Stable Cocrystals and Salts of the Antineoplastic Drug Apatinib with

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Stable Cocrystals and Salts of the Antineoplastic Drug Apatinib with Improved Solubility in the Aqueous Solution Bin Zhu, Jian-Rong Wang, Qi Zhang, Meiqi Li, Chunyang Guo, GuoBin Ren, and Xuefeng Mei Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.8b00684 • Publication Date (Web): 02 Jul 2018 Downloaded from http://pubs.acs.org on July 2, 2018

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Crystal Growth & Design

Stable Cocrystals and Salts of the Antineoplastic Drug Apatinib with Improved Solubility in the Aqueous Solution Bin Zhu, a Jian-Rong Wang, b Qi Zhang, b Meiqi Li, b, d Chunyang Guo, a Guobin Ren, *a, c and Xuefeng Mei* b a

Laboratory of Pharmaceutical Crystal Engineering & Technology, School of

Pharmacy, East China University of Science and Technology, Shanghai, 200237, China b

Pharmaceutical Analytical & Solid-State Chemistry Research Center, Shanghai

Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China c

Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China

University of Science and Technology, Shanghai, 200237, China d

University of Chinese Academy of Sciences, Beijing, 100049, No.19A Yuquan

Road, China

ACS Paragon Plus Environment

Crystal Growth & Design 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract: Apatinib (APA) belongs to the targeted antineoplastic family of drugs by inhibiting the vascular endothelial cell growth factor receptor (VEGFR-2) of tyrosine kinase. APA encounters the poor aqueous solubility problems and its therapeutic dosage form, apatinib mesylate (ATM), is unstable and will be dissociated completely to APA in the aqueous solution. Here, we synthesized and evaluated three new cocrystals of APA with adipic acid (APA+ADA), sebacic acid (APA+SEA), and D/L mandelic

acid

(APA+D/L-MA),

(APA+SUA-H2O),

salicylic

acid

and

four

new

(APA+SA),

salts

with

succinic

1-hydroxy-2-naphthoic

acid acid

(APA+HNA), and saccharin (APA+SAC). All the solid forms were characterized by powder X-ray diffraction, infrared spectroscopy, differential scanning calorimetry, and dynamic vapor sorption. The molecular components and structures were confirmed by single crystal X-ray diffraction. APA+SEA is able to overcome the instability problem and improves solubility compared with ATM. Hence, APA+SEA has the potential to be a superior candidate for this important drug. INTRODUCTION Crystal engineering,1,

2

aiming to understand and control the intermolecular

interactions in the context of crystal packing, is widely utilized in designing solid state of active pharmaceutical ingredients (APIs).3, 4 The screening, characterization, and selection of a suitable solid form are essential in the drug development process5, 6 in order to obtain materials with optimum combinations of important physicochemical properties, such as stability,7, 8 solubility,9, 10 dissolution rate,11, 12 bioavailability,13, 14 and hygroscopicity.15, 16 The quality of the chosen solid form can affect the therapeutic


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efficacy of orally administered drugs, especially for poorly water-soluble or unstable drugs.17 Improving aqueous solubility of insoluble drug has remained a challenge, which in turn leads to a higher dropout rate in drug development programs.18 A number of approaches were used to improve the solubility of active pharmaceutical ingredients (API),19 such as solid dispersions,20 micronization,21 polymorphs,6, 22, 23 and nanocrystals.24-26 For overcoming the solubility challenge, developing new crystalline forms, such as salts, is the most general method without modification of the pharmacophore structure of an API.27-29 More than half of APIs are marketed as salts up to today. However, the utility of salts is at times subjected to their tendency for hygroscopicity due to the ionic nature of their crystals.30-32 Besides, drug salts were reported to encounter the disproportionation, in which the proton exchange involving an acid-base reaction.33, 34 Disproportionation, leading to the conversion of the salt to its neutral form, may render salts more susceptible to chemical degradation and affect the physical integrity of solid dosage forms.35, 36-38 It can adversely affect the performance of drug product, e.g. loss of drug potency, slow dissolution, and reduced bioavailability.39-42 In contrast, cocrystals are stable in ambient conditions and crystalline in nature.8, 30, 43-46 Apatinib (APA) as a free base is practically insoluble in water, hence showing the poor bioavailability.47 Therefore, APA is used as the mesylate salt form to improve the solubility of this alkaline drug.23,

48

However, apatinib mesylate (ATM) was

observed to convert to the insoluble neutral form in the aqueous solution. We intended to design new cocrystal or salt forms to overcome the disproportionation,



