Synthesis and Pharmacokinetic Study of Three Gemfibrozil Salts: An

Article pubs.acs.org/crystal

Synthesis and Pharmacokinetic Study of Three Gemfibrozil Salts: An Exploration of the Structure−Property Relationship Qiuhong Yang,† Tianming Ren,‡ Song Yang,† Xiaoqin Li,† Yingnan Chi,*,† Yan Yang,‡ Jingkai Gu,*,‡ and Changwen Hu*,† †

Key Laboratory of Cluster Science, Ministry of Education of China, Beijing Key Laboratory of Photoelectronic/Electrophotonic, School of Chemistry, Beijing Institute of Technology, Beijing 100081, P. R. China ‡ Research Center for Drug Metabolism, Jilin University, Changchun, 130012, P. R. China S Supporting Information *

ABSTRACT: Three salts, [H3N(CH2)2NH3)][gem]2 (1), [H3N(CH2)3NH3)][gem]2· 2H2O (2), and [H3N(CH2)4NH3)][gem]2·2H2O (3) of the minimally soluble drug gmfibrozil (Hgem), used for the treatment of hyperlipidemia have been synthesized by using a series of diamine with different carbon chain lengths and characterized by single crystal/powder X-ray diffraction, Fourier transform infrared spectroscopy, and 1H nuclear magnetic resonance. In the three salts, two protons of two gmfibrozil molecules transfer to one diamine, and the resulting organic diammonium cation and gmfibrozil anion are assembled by hydrogen bond interactions into a two-dimensional layer. Although the apparent solubility of salts 1−3 is obviously improved compared to that of the original gemfibrozil, pharmacokinetic studies in rats indicate the enhancement of absorption is limited with the relative bioavailability of 104% for 1, 154% for 2, and 108% for 3. It is notable that the rapid dissolution behavior of salt 1−3 leads to the increase of maximal plasma concentration (Cmax) and the dramatic shortening of the time required to reach the Cmax. The investigation of the structure−property relationship shows that there is little correlation of solubility with the carbon chain length of cation which is different from previous observations, and we speculate that both electrostatic attraction and hydrogen bond interaction contribute to the solubility order (2 > 1 > 3) .

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INTRODUCTION Gemfibrozil (in Scheme 1) belongs to the class of fibric acid derivative and is mainly used in the treatment of hyper-

physicochemical properties of an active pharmaceutical ingredient (API).18−22 Here the “solid state form” includes polymorphs, hydrates (or solvates), salts, cocrystals, or amorphous forms. A lot of investigations indicate that the stability,23 solubility,24 hygroscopicity,25,26 and bioavailability27 of an API can be significantly influenced by its solid state. As salt formation is operationally simple and practically effective, it becomes the most preferred method to increase the solubility and dissolution rate of acidic or basic drugs.28−30 In general, the salt forming agents are chosen empirically, and the suitable salt forms are screened out according to their solubility, stability, and bioavailibility with the consideration of cost and toxicity. Although several studies mentioned that properties of pharmaceutical salts varied with salt forming counterions, no predictive relationship between counterion characteristics and salt solubilities has yet been established.31−38 For example, four diclofenac salts using a series of structurally similar primary amines as counter-cations have been synthesized on purpose by O’Connor and Corrigan, but no dependence of solubility on any one parameter was observed.36 In this context, the investigation of the structure−property relationship is very desirable for the development of pharmaceutical salts.

Scheme 1. Molecular Structure of Gemfibrozil

lipidemia.1,2 It was first synthesized in 19723 and screened out as a new lipid-lowering drug by Creger in 19764 and subsequently marketed in the form of gemfibrozil in 1982.5 Clinic studies show that gemfibrozil can prevent cardiovascular events by increasing HDL cholesterol.6 According to the Biopharmaceutical Classification System (BCS), gemfibrozil is a class II drug with low solubility and high permeability.7 The poor aqueous solubility of gemfibrozil (0.03 mg/mL in the pH range of 1.0−5.58 results in its unsatisfactory absorption after oral administration.9 Therefore, to enhance the dissolving behavior, several formulation techniques including solid encapsulation,10 dispersion,11−14 and microemulsion15−17 have been developed. Besides the poor aqueous solubility, another drawback of this drug is the low melting point (61 °C), which is unfavorable for transportation and storage. Currently selecting a suitable solid state form has been proven to be a successful approach to optimize the © XXXX American Chemical Society

