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Reactivity Assays on Metabolites of the Antiplatelet

Drug Clopidogrel

Filipe Miguel Jesus Figueiredo

Instituto Superior Técnico, Universidade de Lisboa

2016, December

Abstract

Clopidogrel is an antiplatelet drug widely used on the prevention or treatment of atherothrombotic

events. It is a thienopyridine, which is an antagonist of the bonding between adenosine 5’-diphosphate and

the P2Y12 and P2Y1 receptors on the platelets’ surface.

The aim of this work was to infer about possible toxicity routes by detecting adduct formation

between reactive metabolites of clopidogrel and endogenous bionucleophiles. As so, it was necessary to

synthetize some of the most relevant metabolites.

It was observed that both 2-oxoclopidogrel and 2-oxoclopidogrelic acid undergo Michael addition

by N-acetyl-L-cysteine, which can deactivate these compounds as thiols are some of the most important

biological nucleophiles.

Adducts with glutathione were not found, and this result suggests that it does not participate in the

excretion of 2-oxoclopiogrel.

Adducts with nucleophilic amines were also not found, suggesting clopidogrel has a low toxicity

potential via covalent adduct formation

Key words: clopidogrel activation, Michael addition, thiols, low toxicity

Abbreviations HPLC High Performance Liquid Chromatography

HRMS High Resolution Mass Spectrometry

LRMS Low Resolution Mass Spectrometry

NAC N-acetyl-L-cysteine

NMR Nuclear Magnetic Resonance Spectroscopy

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Introduction

Clopidogrel [methyl (+)-(S)-(2-chlorophenyl)-2-(6,7-di-hydrothiene[3,2-c]piridin-5(4H)-

yl)acetate] is a drug used on the prevention or treatment of atherothrombotic events. As other

thienopyridine, clopidogrel can irreversibly bind to P2Y12 and P2Y1 receptors on the platelet’

surface, and so, it is used as an antiplatelet drug [1].

Clopidogrel is a prodrug, so it needs to be metabolized to generate the active compound,

a thiol. It has been widely accepted that an oxidation mediated with a CYP P450 enzyme

transforms clopidogrel in the metabolite 2-oxoclopidogrel [2], which generates the active thiol

upon another CYP mediated oxidation [3] or hydrolysis by a paraoxonase [4]. Scheme 1 gives a brief

overview of the metabolism of clopidogrel.

From here, evidences are unclear and most of the scientific community agrees with the

route from 2-oxoclopidogrel undergoing another CYP mediated oxidation with an unstable

ketosulfoxide or epoxide intermediate, followed by hydrolysis to a sulfenic acid, which is reduced

to the active thiol [5].

1 Longo et al (2013). Medicina Interna de Harrison (18ª ed., Vol . 1, translated by Fonseca, A . V. et al), Artmed, Chaps . 115-118 2 Maffrand, J .P. (2012). The story of clopidogrel and its predecesssor, ticlopidine: Could these major antiplatelet and antithrombotic drugs be discovered and developed today? CR CHIM, 15(8), 737–743. 3 Dansette, P. M. et al (2012). Cytochromes P450 catalyze both steps of the major pathway of clopidogrel bioactivation, whereas paraoxonase catalyzes the formation of a minor thiol metabolite isomer . CHEM RES TOXICOL, 25(2), 348–356. 4 Bouman, H . J . et al (2011). Paraoxonase-1 is a major determinant of clopidogrel efficacy . NAT MED, 17(1), 110-116 . 5Dansette, P. M., Bertho, G ., & Mansuy, D. (2005). First evidence that cytochrome P450 may catalyze both S-oxidation and epoxidation of thiophene derivatives. BIOCHEM BIOPH RES CO, 338(1), 450–455

Scheme 1 Simplified metabolism of clopidogrel

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Bouman et al [6] argue that 2-oxoclopidogrel suffers hydrolysis directly yielding the active

thiol by paraoxonase enzymes.

Regardless of the unclear metabolic path to clopidogrel’s bioactivation, it is estimated that

85% of the available drug is hydrolysed by carboxylesterases producing clopidogrelic acid, which

is biologically inactive.

There are some reported methods of clopidogrel oxidation, although more efficient new

methods would have helped this investigation. Lithiation with n-BuLi followed by treatment with

borates and H2O2 oxidation was tried by Shaw et al [7] without success, although Velder et al [8] did

isolate 2-oxoclopidogrel by previously protecting the ester moiety with lithium di-isopropylamide.

