Note: Descriptions are shown in the official language in which they were submitted.
~2586~7~
The present invention relates to opiate
agonists and antagonists.
Back~round
The advantages of irreversible drugs in both
the in vivo and in vitro evaluation of opiate action
has led to the recent development of a num~er of
irreversible opiate agonists and antagonists (Caruso et
al., Science 204:316-318, 1979; Portoghese et al., J.
Med. Chem. 22:168-173, 1978, Portoghese et al., J. Med.
Chem. 22:168-173, 1979; Schultz and Goldstein, ~ife Sc.
16:1843-1848, 1975i Ninter~ and Goldstein, Molec.
Pharmacol. 8:601-611, 1972). One compound, naloxazone,
has been particularly useful in characterizing opiate
receptors and opiate actions because of its ability to
selectively inhibit the high affinity, or mul, bindin~
sites as well as blocking opiate analgesia with little
effect on other classes of binding sites or opiate-
induced lethality tPasternak et alO, Science 208:514-
516, 1980; J. Pharmacol. Exp. Therap. 214:455-462,
1980; Zhang and Pasternak, Life Sc. 29:843-851, 1981;
Pasternak, in press) However, naloxazone requires
high doses both in vivo and in vitro to inactivate
these high affinity, or m,ul, binding sites and the
mechanism of its activity was unrecognized (mu site
classification is described in Wolozin and Pasternak,
Proc. Natl. Acad. Sci., 78: 6181-6185, tl981).
. .
- 2 - ~
~5~6~
_n the Drawinqs
Fig. 1 shows structures of the 14-hydroxy-
dihydromorphinones, their hydrazones and their azines.
Fig. 2 shows the irreversibility of naloxone
and naloxona2ine binding in rat brain membranes.
Rat brain membranes were prepared and
incubated with naloxone ~o) or naloxonazine (~) at the
stated concentration for 30 min. at 25C and then
washed four times and finally assayed with [3H]
dihydromorphine (1.2 nM). Results are from a single
experi~ent which has been replicated three times.
Fig. 3 shows morphine displacement of [3H~ D-
ala2-met5~enkephalinamide binding in naloxone and
naloxonazine treated tissue.
Rat brain me~branes were prepared and
incubated with either naloxone (50 nM; o) or
naloxonazine (50 nM; o) for 30 min. at 25C and washed
three times. Binding of [ H]D-ala2-met5-
enkephalinamide ~1~5 nM) was then measured in the
absence and presence of Yarious concentrations of
morphine sulfate. Values a,re expressed as percent of
binding in the naloxone tré~ted tissue in the absence
of morphine and represent a single experiment which has
been replicated three times.
~2~ 6~
Description
This invention relates to the 14-
hydroxydihydromorphinone hydrazones (naloxazone,
oxymorphazone and naltrexazone) and their irreversible
binding to opiate receptors probably by the formation
of their azines. These azines, naloxonazine,
naltrexonazine and oxymor,phinazine, irreversibly ~lock
opiate binding in vitro 20-40 fold more potently than
their corresponding hydrazones, naloxazone, natrexazone
and oxymorphazone. The blockade of binding by
naloxonazine shows the same selectivity for high
affinity, or mul, sites as naloxazone.
Naloxazone pro`ved very useful in our
understanding of opiate receptor subtypes and their
correlation with analgesia (Pasternak et al., 1980
supra; Pasternak and Hahn, J. Med. Chem. 23:674-~77,
1980; Zhang and Pasternak, Life Sci. 29;843-851, 1981;
Hazum et al., Life Sci. 28:2973-2979, 1981; Pasternak,
Proc. Nat. Acad. Sci. USA 77:3691-3694, 1980; Pasternak
et al., in press), even though its mechanism of action
was not known. The high concentrations and doses
required for irreversible activity led us to the
conclusion that a more active component was being
formed. This active compone~t is believed to be azine,
one of the inventive compounds.
Although previously unrecognized, it has
now been found that although the naloxazone
used was cnemically pure, a small amount
rearranged to the a~ine in solution. This explains,
in part, the low potency of naloxazone. First,
-- 4
.
~25~
only ~ port~on of naloxazone was conver-ted to its azine and
the remaininc1 naloxazone probably competitively inhibited
naloxonazille's action. Thus, the irreversible blockade of
opiate receptor binding requiring high doses of naloxazone
(2 ~M) is reproduced by low (50 nM) doses of naloxonazine.
