Characterization and structure analysis of the heterosolvate of erythromycin thiocyanate

Yuanjie Li,Qiuxiang Yin,2,Meijing Zhang,2,Ying Bao,2,Baohong Hou,2,Jingkang Wang,2,Jiting Huang,Ling Zhou,*

1 School of Chemical Engineering and Technology,State Key Laboratory of Chemical Engineering,Tianjin University,Tianjin 300350,China

2 Collaborative Innovation Center of Chemical Science and Chemical Engineering (Tianjin),Tianjin 300072,China

3 Luoxin Pharmaceutical (Shanghai) Co.,Ltd.,Shanghai 201203,China

Keywords:Erythromycin thiocyanate Heterosolvate Structure analysis Crystallization Thermodynamic properties Transition

ABSTRACT Erythromycin thiocyanate is widely used for the production of other macrolide antibiotics.In this work,a novel heterosolvate of this pharmaceutical compound has been obtained and characterized for the first time,which was transformed from the dihydrate form in the acetone solvent through evaporation crystallization.Thermal behavior together with compositional analysis revealed that both water and acetone molecules participated in the formation of the crystal lattice which is rarely reported before.The general chemical name of the heterosolvate may be defined as erythromycin thiocyanate sesquihydrate hemiacetonate.Furthermore,studies on solid-state spectral analysis provided strong evidence of intermolecular hydrogen bonds in heterosolvate crystals.According to the crystal structure determined by single crystal X-ray diffraction,the formation mechanism of the heterosolvate is proposed in which strong multihydrogen bondings between water and solute molecules form the layer structure.While acetone molecules form single-hydrogen bonds with solutes and reside in channels between layers.This well explains why acetone solvent is easy to escape from the crystal structure during desolvation.

1.Introduction

In recent years,crystal engineering has received more and more attention in the process of drug development.Different crystal forms make the active pharmaceutical ingredients have different physical and chemical properties,bioavailability,etc.,thus affecting the properties of drugs.The search for new solid forms is a hot topic of current research.Solvates are generally referred to the molecular adducts containing a solute compound and a solvent in the same crystal lattice and among which hydrates are the special type of solvates since the solvent is water[1–4].The formation of solvates is a common phenomenon,especially for pharmaceutical materials [5–10].Solvates with better physicochemical property,such as better solubility,particular biochemical activity and so on,can also be used as medicine.Moreover,solvates can produce various crystal forms by desolvation[11,12].Supramolecular chemistry is a new perspective of chemical research in the new century,which refers to the binding between molecules by means of non-covalent bonding force.Solvates has been always the research hotspot of supramolecular chemistry,and the study of their microstructure and macroscopic properties is of great significance for the development and application of supramolecular chemistry.Heterosolvates [13] are defined as special forms of solvates,which are referred to the crystal structures that contains more than one type of solvent.Special interest has been focused on the existence of heterosolvates,and more than 130 cases are found in the Cambridge Structural Database[14–17].Up to five different types of solvent molecules were found in a single crystal structure [17].Heterosolvated pharmaceutical-based materials can pose potential problems throughout the development,as for their dramatically different solid-state properties,solubility,dissolution rate and bioavailability [18,19].The determination of the structure of heterosolvates are more difficult and complex than that of homosolvates (only one kind of solvent in the unit cell).The characterization and analysis can help us better understand the formation process of heterosolvates,obtain new crystal and break the patent monopoly.Overall,the research of heterosolvates is one of the most important projects in crystal engineering and supramolecular chemistry.What’s more,the discovery and fully characterization of heterosolvates of active pharmaceutical ingredients are also vital in the determination of the optimum conditions for crystallization and storage [20,21].At the same time,it is conducive to the understanding of crystallization process,and helps us to limit the choice of solvents in the crystallization process[22],or avoid the formation of unwanted solids,etc.

The target compound,erythromycin thiocyanate(C37H67NO13?-HSCN,CAS Registry No:7704-67-8,hereafter abbreviated as EMTC),is a salt form of erythromycin.As shown in Fig.1,it possesses a 14-membered lactone ring,a cladinose moiety and a desosamine moiety.Due to the highly efficiency against Gram-positive bacteria and mycoplasma infection,it is widely used as a veterinary medicine and raw material for the production of other macrolide antibiotics.It was crystallized from acetone-aqueous mixtures solution through a reactive combined with anti-solvent crystallization [23].Thus,to understand how EMTC interacts with solvents and its ability to form solvate with these solvents becomes vital.The polymorphism of EMTC was rarely reported in literature and only three hydrate forms (hemihydrate,dihydrate and trihydrate)of EMTC are described in patents so far [24].

2.Experimental

2.1.Materials

Erythromycin was purchased from Shanghai Aladdin-Reagent Technology Co.Ltd.(China),with a purity of 98% in mass fraction.Acetone,sodium thiocyanate and acetic acid were provided by Tianjin Kewei Chemical Ltd.(China) with gas–liquid chromatography,guaranteed reagent and analytical reagent grade,respectively.The distilled water was used throughout all experiments.

Fig.1.Chemical structure of EMTC.

