Differential properties of the optical-isomers of pranidipine, a 1,4-dihydropyridine calcium channel modulator

Fundum. Clin. Pliurmucol. 13 ( I 999) 650-655 0 1999 Editions scientifiques et medicales Elsevier SAS. All rights reserved PII: S 0 7 6 7 - 3 9 8 1 ( 9 9 ) 0 0 1 0 5 - X FLA

Original article

Differential properties of the optical-isomers of pranidipine, a 1,4-dihydropyridine calcium channel modulator

Takahiro Hirano, Toyoki Mori*, Masaru Kido, Kiyotaka Toide, Makoto Ohura, Hiroyuki Fujiki, Kensuke Orito, Kenji Yoshida, Katsumi Ikezono, Takumi Sumida,

Natsuki Nakayama, Youichi Yabuuchi

2nd Tokushimu Institute of’ New Drug Research, Otsuku Piiurmuceuticul Co. Ltd.. 463- 10 Kupsuno. Kuivauchi-dio. Tokushimu 771-0192, Jupuii

(Received 9 April 1999; revised 17 May 1999; accepted 13 August 1999)

Abstract - Pranidipine is an optically-active I ,4-dihydropyridine (DHP) voltage-dependent L-type calcium channel inhibitor. Certain enantiomeric pairs display opposite effects, i.e., inhibition and activation of the calcium channel while others exhibit the same qualitative actions. We investigated pranidipine, a new DHP, using a paradigm of vascular smooth muscle reactivity. In isolated rat aorta, depolarized with 80 mM KCI. both isomers of pranidipine caused a right-ward shift of the concentration-contraction curves for extracellular Ca’ + . The apparent pAZ values of the S-isomer and R-isomer were 10.03 and 8.36. respectively, providing evidence that the calcium channel blocking action of the S-isomer was 50 times more potent than that of the R-isomer. Antihypertensive actions of these two isomers studied in pentobarbital-anaesthetized spontaneously hypertensive rats, revealed that the S-isomer, at doses of 3-30 pg/kg i.v. decreased blood pressure in a dose-dependent manner. while the R-isomer had no effect on blood pressure at those doses. We conclude that the pair of enantiomers of pranidipine qualitatively display the same Ca’ + channel blocking action and that neither, isomer exhibits Bay K 8644-like activation. Pranidipine may be useful in studies on the architecture of the D H P receptor ‘pocket’. 0 1999 Editions scientifiques et medicales Elsevier SAS

pranidipine / optical-isomers / calcium channel blocking action / isolated aorta / anaesthetized rat / structure-activity relationship / spontaneously hypertensive rats

1. Introduction

The 1,4-dihydropyridine (DHP) derivatives are useful drugs that specifically inhibit the L-type voltage-dependent calcium channels (L-VDCC). Pranidipine (methyl-3-phenyl-2( E)-propenyl- 1,4-di- hydro-2,6-dimethyl-4-( 3-nitrophenyl)-3,5-pyridinedi- carboxylate,figure I ) is a DHP that is classified as a ‘new generation’ L-VDCC blocker. Pranidipine has a potent and long-lasting antihypertensive and anti- anginal action [l-111. It was suggested that highly lipophilic properties of pranidipine contributed to its potent and long-lasting action [4]. In addition, pranidipine, unique among DHPs, enhances nitric oxide-induced vascular relaxation, via a cyclic GMP-

independent mechanism [7]. Consequently, pranidipine exerts a stronger venodilator effect than that of nifedipine and amlodipine [5].

Structure-activity studies have been carried out for the calcium channel blocking action of DHPs. The calcium channel effects are most evident when the 4-aryl group is placed axially rather than equatori- ally [12]. The ester groups in the 3 and 5 positions are crucially important with regard to all pharmacological effects. DHPs with asymmetric ester substitutions are generally more active pharmacologically than those with symmetric substitutions. Moreover, it is interesting that some DHPs such as Bay K 8644, PN 202-791 or H 160/51 activate rather than inhibit the L-VDCC [13]. Studies using optical isomers of

Ahhreoiurions: DHP: dihydropyridine; L-VDCC: L-type voltage-dependent calcium channel; EC,,,: fifty percent effective concentration; E D 27 mm * Correspondence and reprints

dose producing a 25 mmHg decrease in blood pressure; SHR: spontaneously hypertensive rats.

T. Hirono ('t ul. Fundunimtul & Cliniccrl Pharmacology 13 (1999) 650 655 65 I

such compounds reveal that the S-isomers are L- VDCC activators while the R-isomers are inhibitors [ 14- 161. The use of isomeric pairs in structure-activity mutagenic studies of recombinant calcium channels has revealed, indirectly, the putative architecture of features of the 'drug pocket' [17-201.