H~ Dihydromorphine, ~3H~ D-ala2-D-leu5-enkephalin,
~3~ D-ala -met5-enkephalinamide, and Formula 963 scintilla-
tion fluor were purchased from New England Nuclear Corp.
Inc. (Boston, MA), naloxone, naltrexone and oxymorphone from
Endo (Garden City, NY) and naloxazone, naltrexazone, and oxymor-
phazone synthesized as previously described (Pasternak and
Hahn, J. Med. Chem 23:674-677, 1980).
The dihydromorphone compounds of the present invention
are synthesized in the following manner. In the case of the
azine derivatives of a dihydromorphone, the synthesis can
be carried out by reacting a three-fold excess of the parent
al'~loid in meth~nol with hydra~.ine or ~y reacti~ the
hyarazine aerivative of the alkaloid ~ith an excess of the
dihydromorphone. The reactions are monitored by thin layer
chromatography and the products are purified in known
manner such as by preparative thin l~yer chromatography.
For the preparation of the non-symmetrical dihydromorphone
compounds~ this procedure is modified by replacing the
azones with a compound having free hydrazine or hydrazide
or other free N-NH2 group, such as H2N-NH ~
H2 NH ~----NO2, H2N-NH-CH3, H2N-N=C , etc.
H3
For example, the azine derivatives of naloxone, naltre-
xone and oxymorphone (Fig. 1) were synthesized by reacting
a three-fold molar excess of the parent alkaloid in ethanol
3~
~ 5 -
. . .
~L~5~7~
with naloxa7.0ne, naltrexazone and oxymorphazone respec-tively.
The individual reactions which were moni-tored by thin layer
chromatography (TLC) (silica gel, CHC13:CH30H:NH40H,90:10:1~
were complete within 3 hours. Af-ter evaporation of the solvent
in vacuo, products were purified by preparative TLC using
the above solvent system. The isolated compounds had an Rf
identical with the product in the unpurified reaction mixture,
suggesting that rearrangement did not occur during purifi-
cation. The individual alkaloids, rlalo~onazine, naltrexonazine,
and oxymorphonazine, were further characterized by their mass,
nuclear magnetic resonance (NMR) and infrared (IR) spectra.
Mass spectroscopy using chemical ionization detection showed
M~l ions a-t 651, 679 and 599 respectively. In the NMR spectrum,
the downfield shift in the C-5 hydrogen absorption recorded
for the hydrazone derivatives (Pasternak and Hahn, J. Med.
Chem. 23:674-677, 1980) was also observed for -the azine deriva-
tives. The absence oE a carbonyl absorption in the IR spectra
of the individual compounds further confirmed reaction at
C-6 in each of the 14-hydroxydihydrornorphinone derivatives.
Microanalysis (CHN0- Rockfeller University Microanalytical
Laboratory) of each compound also verified the proposed structu-
res. The absence of a Eree NH2 group was indicated by a nega-
tive TNsS test (Pasternak and Hahn, 1980 supra). In contrast
to the parent hydrazones which produced a reddish-brown color
on standing with TNBS, no color change was seen with any o-f
the azine derivatives.
The invention is illustrated by means of the following
examples of the synthesis of symmetric azines and hydrazones.
EXAMPLE 1
Naloxonazine
Naloxone (2.5 g of free base) is dissolved in 40 ml of dry
.; ., ~
~5~gi7~
methanol and anhydrous hydrazine (0.12 ml) is dissolved in
5 ml of dry methanol and added dropwise to the solution of
the alkaloid. The reaction is stirred for 15 hours at room
temperature after which time the product is removed by
filtration (3.5 g). The reaction is stirred for an additional
24 hours and another 1.2 g of product is collected. Yield:
approximately 90~.
Naloxonazine precipitates from methanol. Some of
the other symmetric compounds need to be purified by preparative
thin-layer chromatography.
EXAMPLE 2
p-NO2-phenylhydrazone derivative of naloxone
To naloxone (250 mg free base) in 25 ml of absolute ethanol
is added p-nitrophenylhydrazine (235 mg) and the solution
is stirred for 1 hour. The solvent is evaporated in vacuo
and the residue is purified by preparative thin layer chroma-to-
graphy on silica gel using chloroform:methanol:ammonia (95:5:
0.5) as solvent. The product is isolated as an oil which
precipitates from methanol with the addition of ether. Yield:
300 mg, which is approximately 85~.
