Journal of Pharmaceutical Sciences
FEBRUARY 1977 VOLUME 66 NUMBER 2
RESEARCH ARTICLES
Nucleophilic Addition of Bisulfite Ion to Prostaglandins Ez and Az: Implication in Aqueous Stability M. J. CHO", W. C. KRUEGER, and T. 0. OESTERLING Abstract Evidence is presented t o indicate that the bisulfite ion (HS03-1 adds across the C-9 carbonyl group of dinoprostone (prostaglandin Ez) and across the AIoJ1-bond of prostaglandin A2. At room temperature, the apparent equilibrium constant, determined by phase solubility analysis, circular dichroism, UV spectroscopy, and partitioning, for the formation of the bisulfib adduct of dinoprostone is about 7.5 M-' at neutral pH. From this result and a free energy relationship reported in the literature for the thermodynamics of nucleophilic addition to carbonyl groups, it is concluded that the chemical reactivity of the C-9 carbonyl group of dinoprostone is not high enough to improve aqueous stability through reversible one-step nucleophilic reactions. However, from a series of kinetic experiments, it is concluded that the equilibrium is extremely favorable for the formation of the bisulfite adduct of prostaglandin A2 over pH 4-8 a t room temperature. The second-order rate constant for the attack of sulfite ion (SOi-) to prostaglandin Az is 1.75 sec-1 A4-I. Keyphrases 0 Prostaglandins-dinoprostone and prostaglandin Az, nucleophilic addition of bisulfite ion, effect on aqueous stability 0 Dinoprostone-nucleophilic addition of bisulfite ion, effect on aqueous stability 0 Nucleophilic addition-bisulfite to dinoprostone and prostaglandin Az, effect on aqueous stability Bisulfite ion-nucleophilic addition to dinoprostoneand prostaglandinAz, effect on aqueous stability Stability, aqueous-bisulfite adducts of dinoprostone and prostaglandin A2
Prostaglandins are analogs of the parent 20.-carbon prostanoic acid found in virtually all mammalian cells. After Bergstrom's pioneering work on the structural characterization and biosynthsis of prostaglandins (l), numerous articles have appeared defining the physiological role and total synthesis of prostaglandins (2). Recently, their general biology (3-5) and chemistry (6-8) were extensively reviewed.
induction (10-12). However, the chemical instability of dinoprostone has limited the development of dosage forms, particularly those in aqueous solutions. The instability problem has resulted in a substantial challenge to the pharmaceutical scientist, and this work is part of a fundamental study aimed a t exploring the utility of reversible chemical reactions in improving the stability of the E series prostaglandins. As shown in Scheme I, dinoprostone, being a 8-ketol, readily undergoes dehydration to produce prostaglandin Az (11) (13-18), which has a different spectrum of biological activity. In aqueous solutions, dinoprostone shows maximum stability at pH 3.5, but the half-life is only about 40 days at 2 5 O (18);prostaglandin Az, in turn, isomerizes to prostaglandin Bz (111) in alkaline conditions. In addition to this major degradation reaction, the compound epimerizes a t C-8 in the presence of general base (19), presumably because of the high acidity of the proton a t C-8 (20). It also epimerizes a t C-15 in acid, apparently because of the easy formation of a stable allylic cation a t C-15 (21).
