Transformation of μ-opioid receptor agonists into biologically potent μ-opioid receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2006.11.019

N-Allylation (-CH₂-CH{double bond, long}CH₂) of [Dmt¹]endomorphins yielded the following: (i) [N-allyl-Dmt¹]endomorphin-2 (Dmt = 2′,6′-dimethyl-l-tyrosine) (12) and [N-allyl-Dmt¹]endomorphin-1 (15) (K_iμ = 0.45 and 0.26 nM, respectively) became μ-antagonists (pA₂ = 8.59 and 8.18, respectively) with weak δ-antagonism (pA₂ = 6.32 and 7.32, respectively); (ii) intracerebroventricularly administered 12 inhibited morphine-induced CNS-mediated antinociception in mice [AD₅₀ (0.148 ng/mouse) was 16-fold more potent than naloxone], but not spinal antinociception, and (iii) 15 reversed the alcohol-elevated frequency in spontaneous inhibitory post-synaptic currents (IPSC) in hippocampal CA1 pyramidal cells in rat brain slices (P = 0.0055). Similarly, N-allylation of the potent μ-opioidmimetic agonists, 1,6-bis-[H-Dmt-NH]-hexane and 3,6-bis-[Dmt-NH-propyl]-2(1H)-pyrazinone, converted them into μ-antagonists (pA₂ = 7.23 and 7.17 for the N-allyl-derivatives 17 and 19, respectively), and exhibited weak δ-antagonism. Thus, N-allylation of Dmt containing opioid peptides or opioidmimetics continues to provide a facile means to convert selective μ-opioid agonists into potent μ-opioid antagonists.

Full text of DOI:10.1016/j.bmc.2006.11.019

Bioorganic & Medicinal Chemistry 15 (2007) 1237–1251
Transformation of l-opioid receptor agonists into biologically
potent l-opioid receptor antagonists
Tingyou Li,a,ꢀ Yunden Jinsmaa,b Masahiro Nedachi,c Anna Miyazaki,c Yuko Tsuda,a,c
Akihiro Ambo,d Yusuke Sasaki,d Sharon D. Bryant,b Ewa Marczak,b Qiang Li,e
H. Scott Swartzwelder,f Lawrence H. Lazarusb,* and Yoshio Okadaa,c,
*
aThe Graduate School of Food and Medicinal Sciences, Kobe Gakuin University, Nishi-ku, Kobe 651-2180, Japan
bMedicinal Chemistry Group, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences,
Research Triangle Park, NC 27709, USA
cFaculty of Pharmaceutical Sciences, Kobe Gakuin University, Nishi-ku, Kobe 651-2180, Japan
dDepartment of Biochemistry, Tohoku Pharmaceutical University, Aoba-ku, Sendai 981-8558, Japan
eDepartment of Pharmacology and Cancer Biology, and Department of Psychiatry, Duke University Medical Center,
Durham, NC 27710, USA
fNeurobiology Research Laboratory, VA Medical Center, Durham, NC 27705, USA
Received 1 October 2006; revised 9 November 2006; accepted 10 November 2006
Available online 14 November 2006
Abstract—N-Allylation (–CH2–CH@CH2) of [Dmt1]endomorphins yielded the following: (i) [N-allyl-Dmt1]endomorphin-2
(Dmt = 20,60-dimethyl-L-tyrosine) (12) and [N-allyl-Dmt1]endomorphin-1 (15) (Kil = 0.45 and 0.26 nM, respectively) became l-an-
tagonists (pA2 = 8.59 and 8.18, respectively) with weak d-antagonism (pA2 = 6.32 and 7.32, respectively); (ii) intracerebroventricu-
larly administered 12 inhibited morphine-induced CNS-mediated antinociception in mice [AD50 (0.148 ng/mouse) was 16-fold more
potent than naloxone], but not spinal antinociception, and (iii) 15 reversed the alcohol-elevated frequency in spontaneous inhibitory
post-synaptic currents (IPSC) in hippocampal CA1 pyramidal cells in rat brain slices (P = 0.0055). Similarly, N-allylation of the
potent l-opioidmimetic agonists, 1,6-bis-[H-Dmt-NH]-hexane and 3,6-bis-[Dmt-NH-propyl]-2(1H)-pyrazinone, converted them
into l-antagonists (pA2 = 7.23 and 7.17 for the N-allyl-derivatives 17 and 19, respectively), and exhibited weak d-antagonism. Thus,
N-allylation of Dmt containing opioid peptides or opioidmimetics continues to provide a facile means to convert selective l-opioid
agonists into potent l-opioid antagonists.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
these receptors, but also to serve as clinically and thera-
peutically relevant agents.1 Interestingly, the addictive
behavior associated with morphine in animal studies
when treated by some alkaloid-derived antagonists can
result in severe withdrawal symptoms due to their ability
to act as inverse agonists.1 Furthermore, the predilection
to alcoholism, in part, depends on the presence of intact
l-opioid receptors evidenced by the existence of sub-
stantial data using knock-out mice and selective opiate
antagonists which verify that these receptors are inti-
mately associated with a neural reward pathway(s) in
the CNS (central nervous system) that is affected by
alcohol consumption2 as well as morphine and its
cognates.
Whereas the existence of potent opioid peptide agonists
is well known for the major opioid-receptor classes
(l, d, j), the development of selective opioid antagonists
continues to be a key objective in pharmacology. In par-
ticular, l-opioid antagonists are important pharmaco-
logical tools not only to delineate critical biochemical,
pharmacological, and physiological roles played by
Keywords: Opioid; l-Opioid receptor antagonists; Endomorphin; Dmt;
Antinociception; Ethanol; Spontaneous IPSC.
*
Corresponding authors. Tel.: +81 78 974 1551; fax: +81 78 974 5689
(Y.O.), tel.: +1 919 541 3238; fax: +1 919 541 5737 (L.H.L).; e-mail
addresses: lazarus@niehs.nih.gov; okada@pharm.kobegakuin.ac.jp
Present address: College of Chemistry, Jilin University, Changchun
130012, China.
ꢀ
Opioid antagonists do not occur naturally and modifica-
tion of the alkaloid architecture of morphine led to the
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.11.019

1238
T. Li et al. / Bioorg. Med. Chem. 15 (2007) 1237–1251
elucidation of structural requirements for their develop-
ment; however, the conversion was neither straightfor-
ward nor predictable. In particular, an allyl (–CH2–
CH@CH2) substitution3 yielded either non-specific
antagonist for l-, d-, and j-opioid receptors (naloxone),
or a mixed agonist/antagonist (nalorphine); a compara-
ble dichotomy in pharmacological activity with
N-allylated opioid peptides occurred: for example
N,N-diallylation of the multisubstituted [Leu5]enkepha-
lin framework yielded two distinct d-opioid antagonists,
namely N,N-(allyl)2-Tyr-Gly-Gly-w-(CH2S)-Phe-Leu-
OH (ICI 154129)4 and N,N-(allyl)2-Tyr-Aib-Aib-Phe-
Leu-OH (ICI 174864),5 while N-allylation produced
mixed agonist/antagonist opioids.6,7 Similarly, [N,N-(al-
lyl)2-Tyr1,D-Pro10]dynorphin A (1-11) imparted antago-
nism toward both j- and l-opioid receptors with weak
j-opioid receptor selectivity;8 however, [N-allyl-Tyr1,
D-Pro10]dynorphin A (1-11) retained the j-agonism of
the parent compound.3
other opioid peptides sporadically exhibit weak l-antag-
onism, but fail to meet the criteria for potent bio-
activity.13–15
The l-opioid agonists endomorphin-1 (EM-1: H-Tyr-
Pro-Trp-Phe-NH2) and endomorphin-2 (EM-2: H-Tyr-
Pro-Phe-Phe-NH2) exhibit the highest selectivity for
the l-opioid receptor,16 and thus represent a potential
opioid framework for modification into l-antagonists.
In this communication, l-agonists containing 20,60-di-
methyl-L-tyrosine (Dmt) in lieu of Tyr, such as [Dmt1]-
endomorphin,17 the bis-Dmt opioidmimetic ligands
linked by an alkyldiamine18 or 3,6-bis-(aminoalkyl)-
2(1H)-pyrazinone,19 were transformed by N-allylation
into potent l-opioid antagonists.
2. Chemistry
The key intermediates (Scheme 1) of N-allyl and N,N-
(allyl)2-Xaa-OCH3 (3 and 2, Xaa = Tyr or Dmt) were
prepared in one pot from allylbromide (2.5 equiv) and
H-Xaa-OMe (1) in the presence of diisopropylethyl-
amine (DIPEA) (2.7 equiv) at 50 ꢁC for 5 h. N,N-(Al-
lyl)2-Xaa-OCH3 (2) was hydrolyzed to give
N,N-(allyl)2-Xaa-OH (4). Na-H of N-allyl-Xaa-OCH3
(3) was protected with tert-butyloxycarbonyl (Boc)
group in dioxane to produce N-allyl-N-Boc-Xaa-OCH3
(5), and then hydrolyzed with NaOH to N-allyl-N-
Boc-Xaa-OH (6). As shown in Scheme 2, [N,N-(allyl)2-
Inasmuch as that many specific d-antagonists exist (e.g.,
ICI 174864,5 naltrindole,9 the TIP(P) family,10 and
Dmt-Tic pharmacophore derivatives11), a majority of
the l-antagonists used pharmacologically lack a high
degree of biological specificity or potency, such as nalox-
one, naltrexone, nalmefene or CTAP (D-Phe-Cys-Tyr-D-
Trp-Arg-Thr-Pen-Thr-NH2).12 On the other hand, while
the alkaloids naloxone and naltrexone are non-selective
opioid receptor antagonists, b-funaltrexamine behaves
as an irreversible l-antagonist. Nonetheless, a host of
iii
N,N-(Allyl)2-Xaa-OH
N,N-(Allyl)2-Xaa-OCH3
ii
i
2
4
H-Xaa-OCH3
H-Xaa-OH
1
iv
N-Allyl-Xaa-OCH3
N-Allyl-N-Boc-Xaa-OCH3
Xaa = Tyr (a) or Dmt (b)
3
5
iii
N-Allyl-N-Boc-Xaa-OH
6
Scheme 1. Synthesis of N-terminal mono- and diallyl-substituted amino acid derivatives. Reagents and conditions: (i) SOCl2/MeOH; (ii) allyl-Br,
DIPEA, MeOH, 50 ꢁC, 5 h; (iii) 1 N NaOH; (iv) (Boc)2O, Et3N, dioxane.
i
N,N-(Allyl)2-Xaa-OH
[N,N-(Allyl)2-Xaa1]EM
4a Xaa = Tyr
4b Xaa = Dmt
7 Xaa = Tyr (EM-2)
8 Xaa = Dmt (EM-2)
13 Xaa = Dmt (EM-1)
i
ii
[N-Allyl-N-Boc-Xaa1]EM
[N-Allyl-Xaa1]EM
N-Allyl-N-Boc-Xaa-OH
6a Xaa = Tyr
6b Xaa = Dmt
9 Xaa = Tyr (EM-2)
10 Xaa = Dmt (EM-2)
14 Xaa = Dmt (EM-1)
11 Xaa = Tyr (EM-2)
12 Xaa = Dmt (EM-2)
15 Xaa = Dmt (EM-1)
Scheme 2. Synthesis of N-terminal N-allyl- and N,N-(allyl)2-substituted endomorphin-1 and endomorphin-2 analogues. Reagents: (i) H-Pro-Phe/
Trp-Phe-NH2, DIPEA, PyBop, DMF; (ii) TFA/anisole.

