Significant Improvement of Metabolic Characteristics and Bioactivities of Clopidogrel and Analogs by Selective Deuteration

In the search for prodrug analogs of clopidogrel with improved metabolic characteristics and antiplatelet bioactivity, a group of clopidogrel and vicagrel analogs selectively deuterated at the benzylic methyl ester group were synthesized, characterized, and evaluated. The compounds included clopidogrel-d3 (8), 2-oxoclopidogrel-d3 (9), vicagrel-d3 (10a), and 12 vicagrel-d3 analogs (10b–10m) with different alkyl groups in the thiophene ester moiety. The D3C-O bond length in 10a was shown by X-ray single crystal diffraction to be shorter than the H3C-O bond length in clopidogrel, consistent with the slower rate of hydrolysis of 8 than of clopidogrel in rat whole blood in vitro. A study of the ability of the compounds to inhibit ADP-induced platelet aggregation in fresh rat whole blood collected 2 h after oral dosing of rats with the compounds (7.8 μmol/kg) showed that deuteration increased the activity of clopidogrel and that increasing the size of the alkyl group in the thiophene ester moiety reduced activity. A preliminary pharmacokinetic study comparing 10a with vicagrel administered simultaneously as single oral doses (72 μmol/kg of each drug) to male Wistar rats showed 10a generated more of its active metabolite than vicagrel. These results suggest that 10a is a potentially superior antiplatelet agent with improved metabolic characteristics and bioactivity, and less dose-related toxicity.


Introduction
Clopidogrel (1) is a thienopyridine antiplatelet agent approved by the US Food and Drug Administration (FDA) for the treatment of cardiovascular diseases, including atherothrombosis, unstable angina and myocardial infarction [1]. Clopidogrel is an inactive prodrug that requires conversion to an active metabolite (AM) by cytochrome P450 (P450) enzymes in the liver to exhibit an antiplatelet effect [2,3]. The AM has a thiol group that irreversibly inhibits the binding of 2MeS-ADP to P2Y12 by covalent binding to a cysteine residue in the receptor through a disulfide bond [4,5]. However, the process is inefficient for two reasons. Firstly, as shown in Scheme 1, the majority (85%) of clopidogrel 2 of 14 is hydrolyzed by esterases to inactive clopidogrel acid [6], and secondly, only a small proportion of the remaining clopidogrel is converted to the AM by two reactions involving 2-oxoclopidogrel as an intermediate [7,8]. CYP2C19, a P450 isoform, contributes to both these reactions (44.9% to the first step, and 20.6% to the second) [9] but poor metabolizers (PMs) for CYP2C19 produce less AM with consequently little inhibition of platelets. This so-called clopidogrel resistance of PMs leads them to have a one-to five-fold higher risk of death, myocardial infarction and stroke than CYP2C19 extensive metabolizers [10,11] prompting the FDA to assign a blackbox warning to clopidogrel.
Molecules 2016, 21,704 2 of 14 of clopidogrel is hydrolyzed by esterases to inactive clopidogrel acid [6], and secondly, only a small proportion of the remaining clopidogrel is converted to the AM by two reactions involving 2oxoclopidogrel as an intermediate [7,8]. CYP2C19, a P450 isoform, contributes to both these reactions (44.9% to the first step, and 20.6% to the second) [9] but poor metabolizers (PMs) for CYP2C19 produce less AM with consequently little inhibition of platelets. This so-called clopidogrel resistance of PMs leads them to have a one-to five-fold higher risk of death, myocardial infarction and stroke than CYP2C19 extensive metabolizers [10,11] prompting the FDA to assign a blackbox warning to clopidogrel.

