0270.6474/82/0205-0572$02.00/O The Journal of Neuroscience Copyright 0 Society for Neuroscience Vol. 2, No. 5, pp. 572-576 Printed in U.S.A. May 1982

IRREVERSIBLE OPIATE AGONISTS AND ANTAGONISTS: THE 14-HYDROXYDIHYDROMORPHINONE AZINES

ELLIOT F. HAHN,* MARY CARROLL-BUATTI,$ AND GAVRIL W. PASTERNAKt, §,’

*Laboratory of Biochemical Endocrinology, Rockefeller University, New York, New York 10021,*George C. Cotzias Laboratory of Neuro-Oncology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021,and SDepartmentsof Neurology and Pharmacology, Cornell University Medical College, New York, New York 10021

Received September 11, 1981; Revised December 11, 1981; Accepted December 29, 1981

Abstract Further investigations into the molecular actions of the 14-hydroxydihydromorphinone hydra- zones (naloxazone, oxymorphazone, and naltrexazone) have suggested that their irreversible actions can be explained by the formation of their azines. These azines, naloxonazine, naltrexonazine, and oxymorphonazine, irreversibly block opiate binding in vitro 20- to 40-fold more potently than their corresponding hydrazones, naloxazone, naltrexazone, and oxymorphazone. The blockade of binding by naloxonazine shows the same selectivity for high affinity, or ~1, sites as naloxazone.