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hygroscopicity, and thermostability problems of ATM. The properties of coformers or counter ions may affect physicochemical property and the formation complexity of cocrystals or salts. A hydrophobic insoluble drug usually form cocrystal with a hydrophilic coformer for the improved physicochemical properties such as ionization, hydrophobicity, and diffusivity.33 The success rate for the pyridine group form synthons with the carboxylic group or hydroxy groups is 91.7% and 89.9% based on the Cambridge Structural Database,49 suggesting the synthons were robust. Saccharin (SAC) was also reported as the reliable coformer to produce admirable cocrystals.50, 51 Meanwhile, the diprotic aliphatic acids and SAC have the excellent water solubility.30 In this contribution, we report three new cocrystals of APA with adipic acid (ADA), sebacic acid (SEA), and D/L mandelic acid (D/L-MA), and four new salts with succinic acid (SUA), salicylic acid (SA), 1-hydroxy-2-naphthoic acid (HNA), and saccharin (SAC) (Scheme 1). All the new solid phases were characterized by powder X-ray diffraction, infrared spectroscopy, differential scanning calorimetry, and dynamic vapor sorption. The molecular components and structures were confirmed by single crystal X-ray diffraction. The powder dissolution and pharmacokinetic profiles showed APA+SEA has the improved solubility and accelerate the absorption in vivo compared with ATM. APA+SEA is also able to overcome the instability problems, such

as

disproportionation

in

the

aqueous

solution,

thermostability of ATM. Scheme 1. Molecular Structures of APA and Coformers.


hygroscopicity,

and

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succinic acid (SUA)

sebacic acid (SEA)

D/L mandelic acid (D/L-MA)

adipic acid (ADA)

salicylic acid (SA)

1-hydroxy-2-naphthoic acid (HNA)

apatinib (APA)

saccharin (SAC)

EXPERIMENTAL SECTION Materials. Apatinib was obtained from BioChemPartner Company, Ltd. (Zhangjiang Road, Pudong New Area, Shanghai, China) with purity greater than 98%. All carboxylic acids and analytical grade organic solvents were purchased from the Sinopharm Chemical Reagent Company. All chemicals were used without further purification. Synthesis of Cocrystals or Salts via Reaction Crystallization Method (RCM). 50-75 mg of APA was added to 1 mL of presaturated ethyl acetate (EA) or ethanol (EtOH) solutions of coformers. The suspensions were stirred at atmospheric condition overnight with the help of a magnetic stir bar on a plate and the multicomponent crystals were obtained.



Preparation of Single crystals. Single crystals of APA+SUA-H2O were obtained by slow evaporation of solutions containing APA and SUA (1:1 molar ratio) in ethyl acetate solvent at room temperature. Single crystals of APA+ADA and APA+SEA were obtained by slow evaporation of solutions containing API and coformer (2:1 molar ratio) in ethyl acetate solvent at the room temperature. Single crystals of the other cocrystals were also successfully obtained by slow evaporation of solutions containing APA and the corresponding hydroxy acidic coformer (at 1:1 molar ratio) in appropriate solvents. For D/L-MA and SA, the solvent used was EA and EtOH. The mixture of ethyl acetate and ethanol were used for APA+HNA and APA+SAC. Powder X-ray Diffraction (PXRD). All PXRD measurements were performed at ambient temperature by a Bruker AXS D8 powder diffractometer with Cu-Kα radiation (λ = 1.54056Å). Voltage and current of the generator were set to 40 kV and 40 mA. Scanning interval was 3-40° 2θ with time (0.05 s) per step. Single-Crystal X-ray Data Collection and Structure Determinations. Suitable crystals for X-ray crystallography were selected using an optical microscope. The X-ray diffraction data were collected on a Bruker-AXS SMART-APEXII CCD diffractometer using MoKα (λ = 0.71073 Å) (Table 2). Indexing was performed using APEX2 (difference vectors method).52 Data integration and reduction were performed using SaintPlus.53 Absorption correction was performed by a multiscan method implemented in SADABS.54 Space groups were determined using XPREP implemented in APEX2.32 The structures were solved by direct methods using


