Vol. 183, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
March 16, 1992
Pages 462-467
SYNTHESIS OF AN
1251 -LABELLED
Dale E. Mais, Lilly
Received
January
AND BIOCHEMICAL
Nancy
PROPERTIES
RYANODINE
Bowling
and August
Research Laboratories, Cardiovascular Eli Lilly and Company, Lilly Corporate Indianapolis, Indiana 46285 10,
DERIVATIVE
M.Watanabe Department Center
1992
The synthesis of a novel radioiodinated ryanodine-OlOeq-N-acylamino acylate along with biological data are reported. The affinity of the iodinated product, 7, was comparable to ryanodine, 7.97 nM and 6.47 nM, respectively. Conversion of the non-radioactive iodinated ryanodine analog to the [1251] isotope was accomplished by conversion of 7 to the trimethyltin derivative followed by [ 12511 exchange using chloramine-T in organic solvent. The radioiodinated ryanodine analog, 9, bound to cardiac membrane preparations in a protein dependent and saturable manner indicating that this analog may represent a useful new tool for the study of ryanodine receptors and that modifications about the C-10 hydroxy group of ryanodine may be carried out without loss in biological activity. 0 1992 AcademicP?xSS,Inc. Ryanodine, 1, and 9,21-didehydroryanodine, 2, are the principle active constituents of the stem wood of the shrub Ryania speciosa(l-4). Ryanodine and its dehydro form are muscle poisons which act by uncoupling the electrical signal of the transverse tubule from the calcium release mechanism of the sarcoplasmic reticulum(5-6). The toxicity associated with the constituents of this shrub is due to their binding to the calcium activated open form of the channel involved in the release of change in the calcium from the sarcoplasmic reticulum(7). The resulting closure(8). The structure of the channel presumably prevents its complete protein with which ryanodine interacts is known as the ryanodine receptor The ryanodine and currently can be assayed using [3H]-ryanodine(3). receptor consists of four identical monomers which form a tetrameric structure(9). The ryanodine receptor has recently been cloned from rabbit cardiac muscle sarcoplasmic reticulum(9). The cDNA encodes a protein with a molecular weight of 560 kdaltons(9) (Fig. 1). 0006-29 IX/92 $1.50 Copyright 0 1992 by Academic Press. All rights of reproduction in an! form
Inc. reserved.
462
Vol.
183,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
1
Figure
1.
Chemical
RESEARCH
COMMUNICATIONS
2
structures of Ryanodine,
1, and Didehydrorynnodine,
2.
The limitations inherant in the use of tritium in probes for the study of receptors, i.e. low specific activity, prompted us to synthesize an analog that would possess greater specific activity and thus allow for the detection of ryanodine receptors in tissues with a lower receptor density. The high specific activity of [ 12511 labelled analogs suggested that analogs with this moiety present may be useful probes for the study of ryanodine receptors. This communication describes the synthesis and biochemical evaluation of a new high affinity [1251]-labelled ryanodine analog via modification at the Cloeq position of ryanodine.
METHODS
Svnthesis. Scheme I shows the synthetic route used to make the iodinatec ryanodine analog. Meta-iodobenzyl alcohol was reacted at 4() overnight with phosgene in toluene to give the chloroformate 4. This was then reacted with beta-alanine ethyl ester in toluene with one equivalent of triethylamine to give 5. Purification of 5 was carried out with flash chromatography in 95% CHC13, 5% methanol. Hydrolysis of the ester in 2N LiOH: THF (1:l) followed by chromatography (85% CHCl3, 15% MeOH) gave the free acid, 6, in an overall yield from the iodobenzyl alcohol of 32%. 1 Ryanodine, 6, dicyclohexylcarbodiimide (DCC) and dimethylaminopyridine (DMAP) were dried over P2O5 under vacuum before the coupling of 1 and 6 was attempted. Ryanodine (100 mg, 0.202 mmole), 6 ( 87 mg, 0.25 mmole), DCC (61 mg, 0.3 mmole) and 3 mg of DMAP were mixed together in 5 ml of methylene chloride dried over sieves. The reaction was allowed to proceed at room temperature for three hours at which time another 20 mg of DCC was added. At the end of another hour the methylene chloride was removed under reduced pressure and the mixture chromatographed using 85% CHC13 and 15% MeOH as the mobile phase to give 13 mg of 7 ( 8% yield).