T. Li et al. / Bioorg. Med. Chem. 15 (2007) 1237–1251
1239
ii
i
N-Allyl-N-Boc-Dmt-OH
Bis-(N-allyl-N-Boc-Dmt-NH)-hexane
6b
16
Bis-(N-allyl-Dmt-NH)-hexane
17
iii
N-Allyl-N-Boc-Dmt-OH
3,6-Bis-(N-allyl-N-Boc-Dmt-NH-propyl)-pyrazinone
6b
18
ii
3,6-Bis-(N-allyl-Dmt-NH-propyl)-pyrazinone
19
Scheme 3. Synthesis of N-terminal bis-(allyl-Dmt)-opioidmimetic ligands. Reagents and conditions: (i) 1,6-diaminohexane, DIPEA, PyBop, DMF;
(ii) TFA/anisole; (iii) 3,6-bis-(30-aminopropyl)-pyrazinone, DIPEA, PyBop, DMF.
Xaa1]- (7 and 8) and (N-allyl-Xaa1)EM-2 analogues (11
and 12) were prepared in solution using Boc-protection;
[N,N-(allyl)2-Dmt1]- (13) and [N-allyl-Dmt1]EM-1 (15)
receptor,20 since deletion reduces ligand efficacy.13 On
the other hand, the impairment of binding by allyl
groups3–5,7–9 might be attributed to steric hindrance
preventing the formation of an ionic bond required
for agonism, yet permitting the ligand to function as
an antagonist through interaction with a different bind-
ing region within the receptor.11a Furthermore, that
Dmt overcame the detrimental effect of N-allylation
(Table 1) implies that hydrophobic factors also play a
dominant role in anchoring the opioid in the receptor
by aligning or stabilizing the hydroxyl group to form
a H-bond, enhancing p–p interactions or aromatic ring
stacking.11a
were prepared identically. After deprotection of Boc-
17a
Pro-Phe-Phe-NH2
with HCl/dioxane, the resulting
amino component was condensed with 4 or 6 using ben-
zotriazol-1-yloxytris-(pyrrolidino)phosphate (PyBop) as
the coupling reagent to give 7 and 8, and Boc-protected
9 and 10, respectively. Removal of the Boc-group (9 and
10) with trifluoroacetic acid (TFA) gave 11 and 12.
Compound 15 was synthesized using the same method
for 11, but using [N-allyl-N-Boc-Dmt1]EM-1 (14). As
shown in Scheme 3, N-allyl-N-Boc-Dmt-OH (6b) was
coupled with 1,6-diaminohexane or 3,6-bis-(aminopro-
pyl)-2(1H)-pyrazinone to produce bis(N-allyl-N-Boc-
Dmt)-compounds, followed by removal of Boc group
with TFA to give 17 and 19, respectively. All final com-
pounds were purified by semi-preparative reversed-
phase HPLC to >98% purity, and verified using mass
spectrometry, 1H and 13C NMR, optical rotation,
thin-layer chromatography, analytical HPLC, and ele-
mental analysis.
3.2. Functional bioactivity
N-Allylation completely eliminated the l-agonism of
[H-Dmt1]EM-1 (40) and [H-Dmt1]EM-2 (20) to yield 15
and 12 with potent l-antagonism (pA2 = 8.18–8.59)
(Table 1). Furthermore, both compounds are 6.6- and
17-fold, respectively, more effective than the published
data on CTAP;12 even the N,N-diallyl ligand 13 has
nearly twice the potency of this molecule.12 As shown
in Table 1, [H-Dmt1]EM-2 (20) is an agonist for both
l- and d-opioid receptors, while [H-Dmt1]EM-1 (40)
exhibited potent l-agonism/d-antagonism (GPI:
IC50 = 0.27 nM; MVD: pA2 = 8.60). Aromatic moiety
of Trp at position 3 in 40 is bulkier and more hydropho-
bic than that of Phe3 in 20. It can be deduced that
aromatic properties of the moiety at position 3 of
[H-Dmt1]EM-1 or -2 are an important factor that deter-
mines whether [H-Dmt1]EM exhibits d-agonism or d-an-
tagonism. This phenomenon is compatible with the
following results: ligand 12 exhibited high l-opioid
antagonism with an A2 ratio of 186, while ligand 15
had only an A2 ratio of 7.2. This difference in the A2 ra-
tio (26-fold) between 12 and 15 is mainly due to the
weaker d-antagonism exhibited by 12 (pA2 = 6.32) com-
pared with that of 15 (pA2 = 7.32) which can be ex-
plained by the hypothesis given above.
3. Results and discussion
3.1. Opioid receptor affinity
Compared to the standard opioids (10–60, Table 1), the
allylated derivatives decreased affinity toward l-opioid
receptors in the following order: parent peptides > N-al-
lyl > N,N-diallyl derivatives; of these analogues, howev-
er, 12 and 15 exhibited the highest l-opioid affinities
(Kil = 0.45 and 0.26 nM, respectively) (Table 1). The ef-
fect of N-allylation of EM-2 on l-opioid affinity was
more sensitive than that of [H-Dmt1]EM-2; that is, N-al-
lyl-EM-2 (11) fell 98-fold compared to only 3-fold with
[N-allyl-Dmt1]EM-2 (12). Although the d-opioid recep-
tor affinities are lower than those for the l-opioid recep-
tor, the order was similar, except 7 and 11. In all cases,
the d-opioid affinities were substantially less than those
observed for the l-opioid receptor (Table 1).
In general, N-modification profoundly affects opioid
peptide activities: for example, elimination of the
N-terminal amino group of [Met5]enkephalin abolished
d-opioid agonism;21 N-formylation of H-Dmt-c[-D-
Orn-2-Nal-D-Pro-Gly] resulted in a moderately active
l-opioid antagonist;13 N-allyl-enkephalin7 and N-allyl-
The primary, secondary or tertiary amino function at
the N-terminus is apparently necessary for the expres-
sion of high affinity and bioactivity presumably due
to the formation of an ionic bond to a group in the

Table 1. Receptor affinities and functional bioactivities of opioid ligands
No. Compound Kil (nM)
GPI (l)d IC50, nM pA2
MVD (d)d
IC50, nM
pA2
[A2] ratiof
MVD/GPI
e
e
Kid (nM)
d/l
10
7
Endomorphin-2 (EM-2)
[N,N-(Allyl)2]EM-2
1.33 0.15 (3) 6080 1,215 (3) 4571 6.88 0.94
1045 175 (6) 5755 1224 (4) NT
—
—
—
—
344 93
NT
NT
—
—
—
—
—
—
—
—
8.7
6
57 NT
11
20
N-Allyl-EM-2
[H-Dmt1]EM-2a
[N,N-(Allyl)2-Dmt1]EM-2
[N-Allyl-Dmt1]EM-2
130 9.3 (6)
0.15 0.004
13.1 1.3 (6)
0.45 0.08 (5)
7358 259 (3)
28.2 8.1
188 0.07 0.016
108 >10,000 (33.9%)
1244 >10,000 (49.3%)
3125 4.03 1.14
104 0.27 0.08
1.87 0.61
8
1416 50 (4)
560 49 (4)
7.07 (7.33–7.02)
8.59 (9.05–8.33)
>10,000 (13.3%) 6.13 (6.34–5.72)
12
>10,000 (42.8%) 6.32 (7.35–5.48) 186
30
40
Endomorphin-1 (EM-1)
[H-Dmt1]EM-1
[N,N-(Allyl)2-Dmt1]EM-1
[N-Allyl-Dmt1]EM-1
1,6-Bis-[H-Dmt-NH]-hexaneb
1,6-Bis-[N-allyl-Dmt-NH]-hexane
3,6-Bis-[H-Dmt-NH-propyl]-2(1H)-pyrazinonec
3,6-Bis-[N-allyl-Dmt-NH-propyl]-2(1H)-pyrazinone
0.56 0.04 (4) 1750 146 (4)
0.054 0.01 (5) 5.60 0.67 (3)
—
—
283 39 —
>10,000 (21.6%) 8.60 (9.58–8.01)
—
—
13
15
50
17
60
19
4.60 0.32 (4)
217 23 (5)
47 >10,000 (23.4%)
40 >10,000 (49.0%)
870 3.08 0.53
7.60 (7.68—7.51) >10,000 (19.8%) 7.15 (7.40–6.90)
>10,000 (37.5%) 7.32 (7.48–7.16)
2.8
7.2
0.26 0.01 (4) 10.3 1.1 (3)
0.053 0.01 (6) 46.1 8.8 (5)
8.18 (8.28–8.16)
—
>10,000
6.1
—
12.4 1.1 (3)
0.042 0.003
6.94 1.4 (3)
51.5 4.1 (3)
13.2 1.7
77.8 14 (4)
4 >10,000 (<1%)
314 1.33 0.2
11 >10,000 (<1%)
7.23 (7.36–6.99)
—
7.17 (7.82–6.84)
>10,000 (<1%)
>10,000
>10,000 (<1%)
6.83 (7.56–5.58)
—
6.38 (6.56–6.21)
2.5
—
6.2
The affinity of each compound was assayed with 5–8 graded dosages of peptide in independent repetitions (n) and data are presented as means SE. The functional bioactivity is the mean SE (n = 5–6
separate tissue preparations). NT, not tested (due to the very weak interaction with opioid receptors, Ki). A dash (ꢀ) indicates non-relevancy.
a Data from Ref. 17.
b Data from Ref. 18.
c Data from Ref. 19.
d Values in parentheses are maximum inhibition of the tissue contraction at the concentration of 10,000 nM.
e pA2 is the negative log of the molar concentration required to double the agonist’s IC50 value in order to achieve the original response, and the 95% confidence limits are in parentheses.
f [A2] ratio is the ratio of molar concentrations at the pA2 values (MVD/GPI). Antagonism was determined using endomorphin-2 and deltorphin II as l- and d-receptor agonists, respectively.

T. Li et al. / Bioorg. Med. Chem. 15 (2007) 1237–1251
1241
dynorphin A-(1-11)8 had mixed biological properties;
Mor
Mor + 0.067 ng 12
Mor + 0.67 ng 12
Mor + 6.7 ng 12
Mor + 67 ng 12
a
and N,N-dimethylation of bis-Dmt-Tic-derivatives (d-
opioid antagonists) yielded potent dual l-/d-opioid
antagonists.22 On the other hand, replacement of Tyr1
in endomorphin by Mdp [(2S)-2-methyl-3-(20,60-dimeth-
yl-40-hydroxyphenyl)-propionic acid] or Dhp [3-(20,60-
100
80
60
40
20
0
dimethyl-40-hydroxyphenyl)-propionic
acid]
gave
moderately weak l-opioid antagonists.23 The parent
compounds of 17 and 19 are l-opioid agonists18,19 and
the allylated derivatives exhibited l-opioid antagonism
(pA2 = 7.23 and 7.17, respectively) (Table 1) comparable
to that of CTAP (pA2 = 7.36).12 While the d-opioid
affinity of 17 remained similar to its parent ligand, the
6.6-fold improvement in d-opioid antagonism yielded a
l-/d-opioid antagonist profile similar to that of 19.
Our group,17–19 as well other investigators,11 published
considerable data demonstrating discrepancies and
inconsistencies between receptor affinity and bioactivity
without an acceptable explanation; however, tentative
answers may, in part, involve inherent differences be-
tween the assay systems, which include the artificial nat-
ure of the synaptosomes used to determine affinity (Ki)
and the bioactivity in isolated tissue preparations for
estimation of functional bioactivity, and the differential
solubility of the ligand in the membrane lipid milieu that
depends on the hydrophobic properties of opioids.
Another possible reason for the inconsistencies regard-
ing endomorphin analogues may be related to their
interactions with tachykinin NK1 and NK2 receptors,24
which mediate antiopioid activity.
10 20 30 40 50 60 70
Time (min)
0
1400
1200
1000
800
600
400
200
0
b
Mor (4 mg/kg)
12
0.07
0.67
6.7
67
ng/mouse
c
100
80
60
40
20
0
12
3.3. Inhibition of morphine antinociception
naloxone
The intracerebroventricular (icv) administration of a
drug is the first analysis of its potential central role in
affecting morphine-induced antinociception using the
classic hot-plate or tail-flick tests. [N-Allyl-Dmt1]EM-2
(12) alone failed to show any significant icv effect at
the same dosages used under identical conditions (hot-
plate test) for the morphine-induced antinociception;
on the other hand, 12 was a potent l-opioid antagonist
in a dose-dependent manner against morphine, such that
the observed effect implies that supraspinally (CNS) l-
opioid receptors are involved (Fig. 1) (infra vide).25
The %MPE (percent of maximal possible effect) at
-13 -12 -11 -10 -9 -8 -7 -6
Log dose (g)
Figure 1. The effect of intracerebroventricularly injected [N-allyl-
Dmt1]EM-2 (12) on morphine (Mor)-induced antinociception (4 mg/kg
sc) in the hot-plate test in mice (a) Time course. (b) Area under the
dose–response curve (AUC). (c) Dose–response curve of 12 and
naloxone at 10 min after icv injection. Each value is the mean SE
(n = 10–17 mice per point). ANOVA and Dunnett’s test were used to
analyze the data. ***P < 0.001 and *P < 0.05 represent significant
differences in the AUC from morphine-treated mice.
12
10 min post-injection of 12 revealed that the AD50
(0.148 ng/mouse, 95% CL95: 0.02–0.993 ng/mouse) was
naloxone
16 times more potent than naloxone (AD50
¼
2:38 ng=mouse, 95% CL95: 0.935–6.00 ng/mouse).
Although, there is little difference between the antinoci-
ception produced by the highest dose for 12 and nalox-
one in Figure 1c, a further increase in naloxone
completely blocked morphine antinociception; however,
12 failed to show this effect. Moreover, in contrast to
many alkaloid opiates and peptidic l-opioid agonists,
12 only acts supraspinally (CNS) due to the absence of
l-opioid antagonism in the classic and accepted
pharmacological tail-flick test which measures spinal
nociceptive mechanisms (Fig. 2a and b); that is, the
tail-flick test indicates whether a spinal mechanism is in-
volved in pain perception by activating a descending
mechanism regardless of the type of injection or route
of administration.
3.4. Changes in spontaneous IPSC frequency
The inhibition of ethanol-induced frequency of sponta-
neous IPSC (a constituent component in the promotion
of GABAA receptor-mediated neuronal function) in
vitro (patch-clamp analysis of isolated neurons in hippo-
campal slices) verified the direct action of 15 as a l-opioid
antagonist on neuronal activity (Fig. 3). Spontaneous
IPSC is one of the most potent CNS actions initiated
by ethanol (EtOH) and constitutes an underlying mech-
anism of the sedative26 and anxiolytic properties27 of
EtOH. In the presence of glutamatergic receptor block-
ers 50 lM AVP (D-2-amino-5-phosphonovalerate) and