The advantages of irreversible drugs in both the in Materials and Methods vivo and in vitro evaluation of opiate action have led to [“HlDihydromophine, n-Ala2-n-Leu5-[3H]enkephalin, the recent development of a number of irreversible opiate n-Ala-[“Hlenkephalinamide, and Formula 963 scintilla- agonists and antagonists (Caruso et al., 1979; Portoghese tion fluor were purchased from New England Nuclear et al., 1978, 1979, 1980; Schultz and Goldstein, 1975; Corp., Inc. (Boston, MA); naloxone, naltrexone, and ox- Winter and Goldstein, 1972). One compound, naloxazone, ymorphone were from Endo (Garden City, NY); and has been particularly useful in characterizing opiate re- naloxazone, naltrexazone, and oxymorphazone were syn- ceptors and opiate actions because of its ability to inhibit thesized as previously described (Pasternak and Hahn, selectively the high affinity, or ~1, binding sites and to 1980). block opiate analgesia with little effect on other classes The azine derivatives of naloxone, naltrexone, and of binding sites or on opiate-induced lethality (Pasternak oxymorphone (Fig. 1) were synthesized by reacting a 3- et al., 1980a, b; Pasternak, 1980, 1981; Zhang and Paster- fold molar excess of the parent alkaloid in ethanol nak, 1981). The need for high doses of naloxazone both with naloxazone, naltrexazone, and oxymorphazone, re- in vivo and in vitro to inactivate these high affinity, or spectively. The individual reactions which were moni- ~1, binding sites raised many questions and suggested tored by thin layer chromatography (tic) (silica gel, that naloxazone’s activity might result from its conver- CHC13:CH30H:NH40H, 9O:lO:l) were complete within 3 sion to a more active compound. In addition, the molec- hr. After evaporation of the solvent in vacua, the prod- ular structure of naloxazone did not give a clear insight ucts were purified by preparative tic using the above into the mechanism of its irreversible actions. We now solvent system. The isolated compounds had an Rf iden- report the isolation, synthesis, and chemical characteri- tical with the product in the unpurified reaction mixture, zation of a series of compounds formed from naloxazone, suggesting that rearrangement did not occur during pu- naltrexazone, and oxymorphazone which may be respon- rification. The individual alkaloids, naloxonazine, nal- sible for their irreversible actions both in vitro and in trexonazine, and oxymorphonazine, were characterized vivo. further by their mass, nuclear magnetic resonance (mm), and infrared (ir) spectra. Mass spectroscopy using chem- ’ We wish to thank Drs. David Ahern and Jerome B. Posner for their ical ionization detection showed M+l ions at 651, 679, assistance in these studies. This work was supported in part by Grant and 599, respectively. In the nmr spectrum, the downfield DA 02615 from the National Institute on Drug Abuse. shift in the C-5 hydrogen absorption recorded for the ’ Recipient of National Institute of Neurological and Communicative Disorders and Stroke Teacher-Investigator Award 5 K07NS415. To hydrazone derivatives (Pasternak and Hahn, 1980) also whom correspondence should be addressed at Department of Neurol- was observed for the azine derivatives. The absence of a ogy, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New carbonyl absorption in the ir spectra of the individual York, NY 10021. compounds further confirmed reaction at C-6 in each of 572 The Journal of Neuroscience Irreversible Opiates 573 the 14-hydroxydihydromorphinone derivatives. Microa- TNBS (Pasternak and Hahn, 1980), confirmed the for- nalysis (CHNO, Rockefeller University Microanalytical mation of a single product and excluded the presence of Laboratory) of each compound also verified the proposed an azine in each case. However, we soon discovered that structures. The absence of a free NH2 group was indi- solutions of the 14-hydroxydihydromorphinone hydra- cated by a negative 2,4,6-trinitrobenzenesulfonate zones rapidly undergo a reaction, particularly in acidic (TNBS) test (Pasternak and Hahn, 1980). In contrast to aqueous solutions. Isolation