Chun et al [9] reported a process via clopidogrel diazonium salt. These authors also describe a

process starting on 2-bromination of thiophene, following by metoxification and hydrolysis. Shaw

et al [5] also reported that oxidation of clopidogrel with 2-chloroperoxibenzoic acid, n-BuLi with

Davis oxaziridine, H2O2 with NaHCO3 and borylation mediated by an iridium catalyst as not

successful. Treiber [10] reported oxidation of thiophene with H2O2 and a carboxylic acid which led

to moderate yields of the thiolactone. An adaptation of this method will be the favourite oxidation

strategy used on this work.

The metabolite 2-oxoclopidogrel is widely reported as stable and so was planned to be

used in the reactivity assays with nucleophiles. As a Michael acceptor, it was expected to react

preferably with soft nucleophiles, like thiols, at the beta carbon.

The active thiol metabolite is not reported as a stable compound, therefore its reactivity

was not be tested with biomolecules at this point.

Results and Discussion

To understand which 2-oxoclopidogrel tautomer is more stable in an aqueous solution, a

computational simulation was performed. Their energies were compared after the geometry had

been optimized for each isomer. It was possible to compute equilibrium composition knowing the

Gibbs free energy of formation of each molecule.

6 Bouman, H . J . et al (2011). Paraoxonase-1 is a major determinant of clopidogrel efficacy . NAT MED, 17(1), 110–116 7 Shaw, S. A. et al (2015). Synthesis of Biologically Active Piperidine Metabolites of Clopidogrel: Determination of Structure and Analyte Development . J ORG CHEM, 80(14), 7019–7032. 8 Velder, J . et al . (2010). A Scalable Synthesis of (±)-2-Oxoclopidogrel . SYNLETT, 2010(3), 467–469 . 9 Chun, G ., et al (2014). Preparation method of thienopyridine compound, CN103864817A 10 Treiber, A . (2002). Mechanism of the aromatic hydroxylation of thiophene by acid-catalyzed peracid oxidation. J ORG CHEM, 67(21), 7261–7266.

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Table 1 Determined electronic E and Gibbs free G energies for each isomer of 2-oxoclopidogrel

E (Hartree) E (MJ/mol) G

Correction (MJ/mol)

G (MJ/mol)

2-Hidroxithiophene

-1758.03 -4615.711 0.643 -4615.062

(7aR)-Tiolact-2-en-3-one

-1758.04 -4615.733 0.642 -4615.084

Tiolact-2-en-4-one

-1758.04 -4615.736 0.641 -4615.089

(7aS)-Tiolact-2-en-3-one

-1758.05 -4615.749 0.644 -4615.098

Table 2 ∆G of formation from the 2-hidroxithiophene isomer and respective equilibrium constant K

∆Grefined (kJ/mol)

Krefined

2-hidroxithiophene → (7aR)-Tiolact-2-en-3-one -22.29 5.68 x 103

2-hidroxithiophene → Tiolact-2-en-4-one -26.76 3.21 x 104

2-hidroxithiophene → (7aS)-Tiolact-2-en-3-one -37.23 1.86 x 106

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By this method, the equilibrium molar composition was estimated as: 0,98 (7aS-thiolact-

-2-en-3one), 2x10-2 (thiolact-2-en-4-one), 3x10-3 (7aR-thiolact-2-en-3-one) e 5x10-7 (2-

hidroxithiophene).

To test 2-oxoclopidogrel reactivity with bionucleophiles in vitro, oxidation with peracetic

acid was favoured. The top advantages of this method are to be a one-step synthesis and not

requiring harsh conditions like the n-BuLi synthesis’ strategy. On the other hand, this method

proved to yield many oxidation products by thin layer chromatography.

Although oxidation with peracetic acid was performed in many different conditions,

isolation of 2-oxoclopidogrel was not possible and the results on adduct formation were

inconclusive. Therefore, experiments with biological nucleophiles were performed directly with

the reaction mixture after its drying. HPLC-MS was used to detect adduct formation, having Low

Resolution Mass Spectrometry (LRMS) results suggested the formation of a bis-adduct of N-

acetyl-L-cysteine. However, only a mono-adduct was detected by High Resolution Mass

Spectrometry (HRMS). A proposal for MS2 fragments of these species is shown in Schemes 2 and

3, respectively for the mono-adduct and the bis-adduct.

Evidences of adducts with glutathione were not found by MS/MS, probably due to

stereochemical hindrance, suggesting that glutathione does not participate in the 2-

-oxoclopidogrel’s excretion mechanism.