1242
T. Li et al. / Bioorg. Med. Chem. 15 (2007) 1237–1251
and 15 are at least two orders of magnitude more potent
(Li; Swartzwelder, et al., unpublished data, in
preparation).
a
100
90
80
70
60
50
40
30
20
10
0
Mor
Mor+ 0.07ng 12
Mor+ 0.67ng 12
Mor+ 6.7ng 12
Mor+ 67ng 12
4. Conclusions
The present study validates the conversion of l-opioid
receptor agonists to potent l-opioid antagonists by
N-allylation; and the N-allyl-derivatives of [H-Dmt1]
endomorphins,17a bis-[H-Dmt-amino]-alkane,18 and
3,6-bis-[H-Dmt-aminoalkyl]-2(1H)-pyrazinone19 could
generate new drug leads. Moreover, this approach
would be applicable to producing analogues on large-
scale synthesis with considerable ease relative to the
replacement of the C-terminal phenylalanyl in endomor-
phin that only yielded weak l-opioid antagonists.15 Our
data provided evidence that [N-allyl-Dmt1]endomor-
phin-1 (15) and -2 (12) are pharmacological probes that
could be used to study the role of l-opioid receptors in
mediating physiological and pharmacological processes
in the CNS. Their potential value could be to effectively
combat alkaloid drug addiction or alleviate alcohol
dependency or abuse (Figs. 1–3) due to the involvement
of the l-opioid receptor in the neural reward pathway2
and the abolishment of spontaneous IPSC (Fig. 3). Fur-
ther studies are underway to define the biochemical
mechanisms of their l-opioid antagonist action, and
the physiological abatement of morphine and alcohol-
mediated changes in vitro and in vivo.
pre 10 20 30 40 50 60 70
time after administration (min)
300
b
250
200
150
100
50
0
Mor (0.5 μg/mouse)
12
0.07 0.67
6.7
ng/mouse
67
Figure 2. The effect of intracerebroventricularly injected [N-allyl-
Dmt1]EM-2 (12) on morphine (Mor)-induced antinociception (0.5 lg/
mouse icv) in the tail-flick test in mice. (a) Time course. (b) Area under
the dose–response curve (AUC). Each value is the mean SE (n = 5–6
mice per point). ANOVA and Dunnett’s test were used to analyze the
data.
5. Experimental
5.1. Chemistry general
20 lM DNOX (6,7-dinitroquinoxaline-2,3-dione) to
block excitory synaptic transmission, spontaneous IPS-
Cs were isolated from hippocampal CA1 pyramidal cells
held at ꢀ70 mV. Similar to our previous report,28 after
the baseline (control) spontaneous IPSCs were estab-
lished, addition of 60 mM EtOH to the infusion medium
significantly increased average frequency of spontaneous
IPSCs of hippocampal CA1 pyramidal cells by 26% of
control (t[5] = 4.40, P = 0.007) and was reversed with
300 nM 15 (t[5] = 2.87, P = 0.0055) (Fig. 3). Further-
more, 15 did not reduce the baseline recordings of the
spontaneous IPSC frequency (t[5] = 1.05, P = 0.341)
indicative that its effect was only on the elevation elicited
by EtOH and complements the in vivo antagonism of
morphine antinociception that it acted as an antagonist
(Fig. 1). Sedative and anxiolytic actions are involved in
the reinforcing properties and addictive liability of etha-
nol, and drugs that attenuate these actions at the l-opi-
oid receptor2 could be used therapeutically for the
treatment of EtOH dependency. Furthermore, 15 was
potent in the nanomolar range in contrast to naloxone
and naltrexone, which are well known to inhibit GABA
(c-aminobutyric acid)-mediated chloride uptake into
hippocampal synaptoneurosomes at micromolar con-
centrations.29 In fact, similar results were also obtained
with 12 at a comparable concentration in which naltrex-
one was active only in the 30–60 lM range (based on
data available in the literature), demonstrating that 12
Melting points were determined on a Yanagimoto
micromelting point apparatus and are uncorrected.
TLC was performed on precoated plates of silica gel
F254 (Merck, Darmstadt, Germany). Rf values refer
to the following solvent systems: (1) AcOEt/hex-
ane = 1:1, (2) AcOEt/hexane = 1:2, (3) AcOEt/hexn-
ane = 10:1, (4) AcOEt, (5) AcOEt/MeOH = 10:1, (6)
AcOEt/MeOH = 20:1, (7) AcOEt/hexane = 2:1, (8)
n-BuOH/H2O/AcOH = 4:1:5 (upper layer), (9) CHCl3/
MeOH/H2O = 8:3:1 (lower layer). Optical rotations
were determined with a DIP-1000 automatic polarime-
ter (Japan Spectroscopic Co.). Analytical RP-HPLC
and semi-preparative RP-HPLC used are Waters Delta
600 with COSMOSIL C18 column (4.6 mm · 250 mm)
and COSMOSIL C18 column (20 mm · 250 mm),
respectively. The solvent for analytical HPLC was as
follows: A, 0.05% TFA in water; B, 0.05% TFA in
CH3CN. The column was eluted at a flow rate of
1 mL/min with a linear gradient of 90% A to 10% A
in 30 min; the retention time is reported as tR (min).
Mass spectra were measured with a KRATOS MAL-
DI-TOF (matrix-assisted laser desorption ionization
time-of-flight mass spectrometry). 1H and 13C NMR
spectra were measured on a Bruker DPX-400 spec-
trometer at 25 ꢁC. Chemical shift values are expressed
as ppm downfield from tetramethylsilane, used as an
internal standard (d value).

T. Li et al. / Bioorg. Med. Chem. 15 (2007) 1237–1251
1243
Control
EtOH (60 mM)
EtOH (60 mM) and 15 (300 nM)
140
120
100
80
*
60
n=6
n=6
n=6
40
20
0
Control
EtOH (60 mM)
EtOH (60 mM)
15 (300 nM)
Figure 3. Changes in spontaneous IPSC frequency in the presence of ethanol and ethanol plus [N-allyl-Dmt1]endomorphin-1 (15). All the recordings
in this figure were recorded from single neurons in hippocampal slices in vitro using patch-clamp methods. Upper figure: from top to bottom, the
recordings refer to baseline spontaneous IPSC recordings, plus 60 mM EtOH, and with 300 nM 15 in 60 mM EtOH. Scale bar = 500 ms/50 pA.
Bottom figure: the bars represent the percentage changes in mean spontaneous IPSC frequency (mean SE) with n = 6 tissue slices per measurement.
Left, baseline; middle, plus 60 mM EtOH (26%, t[5] = 4.40, P = 0.007); and right, the addition of 300 nM 15 in 60 mM EtOH (t[5] = 2.87, P = 0.0055;
denotes significant difference).
*
5.2. General methods for peptide synthesis
The AcOEt layer was dried over Na2SO4 and evaporat-
ed. Petroleum ether was added to the residue to give
crystals, which were collected by filtration. The crude
Boc-Dmt-Pro-OMe was purified by flash chromatogra-
Gram quantities of H-L-Dmt-OH were prepared
according to Dygos et al.,30 and the syntheses of the
endomorphins,17 1,6-bis-[H-Dmt-NH]-hexane,18 and
3,6-bis-[H-Dmt-aminopropyl]-2(1H)-pyrazinone19 followed
published methods. The bold numbers in the text refer
to the synthetic products (Schemes 1–3).
phy (SiO2, AcOEt/hexane = 1:1). Yield 2.16 g (66.3%),
20
D
Anal. Calcd for C22H32N2O6: C, 62.84; H, 7.67; N, 6.66.
mp 144–145 ꢁC, Rf1 = 0.45, ½aꢁ +16.5ꢁ (c 0.63, MeOH).
1
Found: C, 62.72; H, 7.68; N, 6.65. H NMR (CDCl3)d:
6.51 (s, 2H), 5.54 (d, 0.4H, J = 8.4 Hz), 5.31 (d, 0.6H,
J = 8.9 Hz), 4.72 (q, 0.6H, J = 8.7 Hz), 4.49 (br, 0.6H),
4.40 (q, 0.4H, J = 7.0 Hz), 3.73 and 3.69 (s · 2, 3H),
3.65–3.55 (m, 1H), 3.38–3.30 (m, 0.8H), 3.13–2.89 (m,
2.6H), 2.34 and 2.31 (s · 2, 6H), 2.15–1.50 (m, 4H),
1.43 and 1.34 (s · 2, 9H).
5.3. Synthesis of [H-Dmt1]endomorphin-1 ([H-Dmt1]EM-
1)
5.3.1. Boc-Dmt-Pro-OCH3. To a solution of HClÆH-Pro-
OMe (1.28 g, 7.75 mmol) in N,N-dimethylformamide
(DMF) (50 mL) were added Boc-Dmt-OH (tert-butyl-
oxycarbonyl-Dmt-OH, 2.39 g, 7.75 mmol), benztriazol-
5.3.2. Boc-Dmt-Pro-OH. Boc-Dmt-Pro-OCH3 (1.89 g,
4.5 mmol) in methanol (MeOH; 10 mL) was treated with
1.0 N NaOH (11.2 mL, 11.2 mmol) for 2 h at room tem-
perature. MeOH was removed in vacuo, the pH of the
residue was adjusted to 3 with 10% citric acid, and the
resulting precipitate was extracted with AcOEt (3·
30 mL). The combined organic phase was washed with
saturated NaCl (3· 30 mL), dried over Na2SO4, and
1-yloxytris-(pyrrolidino)phosphate
(PyBop) (4.4 g,
8.52 mmol), and diisopropylethylamine (DIPEA)
(3.37 mL, 19.4 mol) at 0 ꢁC. The reaction mixture was
stirred at room temperature overnight. After removal
of DMF, the residue was dissolved in ethyl acetate
(AcOEt) and washed with 10% citric acid solution, 5%
Na2CO3 solution, and saturated NaCl aqueous solution.