Received: July 25, 2016 Revised: September 12, 2016

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DOI: 10.1021/acs.cgd.6b01100 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

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Gemfibrozil as an acidic drug (pKa = 4.7)39 can easily form salt with alkaline compounds. Until now the butylamine, hexylamine, octylamine, benzylamine, cyclohexylamine, tertbutylamine, hydroxy derivatives of t-butylamine, and choline salts of gemfibrozil were prepared,40−43 but no single crystal or pharmacokinetic study was reported in the literature or patent. The unambiguous structural characterization is important for a drug, which helps us to understand the structure−property relationship. In our investigation, three organic diamines, ethylenediamine, 1,3-diaminopropane, and 1,4-butanediamine, having different carbon chain lengths were used as salt forming agents, and as a result three structurally related gemfibrozil salts, namely, [H 3 N(CH 2 ) 2 NH 3 )][gem] 2 (1), [H 3 N(CH2)3NH3)][gem]2·2H2O (2), and [H3N(CH2)4NH3)][gem]2·2H2O (3) (Hgem = gemfibrozil), were synthesized and thoroughly characterized by single-crystal/powder X-ray diffraction, Fourier transform infrared (FTIR) spectroscopy, 1H NMR, thermogravimetry (TG), and differential scanning calorimetry (DSC). All of these salts are more soluble than API, and salt 2 exhibits an improved pharmacokinetic profile. Moreover, the impact of counterions on melting point and aqueous solubility was discussed.

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(6H, s, phenmethyl), 2.07 (6H, s, phenmethyl), 1.62−1.69 (6H, m, methylene), 1.51−1.55 (4H, m, methylene), 1.05 (12H, s, methyl) (Figure S1). Elemental analysis Calc. for C33H58N2O8: C, 64.92; H, 9.51; N, 4.59%; found: C, 65.15; H, 9.79; N, 4.83%. Synthesis of [H3N(CH2)4NH3)][gem]2·2H2O (3). When using a solution of 1,4-butanediamine (10.0 mL, 0.1 M), we prepared the colorless block single crystals of 3. Yield: 74% based on gemfibrozil. 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 6.97 (2H, d, J = 8.0 Hz, phenyl), 6.69 (2H, s, phenyl), 6.61 (2H, d, J = 8.0 Hz, phenyl), 4.21 (12H, s, ammonium), 3.88 (4H, t, J = 6.0 Hz, methylene), 2.64 (4H, s, methylene), 2.24 (6H, s, phenmethyl), 1.62−1.70 (4H, m, methylene), 1.51−1.56 (4H, m, methylene), 1.46−1.49 (4H, m, methylene), 1.05 (12H, s, methyl) (Figure S1). Elemental analysis Calc. for C34H60N2O8: C, 65.39; H, 9.62; N, 4.49%; found: C, 67.78; H, 9.84; N, 4.92%. Single Crystal X-ray Diffraction. Single crystal X-ray data of three salts were collected at 298 K on a Bruker-AXS CCD diffractometer equipped with a graphite-monochromated Mo Kα radiation (λ = 0.71073 Å). The SADABS multiscan absorption correction was applied, and the structures were solved by direct methods using the SHELXTL-97 program.44 All the non-hydrogen atoms were anisotropically refined. H atoms were placed in the calculated positions and refined isotropically except for those on the water molecules that were located from the difference Fourier maps. The crystallographic data and refinements parameters are summarized in Table 1, and the selected hydrogen bonding distances and angles as well as bond lengths and bond angles are listed in Table 2 and Table S1, respectively.