1 All new compounds reported herein exhibited agreement with the assigned structures. 463
mass spectra
in
Vol. 183, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
4
6
1
Y
8 R = Sn(CH,), 9 R = “‘1
Scheme I
In order to convert 7 to the radiolabelled form, advantage was taken of recent advances in radioiododestannylation(l0). In this particular case, the trimethyltin derivative offers the advantage in that a very high degree of site selectivity could be obtained in the introduction of the radiolabel. Under the conditions of radioiodination, the iodine atom can be introduced at the trimethyltin point in the molecule in preference to introduction at the pyrole ring. 7 was reacted with hexamethylditin and tetrakis(triphenylphosphine) palladium(O) in refluxing dioxane for five hours and the product, 8, was purified by flash chromatography (85% CHC13: 15% MeOH) on silica gel. The analog 8 served as the precursor for the introduction of [12511 to give 9. 10 Nmoles of 8 was dissolved in 25 ul of MeOH and 1 mCi of sodium [ 12511 iodide was added, followed by the addition of 5 ul of chloramine-T ( 5 mg/ml in 200 mM phosphate buffer, pH = 7.5). The reaction was allowed to proceed for 4 min at room temperature and then injected onto an ODS-3 reverse-phase column
Vol.
183,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
utilizing a mobile phase of 70 % MeOH and 30 % 0.1 M ammonium acetate at a flow rate of 1 ml per minute. The product elutes under these conditions at lo-11 min and co-elutes with the [1271] isotope, 7. Bindinp assavs. Canine cardiac membranes were prepared as previously described (11). Assay buffer consisted of 20mM Hepes, 250mM KCl, 15mM NaCl, 1mM MgCl2 and 1OOuM CaC12. Membranes (25 ug of protein) were incubated with 9 (approx. 200 PM) along with varying concentrations of ryanodine or 7 for 3 hours at 370 C and terminated by rapid filtration onto Whatman GF/C filter paper. Nonspecific binding was defined as the binding remaining in the presence oflOuM ryanodine.
RESULTS AND DISCUSSION In order to demonstrate the utility of 9 as a probe for the ryanodine receptor, radioligand binding assays were performed using 9 as the ligand. Figure 2 shows the results of the competition assays carried out using 9 as the ligand and competing at the receptor with both ryanodine and the nonradioactive compound 7. The Kd values obtained for ryanodine and 7 were essentially identical, 6.47 f 0.61 nM and 7.97 f1.09 nM, respectively. Figure 3 shows the results of a representative saturation isotherm using 9 as the ligand. As can be be seen, binding is saturable and a replot of the data in the inset indicates a best fit for a single class of binding sites. The mean of three experiments gives a Kd of 4.5 k1.7 nM and a Bmax of 8.6k2.0 pmoles/mg protein for 9. By way of comparison, the Kd and Bmax for
110 100 90
$j
80 70 60
8
50
2
40 30 20 10 0 -10
+ 10-L
n
lo-’
10”
10’ CONC,
Figure 2. Competition membranes. Membranes (approx. 200 PM) along 7(A) in a final volume experiments performed
10’
103
nM
of 9 for ryanodine binding sites in canine ( 25 pg of protein) were incubated with with varying concentrations of ryanodine of 200 pL. The curves represent the mean in duplicate. 465
cardiac 9 (*) or of three
Vol.
183,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Figure 3. Representative saturation isotherm using 9 as the radioligand diluted with 7 to a specific activity of 64 Ci/mmole. The inset shows a replot of the data in a Scatchard format. A Kd and Bmax for this experiment was found to be 3.2 nM and 7.46 pmoles/mg protein, respectively. Binding conditions were performed as described under figure 2 legend.