1244
T. Li et al. / Bioorg. Med. Chem. 15 (2007) 1237–1251
evaporated down. Petroleum ether was added to the res-
idue to produce crystals, which were filtered and dried in
5.4. Synthesis of allylated derivatives
vacuo. Yield 1.79 g (98.0%), mp 84–87 ꢁC, Rf2 = 0.25,
5.4.1. N,N-(Allyl)2-Tyr-OMe (2a) and N-allyl-Tyr-OMe
(3a). To a solution of HClÆH-Tyr-OMe (2.0 g, 8.6 mmol)
in MeOH (20 mL), DIPEA (4.8 mL, 27.5 mmol) and ally-
lbromide (1.86 mL, 21.5 mmol) were added in sequence.
After stirred at room temperature for 20 min, the temper-
ature was raised to 50 ꢁC and the reaction mixture kept at
this temperature for 5 h. The solvent was removed in vac-
uo, and the residue was extracted with AcOEt (3· 30 mL).
The combined extracts were washed with saturated NaCl,
dried over Na2SO4, filtered, and concentrated under re-
duced pressure. The residue was purified by flash chroma-
tography (SiO2, AcOEt/hexane = 1:1) to give pure 2a
20
½aꢁ
ꢀ14.8ꢁ (c 0.54, MeOH). Anal. Calcd for
D
C21H30N2O6: C, 62.05; H, 7.44; N, 6.89. Found: C,
61.99; H, 7.70; N, 6.76. 1H NMR (CDCl3) d: 6.52
(s · 2, 2H), 5.80 (d, 0.3H, J = 8.2 Hz), 5.45 (d, 0.7H,
J = 8.7 Hz), 4.72 (br, 0.7H), 4.56 (dd, 0.7H, J = 8.0,
3.8 Hz), 4.40 (br, 0.3H), 3.64–3.53 (m, 1H), 3.41–3.29
(m, 0.6H), 3.14–2.85 (m, 2.7H), 2.34 and 2.31 (s · 2,
6H), 2.15–1.50 (m, 4H), 1.42 and 1.37 (s · 2, 9H).
5.3.3. Boc-Dmt-Pro-Trp-Phe-NH2. To a solution of
HClÆH-Trp-Phe-NH2 [prepared from Boc-Trp-Phe-
NH2 (200 mg, 0.44 mmol), 7.7 N HCl/dioxane
(570 lL, 4.4 mmol)] in tetrahydrofuran (THF; 30 mL)
containing DIPEA (230 lL, 1.32 mmol), Boc-Dmt-
Pro-OH (180 mg, 0.44 mmol) and PyBop (250 mg,
0.48 mmol) were added. The reaction mixture was stir-
red for 6 h at 35 ꢁC. After removal of the solvent, the
residue was extracted with AcOEt (50 mL), and the
extract was washed with 10% citric acid, 5% Na2CO3,
and saturated NaCl, dried over Na2SO4, and evapo-
rated. The residue was purified by flash chromatogra-
(1.34 g, 59.3%) and 3a (740 mg, 36.4%). Compound 2a:
20
D
Anal. Calcd for C16H21NO3: C, 69.79; H, 7.69; N, 5.09.
mp 127–128 ꢁC, Rf1 = 0.79, ½aꢁ ꢀ198.0ꢁ (c 0.63, MeOH).
1
Found: C, 69.51; H, 7.81; N, 5.22. H NMR (400 MHz,
CDCl3) d: 7.04 (d, 2H, J = 8.32 Hz), 6.73 (d, 2H,
J = 8.32 Hz), 5.77–5.58 (m, 2H), 5.20–5.00 (m, 4H), 4.68
(br, 1H), 3.70–3.55 (m, 4H), 3.44–3.30 (m, 2H), 3.17–
2.93 (m, 3H), 2.90–2.75 (m, 1H). Compound 3a: mp
20
D
Anal. Calcd for C13H17NO3: C, 66.36; H, 7.28; N, 5.95.
117–118 ꢁC, Rf4 = 0.74, ½aꢁ +27.0ꢁ (c 0.51, MeOH).
1
Found: C, 66.14; H, 7.13; N, 5.94. H NMR (400 MHz,
phy (SiO2, AcOEt/MeOH = 10:1). Yield 223 mg
20
D
(68.6%), mp 150–152 ꢁC, Rf3 = 0.57, ½aꢁ
ꢀ34.9ꢁ
CDCl3) d: 7.02 (d, 2H, J = 8.56 Hz), 6.71 (d, 2H,
J = 8.56 Hz), 5.88–5.76 (m, 1H), 5.18–5.07 (m, 2H), 3.67
(s, 3H), 3.57 (t, 1H, J = 6.6 Hz), 3.34–3.27 (m, 1H),
3.20–3.12 (m, 1H), 2.94 (d, 2H, J = 6.6 Hz).
(c 0.49, MeOH). Anal. Calcd for C41H50N6O7Æ1.5H2O:
C, 64.30; H, 6.97; N, 10.97. Found: C, 64.47; H, 6.89;
N, 10.75. 1H NMR (400 MHz, DMSO-d6)d: 9.28 (s,
0.63H), 8.85–8.47 (m, 1.2H), 8.22 (s, 0.53H), 8.00–
7.87 (m, 0.73H), 7.63–6.82 (m, 11.32H), 6.82–6.60
(m, 0.23H), 6.60–6.33 (m, 1.64H), 6.25 (s, 0.44H),
6.15–5.90 (m, 0.5H), 5.66 (d, 0.16H, J = 8.03 Hz),
5.67 (br, 0.48H), 5.26 (br, 0.27H), 4.80–4.59 (m,
1H), 4.52 (br, 1H), 4.46–4.10 (m, 1.24H), 4.10–3.84
(m, 1H), 3.60–3.24 (m, 1.41H), 3.24–2.45 (m, 5.5H),
2.45–2.00 (m, 5H), 1.90–0.90 (m, 13.27H), 0.70–0.40
(m, 1.46H).
5.4.2. N,N-(Allyl)2-Dmt-OMe (2b) and N-allyl-Dmt-
OMe (3b). To a solution of HClÆH-Dmt-OMe (3.0 g,
11.5 mmol) in MeOH (60 mL), DIPEA (6.0 mL,
34.6 mmol) and allylbromide (2.0 mL, 23.1 mmol) were
added in sequence. After stirred at room temperature
for 20 min, the temperature was raised to 50 ꢁC and
the reaction mixture kept at this temperature for 5 h.
The solvent was removed in vacuo, the residue extracted
with AcOEt (3· 50 mL). The combined extracts were
washed with saturated NaCl, dried over Na2SO4, fil-
tered, and concentrated under reduced pressure. The
residue was purified by flash chromatography (SiO2,
5.3.4. H-Dmt-Pro-Trp-Phe-NH2ÆTFA (40). Boc-Dmt-
Pro-Trp-Phe-NH2 (100 mg, 0.136 mmol) was treated
with 7.7 N HCl/dioxane (0.18 mL, 1.36 mmol) and
anisole (0.15 mL, 1.36 mmol) for 1 h at room temper-
ature. Et2O was added to the reaction mixture to pre-
cipitate the product. The crude product was collected
by centrifugation, dried over KOH pellets, and puri-
fied by semi-preparative reverse phase high pressure li-
AcOEt/hexane = 1:1) to give pure 2b 1.05 g (30%) and
3b 1.72 g (56.7%). Compound 2b: oil, Rf1 = 0.76; ½aꢁ
20
D
+2.5ꢁ (c 0.55, MeOH). Anal. Calcd for C18H25NO3: C,
71.26; H, 8.31; N, 4.62. Found: C, 71.04; H, 8.38; N,
1
4.65. H NMR (400 MHz, DMSO-d6) d: 8.93 (s, 1H),
quid chromatography (RP-HPLC). Yield 86.8 mg
6.37 (s, 2H), 5.74-5.63 (m, 2H), 5.20-5.04 (m, 4H), 3.50
(s, 3H), 3.46 (dd, 1H, J = 5.41, 8.74 Hz), 3.93-3.43 (m,
1H), 3.39-3.36 (m, 1H), 3.04-2.95 (m, 3H), 2.70 (dd,
20
D
(85.1%), tR 16.8 min, ½aꢁ ꢀ8.8ꢁ (c 0.41, H2O), m/z
[M+H]+ 640. Anal. Calcd for C46H54N4O8ÆTFAÆ3H2O:
C, 56.57; H, 6.12; N, 10.42. Found: C, 56.31; H, 5.98;
1H, J = 5.40, 14.12 Hz), 2.16 (s, 6H). Compound 3b:
20
D
1
N, 10.28. H NMR (400 MHz, DMSO-d6)d: 10.83 and
oil, Rf1 = 0.53; ½aꢁ +42.1ꢁ (c 0.34, MeOH). Anal. Calcd
10.81 (s · 2, 1H), 9.15 (br, 1H), 8.36 and 8.25 (br,
s · 2, 3H), 8.07–7.97 (m, 1.6H), 7.79 (d, 0.4H,
J = 8.1 Hz), 7.58 (d, 1H, J = 7.81 Hz), 7.46 (s, 0.5H),
7.36–7.30 (m, 1H), 7.27–7.10 (m, 6.5H), 7.10–7.03
(m, 2H), 7.03–6.95 (m, 1H), 6.44 and 6.42 (s · 2,
2H), 4.53–4.32 (m, 2.4H), 4.20–4.10 (m, 0.6H), 3.60–
3.50 (m, 0.6H), 3.42–3.30 (m, 1H), 3.21–3.12 (m,
0.7H), 3.08–2.76 (m, 6.7H), 2.34–2.23 (m, 0.4H), 2.16
(s, 2.5H), 1.98 (s, 3.5H), 1.87–1.73 (m, 0.4H), 1.67–
1.52 (m, 1.6H), 1.50–1.39 (m, 0.6H), 1.39–1.20 (m,
1H).
for C15H21NO3Æ0.17H2O: C, 67.64; H, 8.07; N, 5.26.
Found: C, 67.52; H, 8.08; N, 5.27. 1H NMR
(400 MHz, DMSO-d6) d: 8.93 (s, 1H), 6.38 (s, 2H),
5.78–5.60 (m, 1H), 5.08–4.95 (m, 2H), 3.53 (m, 3H),
3.29 (t, 1H, J = 9.28 Hz), 3.17–3.08 (m, 1H), 2.98–2.90
(m, 1H), 2.83–2.70 (m, 2H), 2.16 (s, 6H).
5.4.3. N,N-(Allyl)2-Tyr-OH (4a). In an ice bath, 1 N
NaOH (11 mL, 11 mmol) was added to a solution of
N,N-(allyl)2-Tyr-OMe (2a) (1.31 g, 4.8 mmol) in MeOH
(15 mL) and the resulting solution was stirred at room

T. Li et al. / Bioorg. Med. Chem. 15 (2007) 1237–1251
1245
temperature overnight. The solvent was removed in vac-
uo, the pH of the residue was adjusted to 3, and the
resulting precipitate was filtered, washed with cold
and saturated NaCl (2· 15 mL), dried over Na2SO4, fil-
tered, and concentrated under reduced pressure. The
residue was purified by flash chromatography (SiO2,
water, and dried in vacuo. Yield 866 mg (69%), mp
AcOEt/hexane = 1:1) to give pure 5b. Yield 1.12 g
20
D
20
D
214–216 ꢁC, Rf8 = 0.43, ½aꢁ ꢀ7.5ꢁ (c 0.48, MeOH).
(53%), oil, Rf1 = 0.65, ½aꢁ ꢀ174.5ꢁ (c 0.49, MeOH).
Anal. Calcd for C15H19NO3Æ0.5H2O: C, 66.65; H, 7.46;
N, 5.18. Found: C, 66.72; H, 7.25; N, 5.13. H NMR
Anal. Calcd for C20H29NO5Æ0.25H2O: C, 65.28; H,
8.08; N, 3.81. Found: C, 65.27; H, 8.10; N, 3.80. H
1
1
(400 MHz, DMSO-d6) d: 12.20 (br, 1H), 9.20 (br, 1H),
6.95 (d, 2H, J = 8.4 Hz), 6.63 (d, 2H, J = 8.4 Hz),
5.73–5.56 (m, 2H), 5.11 (d, 2H, J = 17.24 Hz), 5.04 (d,
2H, J = 10.24 Hz), 3.43 (t, 1H, J = 7.54 Hz), 3.28 (dd,
2H, J = 5.05, 14.8 Hz), 3.06 (dd, 2H, J = 6.92,
14.80 Hz), 2.82 (dd, 1H, J = 7.82, 13.65 Hz), 2.67 (dd,
1H, J = 7.31, 13.65 Hz).
NMR (400 MHz, DMSO-d6) d: 8.98 (s, 1H), 6.41 (s,
2H), 5.40–5.20 (m, 1H), 5.00–4.83 (m, 2H), 3.96–3.83
(m, 1H), 3.80–3.53 (m, 4H), 3.22–2.96 (m, 3H), 2.12 (s,
6H), 1.36 (s, 9H).
5.4.7. N-Allyl-N-Boc-Tyr-OH (6a). 1 N NaOH
(9.33 mL, 9.33 mmol) was added to a solution of N-al-
lyl-N-Boc-Tyr-OMe (5a) (1.36 g, 4.05 mmol) in MeOH
(10 mL) at 0 ꢁC. After stirred at 0 ꢁC for 20 min, the
solution was stirred at room temperature overnight.
MeOH was removed in vacuo, and the pH of the residue
was adjusted to 3 with 10% citric acid. The resulting pre-
cipitate was extracted with AcOEt (2· 40 mL), and the
combined extracts were washed with saturated NaCl
solution (2· 20 mL), dried over Na2SO4, filtered, and
5.4.4. N,N-(Allyl)2-Dmt-OH (4b). In an ice bath, 1 N
NaOH (8 mL, 8 mmol) was added to a solution of
N,N-(allyl)2-Dmt-OMe (2b) (967 mg, 3.19 mmol) in
MeOH (10 mL) and the resulting solution was stirred
at room temperature overnight. The solvent was re-
moved in vacuo, the pH of the residue was adjusted to
3, the resulting precipitate was extracted with AcOEt
(3· 50 mL), the combined extracts were washed with
saturated NaCl (2· 50 mL), dried over Na2SO4, filtered,
and purified by flash chromatography (SiO2, AcOEt/
concentrated in vacuo to give 6a as a clear oil. Yield
20
D
1.09 g (84%), Rf5 = 0.70, ½aꢁ ꢀ153.2ꢁ (c 0.83, MeOH).
Anal. Calcd for C17H23NO5Æ0.2H2O: C, 62.83; H, 7.26;
N, 4.31. Found: C, 63.06; H, 7.48; N, 4.06. H NMR
1
MeOH = 10:1). Yield 428 mg (46.5%), mp 205–207 ꢁC,
20
Rf5 = 0.50, ½aꢁ +25.2ꢁ (c 0.68, MeOH). Anal. Calcd
(400 MHz, DMSO-d6) d: 12.55 (s, 1H), 9.17 (s, 1H),
6.95 (d, 2H, J = 8.4 Hz), 6.83–6.58 (m, 2H), 5.54–5.35
(m, 1H), 5.01–4.87 (m, 2H), 4.35–4.24 (m, 0.36H),
4.05–3.95 (m, 0.57H), 3.79–3.60 (m, 1H), 3.35–3.25 (m,
0.5H), 3.18–2.87 (m, 2.57H), 1.35 (s, 9H).
D
for C17H23NO3Æ0.1H2O: C, 70.12; H, 8.03; N, 4.81.
Found: C, 69.91; H, 7.94; N, 4.77. 1H NMR
(400 MHz, DMSO-d6) d: 12.20 (br, 1H), 8.89 (s, 1H),
6.37 (s, 1H), 5.74–5.62 (m, 2H), 5.13 (dd, 2H, J = 1.29,
17.21 Hz), 5.05 (dd, 2H, J = 1.29, 10.23 Hz), 3.42–3.34
(m, 3H), 3.06–2.90 (m, 3H), 2.66 (dd, 1H, J = 5.70,
14.1 Hz).
5.4.8. N-Allyl-N-Boc-Dmt-OH (6b). 1 N NaOH
(7.15 mL, 7.15 mmol) was added to a solution of N-al-
lyl-N-Boc-Dmt-OMe (5b) (1.04 g, 2.86 mmol) in MeOH
(10 mL) at 0 ꢁC. After stirred at 0 ꢁC for 20 min, the
solution was stirred at room temperature overnight.
MeOH was removed in vacuo, and the pH of the residue
was adjusted to 3 with 10% citric acid. The resulting pre-
cipitate was extracted with AcOEt (2· 40 mL), and the
combined extracts were washed with saturated NaCl
solution (2· 20 mL), dried over Na2SO4, filtered, and
concentrated in vacuo. The residue was purified by flash
chromatography (SiO2, AcOEt/hexane = 1:1) to give the
5.4.5. N-Allyl-N-Boc-Tyr-OMe (5a). N-Allyl-Tyr-OMe
(3a) (580 mg, 2.5 mmol) was reacted with di-tert-butyl
dicarbonate (Boc2O, 600 mg, 2.75 mmol) in dioxane
(15 mL) containing triethylamine (Et3N) (380 lL,
2.75 mmol) at room temperature overnight. After
removal of the solvent, the residue was extracted with
AcOEt (60 mL), and the extract was washed with 10%
citric acid (3· 10 mL), 5% NaHCO3 (3· 10 mL), and sat-
urated NaCl (3· 10 mL), dried over Na2SO4, filtered,
and concentrated under reduced pressure. The residue
was purified by flash chromatography (SiO2, AcOEt/
title compound. Yield 830 mg (83%), mp 150–152 ꢁC,
20
Rf1 = 0.35, ½aꢁ ꢀ185.8ꢁ (c 0.61, MeOH). Anal. Calcd
D
for C19H27NO5: C, 65.31; H, 7.79; N, 4.01. Found: C,
hexane = 1:3) to give pure 5a. Yield 580 mg (69.4%),
20
D
1
65.20; H, 7.58; N, 3.98. H NMR (400 MHz, CDCl3)
oil, Rf1 = 0.82, ½aꢁ ꢀ13.5ꢁ (c 0.83, MeOH). Anal. Calcd
for C18H25NO5Æ0.2H2O: C, 63.77; H, 7.55; N, 4.13.
Found: C, 63.76; H, 7.51; N, 4.00. 1H NMR
(400 MHz, CDCl3) d: 7.10-6.97 (m, 2H), 6.80–6.70 (m,
2H), 5.65–5.44 (m, 1.3H), 5.01 (d, 2H, J = 14.52 Hz),
4.47–4.37 (m, 0.3H), 4.00–3.85 (m, 1.2H), 3.80-3.62
(m, 3.3H), 3.46–3.36 (m, 0.4H), 3.27–3.03 (m, 2.5H),
1.45 (s, 9H).
d: 6.52 (s, 2H), 5.56–5.35 (m, 1H), 5.00–4.85 (m, 2H),
4.10–3.80 (m, 2H), 3.50–2.80 (m, 3H), 2.24 (s, 6H),
1.48 (s, 9H).
5.4.9. [N,N-Diallyl-Tyr1]EM-2ÆHCl (7). Boc-Pro-Phe-
Phe-NH2 (220 mg, 0.433 mmol) was treated with 6.7 N
HCl/dioxane (1.3 mL, 8.66 mmol) at room temperature
for 30 min to remove the Boc group. The product was
precipitated with ether, filtered, and dried in vacuo.
The resulting hydrochloride salt was dissolved in
DMF (10 mL) containing DIPEA (190 lL 1.09 mmol),
and to this solution N,N-(allyl)2-Tyr-OH (4a) (124 mg,
0.476 mmol) and PyBop (260 mg, 0.5 mmol) were add-
ed. The reaction mixture was stirred at 0 ꢁC for
10 min, then at room temperature for 4 h. After removal
5.4.6. N-Allyl-N-Boc-Dmt-OMe (5b). N-Allyl-Dmt-OMe
(3b) (1.53 g, 5.79 mmol) was reacted with Boc2O (1.39 g,
6.37 mmol) in dioxane (30 mL) containing Et3N
(890 lL, 6.37 mmol) at room temperature overnight.
After removal of the solvent, the residue was extracted
with AcOEt (100 mL), and the extract was washed with
10% citric acid (2· 15 mL), 5% NaHCO3 (2· 15 mL),