I
OH
dH
1
dH 11
BACKGROUND Some of the naturally occurring E and F prostaglandins have found wide clinical application in human reproduction (9). In particular, dinoprostone (prostaglandin Ez) (I)has been successfully used for labor
dH
111 Scheme 1
Vol. 66, No. 2, February 1977 I149
Kinetically, dinoprostone appears to undergo dehydration through the ElcB mechanism, with either the formation of enolate a t C-9 or the expulsion of OH- from C-11 as the rate-determining step. Although a thorough kinetic study has to be carried out to identify this proposed mechanism, it is strongly supported by a series of studies (22,23) on the &elimination reactions of B-oxy cyclic ketones with various leavinggroups of pKa ranging from 5 to 16, including OH-. Thermodynamically, the fundamental driving force for the dehydration, which is practically irreversible,appears to be the reduction in free energy content derived from an extended conjugation present in prostaglandin Az. Therefore, any reversible chemical reactions saturating the carbonyl group at C-9 of dinoprostoneshould enhance the overall stability through the relationships:
0
HO
OH
-SO,-
OH I
0
0
(Eq. l a ) -d(total dinoprostone) -(total dinoprostone) (Eq. lb) dt 1 + Keq where k is the apparent dehydration rate constant of dinoprostone, so long as the attainment of the reaction equilibrium is fast enough that it can be considered as an equilibrium reaction (with an equilibrium constant Keq)prior to the dehydration of dinoprostone and the product of the reversible reaction is chemically stable. The nucleophilic addition reaction of the bisulfite ion (HSOa-) to the carbonyl group a t C-9 of dinoprostone was investigated to see if the reaction could be utilized to improve the aqueous stability of dinoprostone. Bisulfite was chosen, among many nucleophilic reagents, because it is relatively nontoxic and hence can be used in pharmaceutical formulations (24) and also because i t is an extremely good nucleophile in a thermodynamic as well as a kinetic sense (25, 26). Thus, reactivity of bisulfite with dinoprostone can serve as a measure of the chemical reactivity (i.e., electrophilicity) of the carbonyl group of C-9 of the E~prostaglandinsin general. The enone system present in prostaglandin Az was expected to be very susceptible to nucleophilicaddition reactions, as frequently found in many Michael-type 1,4-additions (27). Therefore, it was necessary to study the extent of bisulfite addition to prostaglandin A2 to draw a complete picture of an aqueous system of dinoprostone containing the nucleophilic reagent.
RESULTS AND DISCUSSION Characterization of Reaction System-It is often difficult to identify chemically a facile equilibrium reaction, especially when the reaction product cannot he easily isolated. Consequently,various physical methods such as UV, IR, and NMR spectroscopic techniques are frequently employed, since in most cases the equilibrium condition is not to be disturbed. In aqueous solutions, dinoprostone exhibits a negative circular dichroism band near 292 nm with [el = -1.35 X lo* due to the n + ?r* transition of the C-9 carbonyl chromophore (5,28). As shown in Fig. 1, the negative molar rotation was reduced in the presence of the bisulfite ion. A t equilibrium, the reduction was a function of the total bisulfite concentration, indicating that the reaction was reversible in nature. If the reaction went to completion,the final spectrum would be independent of the total bisulfite concentration, unless its stoichiometric ratio to dinoprostone concentration was below 1:l. The ketone carbonyl chromophore of dinoprostone absorbs UV energy very weakly (€288 50),so its absorption band is easily swamped by the bisulfite absorption a t the bisulfite concentration level where the reaction can occur to a significant extent. Nevertheless, the absorption peak was significantly reduced upon addition of sodium bisulfite. As in the case of the circular dichroism spectrum, a t equilibrium the decrease in absorbance a t 288 nm was a function of the total bisulfite concentration. In identifying the reaction, attempts were also made to utilize the carbony1 stretching vibration a t 1725cm-' (5.80 pm) in the IR spectrum of dinoprostone, the C-9 signal a t 215.4 ppm (tetramethyhilane) in the I3C-NMR spectrum of dinoprostone, and the quartet a t 2.70 ppm due to the equatorial proton a t C-10 in the PMR spectrum of dinoprostone. In all cases, supportive but inconclusive data were obtained, mainly because of the small magnitude of the equilibrium constant for the bisulfte adduct formation. From the results obtained from the spectroscopic studies together yith the fact that cyclopentanone adds bisulfite (29), it was concluded that the bisulfite ion adds across the C-9 carbonyl group of prostaglandin Ez in a reversible fashion (Scheme 11). At present, the stereochemistry of the addition is not established. A brief attempt was made to follow the kinetics of bisulfite addition to dinoprostone by monitoring the change in molar rotation a t 292 nm,
