); 13C NMR (CDCl3) δ 18.4 (p,
5-CH3), 26.7 (s, CH2CH2CH2NH-Z), 27.0 and 28.5 (s × 2, 2 ×
CH2CH2CH2NH-Z), 29.5 (s, CH2CH2CH2NH-Z), 39.4 and 40.4
(s × 2, 2 × CH2CH2CH2NH-Z), 66.5 and 66.8 (s × 2, 2 ×
benzyl), 127.7-128.5 (t, aryl C), 130.6, 134.4, 136.6, 136.8,
155.3, 156.5, 156.8, and 157.7 (q × 8).
3,6-Bis[3′-(Nr-Boc-Tyr )-a m in op r op yl]-5-m eth yl-2(1H)-
p yr a zin on e (5c). To a solution of 4c (74.3 mg, 0.33 mmol) in
DMF (20 mL) were added Boc-Tyr-OH (205.3 mg, 0.73 mmol),
Bop (336.6 mg, 0.76 mmol), and DIEA (0.29s mL, 1.71 mmol)
at 0 °C. The reaction mixture was stirred at room temperature
for 4 h. After removal of DMF, the residue was dissolved in
AcOEt and washed with water, 5% NaHCO3, 10% citric acid,
and saturated sodium chloride solution. The AcOEt layer was
dried over Na2SO4 and evaporated. Ether was added to the
residue to give crystals, which were collected by filtration. The
crude product was purified by column chromatography. Yield
31.5%, mp 125-130 °C, [R]25D -2.57° (c ) 0.2, CHCl3), Rf 0.68
(F), MS [M + H]+ 751.8. Anal. Calcd for C39H54N6O9‚H2O: C,
60.9; H, 7.34; N, 10.9. Found: C, 60.7; H, 7.24; N, 11.1. 1H
NMR (pyridine-d5) δ 1.44 (s, 18H, 2 × tert-butyl), 1.90 (br, 2H,
CH2CH2CH2NH), 2.14-2.21 (m, 2H, CH2CH2CH2NH), 2.28 (s,
3H, 5-CH3), 2.59 (t, J ) 7.5 Hz, 2H, CH2CH2CH2NH), 3.06 (t,
J ) 7.3 Hz, 2H, CH2CH2CH2NH), 3.15-3.32 (m, 2H, â-CH2),
3.40-3.52 (m, 4H, CH2CH2CH2NH and â-CH2), 3.56 (m, 1H,
CH2CH2CH2NH), 3.68 (m, 1H, CH2CH2CH2NH), 4.89-4.96 (m,
2H, 2 × R-CH), 7.06-7.10 (m, 4H, ArH), 7.28-7.33 (m, 4H,
ArH), 7.88 (d, J ) 7.8 Hz, 1H, NH of Tyr), 8.09 (d, J ) 7.9 Hz,
1H, NH of Tyr), 8.77 (br, 1H, C3H6NH), 8.90 (br, 1H, C3H6NH);
13C NMR (pyridine-d5) δ 18.9 (p, 5-CH3), 27.4 (s, CH2CH2CH2-
NH), 28.5 (p, tert-butyl), 29.2 (s, CH2CH2CH2NH), 30.4 (s, CH2-
CH2CH2NH), 38.7 (s, â-CH2), 38.9 (s, CH2CH2CH2NH), 39.0
(s, CH2CH2CH2NH), 39.1 (s, â-CH2), 39.6 (s × 2, 2 × CH2-
CH2CH2NH), 57.3 and 57.5 (t × 2, 2 × R-CH), 78.6 and 78.8
(q × 2, 2 × tert-butyl), 116.2 (t, aryl C), 128.6 (q), 131.0 and
131.1 (t × 2, aryl C), 156.5, 156.9, 157.7, 172.5, and 173.1 (q
× 5).
Syn t h esis of 3,6-Bis[a m in o-(CH 2)n ]-5-m et h yl-2(1H )-
p yr a zin on e (4a : n ) 1 or 4b: n ) 2). Compound 3a or 3b
(0.41 mmol) was treated with 25% HBr/AcOH (4.12 mmol) for
2 h at room temperature. Dry ether was added to the solution
until the product precipitated; the precipitate was collected
by filtration and washed with ether. 4a : Rf 0.49 (E), MS [M +
H]+ 169.2, C7H12N4O‚2 HBr; 4b: Rf 0.26 (D), MS [M + H]+
197.3, C9H16N4O‚2HBr.
3,6-Bis(3′-a m in op r op yl)-5-m et h yl-2(1H )-p yr a zin on e‚
2AcOH (4c). A solution of 3c (419 mg, 0.91 mmol) in 60%
AcOH (20 mL) was hydrogenated for 1 h in the presence of Pd
black. After removal of Pd and the solvent, ether was added
to the residue resulting in crystals, which were collected by
filtration and lyophilized from water. Rf 0.12 (D), MS [M +
H]+ 225.6, C11H20N4O‚2AcOH.