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SHELXTL55 and were refined on F2 using the full-matrix least-squares technique in the SHELXL-97 and SHELXL-2014 program packages.56 All non-hydrogen atoms were refined anisotropically. Hydrogen atoms were placed in geometrically calculated positions and included in the refinement process using a riding model with isotropic thermal parameters: Uiso(H) = 1.5Ueq(-CH3, -OH), Uiso(H) = 1.2Ueq(-CH). Hydrogen atoms of the NH2 group were found on a difference Fourier map and were freely refined. Fourier-Transform Infrared (FTIR) Spectroscopy. Fourier-transform infrared (FTIR) spectra were collected by a Nicolet-Magna FTIR 750 spectrometer in the range from 4000 to 350 cm−1, with a resolution of 4 cm−1 at ambient conditions. Differential Scanning Calorimetry (DSC). Thermal analysis was performed on a TA Instruments DSC Q2000 differential scanning calorimeter. An empty pan sealed was used as the reference and the instrument was calibrated using an indium standard. The samples were weighted and placed in the sealed aluminum pans in the same way. The original temperature was equilibrated at 25 °C, then heated to the required temperature at a rate of 10 °C/min under a nitrogen gas flow of 50 mL/min. Thermogravimetric Analysis

(TGA). Thermogravimetric

analysis was

performed on a Netzsch TG 209F3 instrument. The samples were placed in open aluminum oxide pans and heated at 10 °C/min to 400 °C. Nitrogen was used as the purge gas at 20 mL/min. Two-point calibration of the temperature was performed with ferromagnetic materials (Alumel and Ni, Curie-point standards, PerkinElmer).



Dynamic Vapor Sorption (DVS). Moisture sorption/desorption data were collected on an Intrinsic DVS instrument from Surface Measurement Systems, Ltd. Moisture sorption/desorption was analyzed from one cycle over a range of 0-95-0% relative humidity (RH) at 5% RH intervals under a nitrogen purge of 15 mL/min. The samples were mounted and scaled on a balance, and the RH was kept at 0% for equilibrium at 25 °C until dm/dt ≤ 0.001. Samples were not dried prior to analysis. Equilibrium criteria used for analysis were either less than 0.0200% weight change occurring over 10 mins or a maximum time limit of 360 min for each step.

In Vitro Powder Dissolution. Each of the cocrystals or salts was synthesized in bulk by slurring stoichiometric ratios of the starting materials in EA or EtOH for 24 h. To minimize the size effect on dissolution results, APA salts and cocrystals were sieved through 100- mesh sieves. Accurately weighed powders of 30 mg APA (or corresponding to, for salts and cocrystals) were added to dissolution vessels containing 15 mL of pH 2.0 hydrochloric acid (HCl) buffer. The dissolution studies were conducted at a rotation speed of 100 rpm at 37 °C. Sampling was performed at 3, 5, 10, 15, 20, 30, 45, 60, 90, 120, 180, 240, and 330 min. Then the suspensions were centrifuged at 14000 rpm for 5 min, and the supernatant was diluted appropriately with pH 2.0 HCl buffer prior to HPLC analysis. The solubility measurements were done in replicates of three. High Performance Liquid Chromatography (HPLC) Analysis. The contents of APA were determined by an Agilent 1260 series HPLC (Agilent Technologies), equipped with a quaternary pump (G1311C), diode-array detector (G1315D) set at


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260 nm. The analytes were separated on a 4.6 × 75 mm, 3.5 µm particle size Agilent Eclipse plus C18 column. A mobile phase consisting of solvent A (water) and solvent B (acetonitrile) was run at 1.0 mL/min with the ratio of 65/35 (v/v) for 0-5 min, 50/50 (v/v) for 5-10 min, 40/60 (v/v) for 10-10.01 min, and 65/35 (v/v) for 10.01-12 min. The column temperature was set at 30 °C and the injection volume was 15 µL.

In Vivo Pharmacokinetic (PK) Analysis. PK study of ATM and APA+SEA were utilized in rats. The PK experimental protocol was approved by the Institutional Animal Care and Use Committee of Shanghai Institute of Materia Medica and conformed to the Guide for Care and Use of Laboratory Animals. Twelve male Sprague−Dawley rats (200−230 g) were randomly divided into two groups, and suspension formulation (aqueous solution) was delivered as oral administration at a dose of 40 mg/kg APA (expressed as APA equivalents). The rats were allowed to have free access to water and fasted overnight before drug administration. Freshly prepared suspension formulation was immediately delivered orally to rats. Then 200 µL blood samples were obtained from orbital sinus and placed into heparinized tubes at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after dosing. Plasma was immediately separated by centrifugation (10 °C, 15000 rpm, 5 min) and kept frozen at −20 °C until analysis. The plasma samples (40 µL) were transferred to 1.5 mL tubes and mixed with 40 µL of internal standard solution (carbamazepine), 50 µL of 50% aqueous methanol, and 160 µL of acetonitrile. The samples were vortex-mixed for 5 min and centrifuged for 10 min (4 °C, 15000 rpm). The supernatant was transferred for analysis. The Agilent 6460 QQQ/1290 UPLC LC−MS/MS was utilized for the separation and determination



