English

• 1. INTRODUCTION

  In the pharmaceutical industry, the biocompatibility of drugs is more important than the toxicity or efficacy of drugs, and the solubility is a key issue to be solved^[1-5]. In the preparation of drugs, it is often necessary to give up some drug production because of poor solubility and the like^[6]. In the short term, the problems of solubility and bioavailability have become a bottleneck that severely restricts the scope of drug application. Therefore, changing the solubility of raw materials and improving the bioavailability and efficacy of drugs have become an important driving force and support for the research and development of new drugs^{[7, 8]}.

  As a new drug solid form, multidrug cocrystals (MDCs) have been the subject of growing interest in the pharmaceutical industry^[9-15]. The drug forms a drug eutectic by introducing CCF, which can safely regulate and improve the solubility, stability and bioavailability^[16-21]. It can achieve the physicochemical properties of modified active pharmaceutical ingredients without destroying the properties of the drug itself^{[22, 23]}. Therefore, it has a good development prospect in the pharmaceutical industry and is one of the research hotspots in crystal engineering.

  In view of the research status of drug eutectic at home and abroad, we select the antimalarial drug-artesunate as API and 4, 4΄-bipyridine as CCF to improve the solubility and bio-availability of artesunate. A cocrystal of artesunate and 4, 4΄-bipyridine was successfully obtained by solvent evaporation. The chemical structures of artesunate and 4, 4΄-bipyridine are shown in Scheme 1.

  Scheme 1

  

  Scheme 1.  Molecular structures of artesunate and 4, 4΄-bipyridine

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  2. EXPERIMENTAL

  2.1 Materials and physical measurements

  All analytical reagent grade chemicals were obtained from commercial sources and used without further purification. Artesunate (76.89 mg, 0.2 mmol) and 4, 4΄-bipyridine (31.24 mg, 0.2 mmol) were placed in a 25 mL glass vial, and 15 mL of diethyl ether was added followed by stirring at room temperature for 24 hours. After reaction, it was sealed with a plastic wrap and placed in a room for several days to obtain a colorless crystal. Yield: 65.3%. Elemental analysis for C₄₈H₆₄N₂O₁₆ (%), Found: C, 62.32; H, 6.97; N, 3.03. Calcd. (%): C, 62.31; H, 6.95; N, 3.02. IR (KBr plate, cm^–1): 2940m, 1746s, 1597s, 1407m, 1315w, 1164m, 1005s.

  IR spectra were recorded on a Perkin Elmer Spectrum one FT-IR spectrometer with KBr pellets from 3200 to 750 cm^–1. Powder X-ray diffraction (XRD) intensities were measured on a Rigaku D/max diffractometer (CuKα, λ = 1.54056 Å). The single crystalline powder samples were prepared by crushing the crystals and scanned from 2° to 50° with a step of 5 °·min^–1. Thermogravimetric analyses (TGA) were performed using a Perkin-Elmer thermal analyzer from 30 to 1000 ℃ under dry N₂ at a heating rate of 5 ℃·min^-1. All nonhydrogen atoms were refined anisotropically, and hydrogen atoms were refined isotropically. The hydrogen atoms were placed at the calculated positions.

  2.2 X-ray crystallographic determination

  X-ray single-crystal diffraction data for the cocrystal were collected with a Bruker APEX-II CCD instrument using graphite-monochromated MoKα radiation (λ = 0.71073 Å). These data were obtained using SAINT software and an absorption correction was applied with SADABS to the collected reflections^[24]. Structure determination and refinement were performed with XS and XL (with OLEX2 graphical user interface) programs, respectively^{[25, 26]}. Crystal data for the title compound: orthorhombic system, space group P2₁2₁2₁ with a = 10.5075(15), b = 15.207(2), c = 29.899(4) Å, M_r = 925.01, V = 4777.5(11) ų, D_c = 1.286 g⋅cm^–3, Z = 4, S = 1.003, the final R = 0.0456, wR = 0.0866, (Δ/σ)_max = 0.001, (Δρ)_max = 0.170 and (Δρ)_min = –0.149 e/ų.