EXPERIMENTAL SECTION

Materials and General Methods. All the chemical reagents were purchased from commercial sources and used as received. The FT-IR spectra were measured on a Nicolet 170SXFT/IR spectrometer using KBr pressed pellets. Elemental analyses of C, H, and N were performed on a EUROVECTER EA 3000 CHN elemental analyzer. Bulk samples were characterized by powder X-ray diffraction (PXRD) on a Bruker D8 advance powder diffractometer. Experimental conditions: graphite-monochromatized Cu Kα radiation (λ = 0.154060 nm), 2θ: 5−35°, the scan speed: 10°/min and stepsize: 0.019762. Thermogravimetry (TG) data were collected on a Q50 TGA simultaneous thermal analyzer at nitrogen atmosphere in the temperature range of 30 and 300 °C with a heating rate of 10 °C/min. DSC measurement was conducted on a Q100 DSC module from 30 to 300 °C (heating rate: 10 °C/min and nitrogen flow rate: 50 mL· min−1). The heating and cooling DSC experiment of salt 3 was performed in the temperature range of −20 to 120 °C at a heating/ cooling rate of 10 °C/min, and the sample after the heating and cooling DSC measurement was collected and used for PXRD and FTIR characterization. 1H NMR spectra were obtained on a Bruker-400 NMR spectrometer using DMSO-d6 as solvent. Synthesis of [H3N(CH2)2NH3)][gem]2 (1). An aqueous ethylenediamine solution (8 mL, 0.1 M) was mixed with gemfibrozil (50 mg, 0.20 mmol), and the mixture was stirred at room temperature for 2 h. Then 10 mL of ethanol was added, and the solution was stirred for an additional 0.5 h. The resulting solution was filtered and evaporated under ambient conditions. After about 6 days, we harvested the colorless needle-like single crystals of 1. Yield: 86% based on gemfibrozil. 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 6.97 (2H, d, J = 7.2 Hz, phenyl), 6.69 (2H, s, phenyl), 6.61 (2H, d, J = 7.2 Hz, phenyl), 4.93 (6H, s, ammonium), 3.88 (4H, t, J = 6.0 Hz, methylene), 2.70 (4H, s, methylene), 2.24 (6H, s, phenmethyl), 2.08 (6H, s, phenmethyl), 1.63−1.70 (4H, m, methylene), 1.53−1.57 (4H, m, methylene), 1.07 (12H, s, methyl) (Figure S1). Elemental analysis Calc. for C32H52N2O6: C, 68.57; H, 9.29; N, 5.00%; found: C, 68.63; H, 9.29; N, 5.21%. Synthesis of [H3N(CH2)3NH3)][gem]2·2H2O (2). When a 1,3diaminopropane solution (10.0 mL, 0.1 M) was used instead of ethylenediamine, salt 2 was obtained using a similar procedure. After 8 days of evaporation, colorless cubic block single crystals of 2 were generated. Yield: 80% based on gemfibrozil. 1H NMR (DMSO-d6, 400 MHz) δ (ppm) 6.96 (2H, d, J = 7.2 Hz, phenyl), 6.63 (2H, s, phenyl), 6.60 (2H, d, J = 7.2 Hz, phenyl), 4.45 (10H, s, ammonium), 3.87 (4H, t, J = 6.4 Hz, methylene), 2.75 (4H, t, J = 6.4 Hz, methylene), 2.23

Table 1. Crystallographic Parameters for Gemfibrozil Salts formula formula weight crystal system space group a (Å) b (Å) c (Å) α (deg) β (deg) γ (deg) Z volume (Å3) Dcalc (g cm−3) F(000) refns collected unique reflns Rint R1 [I > 2σ(I)] wR2 [I > 2σ(I)] R1 (all data) wR2 (all data) GOF on F2 a

1

2

3

C32H52N2O6 560.76 monoclinic P21/c 23.5885(18) 6.5543(5) 21.6834(16) 90 98.249(3) 90 4 3317.7(4) 1.123 1224 39099 7960 0.0428 0.0590 0.1507 0.1146 0.1864 1.015

C33H58N2O8 610.81 triclinic P1̅ 8.1777(7) 11.4827(11) 20.919(2) 74.116(3) 83.301(4) 75.865(3) 2 1829.5(3) 1.109 668.0 19530 7184 0.0266 0.0493 0.1467 0.0724 0.1601 1.098

C34H60N2O8 624.84 monoclinic P21/c 9.5307(8) 39.909(3) 9.9713(8) 90 91.480(3) 90 4 3791.4(5) 1.095 1368 40201 7775 0.0658 0.0728 0.1785 0.1353 0.2118 1.034

R1 = Σ||F0| − |Fc||/Σ|F0|; wR2 = Σ[w(F02 − Fc2)2]/Σ[w(F02)2]1/2.