-
150 /Journal of Pharmaceutical Sciences
so,Scheme I I but no reliable data were obtained because the reaction occurred too rapidly. For example, the rotation a t 292 nm of a l.W-mg/ml solution of dinoprostone containing 0.692 mole of total bisulfite (spectrum C in Fig. 1)was identical after 4 and 20 min of the sample preparation. From this observation and the equilibrium constants measured, it was concluded that the bisulfite addition to dinoprostone is kinetically favorable, even though it is not so favorable thermodynamically. The high reactivity of the enone system in prostaglandin A2 is well exemplified by the easy formation of 11-substituted E prostaglandins from the reactions of nucleophiles with the A prostaglandins (28,30). For instance, hydrogen peroxide adds to C-11 of prostaglandin Az to form eventually prostaglandin Az lO,ll-epoxide, an important intermediate in the process of converting prostaglandin Az to dinoprostone (31). According to the Sander and Jencks (32) y-parameter, a measure of thermodynamic nucleophilicity originally derived from a series of reversible nucleophilic addition reactions across a carbonyl group, any nucleophiles of y-value greater than that of hydrogen peroxide (-0.65) are expected to add to C-11 of prostaglandin Az, as long as a proper reaction condition is provided. This hypothesis is based on the fact that the thermodynamics governing the nucleophilic addition across a carbonyl group were proved to predict the nucleophilic addition of the reactions across >C=N- (25) and >C==C< (26) bonds. However, no evidence can be found to support that water (y = -3.58) adds to prostaglandin A2 to form dinoprostone. In practice, dehydration of dinoprostone can be considered as an irreversible reaction. On the other hand, the addition of bisulfite (y = 4.02) OL
-0.2
-
-0.4
-
-0.6
-
m -0.8
-
7
I1
X
Y
-1.4
1
240
,
,
,
,
,
,
,
,
,
260
280 300 320 WAVELENGTH, nm
Figure 1-Circular dichroism spectrum of dinoprostone (I)and its change in the presence of the bisulfite ion a t 2 5 O : dinoprostone = 1.00 rng/ml= 2.84 X M in a phosphate buffer of pH 6.5. Total bisulfite concentrations were 1.042,0.277, and 0.69 M for a, b, and c, respectively. For comparison, spectrum a in Fig. 2 is shown as a dotted line.
0 220
240
260 280 300 320 WAVELENGTH, nrn
340
360
Figure %-Circular dichroism spectrum of prostaglandin A2 ( I I ) and its change in the presence of the bisulfite ion at 25°:prostaglandin A2 = 0.02 mglml= 5.98 X M in a phosphate buffer of pH 6.5 and I = 0.1 M. Total bisulfite concentrations were5.38 X 10-3and 2.99 X M for a and b, respectively.
to C-11of prostaglandin A2 was expected to be very favorable, and even the formation of the 9,ll-bis derivative was considered to be possible. The PMR spectrum of prostaglandin A2 in deuterated bisulfite buffer of pD = 7.0 (pD = pH read on a meter + 0.4) (33) showed neither the two doublets at 7.6 ppm due to the proton at C-11nor the two doublets a t 6.2 ppm due to the proton at C-10, indicating that bisulfite either adds across the A1OJ1-bondor attacks a t C-9 or both. However, the overall spectrum looked similar to that of dinoprostone. Especially at 2.9 ppm, where the equatorial proton a t C-11 of dinoprostone was expected to show up as a quartet, poorly resolved but distinctive peaks were still observed, implying the presence of the proton at the carbon alpha to a carbonyl group. That the C-9 carbonyl group of prostaglandin A2 is intact in the presence of bisulfite, i.e., that the 9,ll-bis derivative is not formed, was also supported by the change in the circular dichroism spectrum of prostaglandin A2 in the presence of bisulfite. As shown in Fig. 2, prostaglandin A2 exhibits a strong positive band a t 235 nm ([8] = 4.72 X lo4)and a negative band a t 312 nm ( [ O ] = -0.69 X lo4) due to the ?r ?r+ and n T* transitions of the enone chromophore, respectively. When bisulfite was introduced to a prostaglandin A2 solution, the spectrum underwent a hypsochromic shift of about 20 nm, and the final spectrum became similar to that of dinoprostone (Fig. 1). This type of spectral change arising from breaking a conjugated system is well known in circular dichroism spectroscopy (34). The similarity of the final PMR and circular dichroism spectra of prostaglandin A2 in the presence of bisulfite to those of dinoprostone unambiguously indicates that bisulfite adds across the A'O."