The opioid μ agonist/δ antagonist DIPP-NH2[Ψ] produces a potent analgesic effect, no physical dependence, and less tolerance than morphine in rats

Article abstract of DOI:10.1021/jm980724+

Opioid compounds with mixed μ agonist/δ antagonist properties are expected to be analgesics with low propensity to produce tolerance and dependence. In an effort to strengthen the μ agonist component of the mixed μ agonist/δ antagonist H-Tyr-Tic-Phe-NH

Full text of DOI:10.1021/jm980724+

3520
J . Med. Chem. 1999, 42, 3520-3526
Th e Op ioid µ Agon ist/δ An ta gon ist DIP P -NH2[Ψ] P r od u ces a P oten t An a lgesic
Effect, No P h ysica l Dep en d en ce, a n d Less Toler a n ce th a n Mor p h in e in Ra ts
Peter W. Schiller,*,† Marian E. Fundytus,‡,∇ Lisa Merovitz,‡ Grazyna Weltrowska,† Thi M.-D. Nguyen,†
Carole Lemieux,† Nga N. Chung,† and Terence J . Coderre‡
Laboratory of Chemical Biology and Peptide Research and Pain Mechanisms Laboratory,
Clinical Research Institute of Montreal,§ 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7
Received April 23, 1999
Opioid compounds with mixed µ agonist/δ antagonist properties are expected to be analgesics
with low propensity to produce tolerance and dependence. In an effort to strengthen the µ
agonist component of the mixed µ agonist/δ antagonist H-Tyr-Tic-Phe-Phe-NH2 (TIPP-NH2),
analogues containing structurally modified tyrosine residues in place of Tyr1 were synthesized.
Among the prepared compounds, H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2; Dmt ) 2′,6′-dimethyl-
tyrosine) and H-Dmt-TicΨ[CH2NH]Phe-Phe-NH2 (DIPP-NH2[Ψ]) retained a mixed µ agonist/δ
antagonist profile, as determined in the guinea pig ileum and mouse vas deferens assays,
whereas H-Tmt-Tic-Phe-Phe-NH2 (Tmt ) N,2′,6′-trimethyltyrosine) was a partial µ agonist/δ
antagonist and H-Tmt-TicΨ[CH2NH]Phe-Phe-NH2 was a µ antagonist/δ antagonist. DIPP-NH2-
[Ψ] showed binding affinities in the subnanomolar range for both µ and δ receptors in the rat
brain membrane binding assays, thus representing the first example of a balanced µ agonist/δ
antagonist with high potency. In the rat tail flick test, DIPP-NH2[Ψ] given icv produced a
potent analgesic effect (ED50 ) 0.04 µg), being about 3 times more potent than morphine (ED50
) 0.11 µg). It produced less acute tolerance than morphine but still a certain level of chronic
tolerance. Unlike morphine, DIPP-NH2[Ψ] produced no physical dependence whatsoever upon
chronic administration at high doses (up to 4.5 µg/h) over a 7-day period. In conclusion, DIPP-
NH2[Ψ] fulfills to a large extent the expectations based on the mixed µ agonist/δ antagonist
concept with regard to analgesic activity and the development of tolerance and dependence.
In tr od u ction
showed modest µ agonist potency in the guinea pig
ileum (GPI) assay (IC50 ) 1.70 ( 0.22 µM) and quite
high δ antagonist potency in the mouse vas deferens
(MVD) assay against the δ agonist DPDPE (Ke ) 18.0
( 2.2 nM). In agreement with the bioassay data, TIPP-
NH2 displayed relatively low affinity for µ receptors (Kiµ
) 78.8 ( 7.1 nM) and high affinity for δ receptors (Kiδ
) 3.0 ( 1.5 nM) in the rat brain membrane binding
assays, indicating that it was quite δ-selective (Kiµ/Kiδ
) 26.3). It showed no affinity for κ receptors in the
guinea pig brain membrane assay at concentrations up
to 10 µM. Furthermore, TIPP-NH2 was shown to under-
go spontaneous degradation via diketopiperazine forma-
tion in organic solvents but not in aqueous solution.5
The development of tolerance and physical depen-
dence induced by chronic administration of opioids, such
as morphine, limits their use in the treatment of pain.
A report by Abdelhamid et al.2 first described that
selective δ opioid receptor blockade with the δ antago-
nist naltrindole in mice greatly reduced the development
of morphine tolerance and dependence in both the acute
and chronic models. In a subsequent study, it was shown
that continuous infusion (icv) of the highly selective δ
opioid antagonist TIPP[Ψ] in parallel with continuous
delivery of morphine (sc) to rats also attenuated the
development of morphine tolerance and dependence to
a large extent.3 These results suggest the possibility of
the combined use of a µ type opioid analgesic and a δ
opioid antagonist in chronic pain treatment. Alterna-
tively, the development of a single opioid compound
acting as an agonist at the µ receptor and as an
antagonist at the δ receptor might be of benefit in the
management of chronic pain. Such a mixed µ agonist/δ
antagonist would be expected to be an analgesic with
low propensity to produce analgesic tolerance and
physical dependence.
In this report, we describe attempts to strengthen the
µ agonist component of TIPP-NH2 without compromis-
ing its δ antagonist properties. Since replacement of the
N-terminal tyrosine residue in opioid peptides with 2′,6′-
dimethyltyrosine (Dmt) has been known to produce a
more dramatic binding affinity increase at the µ receptor
than at the δ receptor,6,7 the Dmt1 analogue of TIPP-
NH2, H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2), was pre-
pared. Furthermore, we synthesized the pseudopeptide
analogue H-Dmt-TicΨ[CH2NH]Phe-Phe-NH2 (DIPP-
NH2[Ψ]), containing a reduced peptide bond between
the Tic2 and Phe3 residues, to exclude the possibility of
chemical degradation via diketopiperazine formation.5,8
Analogues of DIPP-NH2 and DIPP-NH2[Ψ] containing
N,2′,6′-trimethyltyrosine (Tmt) in place of Dmt1 were
also prepared, since it was expected that methylation
of the N-terminal amino group would render these
The first known compound with mixed µ agonist/δ
antagonist properties was the tetrapeptide amide H-
Tyr-Tic-Phe-Phe-NH2 (TIPP-NH2).4 This compound
* To whom correspondence should be addressed.