Scheme 1. Metabolic pathway of clopidogrel and vicagrel.
Prasugrel is a new antiplatelet agent which also exists as a prodrug that achieves greater and faster P2Y12 receptor-mediated platelet inhibition, but does not require conversion to its AM by CYP2C19. Instead it generates a thiolactone intermediate analogous to 2-oxoclopidogrel exclusively by esterase-mediated hydrolysis (Scheme 2) [12]. In addition, it has a cyclopropylketone group in place of the methyl ester in clopidogrel and, thus, neither it nor its thienolactone intermediate is deactivated via ester hydrolysis to a carboxylic acid analogous to clopidogrel acid. This means that prasugrel provides more pronounced platelet inhibition than clopidogrel with less intersubject variability. Despite these advantages, the FDA also assigned a blackbox warning to prasugrel because of its ability to cause significant, sometimes fatal, bleeding in patients with active pathological bleeding or a history of transient ischemic attack or stroke. Inspired by prasugrel, vicagrel (2) was designed partially to overcome clopidogrel resistance [13]. Like prasugrel, it forms a thienolactone (in this case 2-oxoclopidogrel) by esterase-mediated hydrolysis rather than by oxidative metabolism (Scheme 1) [13,14]. However, it is also susceptible to hydrolysis by serum and intestinal esterases to analogues of clopidogrel acid and 2-oxoclopidogrel acid [15,16]. Hence, a strategy to enhance the resistance of the benzylic methyl esters of clopidogrel and vicagrel to hydrolysis has the potential to produce significantly improved antiplatelet agents.
Deuterium is a non-radioactive isotope of hydrogen that replaces it in deuterated compounds. Such deuteration causes minimal structural perturbation and has little effect on the pharmacological activity of physiologically-active compounds [17]. However, it can affect the rate of metabolism of Scheme 1. Metabolic pathway of clopidogrel and vicagrel.
Prasugrel is a new antiplatelet agent which also exists as a prodrug that achieves greater and faster P2Y12 receptor-mediated platelet inhibition, but does not require conversion to its AM by CYP2C19. Instead it generates a thiolactone intermediate analogous to 2-oxoclopidogrel exclusively by esterase-mediated hydrolysis (Scheme 2) [12]. In addition, it has a cyclopropylketone group in place of the methyl ester in clopidogrel and, thus, neither it nor its thienolactone intermediate is deactivated via ester hydrolysis to a carboxylic acid analogous to clopidogrel acid. This means that prasugrel provides more pronounced platelet inhibition than clopidogrel with less intersubject variability. Despite these advantages, the FDA also assigned a blackbox warning to prasugrel because of its ability to cause significant, sometimes fatal, bleeding in patients with active pathological bleeding or a history of transient ischemic attack or stroke. of clopidogrel is hydrolyzed by esterases to inactive clopidogrel acid [6], and secondly, only a small proportion of the remaining clopidogrel is converted to the AM by two reactions involving 2oxoclopidogrel as an intermediate [7,8]. CYP2C19, a P450 isoform, contributes to both these reactions (44.9% to the first step, and 20.6% to the second) [9] but poor metabolizers (PMs) for CYP2C19 produce less AM with consequently little inhibition of platelets. This so-called clopidogrel resistance of PMs leads them to have a one-to five-fold higher risk of death, myocardial infarction and stroke than CYP2C19 extensive metabolizers [10,11] prompting the FDA to assign a blackbox warning to clopidogrel.