of APA in plasma. The separation was operated on a Agilent Poroshell EC C18 column (50 × 3.0 mm, 2.7 µm) with solvent A (5 mM ammonium acetate solution with 1‰ formic acid) and solvent B (acetonitrile). The liner gradient was performed with mobile phase B increasing from 22% to 90% within 2.4 min and held for 1.3 min. The mobile phase was returned to the initial condition and re-equilibrated for 1.4 min. The flow rate was set at 0.4 mL/min. The injection volume was 1 µL. Mass spectrometer was operated in the positive ion detection. The heat block temperature was maintained at 400 °C. Nitrogen was used as the nebulizing gas and drying gas. Detection and quantification were conducted in the multiple reactions monitoring mode (MRM), with m/z 397.7 → 211.6 for APA and m/z 237.1 → 194.1 for IS. Pharmacokinetic parameters were determined using a commercially available computer modeling program, DAS 2.0. The reported pharmacokinetic parameters included Cmax, Tmax, and area under curve (AUC). RESULTS AND DISCUSSION Identification of the Disproportionation of ATM. In this contribution, the disproportionation of ATM converting to the freebase drug APA in aqueous media (H2O) was verified by PXRD, 1H-NMR, and FTIR. Adding 10 mg ATM monohydrate (HA) powder into H2O and slurring for 10 min. The excess solid was collected by centrifugation and dried in the vacuum oven. The excess solid powder was measured by PXRD and determined to convert to APA free base (Figure 1a). The characteristic peaks of ATM HA at 2θ =5.38, 10.41°disappeared and the characteristic peaks of APA at 2θ = 5.94, 11.82, 17.78° appeared. The FTIR spectra were measured (Figure 1b)


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and the differences between ATM and APA were obvious. The FTIR spectra of excess solid after slurring ATM HA in H2O were similar to that of APA. The O=S=O stretching vibration (1162 and 1228 cm−1 for ATM HA) disappeared after slurring in H2O, suggesting ATM HA had transformed to APA. 1H-NMR (Figure S1) was also conducted by dissolving the solid powder into CDCl3. The –CH3 peaks at chemical shift in the sulfate anion (δCH3 = 2.78 ppm) disappear, which also suggested ATM was unstable and convert to APA. Therefore, it’s requisite to develop a more stable APA cocrystal or salt as an alternative solid form.

(a)

(b)

Figure 1. (a) PXRD and (b) FTIR patterns of ATM HA, excess solid after ATM HA slurry in H2O, and APA. Preparation of Multicomponent Crystals. For the sake of the solid stability of ATM, seven new multicomponent crystals of APA with various coformers were designed and prepared by RCM. The purity of the bulk materials were tested by PXRD (Figure 2). The PXRD patterns of the bulk materials were compared to the simulated PXRD obtained from the single crystal X-ray diffraction, confirming the



composition of bulk materials are of high crystalline purity and same with the single crystal sample.


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Figure 2. The comparison of the experienced PXRD patterns, simulated PXRD patterns of cocrystals or salts, PXRD patterns of APA and coformers. Salts or Cocrystals. The pKa1 of apatinib was measured as 6.04 ± 0.01 and the pKa1 parameters of coformers were listed in the Table 1. According to the ∆pKa rule of salt formation, the difference between APA and coformers suggests APA form salt with HNA and SA, and form cocrystal with other coformers. However, the extent of proton transfer in the crystalline phase is difficult to rationalize based on pKa values merely.57 In the solid state, formation of cocrysatls or salts can be determined by analyzing proton location or position and bond lengths of atoms involved, for example, C-O distances of carboxyl groups.58 In carboxylic acids, the average C-O distance is 1.31 Å, whereas the C=O distance average is 1.21 Å. On the other hand, the average C-O distance in the carboxylate moiety is 1.25 Å.59 The asymmetric units of each multicomponent crystal were plotted in the Figure S2 and the bond length change of carboxylic coformers in multicomponent crystals was calculated (Table 1). The C-O distance and C=O distance of APA+ADA, APA+SEA, and APA+D/L-MA are longer than 1.31 Å and shorter than 1.25Å. Therefore, they can be identified as cocrystals. While the C-O distance of APA+SUA-H2O and APA+SA are close to 1.25 Å, which are determined as salts. The C-O bond length of carboxylic group in the APA+HNA suggests a partial proton transfer. The delocalizing hydrogen atom is shared by O(2) in the carboxylic group and N(3) in the protonated pyridine ring B. Interestingly, the keto-enol tautomerization for SAC was found in APA+SAC salt (Scheme 2), and APA formed salt with SAC enol.