† Laboratory of Chemical Biology and Peptide Research.
‡ Pain Mechanisms Laboratory.
∇ Present address: Division of Palliative Care, Department of
Oncology, McGill University, Montreal, Quebec, Canada H3A 1A1.
§ Affiliated with the University of Montreal.
10.1021/jm980724+ CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/05/1999

Opioid µ Agonist/ δ Antagonist DIPP-NH2[Ψ]
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3521
Ta ble 1. Guinea Pig Ileum (GPI) and Mouse Vas Deferens (MVD) Assays of TIPP-NH2 Analogues
MVD Ke, nMa
compd
GPI IC50, nMa
DPDPE
deltorphin I
H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2)
H-Dmt-TicΨ[CH2NH]Phe-Phe-NH2 (DIPP-NH2[Ψ])
H-Tmt-Tic-Phe-Phe-NH2
H-Tmt-TicΨ[CH2NH]Phe-Phe-NH2
H-Tyr-Tic-Phe-Phe-NH2 (TIPP-NH2)
18.2 ( 1.8
7.71 ( 0.31
PA (38%)b
antagonistc
1700 ( 220
0.209 ( 0.037
0.537 ( 0.026
0.235 ( 0.032
22.1 ( 3.5
0.260 ( 0.064
0.486 ( 0.058
0.233 ( 0.010
54.4 ( 6.0
18.0 ( 2.2
14.4 ( 2.2
a
b
Mean of three to six determinations ( SEM. Partial agonist; value in parentheses indicates maximal inhibition of the contractions
(%) obtained at high concentrations. c Ke ) 64.5 ( 5.8 nM (determined against TAPP).
peptides more lipophilic and would improve their ability
to cross the blood-brain barrier (BBB). In the present
paper, we report on the syntheses and in vitro opioid
activity profiles of these analogues. We also describe the
analgesic activity of the most promising compound,
DIPP-NH2[Ψ], and its propensity to produce physical
dependence and tolerance.
An a lgesic Testin g a n d Assessm en t of Toler a n ce
a n d Dep en d en ce Develop m en t. Drugs were admin-
istered to rats by the intracerebroventricular (icv) route.
Analgesic potencies (ED50’s) were determined using the
rat tail flick test. To assess the development of acute
tolerance, rats were infused (icv) with the opioid drug
at two different high-dose levels and the period during
which the animals stayed analgesic was determined.
The development of chronic tolerance was assessed by
determining the ED50 at the end of a 7-day period during
which DIPP-NH2[Ψ] or morphine had been injected (icv)
twice daily at a high dose. To determine the develop-
ment of physical dependence, the same rats that had
been used in the acute tolerance study were chronically
infused (icv) with the opioid compound at three different
high-dose levels for 7 days. At the end of the drug
treatment, the abstinence syndrome was precipitated
by subcutaneous (sc) injection of naloxone and with-
drawal symptoms were assessed quantitatively by mea-
suring the amount of time spent teeth chattering and
writhing and by determining the frequency of jumps and
wet dog shakes.
Ch em istr y
Peptides were synthesized by the solid-phase method
using tert-butyloxycarbonyl (Boc)-protected amino acids
and 1,3-diisopropylcarbodiimide (DIC)/1-hydroxybenzo-
triazole (HOBt) as coupling agents. Introduction of the
reduced peptide bond between the Tic2 and Phe3 resi-
dues required a reductive alkylation reaction9 between
2-Boc-1,2,3,4-tetrahydroisoquinoline-3-aldehyde and the
amino group of the resin-bound H-Phe-Phe dipeptide.
2-Boc-1,2,3,4-tetrahydroisoquinoline-3-aldehyde was syn-
thesized via preparation of 2-Boc-1,2,3,4-tetrahydroiso-
quinoline-3-(N-methoxy-N-methylamide) as described in
the literature.8,10 The Boc derivative of commercially
available D,L-2′,6′-dimethyltyrosine was prepared and
incorporated into the peptides in racemic form. Boc-D,L-
N,2′,6′-trimethyltyrosine was obtained by N-methylation
of Boc-D,L-2′,6′-dimethyltyrosine and was also used in
racemic form in the peptide synthesis. Peptides were
cleaved from the resin by HF/anisole treatment in the
usual manner. Peptide purification and separation of
the diastereomeric peptides were achieved by prepara-
tive reversed-phase HPLC. The stereochemical assign-
ment of the diastereomeric peptides was based on
analysis of their acid hydrolysates by chiral TLC.
Resu lts a n d Discu ssion
Substitution of Dmt for Tyr1 in the TIPP-NH2 parent
peptide resulted in a compound, H-Dmt-Tic-Phe-Phe-
NH2 (DIPP-NH2), which showed a 93-fold potency
increase in the GPI assay (Table 1). This effect was
reversed by naloxone at low concentration (Ke ) 2.42 (
0.27 nM), indicating that it was mediated by µ opioid
receptors and not by κ receptors.12 In comparison with
the parent peptide, DIPP-NH2 also displayed 86- and
55-fold increased δ antagonist potency against the δ
agonists DPDPE and deltorphin I, respectively, in the
MVD assay. In agreement with the results observed in
the functional assays, DIPP-NH2 showed corresponding
increases in µ and δ receptor affinities in the rat brain
membrane binding assays (Table 2). The binding data
indicate that this compound is still somewhat δ receptor-
selective, as indicated by the ratio of the binding
inhibition constants (Kiµ/Kiδ ) 10.1).
Biology
Op ioid R ecep t or Bin d in g Assa ys a n d in Vit r o
Bioa ssa ys. Binding affinities for µ and δ opioid recep-
tors were determined by displacing, respectively, [3H]-
DAMGO and [3H]DSLET from rat brain membrane
binding sites, and κ opioid receptor affinities were
measured by displacement of [3H]U69,593 from guinea
pig brain membrane binding sites. For the determina-
tion of their in vitro opioid activities, analogues were
tested in bioassays based on inhibition of electrically
evoked contractions of the GPI and MVD. The GPI assay
is usually considered as being representative for µ
receptor interactions, even though the ileum does also
contain κ receptors. In the MVD assay opioid effects are
primarily mediated by δ receptors, but µ and κ receptors
also exist in this tissue. δ Antagonist potencies in the
MVD assay were determined against the δ agonists
DPDPE and deltorphin I, and the highly selective µ
agonist TAPP (H-Tyr-D-Ala-Phe-Phe-NH2)11 was used
for µ antagonist potency determinations in the GPI
assay.
