Efficient Synthesis of (S)-(+)-Clopidogrel Bisulfate-Capped Silver Nanoparticles

In this work primarily one-pot synthetic development in the preparation of clopidogrel bisulfate with a polymorphic crystalline form II in 90% yield was developed. This premade antiplatelet drug has been used to protect starch-stabilized silver nanoparticles (AgNPs).


Introduction
(S)-(C)-Clopidogrel bisulfate 1 (CLP) (Figure 1) is a thienopyridine derivative as a potent oral antiplatelet agent often used in the treatment of coronary artery, peripheral vascular and cerebrovascular diseases. [1] This drug is one of the biggest selling drugs in the world. It can crystallize in various polymorphic crystalline forms and amorphous forms, but only forms I and II are used in pharmaceutical products. [2,3] Nanoparticles (NPs) are promising as talented candidates for various biomedical applications such as enhanced resolution magnetic resonance imaging (MRI), drug delivery, tissue repair, and cell and tissue targeting and transfection. [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] Silver NPs (AgNPs) are a significant class of nanomaterial for a wide range of industrial and biomedical applications. The unique chemical properties of AgNPs make it a promising targeted delivery approach for drugs or gene specific cells. AgNPs also effectively inhibited integrin-mediated platelet functional responses such as aggregation, secretion, adhesion to immobilized fibrinogen or collagen, and retraction of fibrin clot in a dose-dependent manner, irrespective of the nature of agonists used. [21] With increase use of AgNPs as drug carriers and their accepted antiplatelet property, it would be interesting to investigate its interaction with CLP. Here, synthetic improvement in the preparation of CLP also is described.

Material and Measurements
AgNO 3 (99.9%), paraformaldehyde (PFA; 99%), L-camphorsulfonic acid (L-CSA), soluble starch, and NaBH 4 (99%) were purchased from Merck. The FT-IR spectra for the samples were obtained using Shimadzu FT-IR-8900 spectrophotometer by using KBr pellets. All NMR data were recorded in DMSO-d 6 using Bruker Avance 500 MHz spectrometer. Chemical shifts are reported in ppm (d) using deuterated solvents as internal references. Elemental analyses were made by a Carlo-Erba EA1110 CNNO-S analyzer and agreed with the calculated values. UV-Vis absorption spectra in the range 200-500 nm in EtOH were measured with a Shimadzu UV-2100 spectrophotometer. Melting points are uncorrected and determined by electrothermal 9100 melting point apparatus. The X-ray diffraction (XRD) patterns were recorded in a wide-angle range (2u D 10-70) by Phillips (pw-1840) Xray diffractometer with Cu-Ka radiation. The morphology and particle sizes of synthesized powder were characterized by transmission electron microscope (TEM) images on a Phillips CM-10 instrument with an accelerating voltage of 100 kV.
One-Pot Procedure for the Synthesis of Racemic Clopidogrel Bisulfate (2) To a vessel equipped with Dean-Stark assembly, 2-(thiophen-2-yl)ethanamine 5 (0.48 g, 4 mmol), PFA (0.13 g, 4.3 mmol), and toluene (6 mL) were added and the reaction mixture was refluxed for 2 h to obtain a yellow solution contain imine. After cooling to 20 C, a solution of 6N HCl (0.7 mL) in DMF was added into the reaction mixture and continued to heat at 50 C for 1 h to produce 4 as a white precipitate. The reaction was cooled to 25 C and aqueous solution of 10% Na 2 CO 3 was added and stirred for 30 min. Then a solution of methyl-2-bromo-2-(2-chlorophenyl)acetate 3 (1.04 g, 4 mmol) in toluene (7 mL) was added to the reaction mixture. The reaction mixture stirred at room temperature for 12 h. The aqueous layer is discarded and the organic layer was washed with water. The organic phase was evaporated, to afford viscose oil. To this oil, conc. H 2 SO 4 (0.42 mL) and EtOAc (8 mL) was added. The mixture was stirred for 1 h. The precipitated crystals filtered off and washed with cool acetone. The pure racemic clopidogrel bisulfate 2 was dried in oven at 50 C (1.35 g, 90% yield). White powder; mp 220-222 C; IR: v (KBr, cm -1 ) 3434 (OH stretch), 3100, 2989, 2953, 1755 (C D O stretch), 1239, 1222 (C-O stretch), 1063.

Preparation of (S)-(C)-Clopidogrel Camphorsulfonate Salt
The racemic clopidogrel bisulfate 2 (1.5 g, 3.75 mmol) and L-CSA (0.88 g, 3.75 mmol) were dissolved in acetone and stirred at room temperature for 12 h. The formed precipitate was filtered, washed and dried to give a white solid as a (S)clopidogrel camphor sulfonate salt (0.96 g, 75% yield),