and characterization of the the parent hydrazones which produced a reddish brown product suggested the formation of azines (Fig. 2, scheme color on standing with TNBS, no color change was seen I). The direct synthesis of the various 14-hydroxydihy- with any of the azine derivatives. dromorphinone azines showed that they were identical Radiolabeled [3H]naloxazone (4 mCi/mmol) was syn- with the products formed in the various hydrazone so- thesized as previously described (Pasternak et al., 1980b). lutions. To quantitate the extent of azine formation, we [3H]Naloxonazine (2 mCi/mmol) was prepared by react- incubated 2 PM [3H]naloxazone (4 mCi/mmol) at 25°C in ing naloxazone with [3H]naloxone (2 mCi/mmol) and 1% acetic acid, the conditions used in previous studies to separating the products by thin layer chromatography as inhibit p1 or high affinity sites in d-o. Prior to the described above. The purity of all radiolabeled com- incubation, the [3H]naloxazone was pure, with no evi- pounds was established by co-migration with authentic dence of any [3H]naloxonazine as determined by radio- compounds on thin layer chromatography, comparing scans of thin layer chromatograms. After 15 min in the both radioactivity scans of the plates and UV absorption aqueous acetic acid solution, approximately 35% of the of the “H compounds. [“Hlnaloxazone was converted to [3H]naloxonazine, with Binding experiments were performed as previously the remaining 65% of the [3H]naloxazone unchanged as described using 20 mg/ml of tissue (Pasternak et al., determined by integrating the areas under the radioscan 1975). In brief, brain membranes were prepared and peaks. After 3 hr, the conversion to [3H]naloxonazine was triplicate samples were incubated with 3H-labeled ligands still 35%, implying that the conversion was complete after and designated drugs for 30 min at 25°C and then filtered 15 min. Since 1 mol of naloxonazine is formed from 2 mol over Whatman GF/B filters. The filters then were of naloxazone, the actual concentration of naloxonazine counted in Formula 963 scintillation fluor. Specific bind- formed by 2 pM naloxazone under these conditions is ing is defined as the difference in binding in the presence approximately 300 nM. Presumably, the reaction does not and absence of levallorphan (1 PM). Irreversible inhibition proceed further due to the formation of free hydrazine. of opiate receptor binding was established by the persist- Hydrazine can convert naloxonazine back to naloxone ence of binding inhibition despite extensive washes which (Fig. 2, the reverse of scheme I). did reverse any inhibition by the same concentration of We next investigated the stability of naloxonazine in naloxone. Each tissue wash included incubation for 10 aqueous solutions (Fig. 2, scheme II). [3H]Naloxonazine min at 37”C, centrifugation, and resuspension. Irreversi- (2 mCi/mol; 1 mg/ml of 1% acetic acid) was incubated at bility experiments included four washes before binding room temperature for 60 min and then run on a thin assays were performed unless otherwise stated. layer chromatogram. A single peak was found which co- migrated with authentic naloxonazine, suggesting little, Results if any, dissociation of the azine into the hydrazone and Chemistry. The synthesis of monosubstituted hydra- ketone under these conditions. zones, such as naloxazone, oxymorphazone, and naltrex- To investigate further whether naloxonazine is actually azone, may be complicated by the formation of azines in equilibrium with naloxazone and naloxone (Fig. 2, (Fig. 1). To avoid this problem, a large excess of hydrazine scheme III), we incubated equimolar amounts of nalox- was used in the synthesis. Analysis of the compounds, onazine (2 mg/ml) with [3H]naloxone (2 mCi/mmol; 1 including nuclear magnetic resonance, mass and infrared mg/ml) in aqueous 1% acetic acid at room temperature spectroscopy, thin layer chromatography, CHNO analy- for 3 hr. If the naloxonazine were truly in equilibrium sis, and chemical titration of free --NH2 groups with with naloxazone and naloxone, some [“Hlnaloxone should