Reduction of the peptide bond between Tic2 and Phe3
of DIPP-NH2 produced a compound, H-Dmt-TicΨ-
[CH2NH]Phe-Phe-NH2 (DIPP-NH2[Ψ]), which showed
further enhanced µ agonist potency in the GPI assay
(IC50 ) 7.71 ( 0.31 nM). This effect was again µ recep-
tor-mediated, as indicated by the low Ke value (1.25 (
0.14 nM) determined for naloxone as antagonist. In the
MVD assay, DIPP-NH2[Ψ] turned out to be a δ antago-
nist with about one-half the potency of DIPP-NH2, but
showing Ke values against the two δ agonists that were
still in the subnanomolar range. At high concentrations
(>1 µM) DIPP-NH2[Ψ] produced a partial agonist effect
(20% inhibition of the electrically evoked contractions)

3522 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Schiller et al.
Ta ble 2. Binding Affinities of TIPP-NH2 Analogues at µ and δ Receptors in Rat Brain Homogenates
[3H]DAMGO
[3H]DSLET
rel potencyb
compd
Kiµ,a nM
rel potencyb
Kiδ,a nM
Kiµ/Kiδ
H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2)
H-Dmt-TicΨ[CH2NH]Phe-Phe-NH2 (DIPP-NH2[Ψ])
H-Tmt-Tic-Phe-Phe-NH2
H-Tmt-TicΨ[CH2NH]Phe-Phe-NH2
H-Tyr-Tic-Phe-Phe-NH2 (TIPP-NH2)
[Leu5]enkephalin
1.19 ( 0.11
0.943 ( 0.052
4.08 ( 0.30
76.2 ( 2.0
7.92 ( 0.73
10.0 ( 0.6
2.31 ( 0.17
0.124 ( 0.003
0.120 ( 0.011
1
0.118 ( 0.016
0.447 ( 0.007
0.393 ( 0.070
61.0 ( 17.9
3.0 ( 0.15
21.4 ( 2.9
5.66 ( 0.09
6.44 ( 1.15
0.0415 ( 0.0122
0.843 ( 0.043
1
10.1
2.11
10.4
1.25
26.3
3.73
78.8 ( 7.1
9.43 ( 2.07
2.53 ( 0.35
a
b
Mean of three determinations ( SEM. Potency relative to that of [Leu5]enkephalin.
in this assay. This effect was fully reversible with
naloxone (100 nM) but not at all reversible with the
highly δ-selective opioid antagonist TIPP[Ψ]8 (100 nM),
indicating that it was entirely mediated by µ opioid
receptors which are also present in the MVD aside from
the predominant δ receptors. This result indicates that
DIPP-NH2[Ψ] has no δ agonist properties. In the recep-
tor binding assays, DIPP-NH2[Ψ] displayed subnano-
molar affinity for both µ and δ receptors and a Kiµ/Kiδ
selectivity ratio of 2.11. Taken together, these data
indicate that DIPP-NH2[Ψ] represents the first known
example of a balanced mixed µ agonist/δ antagonist with
high potency.
Replacement of Tyr1 in TIPP-NH2 with Tmt led to a
compound, H-Tmt-Tic-Phe-Phe-NH2, which behaved as
a partial µ agonist in the GPI assay (Table 1). The max-
imal inhibition of the electrically evoked contractions
that could be achieved with this compound amounted
to 38%. In the receptor binding assay it displayed high
µ receptor affinity, being about 20 times more potent
than TIPP-NH2. These results and the fact that the
ileum preparation contains a large number of spare µ
receptors13 indicate that H-Tmt-Tic-Phe-Phe-NH2 is a
partial µ agonist with low intrinsic efficacy. In the MVD
assay, this analogue showed 60-75 times higher δ
antagonist potency against the two δ agonists, as
compared to the parent peptide. In agreement with the
latter result, its δ receptor affinity was also found to be
high (Kiδ ) 0.393 ( 0.070 nM). Interestingly, the corre-
sponding analogue with a reduced peptide bond, H-Tmt-
TicΨ[CH2NH]Phe-Phe-NH2, turned out to be a moder-
ately potent µ antagonist (Ke ) 64.5 ( 5.8 nM) against
the highly specific µ agonist TAPP in the GPI assay.
This compound also showed only moderate δ antagonist
potency, being somewhat less potent than the TIPP-NH2
parent peptide. Its moderate µ and δ antagonist poten-
cies are in agreement with the relatively low µ and δ
receptor affinities determined in the binding assays. All
five peptides showed Kiκ values > 1 µM in the binding
assay based on displacement of [3H]U69,593 from
guinea pig brain membranes and, thus, did not have
any significant affinity for κ opioid receptors.
On the basis of its high potency, balanced µ agonist/δ
antagonist properties, and stability, DIPP-NH2[Ψ] was
selected for a determination of its analgesic potency and
propensity to produce tolerance and dependence. The
analgesic potencies of DIPP-NH2[Ψ] and morphine
given icv were determined in the rat tail flick test. Tail
flick latencies were measured and % MPE (maximum
possible effect) scores were calculated. The area under
the curve (AUC) score was determined for each rat, and
a log dose-response (AUC) curve was constructed
(Figure 1). For DIPP-NH2[Ψ] an ED50 of 0.04 µg was
determined from the log dose-response curve (Table 3).
F igu r e 1. Log dose-response curves of morphine (A) and
DIPP-NH2[Ψ] (B) determined in the rat tail flick test.
Ta ble 3. Analgesic Potency of DIPP-NH2[Ψ] in Comparison
with Morphine
compd
ED50, µg (95% CI)a
DIPP-NH2[Ψ]
morphine
0.04 (0.006-0.25)
0.11 (0.01-3.24)
a
Maximal AUC ) 1100.
This ED50 is about 3 times lower than that determined
for morphine (ED50 ) 0.11 µg) in the same assay system,
indicating that the mixed µ agonist/δ antagonist has
potent analgesic properties. The high analgesic potency
of DIPP-NH2[Ψ] is in agreement with its high µ receptor
affinity and high µ opioid agonist potency determined
in the in vitro assays.