[3H]ryanodine binding is 6.3 It 0.6 nM and 6.1 + 0.3 pmoles/mg protein, respectively. In generaI, structure activity studies of ryanodine derivatives have been limited due to the complex nature of this natural product. The presence of multiple hydroxy groups, for example, makes selective synthesis of novel molecules very difficult. Nevertheless, some studies have attempted to evaluate ryanodine analogs in order to examine the crucial aspects of the molecule and its interaction with its receptor. It has been found that ryanodine and its dehydro analog are very sensitive to alterations in most parts of the molecule(l2). Removal of, or electrophilic substitution on, the pyrole ring results in loss of all activity. N-substitution also rest&s in loss of activity(l2). The presence of the C-10 hydroxy group, the only such secondary hydroxy group in ryanodine, offers the oppurtunity to selectively attach a variety of groups. This type of chemistry was described earlier(12) and has been expanded more recently(l3-14). In these latter works, a series of ryanodineand dehydroryanodine-Otueq-N-acylamino acylates were synthesized and shown to retain the parent agent’s affinity to bind to the ryanodine receptor(l3-14). Using the reaction conditions previously reported, we were interested in synthesizing novel probes to the ryanodine receptor which would incorporate 112511 into the base ryanodine-dehydroryanodine structure. The use of [t251] as the radioactive
Vol.
183,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
moiety offers several advantages over tritium, one of which is the much greater specific activity of [ 1251] over [3H]. The advantages of this greater specific activity are reflected in the ease of detecting receptors of much lower density in a tissue preparation, and in autoradiographic studies, exposure time on film is reduced from weeks to only hours or days. In this communication we have described a synthesis of an [I2511 labelled ryanodine analog of high affinity for the ryanodine receptor. The high affinity and high specific activity of this probe should make it a useful tool for the study of this receptor. This synthesis was accomplished via an exchange of the [ 12711 isotope with a trimethytin group followed by an electrophilic destannylation using [ 1251]-ICl generated with chloramine-T. Selectivity of introducing [ 12511 was accomplished by using the tin exchange even in the presence of the pyrole ring.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Rogers, E.F., Koniuszy, F.R., Shavel, J., Jr., and Folkers, K. (1948) .I. Am Chem. Sot. 70: 3086-3091. Wiesner, K. (1972) Adv. Org. Chem. 8: 295-298. Waterhouse, A.L., Holden, I., and Casida, J.E. (1984) J. Clzenz. Sot., Chem. Commun. 1265-1270. Waterhouse, A.L., Holden, I., and Casida, J.E. (1985) J. Chenz. Sot., Perkin Trans. 2: 1011-1015. Jenden, D.J., and Fairhurst, AS. (1969) Pharmacol. Rev. 21: l-5. Sutko, J.L., and Kenyon, J.L. (1983) J. Gen. Physiol. 82: 385-388. Meissner, G. (1986) J. Biol. Chem. 261: 6300-6305. Pessah, I.N., Anderson, K.W., and Casida, J.E. (1986) Biochem. Biophys. Res. Commun. 139: 235-239. Otsuse, K., Willard, H.F., Khanna, V.K., Zorzato, F., Green, N.M., and MacLennan, D.H. (1990) J. Biol. Chem. 265: 13472-13477. Napolitano, E, Fiaschi, R., Hanson, R.N. (1991) J&fed. Chem. 34: 2754-2759. Jones, L.R., Besch, H.R., Jr., and Fleming, J.M. (1979) J. Biol. Chem. 254: 530-535. Waterhouse, A.L., Pessah, I.N., Francini, A.O., and Casida, J.E. (1987) J. Med. Chem. 30: 710-714. Gerzon, K., Besch, H.R., and Humerickhouse, R. A. (1991) Regional Meeting , American Chemical SoCiety, Indianapolis, IN May29-3 1, Abstract 342. Gerzon, K., (1991) Slide presentation, May 31, Receptor binding of ryanodineand dehydroryanodine-0 loeq-N-acylamino-acylates.