-bond of prostaglandin A2 (Scheme 11). Because dinoprostone and the bisulfite adduct a t C-11 of prostaglandin An are similar to each other in chemical structure, a nearly identical spectroscopic behavior is expected. The possibility of forming the 9,ll-bis derivative was ruled out not only by the presence of the C-9 carbonyl group at equilibrium, which was indicated by the PMR study, but also by the fact that only one molecule of bisulfite adds to the enone system in cortisone (35) and to the 1,4-dien%one system in dexamethasone (36). Formation of the bis derivative is probably hindered by the electrostatic repulsion between the negative charge on the sulfonate group a t C-11 of the monoadduct and the oncoming second molecule of bisulfite. According to Crabb6 (28), various Ilu-substituted derivatives of dinoprostone display a more negative Cotton effect with [O] -10,000 at ,,,X 298 nm than their corresponding 110-substituted derivatives. As shown by curve a in Fig. 2, the circular dichroism spectrum of the bisulfite adduct of prostaglandin A2 is in accord with that of 110-sulfonate.If this is the case, the stereochemistryinvolved in the nucleophilic attack at C-11 of prostaglandin A2 appears to be sterically controlled by the &side chain at C-12. The PMR study could have given additional information on the stereochemistry of the reaction if an accurate value of '75 for the coupling of the protons a t C-10 and C-11 of the adduct had been available. Such data would give an estimate of the dihedral angle between the deuterium
-
-
-
-
1
2 3 4 T O T A L BISULFITE, %
5
Figure 3-Aqueous solubility of dinoprostone at 25" as a function of the total bisulfite concentration at pH 3 ( A ) and 4 ( B ) . The portion indicates the contribution from showing deviation from linearity (M) the micelle formation.
at C-10 and the sulfonate a t C-11. However, as pointed out earlier, the resolution was unfortunately poor a t about 3 ppm. Measurement of Equilibrium Constant for Formation of Bisulfite Adduct of Dinoprostone-Of the many methods commonly employed in determining the equilibrium constant for a reversible reaction or complex formation, phase solubility analysis (37-40), UV and circular dichroism spectroscopic techniques (38,39,41),and the partition technique (39,42) were chosen for the present study. The apparent equilibrium constant is defined as: (Eq. 2) where [ES],[El,and [S]are the concentrations of all possible species of the adduct, dinoprostone, and bisulfite, respectively, at equilibrium. In solubility analysis, if K [ E ]
FEBRUARY 1977 VOLUME 66 NUMBER 2
RESEARCH ARTICLES
Nucleophilic Addition of Bisulfite Ion to Prostaglandins Ez and Az: Implication in Aqueous Stability M. J. CHO", W. C. KRUEGER, and T. 0. OESTERLING Abstract Evidence is presented t o indicate that the bisulfite ion (HS03-1 adds across the C-9 carbonyl group of dinoprostone (prostaglandin Ez) and across the AIoJ1-bond of prostaglandin A2. At room temperature, the apparent equilibrium constant, determined by phase solubility analysis, circular dichroism, UV spectroscopy, and partitioning, for the formation of the bisulfib adduct of dinoprostone is about 7.5 M-' at neutral pH. From this result and a free energy relationship reported in the literature for the thermodynamics of nucleophilic addition to carbonyl groups, it is concluded that the chemical reactivity of the C-9 carbonyl group of dinoprostone is not high enough to improve aqueous stability through reversible one-step nucleophilic reactions. However, from a series of kinetic experiments, it is concluded that the equilibrium is extremely favorable for the formation of the bisulfite adduct of prostaglandin A2 over pH 4-8 a t room temperature. The second-order rate constant for the attack of sulfite ion (SOi-) to prostaglandin Az is 1.75 sec-1 A4-I. Keyphrases 0 Prostaglandins-dinoprostone and prostaglandin Az, nucleophilic addition of bisulfite ion, effect on aqueous stability 0 Dinoprostone-nucleophilic addition of bisulfite ion, effect on aqueous stability 0 Nucleophilic addition-bisulfite to dinoprostone and prostaglandin Az, effect on aqueous stability Bisulfite ion-nucleophilic addition to dinoprostoneand prostaglandinAz, effect on aqueous stability Stability, aqueous-bisulfite adducts of dinoprostone and prostaglandin A2
Prostaglandins are analogs of the parent 20.-carbon prostanoic acid found in virtually all mammalian cells. After Bergstrom's pioneering work on the structural characterization and biosynthsis of prostaglandins (l), numerous articles have appeared defining the physiological role and total synthesis of prostaglandins (2). Recently, their general biology (3-5) and chemistry (6-8) were extensively reviewed.
