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Chloroacryloyl amides and alpha-methylenelactones from naltrexone, oxymorphone and fentanyl
Neuropeptides 5: 395-398, 1985
CHLOROACRYLOYL AMIDES AND ALPHA-METHYLENELACTONES FROM -_ --NALTREXONE, OXYMORPHONE AND FENTANYL Sydney Archer, Josephine Michael and Maged Michael (Reprints to S.A.) Chemistry Department, Rensselaer Polytechnic Institute, Troy, NY 12180 USA Eric J. Simon and Essam M.E. Abdelhamid Department of Psychiatry and Pharmacology, New York University, School of Medicine, 550 First Ave., New York, NY 10016 USA Wendell L. Nelson and Gary A. Koolpe Department of Medicinal Chemistry, School of Pharmacy, University of Washington Seattle, WA 98195 USA A series of chloroacryloyl amides and alpha-methylenelactones were prepared from naltrexone and oxymorphone and some chloroacryloyl amides from fentanyl were prepared as potential Michael acceptors and irreversible ligands. The relative rates of Michael additions of p-methoxybenzenethiol to the double bonds were measured in an NMR spectrometer. The IC5U's in rat brain homogenates and the irreversible binding to rat brain membranes were determined. In the methylenelactone series, it was found that both the alpha and beta isomers derived from naltrexone and oxymorphone were excellent Michael acceptors, but only the beta isomers were more active in the opioid binding assays. The beta isomer derived from naltrexone was an irreversible ligand whereas the oxymorphone analogue was active only in the presence of 100 mM NaCl. In the chloroacryloyl series, only the alpha-chloroacryloyl amide derived from beta naltrexamine proved to be an irreversible binding ligand in the absence of NaCl. It was an excellent Michael acceptor. Under the conditions of our experiments, beta-FNA was a poor Michael acceptor and did not behave as an irreversible ligand in rat membranes. Introduction and Results. As part of our continuing investigations on the preparation of affinity chromatography ligands for use in the isolation of opioid receptors (1,2), we prepared a series of chloroacryloxyl amides and alpha-methylenelactones derived from naltrexone, 1, and oxymorphone, 2, and alpha and beta chloroacryloyl amides derived from fentanyl, I. Alpha chloroacryloyl amides were selected because negative substituents on the alpha carbon increase the electrophilic character of the beta carbon thus making the compounds better Michael acceptors. It is well-known that alpha-methvlenelactones are excellent Michael acceptors. Michael addition of a thiol group to a beta-chloroacryloyl amide followed by elimination of HCl furnishes a beta-substituted acryloylamide as shown in equation 1. 4 RNHC-CH=CH2Cl + R'SH 4
-HCl $ g RNHCCH-CHClSR' + RNHCCH=CHSR' (eq. 1) 395
The target compounds 2 and 5 were prepared from the amines 1 and 2 (3,4) and the fentanyl derivatives 2 and 2 were prepared from the approprTate acids using the DCC procedure as illustrated in equation 2.
RNH2 + HOOC-y=CH2 ----,RNHC-$=CH2 Cl
(eq.
2)
Cl
1,2 or 7 The methylene lactones 10, 11, 12, and gwere prepared as described elsewhere (5). For comparative purposes, the known amide 2 (beta-FNA) and 6 (3) were synthesized by the literature procedure and by the DCC method (eq. 2) using crystalline methyl hydrogen fumarate as the acid component.