467
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
March 16, 1992
Pages 462-467
SYNTHESIS OF AN
1251 -LABELLED
Dale E. Mais, Lilly
Received
January
AND BIOCHEMICAL
Nancy
PROPERTIES
RYANODINE
Bowling
and August
Research Laboratories, Cardiovascular Eli Lilly and Company, Lilly Corporate Indianapolis, Indiana 46285 10,
DERIVATIVE
M.Watanabe Department Center
1992
The synthesis of a novel radioiodinated ryanodine-OlOeq-N-acylamino acylate along with biological data are reported. The affinity of the iodinated product, 7, was comparable to ryanodine, 7.97 nM and 6.47 nM, respectively. Conversion of the non-radioactive iodinated ryanodine analog to the [1251] isotope was accomplished by conversion of 7 to the trimethyltin derivative followed by [ 12511 exchange using chloramine-T in organic solvent. The radioiodinated ryanodine analog, 9, bound to cardiac membrane preparations in a protein dependent and saturable manner indicating that this analog may represent a useful new tool for the study of ryanodine receptors and that modifications about the C-10 hydroxy group of ryanodine may be carried out without loss in biological activity. 0 1992 AcademicP?xSS,Inc. Ryanodine, 1, and 9,21-didehydroryanodine, 2, are the principle active constituents of the stem wood of the shrub Ryania speciosa(l-4). Ryanodine and its dehydro form are muscle poisons which act by uncoupling the electrical signal of the transverse tubule from the calcium release mechanism of the sarcoplasmic reticulum(5-6). The toxicity associated with the constituents of this shrub is due to their binding to the calcium activated open form of the channel involved in the release of change in the calcium from the sarcoplasmic reticulum(7). The resulting closure(8). The structure of the channel presumably prevents its complete protein with which ryanodine interacts is known as the ryanodine receptor The ryanodine and currently can be assayed using [3H]-ryanodine(3). receptor consists of four identical monomers which form a tetrameric structure(9). The ryanodine receptor has recently been cloned from rabbit cardiac muscle sarcoplasmic reticulum(9). The cDNA encodes a protein with a molecular weight of 560 kdaltons(9) (Fig. 1). 0006-29 IX/92 $1.50 Copyright 0 1992 by Academic Press. All rights of reproduction in an! form
Inc. reserved.
462
Vol.
183,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
1
Figure
1.
Chemical
RESEARCH
COMMUNICATIONS
2
structures of Ryanodine,
1, and Didehydrorynnodine,
2.
The limitations inherant in the use of tritium in probes for the study of receptors, i.e. low specific activity, prompted us to synthesize an analog that would possess greater specific activity and thus allow for the detection of ryanodine receptors in tissues with a lower receptor density. The high specific activity of [ 12511 labelled analogs suggested that analogs with this moiety present may be useful probes for the study of ryanodine receptors. This communication describes the synthesis and biochemical evaluation of a new high affinity [1251]-labelled ryanodine analog via modification at the Cloeq position of ryanodine.
METHODS
Svnthesis. Scheme I shows the synthetic route used to make the iodinatec ryanodine analog. Meta-iodobenzyl alcohol was reacted at 4() overnight with phosgene in toluene to give the chloroformate 4. This was then reacted with beta-alanine ethyl ester in toluene with one equivalent of triethylamine to give 5. Purification of 5 was carried out with flash chromatography in 95% CHC13, 5% methanol. Hydrolysis of the ester in 2N LiOH: THF (1:l) followed by chromatography (85% CHCl3, 15% MeOH) gave the free acid, 6, in an overall yield from the iodobenzyl alcohol of 32%. 1 Ryanodine, 6, dicyclohexylcarbodiimide (DCC) and dimethylaminopyridine (DMAP) were dried over P2O5 under vacuum before the coupling of 1 and 6 was attempted. Ryanodine (100 mg, 0.202 mmole), 6 ( 87 mg, 0.25 mmole), DCC (61 mg, 0.3 mmole) and 3 mg of DMAP were mixed together in 5 ml of methylene chloride dried over sieves. The reaction was allowed to proceed at room temperature for three hours at which time another 20 mg of DCC was added. At the end of another hour the methylene chloride was removed under reduced pressure and the mixture chromatographed using 85% CHC13 and 15% MeOH as the mobile phase to give 13 mg of 7 ( 8% yield).