Example 3
Example 2 can be repeated using alkylhydrazine having
1 to 10 carbon atoms, phenylhydrazine and substituted phenyl-
hydrazine to give alkyl-, phenyl- and substituted phenyl-
hydrazone derivative of naloxone.
Example 4
By repeating the procedure of example 1, bu-t starting
from oxymorphone, there is obtained oxymorphonazine.
Binding experiments were performed as previously
described using 20 mg/ml of tissue (Pasternak et al, Molec
Pharmacol 11:340-351, 1975). In brief, brain membranes were
- 6a -
prepared and tripllcate sample incubated wi-th / H/ labelled
ligands and designated drugs for 30 minutes at 25~CI followed
by filtration over Whatman GF/B filters. The filters were
then counted in Formula 963 scintillation fluor. Specific
binding is defined as the difference in binding in the presence
and absence of levallorphan (luM). Irreversible inhibition
of opiate receptor binding was established by the persistence
, ~
- 6b -
~X~5~67~
of binding inhibition despite extensive washes which
did reverse any inhibition by the same concentration of
naloxone. Each tissue wash included incubation for-10
min. at 37C, centrifugation and resuspension.
Irreversibility experiments included four washes before
binding assays were performed, unless otherwise stated.
The synthesis of monosubstituted hydrazones,
such as naloxazone, oxymorphazone and naltrexazone, may
be complicated by the formation of azines (Fig. 1). To
avoid this problem a large excess of hydrazine was used
in the synthesis. Analysis of the compounds, including
nuclear magnetic resonance, mass and infra red
spectroscopy, thin layer chromatography, CHNO analysis
and chemical titration of free -NH2 groups with TNBS
(Pasternak and Hahn, J. Med~ Chem. 23:674-677, 1980)
confirmed the formation of a single product and
excluded the presence of an azine in each caseO
However, it is now realized that solutions of the 14-
hydroxydihydromorphinone hydrazones rapidly undergo a
reaction particularly in acidic a~eous solutions.
Isolation and characterization of the product suggested
the formation of azines. The direct synthesis of the
various 14-hydroxydihydromorphinone azines showed that
they were identical to the products formed in the
various hydrazone solution,s. Having isolated and
established the structures ~f the products as azines,
we then determined that they were responsible for the
apparent irreversible actions of naloxazone,
naltrexazone and oxymorphazone.
Pharmacoloqical ComParisons Between the 14-
hydroxydihydromorPhinones, Their Hydrazones and Their
Azines.
We first tested the affinity of naloxone and
naltrexone, their hydrazones and their azines for
opiate receptors (Table 1). Interestingly~ the
hydrazones and azines displace [3H] dihydromorphine
binding less potently than their parent ketones. In
both groups, however, the azine is more potent than the
hydrazone.
To determine which compounds did irreversi~ly
bind to the receptor, membranes were incubated with the
various drugs (2~M) and then the free and reversibly
bound drug washed away ~Table 23. Special care was
taken with the hydrazones to use conditions under which
little or no azine formation could be detected. Under
these conditions neither naloxazone nor ox~morphazone
irreversibly inhibited the binding of either [3H] D-
ala2-D-leu5-enkephalin or [ H] dihydromorphine. The
small amount of inhibition by naltrexazone reflects the
difficulty in totally eliminating azines from its
solutions. Making concen-trated naloxazone solutions
using acetic acid to dissolve the free base as
previously reported (Childerjs and Pasternak, in press),
results in a significant amount of azine formation and
does irreversibly block the specific binding of both
[3H] ligands. However, the far greater potency of the
azines sugges-ts that they are the active compound. --
;~
~25~367q~
Table 1: Dir~ct inhibition of [3H] dihydroxymorphine
binding by naloxone, naltrexone and their derivatives.
Compound lC50 (nM)
Naloxone 4.4 + 0.7
Naloxazone 16.3 + 4.9
Naloxonazine 5.4 + 1.3
Naltrexone 0.77 + O.OS
Naltrexazone 1.7 + 0.2
Naltrexonazine - 1.16 + 20.3
Rat brain homogenates were prepared as described and
the above drugs at 4 concentrations were incubated with
[3H]dihydromorphine (0.8 nM) for 45 min. and filtered.
IC50 values were det2rmined by least square fits of a
log-probit curve. The results are the means + s.e.m.
of three IC50 values determined in separate
experiments.