1246
T. Li et al. / Bioorg. Med. Chem. 15 (2007) 1237–1251
of the solvent, the residue was diluted with AcOEt
(80 mL). The dilution was washed with cold 10% citric
acid (3· 10 mL), 5% Na2CO3 (3· 10 mL), and saturated
NaCl (3· 10 mL), dried over Na2SO4, and evaporated to
dryness. The residue was purified by flash chromatogra-
phy (SiO2, AcOEt/MeOH = 10:1). The compound was
precipitated with hexane, filtered, and dried in vacuo.
The solid was dissolved in MeOH, and TFA (36 lL,
0.472 mmol) was added. After concentrated to dryness,
the residue was purified by semi-preparative HPLC,
and the purified peptide was lyophilized from 1 N HCl
(m, 5.5H, CH2@CH–CH2–, CH2@CH–CH2–), 4.46–
4.35 (m, 2.0H, Phe aH), 4.35–4.28 (m, 0.27H, Pro
trans-aH), 4.17–3.70 (m, 3.5H, Dmt trans-aH,
CH2@CH–CH2–), 3.41–2.74 (m, 10.23H, Pro cis-aH,
Dmt cis-aH, CH2@CH–CH2–, Pro dH, Phe bH, Dmt
bH), 2.15 and 2.08 (s · 2, 6.0H, Dmt CH3), 1.83–1.13
(m, 4.0H, Pro bH, Pro cH). 13C NMR d: 172.55,
170.66, 170.32, 170.25, 165.88, 156.03, 138.45, 138.42,
138.02, 137.62, 137.54 (11q), 129.11, 129.07, 128.96,
127.97, 127.93 (5t, Phe Ar-C), 127.87 (t, CH2@CH–
CH2–), 126.24, 126.10 (2t, Phe Ar-C), 125.40
(s, CH2@CH–CH2–), 119.69 (q), 115.18, 115.05 (2t,
Dmt Ar-C), 60.77 (t, Pro cis-aC), 59.95 (t, Por trans-
aC), 59.13 (t, Dmt trans-aC), 58.87 (t, Dmt cis-aC),
55.12 (t, Phe cis-aC), 54.20 (s, CH2@CH–CH2–), 54.04
(t, Phe trans-aC), 53.80 (t, Phe cis-aC), 53.62 (t, Phe
trans-aC), 53.22, 51.41 (2s, CH2@CH–CH2–), 46.99 (s,
Pro cis-dC), 45.99 (s, Pro trans-dC), 37.45, 37.22, 36.69
(3s, Phe bC), 31.33 (s, Pro cis-bC), 28.67 (s, Pro trans-
bC), 27.71 (s, Dmt bC), 23.88 (s, Pro trans-cC), 21.17
(s, Pro cis-cC), 20.22 (p, Dmt trans-CH3), 19.76 (p,
Dmt cis-CH3).
(3· 320 lL) to give an amorphous powder. Yield
20
220 mg (73.8%), Rf9 = 0.75, tR 16.40, ½aꢁ ꢀ30.1ꢁ
D
(c 0.57, H2O), m/z [M+1]+ 653. Anal. Calcd for
C38H45N5O5ÆHClÆ2H2O: C, 63.01; H, 6.96; N, 9.67.
Found: C, 63.02; H, 6.68; N, 9.64. 1H NMR
(400 MHz, DMSO-d6) d: 11.55 (br, 0.3H, Dmt NH+),
10.50 (br, 0.2H, Dmt NH+), 9.63 and 9.42 (br · 2,
0.6H, Dmt OH), 8.40 (d, 0.40H, J = 7.95 Hz, Phe cis-
NH), 8.15 (d, 0.40H, J = 8.15 Hz, Phe cis-NH), 8.11
(d, 0.54H, J = 8.22 Hz, Phe trans-NH), 7.99 (d, 0.52H,
J = 7.76 Hz, Phe trans-NH), 7.38–7.04 (m, 13.1H, Phe
Ar-H, CONH2, Tyr Ar-H), 6.94 (d, 0.90H,
J = 8.44 Hz, Tyr Ar-H), 6.76 (d, 0.90H, J = 8.44 Hz,
Tyr Ar-H), 6.69 (d, 1.1H, J = 8.42 Hz, Tyr Ar-H),
6.15–5.97 (m, 1.0H, CH2@CH–CH2–), 5.86–5.72 (m,
5.4.11. [N-Allyl-N-Boc-Tyr]EM-2 (9). Boc-Pro-Phe-Phe-
NH2 (220 mg, 0.433 mmol) was treated with 6.7 N HCl/
dioxane (1.3 mL, 8.66 mmol) at room temperature for
30 min to remove the Boc group. The product was pre-
cipitated with ether, filtered, and dried in vacuo. The
resulting hydrochloride salt was dissolved in DMF
(10 mL) containing DIPEA (190 lL, 1.09 mmol), and
to this solution N-allyl-N-Boc-Tyr-OH (6a) (153 mg,
0.476 mmol) and PyBop (260 mg, 0.5 mmol) were add-
ed. The reaction mixture was stirred at 0 ꢁC for
10 min, then at room temperature for 4 h. After removal
of the solvent, the residue was diluted with AcOEt
(80 mL). The dilution was washed with cold 10% citric
acid (3· 10 mL), 5% Na2CO3 (3· 10 mL), and saturated
NaCl (3· 10 mL), dried over Na2SO4, and evaporated to
dryness. The residue was purified by flash chromatogra-
phy (SiO2, AcOEt). The compound was precipitated
1.0H, CH2@CH–CH2–), 5.72–5.20
(m, 4.0H,
CH2@CH–CH2–), 4.50–4.35 (m, 2.0H, Phe aH), 4.35–
4.28 (m, 0.57H, Pro trans-aH), 4.15–3.57 (m, 5.0H,
Tyr aH, CH2@CH–CH2–), 3.50–3.20 (m, 2.43H, Pro
cis-aH, Pro trans-dH, Pro cis-dH, Tyr trans-bH, Tyr
cis-bH), 3.20–2.73 (m, 5.54H, Pro cis-dH, Phe bH, Tyr
trans-bH, Tyr cis-bH), 2.23 (br, 0.46H, Pro trans-dH),
1.92–1.78 (m, 0.54H, Pro trans-bH), 1.66–1.45
(m, 2.08H, Pro cis-bH, Pro trans-bH, Pro trans-cH),
1.45–1.26 (m, 0.92H, Pro cis-cH), 1.27–1.10 (m, 0.46H,
Pro cis-bH). 13C NMR d: 172.54, 172.50, 170.56,
170.46, 165.50, 156.84, 156.39, 137.83, 137.72, 137.59
(11q), 130.62, 130.41 (2t, Tyr Ar-C), 129.13, 129.09,
129.01, 128.00, 127.96, 127.94, 127.89, 126.31, 126.13
(9t, Phe Ar-C), 124.40 (s, CH2@CH–CH2–), 115.41,
115.26 (2t, Tyr Ar-C), 63.46 (t, Tyr trans-aC), 62.03
(t, Tyr cis-aC), 59.85 (t, Pro trans-aC), 59.20 (t, Pro
cis-aC), 54.82 (t, Phe cis-aC), 54.43 (t, Phe trans-aC),
53.84 (t, Phe cis-aC), 53.74 (t, Phe trans-aC), 53.28
(s, CH2@CH–CH2-), 46.80 (s, Pro trans-dC), 46.54
(s, Pro cis-dC), 37.38, 37.21, 36.88 (3s, Phe bC), 33.68
(s, Tyr trans-bC), 32.74 (s, Tyr cis-bC), 30.98 (s, Pro
cis-bC), 28.63 (s, Pro trans-bC), 23.99 (s, Pro trans-
cC), 21.14 (s, Pro cis-cC).
with hexane, filtered, and dried in vacuo. Yield 247 mg
20
D
(80%), mp 121-122 ꢁC, Rf4 = 0.42, ½aꢁ ꢀ75.1ꢁ (c 0.58,
MeOH). Anal. Calcd for C40H49N5O7Æ0.9H2O: C,
65.99; H, 7.03; N, 9.62. Found: C, 65.99; H, 6.85; N,
1
9.61. H NMR (400 MHz, DMSO-d6) d: 9.14 and 9.07
(s · 2, 1.0H, Dmt OH), 8.02–7.88 (m, 1.0H, Phe NH),
7.85–7.76 (m, 1.0H, Phe NH), 7.30–7.08 (m, 12.0H,
Phe Ar-H, CONH2), 6.98–6.89 (m, 2.0H, Tyr Ar-H),
6.69–6.57 (m, 2.0H, Tyr Ar-H), 5.73–5.59 (m, 1.0H,
CH2@CH–CH2–), 5.07–4.93 (m, 2.4H, CH2@CH–
CH2–, Tyr cis-aH), 4.83–4.71 (m, 0.6H, Tyr trans-aH),
4.47–4.35 (m, 2.0H, Phe aH), 4.16–4.06 (m, 1.0H, Pro
aH), 3.94–3.82 (m, 0.6H, trans CH2@CH–CH2–),
3.82–3.71 (m, 0.4H, cis CH2@CH–CH2–), 3.63–3.37
(m, 2.0H, CH2@CH–CH2–, Pro cis-dH, Pro trans-dH),
3.35–3.18 (m, 1.0H, Pro cis-dH, Pro trans-dH), 3.14–
2.95 (m, 2.0H, Phe bH), 2.95–2.63 (m, 4.0H, Phe bH,
Tyr bH), 1.90–1.57 (m, 4.0H, Pro bH, Pro cH), 1.25
and 1.14 (s · 2, 9.0H, Boc). 13C NMR d: 172.48,
171.31, 170.47, 169.25, 168.72, 155.75, 155.58, 154.36,
153.45, 137.82, 137.72 (11q), 134.70 (t, cis CH2@CH–
CH2–), 134.15 (t, trans CH2@CH–CH2–), 130.28,
130.05 (t, Tyr Ar-C), 128.98, 127.99, 127.89 (3t, Phe
5.4.10. [N,N-(Allyl)2-Dmt1]EM-2ÆHCl (8). The title com-
pound was synthesized using the same method for 7.
20
Yield 200 mg (71.3%), Rf9 = 0.73, tR 17.28, ½aꢁ ꢀ15.6ꢁ
D
(c 0.44, H2O), m/z [M+1]+ 681. Anal. Calcd for
C40H49N5O5ÆHClÆ3H2O: C, 62.36; H, 7.33; N, 9.09.
Found: C, 62.59; H, 7.08; N, 9.13. 1H NMR
(400 MHz, DMSO-d6) d: 10.60 (br, 1H, Dmt NH+),
9.40 and 9.16 (br · 2, 1H, Dmt OH), 8.42 (br, 0.7H,
Phe NH), 8.19 (br, 0.7H, Phe NH), 7.99 (br, 0.6H,
Phe NH), 7.44 (s, 0.7H, CONH2), 7.37–7.00 (m,
11.3H, Phe Ar-H, CONH2), 6.47 and 6.41 (s · 2, Dmt
Ar-H), 6.15 (br, 0.5H, CH2@CH–CH2–), 5.92–5.28