To assess the development of acute analgesic toler-
ance, rats were continuously infused icv with DIPP-NH2-
[Ψ] at a dose of 0.15 µg [(ED50 × 3.7)/h] or with mor-
phine at a dose of 0.3 µg [(ED50 × 2.7)/h] and the mean
time after initiation of drug treatment for tail flick laten-
cies to return to baseline levels was determined. As
shown in Figure 2, the DIPP-NH2[Ψ]-treated rats stayed

Opioid µ Agonist/ δ Antagonist DIPP-NH2[Ψ]
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3523
F igu r e 2. Mean time intervals of analgesic efficacy of
morphine and DIPP-NH2[Ψ] at two different doses adminis-
tered icv, as determined in the rat tail flick test. Planned
comparisons revealed that rats given DIPP-NH2[Ψ] at a dose
of 0.15 µg/h were analgesic for a significantly longer period of
time than control rats (F ) 11.78(1,65), p < 0.05). Planned
comparisons revealed that rats given DIPP-NH2[Ψ] (0.15 µg/
h) were analgesic for a significantly longer period of time than
rats given morphine at a dose of 0.3 µg/h (F ) 6.674(1,65), p <
0.05). *Significant difference from 0.3 µg/h determined for
morphine. @Significant difference from control (p < 0.05, post-
hoc LSD t-tests).
F igu r e 3. AUC scores determined in the rat tail flick test of
rats that had been injected icv twice daily for 7 days with
either morphine or DIPP-NH2[Ψ] at respective doses of 0.3 and
0.15 mg/h and then were given various doses (icv) of either
morphine or DIPP-NH2[Ψ]. An ED50 of 48.5 µg (R2 ) 0.37) was
calculated from the AUC scores for rats treated with morphine.
At a dose of 1 µg the AUC score of DIPP-NH2[Ψ] was higher
than that of morphine, but at higher doses DIPP-NH2[Ψ]
produced seizures which prevented the calculation of an ED50
.
as follows: 0.3 µg [(ED50 × 2.7)/h], 4.5 µg [(ED50 × 41)/
h], and 9.0 µg [(ED50 × 82)/h]; DIPP-NH2[Ψ] doses were
as follows: 0.15 µg [(ED50 × 3.7)/h], 2.25 µg [(ED50
×
analgesic for a significantly longer time interval (15 h)
than the morphine-treated rats (3 h), indicating that
DIPP-NH2[Ψ] produced less acute tolerance at this dose
level than morphine. However, when the doses of
infused DIPP-NH2[Ψ] and morphine were increased 30-
fold to 4.5 µg [(ED50 × 112)/h] and 9 µg [(ED50 × 82)/h],
respectively, both drugs showed a very short time
interval of analgesic efficacy. Thus, at this very high
dose level both DIPP-NH2[Ψ] and morphine produced
acute tolerance to a similar degree.
56)/h], and 4.5 µg [(ED50 × 112)/h]. At the end of the
drug treatment, the abstinence syndrome was precipi-
tated by injection of naloxone (1 mg/kg, sc). Abstinence
symptoms that were quantitatively assessed included
teeth chattering, writhing, jumps, wet dog shakes,
ptosis, lacrimation, eye twitch, and scream on touch.
Rats in each of the three treatment groups, which had
been administered morphine chronically at the three
different dose levels, all spent a large amount of time
teeth chattering and writhing (Figure 4a). Thus, even
at the lowest dose used in the chronic treatment,
morphine produced an intense withdrawal syndrome.
On the other hand, DIPP-NH2[Ψ]-treated animals in all
three treatment groups spent no more time in with-
drawal than control animals that had received saline
during the 7-day period. This result indicates that
chronically administered DIPP-NH2[Ψ] had no effect on
time spent in withdrawal, even at the highest dose level
used. Similar observations were made when the mean
frequency of jumps and wet dog shakes combined was
determined in each treatment group (Figure 4b). Again,
a very significant increase in the frequency of counted
symptoms was observed in the case of the morphine-
treated rats, whereas the DIPP-NH2[Ψ]-treated animals
showed no increase in the frequency of these symptoms
as compared to control rats. Finally, the average sever-
ity of ptosis, lacrimation, eye twitch, and scream on
touch was significantly increased in the case of the
morphine-treated rats as compared to control rats, but
not at all in the case of the DIPP-NH2[Ψ]-treated rats
(data not shown). Taken together, these results indicate
that, in sharp contrast to morphine, DIPP-NH2[Ψ]
produced no physical dependence, even at the highest
dose [(ED50 × 112)/h] used in the 7-day drug treatment.
The propensities of DIPP-NH2[Ψ] and morphine to
produce chronic tolerance were determined by injecting
the drugs icv at doses of 0.15 and 0.3 µg, respectively
(corresponding to the ED50 × 3.7 and ED50 × 2.7,
respectively), twice daily for 7 days. At the end of the
drug treatment, various doses of either DIPP-NH2[Ψ]
or morphine were given icv and tail flick latencies were
measured at 10-min intervals for 2 h. A plot of the mean
AUC as a function of drug dose (Figure 3) revealed that
neither drug produced a significant analgesic effect in
the dose range 0.0001-0.1 µg. A significant analgesic
effect was observed with DIPP-NH2[Ψ] at a dose of 1
µg but not with morphine at that same dose. Thus, the
results obtained at this particular dose level indicated
that DIPP-NH2[Ψ] had produced less chronic tolerance
than morphine. However, at higher doses (10-400 µg)
DIPP-NH2[Ψ] produced seizures and it was no longer
possible to accurately determine tail flick latencies.
Therefore, it was not possible to determine an ED50 for
DIPP-NH2[Ψ]. The nature of the DIPP-NH2[Ψ]-induced
seizures needs to be further investigated. No seizures
were observed with the morphine-treated rats in the
higher dose range, and an ED50 (48.5 [7.52-1130] µg)
could be calculated by performing a linear regression
analysis on the AUC scores.
Con clu sion s
To assess the development of dependence, rats were
infused icv either with DIPP-NH2[Ψ] or with morphine
at three different high-dose levels. Morphine doses were
The performed structural modifications of the parent
peptide TIPP-NH2 resulted in mixed µ agonist/δ an-

3524 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Schiller et al.