14-Hydroxydihydromorphinone 14- Hydroxydihydromorphinone 14-Hydroxydihydromorphinone azine R Series Hydrazone Series Series

-CH3 oxymorphone oxymorphozone oxymorphonozine

- CH2 - CH ‘Cl-+ noloxone noloxozone noloxonazine ,CH2 -CH2 -CH 1 noltrexone noltrexazone noltrexonazine ‘CH2 Figure 1. Structures of the 14-hydroxydihydromorphinones, their hydrazones, and their azines. 574 Hahn et al. Vol. 2, No. 5, May 1982

+ NH,NH,

CH, CH=Cti2 CH,CH=CH, CH,CH=CH, I I

be incorporated into naloxonazine. Radioscans of thin TABLE I layer chromatograms of the solutions demonstrated no Direct inhibition of [3H]dihydromorphine binding by naloxone, detectable incorporation of [“Hlnaloxone into naloxona- naltrexone, and their derivatives Rat brain homogenates were prepared as described and four concen- zine. Thus, naloxonazine appears to be quite stable under trations of the drugs listed below were incubated with [“Hldihydro- these conditions. Having isolated and established the morphine (0.8 nM) for 45 min and filtered. I&O values were determined structures and the stability of the azines, we then inves- by least square fits of a log-probit curve. The results are the means f tigated whether they were responsible for the irreversible SEM of three If& values determined in separate experiments. actions of naloxazone, naltrexazone, and oxymorphazone. Compound IC, Pharmacological comparisons between the 14-hy- l&M droxydihydromorphinones, their hydrazones, and their Naloxone 4.4 * 0.7 azines. We first tested the affinity of naloxone and nal- Naloxazone 16.3 -t 4.9 trexone, their hydrazones, and their azines for opiate Naloxonazine 5.4 + 1.3 receptors (Table I). Interestingly, the hydrazones and azines displace [3H]dihydromorphine binding less po- Naltrexone 0.77 * 0.05 tently than their parent ketones. In both groups, how- Naltrexazone 1.7 + 0.2 ever, the azine is more potent than the hydrazone. This Naltrexonazine 1.16 +- 0.3 study does not address the question of irreversibility since the displacer was not washed away. tions. Making concentrated naloxazone solutions using In an effort to determine which compounds did irre- acetic acid to dissolve the free base as previously reported versibly bind to the receptor, we incubated membranes (Childers and Pasternak, 1981) results in a significant with the various drugs (2 PM) and then washed the free amount of azine formation (about 300 nM) and does and reversibly bound drug away (Table II). Special care irreversibly block the specific binding of both 3H-ligands. was taken with the hydrazones to use conditions under However, the far greater potency of the azines suggests which little or no azine formation could be detected. that they are the active component. Under these conditions, neither naloxazone nor oxymor- Since naloxonazine at 2 PM eliminated over 90% of [3H] phazone irreversibly inhibited the binding of either D- dihydromorphine and D-Ala2-D-Leu5-[3H]enkephalin Ala’-n-Let?-[“Hlenkephalin or [3H]dihydromorphine. binding, we next examined its irreversible effects at a The small amount of inhibition by naltrexazone reflects variety of concentrations (Fig. 3). After incubating mem- the difficulty in totally eliminating azines from its solu- branes with the specified drug, the membranes were The Journal of Neuroscience Irreversible Opiates 575 TABLE II puter analysis, one site appears to be quite sensitive to Irreversible inhibition of receptor binding by naloxone, naltrexone, naloxonazine (IC50, -10 to 15 nM), while the other re- oxymorphone, and their derivations quires concentrations approximately 40-fold higher (I&, Rat brain membranes were prepared and incubated with the above 400 to 500 nM). drugs (2 PM) for 30 min at 25°C and the tissue then was washed four Previous studies have demonstrated naloxazone’s se- times as described in the text to remove reversibly bound material. The results represent the means f SEM of three separate experiments lective inhibition of high affinity, or pl, binding sites utilizing n-Ala’-n-Leu”-[“H]enkephabn (1 nM) and [3H]dihydromor- (Pasternak et al., 1980a, b; Zhang and Pasternak, 1981; phine (0.8 nM). With the exception of naloxazone and acetic acid, all Childers and Pasternak, 1981). To determine whether drugs were dissolved in absolute ethanol immediately before dilution naloxonazine demonstrated the same selectivity de- and addition to tissue. Under these conditions, no azine formation could scribed with naloxazone treatment, we incubated tissue be detected for naloxazone and oxymorphazone as demonstrated by with either naloxone or naloxonazine (50 nM), washed thin layer chromatography. A trace amount of azine was present in the the tissue, and examined morphine’s displacement of D- naltrexazone solutions. The naloxazone and acetic acid sample (10 mg/ Ala2-Met5-[3H]enkephalinamide binding (Fig. 4). Mor- kg) was dissolved in water/acetic acid (1%). After 15 min, the solution phine has the same biphasic displacement in naloxone- was added to tissue. Under these conditions, a significant portion of the treated tissue as previously published (Chang and Cua- naloxazone had reacted to its azine. trecasas, 1978; Zhang and Pasternak, 1981). The initial Change in Binding morphine displacement represents n-Ala2-Met”-[“HIen- Compound [“HlDihydro- kephalinamide binding to high affinity, or ~1, receptors n-Ala’-n-Leu”-[“HlEnkephalin morphine (Wolozin and Pasternak, 1981; Zhang and Pasternak, w % 1981). Treating the tissue with naloxonazine eliminates Naloxone 0 - this initial displacement, yielding results virtually the Naloxazone 15 +- 5 2 f 11 same as those using naloxazone. Naloxazone + -48 +- 11 -41 f 13 We also performed saturation studies with [“Hlnalox- acetic acid one in control tissue and tissue treated with naloxonazine Naloxonazine -93 +- 4 -89 + 4 (50 nM) and then washed. Previous studies using nalox- Naltrexone - - azone (2 PM) clearly demonstrated the loss of the high Naltrexazone -17 + 19 -31+ 26 affinity, or pl, binding sites. Similarly, naloxonazine treat- Naltrexonazine -97 * 3 -88 + 12 ment irreversibly inhibited the high affinity, or ~1, sites with little effect on low affinity binding. Thus, naloxon- Oxymorphone - - azine has the same selectivity but far greater potency Oxymorphazone 3 + 24 3 + 21 than naloxazone. Oxymorphonazine -59+-3 -71 f 4 u -, no change. Discussion Naloxazone has proven very useful in our understand- ing of opiate receptor subtypes and their correlation with analgesia (Pasternak et al., 1980a, b, 1981; Pasternak and Hahn, 1980; Zhang and Pasternak, 1981; Hazum et al., 1981; Pasternak, 1980), even though its mechanism of