Dissolution and Solubility. Seven standard solutions of API in phosphate buffer (pH = 6.8 and total concentration = 0.05 M) were prepared, and their absorbance at the given λmax (274 nm) was measured using a TU-1901 UV−vis spectrometer. A calibration curve of absorbance vs concentration was built, and the R2 of our curve is 0.9994. According to Beer−Lambert’s law, the molar extinction coefficient (ε) of gemfibrozil was read from the slope of the standard curve. The solids used in this part were ground and sieved providing samples with the particle size in the range of 75−150 μm. In the measurement of equilibrium solubility, samples (500 mg) and phosphate buffer (10 mL) were mixed in a 50 mL round-bottom flask B



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intervals: 0.17, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24, and 36 h after doing. Plasma was separated by centrifugation (5 min, 13300 rpm) at ambient temperature and stored at −20 °C until the LC−MS analysis. Finally, the separation and quantitative analysis of each blood sample were conducted on a LC-20a HPLC (Shimadzu, Japanese) and an API4000 MS (Applied BiosystemsSciex, Canada), respectively. The HPLC system was equipped with an Ascentis Express C18 column (50 mm × 4.6 mm I.D, 2.7 μm) and chromatography was performed at 40 °C and the mobile phase, a mixture of acetonitrile/water (70:30, v/v), was delivered at a flow rate of 1 mL/min. The MS using an electrospray ionization (ESI) source in the negative ion mode was controlled by Analyst Software for data acquisition and integration. The used pharmacokinetic parameters were calculated by Software DAS 3.0.

Table 2. Hydrogen Bonds in Crystal Structures D−H···A

d(D···A) (Å)

N1−H1A···O4 N1−H1 B···O5 N1−H1C···O4 N2−H2A···O1 N2−H2B···O2 N2−H2C···O1

2.741 2.676 2.726 2.738 2.671 2.738

N1−H1A···O2 N1−H1B···O2w N1−H1C···O1 N2−H2A···O4 N2−H2B···O5 N2−H2C···O1 O1W−H1WB···O5 O1W−H1WA···O2 O2W−H2WB···O2 O2W−H2WA···O4

2.787 2.769 2.778 2.764 2.723 2.939 2.870 3.321 2.806 2.662

N1−H1A···O2 N1−H1 B···O1w N1−H1C···O2w N2−H2A···O1 N2−H2B···O4 N2−H2C···O5 O 1W−H1WA···O1 O1W−H1WB···O4 O2W−H2WA···O5 O2W−H2WB···O2

2.770 2.768 2.763 2.761 2.741 2.739 2.750 2.789 2.726 2.818

∠D−H···A (deg) Salt 1 168.06 177.95 158.54 162.02 173.21 163.70 Salt 2 174.07 164.04 176.19 164.69 154.51 174.61 175.23 145.09 171.24 168.47 Salt 3 162.73 175.16 160.77 168.89 158.65 159.87 166.78 160.13 160.14 166.20

symmetry code −x+1, y+1/2, −z+1/2 −x+1, y−1/2, −z+1/2 intramolecular −x+1, y−1/2, −z+1/2 x, −y+3/2, z−1/2 x, −y+1/2, z−1/2

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x−1, y, z intramolecular −x, −y+1, −z+1 intramolecular −x+1, −y, −z+1 −x+1, −y+1, −z+1 x−1, y, z x−1, y, z −x+1, −y+1, −z+1 −x, −y+1, −z+1