DIPP-NH2[Ψ] (icv) for 7 days at very high dose levels
showed no withdrawal symptoms upon injection of
naloxone at the end of the treatment period. Thus,
unlike morphine, DIPP-NH2[Ψ] produced no physical
dependence whatsoever after chronic administration at
doses much higher than needed for producing analgesia.
The results of the analgesic test and of the tolerance
and dependence studies indicate that DIPP-NH2[Ψ], to
a large extent, fulfills the expectations based on the
mixed µ agonist/δ antagonist concept with regard to
analgesic activity and the development of tolerance and
dependence. However, an effort will have to be made to
develop DIPP-NH2[Ψ] analogues with a further reduced
propensity to produce acute and chronic tolerance. This
could possibly be achieved by varying the ratio of µ
agonist potency to δ antagonist potency through ap-
propriate structural modifications. Other types of mixed
µ agonist/δ antagonists developed in our laboratory,
including analogues of the cyclic â-casomorphin peptide
H-Tyr-c[-D-Orn-2-Nal-D-Pro-Gly-]14 and of the dipeptide
derivative H-Dmt-Tic-NH-(CH2)3-Ph,15 are also in the
process of being examined for their analgesic properties
and propensities to produce tolerance and dependence.
Exp er im en ta l Section
Gen er a l Meth od s. Precoated plates (silica gel 60 F254, 250
µm; Merck, Darmstadt, FRG) were used for ascending TLC in
the following solvent systems (all v/v): (I) EtOAc/hexane (2:
1), (II) CH3Cl/MeOH (9:1), (III) CH3Cl/MeOH/AcOH (85:10:
5), (IV) n-BuOH/AcOH/H2O (4:1:1). The HPLC system GOLD
(Beckman) consisting of a programmable solvent module 126,
and a diode array detector module 168 was used for the
purification and the purity control of the peptides. Recording
and quantification were accomplished using the GOLD soft-
ware.
Preparative reversed-phase HPLC was performed on a
Vydac 218-TP column (22 × 250 mm) with a linear gradient
of 30-50% MeOH in 0.1% TFA at a flow rate of 13 mL/min,
absorption being measured at 216 and 280 nm. Analytical
reversed-phase HPLC chromatography was carried out on a
Vydac 218-TP column (4.6 × 250 mm) using a linear gradient
of 15-50% acetonitrile in 0.1% TFA at a flow rate of 1.0 mL/
min. Proton magnetic resonance spectra were recorded at 25
°C on a Varian VXR-400S spectrometer using tetramethylsi-
lane as internal standard. Molecular weights of compounds
were determined by FAB mass spectrometry on an MS-50
HMTCTA mass spectrometer interfaced to a DS-90 data
system (Dr. M. Evans, Department of Chemistry, University
of Montreal).
F igu r e 4. Evaluation of withdrawal symptoms of rats that
had been infused (icv) at three different dose levels with either
morphine (0.3, 4.5, and 9.0 µg/h) or DIPP-NH2[Ψ] (0.15, 2.25,
and 4.5 µg/h) for 7 days and then received naloxone (1 mg/kg,
sc). (A) Mean time spent in withdrawal (teeth chattering and
writhing combined) during the 40-min withdrawal period.
ANOVA indicated a significant dose effect for morphine (F(3,40)
) 9.16, p < 0.01). There was no effect of DIPP-NH2[Ψ] on time
spent in withdrawal (F(3,44) ) 2.84, p ) 0.05). *Significant
difference from the vehicle control group (post-hoc LSD test).
(B) Mean frequency of counted symptoms (jumps and wet dog
shakes combined) during the 40-min withdrawal period.
Kruskal-Wallis ANOVA for nonparametric data indicated a
significant dose effect for morphine (H(3,44) ) 23.47, p < 0.01).
There was no effect of DIPP-NH2[Ψ] on the frequency of
counted symptoms (H(3,48) ) 2.99, p < 0.05). *Significant
difference from the vehicle control group (Mann-Whitney
U-test).
tagonists with increased µ agonist potency, a partial µ
agonist/δ antagonist, and a µ antagonist/δ antagonist.
DIPP-NH2[Ψ] was found to be a potent µ agonist and a
potent δ antagonist in the functional in vitro assays and
displayed subnanomolar affinity for both µ and δ recep-
tors in the rat brain membrane binding assays. Thus,
DIPP-NH2[Ψ] turned out to be the first known opioid
compound with balanced µ agonist/δ antagonist proper-
ties. Furthermore, this compound is highly stable
against chemical and enzymatic degradation, as dem-
onstrated for the structurally analogous δ antagonist
TIPP[Ψ].8
In the rat tail flick test, DIPP-NH2[Ψ] produced a
potent analgesic effect when given icv. It was about 3
times more potent than morphine in this test system.
DIPP-NH2[Ψ] produced less acute tolerance than mor-
phine upon continuous infusion at the lower of the two
dose levels used. It did produce a certain level of chronic
tolerance which, however, appeared again to be some-
what less pronounced than in the case of morphine. In
the dependence studies, rats having been infused with
Am in o Acid s a n d Der iva tives. Boc amino acids were
purchased from Bachem Bioscience. A sample of optically pure
H-L-Dmt-OH was a gift from Dr. Henry Mosberg, University
of Michigan. Racemic H-D,L-Dmt-OH was obtained from Syn-
thelec, Lund, Sweden. 2-Boc-1,2,3,4-tetrahydroisoquinoline-3-
aldehyde was synthesized as described.8
P r ep a r a tion of Boc-D,L-Dm t(Boc)-OH. To a solution of
H-D,L-Dmt-OH (639 mg, 3 mmol) in 20 mL of THF/H2O (1:1,
v/v) were added di-tert-butyl dicarbonate (1.57 g, 7.2 mmol),
triethylamine (834 µL, 7.2 mmol), and (dimethylamino)-
pyridine (36 mg, 0.30 mmol). The solution was stirred at room
temperature for 18 h. After solvent evaporation, the crude
product was dissolved in 30 mL of EtOAc, and the solution
was washed with 5% citric acid (3 × 30 mL) and H2O (3 × 30
mL) and was dried over MgSO4. After filtration and solvent
evaporation, the residue was crystallized from EtOAc/hexane
to yield 768 mg (65%) Boc-D,L-Dmt(Boc)-OH: TLC Rf 0.42 (II);
1H NMR (CDCl3) δ 1.30 (m, 9H, C(CH3)3), δ 1.46 (s, 9H,
C(CH3)3), δ 2.26 (s, 6H, CH3 ar), δ 2.85-3.05 (m, 2H, CH2), δ
3.99-4.05 (m, 1H, CH), δ 6.78 (s, 2H, ar), δ 7.21 (d, 1H, NH);
FAB-MS m/e 410.