One of the problems in interpreting data derived from the above studies is the purity of the optically active enatiomeric pairs. The S- and R-isomers of pranidipine are 100% pure and thus afford a valuable reagent for study. One of the aims of this study is the examination of the structure-activity relationship of DHP using the pure optical-isomers of pranidipine. Another aim is to determine the contri- bution of isomers to the in vivo action of racemic pranidipine.

2. Materials and methods

2.1. Inhibition of calciunz-induced contraction in 80 mM KCI-depolarized isolated rat aorta

Male Wistar rats (Charles River Japan, Tokyo, Japan) weighing 510-620 g were sacrificed by a blow on the neck and the thoracic aorta was immediately excised. The excised tissue was washed with a buffered Krebs-Henseleit solution (NaCI 1 18, CaCI,

2.5, KCI 4.7, MgCl, 1.2, KH,PO, 1.2, NaHCO, 25.0, EDTA-2Na 0.03, ascorbate 0.06, and glucose 1 1 .O, in mM) bubbled with 95% 0, and 5% CO, at 37 "C and the pH was adjusted to 7.3-7.5. After adhering fat and connective tissues were removed, the isolated aortic tissue was cut into rings of 3-4 mm in length and opened into strips. These preparations were suspended in an organ bath of 30 mL volume containing a buffered Krebs-Henseleit solution. The preparations were preloaded with a tension of 1 g.

Developed tension was measured with a force-dis- placement transducer (UL-20G, Minebea, Nagano, Japan) and recorded with a thermal pen recorder (Recti-Horiz 8K, NEC-San-ei, Tokyo, Japan). In all experiments, the preparations were equilibrated for 60 min before the experiment was started. Endothe- lial cells of these aortic preparations were kept intact, which was confirmed by the appearance of relaxation with addition of lo-' M acetylcholine. After this confirmation, the preparation was washed three times at 10 min intervals with Ca2 + -free buffered Krebs- Henseleit solution (NaCl 118, KCI 4.7, MgCI, 1.2, KH,PO, 1.2, NaHCO, 25.0, EDTA-2Na 1.0, ascorbate 0.06, and glucose 1 1 .O, in mM) and then washed three times at 10 min intervals with high-K-Ca'+- free buffered Krebs-Henseleit solution (NaCl 43.9, KCl 80.0, MgCI, 1.2, KH,PO, 1.2, NaHCO, 25.0, ascorbate 0.06, and glucose 11.0, in mM). After

Spranidipine R-pranidipine

Port Figure 1. Structures of pranidipine optical-isomers. (a) Chemical structures of S - and R-prdnidipine. (b) Stable conformers of the isomers.

652 T. Hiruno 1’1 ul. ’ FundunienIiil & Clinicul Pliummcologj 1 3 (1999) 650-655

120’

100’

80’

60’

40’ 20’

equilibration, cumulative concentration-contraction curves for calcium were obtained following the addition of CaCl, (0.01 -30 mM). The preparations were then washed with Ca2 + -free buffered Krebs- Henseleit solution and allowed to recover to the basal level. After stabilization for 60 min, the preparations were again depolarized with high-K-Ca2 + - free buffered Krebs-Henseleit solution, and pranidipine or the vehicle was added, and the drug allowed to remain for 30 min in order to obtain data for construction of concentration-contraction curves. The vehicle had no effect on the concentration-contraction curves.

2.2. Antihypertensive action in spontaneously hypertensive rats (SHR)

Male SHRs (Hoshino Animal, Yashio, Japan) at the age of 19 weeks, weighing 271-330 g were anaesthetized with pentobarbital (40 mg/kg i.p.). A polyethylene tube (SP-55, Natsume, Tokyo, Japan) was inserted into the femoral artery and vein for monitoring blood pressure and injection of drugs, respectively. The arterial tubes were connected to a pressure transducer (SPB- 105, NEC-San-ei). Blood pressure and heart rate were measured with an am- plifier (4441, NEC-San-ei) and a tachometer (4778, NEC-San-ei), respectively, and recorded with a thermal pen recorder (Recti-Horiz 8K, NEC-San-ei). Racemic and optical-isomers of pranidipine were injected at cumulative doses of 1-30 pg/kg.