1 All new compounds reported herein exhibited agreement with the assigned structures. 463
mass spectra
in
Vol. 183, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
4
6
1
Y
8 R = Sn(CH,), 9 R = “‘1
Scheme I
In order to convert 7 to the radiolabelled form, advantage was taken of recent advances in radioiododestannylation(l0). In this particular case, the trimethyltin derivative offers the advantage in that a very high degree of site selectivity could be obtained in the introduction of the radiolabel. Under the conditions of radioiodination, the iodine atom can be introduced at the trimethyltin point in the molecule in preference to introduction at the pyrole ring. 7 was reacted with hexamethylditin and tetrakis(triphenylphosphine) palladium(O) in refluxing dioxane for five hours and the product, 8, was purified by flash chromatography (85% CHC13: 15% MeOH) on silica gel. The analog 8 served as the precursor for the introduction of [12511 to give 9. 10 Nmoles of 8 was dissolved in 25 ul of MeOH and 1 mCi of sodium [ 12511 iodide was added, followed by the addition of 5 ul of chloramine-T ( 5 mg/ml in 200 mM phosphate buffer, pH = 7.5). The reaction was allowed to proceed for 4 min at room temperature and then injected onto an ODS-3 reverse-phase column
Vol.
183,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
utilizing a mobile phase of 70 % MeOH and 30 % 0.1 M ammonium acetate at a flow rate of 1 ml per minute. The product elutes under these conditions at lo-11 min and co-elutes with the [1271] isotope, 7. Bindinp assavs. Canine cardiac membranes were prepared as previously described (11). Assay buffer consisted of 20mM Hepes, 250mM KCl, 15mM NaCl, 1mM MgCl2 and 1OOuM CaC12. Membranes (25 ug of protein) were incubated with 9 (approx. 200 PM) along with varying concentrations of ryanodine or 7 for 3 hours at 370 C and terminated by rapid filtration onto Whatman GF/C filter paper. Nonspecific binding was defined as the binding remaining in the presence oflOuM ryanodine.
RESULTS AND DISCUSSION In order to demonstrate the utility of 9 as a probe for the ryanodine receptor, radioligand binding assays were performed using 9 as the ligand. Figure 2 shows the results of the competition assays carried out using 9 as the ligand and competing at the receptor with both ryanodine and the nonradioactive compound 7. The Kd values obtained for ryanodine and 7 were essentially identical, 6.47 f 0.61 nM and 7.97 f1.09 nM, respectively. Figure 3 shows the results of a representative saturation isotherm using 9 as the ligand. As can be be seen, binding is saturable and a replot of the data in the inset indicates a best fit for a single class of binding sites. The mean of three experiments gives a Kd of 4.5 k1.7 nM and a Bmax of 8.6k2.0 pmoles/mg protein for 9. By way of comparison, the Kd and Bmax for
110 100 90
$j
80 70 60
8
50
2
40 30 20 10 0 -10
+ 10-L
n
lo-’
10”
10’ CONC,
Figure 2. Competition membranes. Membranes (approx. 200 PM) along 7(A) in a final volume experiments performed
10’
103
nM
of 9 for ryanodine binding sites in canine ( 25 pg of protein) were incubated with with varying concentrations of ryanodine of 200 pL. The curves represent the mean in duplicate. 465
cardiac 9 (*) or of three
Vol.
183,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Figure 3. Representative saturation isotherm using 9 as the radioligand diluted with 7 to a specific activity of 64 Ci/mmole. The inset shows a replot of the data in a Scatchard format. A Kd and Bmax for this experiment was found to be 3.2 nM and 7.46 pmoles/mg protein, respectively. Binding conditions were performed as described under figure 2 legend.