Syn th esis of 3,6-Bis[Nr-Boc-Tyr -NH-(CH2)n ]-5-m eth yl-
2(1H)-p yr a zin on e (5a : n ) 1 or 5b: n ) 2). To a solution
of Boc-Tyr-N2H3 (0.65 mmol) in DMF (6 mL) were added 7.3
N HCl/dioxane (1.30 mmol) and isopentyl nitrite (0.78 mmol)
under cooling to -15 °C; the solution was stirred for 10 min
at the same temperature, and NMM (1.37 mmol) was added
to adjust the pH to 8. This solution was added to a cooled
solution of 4a or 4b (0.26 mmol) and NMM (0.52 mmol) in
DMF (6 mL). The reaction mixture was stirred at 0 °C for 24
h, before adding an azide solution prepared from Boc-Tyr-N2H3
(69.6 µmol) in DMF (2 mL). The reaction mixture was stirred
for another 24 h under the same conditions and the pH
maintained at 8. After removal of DMF, the residue was
dissolved in AcOEt and washed with water, 5% NaHCO3, 10%
citric acid, and saturated sodium chloride solution. The AcOEt
layer was dried over Na2SO4 and evaporated. The product was
crystallized from ether, and the crystals were collected by
filtration.
Syn th esis of 3,6-Bis[Tyr -NH-(CH2)n ]-5-m eth yl-2(1H)-
p yr a zin on e (1′: n ) 1; 2′: n ) 2; 3′: n ) 3). Compounds 5a ,
5b, or 5c (0.13 mmol) were treated with TFA (3.75 mmol)
containing anisole (0.38 mmol) for 1 h at room temperature.
Dry ether was added to the solution until the product
precipitated. The precipitate was collected by filtration, puri-
fied by reverse-phase HPLC, and lyophilized from water
containing 1 N HCl to give an amorphous powder. Yield, [R]25
D
and mass spectrum analyses of 1′-3′ are summarized in
3,6-Bis[(Nr-Boc-Tyr )-a m in om e t h yl]-5-m e t h yl-2(1H )-
p yr a zin on e (5a ). Yield 53.6%, mp 143-144 °C, [R]25D -27.5°
(c ) 0.1, CHCl3), Rf 0.57 (F), MS [M + H]+ 696.2. Anal. Calcd
for C35H46N6O9‚1.25H2O: C, 58.6; H, 6.82; N, 11.7. Found: C,
Supporting Information (Table 1).
3,6-Bis(Tyr -am in om eth yl)-5-m eth yl-2-(1H)-pyr azin on e‚
2HCl (1′). Anal. Calcd for C25H30N6O5‚2HCl‚2H2O: C, 49.8;
H, 6.01; N, 11.8. Found: C, 49.6; H, 6.15; N, 11.8. 1H NMR
(pyridine-d5) δ 2.37 (3H, s, 5-CH3), 3.77 (d, J ) 5.0 Hz, 2H,
â-CH2), 3.84 (d, J ) 6.2 Hz, 2H, â-CH2), 4.61 (bm, 2H, CH2-
NH), 4.81 (bm, 1H, CH2NH), 4.99 (bm, 1H, CH2NH), 5.24 (br,
2H, 2 × R-CH), 7.00-7.57 (m, 8H, ArH), 9.62 (br, 1H, CH2NH),
10.33 (br, 1H, CH2NH).
1
58.8; H, 6.85; N, 11.3. H NMR (pyridine-d5) δ 1.44 (s, 18H, 2
× tert-butyl), 2.39 (s, 3H, 5-CH3), 3.21 (m, 1H, â-CH2), 3.31
(m, 1H, â-CH2), 3.41 (m, 1H, â-CH2), 3.59 (m, 1H, â-CH2), 4.56
(d, J ) 4.1 Hz, 2H, CH2NH), 4.84 (bm, 1H, CH2NH), 4.91 (bq,
J ) 7.3 Hz 1H, R-CH), 5.00 (bm, 1H, CH2NH), 5.12 (bq, J )
7.1 Hz, 1H, R-CH), 7.02-7.41 (m, 8H, ArH), 8.08 (d, J ) 9.6
Hz, 1H, NH of Tyr), 8.10 (d, J ) 9.8 Hz, 1H, NH of Tyr), 8.88
(br, 1H, CH2NH), 9.22 (br, 1H, CH2NH); 13C NMR (pyridine-
d5) δ 19.0 (p, 5-CH3), 28.5 (p, 2 × tert-butyl), 38.6 and 38.7 (s
× 2, 2 × â-CH2), 40.7 and 41.2 (s × 2, 2 × CH2NH), 57.2 and
57.3 (t × 2, 2 × R-CH), 78.7 and 78.8 (q × 2, 2 × tert-butyl),
116.1 (t, aryl C), 128.3 and 128.8 (q × 2), 131.0 and 131.3 (t ×
2, aryl C), 156.1, 156.5, 157.6, 157.7, 172.9, and 173.3 (q × 7).