Opioid µ Agonist/ δ Antagonist DIPP-NH2[Ψ]
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3525
P r ep a r a t ion of Boc-D,L-Tm t (Boc)-OH. NR-Methylation
was performed according to a published procedure.16 Boc-D,L-
Dmt(Boc)-OH (409 mg, 1 mmol) and iodomethane (512 µL, 8
mmol) were dissolved in 10 mL of THF/DMF (9:1,v/v). The
solution was cooled to 0° C, and sodium hydride (48 mg, 1.2
mmol; 60% dispersion in mineral oil) was added slowly. The
suspension, protected from the atmosphere by a drying tube,
was stirred at room temperature for 24 h. The reaction was
terminated by addition of 20 mL of EtOH, and then water was
added drop by drop to destroy the excess of sodium hydride.
After solvent evaporation, the oily residue was partitioned
between ether (20 mL) and H2O (30 mL). The organic phase
was washed with three 20-mL portions of aqueous NaHCO3
(10%), and the combined aqueous extracts were acidified to
pH 3 with aqueous citric acid (5%). The precipitated product
was extracted with three 20-mL portions of EtOAc; the extract
was washed with water (3 × 20 mL), dried over MgSO4, and
evaporated. The crude product was crystallized from EtOAc/
hexane to yield 381 mg (90%) of Boc-D,L-Tmt(Boc)-OH: TLC
acetonitrile/MeOH/H2O (6:1:1): L-Dmt, Rf 0.75; D-Dmt, Rf 0.63;
L-Tmt, Rf 0.76; D-Tmt, Rf 0.64. Each peptide was 98% pure as
assessed by analytical RP-HPLC and by TLC. Molecular
weights were confirmed by FAB-MS.
H-Dm t-Tic-P h e-P h e-NH2: HPLC K′ 4.75; TLC Rf 0.17 (III),
Rf 0.67 (IV); FAB-MS m/e 662 (M+).
H-Dm t-TicΨ[CH2NH]P h e-P h e-NH2: HPLC K′ 3.37; TLC
Rf 0.24 (III), Rf 0.40 (IV); FAB-MS m/e 648 (M+).
H-Tm t-Tic-P h e-P h e-NH2: HPLC K′ 4.54; TLC Rf 0.25 (III),
Rf 0.57 (IV); FAB-MS m/e 676 (M+).
H-Tm t-TicΨ[CH2NH]P h e-P h e-NH2: HPLC K′ 4.52; TLC
Rf 0.24 (III), Rf 0.29 (IV); FAB-MS m/e 662 (M+).
In Vitr o Bioa ssa ys a n d Recep tor Bin d in g Assa ys. The
GPI17 and MVD18 bioassays were carried out as reported in
detail elsewhere.19,20 A dose-response curve was determined
with [Leu5]enkephalin as standard for each ileum and vas
preparation, and IC50 values of the compounds being tested
were normalized according to a published procedure.21 Ke
values for antagonists were determined from the ratio of IC50
values obtained with an agonist in the presence and absence
of a fixed antagonist concentration.22 δ Antagonist Ke values
of all compounds were determined in the MVD assay against
the δ agonists DPDPE and deltorphin I using antagonist
concentrations ranging from 0.5 to 100 nM. The µ antagonist
Ke value of H-Tmt-TicΨ[CH2NH]Phe-Phe-NH2 was determined
in the GPI assay against the µ agonist TAPP with an
antagonist concentration of 500 nM.
1
Rf 0.70 (II); H NMR (CDCl3) δ 1.30 (m, 9H, C(CH3)3), δ 1.54
(s, 9H, C(CH3)3), δ 2.32 (s, 6H, CH3 ar), δ 2.65 (s, 3H, NCH3),
δ 3.08-3.27 (m, 2H, CH2), δ 4.26-4.44 (m, 1H, CH), δ 6.89 (s,
2H, ar); FAB-MS m/e 424.
P ep tid e Syn th esis. Peptide synthesis was performed by
the manual solid-phase technique using a p-methylbenzhy-
drylamine resin (1% cross-linked, 100-200 mesh, 0.54 mequiv/g
of titratable amine) obtained from Bachem Bioscience. Pep-
tides were assembled using Boc-protected amino acids and 1,3-
diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole
(HOBt) as coupling agents. The side chains of Dmt and Tmt
were also Boc-protected. Optically pure Boc-L-Dmt(Boc)-OH
was used for the preparation of H-Dmt-Tic-Phe-Phe-NH2,
whereas racemic Boc-D,L-Dmt(Boc)-OH and Boc-D,L-Tmt(Boc)-
OH were used in the syntheses of the other peptides. The
following steps were performed in each cycle: (1) addition of
Boc amino acid in CH2Cl2 (2.5 equiv); (2) addition of HOBt
(2.5 equiv); (3) addition of DIC (2.5 equiv) and mixing for 2-3
h; (4) washing with CH2Cl2 (3 × 1 min); (5) washing with EtOH
(1 min); (6) monitoring completion of the reaction with the
ninhydrin test; (7) Boc deprotection with 50% (v/v) TFA in
CH2Cl2 (30 min); (8) washing with CH2Cl2 (5 × 1 min); (9)
neutralization with 10% (v/v) DIEA in CH2Cl2 (2 × 5 min);
and (10) washing with CH2Cl2 (5 × 1 min).
Opioid receptor binding studies were performed as described
in detail elsewhere.19 Binding affinities for µ and δ receptors
were determined by displacing, respectively, [3H]DAMGO
(Multiple Peptide Systems, San Diego, CA) and [3H]DSLET
(Multiple Peptide Systems) from rat brain membrane binding
sites, and κ opioid receptor affinities were measured by
displacement of [3H]U69,593 (Amersham) from guinea pig
brain membrane binding sites. Incubations were performed
for 2 h at 0° C with [3H]DAMGO, [3H]DSLET, and [3H]U69,-
593 at respective concentrations of 0.72, 0.78, and 0.80 nM.