2.3. Drugs

The drugs used in these experiments were racemic and optical-isomers of pranidipine (Otsuka Pharma- ceutical Co. Ltd., Tokyo, Japan). Their enantiomeric purities as checked by an HPLC method were 100‘%~ All other chemicals used were of analytical grade and obtained from commercial sources such as Sigma (St Louis, MO, USA) and Dojin Chemical (Kumamoto, Japan). Optical-isomers of pranidipine were dissolved in 100% dimethylsulfoxide (DMSO, Wako Pure Chemical, Osaka, Japan) at a concentration of 10 mM and diluted with DMSO containing distilled water and added to an organ bath in a volume of 100 pL. The final concentrations of DMSO were 0.17-0.23%,.

For the in vivo study, racemic and optical-isomers of pranidipine were dissolved at a concentration of 0.5 and 0.05 mg/mL in 50% dimethylformamide (DMF, Wako Pure Chemical)/SO‘%, saline and injected at the appropriate concentration level.

0.01 0.03 0.1 0.3 1 3 10 30 Ca2+ concentration (mM)

Figure 2. Effects of S-isomer (a) and R-isomer (b) of pranidipine on calcium-induced vascular contraction in the isolated rat aorta. Contraction was normalized with percentage of maximum tension of isomer-untreated contraction. Values are expressed as means

SEM of six to eight preparations from different rats.

2.4. Data analyses and statistics

All values were expressed as means SEM. Con- traction was normalized as a percentage of maximum tension of isomer-untreated contraction. Fifty percent effective concentrations (EC,,) of each concentration-contraction curve for calcium were obtained by regression analysis, and the apparent pA2 values were calculated by the method of Arunlak- shana and Schild [21] and Van Rossum [22].

The difference between the treated group and control group was analysed by ANOVA (repeated mea- surement), followed by a multiple comparison test (Dunnett’s method). The values were considered sig- nificant at the P < 0.05 level. Calculation of the dose producing a 25 mm Hg decrease in blood pressure (ED - 25 mm Hg) using regression analysis of the dose- response curve was used. SAS (Statistical Analysis System, SAS Institute Japan Ltd., Tokyo, Japan) was used for the analysis.

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2.5. Calculation of' the stable confiirmation of prunidipine

Modelling was carried out with the molecular modelling package QUANTA 97. The calculation of the stable conformation was carried out by PM3 [23] approximation using the MOPAC (the semi-empiri- cal molecular orbital program Version 6.0, Quantum Chemistry Program Exchange, Indiana University, Bloomington, IN, USA). The interactive modelling and display were carried out on the computer (In- digo 2, Silicon Graphics, Mountain View, CA, USA).

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3. Results

0 Control 0 Racemic

3.1. Ejjkcts of pranidipine isomers on calcium-induced contraction in KCI-depolariied isolated rat aorta

We used six to eight preparations for each concentration of the pranidipine isomers. In the isolated rat aorta, depolarized with 80 mM KC1, increment addition of extracellular calcium produced contraction in a concentration-dependent manner. Treatment with the S-isomer of pranidipine at concentrations of 10- ") to M shifted the concentration-contraction curves for calcium to the right in a concentration-dependent manner (figure 2a). Addition of the R-isomer of pranidipine also shifted concentration- contraction curves for calcium in a right-ward direc- tion but the concentrations required were lo-* to lo- ' M (figure 2b). From Schild plot analyses, the apparent PA, values for the S-isomer and R-isomer were 9.90 and 8.27, respectively. Using Van Rossum analyses, the values for the S-isomer and R-isomer were 10.15 and 8.44, respectively. The average PA, values for the S-isomer and R-isomer were 10.03 and 8.36, respectively. We conclude that the calcium channel blocking action of the S-isomer was about 50 times more potent than that of the R-isomer.

3.2. Antihypertensive action of pranidipine isomers

Twenty-four SHR were stratified according to blood pressure and randomized into four groups ( n = 6 in each group). Basal values of the systolic- blood pressure were 174 f 9, 168 f 4, 174 f 9, and 176 & 6 mm Hg in control, racemic, S - , and R-isomer, respectively, and there were no statistical differences among the groups (figure 3 ) . Basal values for diastolic-blood pressure and heart rate were also

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Figure 3. Effects of racemic and optical-isomers of pranidipine on systolic (upper) and diastolic (middle) blood pressure and heart rate (lower) in spontaneously hypertensive rats anesthetized with pentobarbital. Values are expressed as means f SEM of six rats. * P < 0.05, * * P < 0.01 compared with the vehicle-treated group.

comparable in all groups, and there were no signifi- cant statistical differences among the groups (/igure 3 ) . Racemic pranidipine and the S-isomer of pranidipine at doses of 3-30 pg/kg i.v. decreased blood pressure in a dose-dependent manner, while the R-isomer of pranidipine and vehicle did not exert any hypotensive action even at doses up to 30 pg/kg i.v. (figure 3 ) . ED 25 m,ll Hg values for systolic blood pressure of racemic pranidipine and the S-isomer of pranidipine were 2.5 pg/kg i.v. and 1.4 pg/kg i.v., respectively, and ED-,, mm He values for diastolic blood pressure of racemic and the S-isomers of pranidipine were 2.2 pg/kg i.v. and 1.2 pg/kg i.v., respectively. Thus we conclude that the S-isomer was about 1.8 times more potent as an antihypertensive than the racemate. The racemic and optical-isomers of pranidipine did not significantly affect heart rate.