Scheme 1. Metabolic pathway of clopidogrel and vicagrel.
Prasugrel is a new antiplatelet agent which also exists as a prodrug that achieves greater and faster P2Y12 receptor-mediated platelet inhibition, but does not require conversion to its AM by CYP2C19. Instead it generates a thiolactone intermediate analogous to 2-oxoclopidogrel exclusively by esterase-mediated hydrolysis (Scheme 2) [12]. In addition, it has a cyclopropylketone group in place of the methyl ester in clopidogrel and, thus, neither it nor its thienolactone intermediate is deactivated via ester hydrolysis to a carboxylic acid analogous to clopidogrel acid. This means that prasugrel provides more pronounced platelet inhibition than clopidogrel with less intersubject variability. Despite these advantages, the FDA also assigned a blackbox warning to prasugrel because of its ability to cause significant, sometimes fatal, bleeding in patients with active pathological bleeding or a history of transient ischemic attack or stroke. Inspired by prasugrel, vicagrel (2) was designed partially to overcome clopidogrel resistance [13]. Like prasugrel, it forms a thienolactone (in this case 2-oxoclopidogrel) by esterase-mediated hydrolysis rather than by oxidative metabolism (Scheme 1) [13,14]. However, it is also susceptible to hydrolysis by serum and intestinal esterases to analogues of clopidogrel acid and 2-oxoclopidogrel acid [15,16]. Hence, a strategy to enhance the resistance of the benzylic methyl esters of clopidogrel and vicagrel to hydrolysis has the potential to produce significantly improved antiplatelet agents.
Deuterium is a non-radioactive isotope of hydrogen that replaces it in deuterated compounds. Such deuteration causes minimal structural perturbation and has little effect on the pharmacological activity of physiologically-active compounds [17]. However, it can affect the rate of metabolism of Inspired by prasugrel, vicagrel (2) was designed partially to overcome clopidogrel resistance [13]. Like prasugrel, it forms a thienolactone (in this case 2-oxoclopidogrel) by esterase-mediated hydrolysis rather than by oxidative metabolism (Scheme 1) [13,14]. However, it is also susceptible to hydrolysis by serum and intestinal esterases to analogues of clopidogrel acid and 2-oxoclopidogrel acid [15,16]. Hence, a strategy to enhance the resistance of the benzylic methyl esters of clopidogrel and vicagrel to hydrolysis has the potential to produce significantly improved antiplatelet agents.
Deuterium is a non-radioactive isotope of hydrogen that replaces it in deuterated compounds. Such deuteration causes minimal structural perturbation and has little effect on the pharmacological activity of physiologically-active compounds [17]. However, it can affect the rate of metabolism of drugs that undergo metabolism involving C-H bond scission, although the effect is highly unpredictable and dependent on the specific compound and its deuterium substitution pattern. Irrespective of whether deuteration affects the rate of metabolism, it does not appear to result in unique metabolites that are not observed for all-hydrogen analogs [18].
The subtle, but sometimes powerful, effect of deuteration has the potential to positively affect the safety, efficacy, and/or tolerability of drugs [17,19]. For example, SD-809, an analog of tetrabenazine with deuterated methoxy groups, forms an active metabolite that undergoes CYP2D6-mediated O-dealkylation at almost half the rate of the active metabolite of tetrabenazine, itself [20]. This provides a superior pharmacokinetic profile which has led to a New Drug Application (NDA) for SD-809 to the FDA for the treatment of chorea associated with Huntington's disease.
On this basis, we envisaged that deuteration of the benzylic methyl ester group in clopidogrel and vicagrel to give clopidogrel-d 3 (8) and vicagrel-d 3 (10a) would reduce their susceptibility to inactivation, increase formation of their AM, and increase their antiplatelet potency. We also hypothesized that the antipode of vicagrel-d 3 (R-10a) would be inactive and that changing the alkyl group in the thiophene ester moiety of vicagrel-d 3 would change the rate of formation of the AM with potential improvement of antiplatelet activity.
On this basis, we envisaged that deuteration of the benzylic methyl ester group in clopidogrel and vicagrel to give clopidogrel-d3 (8) and vicagrel-d3 (10a) would reduce their susceptibility to inactivation, increase formation of their AM, and increase their antiplatelet potency. We also hypothesized that the antipode of vicagrel-d3 (R-10a) would be inactive and that changing the alkyl group in the thiophene ester moiety of vicagrel-d3 would change the rate of formation of the AM with potential improvement of antiplatelet activity.

X-ray Single Crystal Diffraction Studies
Single crystals of clopidogrel besylate and 10a were subjected to X-ray Single Crystal Diffraction. Computer-generated drawings based on the results are shown on Figure 1 where it can be seen that the D 3 C-O bond length in 10a (1.448 Å) is shorter than the corresponding H 3 C-O bond in clopidogrel besylate (1.466 Å) suggesting that the deuterated benzylic methyl ester would be more stable to esterase-mediated hydrolysis.

X-Ray Single Crystal Diffraction Studies
Single crystals of clopidogrel besylate and 10a were subjected to X-Ray Single Crystal Diffraction. Computer-generated drawings based on the results are shown on Figure 1 where it can be seen that the D3C-O bond length in 10a (1.448 Å) is shorter than the corresponding H3C-O bond in clopidogrel besylate (1.466 Å) suggesting that the deuterated benzylic methyl ester would be more stable to esterase-mediated hydrolysis.

In Vitro Hydrolysis of Clopidogrel and 8 in Rat Whole Blood
The first order rate of hydrolysis of clopidogrel-d 3 (8) (0.0919 min´1) was significantly slower than that of clopidogrel (0.0219 min´1) in rat whole blood in vitro at 37˝C and an initial concentration of 1000 ng/mL ( Figure 2). In fact, the concentration of clopidogrel was below the limit of detection after 70 min in contrast to that of 8 which was still detectable after 2 h. This reduced rate of hydrolysis is consistent with the results of the X-ray single crystal diffraction study reported in Section 2.2.

In Vitro Hydrolysis of Clopidogrel and 8 in Rat Whole Blood
The first order rate of hydrolysis of clopidogrel-d3 (8) (0.0919 min −1 ) was significantly slower than that of clopidogrel (0.0219 min −1 ) in rat whole blood in vitro at 37 °C and an initial concentration of 1000 ng/mL ( Figure 2). In fact, the concentration of clopidogrel was below the limit of detection after 70 min in contrast to that of 8 which was still detectable after 2 h. This reduced rate of hydrolysis is consistent with the results of the X-ray single crystal diffraction study reported in Section 2.2. Data are means ± SD, n = 3, for reactions at 37 °C and an initial concentration of 1000 ng/mL.