_ g _
6~
Table ~: Irreversible inhibition of receptor binding by
naloxone, naltrexone and oxymorphone and their
derivations.
Chanqe in Bindinq (Percent~
t3H]D-ala2-D-leu 3
Compound (2~M) _ -enkephalin[ HldihYdromorphine
Naloxone --- ___
Naloxazone +15 + 5% ~2 + 11%
Naloxazone +
acetic acid -48~ + 11% -41 + 13%
Naloxonazine-93% + 4% -89 + 4%
Naltrexone --- ---
Naltrexazone-17 + 19% -31 + 26%
Naltrexonazine-97 + 3% -88 ~ 12%
Oxymorphone -~
Oxymorphazone+3% + 24% +3 + 21%
Oxymorphonazine-59 + 3~ -71 + 4%
Rat brain membranes were prepared and incubated with
the above drugs (2~M) for 30 min at 25C and the tissue
was then washed four times as dPscribed in the text to
r~move r~versibly bound material. The results
represent the means + s.e.m. of three separate
experiments utilizing [ H]D~ala2-D-leu5-enkephalin 5 1
nM~ and ~3H]dihydromorphine (0.8 nM). With the
exception of naloxazone and acetic acid, all drugs were
dissolved in absolute ethanol immediately ~efore
dilution and addition to tissue. Under these
conditions, ~o azine formation could be detected for
naloxazone and oxymorphazo~e as demonstrated by thin
layer chromatomography. A itrace amount of azine was
present in the naltrexazone solutions. The naloxazone
and acetic acid sample (10 mg/kg) was dissolved in
water/acetic acid (1%). After 15 min. the solution was
added to tissue. Under these conditions a significant
portion of the naloxazone had reacted to its azine.
--10--
~l~$~3~i7~
Since naloxonazine at 2 ~M eliminated over 90%
of [3H] dihydromorphine and ~3~I] D-ala2-D_leu5_
enkephalin binding, we next examined its irreversible
effects at a variety of concentrations (Fig. 2). After
incubating membranes with the specified drug, the
membranes were extensively washed and a binding assay
with [3H] dihydromorphine perfor~ed. As expected,
naloxone at high concentrations does not inhibit
binding irreversibly. Naloxonazine potently inhihits
binding in a dose dependent manner. The inhibition
curve appears biphasic, suggesting more than one site
with differing sensitivities to naloxonazine's
irreversible actions. Although it is difficult to
accurately determine IC50 values without computer
analysis, one site appears to be quite sensitive to
naloxonazine (IC50 about 10-15 nM) while the other
requires concentrations approximately 40-fold higher
(IC50 400 500 nM).
Previous studies have demonstrated
naloxazone's selective inhibition of high affinity,
or mul, binding sites (Pasternak, et al. J. Med.
Chem. 23.674-677, 1980, Zhang and Pasternak, Life
Sci. 29:843-851, 1981; Childers and Pasternak, in
press). To determine whether naloxonazine demon-
strated the same selectivit~ described with naloxazone
treatment, we incubated' tissue with either
naloxone or naloxonazine (50 nM), washed the
tissue, and examined morphine's displacement of ~3H]D-
ala2-met5-enkephalinamide binding (Fig. 3). Morphine
has the same biphasic displacement in naloxone-treated
--11--
tissue as previously published (Chang and Cuatrecases,
1978; Zhang and Pasternak, Life Sci. 29:843-851, 1981).
The initial morphine displacement represents [3H]D-
ala2-met5-enkephalinamide binding to high affinity, or
mu1 receptors (Wolozin and Pasternak, in press; Zhang
and Pasternak, 1981, supra). Treating the tissue with
naloxonazine eliminates this initial displacement,
yielding results virtually the same as those using
naloxazone. Thus, naloxonazine has the same selec-
tively but far greater potency than naloxazone.
The Use of the Antaqonists Naloxonazine and
Naltrexonazine as Appetite Suppressants.