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Table 1. The pKa Parameters of the Coformers and Distribution of C-O Bond Lengths for APA and Carboxylic Acid Coformers in New Forms. Form

pKa1

∆pKa

d(C-O)/Å

d(C=O)/Å

Nature of form

APA+ADA

4.44

1.6

1.32

1.21

cocrystal

APA+SEA

4.59

1.45

1.333

1.20

cocrystal

APA+D/L-MA

3.37

2.67

1.33

1.19

cocrystal

APA+HNA

2.70

3.34

1.31

1.24

H-atom delocalization

APA+SA

2.97

3.07

1.28

1.26

salt

APA+SUA-H2O

4.21

1.83

1.26

1.25

salt

Scheme 2. Molecular Structures of (a) SAC keto and (b) SAC enol.

(a) SAC keto

(b) SAC enol

Spectroscopic Analysis. Besides the crystal structure analysis, the spectroscopic technology can also be used to determine the formation of salt or cocrystal. FTIR spectroscopy were used to characterize the vibrational energy of hydrogen bonding and crystal packing in the solid state.59, 60 The carbonyl group of aromatic carboxylic acids absorbs at lower frequencies (1710-1680 cm-1) than aliphatic of carboxylic acids (1750-1700 cm-1) due to resonance.34, 61 APA+SUA-H2O, APA+ADA, APA+SEA, and APA+D/L-MA exhibit a strong IR band at frequency >1700 cm-1 corresponding to


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aliphatic carboxylic acid peak, suggesting there is no proton transfer between APA and coformers (Figure 3). One of SUA carboxylic acid groups in APA+SUA-H2O remains the free acid, another form salt with the pyridine in the form of carbonate ion. The IR stretching vibration peak of carboxylic group in the APA+SA and APA+HNA disappear, which indicates the proton transfers from the acid coformers to APA. FTIR spectra also suggest that APA+ADA, APA+SEA, and APA+D/L-MA are cocrystals, and APA+SUA-H2O, APA+SA, and APA+HNA are salts.

Figure 3. FTIR spectra for APA cocrystals and salts. Crystal Structures Analysis. The single crystal photos of APA cocrystals and salts were collected using a XPV-400E polarizing microscope coupled with a JVC TK-C9201 EC digital video recorder (Figure S3). Polarizing microscope images showed APA+SUA-H2O and APA+SAC are needle-shaped. APA+ADA, APA+SEA, and APA+D/L-MA are trapezoid-shaped. APA+HNA and APA+SA are block-like. The single crystal data of APA cocrystals and salts were collected at 173(2), 206(2), or 276(2) K. Crystallographic data was summarized in Table 2. On the basis of the unit



cell parameters, APA+SAC, APA+SA and APA+HNA have isostructural properties. Only APA+D/L-MA crystalized in the monoclinic P21/n spaceg group. The hydrogen bond data of different solid forms was summarized in Table S1. The conformations of APA molecules in asymmetric units were analyzed on the basis backbone of ring A (Figure S4). The τ1−τ3 (τ1, τ2, and τ3 representing the torsion angles along C1−N2−C6−C7, C13−N4−C12−C2, and C17−C18−C19−C21 respectively) were compared (Table S2), revealing that the conformations in different multicomponent forms are similar except for APA+ADA. The discrepant conformation of APA results in no dimers in the APA+ADA crystal structure. In other salts and cocrystals, the common R22 (18) dimers are formed by hydrogen bonds between two APA molecules as the fundamental building block. Apatinib+Adipic Acid Cocrystal (APA+ADA, 2:1). APA+ADA cocrystal crystallizes in the triclinic P-1 space group with one APA molecule and a half equivalent of ADA molecule in the asymmetric unit. The coformer ADA is located on a crystallographic inversion center. Two APA molecules are connected with one ADA molecule via the hydrogen bond between N(3) in pyridine group and O(3) in the carboxylic group (O(3)−H···N(3), 2.673 Å, 179°) to form a zigzag-liked basic building block (the blue area in the Figure 4). Two zigzag units are stacked head-to-head, and extend to form a tape structure with two obvious hydrophobic and hydrophilic chains along the a-axis direction.