[Drug] nM

Figure 3. Irreversibility of naloxone and naloxonazine bind- ing in rat brain membranes. Rat brain membranes were pre- pared and incubated with naloxone (0) or naloxonazine (0) at the stated concentration for 30 min at 25°C then washed four times, and finally assayed with [“Hldihydromorphine (1.2 nM). The results are from a single experiment which has been replicated three times. ,’ [Morphine] nM Figure 4. Morphine displacement of n-Ala”-Met”-[“H]en- washed extensively and a binding assay with [3H]dihy- kephalinamide binding in naloxone- and naloxonazine-treated dromorphine was performed. As expected, naloxone at tissue. Rat brain membranes were prepared and incubated with either naloxone (50 nM) or naloxonazine (50 nM) for 30 min at high concentrations does not inhibit binding irreversibly. 25°C and washed three times. The binding of n-Ala”-Met”-[“HI Naloxonazine potently inhibits binding in a dose-depen- enkephalinamide (1.5 nM) then was measured in the absence dent manner. The inhibition curve appears biphasic, and presence of various concentrations of morphine sulfate. suggesting more than one site with differing sensitivities The values are expressed as the percentage of binding in the to naloxonazine’s irreversible actions. Although it is dif- naloxone-treated tissue in the absence of morphine and repre- ficult to determine IC& values accurately without com- sent a single experiment which has been replicated three times. 576 Hahn et al. Vol. 2, No. 5, May 1982 action was not known. The high concentrations and doses Hazum, R., K. J. Chang, P. Cuatrecasas, and G. W. Pasternak required for irreversible activity suggested that it was (1981) Naloxazone irreversibly inhibits the high affinity bind- converted to a more active component. This active com- ing of [‘251]n-aIa2-n-Ieu5-enkephahn. Life Sci. 28: 2973-2979. pound appears to be the azine. It has the same pharma- Pastemak, G. W. (1980) Multiple opiate receptors: 3H-Ethyl- ketocyclazocine receptor binding and ketocyclazocine anal- cological specificity for the pl, or high affinity, sites as gesia. Proc. Natl. Acad. Sci. U. S. A. 77: 3691-3694. naloxazone. Although the naloxazone used was chemi- Pastemak, G. W. (1981) Opiate, enkephahn and endorphin cally pure, it rearranged to the azine in solution, explain- analgesia: Relations to a single subpopulation of opiate re- ing, in part, the low potency of naloxazone. First, only a ceptors. Neurology 31: 1311-1315. portion of naloxazone (about 35%) is converted to its Pastemak, G. W., and E. F. Hahn (1980) Long-acting opiate azine and, second, the remaining unchanged naloxazone agonists and antagonists: 14-Hydroxydihydromorphinone hy- probably competitively inhibits naloxonazine binding. drazones. J. Med. Chem. 23: 674-677. Thus, the irreversible blockade of opiate receptor binding Pastemak, G. W., H. A. Wilson, and S. H. Snyder (1975) requiring high doses of naloxazone (2 PM) is reproduced Differential effects of protein modifying reagents on receptor by low (50 nM) doses of naloxonazine. binding of opiate agonists and antagonists. Mol. Pharmacol. Merely demonstrating that the azines have a very high 11: 340-351. Pastemak, G. W., S. R. ChiIders, and S. H. Snyder (1980a) potency does not prove that they are actually the molec- Opiate analgesia: Evidence for mediation by a subpopulation ular species responsible for the irreversible inhibition of of opiate receptors. Science 208: 514-516. binding sites. However, naloxonazine does appear to be Pasternak, G. W., S. R. Childers, and S. H. Snyder (1980b) stable chemically in our system. More important, we still NaIoxazone, a long-acting opiate antagonist: Effects on anal- do not know the mechanism of the irreversible inhibition gesia in intact animals and on opiate receptor binding in of binding. Many irreversible compounds form covalent vitro. J. Pharmacol. Exp. Ther. 214: 455-462. linkages with the binding site, and this possibility still Pasternak, G. W., M. Carroll-Buatti, and K. Spiegel (1981) The must be considered for the azines. However, it should be binding and analgesic properties of a sigma opiate, SKF stressed that irreversibility in these studies is being de- 10,047. J. Pharmacol. Exp. Ther. 219: 192-198. termined experimentally. A noncovalently bound drug Portoghese, P. S., D. L. Larson, J. B. Liang, A. E. Takemori, and T. P. Caruso (1978) S-/3-[ N-N-bis(P-chIoroethyl)amino]- which dissociates very slowly might appear to be irre- 17(cyclopropylmethyl) -4 -5 -/3 -Epoxy -3,14 -dihydroxy- versible in a practical sense. An example would be the morphinan (Chloronaltrexamine) a potent opioid receptor binding of a-bungarotoxin to nicotinic acetylcholine re- alkylating agent with ultra long narcotic antagonist activity. ceptors (Changeux et al., 1970). Neither possibility can J. Med. Chem. 21: 598-599. be dismissed. Since the azines might be considered bi- Portoghese, P. S., D. L. Larson, and J. B. Liang (1979) Synthesis functional molecules, they might bind to two different and pharmacological characterization of an alkylating ana- sites at once, greatly enhancing the affinity and decreas- logue (chloronahrexamine) of naltrexone with ultra long-last- ing the rate of dissociation. Further characterization of ing narcotic antagonist properties. J. Med. Chem. 22: 168- the mechanism of azine action is needed but will have to 173. wait for the development of radiolabeled azines of high Portoghese, P. S., D. L. Larson, L. M. Sayre, B. S. Fries, and A. E. Takemori (1980) A novel opioid receptor site-directed specific activity. alkylating agent with irreversible narcotic antagonist and reversible agonist activities. J. Med. Chem. 23: 233-234. References Schultz, R., and A. Goldstein (1975) Irreversible alteration of opiate receptor function by a photoaffnity labeling reagent. Caruso, T. P., A. E. 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