Preparation of (S)-(C)-Clopidogrel Bisulfate-Coated Silver Nanoparticles (Ag@CLP)
A solution of AgNO 3 (0.082 g, 0.48 mmol) in ultrapure H 2 O (10 mL) was prepared. To this solution were added 0.03 g soluble starch in 5.0 mL ultrapure H 2 O and then the mixture was stirred heavily for 30 min in an ice bath. A solution of NaBH 4 (0.018 g, 0.48 mmol) in ultrapure H 2 O (6 mL) was added dropwise to the aqueous solution, and the color of solution suddenly turned to bright yellow, attributed to the formation of AgNPs. Then, the color of solution turned to black. On complete addition of NaBH 4 , the resulting mixture was further stirred for 30 min at room temperature. Then, a solution of CLP (0.403 g, 0.96 mmol) in EtOH (10 mL) was added to the reaction vessel, and then the mixture was stirred for further 4 h at this stage the color of solution was turned to dark brown. The suspension obtained was then centrifuged at 10,000 rpm for 15 min and the precipitate washed three times with double distilled H 2 O to remove any water-soluble impurity. After that, the precipitate was washed 3 times by Synthesis of (S)-(C)-Clopidogrel Bisulfate-@Ag dispersion and centrifugation using EtOH to remove excess CLP and excess reducing agent. The precipitate was then dried in an oven at 60 C for 10 h and the pale gray powder was obtained.
The mp, IR, 1 H NMR, 13 C NMR ( Figures S1 and S2), XRD, [a] D , and elemental analysis confirm that a polymorphic crystalline form II of 1 was produced. [3] The synthesis of tetrahydrothieno pyridine 4 was optimized and the results are shown in Table 1. When this reaction was carried out in the presence of DMF.HCl and toluene, the yield of 4 was increased up to 90% (Table 1, entry 2).
In other efforts, we examined several other bases and solvents to optimize the reaction conditions for preparation of 1. The results are listed in Table 2. It was found that using toluene along with Na 2 CO 3 led to higher yields in shorter reaction times ( Table 2, entry 5).
The kinetic resolution of prepared racemic clopidogrel bisulfate 1 was completed by using L-CSA. An optimization of the reaction parameter such as utilized L-CSA (in mmol), solvent and yield with respect to the final product concentration were performed. It was found that using 0.012 mmol L-CSA in acetone led to higher yields (Table 3, entry 3). Subsequently, the obtained (S)-clopidogrel camphor sulfonate salt, was converted to (S)-(C)-clopidogrel bisulfate 1 in H 2 SO 4 / EtOAc condition (Table 4, entry 5).
For preparation of Ag@CLP, solution of AgNO 3 in ultrapure H 2 O and starch as a capping agent under vigorous stirring was added to the round bottom flask. Subsequently, a modified Brust reaction [33] was carried out by addition of excess amount of NaBH 4 as reducing agent. To reduce all of the Ag C ions to metallic silver, the molar ratio of metal to the reducing agent was selected as 1:2 respectively. Then, EtOH solution of CLP (molar ratio CLP/Ag D 2:1) was added to the reaction vessel. The sulfur of CLP bind strongly to AgNPs surface by self-assembly. [25,34,35] Formation of Ag@CLP was monitored by IR, X-ray diffraction, UV-Vis, and TEM. To achieve a fine nanosized particle the amylose was used as a green capping agent. [36] The interaction between CLP and AgNPs was recognized by IR spectra of CLP and Ag@CLP ( Figures S3 and S4). In both spectra the C D O, O-H of the HSO 4 -, N C -H, and aromatic C-H stretching vibration bands at 1753, 3435, 2552, and 3121 cm -1 were observed, respectively. The C D C sym and C D C asym bands of thiophene for CLP and Ag@CLP were observed at 1439, 1591 cm -1 and 1444, 1590 cm -1 , respectively, indicating that sulfur is attached to the AgNPs surface. [34]    Mahmoodi et al.
The powder XRD patterns of the CLP and Ag@CLP are shown in Figure 2. The peaks position of the CLP were observed at 2u D 8.87 , 13.04 , 17.76 , 18.59 , 20.24 , 21.69 , 23.04 , 23.86 , 24.79 , 26.61 , 27.72 , 29.44 , 34.30 , 35.76 , 41.15 , and 48.68 (Figure 2a). This pattern correlated well with the crystalline structure of form II. 3 In Figure 2b, the characteristic peaks of AgNPs were at 2u D 38.24 , 44.43 , and 64.56 , respectively, assigned as (111), (200), and (220) reflection lines of the face centered cubic (fcc) structure of metallic silver. The average crystallite size, D, was calculated from Scherrer's equation to be 29 nm while D D Kl/(bcosu) and λ is the wavelength of Cu-Ka radiation used (λ D 1.54 A ), b is the full width at half-maximum intensity (0.31) of the diffraction line, u is the Bragg angle for the measured hkl peak, and K is a constant equal to 0.94. This value is in good agreement with the TEM image. The intensity of peaks reflected the high degree of crystallinity of the AgNPs. Figure 3 illustrates the UV-Vis absorption spectra of CLP and Ag@CLP in EtOH solution. The pure CLP, shows a λ max at 243 nm corresponds to the p-p* transition of the chlorobenzene and thiophene moiety, while solution of Ag@CLP shows absorption peaks at 243 and 412 nm corresponds to CLP and surface plasmon resonance absorption band (SPRAB) of AgNPs, respectively. The weaker absorption band of Ag@CLP, compared to absorption band of free CLP at 243 nm, confirms a lower concentration of CLP on AgNPs. The appearance of two new peaks at 297 and 346 nm probably are related to the formation of Ag-CLP complex.

TEM Imaging
The TEM of Ag@CLP image reveals that particles are spherical shape with approximate size of <40 nm; Scherrer's calc. D 29 nm (Figure 4).

Conclusions
We developed one-pot process for the synthesis of polymorphic crystalline form II CLP and utilizing a novel   Synthesis of (S)-(C)-Clopidogrel Bisulfate-@Ag method for adsorption of premade CLP molecule on AgNPs surface. The sulfur of CLP binds strongly to AgNPs surface by self-assembly as confirmed by IR and UV-Vis. This Ag@CLP system may provide an advantage model system for the development of new effective antiplatelet drug.

Funding
The authors are grateful to the Research Council of University of Guilan & Iranian Nanotechnology Initiative Council (INIC) for the financial support of this research work.