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Table 2. The crystallographic data of APA salts and cocrystals. Form

APA+SUA-H2O APA+ADA

a

b

Radiation

MoKα

MoKα

MoKα

Empirical formula

C28H31N5O6

C27H28N5O3

Formula weight

533.58

Temperature/K

APA+SEA

APA+SEA

APA+D/L-MA APA+SA

APA+HNA

APA+SAC

MoKα

MoKα

MoKα

MoKα

MoKα

C29H32N5O3

C29H26N5O3

C32H31N5O4

C31H29N5O4

C35H31N5O4

C31H28N6O4S

470.54

498.59

492.55

549.62

535.59

585.65

580.65

173(2)

173(2)

173(2)

276(2)

173(2)

206(2)

206(2)

173(2)

Crystal system

triclinic

triclinic

triclinic

triclinic

monoclinic

monoclinic

monoclinic

monoclinic

Space group

P-1

P-1

P-1

P-1

P21/n

C2/c

C2/c

C2/c

a/Å

8.9034(7)

7.2695(3)

8.8777(2)

8.9892(6)

8.6434(19)

17.657(4)

18.0080(7)

18.1887(17)

b/Å

9.2809(6)

8.9956(4)

9.0347(2)

9.1111(6)

37.502(9)

8.5850(17)

8.4825(3)

8.4040(11)

c/Å

18.6643(13)

18.8041(7)

18.6242(4)

18.7864(12)

9.602(2)

35.632(7)

39.5323(15)

36.630(4)

α/deg

78.137(4)

101.793(2)

88.0740(10)

78.988(4)

90

90

90

90


Crystal Growth & Design 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 18 of 41

β/deg

83.184(4)

97.802(2)

82.2220(10)

76.935(5)

114.867(14)

100.651(5)

101.8750(10)

98.093(6)

γ/deg

62.721(4)

100.984(2)

61.1970(10)

62.674(4)

90

90

90

90

Volume/Å3

1340.93(17)

1161.97(8)

1296.09(5)

1324.77(16)

2823.8(11)

5308.2(19)

5909.4(4)

5543.5(10)

Z

2

2

2

2

4

8

8

8

ρcalc /g·cm-3

1.322

1.345

1.278

1.235

1.293

1.34

1.317

1.391

4712

5406

5933

4562

4984

6118

6755

4739

0.0522

0.0264

0.0221

0.0451

0.1097

0.0873

0.04

0.0436

Goodness-of-fit on F2 0.971

1.039

1.034

1.080

0.923

0.992

1.052

1.024

R1 (I > 2σ(I))

0.0464

0.0397

0.0403

0.075

0.066

0.0569

0.0634

0.0493

wR2

0.1013

0.1049

0.0953

0.2268

0.1082

0.1235

0.1501

0.1145

Independent reflections Rint

The superscripts a and b represent the single crystal structure of APA+SEA measured at 173(2) and 276(2) K respectively.


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Figure 4. The hydrogen bonding pattern in the crystal structure of APA+ADA along b-axis. ADA interacts with APA through N–H···O hydrogen bonds to form a tape stucture. Apatinib+Sebacic Acid (APA+SEA, 2:1) Cocrystal. The single crystal diffraction were performed at 173 and 276 K, suggesting the crystal structure of APA+SEA was changed from the measurement temperature. The simulated PXRD pattern of APA+SEA at 276 K differ from that at 173 K (Figure S5). However, the hydrogen bonding network and steric packing pattern are similar. Due to the crystal structural disorder of APA+SEA at 276 K resulted from the thermal vibration of atoms, the crystal structure of APA+SEA at 173 K was presented (Figure 5). APA+SEA cocrystal crystallizes in the triclinic P-1 space group with one APA molecule and a half equivalent of SEA molecule in the asymmetric unit. SEA is located on a crystallographic inversion center. A R22 (18) supramolecular dimer formed as the basic building

block

via

the

hydrogen

bonds

between

two

APA

molecules

((N(4)−H(4)···N(5), 3.135Å, 159°). Two dimers are connected with one SEA molecule via the hydrogen bonds between N(3) in pyridine group and O(2) in the



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carboxylic group (O(2)−H···N(3), 2.670 Å, 165°) to form a liner chain. The liner chains are linked to a 2D network via the hydrogen bond between the O(2) in carboxylic group and the N(2) in the amide group (N(2)–H···O(2), 3.249 Å, 140°).