NHR' HO
CH2CH2C6H5
H
I,R,CPM, R',H
7, R,H
&R,CH3,
: 2% R,C-y=CH2
R',H E L,R,CPM, R',C-$=CH2 Cl 0 L,R,CH3,R',&C=CH2
0
10, R,CPM 11, R,CH3
c1
Cl 2, R,:-CH=( (Trans) 'H
L1 HO 0
9 L,R,CPM, R',C-CH=CHCOOCH3(&FNA)
&R,CPM,
P R',C-CH=CH2
L?_, R,CPM 13, R,CH3
We compared the relative rates of triethylamine-catalysed addition of equimolar quantities of p-methoxybenzenethiol to the target compounds with the TC5C values obtained in displacing 3H-naltrexone from rat brain homogenates (6) and with the ability of the drugs to produce irreversible binding. The target molecules were incubated for 15 minutes with rat brain membranes. The latter were washed thoroughly (see Table I) and incubated with 3H-naltrexone to determine the amount of binding capacity restored by the washing procedure. The results are summarized in Table I. Discussion. Several observations are worthy of comment. First, with respect to opioid activity, chirality at C-6 takes precedence over the rate of Michael addition (12 and 13 vs 10 and 11). This result confirms previous findings of Sayre et aF(4). Seconz there is a correlation among the antagonists between the rate of Michael addition and the degree of irreversible binding in the absence of NaCl (2 and lo). Third, in the agonist group (i.e., 5 and fi), irreversible binding occurs only in the presence of NaCl and in the antagonist group. Irreversible binding may be increased in the presence of NaCl. These 396
findings lend further support to the view that a conformational change occurs in the receptor in the presence of 100 mM NaCl which results in availability of SH groups for specific ligands, but decreases availability for non-specific ligands (e.g., NEM) (7). Fourth, the rate of Michael addition to B-FNA at 35OC is quite slow and is reflected in the lack of irreversible binding of 5. The irreversible binding noted in -in vivo systems (3,4) cannot be attributed to interaction with receptor thiols. TABLE I
Compound
IC50 - VALUES, IRREVERSIBLE BINDING AND RELATIVE RATES OF MICHAEL ADDITION OF TARGET MOLECULES Michael Cont. Additionsb Binding, % Controla T1 /p(hrs) ICsn(nM) (nM) -NaCl
No
+NaCl
NaCl
Unwashed 3 4 5 (p-FNA) 6 8 9 10 11 12 13
1.8 :"2 3.5 69 60 5.0 35 -13 125
2.0 140 ----2.0 200 -15 880
10 1000 10 10 100 1000 10 200 500 30
Washed
100 mM NACl
Unwashed
9 73 101 25 107 18 48 101 100 65 100 75 66 25 -90 -41 Completely reversible Not tested 21 8 22.5 34 40 34 39 49 -
Washed 39 75 85 98 95 110 40 -_ 68
0.25 2.17 24 NR @ 35.20 2.5 3.5 co.15 co.15 _0.15 0.15.
a.
Crude rat brain membrane fractions were pre-incubated with opiate ligands at 250C for 45 minutes in the presence and absence of 100 mM NaCl and washed extensively. The washing procedure consisted of a 6-fold dilution, centrifugation at 20,000 g for 15 minutes, incubation at 370C for 15 minutes, a second centrifugation at 20,000 g for 15 minutes followed by resuspension in the original volume. Specific binding was then determined by incubating 1 nM 3H-naltrexone (9.8 Ci/mmole) with 2 mL aliquots of washed and unwashed membrane preparation at 250C for 45 minutes in the presence and absence of 1 mM unlabelled naltrexone. Excess naltrexone protected against irreversible inhibition in all relevant cases. The results in Table I are means of at least 3 experiements.
b.
Equimolar quantities of the ligands and p-methoxybenzenethiol were dissolved in CHC13, placed in an NMR tube and the peak heights of the vinyl proton resonances were measured. Then an equimolar quantity of triethyl amine was added and the NMR spectra were recorded every five minutes until the initial reaction slowed down. Thereafter, spectra were run every half hour. The probe temperature was 35.2oC. When the reaction was complete, the contents of the tube were removed and the Michael adducts were isolated and characterized by spectroscopic methods and elemental analyses.
397
Acknowledgements. This work was supported by research grants DA-1674 (S.A.), DA-0017 (EJS), and DA-2370 (WLN) from the National Institute on Drug Abuse.
REFERENCES 1.
Bidlack, J.M., Abood, L.G., Osei-Gyimah, P. and Archer, S., (1981). Purification of the opiate receptor from rat brain. Proc. Natl. Acad. Sci. USA -78 636-639.
2.