654 T. Hircmo et d. Fundunientul & Clmicul Pharniucologv 13 (1999) 650-655

3.3. Calculation of pranidipine conformation

The minimum energy of each conformer of pranidipine was calculated by a PM3 program. The stable structure is shown in figure lb. The shape of the stable conformer showed that the DHP ring is in a flattened boat conformation, with the 4-phenyl group in the pseudoaxial position and orthogonal to the plane of DHP ring. The carbonyl groups of the cinnamoyl and the methyl ester groups are trans and cis, respectively, to the double bonds of the DHP ring.

4. Discussion

DHPs with different ester groups in the 3 and 5 positions are generally more active pharmacologically than the same ester groups in those positions. Pranidipine has a potent and long-lasting action, probably because of the two unsymmetrical ester groups, a bulky cinnamoyl group and a methyl ester group. We found that the inhibitory activity of the S-isomer was about 50 times more potent than that of the R-isomer, but both isomers exerted approxi- mately equal L-VDCC blocking action. The S-isomer was about 1.8 times more potent than the racemic pranidipine in reducing blood pressure in the rats. It is of interest that the R-isomer did not exert any antihypertensive action at doses in the range used for the S-isomer and the racemic com- pound. We conclude that the antihypertensive action of pranidipine is mainly contributed by the S-isomer via its blocking action on the L-VDCC.

It is suggested by the studies of the structure-activity relationship that the stereochemistry of DHPs is of crucial importance to their pharmacological effect. The structure of the S-isomer, evaluated by PM3 calculations, appears to be most stable when the DHP ring is in a flattened boat conformation, with the phenyl group in the pseudoaxial position that is similar to other 1,4-DHP compounds [12, 241. The fact that the antihypertensive action of pranidipine is mainly contributed by the S-isomer is consistent with the structure-activity relationship of DHPs, as postulated by Goldman & Stoltefuss [12]. For example, DHPs with a non-symmetrical ester substitution are more potent than those with a symmetrical substitution. Also, more potent activity is exerted when the larger ester group is on the port side of the boat form.

The S-isomer of pranidipine has a cinnamoyl ester group on the port side and a methyl group on the

starboard side. It has been suggested that the smaller ester group on the starboard side is important for formation of a hydrogen bond with a binding site in the L-VDCC and the large ester group on the port side with its lipophilic group may occupy a lipophilic domain at the binding site [12]. We presume that the methyl group participates in hydrogen bond formation and the cinnamoyl ester group occupies the lipophilic domain.

The activators of DHP, such as Bay K 8644 or YC-170, lack a large lipophilic alkyl group on the port side or in its place they have a partial negative charge. Although YC-170 has large moieties on both sides of 3 and 5 positions of DHP, this drug exerts weak L-VDCC activator action [25]. Thus since pranidipine has a large cinnamoyl ester group on the port side and does not have a negative charge, any activator effect is unlikely.

L-VDCC inhibitors usually produce negative chronotropic and negative inotropic effects. However, in the intact organisms, the negative chronotropic effect is generally offset by a baroreflex response with a resulting tachycardia. The racemic and optical-isomers of pranidipine, however, did not significantly affect heart rate in in vivo experiments in the present study. We suggest that the reason is that pranidipine has a stronger negative chronotropic action than a negative inotropic action [26].

In summary, both optical-isomers of pranidipine are inhibitors of the calcium channel, and its effects are consistent with the generally accepted structure- activity relationship of dihydropyridine calcium channel blockers and activators. Pranidipine and its isomers will be useful in future mechanistic studies and in clinical medicine.

Acknowledgements

Authors express sincere thanks to Dr S. Tamada and Mr K. Abe, Otsuka Pharmaceutical Co. Ltd., for providing optical-isomers of pranidipine, and Dr A. Schwartz, University of Cincinnati, College of Medicine, for valuable comments and checking the manuscript.

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Differential properties of the optical-isomers of pranidipine, a 1,4-dihydropyridine calcium channel modulator