It is known that opiate antagonists such as
naloxone and naltrexone effectively suppress eating
behavior in rats. In view of the generally good
correlation between the pharmacological actions of
opiates in animal models and their effects in man, it
is therefore believed that these agents will also
suppress appetite in man. The major disadvantage of
the currently available agents (naloxone and
naltrexone) is their short half-life and the need for
multiple doses each day. A long-acting agent would
have many a~vantages, especially compliance by the
patient and convenience. Inj addition, whereas naloxone
and naltrexone interact wikh a number of different
classes of opiate receptors, naltrexonazins and
naloxonazine are far more selective for the mul class
of opiate binding sites and therefore will have far
more selective pharmacological actions in vivo. The
major question relevant to this use of
-12-
~he in~entive compourlds is whether tlle subpopulat:ion oE
sites affected by naloxorlazine and naltrexonazine are
involved in the suppression of appetite. Although direct
tests have not been done,-tests have been run using
naloxazone. As explained above, the actions of naloxazone
are believed to result from the conversion of -this drug to
naloxonazine. It is therefore predic-ted -tha-t all the
actions seen with naloxazone will be ~roduced by
naloxonazine at far lower doses. The effec-tiveness of
naloxazone was shown, as follows:
Rats were administered elther saline or naloxazone was--
(50 mg/kg intravenously) through cannulae placed in the
jugular vein. They were then food deprived for 24 hours but
allowed free access to water. Five rats received saline,
four received naloxone and three naloxazone. Both naloxone
and naloxazone were given at 50 mg/kg intravenausly. The
animals were then given free access to food for 1 hour and
their weight gain noted. Thus, ea-ting was measured 24 hours
after the administration of the drugs and after 24 hours of
food deprivation.
.
Amounts eaten in gcall~s:
Saline l~laloxone llaloxazone
4.1 ~.1 . 3.3
3.9 5.3 Z.~
.4 ~.~ , 3.5
6.1~ 3.7
5.4
~lean~SEIl 4.78+0.42 ~.38+~.34 3.~7~.34
en analyze~ by analysls Or variance, the a~ove results ~ere signi~icallt
at the p 0.05 level [F~ 9=4.56J
.
- -13-
These results indicate that naloxazone has a significant
effect as an appetite suppressant. The inability of
naloxone to effect feeding behavior does not imply that i-t
has no actions on feeding, but merely emphasizes that its
actions are short-lived and clearly illustrates the ~arked
advantages of naloxazone, and thus naloxonazine. In vivo
work with naloxonazine has demonstrated that it is 5-fold
more active on a mg/kg basis than naloxazone and bo-th drugs
have the same ac-tions.
The Use of the;Azines as Selective Agents in Modulating
Hormone Release.
The two antagonists naloxonazine and naltrexonazine and
the agonist oxymorphonazine have ac-tions lasting far lonyer
than conventional opiates, In addition we have found that
the~selective interactions of these drugs with the mul class
of opia~e receptors has a selective action on hormone
release. An example of this selectivity are the effects of ~
morphine on the release of prolactin and growth hormone.
Although morphine releases both hormones it utilizes
different classes of opiate receptors. Thus, the selective
blockage of the mul class can inhibit morphine's action on
prlactin release but not growth hormone.
-14-
~5~
The selective blockade of the mul sites in vivo by the
prior administration of naloxazone depressed -the
morphille-induced release of prolactin over 80%. Morphine-
induced growth hormone release, on the other hand, was
actually increasedO The implications of these findings are
that different receptors media-te the release of the
different hormones by a single drug. ~Naloxone inhibits
morphine's release of both hormones. In comparison,
naloxazone has the abillty of selectively lowering prolactin
release without effecting growth hormone release elicited by
morphine and presumably o-ther opiates as well. On the other
hand, one might expec-t the agonist compound oxymorphonazine
to selectively elevate prolactin levels without
substantially increasing growth hormone concentrations.
The release of many hormones~is under the tonic control
of opioid systems. The evidence for this effect comes from
work demonstrating a lowering of the basal levels of
prolactin and growth hormone following the administration of
naloxone. Our work also illustrates a significant reduction
in the basal levels of the drugs after naloxazone.
The use of the Azines in the Classification of Opioid Drugs.
Recent work has suggested that several classes of
opioid receptors exist. One area of immense importancè is
in the new classification of two types of mu receptors. Up
until now, it has been difficult to determine the relative
-15-
i7~
potency o~ agents for the mul and muz class of
receptor. The affinity of naloxonazine and the other
azines for the mul class permits the rapid and simple
estimation of the potency of agents for the mu2 site
and the mu1 by comparison with measures of both types
together.
The Use of Oxymorphonazine as an Analqesic Aqent.