Inhibition of ADP Induced Platelet Aggregation in Rats and SAR Analysis
The antiplatelet effects of clopidogrel, vicagrel, clopidogrel-d3 (8), vicagrel-d3 (10a), 2oxoclopidogrel-d3 (9), and 12-deuterated vicagrel-related compounds (10b-m) were evaluated using Born's method to determine the inhibition of ADP-induced platelet aggregation in rat blood ex vivo [21]. Compounds were administered orally at a dose of 7.8 μmol/kg and compared with the effect of clopidogrel at a dose of 78 μmol/kg. The results are summarized in Table 1 where it can be seen that clopidogrel shows a strong inhibitory effect at a dose of 78 μmol/kg, but only low activity at a dose of 7.8 μmol/kg. In contrast, clopidogrel-d3 (8), vicagrel, and vicagrel-d3 (10a) are potent inhibitors at this low dose. Not surprisingly, 2-oxoclopidogrel-d3 (9), the metabolite of 10a, is also a potent antiplatelet agent.
The n-alkyl R group of the thiophene ester moiety has a significant impact on antiplatelet potency with an inverse relationship between the length of the linear chain and potency in the order 10a > 10b > 10c > 10g > 10i. In addition, compounds with linear alkyl groups appeared to be more potent than those with branched ones (e. g., 10c > 10d and 10g > 10f and 10h) with the exception of the compound with R = cyclopropyl (10e) which was anomalously potent. Esters of aromatic acids (e.g., 10j, 10k, 10l) appeared to be less potent than those of aliphatic acids except for the nicotinate ester 10m which also exhibited strong potency. These results indicate that steric hindrance at the thiophene ester carboxylate group reduces the rate of formation of the AM and reduces antiplatelet potency.
These preliminary results suggest that 10a is the most promising drug candidate of the compounds tested. Accordingly, its antipode (R-10a) was synthesized in order to investigate the effect of configuration on potency. It was found that, in contrast to 10a, R-10a was almost inactive suggesting inhibition of platelet inhibition by the AM is stereoselective. On the basis of the above results, 10a was selected for a study of its metabolism in vivo.

Pharmacokinetic Study of AM Generated from 10a or Vicagrel in Rat
The AM produced from the deuterated analogs differs from that produced from clopidogrel and vicagrel only in deuteration of the benzylic methyl ester. Vicagrel has previously been shown to produce a four-fold higher level of AM than clopidogrel in rat [22] and, to compare the effect of deuteration, a pharmacokinetic study comparing the formation of the corresponding AMs from 10a

Inhibition of ADP Induced Platelet Aggregation in Rats and SAR Analysis
The antiplatelet effects of clopidogrel, vicagrel, clopidogrel-d 3 (8), vicagrel-d 3 (10a), 2-oxoclopidogrel-d 3 (9), and 12-deuterated vicagrel-related compounds (10b-m) were evaluated using Born's method to determine the inhibition of ADP-induced platelet aggregation in rat blood ex vivo [21]. Compounds were administered orally at a dose of 7.8 µmol/kg and compared with the effect of clopidogrel at a dose of 78 µmol/kg. The results are summarized in Table 1 where it can be seen that clopidogrel shows a strong inhibitory effect at a dose of 78 µmol/kg, but only low activity at a dose of 7.8 µmol/kg. In contrast, clopidogrel-d 3 (8), vicagrel, and vicagrel-d 3 (10a) are potent inhibitors at this low dose. Not surprisingly, 2-oxoclopidogrel-d 3 (9), the metabolite of 10a, is also a potent antiplatelet agent.
The n-alkyl R group of the thiophene ester moiety has a significant impact on antiplatelet potency with an inverse relationship between the length of the linear chain and potency in the order 10a > 10b > 10c > 10g > 10i. In addition, compounds with linear alkyl groups appeared to be more potent than those with branched ones (e. g., 10c > 10d and 10g > 10f and 10h) with the exception of the compound with R = cyclopropyl (10e) which was anomalously potent. Esters of aromatic acids (e.g., 10j, 10k, 10l) appeared to be less potent than those of aliphatic acids except for the nicotinate ester 10m which also exhibited strong potency. These results indicate that steric hindrance at the thiophene ester carboxylate group reduces the rate of formation of the AM and reduces antiplatelet potency.
These preliminary results suggest that 10a is the most promising drug candidate of the compounds tested. Accordingly, its antipode (R-10a) was synthesized in order to investigate the effect of configuration on potency. It was found that, in contrast to 10a, R-10a was almost inactive suggesting inhibition of platelet inhibition by the AM is stereoselective. On the basis of the above results, 10a was selected for a study of its metabolism in vivo.