3,6-Bis(2′-Tyr -am in oeth yl)-5-m eth yl-2-(1H)-pyr azin on e‚
2HCl (2′). Anal. Calcd for C27H34N6O5‚2HCl‚4H2O: C, 48.6;
H, 6.64; N, 12.6. Found: C, 48.5; H, 6.38; N, 12.7. 1H NMR
(pyridine-d5) δ 2.18 (s, 3H, 5-CH3), 2.95-3.25 (m, 4H, 2 × CH2-
CH2NH), 3.70-4.07 (m, 8H, 2 × CH2CH2NH and 2 × â-CH2),
5.17 (br, 2H, 2 × R-CH), 7.00-7.67 (m, 8H, ArH), 9.64 (br,
1H, C2H4NH), 10.0 (br, 1H, C2H4NH); 13C NMR (pyridine-d5)
δ 18.7 (p, 5-CH3), 30.8 (s, CH2CH2NH), 33.5 (s, CH2CH2NH),
37.3, 37.4, 37.7 and 38.6 (s × 4, 2 × CH2CH2NH and 2 ×
â-CH2), 56.0 (t, R-CH), 56.2 (t, R-CH), 116.4 and 116.5 (t × 2,
aryl C), 126.0 and 126.5 (q × 2), 131.6 and 131.7 (t × 2, aryl
C), 157.0, 158.2, 158.2, 169.7, and 169.8 (q × 5).
3,6-Bis[2′-(Nr-Boc-Tyr )-a m in oet h yl]-5-m et h yl-2(1H )-
p yr a zin on e (5b). Yield 85.2%, mp 148-150 °C, [R]25D +49.6°
(c ) 0.1, DMF), Rf 0.78 (F), MS [M + H]+ 723.7. Anal. Calcd
for C37H50N6O9‚1.5H2O: C, 59.3; H, 7.12; N, 11.2. Found: C,
1
59.4; H, 6.76; N, 11.4. H NMR (pyridine-d5) δ 1.44 (s, 18H, 2
× tert-butyl), 2.33 (s, 3H, 5-CH3), 3.02-2.75 (m, 2H, CH2CH2-
3,6-Bis(3′-Tyr -a m in op r op yl)-5-m et h yl-2-(1H )-p yr a zi-
n on e‚2HCl (3′). Anal. Calcd for C29H38N6O5‚2HCl‚4H2O C,
NH), 3.32-3.11 (bm, 4H, â-CH2 and CH2CH2NH), 3.50-3.40

Oral Bioavailability of µ-Opioid Receptor Agonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2607
50.1; H, 6.95; N, 12.1. Found: C, 50.1; H, 6.75; N, 12.2. 1H
NMR (pyridine-d5) δ 1.94 (br, 2H, CH2CH2CH2NH), 2.14-2.17
(m, 2H, CH2CH2CH2NH), 2.24 (s, 3H, 5-CH3), 2.62 (t, J ) 7.8
Hz, 2H, CH2CH2CH2NH), 3.00 (t, J ) 7.4 Hz, 2H, CH2CH2-
CH2NH), 3.43-3.68 (m, 8H, 2 × CH2CH2CH2NH- and 2 ×
â-CH2), 4.85 (br, 1H, R-CH), 5.01 (br, 1H, R-CH), 7.02 (d, J )
8.4 Hz, 2H, ArH), 7.04 (d, J ) 8.3 Hz, 2H, ArH), 7.40 (d, J )
7.7 Hz, 4H, ArH), 9.42 (br, 1H, C3H6NH), 9.75 (br, 1H,
C3H6NH); 13C NMR (pyridine-d5) δ 18.8 (p, 5-CH3), 27.0 (s,
CH2CH2CH2NH), 28.5 (s, CH2CH2CH2NH), 28.6 (s, CH2CH2-
CH2NHH), 30.2 (s, CH2CH2CH2NH), 37.9 (s, 2 × â-CH2), 39.4
and 39.8 (s × 2, 2 × CH2CH2CH2NH), 56.1 (t, 2 × R-CH), 116.5
(t, aryl C), 126.0 (q), 131.4 (t, aryl C), 156.9, 158.3, 169.7, and
170.2 (q × 4).
over Na2SO4 and evaporated. Ether was added to the residue
to give crystals, which were collected by filtration and the
crude product purified by column chromatography.