  2.3 Biological activity test

  The antimicrobial activities of 4, 4΄-bipyridine and cocrystal were qualitatively assessed based on standard methods by the agar dilution method^[27-30]. The 4, 4΄-bipyridine and cocrystal were dissolved in dimethylsulfoxide (DMSO) and diluted to prepare different concentrations.

  3. RESULTS AND DISCUSSION

  3.1 Description of the crystal structures

  The single-crystal X-ray analysis confirmed that cocrystal crystallizes in orthorhombic space group P2₁2₁2₁, and the asymmetric unit contains two artesunate and one 4, 4΄-bipyridine molecules. Artesunate molecule in this cocrystal contains a C–O–O–C dihedral angle of 43.81(3)° (C···O 1.46(4) and 1.41(4) Å, O–O 1.48(4) Å). It should be noted that neither π-π stacking nor significant C–H⋅⋅⋅π contacts between the bipyridine molecules are evident in the crystal packing of this cocrystal (Fig. 1). The intermolecular packing is further controlled by two important hydrogen bonds O(8)–H(8)⋅⋅⋅N(2A) and O(16)–H(16)···N(1B) (symmetry codes: A: 2 + x, y, z; B: –1/2 + x, 1/2 – y, 1 – z) between the bipyridine nitrogen and carboxyl oxygen atoms, with the O···N distances of 2.78(5), 2.59(6) Å and O–H···N bond angles of 157(5) and 176(5)°, respectively.

  Figure 1

  

  Figure 1.  Hydrogen bond stacking diagram

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  3.2 IR analysis

  To further confirm the cocrystal structures, IR spectral data for artesunate, 4, 4΄-bipyridine and cocrystal in the range of 3200~750 cm^–1, where the C=O and C=N stretching vibration is presented, are examined in detail for relevant chemical information on these solid forms. The formation of hydrogen bonds leads to the association of carboxyl groups in cocrystals. As shown in Fig. 2(a), the absorbance peak at 2940 cm^–1 due to the formation of hydrogen bonds leads to the stretching vibrations of associated O–H in cocrystal. The absorbance peaks at 1755 and 1746 cm^–1 are assigned to the stretching vibration of carbonyl groups in artesunate and cocrystal respectively because of the hydrogen bonds, leading to a decrease in the frequency of carbonyl groups. The absorbance peaks at 1595 and 1597 cm^–1 result from the stretching vibration of C=N in 4, 4΄-bipyridine and cocrystal, respectively. Therefore, IR data confirm the formation of cocrystals.

  Figure 2

  

  Figure 2.  (a) IR curves of artesunate, 4, 4΄-bipyridine and cocrystal, (b) Powder X-ray pattern of artesunate, 4, 4΄-bipyridine, cocrystal and the simulated cocrystal

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  3.3 PXRD analyses

  Fig. 2(b) shows the XRD spectra of the artesunate drug substance, 4, 4΄-bipyridyl, and artesunate-4, 4΄-bipyridine cocrystal. The XRD peaks of the three curves in the figure are quite different, which proves that a new phase is formed, that is, the prepared artesunate-4, 4΄-bipyridine eutectic structure. In addition, the PXRD patterns of the crystal forms were compared with those simulated from the single-crystal structures. The experimental PXRD patterns of crystal exhibit good agreement with the simulated one, indicating that the crystal forms have been successfully obtained as a pure crystalline phase. PXRD patterns of cocrystal are the same as those simulated from the single-crystal structure determination, indicating high purity of the products.