Gioannini, T.L., Howard, A., Hiller, J.M. and Simon, E.J. (1984). Affinity chromatography of solubilized opioid binding sites using CH-Sepharose modified with a new naltrexone derivative. Biochem. Biophys. Res. Comm. 119 624-629.
3.
Portoghese, P.S., Larson, D.L., Sayre, L.M., Fries, D.S. and Takemori, A.E. (1980). A novel opioid receptor site directed alkylating agent with irreversible narcotic antagonistic and reversible agonistic activities. J. Med. Chem. -23 233-234.
4.
Sayre, L.M., Larson, D-L., Fries, D.S. Takemori, A.E. and Portoghese, P.S. (1983). Importance of C-6 chirality in conferring irreversible opioid antagonism to naltrexone-derived affinity labels. J. Med. Chem., 26, 1229-1235. In this paper the authors reported that "the half-lives for the pseudo-first-order disappearance of these agents (namely, alpha-FNA and beta-FNA) in the presence of 13-fold excess of cysteine at pH 7.4 were found to be nearly identical (approximately 0.5 min)." Neither the temperature at which the reaction was carried out nor the parameter used to follow the reaction was mentioned. The presumed Michael adduct was not isolated or characterized.
5.
Koolpe, G.A., Nelson, W.L., Gioannini, Angel, L. and Simon, E.J. (1984). Diastereomeric 6-desoxy-6-Spiro-a methylene- ?f-butyrolactonederivatives of naltrexone and oxymorphone-selective irreversible inhibition of naltrexone binding in an opioid receptor preparation by a conformationally restricted Michael acceptor legand. J. Med. Chem. in press.
6.
Simon, E.J., Hiller, J.M., Groth, J. and Edelman, I. (1975). Further properties of stereospecific opiate binding sites in rat brain: on the nature of the sodium effect. J. Pharmacol. Exp. Therap. -132 531-537.
7.
Simon, E.J., Groth, J., (1975) Kinetics of opiate receptor inactivation by sulhydryl reagents: evidence for a conformational change in the presence of sodium ions. Proc. Natl. Acad. Sci., USA 72, 2404-2407.
398
CHLOROACRYLOYL AMIDES AND ALPHA-METHYLENELACTONES FROM -_ --NALTREXONE, OXYMORPHONE AND FENTANYL Sydney Archer, Josephine Michael and Maged Michael (Reprints to S.A.) Chemistry Department, Rensselaer Polytechnic Institute, Troy, NY 12180 USA Eric J. Simon and Essam M.E. Abdelhamid Department of Psychiatry and Pharmacology, New York University, School of Medicine, 550 First Ave., New York, NY 10016 USA Wendell L. Nelson and Gary A. Koolpe Department of Medicinal Chemistry, School of Pharmacy, University of Washington Seattle, WA 98195 USA A series of chloroacryloyl amides and alpha-methylenelactones were prepared from naltrexone and oxymorphone and some chloroacryloyl amides from fentanyl were prepared as potential Michael acceptors and irreversible ligands. The relative rates of Michael additions of p-methoxybenzenethiol to the double bonds were measured in an NMR spectrometer. The IC5U's in rat brain homogenates and the irreversible binding to rat brain membranes were determined. In the methylenelactone series, it was found that both the alpha and beta isomers derived from naltrexone and oxymorphone were excellent Michael acceptors, but only the beta isomers were more active in the opioid binding assays. The beta isomer derived from naltrexone was an irreversible ligand whereas the oxymorphone analogue was active only in the presence of 100 mM NaCl. In the chloroacryloyl series, only the alpha-chloroacryloyl amide derived from beta naltrexamine proved to be an irreversible binding ligand in the absence of NaCl. It was an excellent Michael acceptor. Under the conditions of our experiments, beta-FNA was a poor Michael acceptor and did not behave as an irreversible ligand in rat membranes. Introduction and Results. As part of our continuing investigations on the preparation of affinity chromatography ligands for use in the isolation of opioid receptors (1,2), we prepared a series of chloroacryloxyl amides and alpha-methylenelactones derived from naltrexone, 1, and oxymorphone, 2, and alpha and beta chloroacryloyl amides derived from fentanyl, I. Alpha chloroacryloyl amides were selected because negative substituents on the alpha carbon increase the electrophilic character of the beta carbon thus making the compounds better Michael acceptors. It is well-known that alpha-methvlenelactones are excellent Michael acceptors. Michael addition of a thiol group to a beta-chloroacryloyl amide followed by elimination of HCl furnishes a beta-substituted acryloylamide as shown in equation 1. 4 RNHC-CH=CH2Cl + R'SH 4
-HCl $ g RNHCCH-CHClSR' + RNHCCH=CHSR' (eq. 1) 395
The target compounds 2 and 5 were prepared from the amines 1 and 2 (3,4) and the fentanyl derivatives 2 and 2 were prepared from the approprTate acids using the DCC procedure as illustrated in equation 2.