Pharmacokinetic Study of AM Generated from 10a or Vicagrel in Rat
The AM produced from the deuterated analogs differs from that produced from clopidogrel and vicagrel only in deuteration of the benzylic methyl ester. Vicagrel has previously been shown to produce a four-fold higher level of AM than clopidogrel in rat [22] and, to compare the effect of deuteration, a pharmacokinetic study comparing the formation of the corresponding AMs from 10a and vicagrel was carried out. In order to avoid individual differences, this involved simultaneous administration of the compounds to male Wister rats at a dose of 72 µmol/kg and collection of blood samples before the dose and at 0.033, 0.083, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 10, and 12 h after the dose. After reacting with 2-bromo-3 1 -methoxyacetophenone (MPB) to stabilize AMs [23], plasma samples were analyzed for AM derivatives from vicagrel (MP_AM) and 10a (MP_DAM) by LC-MS/MS, which also served to confirm that the AM produced from 10a was greater by three in molecular weight than that produced from vicagrel. At all time points, the level of MP_DAM was higher than that of MP_AM in all 3 rats (Figure 3). Corresponding pharmacokinetic parameters for vicagrel and 10a were, respectively, C max 386.0˘67.9 and 459.5˘65.5 ng/L; AUC 0-24 1166.5˘207.6 and 1346.4˘238.9 ng¨h/L; AUC 0´8 , 1195.7˘211.2 and 1389.0˘231.5 ng¨h/L; Vd 47.2˘8.3 and 40.5˘6.8 L/Kg; t 1/2 2.39˘0.24 and 2.63˘0.14 h. The results indicate that the concentration of AM generated from 10a is higher than that generated from vicagrel at the same dose. and vicagrel was carried out. In order to avoid individual differences, this involved simultaneous administration of the compounds to male Wister rats at a dose of 72 μmol/kg and collection of blood samples before the dose and at 0.033, 0.083, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 10, and 12 h after the dose. After reacting with 2-bromo-3′-methoxyacetophenone (MPB) to stabilize AMs [23], plasma samples were analyzed for AM derivatives from vicagrel (MP_AM) and 10a (MP_DAM) by LC-MS/MS, which also served to confirm that the AM produced from 10a was greater by three in molecular weight than that produced from vicagrel. At all time points, the level of MP_DAM was higher than that of MP_AM in all 3 rats (Figure 3). Corresponding pharmacokinetic parameters for vicagrel and 10a were, respectively, Cmax 386.0 ± 67.9 and 459.5 ± 65.5 ng/L; AUC0-24 1166.5 ± 207.6 and 1346.4 ± 238.9 ng·h/L; AUC0−∞, 1195.7 ± 211.2 and 1389.0 ± 231.5 ng·h/L; Vd 47.2 ± 8.3 and 40.5 ± 6.8 L/Kg; t1/2 2.39 ± 0.24 and 2.63 ± 0.14 h. The results indicate that the concentration of AM generated from 10a is higher than that generated from vicagrel at the same dose.

Materials and General Methods
Solvents and reagents were commercially available and used without further purification. Clopidogrel bisulfate and vicagrel were obtained from Hunan CHEMAPI Biological Technology Company, Changsha, Hunan, China. Melting points were uncorrected. The specific rotation of compounds was determined using a WZZ-2B automatic polarimeter (Shanghai Precision Instrument Co., Ltd, Shanghai, China). 1 H-and 13 C-NMR spectra were recorded on a Bruker AVANCE III 400 spectrometer (Bruker BioSpin AG, Fällanden, Switzerland). Low-and high-resolution mass spectra (LRMS and HRMS) were recorded in ESI mode using a Thermo-Scientific LTQ XL (Thermo Fisher Scientific, San Jose, CA, USA)and Waters Synapt G2 Q-TOF (Waters Corporation, Milford, MA, USA), respectively. Reactions were monitored by TLC on silica gel 60 F254 plates and column chromatography was carried out on silica gel (200-300 mesh) both from Qingdao Ocean Chemical Company, Qingdao, China. Enantiomer excess (ee) values were determined by chiral HPLC (Agilent Technologies, Santa Clara, CA, USA) using an Agilent 1100 system with a G1315A diode array detector. Chiral HPLC conditions are provided for each compound.