  3.4 TGA analyses

  TGA analysis was performed in a N₂ atmosphere on samples of cocrystal, artesunate and 4, 4΄-bipyridine, and the resulting curves are shown in Fig. 3. Further examination of their thermal properties by TGA revealed that cocrystals exhibit different thermal behaviors from single artesunate or 4, 4΄-bipyridine. As shown in Fig. 3, weight loss of 4, 4΄-bipyridine was observed in the temperature range of 100~235 ℃. The first weight loss of artesunate and cocrystal was observed in the temperature ranges of 158~250 and 124~250 ℃, respectively. The second weight loss of artesunate and cocrystal appeared from 250 to 449 ℃. The TGA of cocrystals shows that the formation of hydrogen bonds has an important influence on the thermal decomposition process of cocrystal.

  Figure 3

  

  Figure 3.  TGA curves of original artesunate, 4, 4΄-bipyridine and cocrystal

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  3.5 Antimicrobial activities

  The minimum concentration of antibacterial was evaluated by evaluating the activity of 4΄4-bipyridine and cocrystal on different microorganisms in DMSO solvent using an agar dilution method. The growth of bacteria was examined after incubation at 37 ℃ for 24 h. Gram-negative bacteria Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa and Shigella sp., Gram-positive bacteria Staphylococcus aureus were chosen as model microorganisms, and a series of experiments were implemented.

  It has been reported that artesunate had no antibacterial activity itself^{[31, 32]}. The results showed (Table 1) that 4΄4-bipyridine only showed very little inhibition on Shigella Sp. The minimum inhibitory concentration is 8.58 mg·mL^–1. Cocrystal showed effective inhibition on E. coli, S. aureus, S. enterica, Shigella sp. and P. aeruginosa. Among them, the inhibitory effects on S. enterica and P. aeruginosa were the most obvious. The minimum inhibitory concentration is 4.29 mg·mL^–1. From the observed results, artesunate combined with 4΄, 4-bipyridine against E. coli, S. aureus, S. enterica, Shigella sp. and P. aeruginosa has a certain antibacterial effect. The results show that the synthesis of cocrystal greatly increases the antibacterial properties of artesunate.

  Table 1

  

  Table 1.  Minimum Inhibitory Concentration (mg·mL^–1) of 4΄4-Bipyridine and Cocrystal

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  Compound	S. aureus	S. enterica	E. coli	Shigella sp.	P. aeruginosa
  4΄4-Bipyridine	+	+	+	8.58	+
  Co-crystal	17.16	4.29	8.58	8.58	4.29
  Note: " + "means it has bacterial growth

  4. CONCLUSION

  In this study, artesunate was selected as API and 4, 4΄-bipyridine as CCF. A drug eutectic was successfully designed and synthesized. The results show that the formation of eutectic is mainly caused by weak hydrogen bonding interaction. Its structure was characterized by the formation of O–H···N hydrogen bonds and the asymmetric unit contains two artesunate and one 4, 4΄-bipyridine molecules. The cocrystal is slightly soluble in water. The biological activity test indicated that the cocrystal possessed enhanced antibacterial activities to a few bacteria over the CCF and API. We continue to accumulate experience in the design and synthesis of drug eutectic, providing a reference for the synthesis of more structural and superior drug eutectics in the future, and providing a basis for further bioavailability research. We also expect that this material may open a new way to the fabrication of advanced antibacterial materials.

  
  
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• 

  Scheme 1  Molecular structures of artesunate and 4, 4΄-bipyridine

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  Figure 1  Hydrogen bond stacking diagram

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  Figure 2  (a) IR curves of artesunate, 4, 4΄-bipyridine and cocrystal, (b) Powder X-ray pattern of artesunate, 4, 4΄-bipyridine, cocrystal and the simulated cocrystal

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  Figure 3  TGA curves of original artesunate, 4, 4΄-bipyridine and cocrystal

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  Table 1.  Minimum Inhibitory Concentration (mg·mL^–1) of 4΄4-Bipyridine and Cocrystal

  Compound	S. aureus	S. enterica	E. coli	Shigella sp.	P. aeruginosa
  4΄4-Bipyridine	+	+	+	8.58	+
  Co-crystal	17.16	4.29	8.58	8.58	4.29
  Note: " + "means it has bacterial growth

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•