RNH2 + HOOC-y=CH2 ----,RNHC-$=CH2 Cl
(eq.
2)
Cl
1,2 or 7 The methylene lactones 10, 11, 12, and gwere prepared as described elsewhere (5). For comparative purposes, the known amide 2 (beta-FNA) and 6 (3) were synthesized by the literature procedure and by the DCC method (eq. 2) using crystalline methyl hydrogen fumarate as the acid component.
NHR' HO
CH2CH2C6H5
H
I,R,CPM, R',H
7, R,H
&R,CH3,
: 2% R,C-y=CH2
R',H E L,R,CPM, R',C-$=CH2 Cl 0 L,R,CH3,R',&C=CH2
0
10, R,CPM 11, R,CH3
c1
Cl 2, R,:-CH=( (Trans) 'H
L1 HO 0
9 L,R,CPM, R',C-CH=CHCOOCH3(&FNA)
&R,CPM,
P R',C-CH=CH2
L?_, R,CPM 13, R,CH3
We compared the relative rates of triethylamine-catalysed addition of equimolar quantities of p-methoxybenzenethiol to the target compounds with the TC5C values obtained in displacing 3H-naltrexone from rat brain homogenates (6) and with the ability of the drugs to produce irreversible binding. The target molecules were incubated for 15 minutes with rat brain membranes. The latter were washed thoroughly (see Table I) and incubated with 3H-naltrexone to determine the amount of binding capacity restored by the washing procedure. The results are summarized in Table I. Discussion. Several observations are worthy of comment. First, with respect to opioid activity, chirality at C-6 takes precedence over the rate of Michael addition (12 and 13 vs 10 and 11). This result confirms previous findings of Sayre et aF(4). Seconz there is a correlation among the antagonists between the rate of Michael addition and the degree of irreversible binding in the absence of NaCl (2 and lo). Third, in the agonist group (i.e., 5 and fi), irreversible binding occurs only in the presence of NaCl and in the antagonist group. Irreversible binding may be increased in the presence of NaCl. These 396
findings lend further support to the view that a conformational change occurs in the receptor in the presence of 100 mM NaCl which results in availability of SH groups for specific ligands, but decreases availability for non-specific ligands (e.g., NEM) (7). Fourth, the rate of Michael addition to B-FNA at 35OC is quite slow and is reflected in the lack of irreversible binding of 5. The irreversible binding noted in -in vivo systems (3,4) cannot be attributed to interaction with receptor thiols. TABLE I
Compound
IC50 - VALUES, IRREVERSIBLE BINDING AND RELATIVE RATES OF MICHAEL ADDITION OF TARGET MOLECULES Michael Cont. Additionsb Binding, % Controla T1 /p(hrs) ICsn(nM) (nM) -NaCl
No
+NaCl
NaCl
Unwashed 3 4 5 (p-FNA) 6 8 9 10 11 12 13
1.8 :"2 3.5 69 60 5.0 35 -13 125
2.0 140 ----2.0 200 -15 880
10 1000 10 10 100 1000 10 200 500 30
Washed
100 mM NACl
Unwashed
9 73 101 25 107 18 48 101 100 65 100 75 66 25 -90 -41 Completely reversible Not tested 21 8 22.5 34 40 34 39 49 -
Washed 39 75 85 98 95 110 40 -_ 68
0.25 2.17 24 NR @ 35.20 2.5 3.5 co.15 co.15 _0.15 0.15.
a.