(a)

(b) Figure 5. (a) APA interacts with SEA through the hydrogen bonds and forms the dimer. (b) The 2D hydrogen bonding network pattern in the crystal structure of APA+SEA. Apatinib+Succinic

Acid

Salt

Monohydrate

(APA+SUA-H2O,

1:1:1).

APA+SUA salt monohydrate crystallizes in the triclinic P-1 space group with one apatinib cation, one succinate anion and one water molecule in the asymmetric unit. The proton transfers from the O(2) in carboxylic group of SUA to the N(3) in pyridine ring B of APA, confirming the salt formation. The ionic bond formed between the O(2) in the deprotonated carboxylic group and N(3) in the protonated pyridine ring B (N(3)–H···O(2), 2.572 Å, 170°). The structure of APA+SUA monohydrate is stacked


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with a chain-by-chain pattern (Figure 6). The hydrophobic chains are linked via the dimer consisted two APA molecules, and the hydrophilic chains are linked via two tetramers between SUA and water molecules. A R22 (18) dimer (D1) formed as the basic building block in the hydrophobic chains of APA+SUA monohydrate, which is connected by hydrogen bonds between N(5) in the cyan-group and N(4) in the amide group (N(4)−H(4)···N(5), 3.151Å, 159°) of adjacent APA molecules. D1 is linked by the O(2) in SUA and the N(2) in the amino group (N(2)−H···O(3), 3.100 Å, 133°) to form a one-dimensional (1D) bimolecular layer chain (BLC1) along the a-axis direction. Due to the introduction of water molecules, more complicated H-bond interactions are involved. The water molecules form a R24 (8) tetramer (T1) with adjacent SUA moieties via hydrogen bonds with O(6)–H(6C)···O(3), 2.742 Å, 173° and O(6)–H(6D)···O(3), 2.885 Å, 161°. Another carboxylic group remains free acid, and linked to the T1 to form another R44 (18) tetramer (T2) by hydrogen bonds between O(4) in the carboxylic group and O(6) in the water (O(4)–H···O(6), 2.617Å, 177°). Therefore, the water molecules of APA+SUA monohydrate are in a DDA (D: donor, A: acceptor) environment.62 The hydrophobic/ hydrophilic chains extend along the a-axis direction respectively and are stacked one by one along the c-axis direction. The 2D networks consisted of hydrophobic/ hydrophilic chains are repeated along the b-axis direction.



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(a)

hydrophobic chain

hydrophilic chain

(b) Figure 6. (a) APA interacts with SUA through the hydrogen bonds and forms the dimer. (b) The hydrogen bonding pattern in the crystal structure of APA+SUA-H2O. SUA interacts with H2O through O–H···O hydrogen bonds to form tetrameric motifs. Apatinib+D/L Mandelic Acid Cocrystal (APA+ D/L-MA, 1:1). APA+D/L-MA cocrystal crystallizes in the monoclinic P21/n space group with one APA molecule and one MA molecule in the asymmetric unit. The synthon is composed of the hydroxyl group and pyridine ring via the hydrogen bond (O(2)–H···N(3), 2.721 Å, 167°). Two synthons form a R22 (18) dimer via hydrogen bonds between N(5) in the cyan-group and N(4) in the amide group (N(4)–H(4)···N(5), 3.183Å, 163°). The dimers are linked to a zigzag chain via the hydrogen bond between O(3) in the hydroxyl group and O(2)


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in the carboxylic group of MA (O(3)–H···O(2), 2.811Å, 174°) along b-axis direction (Figure 7). The zigzag chains are connected into the 2D zigzag network via the O(3)– H···O(2) hydrogen bond along the diagonal line of the aoc plane (Figure S6). The 2D zigzag network is stacked layer by layer along the ac diagonal direction.

(a)

(b) Figure 7. (a) APA interacts with D/L-MA through the hydrogen bonds and forms the dimer. (b) The dimers form a 2D zigzag pattern in the crystal structure of APA+



D/L-MA. The enlarged picture of the selected area is the hydrogen bonds patterns among D/L-MA molecules viewing along the diagonal line of the aoc plane. Apatinib+Salicylic Acid Salt (APA+SA, 1:1). APA+SA salt crystallizes in the monoclinic C2/c space group with one apatinib cation and one salicylate anion in the asymmetric unit. The proton transfers from the O(2) in carboxylic group of SA to the N(3) in pyridine ring B of APA, and the ionic bond (N(3)–H···O(2), 2.562 Å, 169°) confirmed the salt formation. The salt component form a R22 (18) dimer via hydrogen bonds between the N(5) in the cyan-group and the N(4) in the amide group (N(4)– H(4)···N(5), 3.143Å, 161°). The dimers are connected to a bimolecular layer chain (BLC2) via the hydrogen bonds between the O(2) in SUA and the N(2) in the amino group (N(2)–H···O(2), 3.091Å, 129°) along the b-axis direction. The bimolecular layer chains are further stacked via Van der Waals force to form a zigzag skeleton along c-axis direction (Figure 8).