3,6-Bis(3′-Nr-Boc-Dm t -a m in op r op yl)-5-m et h yl-2(1H )-
p yr a zin on e (6c). Yield 59.4%, mp 153-155 °C, [R]25D +52.1°
(c ) 0.1, CHCl3), Rf 0.73 (F), MS [M + H]+ 808.5. Anal. Calcd
for C43H62N6O9‚H2O: C, 62.6; H, 7.77; N, 10.2. Found: C, 62.6;
H, 7.80; N, 10.2. 1H NMR (pyridine-d5) δ 1.46 (s, 18H, 2 ×
tert-butyl), 1.85 (br, 2H, CH2CH2CH2NH), 2.05-2.18 (m, 2H,
CH2CH2CH2NH), 2.26 (s, 3H, 5-CH3), 2.38 and 2.43 (2s, 12H,
4 × CH3 of Dmt), 2.53 (t, J ) 7.6 Hz, 2H, CH2CH2CH2NH),
2.96 (m, 2H, CH2CH2CH2NH), 3.14-3.26 (m, 2H, â-CH2), 3.33
(m, 1H, CH2CH2CH2NH), 3.40-3.54 (m, 4H, CH2CH2CH2NH-
and â-CH2), 3.61 (m, 1H, CH2CH2CH2NH), 4.76-4.90 (m, 2H,
2 × R-CH), 6.86 and 6.88 (s, 4H, ArH), 7.81 (d, J ) 8.5 Hz,
1H, NH of Dmt), 8.04 (d, J ) 8.3 Hz, 1H, NH of Dmt), 8.71(br,
1H, C3H6NH), 8.83 (br, 1H, C3H6NH); 13C NMR (pyridine-d5)
δ 18.9 (p, 5-CH3), 20.5 and 20.6 (p, 4 × CH3 of Dmt), 27.1 (s,
CH2CH2CH2NH), 28.5 (s, CH2CH2CH2NH), 28.6 (p × 2, tert-
butyl), 29.0 (s, CH2CH2CH2NH), 30.3 (s, CH2CH2CH2NH), 33.8
(s, â-CH2), 39.2 (s, CH2CH2CH2NH), 39.7 (s, CH2CH2CH2NH),
55.6 and 55.7 (t × 2, 2 × R-CH), 78.6 and 78.7 (q × 2, 2 ×
tert-butyl), 116.2 and 123.0 (t × 2, aryl C), 125.6, 125.7, 139.1,
156.1, 156.2, 156.8, 157.0, 172.5, and 173.2 (q × 9).
Syn th esis of 3,6-Bis[Nr-Boc-Dm t-NH(CH2)n ]-5-m eth yl-
2(1H)-p yr a zin on e (6a : n ) 1 or 6b: n ) 2). To a solution
of Boc-Dmt-N2H3 (0.27 mmol) in DMF (5 mL) were added 7.3
N HCl/dioxane (0.54 mmol) and isopentyl nitrite (0.32 mmol)
under cooling to -15 °C. After the solution was stirred for 10
min at the same temperature, NMM (0.57 mmol) was added
to adjust the pH to 8. This was then added to a cold solution
of 4a or 4b (0.11 mmol) and NMM (0.22 mmol) in DMF (3 mL).
The reaction mixture was stirred at 0 °C for 24 h before adding
the azide solution prepared from Boc-Dmt-N2H3 (95.6 µmol)
in DMF (2 mL). The reaction mixture was stirred for another
24 h under the same conditions and the pH kept at 8. After
removal of DMF, the residue was extracted with AcOEt and
washed with water, 5% NaHCO3, 10% citric acid, and satu-
rated sodium chloride solution. The AcOEt layer was dried
over Na2SO4 and evaporated. The product was crystallized
from ether and collected by filtration.
3,6-Bis(4′-Nr-Boc-Dm t -a m in ob u t yl)-5-m e t h yl-2(1H )-
p yr a zin on e (6d ). Yield 12.7%, mp 124-128 °C, [R]25D +25.3°
(c ) 0.5, MeOH), Rf 0.61 (F), MS [M + H]+ 837.5. Anal. Calcd
for C45H66N6O9‚H2O: C, 63.4; H, 7.98; N, 9.86. Found: C, 63.5;
1
H, 7.99; N, 9.34. H NMR (pyridine-d5) δ 1.35-1.53 (m, 20H,
CH2CH2CH2CH2NH- and 2 × tert-butyl), 1.56-1.72 (m, 4H,
CH2CH2CH2CH2NH and CH2CH2CH2CH2NH), 1.87 (br, 2H,
CH2CH2CH2CH2NH), 2.30 (s, 3H, 5-CH3), 2.38 and 2.41 (2s,
12H, 4 × CH3 of Dmt), 2.53 (br, 2H, CH2CH2CH2CH2NH), 2.98
(t, J ) 7.3 Hz, 2H, CH2CH2CH2CH2NH), 3.14-3.25 (m, 2H,
â-CH2), 3.25-3.39 (m, 3H, CH2CH2CH2CH2NH), 3.40-3.58 (m,
3H, â-CH2 and CH2CH2CH2CH2NH), 4.71-4.88 (m, 2H, 2 ×
R-CH), 6.88 (s, 4H, 2 × ArH), 7.81 (d, J ) 8.6 Hz, 1H, NH of
Dmt), 7.90 (d, J ) 8.5 Hz, 1H, NH of Dmt), 8.61 (br, 1H,
C4H8NH), 8.66 (br, 1H, C4H8NH); 13C NMR (pyridine-d5) δ 18.7
(p, 5-CH3), 20.3 (p, 4 × CH3 of Dmt), 24.6 and 26.0 (s, 2 ×
CH2CH2CH2CH2NH), 28.1 (p, tert-butyl), 29.3 and 29.4 (s ×
2, 2 × CH2CH2CH2CH2NH), 30.6 and 32.4 (s × 2, 2 × CH2-
CH2CH2CH2NH), 33.3 and 33.4 (s × 2, 2 × â-CH2), 39.1 and
39.5 (s × 2, 2 × CH2CH2CH2CH2NH), 55.3 (t, 2 × R-CH), 78.3
and 78.4 (q × 2, 2 × tert-butyl), 115.8 (t × 2, aryl C), 125.4,
138.8, 138.9, 155.9, 156.7, and 172.2 (q × 6).