The advantages of this drug are: i) long
action, ii) presumed decreased respiratory depressant
actions, iii) presumed decreased inhibition of
gastrointestinal motility and thus constipation, iv)
presumed decreased incidence of hypotension. The
evidence for many of these decreased side-effects of
oxymorphonazine come from evidence suggesting that
these other actions reside in receptors other than mul.
Thus, the selective interactions of the drug with mu
sites should result in fewer side-effects.
Evidence of the analgesic effect of
oxymorphona2ine is shown in the following experiment
summary:
Oxymorphonazine analgesia in vivo
Analgesia (percent)
TimeOxymorphone Group Oxymorphonazine Group
1 hr.100 % n=14 100 % n=9
4 hr.78 % n=9 100 % n=5
10 hr.33 % n=9 100 % n=5
20 hr.0 % n=14 44 % n=9 p~0.015
Mice were injected with either oxymorphone or
oxymorphonazine (both 25 ~g in 5 ~1 of artificial CSF)
under Ethrane/oxygen anesthesia and tested for
analyesia at the stated time. Analgesia was determined
by Tail-Flick Assay. Quantal criteria, the doubling of
the baseline latency for each individual animal, was
used. At 20 hours, the oxymorphone and oxymorphonazine
groups differed from each other at p<0.015 level, as
determined by Fisher's Exact Test.
~ -16-
7~
'I`h~ Use o~ t~_e ~tagonlsts Naloxonazine and Naltrexonazine
as Blockers of Morphine and Other Opiates in Maintenance
Pro~rams_from Opioid Abusers.
-
. ~he advantayes of -the agents are -their prolonged
actions (in the order of days) compared to the duration of
curren-tly available drugs (in the order of only a few
hours.)
The following test show t-he effectiveness of this
compound as blockers:
Naloxonazine blockade of morphine analgesia in vLvo
~orphine s~llfate ED50 value
Control group 3.7 mg/kg s.c.
Naloxonazine group
35 mg/kg 76.0 mg/kg s.c.
50 mg/kg 202.7 nng/kg s.c.
Groups of mice (n=10) were treated with either nothing, or
naloxonazine (35 or 50 mg/kg, 5.C.) and tested for morphine
analgesia 24 hours later. Full dose response curves were
performed with at least three separate dpses of morphine
for each curve. ED50values, that dose of-morphlne needed
to produce analgesia in 50X of animals, was determined by
Probit analysis. ~nalgesla was determlned ln the Tall-Fllck
Assay using the quantal measurement of doubllng of basellne
flick as crlteria.
-17-
G7~
In general, opiate receptor binding/blocking has been
shown for a wLde range of symmetric and asymmetric dihydro-
morph~none compounds of the type whLch can be represented as
HDM=N-R
wherein
HDM lS
or
where * indicates binding carbon
Rl is Cl-C10 alkyl, C2-C10 alkenyl, C3-C6 cycloalkyl,
or C3-C6 cycloalkylene
R2 is OH or H
R3 is OH and O O O O
R is N=R4, NH-R5, NHC(CH2)nCNHN=R4 or NHC CH=CHCNHN=
R4
R4 is Cl-C10 alkenylidine, C3-C6 cycloalkenylidene,
or HDM
R5 is phenyl, 4-nitrophenyl, 2,4 dinitrophenyl, Cl
-C6 alkyl, C2-C10 alkenyl, C3-C10 cycloalkyl, C3-C10 cycloal-
kenyl, and
n is 1-10.
-18-
,,, ~
,. ~ .
3679~
Presently preferred o:E the dihydromorphinone compounds
of the present invention are the 14-hydroxydihydromorphino,nes
especially naloxonazine, naltrexonazine and oxymorphonazine
as well as mi~ed azines. The 3-methoxy derivatives of these
compounds are expected to be potent drugs with similar long
acting and selective properties sinOEe the 3-methoxy derivatives
of the ketone derivatives have high oral potency. -
Symmetrical compounds (where R is -N=HDM) as
exemplified above include
oxymorphonazine (Rl=-CH3; R2=OH; R3=OH:
HDM=N-N=HDM)
naloxonazine (Rl=-CH2-CH=CH2; R2=OH; R3=OH;
HDM=N-N=HDM)
C~12
naltrexonazine (Rl--CH2-CH'" ~ ¦ ;
R2=OH; R3=OH: HDM-N-N=HDMJ. CH2
Activity of these compounds was described in de~ail
in the general discussion above.