T. Li et al. / Bioorg. Med. Chem. 15 (2007) 1237–1251
1247
Ar-C), 127.64, 127.27 (2q), 126.16, 126.10 (2t, Phe Ar-
C), 116.09 (s, trans CH2@CH–CH2–), 115.98 (s, cis
CH2@CH–CH2–), 114.77, 114.69 (2t, Tyr Ar-C), 79.04,
78.83 (2q, Boc), 60.04 (t, Pro aC), 58.04 (t, Tyr trans-
aC), 56.28 (t, Tyr cis-aC), 54.08, 53.98, 53.74 (3t, Phe
aC), 46.31 (s, Pro cis-dC), 46.13 (s, Pro trans-dC),
45.42 (s, cis CH2@CH–CH2–), 44.67 (s, trans
CH2@CH–CH2–), 37.27, 36.73, 36.67 (3s, Phe bC),
33.68 (s, Tyr bC), 28.48 (s, Pro bC), 27.73 (p, cis-Boc),
27.41 (p, trans-Boc), 23.98 (s, Pro cC).
J = 8.40 Hz, Tyr Ar-H), 6.75 (d, 1.0H, J = 8.37 Hz,
Tyr Ar-H), 6.69 (d, 1.0H, J = 8.42 Hz, Tyr Ar-H),
5.95–5.71 (m, 1.0H, CH2@CH–CH2–), 5.41–5.22 (m,
2.0H, CH2@CH–CH2–), 4.53–4.32 (m, 2.40H, Pro
trans-aH, Phe aH), 4.13 (br, 0.42H, Tyr cis-aH), 3.49
(br, 0.84H, trans CH2@CH–CH2–), 3.43–3.09 (m,
4.30H, Pro cis-aH, Tyr cis-aH, cis CH2@CH–CH2–,
Pro trans-dH, Pro cis-dH, Tyr cis-bH, Tyr trans-bH),
3.09–2.65 (m, 6.14H, cis CH2@CH–CH2–, Pro trans-
dH, Phe bH, Tyr cis-bH), 2.65–2.53 (m, 0.46H, Pro
trans-dH), 1.97–1.84 (m, 0.44H, Pro trans-bH), 1.71–
1.53 (m, 1.88H, Pro cis-bH, Pro trans-bH, Pro trans-
cH), 1.52–1.38 (m, 0.56H, Pro cis-cH), 1.38–1.20 (m,
1.12H, Pro cis-bH, Pro cis-cH). 13C NMR d: 172.65,
172.57, 170.48, 170.45, 170.40, 169.81, 165.84, 165.81,
156.72, 156.47, 137.92, 137.73, 137.47 (13q), 130.92,
130.84 (2t, Tyr Ar-C), 129.14, 129.11 (2t, Phe Ar-C),
129.09, 128.76 (2t, CH2@CH–CH2–), 127.99, 127.97,
127.94, 127.88, 126.64, 126.20 (6t, Phe Ar-C), 124.35,
123.42 (2q), 122.39, 121.98 (2s, CH2@CH–CH2–),
115.41, 115.15 (2t, Tyr Ar-C), 59.59 (s, Pro trans-aC),
59.19 (s, Pro cis-aC), 58.98 (s, Tyr cis-aC), 58.38
(s, Tyr cis-aC), 54.52, 54.19 (2s, Phe trans-aC), 53.78,
53.75 (2s, Phe cis-aC), 48.06 (s, cis CH2@CH–CH2–),
47.84 (s, trans CH2@CH–CH2–), 46.63 (s, Pro trans-
dC), 46.39 (s, Pro cis-dC), 37.45, 37.39, 37.10 (3s, Phe
bC), 35.58 (s, Tyr cis-bC), 34.84 (s, Tyr trans-bC),
31.11 (s, Pro cis-bC), 28.85 (s, Pro trans-bC), 24.09 (s,
Pro trans-cC), 21.17 (s, Pro cis-cC).
5.4.12. [N-Allyl-N-Boc-Dmt1]EM-2 (10). The title com-
pound was synthesized using the same method for 9.
20
D
Yield 225 mg (70%), mp 131–133 ꢁC, Rf5 = 0.70, ½aꢁ
ꢀ82.9ꢁ (c 0.51, MeOH). Anal. Calcd for
C42H53N5O7Æ0.5H2O: C, 67.36; H, 7.27; N, 9.35. Found:
C, 67.22; H, 7.33; N, 9.06. 1H NMR (400 MHz, DMSO-
d6) d: 8.90 and 8.85 (s · 2, 1.0H, Dmt OH), 8.06–7.81
(m, 2.0H, Phe NH), 7.35–7.00 (m, 12.0H, Phe Ar-H,
CONH2), 6.40 and 6.36 (s · 2, 2.0H, Dmt Ar-H), 5.64
(br, 1.0H, CH2@CH–CH2–), 5.10–4.88 (m, 2.27H,
CH2@CH–CH2–, Dmt cis-aH), 4.88–4.73 (m, 0.73H,
Dmt trans-aH), 4.50–4.30 (m, 2.0H, Phe aH), 4.07–
3.93 (m, 0.73H, trans CH2@CH–CH2–), 3.93–3.82
(m, 0.27H, cis CH2@CH–CH2–), 3.73–3.60 (m, 0.27H,
cis CH2@CH–CH2–), 3.60–3.48 (m, 0.73H, trans
CH2@CH–CH2–), 3.48–3.18 (m, 2.0H, Pro dH), 3.15–
2.62 (m, 6.0H, Phe bH, Dmt bH), 2.16 and 2.12 (s · 2,
6.0H, Dmt CH3), 1.90–1.55 (m, 4.0H, Pro bH, Pro
cH), 1.28 and 1.08 (s · 2, 9.0H, Boc). 13C NMR d:
172.48, 171.15, 170.41, 168.94, 168.29, 155.10, 153.36,
137.77, 137.62, 137.57 (10q), 134.72 (t, cis CH2@CH–
CH2–), 133.85 (t, trans CH2@CH–CH2–), 129.06,
127.95, 127.88, 126.13 (4t, Phe Ar-C), 116.10 (s, trans
CH2@CH–CH2–), 115.79 (s, cis CH2@CH–CH2–),
114.64 (t, Dmt Ar-C), 79.15, 78.84 (2q, Boc), 59.71
(t, Pro aC), 55.47 (t, Dmt trans-aC), 54.74 (t, Dmt,
cis-aC), 54.21, 53.73 (2t, Phe aC), 46.41 (s, Pro cis-
dC), 46.15 (s, Pro trans-dC), 45.36 (s, CH2@CH–CH2–
), 37.35, 36.96 (2s, Phe bC), 28.55 (s, Pro bC), 28.18
(s, Dmt bC), 27.70 (p, trans-Boc), 27.16 (p, cis-Boc),
24.00 (s, Pro cC), 19.85 (p, Dmt CH3).
5.4.14. [N-Allyl-Dmt1]EM-2ÆHCl (12). The title com-
pound was synthesized using the same method for 11.
20
Yield 72 mg (71.6%), Rf9 = 0.68, tR 15.82, ½aꢁ ꢀ5.9ꢁ
D
(c 0.42, H2O), m/z [M+1]+ 641. Anal. Calcd for
C37H45N5O5ÆHClÆ3H2O: C, 60.85; H, 7.18; N, 9.59.
Found: C, 61.19; H, 6.85; N, 9.65. 1H NMR
(400 MHz, DMSO-d6) d: 10.07 (br, 0.90H, Dmt
NH2þ), 9.40–9.10 (m, 1.96H, Dmt NH2þ, Dmt OH),
8.36 (d, 0.85H, J = 8.60 Hz, Phe cis-NH), 8.18 (d,
0.85H, J = 8.28 Hz, Phe cis-NH), 8.05 (d, 0.15H,
J = 8.12 Hz, Phe trans-NH), 8.01 (d, 0.15H,
J = 8.28 Hz, Phe trans-NH), 7.47 (s, 1.0H, CONH2),
7.35–7.05 (m, 11.0H, Phe Ar-H, CONH2), 6.46 and
6.42 (s · 2, 2.0H, Dmt Ar-H), 6.00–5.87 (m, 0.15H, trans
CH2@CH–CH2–), 5.87–5.73 (m, 0.85H, cis CH2@CH–
CH2–), 5.43–5.27 (m, 1.15H, trans CH2@CH–CH2–,
cis CH2@CH–CH2–), 5.23 (d, 0.85H, J = 10.39 Hz, cis
CH2@CH–CH2–), 4.48–4.32 (m, 2.22H, Phe aH, Pro
trans-aH), 4.13–4.04 (m, 0.15H, Dmt trans-aH), 3.64–
3.56 (m, 0.22H, trans CH2@CH–CH2–), 3.54–3.45 (m,
0.22H, trans CH2@CH–CH2–), 3.33–2.73 (m, 11.20H,
Pro cis-aH, Dmt cis-aH, cis CH2@CH–CH2–, Pro dH,
Phe bH, Dmt bH), 2.15 and 2.10 (s · 2, 6.0H, Dmt
CH3), 1.86–1.73 (m, 0.14H, Pro trans-bH), 1.67–1.52
(m, 1.21H, Pro cis-bH, Pro trans-bH, Pro trans-cH),
1.52–1.36 (m, 1.0H, Pro cis-cH, Pro trans-cH), 1.28–
1.12 (m, 1.66H, Pro cis-bH, Pro cis-cH). 13C NMR d:
172.66, 172.50, 170.58, 170.34, 170.16, 169.91, 166.40,
166.07, 155.88, 155.63, 138.50, 138.20, 137.96, 137.65,
137.61, 137.52 (16q), 129.12, 128.90 (2t, Phe Ar-C),
128.80 (t, CH2@CH–CH2–), 128.05, 127.96, 127.92,
127.84, 126.44, 126.08 (6t, Phe Ar-C), 122.20 (s, trans
CH2@CH–CH2–), 121.87 (s, cis CH2@CH–CH2–),
5.4.13. [N-Allyl-Tyr1]EM-2ÆHCl (11). [N-Allyl-N-Boc-
Tyr1]EM-2 (9) (147 mg, 0.206 mmol) was treated with
TFA (320 lL, 4.13 mmol) and anisole (32 lL) for
30 min at room temperature. The reaction solution
was diluted with hexane, the solid was collected by filtra-
tion, dried over KOH pellets, and purified by semi-pre-
parative RP-HPLC. The purified peptide was
lyophilized from 1 N HCl (3· 200 lL) to give amor-
phous powder. Yield 113 mg (84%), Rf9 = 0.68,
20
D
tR 15.29, ½aꢁ ꢀ20.7ꢁ (c 0.46, H2O), m/z [M+1]+ 613.
Anal. Calcd for C35H41N5O5ÆHClÆ2H2O: C, 61.44; H,
6.78; N, 10.24. Found: C, 61.32; H, 6.56; N, 10.26. H
1
NMR (400 MHz, DMSO-d6) d: 9.78 (br, 0.82H,
–NH2þ), 9.59 (s, 0.55H, Dmt cis-OH), 9.44 (s, 0.43H,
Dmt trans-OH), 9.12 (br, 0.83H, –NH2þ), 8.38 (d,
0.5H, J = 8.57 Hz, Phe cis-NH), 8.19 (d, 0.53H,
J = 8.26 Hz, Phe cis-NH), 8.15 (d, 0.42H, J = 8.30 Hz,
Phe trans-NH), 8.02 (d, 0.42H, J = 7.85 Hz, Phe trans-
NH), 7.44 (s, 0.53H, CONH2), 7.37–7.07 (m, 12.47H,
Phe Ar-H, Tyr Ar-H, CONH2), 6.92 (d, 1.0H,