3,6-Bis(Nr-Boc-Dm t -a m in om e t h yl)-5-m e t h yl-2(1H )-
p yr a zin on e (6a ). Yield 49.6%, mp 175-178 °C, [R]25D -10.3°
(c ) 0.1, CHCl3), Rf 0.78 (F), MS [M + H]+ 752.0. Anal. Calcd
for C39H54N6O9‚1.25H2O: C, 60.6; H, 7.37; N, 10.9. Found: C,
1
60.6; H, 7.06; N, 11.2. H NMR (pyridine-d5) δ 1.46 and 1.47
(2s, 18H, 2 × tert-butyl), 2.32, 2.34 and 2.43 (3s, 15H, 4 × CH3
of Dmt and 5-CH3), 3.15-3.54 (m, 4H, 2 × â-CH2), 4.50 (m,
2H, CH2NH), 4.68-4.96 (m, 3H, CH2NH- and R-CH), 5.01 (bq,
J ) 9.9 Hz, 1H, R-CH), 6.83 (bs, 4H, ArH), 7.93 (d, J ) 8.2
Hz, 1H, NH of Dmt), 8.08 (d, J ) 8.1 Hz, 1H, NH of Dmt),
8.51 (br, 1H, CH2NH), 9.09 (br, 1H, CH2NH); 13C NMR
(pyridine-d5) δ 18.8, 20.4 and 20.6 (p × 3, 4 × CH3 of Dmt and
5-CH3), 28.5 (p, tert-butyl), 33.3 and 33.6 (s × 2, 2 × â-CH2),
40.8 and 41.2 (s × 2, 2 × CH2NH), 55.5 and 55.7 (t × 2, 2 ×
R-CH), 78.7 and 78.8 (q × 2, 2 × tert-butyl), 116.0 and 116.1
(t × 2, aryl C), 125.3, 125.8, 139.0, 155.9, 156.2, 156.8, 157.0,
172.8, and 173.2 (q × 9).
Syn th esis of 3,6-Bis[Dm t-NH(CH2)n ]-5-m eth yl-2(1H)-
p yr a zin on e (5′: n ) 1; 6′: n ) 2; 7′: n ) 3; 8′: n ) 4).
Compounds 6a , 6b, 6c, or 6d (1.04 mmol) were treated with
TFA (34.4 mmol) containing anisole (3.44 mmol) for 2 h at
room temperature. Ether was added to the solution until the
product precipitated. The precipitate was collected by filtra-
tion, purified by reverse-phase HPLC, and lyophilized from
water containing 1 N HCl to give an amorphous powder. Yield,
[R]25D, and mass spectrum analyses of 5′-8′ are summarized
in Supporting Information (Table 1).
3,6-Bis(2′-Nr-Boc-Dm t -a m in oe t h yl)-5-m e t h yl-2(1H )-
p yr a zin on e (6b). Yield 53.3%, mp 148-158 °C, [R]25D +89.0°
(c ) 0.1, CHCl3), Rf 0.78 (F), MS [M + H]+ 780.0. Anal. Calcd
for C41H58N6O9‚1.75H2O: C, 60.8; H, 7.65; N, 10.4. Found: C,
1
60.5; H, 7.77; N, 10.2. H NMR (pyridine-d5) δ 1.46 and 1.48
(2s, 18H, 2 × tert-butyl), 2.30 (s, 3H, 5-CH3) 2.40 (2s, 12H, 4
× CH3 of Dmt), 2.68-3.02 (m, 2H, CH2CH2NH), 3.04-3.33 (m,
4H, â-CH2 and CH2CH2NH), 3.42-3.57 (m, 2H, â-CH2), 3.57-
3.80 (m, 2H, CH2CH2NH), 3.81-4.17 (m, 2H, CH2CH2NH),
4.83 (br, 2H, 2 × R-CH), 6.86 and 6.84 (2s, 4H, ArH), 7.77 (d,
J ) 8.3 Hz, 1H, NH of Dmt), 7.94 (d, J ) 8.0 Hz, 1H, NH of
Dmt), 8.73 (br, 1H, C2H4NH), 9.03 (br, 1H, C2H4NH); 13C NMR
(pyridine-d5) δ 19.0 (p, 5-CH3), 20.5 and 20.6 (p × 2, 2 × CH3
of Dmt), 28.5 (p, tert-butyl), 32.0 and 32.7 (s × 2, 2 × CH2-
CH2NH), 33.5 and 33.7 (s × 2, 2 × â-CH2), 37.5 and 38.6 (s ×
2, 2 × CH2CH2NH), 55.4 and 55.6 (t × 2, 2 × R-CH), 78.6 and
78.7 (q × 2, 2 × tert-butyl), 109.2 (q), 116.1 (t, aryl C), 139.0,
139.1, 156.1, 156.9, 157.0, 172.3, and 173.0 (q × 7).