-19- .
67~
Syn1me_rlcaL co~ o~nds w h a br ~e.
The azine linkage itself is not believed to be cri-tical
to the activity of the compounds. Thus compounds of the
following type should be able -to po-tently inhib:i-t binding:
0 11 0 11
HDM=N-NC (CH2) CN-N=R4 or HDM=N-N-C-CH=CH-C-NN=R4
H n H H H
(non-conjugated bridge) ~ (conjugated bridge)
In fact activity has been ~hown in both cases where
Rl is CH2-CH \C~ 2; R2 is OH; R3 is OH; and R4 is HDM;
C~12
and n = 2, 4,6;. Activity for an extended conjugated bridge is
to be expected as well.
As~mmetrical compounds have been shown_to be active.
The symmetrical nature of the molecule is also not
critical. Significiant irreversible inhibition of opia-te
receptor binding under conditions where standard opiates
such as morphine show no irreversible i.nhibition has been
shown for the following compounds: of the general formula
NTX=N-R
C~12
wherein NTX lS naltrexone= HDM where (Rl=-cH2-cH-cH2; and
R2=R3=OH)
NTX=N-N-~ (note: no-t an azine)
NTX=N-N-~N02 (note: not an azine)
NTX=N-N-CH3 (note: not an azine)
C~13 -
N~X=N-N-C ~ I note: an azine)
~13
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~S~;7~
~s~ ric c~ ouncls wiLIl mlxed agonist/alltagonist
__ __
co~ on~llts ar~ also active.
In vivo te.Yts have been run on mixed agonist/antagonist
compounds, viz n~ltrexone-oxymorphonazine. These asymmetric
compounds appear to irreve~sibly bind the opia-te recep-tors
and act as antagonists.
.
Tests were run in binding assays with analogs of
morphine and the endogenous op;oid enkephalin compounds
comparing these asymmetric compounds with symmetric
naltrexonazine -to show irreversible blocking of receptor
sites as follows:
Irreversible Inhibition of H-OpLoid Binding
by Naltrexone-Oxymorphonazine
Treatment 3il-~DL Binding ~cpm) 3H-DIIM Binding (cl~m)
.. .. _ .
Nothing (control)- 1800 + 53 3447 + 273
Naltrexonazine 653 + 85 (-65%) 1183 + 128 (-66~)
~altrexone-oxymorphonazlne 1205 + 157 (-33%) 1487 + 120 (-57%)
Rat brain homogenates were incubated with~either nothing or naltrexonazine
or naltrexone-oxymorphonazine at 100 nM for 30 min at 25C and then washed
twice. Each wash consisted of centrifugation, incubation at 37 for 10 min
and centrifugation again. Under these conditions, reversible ligands such
a~ naloxone, naltrexone and oymorphone are all effectively removed. Bindig
was then perfonned wlth eitller 3H-D-ala2-~-leu5-enkephalin (D~DE) or
3H-dihydromorphine (D~) both at 1 nM. Speclfic bincllng was determined
as that displaceable by levallorphan at 1000 nM. ~ll values represent the
means of tripllcate samples + S.E.M.
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.
7~
~ ction as an antagonist for the asymmetric compound as
compare~ witll the corresponding symmetric compound was shown
as follows:
ln vivo ac~ions of Naltrexone-oxymorphona~ine
Tailfli~k ~atencies (sec)
Drug Prior ~fter 20 min ~ftèr 40 min
naltrexone ( 10 mg/kg ) n=lO 2.63 + 0.27 2.37 + 0.31 2.59 + 0.35
naltrexone-oxymorphonazine 2.44 + b.32 2.20 + 0.35 2.29 + 0.31
( 10 mg/kg ) n-10
naltrexonazine ( lO mg/kg) n=9 2.50 + 0.34 2.50 + 0.36 2.74 + 0.49
,
Groups of mice were inJected subcutaneously with the stated drug and
latencies in the tailflick test determined and compared to those obtained
prior to testing. Using the doubling oE baseline latencies, none of the
animals receiving the mixed azine (naltrexone-oxymorphonazine) were
analgesic. This compares with an analgesic rate of 100 % o~ animals
receiving oxymorphone HCl in other control~experiments.
It will be unders-tood that the specification and
examples are illustrative but ~o-t limitative of the present
invention and that other embodiments within -the spirit and
sçope of the invention will suggest themselves to those
skilled in the art.
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