Evidences of adducts with aminoacids with side amino chain were also not found in MS/MS,

leading to the conclusion that clopidogrel should be well tolerated as it only reacted with very

small and soft nucleophiles.

Scheme 2 Proposed fragmentation pattern for the protonated mono-adduct of clopidogrel with NAC found by HRMS for m/z 501.0925

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Clopidogrelic acid was obtained by alkaline hydrolysis of clopidogrel with a yield of 63%.

An anionic exchange resin was used to isolate the product. 1H NMR clearly showed no methoxy

singlet. This compound was also submitted to HPLC-LRMS which indicated a good purity. Scheme

4 displays proposals for the MS2 fragmentation of the protonated molecule.

Oxidation of clopidogrelic acid was performed with 5 eq of peracetic acid. The work-up

involved the use of KI to reduce the remaining peracid in solution and the extraction of iodine to

n-hexane. After evaporation of the solvent, isolation by an OASIS column was performed, having

the 2-oxoclopidogrelic acid been isolated in methanol. Remains of the clopidogrelic acid were

observed in the aqueous phase. This product was analysed by LRMS; the proposal for its

fragmentation is shown in Scheme 5.

Scheme 4 Proposed fragmentation pattern for the protonated clopidogrelic acid found on LRMS

Scheme 3 Proposed fragmentation pattern for the protonated bis-adduct of clopidogrel with NAC found by LRMS for m/z 662

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The 1H NMR (Figure 1) confirmed that the product was obtained as a 2-hydroxylated

species.

Figure 1 2-oxoclopidogrel (2-hidroxithiophene isomer) proton NMR spectrum of the aromatic region

Scheme 5 Proposed fragmentation pattern for the protonated 2-oxoclopidogrelic acid found by LRMS

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An essay with N-acetyl-L-cysteine was performed with this product and MS/MS analysis

showed evidence of adduct formation. A proposal for its MS/MS fragmentation is shown at

Scheme 6.

Materials and Methods

All computational simulations were performed with Gaussian 09, revision B0.1[11] using the hybrid DFT

M06-2X [12], which is applied to organic molecules, and 6-311++G** as base functions (triple zeta on valence

and diffusional functions, besides (d,p) polarization) adding the SCRF=(PCM,SMD,Solvent=Water)

parameter. Correction from electronic energy to Gibbs free energy was done via frequencies’ computation,

which also proved the estimated values are minima.

11 Frisch, M. J . et all, Gaussian, Inc ., Wallingford CT, 2010 . 12 Zhao, Y. & Truhlar, D. G. (2008). The M06 suite of density functionals for main group thermochemistry, thermochemical kinetics, noncovalent interactions, excited states, and transition elements: two new functionals and systematic testing of four M06-class functionals and 12 other functionals, THEOR CHEM ACC, 120, 215-41.

Scheme 6 Proposed fragmentation pattern for the protonated adduct of clopidogrelic acid with NAC found by LRMS

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HPLC-LRMS analyse were performed on a Dionex Ultimate® 3000 HPLC, composed of a HPG3200

binary pump, a WPS300 automatic sampler and a TCC3000 oven coupled in line with LCQ Feet ion trap mass

spectrometer equipped with an ESI ion source (Thermo Fisher Scientific, Inc.).10 µL of sample were injected

by the means of a Rheodyne injector with a 25 µL loop. A Luna C18 100 A (150 mm x 2 mm, 5 µm,

Phenomenex) was used column at 30 ºC and a flow of 0.20 mL/min. The mobile phase was constituted by an

aqueous buffer of 0.1% formic acid (v/v) (A) and acetonitrile (B), with the following gradient: 0-30 min linear

gradient from 5% to 70% B, 30-32 min linear gradient from 70% to 100% B, 32-40 min isocratic 100% B, 40-

45 min 100% to 5% B. The column was reequilibrated during 10 min. The mass spectrometer was operated

on (+)-ESI ionization mode with the following instrumental parameters: ionization spray voltage, +4,5 kV;

transference capillary voltage, 18V; focus lens voltage, -58V, nebulization gas, N2, 80 arbitrary units; drying

gas, N2, 10 arbitrary units, transference capillary temperature, 270ºC. Tandem mass spectrometry MS2 (n=2)

(collisions induced by dissociation with helium) was tracked using an isolating window of 6 Da, collision

energy around 20-30 V and an activation time of 30 ms.