RESULTS AND DISCUSSION

Crystal Structure of [H3N(CH2)2NH3)][gem]2 (1). The crystal structure of salt 1 crystallizes in the monoclinic P21/c space group. During the formation of 1, two protons transfer from the carboxylic group of gemfibrozil to the nitrogen atoms of ethanediamine. As shown in Figure 1a, the asymmetric unit of 1 contains two gemfibrozil anions which have a similar conformation and one [H3N(CH2)2NH3)]2+ cation. The ring motif R44(18) is generated by the hydrogen bonds (H-bond) between two [gem]− and two [H3N(CH2)2NH3)]2+ ions, and these R(18) rings are fused together by sharing the organic amine to form a ladder along the b axis (Figure 1b). The adjacent ladders are further connected via N1···O4 and N2··· O1 H-bonds into a two-dimensional supermolecular sheet on the bc plane (Figure 1c). In salt 1, each [H3N(CH2)2NH3)]2+ as a H-bond donor interacts with six neighboring API anions, and the average N···O distance is 2.715 Å (Figure S2). Crystal Structure of [H3N(CH2)3NH3)][gem]2·2H2O (2). When gemfibrozil reacted with 1,3-diaminopropane, salt 2 crystallizing in the triclinic P1̅ space group was synthesized. Similar to salt 1, in 2 proton transfer happens between two gemfibrozil and one 1,3-diaminopropane molecule. The single crystal analysis shows that there are two [gem]− anions, one [H3N(CH2)3NH3)]2+ cation, and two lattice water molecules (O1w and O2w) in the asymmetric unit of 2 (Figure 2a). In this case the conformations of two independent [gem]− anions are slightly different. Two different H-bond rings with the R24(12) motif appear in 2, and they are alternately linked by organic amine into a 1D supermolecular chain with one lattice water molecule (O1w) hanging on the O5 atom through the O···O H-bond interaction (Figure 2b). As shown in Figure 2c, the N2···O1 H-bonds (2.939 Å) extend above the 1D chain into a 2D layer. Moreover, the crystallographic water molecule (O2w) acting as glue further reinforces the 2D layer (Figure S3). In salt 2, every organic ammonium interacts with five neighboring [gem]− anions and one crystallographic water molecule (O2w) through H-bonds, and the average N···O distance is 2.793 Å (Figure S2). Crystal Structure of [H3N(CH2)4NH3)][gem]2·2H2O (3). When 1,4-butanediamine was used, salt 3 was obtained. Salt 3 belongs to the monoclinic P21/c space group, and its asymmetric unit consists of two [gem] −, one [H 3 N(CH2)4NH3)]2+, and two lattice water molecules (O1w and O2w) (Figure 3a). As shown in Figure 3b, two kinds of H-bond rings, R42(22) and R42(12), are formed by the H-bond interactions between 1,4-butanediamine cations and drug anions. The R(22) and R(12) rings are alternately assembled by sharing nitrogen atoms into a ribbon along the a axis (Figure 3b). In addition, the lattice water molecules O1w interact with the above 1D chain through O···O and N···O H-bonds (Figure

−x+1, −y, −z+1 −x+1, −y, −z+1 −x+1, −y, −z+1 x, y, z−1 x−1, y, z−1 −x+1, −y, −z+1 x+1, y, z intramolecular x, y, z−1 −x+1, −y, −z+1

and stirred at room temperature for 48 h. The resulting suspension was filtered to remove the precipitate through a 0.22 μm nylon filter and diluted with phosphate buffer. The concentration of the filtered solution was determined according to its absorption using the calibration curve. For measuring the dissolution rate, an excess amount of samples (400−600 mg) was added to a 100 mL beaker containing phosphate buffer (50 mL), and the suspension was stirred at room temperature with a stirring speed of 500 rpm. The suspension (2 mL) was removed from the beaker at different time intervals (3, 8, 13, 18, 23, 30, 45, 60, 90, 120, 180, 240, and 360 min) and filtered through a 0.22 μm nylon filter. The concentration was also determined by the established calibration curve. For both the apparent solution and dissolution rate tests, three parallel experiments were performed, and we used the mean value to plot the dissolution profiles in Figure 6. Stability Experiment. To test the physical stability on storage, samples of 1−3 were stored in desiccators containing saturated sulfuric acid solution (providing 75% RH) at 40 ± 1 °C oven. After one month, PXRD was performed on the collected samples. For slurry experiments, excess salts were added to a beaker containing phosphate buffer (10 mL, pH = 6.8), and the suspension was stirred at room temperature for 3 days and then the left solid samples were collected and characterized by PXRD. Pharmacokinetic Study. The PK studies were performed in Wistar rats. The power samples of original API and its three salts were prepared by grinding, and their phase purities were confirmed by PXRD. First, 16 healthy Wistar rats with the weight of 220−260 g were divided into four groups and each group contains half male and half female. Then after a 12 h fast, the powder sample of each drug (single dose: 10 mg/kg) was given to Wistar rats by gastric perfusion method using a Dry Powder Insufflator. Subsequently, whole blood samples (0.5 mL) were collected from the eyeball veins of each rat into heparinized tubes before administration and at the following time C



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Figure 1. (a) An asymmetric unit of salt 1. (b) 1D supermolecular chain forming along the b-axis in 1. (c) The 2D sheet on the bc plane in 1 formed by hydrogen bond interactions represented by green dashed lines (The atoms not involved in hydrogen bond interactions are present with the transparency of 0.8).