3,6-Bis(Dm t-am in om eth yl)-5-m eth yl-2(1H)-pyr azin on e‚
2HCl (5′). Anal. Calcd for C29H38N6O5‚2.5HCl‚0.5H2O: C, 51.4;
H, 6.61; N, 12.4. Found: C, 51.2; H, 6.34; N, 12.3. 1H NMR
(pyridine-d5) δ 2.31, 2.34 and 2.37 (3s, 15H, 4 × CH3 of Dmt
and 5-CH3), 3.56-3.94 (m, 4H, 2 × â-CH2), 4.50 (bm, 2H, CH2-
NH), 4.73-4.93 (m, 4H, 2 × R-CH and CH2NH), 6.80 (s, 2H,
ArH), 6.84 (s, 2H, ArH), 8.89 (br, 1H, CH2NH), 9.81 (br, 1H,
CH2NH); 13C NMR (pyridine-d5) δ 18.8 (p, 5-CH3), 20.6 (p, 2
× CH3), 31.9 and 32.1 (s × 2, 2 × â-CH2), 40.1 and 41.4 (s ×
2, 2 × CH2NH), 53.9 (t, 2 × R-CH), 116.2 and 116.3 (t × 2,
aryl C), 139.3, 155.8, 157.3, 157.4, 170.0, and 170.5 (q × 6).
Syn th esis of 3,6-bis-[Nr-Boc-Dm t-NH(CH2)n ]-5-m eth yl-
2(1H)-p yr a zin on e (6c: n ) 3 or 6d : n ) 4). To a solution
of 4c or 4d (0.81 mmol) in DMF (20 mL) were added Boc-Dmt-
OH (1.79 mmol), Bop (1.88 mmol) and DIEA (4.10 mmol) at 0
°C. The reaction mixture was stirred at room temperature for
4 h. After removal of DMF, the residue was dissolved in AcOEt
and washed with water, 5% NaHCO3, 10% citric acid, and
saturated sodium chloride solution. The AcOEt layer was dried
3,6-Bis(2′-Dm t-am in oeth yl)-5-m eth yl-2(1H)-pyr azin on e‚
2HCl (6′). Anal. Calcd for C31H42N6O5‚2HCl‚3.5H2O: C,52.1;
H, 7.19; N, 11.8. Found: 52.3, H, 6.80; N, 12.0. 1H NMR
(pyridine-d5) δ 2.19, 2.43 and 2.53 (3s, 15H, 4 × CH3 of Dmt
and 5-CH3), 2.71 (br, 2H, CH2CH2NH), 2.92-3.14 (m, 2H, CH2-
CH2NH), 3.21 (br, 1H, CH2CH2NH), 3.42 (br, 1H, CH2CH2NH),
3.56-3.75 (m, 3H, â-CH2), 3.75-3.92 (m, 2H, â-CH2 and

2608 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
J insmaa et al.
CH2CH2NH), 4.10 (br, 1H, CH2CH2NH), 4.73 (br, 1H, R-CH),
5.20 (br, 1H, R-CH), 6.76 (s, 2H, ArH), 6.85 (s, 2H, ArH), 8.61
(br, 1H, C2H4NH), 9.55 (br, 1H, C2H4NH); 13C NMR (pyridine-
d5) δ 18.5 (p, 5-CH3), 20.7 and 20.8 (p × 2, 4 × CH3 of Dmt),
30.7 (s, CH2CH2NH), 31.3 and 31.4 (s × 2, â-CH2 and CH2CH2-
NH), 31.6 (s, â-CH2), 32.2 (s, CH2CH2NH), 38.4 (s, CH2CH2-
NH), 54.1 and 54.2 (t × 2, 2 × R-CH), 116.1 and 116.2 (t × 2,
aryl C), 139.4, 157.3, 157.3, 169.8, and 169.9 (q × 5).
administration of the compound. Morphine was used as a
positive control, and naloxone was used to antagonize the
effect.20
For the hot-plate test (supraspinal analgesia), mice were
set on an electrically heated plate at 55 ( 0.1 °C (IITC Inc.,
Woodland Hills, CA) following the same drug injection para-
digm above. Hot-plate latency (HPL) was measured as the
interval between the placement of the mice on the hot plate
and observing movements consisting of either jumping, licking,
or shaking their hind paws with a baseline latency of 15 s and
maximal cut off time of 30 s. The area under the curve (AUC)
is derived from data based on the response (mean ( SE) of
five to seven mice per time point. The analgesic effect of
morphine and 7′ are relative to saline or water.
Sta tistica l An a lysis (ANOVA). Asterisks denote values
that are significantly different from the controls. Mice treated
with morphine or 7′ are relative to saline or water controls (*,
p < 0.05; **, p < 0.01; *** p < 0.001) or both substances
relative naloxone treatment (###, p < 0.001).
F u n ction a l Bioa ssa ys in Isola ted Tissu e P r ep a r a tion s.