HPLC-HRMS analysis were performed on a Dionex Ultimate® 3000 RSLCnano coupled in line with

QqTOF Impact IITM mass spectrometer equipped with an ESI ion source (Bruker Daltonics. Chromatography

was performed with a Kinetics C18 100 A, LC column 150 mm x 2.1 mm, 1.7 µm, Phenomenex) at 35 ºC and

a flow of 0,150 mL/min. Mobile phase was the same as described for the HPLC-LRMS. The mass spectrometer

was operated on (+)-ESI ionization mode with the following instrumental parameters: ionization spray

voltage, +4,5 kV; offset transference lens, -500 V, nebulization gas, N2, at 2.8 bar; drying gas, N2, at 8 L/min,

200 ºC. Tandem MS2 spectra were recorded by Multiple Reaction Monitoring with a 7 Da isolation window,

with the collisions being induced by N2 with an energy of 25-27 V. TOF calibrations were performed using a

solution of 10 mM sodium formate.

NMR experiments were performed on Bruker Advance II+ at 300 or 400 MHz for the 1H NMR spectra

and 75 or 100 MHz, respectively, for 13C. Spectra were calibrated with the solvent peak.

(+)-Clopidogrel was gently offered by a pharmaceutical corporation.

Generation of 2-oxoclopidogrel’s adduct with N-acetyl-L-cysteine (NAC) in situ. 222.4 mg of

clopidogrel acid bisulphate were dissolved in 4 mL of acetonitrile and 0.4 mL of 1N HCl and 3.8 eq of peracetic

acid were added. After 22 hours of reaction, 30 mL of a saturated solution of NaHCO3 were added and the

oxidation product was extracted to dichloromethane (40 mL, 3x). The organic phase was dried with

magnesium sulphate, which was removed by filtration and the solvent was evaporated to dryness. The

viscous oil was dissolved in 2 mL of N,N-dimethylformamide and 0.5 mL of tetrahydrofuran, and this solution

was added to 7 mL of a 10 mM bicarbonate buffer with 4.2 eq of NAC. Solid-liquid extraction was performed

and both phases were led to dryness.

Exclusively on the aqueous phase was found:

LRMS-ESI(+): m/z 660/662 [M+H]+; MS2 (m/z): 449/451, 336/338, 276/278. tr =23 min

HRMS-ESI(+): m/z 501.0925 [M+H]+; MS2 (m/z): 372.0500, 243.0080, 209.0199, 162.0230.

tr =6.9 min

Exclusively on the organic phase was found:

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HRMS-ESI(+): m/z 354.0556 [M+H]+; MS2 (m/z): 294.0344, 276.0237, 240.0468, 212.0468, 183.0199,

155.0260, 139.02239. tr = 14,0 min

On both phases were found:

HRMS-ESI(+); m/z 338.0609 [M+H]+; MS2 (m/z): 183.0202, 155.0253, 138.0373. tr = 16.3 min

HRMS-ESI(+): m/z 354.0556 [M+H]+; MS2 (m/z): 294.0348, 276.0239, 240.0470, 200.0466, 183.0203,

155.0243, 137.0076. tr = 10,0 min

Synthesis of clopidogrelic acid hydrochloride, 5-[(S)-carboxy(2-chlorophenyl)methyl]-4,5,6,7-

tetra-hydrothieno[3,2-c]piridin-5-ium hydrochloride. 295.9 mg of clopidogrel were dissolved in 6.5 mL

of methanol and 2.5 mL of a solution of NaOH were added. Reaction proceeded for 6 hours in an oil bath at

49 ºC. The solvent was evaporated under reduced pressure and the solution was treated with an ionic

exchange resin (Amberlite IRA-402). The product was precipitated over diethyl ether as hydrochloride salt

(63% yield).

1H RMN (δ, DMSO-d6, 400 MHz): 7.64 (d*, 1H, 3J=7.2 Hz, Hphenyl), 7.48 (d*, 1H, 3J=7,6 Hz, Hphenyl),

7.36 (qi*, 2H, Hphenyl), 7.26 (d, 1H, 3J= 4.8 Hz, Htiof), 6.76 (d, 1H, 3J= 4.8 Hz, Htiof), 4.71 (s, 1H, O2CCH), 3.64

(qa*, 2H, CCHHN), 2.74-2.90 (m, 4H, SCCH2H2)

13C-CPD RMN (δ, MeOD-d4, 400 MHz): 177.6 (CO2H), 143.9 (Cphenyl), 141.4 (Cphenyl), 141.1 (Cphenyl)