Additionally, the first cooling cycle matches well with the second one. In DSC the peak area of heat flow is proportional to the transition enthalpy value. In most cases, the enthalpy value of the solid−solid phase transition is a fraction of one compared with melting enthaphy. However, in this case the peak area of the second peak (at 100 °C) is basically similar to that of the third one (at 110 °C). Accordingly we speculate that two new phases including semistable 3-I and stable 3-II were formed after salt 3 loses its lattice water molecules, and the endothermal peaks at 100 and 110 °C correspond to the melting points of 3-I and 3-II, respectively. To confirm our speculation, the anhydrous sample of 3 prepared by heating at 70 °C under vacuum conditions and the sample after two DCS cycles were collected (Figure S6), and their PXRD were recorded (Figure S5). The PXRD pattern of the sample after two DCS cycles is apparently distinct from that of 3 (Figure S5), and we think it is the stable phase 3-II. By comparison of PXRD patterns of two anhydrous samples (Figure S5), we find that the anhydrous sample after 70 °C heating contains two phases: one is the stable phase 3-II, and the other is possibly the semistable phase 3-I. Moreover, the absence of the endothermic peak at 100 °C in the second heating cycle suggests that the semistable 3-I transfers to stable 3-II during the heating process and the fact that the peak area at 110 °C in second heating

S4). The adjacent ribbon chains are further connected by the crystallographic water molecule O2w into a 2D layer on the ac plane (Figure 3c). In salt 3, each diammonium cation connects with four neighboring [gem]− anions and two crystallographic water molecules (O1w and O2w) by H-bond interactions and the average N···O distance is 2.757 Å (Figure S2). Thermal Analysis. The thermal behaviors of salts 1−3 were investigated by DSC and TGA, and the results are presented in Figure 4. For salt 1, its melting point is 124 °C. There are two endothermic peaks in the DSC curve of 2. The first small endothermic peak at 74 °C corresponds to the weight loss of two lattice water in the TGA (observed: 5.83%, calculated: 5.89%) and the second big peak at 92 °C is ascribed to the melting of 2. The melting points of 1 and 2 are much higher than that of API (61 °C) due to the formation of salts. The DSC curve of 3 shows three endothermic peaks. The first peak at 56 °C is attributed to the loss of two lattice water molecules (observed: 5.80%, calculated: 5.83%). Different from the DSC of 1 and 2, two endothermic peaks at 100 and 110 °C, respectively, are observed before the decomposition of 3. The presence of multiple peaks is generally associated with the solid-phase transformation,45−47 so heating and cooling cycles of DSC experiments were performed. In the first DSC heating cycle, three endothermic peaks can be clearly seen, but the first two peaks are absent in the second heating one (Figure 5). D



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Figure 2. (a) An asymmetric unit of salt 2. (b) The 1D chain in 2. (c) 2D supermolecular network formed by hydrogen bonds represented by green dashed lines (The atoms not involved in hydrogen bond interactions are given with the transparency of 0.8).

and δ = 4.21 for 3 are observed. In addition, compared with gemfibrozil the H peaks of [gem]− shift a little toward a high field, possibly because of losing its proton. Dissolution and Stability. As the oral absorption of class II drugs such as gemfibrozil is greatly affected by their solubility in water and dissolution rate, powder dissolution experiments of three new salts and API in phosphate buffer (pH = 6.8) were conducted. As shown in Figure 6, both the dissolution rate and the apparent solubility of the three salts are greatly improved relative to API. The equilibrium solubility of salts 1−3 is 7.22, 9.41, and 5.14 mg/mL, respectively, which is enhanced by 13.3, 17.4, and 9.5 times, compared to that of API (0.542 mg/mL). The dissolution rate of three salts and API follows the order of 2 > 1 ≈ 3 > API. It is notable that salt 3 reaches its maximum

cycle is about twice larger than the one in the first heating process further supports the transition of 3-I to 3-II. Spectral Analyses. The proton transformation between gemfibrozil and diamine proved by X-ray single crystal diffraction can be also found in the FT-IR (Figure S7) and 1 H NMR (Figure S1) spectra. The characteristic absorption peak at 1701 cm−1 attributed to the vibration of the −COOH group for API is absent in the three salts. Instead, the absorption peaks of −COO− groups are found at 1618 and 1403 cm−1 (for 1), 1650 and 1403 cm−1 (for 2), and 1620 and 1403 cm−1 (for 3). The comparison of the 1H NMR spectra of API and its salts shows that the active hydrogen of carboxylic group with δ = 12.12 disappears in all three salts, and the broad H peaks of ammonium ions with δ = 4.93 for 1, δ = 4.45 for 2, E



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Figure 3. (a) An asymmetric unit of salt 3. (b) The 1D chain in 2 along the a-axis. (c) 2D supermolecular network formed by hydrogen bond interactions represented by green dashed lines (The atoms not involved in hydrogen bond interactions are given with the transparency of 0.8).