[3H]ryanodine binding is 6.3 It 0.6 nM and 6.1 + 0.3 pmoles/mg protein, respectively. In generaI, structure activity studies of ryanodine derivatives have been limited due to the complex nature of this natural product. The presence of multiple hydroxy groups, for example, makes selective synthesis of novel molecules very difficult. Nevertheless, some studies have attempted to evaluate ryanodine analogs in order to examine the crucial aspects of the molecule and its interaction with its receptor. It has been found that ryanodine and its dehydro analog are very sensitive to alterations in most parts of the molecule(l2). Removal of, or electrophilic substitution on, the pyrole ring results in loss of all activity. N-substitution also rest&s in loss of activity(l2). The presence of the C-10 hydroxy group, the only such secondary hydroxy group in ryanodine, offers the oppurtunity to selectively attach a variety of groups. This type of chemistry was described earlier(12) and has been expanded more recently(l3-14). In these latter works, a series of ryanodineand dehydroryanodine-Otueq-N-acylamino acylates were synthesized and shown to retain the parent agent’s affinity to bind to the ryanodine receptor(l3-14). Using the reaction conditions previously reported, we were interested in synthesizing novel probes to the ryanodine receptor which would incorporate 112511 into the base ryanodine-dehydroryanodine structure. The use of [t251] as the radioactive
Vol.
183,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
moiety offers several advantages over tritium, one of which is the much greater specific activity of [ 1251] over [3H]. The advantages of this greater specific activity are reflected in the ease of detecting receptors of much lower density in a tissue preparation, and in autoradiographic studies, exposure time on film is reduced from weeks to only hours or days. In this communication we have described a synthesis of an [I2511 labelled ryanodine analog of high affinity for the ryanodine receptor. The high affinity and high specific activity of this probe should make it a useful tool for the study of this receptor. This synthesis was accomplished via an exchange of the [ 12711 isotope with a trimethytin group followed by an electrophilic destannylation using [ 1251]-ICl generated with chloramine-T. Selectivity of introducing [ 12511 was accomplished by using the tin exchange even in the presence of the pyrole ring.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Rogers, E.F., Koniuszy, F.R., Shavel, J., Jr., and Folkers, K. (1948) .I. Am Chem. Sot. 70: 3086-3091. Wiesner, K. (1972) Adv. Org. Chem. 8: 295-298. Waterhouse, A.L., Holden, I., and Casida, J.E. (1984) J. Clzenz. Sot., Chem. Commun. 1265-1270. Waterhouse, A.L., Holden, I., and Casida, J.E. (1985) J. Chenz. Sot., Perkin Trans. 2: 1011-1015. Jenden, D.J., and Fairhurst, AS. (1969) Pharmacol. Rev. 21: l-5. Sutko, J.L., and Kenyon, J.L. (1983) J. Gen. Physiol. 82: 385-388. Meissner, G. (1986) J. Biol. Chem. 261: 6300-6305. Pessah, I.N., Anderson, K.W., and Casida, J.E. (1986) Biochem. Biophys. Res. Commun. 139: 235-239. Otsuse, K., Willard, H.F., Khanna, V.K., Zorzato, F., Green, N.M., and MacLennan, D.H. (1990) J. Biol. Chem. 265: 13472-13477. Napolitano, E, Fiaschi, R., Hanson, R.N. (1991) J&fed. Chem. 34: 2754-2759. Jones, L.R., Besch, H.R., Jr., and Fleming, J.M. (1979) J. Biol. Chem. 254: 530-535. Waterhouse, A.L., Pessah, I.N., Francini, A.O., and Casida, J.E. (1987) J. Med. Chem. 30: 710-714. Gerzon, K., Besch, H.R., and Humerickhouse, R. A. (1991) Regional Meeting , American Chemical SoCiety, Indianapolis, IN May29-3 1, Abstract 342. Gerzon, K., (1991) Slide presentation, May 31, Receptor binding of ryanodineand dehydroryanodine-0 loeq-N-acylamino-acylates.
467