1248
T. Li et al. / Bioorg. Med. Chem. 15 (2007) 1237–1251
20
D
120.86, 120.47 (2q), 115.03 (t, Dmt Ar-C), 59.76 (t, Pro
trans-aC), 58.94 (t, Pro cis-aC), 56.26 (t, Dmt cis-aC),
55.66 (t, Dmt trans-aC), 54.84, 53.65 (2t, Phe aC),
47.65 (s, cis CH2@CH–CH2–), 47.56 (s, trans
CH2@CH–CH2–), 46.63 (s, Pro cis-dC), 45.94 (s, Pro
trans-dC), 37.54 (s, Phe cis-bC), 37.44, 37.31 (2s, Phe
trans-bC), 36.83 (s, Phe cis-bC), 31.19 (s, Pro bC),
29.64 (s, Dmt cis-bC), 28.78 (s, Dmt trans-bC), 23.87
(s, Pro trans-cC), 21.09 (s, Pro cis-cC), 20.79 (p, Dmt
trans-CH3), 19.37 (p, Dmt cis-CH3).
Yield 246 mg (72.9%), mp 138-140 ꢁC, Rf6 = 0.69, ½aꢁ
ꢀ71.9ꢁ (c 0.36, MeOH). Anal. Calcd for
C44H54N6O7Æ1.25H2O: C, 65.94; H, 7.11; N, 10.49.
Found: C, 66.03; H, 6.80; N, 10.50. 1H NMR
(400 MHz, DMSO-d6) d: 10.83 (s, 0.91H, Trp Ar-NH),
8.90 (br, 0.70H, Dmt OH), 8.03 (d, 0.65H,
J = 6.98 Hz, Trp trans-NH), 7.96 (d, 0.23H,
J = 7.46 Hz, Trp cis-NH), 7.82–7.71 (m, 0.91H, Phe
NH), 7.51 (d, 1.0H, J = 7.81 Hz, Trp Ar-H), 7.32 (d,
1.0H, J = 8.02 Hz, Trp Ar-H), 7.27–7.14 (m, 6.0H, Phe
Ar-H, CONH2), 7.14–7.01 (m, 3.0H, Trp Ar-H,
CONH2), 7.01–6.92 (m, 1.0H, Trp Ar-H), 6.39 and
6.35 (s · 2, 2.0H, Dmt Ar-H), 5.71–5.55 (m, 1.0H,
CH2@CH–CH2–), 5.07–4.87 (m, 2.30H, CH2@CH–
CH2–, Dmt cis-aH), 4.79 (d, 0.70H, J = 9.29 Hz, Dmt
trans-aH), 4.47–4.34 (m, 2.0H, Phe aH, Trp aH),
4.05–3.93 (m, 0.73H, trans CH2@CH–CH2–), 3.93–3.82
(m, 0.27H, cis CH2@CH–CH2–), 3.70–3.59 (m, 0.27H,
cis CH2@CH–CH2–), 3.53 (dd, 0.73H, J = 7.16,
15.26 Hz, trans CH2@CH–CH2–), 3.47–3.17 (m, 2.0H,
Pro dH), 3.13–2.74 (m, 5.27H, Dmt cis-bH, Dmt trans-
bH, Phe bH, Trp bH), 2.72–2.59 (m, 0.73H, Dmt
trans-bH), 2.15–2.11 (2s, 6.0H, Dmt CH3), 1.98–1.58
(m, 4.0H, Pro cH, Pro bH), 1.27 and 1.07 (s · 2, 9.0H,
Boc). 13C NMR d: 172.50, 171.41, 170.85, 168.91,
168.27, 155.08, 154.84, 153.38, 137.70, 137.57, 135.92
(11q), 134.76 (t, cis CH2@CH–CH2–), 133.88 (t, trans
CH2@CH–CH2–), 129.08, 127.93 (2t, Phe Ar-C),
127.25 (q), 126.13 (t, Phe Ar-C), 124.98 (q), 123.32,
120.75, 118.12 (3t, Trp Ar-C), 116.06 (s, CH2@CH–
CH2–), 114.61 (t, Dmt Ar-C), 111.17 (t, Trp Ar-C),
109.87 (q), 79.12, 78.82 (2q, Boc), 59.74 (t, Pro aC),
55.56 (t, Dmt aC), 53.90 (t, Phe aC), 53.57 (t, Trp
aC), 46.44 (s, Pro cis-aC), 46.19 (s, Pro trans-dC),
45.37 (s, CH2@CH–CH2–), 37.27 (s, Phe bC), 28.73
(s, Pro bC), 28.16 (s, Dmt bC), 27.71, 27.15 (2p, Boc),
26.95 (s, Trp bC), 24.05 (s, Pro cC), 19.83 (p, Dmt CH3).
5.4.15. [N,N-(Allyl)2-Dmt1]EM-1ÆHCl (13). The title
compound was synthesized using the same method for
20
D
7. Yield 115 mg (71.3%), Rf9 = 0.69, tR 18.55, ½aꢁ
ꢀ33.2ꢁ (c 0.36, H2O), m/z [M+1]+ 720. Anal. Calcd for
C42H50N6O5ÆHClÆ3H2O: C, 62.32; H, 7.10; N, 10.38.
Found: C, 62.43; H, 6.99; N, 10.42. 1H NMR
(400 MHz, DMSO-d6) d: 11.66 (br, 0.16H, Dmt trans-
NH+), 11.00 (s, 0.68H, Trp cis Ar-NH), 10.89 (s,
0.25H, Trp trans Ar-NH), 10.52 (br, 0.56H, Dmt cis-
NH+), 9.39 (br, 0.58H, Dmt cis-OH), 9.16 (br, 0.15H,
Dmt trans-OH), 8.39 (d, 0.67H, J = 6.90Hz, Trp cis-
NH), 8.10–8.00 (m, 1.0H, Trp trans-NH, Phe cis-NH),
7.80 (d, 0.27H, J = 8.16 Hz, Phe trans-NH), 7.68 (d,
0.74H, J = 7.79 Hz, Trp Ar-H), 7.58 (d, 0.26H,
J = 7.75 Hz, Trp Ar-H), 7.42 (br, 0.73H, cis-CONH2),
7.38–7.30 (m, 1.0H, Trp Ar-H), 7.30–7.13 (m, 6.0H,
Phe Ar-H, Trp Ar-H), 7.13–6.95 (m, 3.27H, CONH2,
Trp Ar-H), 6.47 and 6.41 (s · 2, 2.0H, Dmt Ar-H),
6.16 (br, 0.48H, trans CH2@CH–CH2–), 5.75 (br,
0.76H, cis CH2@CH–CH2–), 5.66–5.36 (m, 2.54H, cis
CH2@CH–CH2–, CH2@CH–CH2–), 5.23 (d, 2.20H,
J = 10.23 Hz, CH2@CH–CH2–), 4.48–4.36 (m, 2.0H,
Phe aH, Trp aH), 4.35–4.28 (m, 0.29H, Pro trans-aH),
4.17–3.98 (m, 0.73H, Dmt trans-aH, CH2@CH–CH2–),
3.98–3.64 (m, 2.81H, CH2@CH–CH2–), 3.42–3.16 (m,
3.13H, Pro cis-aH, Pro cis-dH, Pro trans-dH, Dmt cis-
bH, CH2@CH–CH2–), 3.16–2.77 (m, 6.71H, Dmt cis-
aH, Pro cis-dH, Phe bH, Dmt cis-bH, Dmt trans-bH,
Trp bH), 2.20–1.94 (m, 6.29H, Dmt CH3, Pro trans-
dH), 1.80–1.72 (m, 0.24H, Pro trans-bH), 1.72–1.63
(m, 0.72H, Pro cis-bH), 1.60–1.44 (m, 0.72H, Pro
trans-bH, Pro trans-cH), 1.44–1.17 (m, 2.28H, Por cis-
bH, Pro cis-cH). 13C NMR d: 172.54, 172.49, 171.00,
170.74, 170.23, 165.85, 156.04, 138.47, 137.64, 137.55,
136.07, 135.99 (12q), 129.15, 127.90 (2t, Phe Ar-C),
127.59 (t, CH2@CH–CH2–), 127.23, 126.96 (2q),
126.13 (t, Phe, Ar-C), 125.16 (t, CH2@CH–CH2–),
124.25, (s, CH2@CH–CH2–), 123.88, 123.48, 118.33,
118.11 (4t, Trp Ar-C), 115.21, 115.05 (t, Dmt Ar-C),
111.32, 115.15 (2t, Trp Ar-C), 109.89, 109.73 (2q),
60.98 (t, Pro cis-aC), 60.03 (t, Pro trans-aC), 59.34 (t,
Dmt trans-aC), 58.86 (t, Dmt cis-aC), 54.42 (t, Phe
aC), 54.19 (s, cis CH2@CH–CH2–), 53.64 (t, Trp cis-
aC), 53.46 (t, Trp trans-aC), 51.48 (s, trans CH2@CH–
CH2–), 47.01 (s, Pro cis-dC), 46.09 (s, Pro trans-dC),
37.39 (s, Phe bC), 31.38 (s, Pro cis-bC), 28.63 (s, Pro
trans-bC), 27.79 (s, Dmt cis-bC), 27.49 (s, Dmt trans-
bC), 27.11 (s, Trp bC), 23.85 (s, Pro trans-cC), 21.24
(s, Pro cis-cC), 19.78, 19.66 (2p, Dmt CH3).
5.4.17. [N-Allyl-Dmt1]EM-1ÆHCl (15). The title com-
pound was synthesized using the same method for 11.
20
Yield 131 mg (77.0%), Rf9 = 0.63, tR 17.10, ½aꢁ ꢀ12.3ꢁ
D
(c 0.34, H2O), m/z [M+1]+ 680. Anal. Calcd for
C39H46N6O5ÆHClÆ2.5H2O: C, 61.61; H, 6.89; N, 11.05.
Found: C, 61.59; H, 6.59; N, 11.09. 1H NMR
(400 MHz, DMSO-d6) d: 11.06 (s, 0.77H, Trp cis Ar-
NH), 10.85 (s, 0.13H, Trp trans Ar-H), 10.08 and 9.88
(br · 2, 0.82H, Dmt OH), 9.27 (s, 1.60H, Dmt
cis-NH2þ), 9.14 (s, 0.28H, Dmt trans-NH2þ), 8.30
(d, 0.81H, J = 8.20 Hz, Trp cis-NH), 8.04 (d, 0.14H,
J = 8.47 Hz, trp trans-NH), 8.00 (d, 0.81H,
J = 8.09 Hz, Phe cis-NH), 7.80 (d, 0.16H, J = 8.10 Hz,
Phe trans-NH), 7.63–7.46 (m, 1.0H, Trp Ar-H), 7.43
(br, 0.84H, cis-CONH2), 7.38–7.30 (m, 1.0H, Trp Ar-
H), 7.27–7.13 (m, 5.19H, Phe Ar-H, trans-CONH2),
7.13–7.03 (m, 3.0H, CONH2, Trp Ar-H), 7.03–6.94 (m,
1.0H, Trp Ar-H), 6.43, 6.42 (2s, 2.0H, Dmt Ar-H),
5.97–5.86 (m, 0.16H, trasn CH2@CH–CH2–), 5.81–
5.76 (m, 0.84H, cis CH2@CH–CH2–), 5.44–5.30 (m,
0.32H, trans CH2@CH–CH2–), 5.21 (d, 0.84H
J = 17.06 Hz, cis CH2@CH–CH2–), 5.09 (d, 0.84H,
J = 10.36 Hz,
cis
CH2@CH–CH2–),
4.50–4.13
5.4.16. [N-Allyl-N-Boc-Dmt1]EM-1 (14). The title com-
pound was synthesized using the same method for 9.
(m, 2.18H, Phe aH, Trp aH, Pro trans-aH), 4.15–4.04
(m, 0.15H, Dmt trans-aH), 3.65–3.55 (m, 0.18H, trans