(a)


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(b) Figure 8. (a) APA interacts with SA through the hydrogen bonds and forms the dimer. (b) The dimers form a 2D zigzag skeleton pattern in the crystal structure of APA+ SA. Apatninib+1-Hydroxy-2-Naphthoic Acid (APA+HNA, 1:1) Salt. APA+HNA salt crystallizes in the monoclinic C2/c space group with one apatinib cation and one 1-hydroxy-2-naphthoate anion in the asymmetric unit. The proton transfers from the O(2) in carboxylic group of HNA to the N(3) in pyridine ring B of APA, and the ionic bond (N(3)–H···O(2), 2.554 Å, 170°) confirmed the salt formation. The salt component form a R22 (18) dimer via hydrogen bonds between N(5) in the cyan-group and N(4) in the amide group (N(4)−H(4)···N(5), 3.143Å, 162°). The dimers take a chain-like arrangement via Van der Waals force along the b-axis direction. The dimers are further stacked via Van der Waals force and form a zigzag skeleton along c-axis direction (Figure 9).



(a)

(b) Figure 9. (a) APA interacts with HNA through the hydrogen bonds and forms the dimer. (b) The dimers form a 2D zigzag skeleton pattern in the crystal structure of APA+ HNA. Apatinib+Saccharin (APA+SAC, 1:1) Salt. Saccharin molecules exist as two tautomers corresponding to SAC keto and enol form (Scheme 2), which is similar to acesulfame.63 Generally, SAC as a coformer was architecture with −NH−C=O group


Page 26 of 41

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of SAC keto. However, SAC form salts with APA by −N=C−OH group of SAC enol. APA+ SAC salt crystallizes in the monoclinic C2/c space group with one apatinib cation and one saccharinate anion in the asymmetric unit. The proton transfers from the O(2) in hydroxyl group of SAC enol to the N(3) in pyridine ring B of APA, and the ionic bond (N(3)–H···O(2), 2.636 Å, 171°) confirmed salt formation. The salt component form a R22 (18) dimer via hydrogen bonds between N(5) in the cyan-group and N(4) in the amide group (N(4)−H(4)···N(5), 3.127Å, 161°). The dimers take a chain-like arrangement via Van der Waals force along the b-axis direction. The dimers are further stacked via Van der Waals force and form a zigzag skeleton along c-axis direction (Figure 10).

(a)



(b) Figure 10. (a) APA interacts with SAC through the hydrogen bonds and forms the dimer. (b) The dimers form a 2D zigzag skeleton pattern in the crystal structure of APA+ SAC. Thermal Analysis. The DSC traces for APA, its salts, and cocrystal are shown in Figure 11. The thermal data are summarized in Table 3. Likewise, DSC of the bulk cocrystal exhibits an endotherm for the melting point of the cocrystals and the desolvation/dehydration.


Page 28 of 41

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Figure 11. DSC figures of the apatinib salts and cocrystals. The DSC plots of APA+SUA-H2O had three endothermic peaks. The Tonset, desolvation

of APA+SUA-H2O was 59.73 °C and Tpeak, desolvation was 105.80 °C, which

showed the wide temperature range of desolvation gradual. The ∆Hfusion,desolvation was 101.91 J/g. The second endothermic peak was the melting point with an onset at 134.60 °C (∆Hfusion = 87.80 J/g). Based on the HSM (Figure S7), there was also an amount of residual solid after APA-SUA melting partially. The third small endothermic peak with an onset at 144.61 °C was the melting of residual solid (∆Hfusion = 3.17 J/g). The residual solid identification need to be further analyzed. The other APA salts or cocrystals show one sharp endothermic melting peak, suggesting all had high purity and crystallinity. The melting point of APA+SUA-H2O, APA+ADA, and APA+D/L-MA were lower than that of APA. The melting point of APA+SEA, APA+SA, APA+HNA, and APA+SAC were higher than that of APA. The order was APA+DL MA < APA+SUA (dehydration) < APA+ADA < APA

Stable Cocrystals and Salts of the Antineoplastic Drug Apatinib with

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