induction (10-12). However, the chemical instability of dinoprostone has limited the development of dosage forms, particularly those in aqueous solutions. The instability problem has resulted in a substantial challenge to the pharmaceutical scientist, and this work is part of a fundamental study aimed a t exploring the utility of reversible chemical reactions in improving the stability of the E series prostaglandins. As shown in Scheme I, dinoprostone, being a 8-ketol, readily undergoes dehydration to produce prostaglandin Az (11) (13-18), which has a different spectrum of biological activity. In aqueous solutions, dinoprostone shows maximum stability at pH 3.5, but the half-life is only about 40 days at 2 5 O (18);prostaglandin Az, in turn, isomerizes to prostaglandin Bz (111) in alkaline conditions. In addition to this major degradation reaction, the compound epimerizes a t C-8 in the presence of general base (19), presumably because of the high acidity of the proton a t C-8 (20). It also epimerizes a t C-15 in acid, apparently because of the easy formation of a stable allylic cation a t C-15 (21).
I
OH
dH
1
dH 11
BACKGROUND Some of the naturally occurring E and F prostaglandins have found wide clinical application in human reproduction (9). In particular, dinoprostone (prostaglandin Ez) (I)has been successfully used for labor
dH
111 Scheme 1
Vol. 66, No. 2, February 1977 I149
Kinetically, dinoprostone appears to undergo dehydration through the ElcB mechanism, with either the formation of enolate a t C-9 or the expulsion of OH- from C-11 as the rate-determining step. Although a thorough kinetic study has to be carried out to identify this proposed mechanism, it is strongly supported by a series of studies (22,23) on the &elimination reactions of B-oxy cyclic ketones with various leavinggroups of pKa ranging from 5 to 16, including OH-. Thermodynamically, the fundamental driving force for the dehydration, which is practically irreversible,appears to be the reduction in free energy content derived from an extended conjugation present in prostaglandin Az. Therefore, any reversible chemical reactions saturating the carbonyl group at C-9 of dinoprostoneshould enhance the overall stability through the relationships:
0
HO
OH
-SO,-
OH I
0
0
(Eq. l a ) -d(total dinoprostone) -(total dinoprostone) (Eq. lb) dt 1 + Keq where k is the apparent dehydration rate constant of dinoprostone, so long as the attainment of the reaction equilibrium is fast enough that it can be considered as an equilibrium reaction (with an equilibrium constant Keq)prior to the dehydration of dinoprostone and the product of the reversible reaction is chemically stable. The nucleophilic addition reaction of the bisulfite ion (HSOa-) to the carbonyl group a t C-9 of dinoprostone was investigated to see if the reaction could be utilized to improve the aqueous stability of dinoprostone. Bisulfite was chosen, among many nucleophilic reagents, because it is relatively nontoxic and hence can be used in pharmaceutical formulations (24) and also because i t is an extremely good nucleophile in a thermodynamic as well as a kinetic sense (25, 26). Thus, reactivity of bisulfite with dinoprostone can serve as a measure of the chemical reactivity (i.e., electrophilicity) of the carbonyl group of C-9 of the E~prostaglandinsin general. The enone system present in prostaglandin Az was expected to be very susceptible to nucleophilicaddition reactions, as frequently found in many Michael-type 1,4-additions (27). Therefore, it was necessary to study the extent of bisulfite addition to prostaglandin A2 to draw a complete picture of an aqueous system of dinoprostone containing the nucleophilic reagent.