140.0 (Cphenyl), 139.5 (Cphenyl), 138.3 (Cfus), 137.9 (Cfus), 137.8 (Cphenyl), 135.0 (Ctiof), 134.5 (Ctiof), 75.3 (2x CH2N),

31.7 (CH2CH2N)

LRMS-ESI(+): m/z 308/310 [M+H]+; MS2 (m/z): 198/200, 172/174, 152/154. tr: 14 min

p.f. = 226-230 ºC.

max = 239.8 nm. tr: 19.8 min

Rf=0.66 (gel de sílica, acetona)

Amberlite IRA-402, a strongly basic anionic exchange resin, was used to isolate clopidogrelic acid

(hydrochloride form). Storage solution was replaced with the aqueous solution of the product and was let to

incubate for an hour. The mixture was filtred at vacuum and the resin was washed with water (20 mL, 3x). 20

mL of methanol and 1 mL of 12M HCl were added for an hour to yield the product in the solution. The liquid

was removed and the resin was rinsed with 20 mL of methanol, which were added to the product solution,

and the washed with water (20mL, 5x). Regeneration was done with a 1M NaOH solution overnight, and after

the resin was washed with water (20 mL, 10x), 10 mL were added to the resin to store it.

Synthesis of 2-oxoclopidogrelic acid, (S)-(2-chlorophenyl)(2-oxo-2,6,7,7a-

tetrahydrothieno[3,2-c]pyridin-5(4H)-yl)ethanoic acid. 54.5 mg of the synthesized hydrochloride of

clopidogrelic acid in 1mL of acetic acid, 1 mL of acetonitrile and 0,5 mL of N,N-dimethylformamide. 5.0 eq

of peracetic acid were added and the mixture was let to react for 2 hours. At this point, 139.2 mg of KI were

added and the mixture was evaporated to dryness. 10 mL of water were added and the produced iodine was

extracted to n-hexane (10 mL, 4x). The aqueous phase was concentrated and submitted to an OASIS MAX

CARTRIDGE column, yielding the product in methanol.

1H RMN (δ, Me2CO-d6, 300 MHz): 7.76 (m, 1H, Hphenyl), 7.52 (m, 1H, Hphenyl), 7.42 (m, 2H, Hphenyl),

6.74 (s, 1H, SCOHCHC), 5.04 (s, 1H, O2CCH), 3.78 (dd, 2H, CCH2N), 3.10 (m, 2H, SCCH2H2), 2.86 (m, 2H,

SCCH2H2)

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13C-APT RMN (δ, Me2CO-d6, 300 MHz): 130.3 (Cphenyl), 129.9 (Cphenyl), 129.8 (Cphenyl), 127.3 (Ctiof),

124.9 (Ctiof), 67.4 (O2CCH), 49.7 (CH2N), 47.9 (CH2N), 24.7 (CH2CH2N)

LRMS-ESI(+): m/z 324/326 [M+H]+; MS2 (m/z): 214/216, 169/171. tr :19.5 min

OASIS column was charge with: methanol, milliQ water, sample, milliQ water, methanol/milliQ

water (1:1) and methanol.

Generation of an adduct of 2-oxoclopidogrelic acid with NAC. The synthetized 2-oxoclopidogrelic

acid was dissolved in 1 mL of a 10 mM bicarbonate buffer at pH 6 and 14.4 mg of NAC were added. The

reaction proceeded for 4 hours.

LRMS-ESI(+): m/z 487/480 [M+H]+; MS2 (m/z): 469/471, 443/445, 441/443, 358/360, 340, 272, 230,

198/200. tr =12-14 min

Conclusions

Though the oxidation of clopidogrel with peracetic acid was possible, this strategy lacked

optimization, which could improve the results. Also, the absence of reliable methods of oxidation

of clopidogrel had hampered this investigation.

Two major clopidogrel metabolites, clopidogrelic acid and 2-oxoclopidogrelic acid, were

isolated and characterized by NMR and HPLC-LRMS.

2-oxoclopidogrel and 2-oxoclopidogrelic acid adducts with NAC were found by mass

spectrometry suggesting that these metabolites can add soft nucleophiles by 1,4-addition.

However, attempts to generate any adducts of 2-oxoclopidogrel with were not successful. This

suggests that excretion is carried by other entities rather than glutathione, like smaller thiols or

even water.

Adducts with nitrogen nucleophiles were also not found, suggesting clopidogrel has a low

toxicity potential via covalent adduct formation.