IC50 values were determined from log dose-displacement
curves, and Ki values were calculated from the obtained IC50
values by means of the equation of Cheng and Prusoff,23 using
values of 1.3, 2.6, and 2.9 nM for the dissociation constants of
[3H]DAMGO, [3H]DSLET, and [3H]U69,593, respectively.
In Vivo Testin g. Male Long Evans rats (280-350 g) were
used for analgesic testing and studies on the development of
tolerance and dependence. Rats were anesthetized with sodium
pentobarbital (Somnotol, MTC Pharmaceuticals; 60 mg/kg, ip),
and 23-gauge stainless steel cannulae were implanted ster-
eotaxically in the lateral ventricle (AP ) -1.3 mm and L )
-1.8 mm from bregma, and V ) -3.8 mm from the skull
surface for chronic infusion and -3.0 mm for acute injections
with the injector extending 1.5 mm beyond).24 For the acute
tolerance and dependence studies, the cannulae were attached
to model 2001 Alzet osmotic mimipumps (ALZA Corp., Palo
Alto, CA) filled with one of the compound solutions or saline.
Analgesic potencies of compounds administered icv to rats
were determined in the radiant heat tail flick test. Baseline
tail flick latencies were measured for each rat prior to drug
injection. Following icv administration of increasing doses of
DIPP-NH2[Ψ] (n ) 5-6/dose) or morphine sulfate (n ) 4-8/
dose), tail flick latencies were measured for 2 h at 10-min
intervals. A percent maximum possible effect (% MPE) score
for each tail flick latency measurement following drug injection
was calculated using the following formula: % MPE ) [(test
latency - baseline latency)/(cutoff latency - baseline latency)]
× 100. A cutoff latency of 10 s was used to prevent tissue
injury. The area under the curve (AUC) for the % MPE scores
over the 2-h test period was calculated for each rat. ED50
values were calculated by performing regression analysis on
AUC scores and interpolating from the regression equation to
find the agonist dose which produced a half-maximal AUC.
ED50 values are presented in Table 3 along with their 95%
confidence intervals (CI).
To introduce the reduced peptide bond between the Tic2 and
Phe3 residues, a reductive alkylation reaction9 between 2-Boc-
1,2,3,4-tetrahydroisoquinoline-3-aldehyde and the amino group
of the resin-bound H-Phe-Phe dipeptide was performed as
follows. After two washes with DMF, 2-Boc-1,2,3,4-tetrahy-
droisoquinoline-3-aldehyde (2.5 equiv) in DMF containing 1%
AcOH was added to the resin. Sodium cyanoborohydride (5.0
equiv) was then added portionwise over a period of 40 min,
and the reaction was allowed to continue for 3 h. The resin
was then washed with DMF (2 × 1 min) and CH2Cl2 (3 × 1
min). Deprotection and coupling of Boc-Dmt(Boc) or Boc-Tmt-
(Boc)-OH were then performed according to the protocol
described above.
After final deprotection with 50% (v/v) TFA in CH2Cl2 (30
min), the resin was washed with CH2Cl2 (3 × 1 min) and EtOH
(3 × 1 min) and was dried in a desiccator. Peptides were
cleaved from the resin by treatment with HF for 60 min at 0°
C (20 mL of HF plus 1 mL of anisole/g of resin). After
evaporation of the HF, the resin was extracted three times
with Et2O and, subsequently, three times with glacial AcOH.
The crude peptide was obtained in solid form through lyo-
philization of the acetic acid extract.
Crude peptides were purified by preparative RP-HPLC. In
the case of H-D,L-Dmt-TicΨ[CH2NH]Phe-Phe-NH2, H-D,L-Tmt-
Tic-Phe-Phe-NH2, and H-Tmt-TicΨ[CH2NH]Phe-Phe-NH2,
separation of the diastereoisomers was achieved using various
gradients of MeOH in 0.1% TFA. For the stereochemical
assignments the peptide isomers (0.2 mg) were hydrolyzed in
6 N HCl (0.5 mL) containing a small amount of phenol for 24
h at 110 °C in deaerated tubes and the hydrolysates were run
on HPTLC-Chir plates (Merck) using the solvent system (v/v)
For the assessment of the development of acute tolerance,
compounds were infused icv at two different dose levels

3526 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Schiller et al.
(5) Marsden, B. J .; Nguyen, T. M.-D.; Schiller, P. W. Spontaneous
Degradation Via Diketopiperazine Formation of Peptides Con-
taining a Tetrahydroisoquinoline-3-carboxylic Acid Residue in
the 2-Position of the Peptide Sequence. Int. J . Pept. Protein Res.
1993, 41, 313-316.
(6) Hansen J r., D. W.; Stapelfeld, A.; Savage, M. A.; Reichman, M.;
Hammond, D. L.; Haaseth, R. C.; Mosberg, H. I. Systemic
Analgesic Activity and δ-Opioid Selectivity in [2,6-Dimethyl-
Tyr1,D-Pen2,D-Pen5]enkephalin. J . Med. Chem. 1992, 35, 684-
687.
(7) Schiller, P. W.; Nguyen, T. M.-D.; Weltrowska, G.; Wilkes, B.
C.; Marsden, B. J .; Schmidt, R.; Lemieux, C.; Chung, N. N. TIPP
Opioid Peptides: Development of Extraordinarily Potent and
Selective δ Antagonists and Observation of Astonishing Structure-
Intrinsic Activity Relationships. In Peptides: Chemistry, Struc-
ture and Biology; Proceedings of the 13th American Peptide
Symposium, Edmonton, J une 20-25, 1993; Hodges, R. S., Smith,
J . A., Eds.; ESCOM Science Publ.: Leiden, The Netherlands,
1994; pp 483-486.
(8) Schiller, P. W.; Weltrowska, G.; Nguyen, T. M.-D.; Wilkes, B.
C.; Chung, N. N.; Lemieux, C. TIPP[Ψ]: A Highly Potent and
Stable Pseudopeptide δ Opioid Receptor Antagonist with Ex-
traordinary δ Selectivity. J . Med. Chem. 1993, 36, 3182-3187.
(9) Sasaki, Y.; Coy, D. H. Solid-Phase Synthesis of Peptides
Containing the CH2NH Peptide Bond Isostere. Peptides 1987,
8, 119-121.