RESULTS AND DISCUSSION Characterization of Reaction System-It is often difficult to identify chemically a facile equilibrium reaction, especially when the reaction product cannot he easily isolated. Consequently,various physical methods such as UV, IR, and NMR spectroscopic techniques are frequently employed, since in most cases the equilibrium condition is not to be disturbed. In aqueous solutions, dinoprostone exhibits a negative circular dichroism band near 292 nm with [el = -1.35 X lo* due to the n + ?r* transition of the C-9 carbonyl chromophore (5,28). As shown in Fig. 1, the negative molar rotation was reduced in the presence of the bisulfite ion. A t equilibrium, the reduction was a function of the total bisulfite concentration, indicating that the reaction was reversible in nature. If the reaction went to completion,the final spectrum would be independent of the total bisulfite concentration, unless its stoichiometric ratio to dinoprostone concentration was below 1:l. The ketone carbonyl chromophore of dinoprostone absorbs UV energy very weakly (€288 50),so its absorption band is easily swamped by the bisulfite absorption a t the bisulfite concentration level where the reaction can occur to a significant extent. Nevertheless, the absorption peak was significantly reduced upon addition of sodium bisulfite. As in the case of the circular dichroism spectrum, a t equilibrium the decrease in absorbance a t 288 nm was a function of the total bisulfite concentration. In identifying the reaction, attempts were also made to utilize the carbony1 stretching vibration a t 1725cm-' (5.80 pm) in the IR spectrum of dinoprostone, the C-9 signal a t 215.4 ppm (tetramethyhilane) in the I3C-NMR spectrum of dinoprostone, and the quartet a t 2.70 ppm due to the equatorial proton a t C-10 in the PMR spectrum of dinoprostone. In all cases, supportive but inconclusive data were obtained, mainly because of the small magnitude of the equilibrium constant for the bisulfte adduct formation. From the results obtained from the spectroscopic studies together yith the fact that cyclopentanone adds bisulfite (29), it was concluded that the bisulfite ion adds across the C-9 carbonyl group of prostaglandin Ez in a reversible fashion (Scheme 11). At present, the stereochemistry of the addition is not established. A brief attempt was made to follow the kinetics of bisulfite addition to dinoprostone by monitoring the change in molar rotation a t 292 nm,
-
150 /Journal of Pharmaceutical Sciences
so,Scheme I I but no reliable data were obtained because the reaction occurred too rapidly. For example, the rotation a t 292 nm of a l.W-mg/ml solution of dinoprostone containing 0.692 mole of total bisulfite (spectrum C in Fig. 1)was identical after 4 and 20 min of the sample preparation. From this observation and the equilibrium constants measured, it was concluded that the bisulfite addition to dinoprostone is kinetically favorable, even though it is not so favorable thermodynamically. The high reactivity of the enone system in prostaglandin A2 is well exemplified by the easy formation of 11-substituted E prostaglandins from the reactions of nucleophiles with the A prostaglandins (28,30). For instance, hydrogen peroxide adds to C-11 of prostaglandin Az to form eventually prostaglandin Az lO,ll-epoxide, an important intermediate in the process of converting prostaglandin Az to dinoprostone (31). According to the Sander and Jencks (32) y-parameter, a measure of thermodynamic nucleophilicity originally derived from a series of reversible nucleophilic addition reactions across a carbonyl group, any nucleophiles of y-value greater than that of hydrogen peroxide (-0.65) are expected to add to C-11 of prostaglandin Az, as long as a proper reaction condition is provided. This hypothesis is based on the fact that the thermodynamics governing the nucleophilic addition across a carbonyl group were proved to predict the nucleophilic addition of the reactions across >C=N- (25) and >C==C< (26) bonds. However, no evidence can be found to support that water (y = -3.58) adds to prostaglandin A2 to form dinoprostone. In practice, dehydration of dinoprostone can be considered as an irreversible reaction. On the other hand, the addition of bisulfite (y = 4.02) OL
-0.2
-
-0.4
-
-0.6
-
m -0.8
-
7
I1
X
Y
-1.4
1
240
,
,
,
,
,
,
,
,
,
260
280 300 320 WAVELENGTH, nm
Figure 1-Circular dichroism spectrum of dinoprostone (I)and its change in the presence of the bisulfite ion a t 2 5 O : dinoprostone = 1.00 rng/ml= 2.84 X M in a phosphate buffer of pH 6.5. Total bisulfite concentrations were 1.042,0.277, and 0.69 M for a, b, and c, respectively. For comparison, spectrum a in Fig. 2 is shown as a dotted line.