The myenteric plexus longitudinal muscle preparations (2-3
cm segments) were surgically removed from the small intestine
of guinea pigs (GPI) and used to measure µ-opioid receptor
agonism. A single mouse vas deferens (MVD), containing
primarily δ-opioid receptors, was used to determine agonism
or antagonism for δ-opioid receptor activity. The isolated
tissues were suspended in organ baths containing balanced
salt solutions in a physiological buffer, pH 7.5. Agonists were
tested for the inhibition of electrically evoked contraction and
expressed as IC50 (nM) obtained from the concentration-
response curves in comparison to dermorphin with GPI and
deltorphin C for MVD. IC50 values represent the mean ( SE
of five to seven separate tissue samples. Antagonism is listed
as the pA2, which is the negative log of the molar concentration
required to double the agonist concentration to achieve the
original response.
Com p etitive Op ioid Recep tor Bin d in g Assa ys. Opioid
receptor binding affinities were determined under equilibrium
conditions (2.5 h at 22 °C) in a competition assay using rat
brain P2 synaptosomes membranes.20,29,35 The synaptosomes
were preincubated to remove endogenous opioids, extensively
washed in ice cold buffer containing protease inhibitor, resus-
pended in buffered 20% glycerol, and stored at -80 °C.37 δ-
and µ-Opioid receptors were radiolabeled with [3H]DPDPE (45
Ci/mmol, Dupont-NEN, Boston, MA) and [3H]DAMGO (50 Ci/
mmol, Amersham Biosciences, Arlington, IL), respectively.
Excess unlabeled peptide (2 µM) established nonspecific bind-
ing background. Radiolabeled membranes were rapidly filtered
on Whatman GF/C glass fiber filters presoaked in 0.1%
polyethylenimine to enhance the signal-to-noise ratio, washed
with ice-cold BSA buffer, and dried at 75 °C for 60 min, and
radioactivity was determined using CytoScint (ICN, Costa
Mesa, CA). The analogues were analyzed in duplicate assays
using 5-8 dosages and 3-5 independent repetitions (n values
noted in parentheses in Table 1) with different synaptosomal
preparations to ensure statistical significance and listed as
mean ( SE (Prism 3.03). The affinity constants (Ki) were
calculated according to Cheng and Prusoff.40
3 ,6 -B i s ( 3 ′-D m t -a m i n o p r o p y l ) -5 -m e t h y l -2 ( 1 H ) -
p yr a zin on e‚2HCl (7′). Anal. Calcd for C33H46N6O5‚2HCl‚
3H2O: C, 54.0; H, 7.42; N, 11.5. Found: C, 54.3; H, 7.15; N,
1
11.8. H NMR (pyridine-d5) δ 1.76 (m, 1H, CH2CH2CH2NH),
1.86-2.10 (m, 3H, CH2CH2CH2NH), 2.14, 2.43 and 2.45 (3s,
15H, 4 × CH3 of Dmt and 5-CH3), 2.64 (m, 1H, CH2CH2CH2-
NH), 2.73-2.94 (m, 3H, CH2CH2CH2NH), 3.16 (m, 1H, CH2-
CH2CH2NH), 3.42 (m, 1H, CH2CH2CH2NH), 3.52 (m, 1H,
CH2CH2CH2NH), 3.57-3.73 (m, 3H, CH2CH2CH2NH- and
â-CH2), 3.73-3.88 (m, 2H, â-CH2), 4.76 (dd, J ) 4.4 Hz and
6.4 Hz, 1H, R-CH), 4.89 (dd, J ) 4.8 Hz and 5.7 Hz, 1H, R-CH),
6.88 (s, 2H, ArH), 6.90 (s, 2H, ArH), 9.20 (br, 1H, C3H6NH),
9.57 (br, 1H, C3H6NH); 13C NMR (pyridine-d5) δ 18.3 (p,
5-CH3), 20.5 (p, 4 × CH3 of Dmt), 26.2 (s, CH2CH2CH2NH),
27.6 (s, CH2CH2CH2NH), 28.2 (s, CH2CH2CH2NH), 29.7 (s,
CH2CH2CH2NH), 31.5 and 31.6 (s × 2, 2 × â-CH2), 38.9 and
39.5 (s × 2, 2 × CH2CH2CH2NH), 53.9 (t, 2 × R-CH), 116.0
and 116.1 (t × 2, aryl C), 139.2, 139.3, 156.4, 157.2, 169.6,
and 169.9 (q × 6).