(morphine: 0.3 µg/h (n ) 10) and 9.0 µg/h (n ) 14); DIPP-
NH2[Ψ]: 0.15 µg/h (n ) 16) and 4.5 µg/h (n ) 14)) continuously
for 4 days. Tail flick latencies were measured every 15 min
during the first 2 h, every 1 h during the next 6 h, and then
every 12 h for the rest of the infusion period. The mean time
in minutes after the initiation of drug treatment for tail flick
latencies to return to baseline levels (e20% MPE), also referred
to as the mean time interval of analgesic efficacy, was then
determined from the collected data. To assess the development
of chronic tolerance, morphine (n ) 60) and DIPP-NH2[Ψ] (n
) 88) were injected icv at
a dose of 0.3 and 0.15 µg,
respectively, twice daily for 7 days. At the end of the drug
treatment, rats were given icv injections of various doses of
morphine sulfate (n ) 5-8/dose) or DIPP-NH2[Ψ] (n ) 7-12/
dose) and tail flick latencies, % MPEs, AUC scores, and, if
possible, ED50 values were determined as described above.
To determine the propensities to produce dependence,
compounds were infused icv for 7 days at three different dose
levels. Morphine doses were as follows: 0.3 µg/h (n ) 9), 4.5
µg/h (n ) 10), and 9.0 µg/h (n ) 10); DIPP-NH2[Ψ] doses were
as follows: 0.15 µg/h (n ) 13), 2.25 µg/h (n ) 10), and 4.5 µg/h
(n ) 10); saline (n ) 15). Precipitated abstinence symptoms
were assessed on the seventh day of treatment after injection
of naloxone (1 mg/kg, sc). For 10 min before and 40 min after
naloxone injection, the amount of time spent teeth chattering
and writhing (combined) and the frequency of jumps and wet
dog shakes (combined) were determined. The average severity
of other checked signs, including ptosis, lacrimation, eye
twitch, and scream on touch, during the 40-min withdrawal
period was rated on a 4-point scale, where 0 ) absent and 3 )
severe.
(10) Fehrentz, J .-A.; Castro, B. An Efficient Synthesis of Optically
Active R-(t-Butoxycarbonylamino)-aldehydes from R-Amino Ac-
ids. Synthesis 1983, 676-678.
(11) Schiller, P. W.; Nguyen, T. M.-D.; Chung, N. N.; Lemieux, C.
Dermorphin Analogues Carrying an Increased Positive Net
Charge in Their “Message” Domain Display Extremely High
µ-Opioid Receptor Selectivity. J . Med. Chem. 1989, 32, 698-
703.
(12) Chavkin, C.; J ames, I. F.; Goldstein, A. Dynorphin Is a Specific
Endogenous Ligand of the κ Opioid Receptor. Science 1982, 215,
413-415.
Ack n ow led gm en t. This work was supported by the
National Institute on Drug Abuse (Grant DA-04443 to
P.W.S.) and the Medical Research Council of Canada
(Grant MT-5655 to P.W.S. and Grant MT-13236 to
T.J .C.). M.E.F. was supported by an FCAR studentship.
We thank Drs. M. Evans and M. Bertrand, Department
of Chemistry, University of Montreal, for performing the
FAB mass spectrometric determinations. Thanks are
also due to M.-A. LeBlanc for the preparation of this
manuscript.
(13) Chavkin, C.; Goldstein, A. Opioid Receptor Reserve in Normal
and Morphine-tolerant Guinea Pig Ileum Myenteric Plexus.
Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 7253-7257.
(14) Schmidt, R.; Vogel, D.; Mrestani-Klaus, C.; Brandt, W.; Neubert,
K.; Chung, N. N.; Lemieux, C.; Schiller, P. W. Cyclic â-Caso-
morphin Analogues with Mixed µ Agonist/δ Antagonist Proper-
ties: Synthesis, Pharmacological Characterization and Confor-
mational Aspects. J . Med. Chem. 1994, 37, 1136-1144.
(15) Schiller, P. W.; Weltrowska, G.; Schmidt, R.; Nguyen, T. M.-D.;
Berezowska, I.; Lemieux, C.; Chung, N. N.; Carpenter, K. A.;
Wilkes, B. C. Analgesia 1995, 1, 703-706.
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Synthesis. V. The Synthesis of N-tert-butyloxycarbonyl,N-meth-
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910.
Refer en ces
(1) Symbols and abbreviations are in accordance with recommenda-
tions of the IUPAC-IUB J oint Commission on Biochemical
Nomenclature: Nomenclature and Symbolism for Amino Acids
and Peptides. Biochem. J . 1984, 219, 345-373. The other
abbreviations are as follows: AUC, area under the curve; BBB,
blood-brain barrier; Boc, tert-butoxycarbonyl; DAMGO, H-Tyr-
D-Ala-Gly-NRMePhe-Gly-ol; DIC, 1,3-diisopropylcarbodiimide;
DIEA, diisopropylethylamine; DIPP-NH2, H-Dmt-Tic-Phe-Phe-
NH2; DIPP-NH2[Ψ], H-Dmt-TicΨ[CH2NH]Phe-Phe-NH2; Dmt,
2′,6′-dimethyltyrosine; DPDPE, H-Tyr-c[-D-Pen-Gly-Phe-D-Pen]-
OH; DSLET, H-Tyr-D-Ser-Gly-Phe-Leu-Thr-OH; FAB-MS, fast
atom bombardment mass spectrometry; GPI, guinea pig ileum;
HOBt, 1-hydroxybenzotriazole; icv, intracerebroventricular; MPE,
maximum possible effect; MVD, mouse vas deferens; sc, subcu-
taneous; TAPP, H-Tyr-D-Ala-Phe-Phe-NH2; TFA, trifluoroacetic
acid; THF, tetrahydrofuran; Tic, 1,2,3,4-tetrahydroisoquinoline-
3-carboxylic acid; TIPP-NH2, H-Tyr-Tic-Phe-Phe-NH2; Tmt,
N,2′,6′-trimethyltyrosine; U69,593, (5R, 7R, 8â)-(-)-N-methyl-
N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]benzeneaceta-
mide.
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Constant (KI) and the Concentration of Inhibitor which Causes
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(4) Schiller, P. W.; Nguyen, T. M.-D.; Weltrowska, G.; Wilkes, B.
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J M980724+