Materials and General Methods
Solvents and reagents were commercially available and used without further purification. Clopidogrel bisulfate and vicagrel were obtained from Hunan CHEMAPI Biological Technology Company, Changsha, Hunan, China. Melting points were uncorrected. The specific rotation of compounds was determined using a WZZ-2B automatic polarimeter (Shanghai Precision Instrument Co., Ltd., Shanghai, China). 1 H-and 13 C-NMR spectra were recorded on a Bruker AVANCE III 400 spectrometer (Bruker BioSpin AG, Fällanden, Switzerland). Low-and high-resolution mass spectra (LRMS and HRMS) were recorded in ESI mode using a Thermo-Scientific LTQ XL (Thermo Fisher Scientific, San Jose, CA, USA)and Waters Synapt G2 Q-TOF (Waters Corporation, Milford, MA, USA), respectively. Reactions were monitored by TLC on silica gel 60 F254 plates and column chromatography was carried out on silica gel (200-300 mesh) both from Qingdao Ocean Chemical Company, Qingdao, China. Enantiomer excess (ee) values were determined by chiral HPLC (Agilent Technologies, Santa Clara, CA, USA) using an Agilent 1100 system with a G1315A diode array detector. Chiral HPLC conditions are provided for each compound.

Cultivation and X-ray Diffraction of Single Crystals of 10a and Clopidogrel Besylate
After dissolving in ethanol, single crystals of 10a were obtained by the slow solvent evaporation method. Clopidogrel bisulfate was suspended in dichloromethane and the mixture slowly mixed with 5% sodium bicarbonate. The organic layer was separated, dried over anhydrous sodium sulfate and concentrated under vacuum. The residue was dissolved in ethyl ether, after which an ethyl ether solution of besylic acid was added to afford a precipitate of clopidogrel besylate. After dissolving the clopidogrel besylate in isopropanol, the solution in a glass tube was placed in a sealed container with tert-butyl methyl ether to afford single crystals of clopidogrel besylate by the solvent diffusion method. X-ray single crystal diffraction of 10a was carried out on a Rigaku MM-007 Saturn 70 instrument.

Conclusions
Although new antiplatelet agents, such as prasugrel, are now in clinical use, clopidogrel remains the most widely used antiplatelet agent because of its greater safety and lower risk of bleeding. However, it still has drawbacks, including reduced effectiveness in CYP2C19 PMs (clopidogrel resistance) and the fact that more than 90% of an oral dose is metabolized to inactive metabolites [15,16] with the potential for dose-related toxicity. The similar prodrug vicagrel [13] partially overcomes the reduced effectiveness of clopidogrel in CYP2C19 PMs, but remains susceptible to inactivation due to esterase-mediated hydrolysis [14]. In the present study, a number of selectively-deuterated clopidogrel and vicagrel-related compounds were synthesized and evaluated with the aim of increasing the resistance of the hydrolytically-susceptible benzylic methyl ester to enzyme-mediated hydrolysis and, thereby, enhancing the formation of the AM. Selective deuteration is known to produce minimal structural modification and retain pharmacological activity, but potentially modify metabolic fate [19]. Encouragingly, X-ray single crystal diffraction data showed the D 3 C-O bond length in 10a was slightly shorter than in clopidogrel and the rate of hydrolysis of clopidogrel-d 3 in rat whole blood in vitro was significantly slower than that of clopidogrel.
The compounds were evaluated for their inhibitory effect on ADP-induced platelet aggregation in rat whole blood ex vivo and some of them (e. g., 10a, 10b, 10c, 10d, 10e, 10g, and 10m) shown to exhibit potent antiplatelet activity. The most active 10a was selected to undergo a pharmacokinetic study in vivo to compare its ability to form its AM with that of vicagrel. After simultaneous oral administration, the concentration of AM generated from 10a was higher than that generated from vicagrel at the same dose suggesting 10a would produce significantly more of its AM than clopidogrel in vivo.
On the basis of these results, selective deuteration of the benzylic methyl ester group in vicagrel produces a potentially superior antiplatelet agent to clopidogrelwith better metabolic characteristics and bioactivity and less dose-related toxicity. Further preclinical studies of 10a are currently underway in our laboratory to consolidate this prediction.