3,6-Bis(4′-Dm t-am in obu tyl)-5-m eth yl-2(1H)-pyr azin on e‚
2HCl (8′). Anal. Calcd for C35H50N6O5‚2HCl‚2H2O: C, 56.5;
H, 7.59; N, 11.3. Found: C, 56.3; H, 7.45; N, 11.4. 1H NMR
(pyridine-d5) δ 1.41-1.72 (m, 6H, CH2CH2CH2CH2NH- and 2
× CH2CH2CH2CH2NH), 1.73-1.83 (m, 2H, CH2CH2CH2CH2-
NH), 2.25, 2.42 and 2.44 (3s, 15H, 4 × CH3 of Dmt and 5-CH3),
2.48-2.65 (m, 2H, CH2CH2CH2CH2NH), 2.86 (t, J ) 7.4 Hz,
2H, CH2CH2CH2CH2NH), 3.14-3.25 (m, 1H, CH2CH2CH2CH2-
NH), 3.28-3.52 (m, 3H, CH2CH2CH2CH2NH), 3.63 (m, 2H,
â-CH2), 3.80 (m, 2H, â-CH2), 4.74 (dd, J ) 4.3, 6.9 Hz, 1H,
R-CH), 4.86 (dd, J ) 4.4, 6.6 Hz, 1H, R-CH), 6.88 (s, 2H, ArH),
6.90 (s, 2H, ArH), 9.14 (br, 1H, C4H8NH), 9.32 (br, 1H,
C4H8NH); 13C NMR (pyridine-d5) δ 18.7 (p, 5-CH3), 20.7 and
20.8 (p × 2, 4 × CH3 of Dmt), 24.6 and 26.2 (s × 2, 2 × CH2CH2-
CH2CH2NH), 28.9 and 29.0 (s × 2, 2 × CH2CH2CH2CH2NH),
29.8 (s, CH2CH2CH2CH2NH), 31.8 (s, 2 × â-CH2), 32.5 (s, CH2-
CH2CH2CH2NH), 39.2 (s, 2 × CH2CH2CH2CH2NH), 53.9 and
54.0 (t × 2, 2 × R-CH), 116.3 (t, aryl C), 139.5, 156.9, 157.4,,
169.8, and 169.9 (q × 5).
An im a ls. Swiss-Webster male mice (20-25 g) (Taconic,
Germantown, NY) were housed under pathogen-free conditions
by the Comparative Medicine Branch at NIEHS, an AALAC
affiliated institution; food and sterile water were freely avail-
able in microisolator cages. Male rats (140-160 g) (Charles
River, CD strain, Wilmington, MA) were euthanized with CO2
and decapitated according to approved protocols by the ACUC
at NIEHS. Guinea pigs and mice for use in functional bioas-
says in vitro were obtained by Tohoku Pharmaceutical Uni-
versity and treated humanely following prescribed protocols.
Deter m in a tion of An a lgesia in Vivo. Tail-flick and hot-
plate latencies in mice measured the effect of icv, sc, and po
administration of morphine or 7′ [3,6-bis(3′-Dmt-aminopropyl)-
5-methyl-2(1H)-pyrazinone] as follows. Compounds dissolved
in physiological saline (pH 7) were injected into mice icv or
sc, or dissolved in water for po administration. Tail-flick tests
(spinal analgesia) were performed by applying radiant heat
to the dorsal surface of the tail (Columbus Instruments,
Columbus, OH). The latency period for removal of the tail,
defined as the tail-flick latency (TFL), was adjusted between
2 and 3 s (pre-response time), and a cutoff time was set at 8 s
to avoid external heat-related damage. The analgesic response
was measured beginning at 10, 15, or 30 min following icv, sc,
or po administration of either morphine or 7′, respectively, and
testing was terminated when TFL approached the pre-
response time. The AUC (area under the curve) was obtained
by plotting the response time (s) versus time (min) after
Ack n ow led gm en t. This work was supported in part
by a Grant-in-Aid for Scientific Research from J apan
Society for the Promotion of Science (C-11694326).
Su p p or tin g In for m a tion Ava ila ble: Yield, [R]25D, tR and
mass spectrum analysis of compounds 3,6-bis[Tyr-NH(CH2)n]-
5-methyl-2(1H)pyrazinone (1′-3′) and 3,6-bis[Dmt-NH(CH2)n]-
5-methyl-2(1H)pyrazinone (5′-8′) as well as the yield, mp,
[R]25D, Rf, and elemental analyses of Boc-X(Z)-CH2Cl (1a -c)
and Boc-X(Z)-X(Z)-CH2Cl (2a -c). This material is available
free of charge via the Internet at http://pubs.acs.org.
Refer en ces
(1) Abbreviations: In addition to the IUPAC-IUB Commission on
Biochemical Nomenclature (J . Biol. Chem. 1985, 260, 14-42),
this paper uses the following symbols and abbreviations: AcOEt,
ethyl acetate; AcOH, acetic acid; BBB, blood-brain barrier; Boc,

Oral Bioavailability of µ-Opioid Receptor Agonists
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2609
tert-butyloxycarbonyl; BOP, benzoltriazol-1-yloxytris(dimethyl-
amino)phosphonium hexafluorphosphate; n-BuOH, n-butanol;
DAMGO, [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin; DALDA, Tyr-
D-Arg-Phe-Lys-NH2; DIEA, diisopropylethylamine; DMF, N,N-
dimethylformamide; DMSO, dimethyl sulfoxide; Dmt, 2′,6′-
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ethanol; GPI, guinea-pig ileum; HPLC, high performance liquid
chromatography; IC50, concentration required for 50% inhibition
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concentration required to double the agonist concentration to
achieve the original response; TFA, trifluoroacetic acid; Tic,
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; TLC, thin-layer
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