0 220
240
260 280 300 320 WAVELENGTH, nrn
340
360
Figure %-Circular dichroism spectrum of prostaglandin A2 ( I I ) and its change in the presence of the bisulfite ion at 25°:prostaglandin A2 = 0.02 mglml= 5.98 X M in a phosphate buffer of pH 6.5 and I = 0.1 M. Total bisulfite concentrations were5.38 X 10-3and 2.99 X M for a and b, respectively.
to C-11of prostaglandin A2 was expected to be very favorable, and even the formation of the 9,ll-bis derivative was considered to be possible. The PMR spectrum of prostaglandin A2 in deuterated bisulfite buffer of pD = 7.0 (pD = pH read on a meter + 0.4) (33) showed neither the two doublets at 7.6 ppm due to the proton at C-11nor the two doublets a t 6.2 ppm due to the proton at C-10, indicating that bisulfite either adds across the A1OJ1-bondor attacks a t C-9 or both. However, the overall spectrum looked similar to that of dinoprostone. Especially at 2.9 ppm, where the equatorial proton a t C-11 of dinoprostone was expected to show up as a quartet, poorly resolved but distinctive peaks were still observed, implying the presence of the proton at the carbon alpha to a carbonyl group. That the C-9 carbonyl group of prostaglandin A2 is intact in the presence of bisulfite, i.e., that the 9,ll-bis derivative is not formed, was also supported by the change in the circular dichroism spectrum of prostaglandin A2 in the presence of bisulfite. As shown in Fig. 2, prostaglandin A2 exhibits a strong positive band a t 235 nm ([8] = 4.72 X lo4)and a negative band a t 312 nm ( [ O ] = -0.69 X lo4) due to the ?r ?r+ and n T* transitions of the enone chromophore, respectively. When bisulfite was introduced to a prostaglandin A2 solution, the spectrum underwent a hypsochromic shift of about 20 nm, and the final spectrum became similar to that of dinoprostone (Fig. 1). This type of spectral change arising from breaking a conjugated system is well known in circular dichroism spectroscopy (34). The similarity of the final PMR and circular dichroism spectra of prostaglandin A2 in the presence of bisulfite to those of dinoprostone unambiguously indicates that bisulfite adds across the A'O."-bond of prostaglandin A2 (Scheme 11). Because dinoprostone and the bisulfite adduct a t C-11 of prostaglandin An are similar to each other in chemical structure, a nearly identical spectroscopic behavior is expected. The possibility of forming the 9,ll-bis derivative was ruled out not only by the presence of the C-9 carbonyl group at equilibrium, which was indicated by the PMR study, but also by the fact that only one molecule of bisulfite adds to the enone system in cortisone (35) and to the 1,4-dien%one system in dexamethasone (36). Formation of the bis derivative is probably hindered by the electrostatic repulsion between the negative charge on the sulfonate group a t C-11 of the monoadduct and the oncoming second molecule of bisulfite. According to Crabb6 (28), various Ilu-substituted derivatives of dinoprostone display a more negative Cotton effect with [O] -10,000 at ,,,X 298 nm than their corresponding 110-substituted derivatives. As shown by curve a in Fig. 2, the circular dichroism spectrum of the bisulfite adduct of prostaglandin A2 is in accord with that of 110-sulfonate.If this is the case, the stereochemistryinvolved in the nucleophilic attack at C-11 of prostaglandin A2 appears to be sterically controlled by the &side chain at C-12. The PMR study could have given additional information on the stereochemistry of the reaction if an accurate value of '75 for the coupling of the protons a t C-10 and C-11 of the adduct had been available. Such data would give an estimate of the dihedral angle between the deuterium
-
-
-
-
1
2 3 4 T O T A L BISULFITE, %
5
Figure 3-Aqueous solubility of dinoprostone at 25" as a function of the total bisulfite concentration at pH 3 ( A ) and 4 ( B ) . The portion indicates the contribution from showing deviation from linearity (M) the micelle formation.
at C-10 and the sulfonate a t C-11. However, as pointed out earlier, the resolution was unfortunately poor a t about 3 ppm. Measurement of Equilibrium Constant for Formation of Bisulfite Adduct of Dinoprostone-Of the many methods commonly employed in determining the equilibrium constant for a reversible reaction or complex formation, phase solubility analysis (37-40), UV and circular dichroism spectroscopic techniques (38,39,41),and the partition technique (39,42) were chosen for the present study. The apparent equilibrium constant is defined as: (Eq. 2) where [ES],[El,and [S]are the concentrations of all possible species of the adduct, dinoprostone, and bisulfite, respectively, at equilibrium. In solubility analysis, if K [ E ]