Figure 4. TG (in black line) and DSC (in red line) plots of salts 1−3 and gemfibrozil. F



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properties of pharmaceutical salts, three gemfibrozil salts [NH3(CH2)nNH3][gem]2 (n = 2−4) were prepared by using a series of diamine with different carbon chain lengths. The structural analyses show that all the three salts have a stoichiometry of 1:2 and 2D H-bond structures. The distinct difference between them is that 1 is an anhydrous salt and 2 and 3 are hydrated ones. We speculate that as the carbon chain of organic diamine increases, the packing between cations and anions is not as close as before so water molecules are involved in the crystal lattice. In addition, the number, connecting motif, and distances of H-bonds varied with the increase of carbon chain length (see Table 3). As shown in Table 3, the melting point of salt 1 is higher than 2. Generally, the melting point is mainly influenced by the structural density together with H-bond interactions.37,49 We think the high melting point of 1 is attributed to its close packing reflected by its high calculated density and relatively strong H-bond interactions reflected by the short average Hbond distance (see Table 3). Because the solid-state phase transformation happens in the DSC experiment of 3, it is impossible to measure the melting point of 3, and this hinders us from finding the relationship between chain length of cations and the melting point. The result obtained by Conway indicates that increasing chain length of alkyl amine counterions leads to a reduction in solubility across three acidic drugs including gemfibrozil.41 If above-mentioned conclusion is followed, the solubility order in our study should be 1 > 2 > 3, while actually the observed one is 2 > 1 > 3. Generally, the aqueous solubility of salts depends on crystal lattice energies and hydration energies of cations and anions. The free energy of the solution is represented by ΔGsolution = ΔG cation hydration + ΔG anion hydration − ΔG lattice. As the chemical changes that increase free engergies of hydration also tend to increase lattice energy, it is difficult to correlate the solubility of a salt with its structure.30,38 In this case, the anions for salts 1−3 are all [gem]−, and the cations are a set of protonated organic diamines which are all miscible with water, so the free energies of hydration of the three salts are very similar and the influence of free energy of hydration on solubility is negligible. Therefore, the difference on solubility of salts 1−3 is mainly due to different lattice energies. As for lattice energy, the contributions of electrostatic attraction between ionic components and H-bond interactions should be considered. The H-bond distances of N+−H···OAPI represent the distance between cation and anion effecting the electrostatic interaction. The long average N+···OAPI distance (2.798 Å) of 2 might lead to its relatively weak electrostatic attraction, and as a result its solubility is higher than those of others. Besides electrostatic attraction, H-bond interactions have some influence on the lattice energy. As shown in Table 3, with the increase of carbon chain length, the number of N+−H··· OAPI H-bonds decreases and the number of N+−H···Ow Hbonds increases. As the introduction of crystallographic water

Figure 5. Heating and cooling DSC thermograms of salt 3.

Figure 6. Dissolution profiles of gemfibrozil and its three salts in phosphate buffer (pH = 6.8).

solubility within 30 min. In general, the solubility of the dehydrated form is higher than that of the hydrated one,48 so we prepared the dehydrated form of salt 3 (Figure S6). The result shows that the solubility of the dehydrated salt (6.01 mg/ mL) is slightly higher than that of the hydrated one (5.14 mg/ mL) (Figure S8). Moreover, the stability of salts 1−3 was studied. The three salts can keep stable under ambient conditions for more than 3 months (Figure S9) and under moisture conditions (40 °C and 75% relative humidity) for one month (Figure S10). In addition, slurry experiments of the three salts were performed, and the PXRD results display that all the salts completely hydrolyzed into API (Figure S11). Investigation of the Structure−Property Relationship. In order to investigate the effect of counterions on the