Crude rat brain membrane fractions were pre-incubated with opiate ligands at 250C for 45 minutes in the presence and absence of 100 mM NaCl and washed extensively. The washing procedure consisted of a 6-fold dilution, centrifugation at 20,000 g for 15 minutes, incubation at 370C for 15 minutes, a second centrifugation at 20,000 g for 15 minutes followed by resuspension in the original volume. Specific binding was then determined by incubating 1 nM 3H-naltrexone (9.8 Ci/mmole) with 2 mL aliquots of washed and unwashed membrane preparation at 250C for 45 minutes in the presence and absence of 1 mM unlabelled naltrexone. Excess naltrexone protected against irreversible inhibition in all relevant cases. The results in Table I are means of at least 3 experiements.
b.
Equimolar quantities of the ligands and p-methoxybenzenethiol were dissolved in CHC13, placed in an NMR tube and the peak heights of the vinyl proton resonances were measured. Then an equimolar quantity of triethyl amine was added and the NMR spectra were recorded every five minutes until the initial reaction slowed down. Thereafter, spectra were run every half hour. The probe temperature was 35.2oC. When the reaction was complete, the contents of the tube were removed and the Michael adducts were isolated and characterized by spectroscopic methods and elemental analyses.
397
Acknowledgements. This work was supported by research grants DA-1674 (S.A.), DA-0017 (EJS), and DA-2370 (WLN) from the National Institute on Drug Abuse.
REFERENCES 1.
Bidlack, J.M., Abood, L.G., Osei-Gyimah, P. and Archer, S., (1981). Purification of the opiate receptor from rat brain. Proc. Natl. Acad. Sci. USA -78 636-639.
2.
Gioannini, T.L., Howard, A., Hiller, J.M. and Simon, E.J. (1984). Affinity chromatography of solubilized opioid binding sites using CH-Sepharose modified with a new naltrexone derivative. Biochem. Biophys. Res. Comm. 119 624-629.
3.
Portoghese, P.S., Larson, D.L., Sayre, L.M., Fries, D.S. and Takemori, A.E. (1980). A novel opioid receptor site directed alkylating agent with irreversible narcotic antagonistic and reversible agonistic activities. J. Med. Chem. -23 233-234.
4.
Sayre, L.M., Larson, D-L., Fries, D.S. Takemori, A.E. and Portoghese, P.S. (1983). Importance of C-6 chirality in conferring irreversible opioid antagonism to naltrexone-derived affinity labels. J. Med. Chem., 26, 1229-1235. In this paper the authors reported that "the half-lives for the pseudo-first-order disappearance of these agents (namely, alpha-FNA and beta-FNA) in the presence of 13-fold excess of cysteine at pH 7.4 were found to be nearly identical (approximately 0.5 min)." Neither the temperature at which the reaction was carried out nor the parameter used to follow the reaction was mentioned. The presumed Michael adduct was not isolated or characterized.
5.
Koolpe, G.A., Nelson, W.L., Gioannini, Angel, L. and Simon, E.J. (1984). Diastereomeric 6-desoxy-6-Spiro-a methylene- ?f-butyrolactonederivatives of naltrexone and oxymorphone-selective irreversible inhibition of naltrexone binding in an opioid receptor preparation by a conformationally restricted Michael acceptor legand. J. Med. Chem. in press.
6.
Simon, E.J., Hiller, J.M., Groth, J. and Edelman, I. (1975). Further properties of stereospecific opiate binding sites in rat brain: on the nature of the sodium effect. J. Pharmacol. Exp. Therap. -132 531-537.
7.
Simon, E.J., Groth, J., (1975) Kinetics of opiate receptor inactivation by sulhydryl reagents: evidence for a conformational change in the presence of sodium ions. Proc. Natl. Acad. Sci., USA 72, 2404-2407.
398