T. Li et al. / Bioorg. Med. Chem. 15 (2007) 1237–1251
1249
CH2@CH–CH2–), 3.55–3.45 (m, 0.18H, trans
CH2@CH–CH2–), 3.45–2.87 (m, 11.41H, cis
DMSO-d6) d: 9.07 (s, 2H, Dmt OH), 8.02 (br, 2H,
NHCH2) 6.39 (s, 4H, Dmt Ar-H), 5.95–5.84 (m, 2H,
CH2@CH–CH2–), 5.46–5.30 (m, 4H, CH2@CH–CH2–),
3.72–3.60 (m, 2H, Dmt aH), 3.48–3.35 (m, 4H,
CH2@CH–CH2–), 3.05–2.93 (m, 6H, Dmt bH,
NHCH2CH2), 2.92–2.80 (m, 2H, Dmt bH), 2.17 (s, 12H,
Dmt CH3), 1.20–1.02 (m, 4H, NHCH2CH2CH2), 0.96–
0.82 (m, 4H, NHCH2CH2CH2). 13C NMR d: 165.40,
155.46, 140.33, 138.11 (4q), 129.50 (t, CH2@CH–CH2–),
122.11 (s, CH2@CH–CH2–), 114.76 (t, Dmt Ar-C),
58.27 (t, Dmt aC), 47.54 (s, CH2@CH–CH2–), 38.52
(s, NHCH2), 28.29 (s, NHCH2CH2), 25.71
(s, NHCH2CH2CH2), 19.93 (p, Dmt CH3).
CH2@CH–CH2–, Pro cis-aH, Dmt cis-aH, Pro dH,
Phe bH, Dmt bH, Trp bH), 2.15, 2.06 (2s, Dmt CH3),
1.87–1.76 (m, 0.16H, Pro trans-bH), 1.68–1.48 (m,
1.32H, Pro cis-bH, Pro trans-bH, Pro trans-cH), 1.48–
1.33 (m, 0.84H, Pro cis-cH), 1.33–1.77 (m, 1.68H, Pro
cis-bH, Pro cis-cH). 13C NMR d: 172.57, 172.42,
170.86, 170.67, 170.19, 169.96, 166.31, 155.81, 155.62,
138.52, 138.27, 137.62, 137.53, 135.98, 135.89 (15q),
129.11 (t, Phe Ar-C), 128.69 (s, CH2@CH–CH2–),
127.83 (t, Phe Ar-C), 127.19, 126.94 (2q), 126.08
(t, Phe Ar-C), 123.43, 122.89 (t, Trp Ar-C), 121.79
(t, CH2@CH–CH2–), 120.83 (t, Trp Ar-C), 120.47 (q),
118.18, 118.12 (2t, Trp Ar-C), 114.96 (t, Dmt Ar-C),
111.35 (t, Trp Ar-C), 110.18, 109.67 (2q), 59.82 (t, Pro
trans-aC), 58.88 (t, Pro trans-aC), 56.29 (t, Dmt cis-
aC), 55.78 (t, Dmt, trans-aC), 53.49 (t, Phe aC), 53.49
(t, Trp aC), 47.64 (s, Pro trans-aC), 46.67 (s, Pro cis-
aC), 45.91 (s, CH2@CH–CH2–), 37.45 (s, Phe bC),
31.27 (s, Pro bC), 29.73 (s, Dmt bC), 27.10 (s, Trp
bC), 24.01 (s, Pro trans-cC), 21.20 (s, Pro cis-cC),
19.34, 19.28 (2p, Dmt CH3).
5.4.20. 3,6-Bis-(N-allyl-N-Boc-Dmt-aminopropyl)-2(1H)-
pyrazinone (18). 3,6-Bis- (Z-aminopropyl)-2(1H)-pyrazi-
none (141 mg, 0.286 mmol) was stirred in 25% HBr/
HOAc (960 lL, 4 mmol) with an ice bath for 10 min and
then at room temperature for 3 h. The resulting amine
was precipitated with ether, collected by filtration, and
dried in vacuo. The amine hydrobromide was dissolved
in DMF (10 mL) containing DIPEA (240 lL,
1.37 mmol), to which N-allyl-N-Boc-Dmt-OH (6b)
(200 mg, 0.572 mmol) and PyBop (312 mg, 0.60 mmol)
were added. The solution was first stirred in an ice bath
for 10 min, then at room temperature for 4 h. After
removal of the solvent in vacuo, the residue was extracted
with AcOEt (30 mL), which was washed with 10% citric
acid (2· 10 mL), 5% NaHCO3 (2· 10 mL), and saturated
aqueous NaCl solution (2· 10 mL), dried over Na2SO4,
filtered, and evaporated in vacuo. The resulting residue
was purified by flash chromatography (SiO2, AcOEt/
MeOH = 10:1) and the compound was precipitated with
5.4.18. 1,6-Bis-(N-allyl-N-Boc-Dmt-NH)-hexane (16). To
a solution of 1,6-diaminohexane (33 mg, 0.286 mmol) in
DMF (10 mL), N-allyl-N-Boc-Dmt-OH (6b) (200 mg,
0.572 mmol), DIPEA (119 lL, 0.687 mmol), and PyBop
(313 mg, 0.60 mmol) were added. The reaction mixture
was stirred at 0 ꢁC for 10 min, then at room temperature
for 4 h. After removal of the solvent, the residue was
extracted with AcOEt (50 mL). The organic phase was
washed with cold 10% citric acid (3· 10 mL), 5%
Na2CO3 (3· 10 mL), and saturated aqueous NaCl (3·
10 mL), dried over Na2SO4, and evaporated to dryness.
The residue was purified by flash chromatography
(SiO2, AcOEt/hexane = 2:1), precipitated with hexane,
ether. Yield 109 mg (43%), mp 122–124 ꢁC, Rf5 = 0.59,
20
½aꢁ
ꢀ57.0ꢁ (c 0.31, MeOH). Anal. Calcd for
D
C49H70N6O9Æ0.5H2O: C, 65.67; H, 7.99; N, 9.38. Found:
C, 65.49; H, 7.91; N, 9.33. 1H NMR (400 MHz, DMSO-
d6) d: 11.89 (br, 1H, Pyrazinone NH), 8.89 (br, 2H, Dmt
OH), 7.66 (s, 2H, NHCH2CH2CH2), 6.37 (s, 4H, Dmt
Ar-H), 5.62 (br, 2H, CH2@CH–CH2–), 5.10–4.70 (m,
4H, CH2@CH–CH2–), 4.45–3.94 (m, 2H, Dmt aH),
3.94–3.20 (m, 4H, CH2@CH–CH2–), 3.20–2.60 (m, 8H,
Dmt bH, NHCH2CH2CH2), 2.60–2.00 (m, 19H, Dmt
CH3, pyrazinone CH3, NHCH2CH2CH2), 1.80–1.05 (m,
22H, Boc, NHCH2CH2CH2). 13C NMR d: 170.01,
169.77, 155.54, 154.97, 153.94, 137.60 (6q), 135.72,
134.81 (2t, CH2@CH–CH2–), 125.51 (q), 116.45, 115.61
(2s, CH2@CH–CH2–), 114.71 (t, Dmt Ar-C), 79.03 (q,
Boc), 58.48 (t, Dmt aC), 48.03 (s, CH2@CH–CH2–),
38.52, 38.01 (2s, NHCH2CH2CH2), 29.21 (s, NHCH2CH2
CH2), 28.51 (s, Dmt bC), 26.09 (s, NHCH2CH2CH2),
19.80 (p, Dmt CH3), 18.02 (p, pyrazinone CH3).
filtered, and dried in vacuo. Yield 132 mg (59.2%), mp
20
90–92 ꢁC, Rf7 = 0.65, ½aꢁ ꢀ57.6ꢁ (c 0.32, MeOH). Anal.
Calcd for C44H66N4O8ÆD0.5H2O: C, 67.06; H, 8.57; N,
7.11. Found: C, 67.09; H, 8.57; N, 7.13. 1H NMR
(400 MHz, CDCl3) d: 8.04 (s, 1.3H, Dmt OH), 6.53 (s,
4.0H, Dmt Ar-H), 5.96–5.52 (m, 4.0H, NH, CH2@CH–
CH2–), 5.26–4.95 (m, 4.0H, CH2@CH–CH2–), 4.55–
4.23 (m, 2.0H, Dmt aH), 4.23–3.85 (m, 4.0H,
CH2@CH–CH2–), 3.53–3.10 (m, 4.0H, Dmt bH), 3.10–
2.55 (m, 4.0H, NHCH2), 2.28 (s, 12.0H, Dmt CH3),
1.50 and 1.46 (s · 2, 18.0H, Boc), 1.40–0.98 (m, 4.0H,
NHCH2CH2), 0.98–0.50 (m, 4.0H, NHCH2CH2CH2).
13C NMR d: 170.75, 156.43, 154.73, 154.10, 138.44
(5q), 135.63 (t, CH2@CH–CH2–), 126.34 (q), 115.64
(s, CH2@CH–CH2–), 115.39, 115.04 (2t, Dmt Ar-C),
81.50 (q, Boc), 59.25 (t, Dmt aC), 46.87 (s, CH2@CH–
CH2–), 39.15 (s, NHCH2), 29.89 (s, Dmt bC), 28.81
(s, NHCH2CH2), 28.38 (p, Boc), 26.29 (s,
NHCH2CH2CH2), 20.31 (p, Dmt CH3).
5.4.21. 3,6-Bis-(N-allyl-Dmt-aminopropyl)-2(1H)-pyrazi-
noneÆ2HCl (19). The title compound was synthesized
using the same method for 11. Yield 31.4 mg (64.6%),
20
Rf9 = 0.60, tR 16.8, ½aꢁ +32.1ꢁ (c 0.36, H2O), m/z
D
5.4.19. 1,6-Bis-(N-allyl-Dmt)-hexaneÆ2HCl (17). The title
compound was synthesized using the same method for 11.
[M+1]+ 688. Anal. Calcd for C39H54N6O5Æ2HClÆ5.5H2O:
C, 54.54; H, 7.86; N, 9.78. Found: C, 54.66; H, 7.50; N,
Yield 36.4 mg (57.3%), Rf9 = 0.66, tR 15.43, ½aꢁ20 +55.0ꢁ (c
9.84. H NMR (400 MHz, DMSO-d6) d: 9.99 (br, 2H,
1
D
0.36, H2O), m/z [M+1]+ 680. Anal. Calcd for
C34H50N4O4Æ2HClÆ2.5H2O: C, 58.21; H, 8.25; N, 8.04.
Found: C, 58.30; H, 7.90; N, 8.03. H NMR (400 MHz,
Dmt NH2þ), 9.33 (br, 2H, Dmt NH2þ), 9.07 (br, 2H,
Dmt OH), 8.23 (t, 1H, J = 4.77 Hz, NHCH2CH2CH2),
8.15 (t, 1H, J = 4.52 Hz, NHCH2CH2CH2), 6.38
1

1250
T. Li et al. / Bioorg. Med. Chem. 15 (2007) 1237–1251
(s, 4H, Dmt Ar-H), 5.98–5.85 (m, 2H, CH2@CH–CH2–),
5.50–5.32 (m, 4H, CH2@CH–CH2–), 3.80–3.30 (m, 6H,
Dmt aH, CH2@CH–CH2–), 3.13–2.85 (m, 8H, Dmt
bH, NHCH2CH2CH2), 2.47–2.42 (m, 2H, NHCH2
CH2CH2), 2.30–2.05 (m, 17H, Dmt CH3, Pyrazinone
tween placement of mice on the hot plate and movement
consisting ofeither licking or shaking their hind paws with
a baseline latency of 15 s and maximal cut off time of 30 s.
Tail-flick latency is the latency for removal of the tail from
the onset of radiant heat with a baseline latency of 2–3 s,
and with a cut-off time of 8 s. The duration time after
icv administration was 10 min and the test was terminated
when hot-plate and tail-flick latencies were close to the
pre-response times. Statistical significance applied one-
way analysis of variance (ANOVA) followed by Dun-
nett’s test (significant at P < 0.05). The area under the
time–response curve (AUC) combines the response time
and time after administration.18,19 The percent of maxi-
mal possible effect (%MPE) = (T1 ꢀ T0)/(T2 ꢀ T0) ·
100, where the T0 and T2 are pre-response and cut off
times, respectively. AD50 presents the dose of 12 or nalox-
one to inhibit 50% of the morphine-induced antinocicep-
tion at 10 min post-injection.
CH3,
NHCH2CH2CH2),
1.70–1.38
(m,
4H,
NHCH2CH2CH2). 13C NMR d: 166.81, 166.70, 155.57,
155.52, 155.40, 138.20 (6q), 128.54, 128.45 (2t,
CH2@CH–CH2–), 122.78, 122.68 (2s, CH2@CH–CH2–),
121.69, 121.64 (2q), 114.82 (t, Dmt Ar-C), 57.90, 57.86
(2t, Dmt aC), 47.28 (s, CH2@CH–CH2–), 38.59, 38.20
(2s, NHCH2CH2CH2), 29.27, 28.76 (2s, NHCH2
CH2CH2), 27.17 (s, Dmt bC), 25.42 (s, NHCH2
CH2CH2), 19.90, 19.87 (2p, Dmt CH3), 17.97
(p, pyrazinone CH3).
5.5. Competitive receptor binding
Rat brain P2 synaptosomal preparations were used in
competitive displacement assays for l- and d-opioid
receptors after removal of endogenous opioids as pub-
lished elsewhere in considerable detail,11,17–19 and using
3.5 nM [3H]DAMGO [(D-Ala2,N-Me-Phe4,Gly-ol5)en-
kephalin] (50.0 Ci/mmol, Amersham Biosciences, Buck-
inghamshire, UK) and 1.9 nM [3H]deltorphin II
(45.0 Ci/mmol, Perkin-Elmer, Boston, MA) to radiola-
bel l- and d-opioid receptors, respectively. After a 2.5-
h incubation at room temperature (22–23 ꢁC), samples
were rapidly rinsed on Whatman GF/C glass fiber filters,
presoaked in 0.1% polyethyleneimine to enhance the re-
sponse : background ratio, washed with ice-cold buffer,
dried, and the radioactivity determined using EcoLume
scintillation fluid.17–19
5.8. Brain slice preparation and whole cell recording
Vibratome-prepared hippocampal slices (300 lm) of rat
brains were incubated in a holding chamber containing
artificial cerebrospinal fluid (ACSF) bubbled with
O2/CO2 (95:5%) at room temperature for 60 min.
Spontaneous GABAA receptor-mediated IPSCs were
pharmacologically isolated by adding 50 lM D-(ꢀ)-2-
amino-5-phosphonovaleric acid and 20 lM 6,7-dinitro-
quinoxaline-2,3-dione.28 Patch-clamp pipettes were
prepared as detailed previously.28 Individual CA1 pyra-
midal cells were visualized using an infrared differential
interference contrast Zeiss Axioskop microscope (40-X
water immersion objective) and spontaneous IPSCs were
recorded at ꢀ70 mV for 10 min periods consistently of
baseline conditions, plus 60 mM EtOH, and with
300 nM 15 in EtOH. Series resistance was monitored
throughout the recording and cells were discarded if they
changed significantly, and the data were analyzed using
paired Student’s t-tests. More comprehensive controls,
dose–response curves, and analyses were conducted
using 12 with comparable data (Li; Swartzwelder, et al.
unpublished data).
5.6. Functional opioid bioassays
Myenteric plexus longitudinal muscle from the small
intestine of male Hartley strain guinea pigs (GPI) mea-
sured l-opioid agonism and a single mouse vas deferens
(MVD) determined d-opioid agonism. The isolated tis-
sues were suspended in organ baths containing balanced
salt solutions in a physiological buffer, pH 7.5.17–19 Ago-
nists inhibited electrically evoked contractions; the IC50
(nM) was obtained from dose–response curves
(mean SE five to six separate tissue preparations).
Antagonism was determined against the d-opioid ago-
nist deltorphin II or l-opioid agonist endomorphin-2
and expressed as the pA2 (Table 1), which was deter-
mined using the Schild Plot.31 The Schild slopes of all
antagonists were 0.917–1.189 in the GPI and 0.883–
1.088 in the MVD assays.
Acknowledgments
These studies were supported in part by a Grant-in-Aid
for Japan Society for the Promotion of the Science
(JSPS) Fellows (1503306) to T.L., in part by the Intra-
mural Research Program of the NIH, and NIEHS,
and in part by NIAAA Grant Nos. 12478 and 14894,
and VA Merit Review and Senior Research Career Sci-
entist awards to H.S.S. The authors appreciate the pro-
fessional expertise of the library staff and the
Comparative Medicine Branch at NIEHS. A U.S. Provi-
sional Patent Application No. 60/714,071 covers the
compounds in Table 1 and derivatives thereof.
5.7. Inhibition of morphine antinociception
Male mice were used according to protocols approved by
the Animal Care and Use Committee at the NIEHS.
Intracerebroventricular (icv) administration was per-
formed as described, and antinociception measured using
a hot-plate (55.0 0.1 ꢁC) and by tail-flick tests.18,19 Five
minutes after subcutaneous administration of morphine
(4 mg/kg), 12 or naloxone was injected icv. As a control
to determine the effect of the compound on mice, only
12was injected icv. Thehot-plate latency is the interval be-
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