Table 3. Selected Physical Properties and Structural Characteristics of Salts 1−3 N+−H···OAPI salt 1 2 3

mp (°C)

solubility (mg/ mL)

Dcalc (g/cm3)

structural type

124 91.9

7.22 9.41 5.14

1.123 1.109 1.095

2D layer 2D layer 2D layer

H-bond motif R44(18) R24(12) R24(22), R24(12)

N+−H···Ow

Ow−H···OAPI

number

average distance (Å)

number


number


6 5 4

2.715 2.798 2.752

0 1 2

2.769 2.766

0 4 4

2.914 2.771

G



Article

of α,ω-diamine with different carbon chain lengths. All of the three salts are composed of one diammonium cation and two API anions and exhibit a two-dimensional hydrogen bond structure, but the motif, number, and distance of hydrogen bond vary with the change of the salt forming agent. Although no correlation between chain length of counterion and solubility is built in our study, we find that the electrostatic and hydrogen bond interactions are two main factors affecting the melting point and the solubility orders of salts 1−3. In addition, the solubility and dissolution rate of the three salts and the oral absorption of salt 2 are improved compared to those of the API.

molecules, four additional Ow−H···OAPI H-bonds appear in 2 and 3, and the average of Ow···OAPI distance in 3 is obviously shorter than that of 2. We speculate that the additional Ow− H···OAPI H-bonds possibly makes the solubility of 3 lower than that of 1. The solubility order of 2 > 1 > 3 is a result of electrostatic attraction combined with H-bond interaction. Pharmacokinetic Study. The oral absorption of three gemfibrozil salts was estimated in Wistar rats. In the pharmacokinetic study (PK), the mean plasma concentration vs time curves for salts 1−3 and API were plotted and presented in Figure 7. The maximal plasma concentration

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.6b01100. Additional PXRD, IR, NMR, the selected bond distances and angles PDF) Accession Codes

CCDC 1494310−1494312 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 nion Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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Figure 7. Mean plasma concentration vs time curves of gemfibrozil and its three salts in Wistar rats.

AUTHOR INFORMATION

Corresponding Authors

*(Y.C.) E-mail: [email protected]. *(J.G.) E-mail: [email protected]. *(C.H.) E-mail: [email protected].

(Cmax), the time required to reach the Cmax (tmax), and the area under the curve (AUC) are recorded in Table 4, where the higher standard deviation values may be the result of individual differences. In pharmacology, bioavailability is measured by the AUC value. Taking the AUC0‑∞ of API as 100%, the relative bioavailability of 1−3 was calculated as 104%, 154%, and 108%, respectively. It is worth mentioning that the tmax was dramatically shortened from the 10.5 h for API to 2.8 h for salts 1, 0.5 h for salts 2, and 3.2 h for salt 3. In addition, the Cmax values of salts 1−3 are increased by 3.49-fold, 2.60-fold, and 2.97-fold, respectively, compared to that of API. For the three salts, the obvious changes of tmax and Cmax closely relate to the improvement of dissolution rate. Although the solubility of salts 1 and 3 was increased by 7.22 and 5.14 times, respectively, their bioavailability is nearly as same as the API. This observation indicates that the solubility is just one of the factors affecting a drug’s absorption, and even for class II drugs the enhancement of solubility does not always lead to the improvement of bioavailability.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank NSFC (21231002, 21276026, 21173021, 21371024, 81430087), the 111 Project (B07012), and 973 Program (2014CB932103) for their financial supporting.

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REFERENCES

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CONCLUSIONS In order to investigate structure−property of pharmaceutical salts, three gemfibrozil salts were synthesized by using a series

Table 4. Pharmacokinetic Parameters of the Gemfibrozil and Its three Salts drug gemfibrozil Salt 1 Salt 2 Salt 3

Cmax (ng/mL) 987.8 3449.5 2570.0 2932.3

± ± ± ±

393.7 2751.4 663.5 2770.1

tmax (h) 10.5 2.8 0.5 3.2 H

± ± ± ±

1.9 2.7 0.0 5.9

AUC (ng·h/mL)

F (%)

± ± ± ±

100 1.04 1.54 1.08

13374 13968 20610 14461

2029.1 1317.2 2326.0 3336.1



Article

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Synthesis and Pharmacokinetic Study of Three Gemfibrozil Salts: An

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