2.2.Preparation

2.2.1.Preparation of EMTC dihydrate

EMTC dihydrate was crystallized from erythromycin acetone solutions through reactive crystallization combined with antisolvent crystallization at 303.15 K[23].The detailed crystallization process is described as follows:Firstly,certain amounts of erythromycin and sodium thiocyanate were dissolved in acetone as solvent.Then acetic acid aqueous solution (15% mass concentration) was added to the acetone solution to adjust the pH value.The pH value was measured by a standard pH meter and the final pH value of the solution was controlled at 6.0 to obtain EMTC salt by the reaction of erythromycin and sodium thiocyanate.Next,distilled water,used as an antisolvent,was added to the solution through a syringe pump.The volume of water was three times of that of acetone [23,24].Crystals were obtained by filtration and washed three times with water to remove the adhered mother liquor.The final solid product was dried in a vacuum oven at 303.15 K for 24 h.Then we explored the effect of experimental conditions on the EMTC dihydrate products,including different concentration of acetic acid(15%mass concentration)and different volume of water (four times of that of acetone) as the antisolvent.

2.2.2.Preparation of EMTC heterosolvate

The single crystal of heterosolvate form was produced through evaporation crystallization.Approximately 1.5 g of EMTC dihydrate was dissolved in around 30 g of pure acetone at 303.15 K.The solution was then cooled to 293.15 K.The heterosolvate crystals were allowed to grow in the solution by slow evaporation.The solid form was vacuum filtered and dried in a vacuum oven at 303.15 K for 24 h before being characterized.For the evaporation process,the effect of rapid evaporation with rotary evaporator on the product was also studied.

2.3.Thermal analysis

The melting properties of EMTC heterosolvate and dihydrate were determined by differential scanning calorimetry (DSC 1/500,Mettler Toledo,Switzerland) under the protection of nitrogen gas (70 ml?min-1).The mass of samples was 5–10 mg.The heating rate of samples was set at 10 K?min-1and the temperature range of the experiment was from 308.15 to 458.15 K.Thermogravimetric analysis was conducted at a TGA 1/SF(Mettler Toledo,Greifensee,Switzerland) instrument.The sample (5–10 mg) was heated up at a rate of 10 K?min-1in the crucibles without a cap.The TG curve was recorded from 293.15 to 473.15 K under nitrogen gas protection (90 ml?min-1).

2.4.Composition

The C,H,N and S contents of the heterosolvate was analyzed using a Vario Micro Cube elemental analyzer(Elementar Analysensysteme GmbH,Germany),and the O content was calculated by the difference.The water contents of the heterosolvate and the dihydrate were analyzed by a Mettler Toledo V20 Volumetric Karl Fischer titrator.

2.5.X-ray diffraction

Powder X-ray diffraction (PXRD) tests (D/max-2500,Rigaku,Japan) were used to distinguish structural differences between the two solid-state forms of EMTC.PXRD was conducted at 40 kV and 100 mA with a Cu Kα radiation source(λ=0.154 nm).The samples were scanned at a step size of 0.02°and at a scanning rate of 1 step?s-1from 2° to 50° (2θ).The single crystal X-ray diffraction data of the heterosolvate was collected on a Rigaku-Rapid II diffractometer with a Mo Kα radiation source (λ=0.071073 nm).

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2.6.Morphology

The morphologies of the heterosolvate and the dihydrate forms were characterized by scanning electron microscope (SEM,X650,Hitachi,Japan) and polarized light microscopy (PLM,BX51,Olympus,Japan).The heterosolvate was heated at a rate of 5 K?min-1between 303.15 and 373.15 K under PLM.

2.7.Spectroscopy

Raman Spectroscopy (RXN2,Kaiser Optical systems,Inc.,USA)was applied to the heterosolvate and the dihydrate.Fourier transform infrared spectroscopy (FTIR) of the two solid samples were also recorded on a Tensor 27 infrared instrument (Brucker Corp.,Germany) with attenuated total reflectance accessory.The scanning range was 4000–400 cm-1.

3.Results and Discussion

3.1.Thermal analysis and composition analysis

Typical DSC and TG data are shown in Table 1 and Fig.2.The peak of desolvation(Tpeak,desolvation)and melting process(Tpeakmelt-ing) of the heterosolvate is 369.3 and 442.6 K,respectively.The peak of the desolvation process is at 369.3 K which is much higher than the boiling point of acetone,indicating that the strong interaction bonds between acetone and the solutes made acetone molecules enter into the crystal lattice.The theoretical acetone content of the heterosolvate is 3.42% and the TGA thermograph indicates 3.47%mass loss of the solvate which is quite close to the expected value.This also implies clearly that only the acetone solvent is lost during the heating process instead of both water and acetone.The peak of dehydration and melting process of the dihydrate is 399.6 and 446.9 K,respectively.The measured mass loss of the dihydrate is 4.88%,which is a little higher than that of theoretical value,4.34%.This may due to the strong hygroscopic property of the dihydrate,as the water molecules can easily adsorb on crystal surface.The heterosolvate releases heat during desolvation(ΔHdesolva-tion),based on the initial mass of the sample,of 77.29 J?g-1which varies from the heat of dehydration of the dihydrate,65.68 J?g-1.In addition,the melting enthalpy (ΔHmelting) of the heterosolvate,72.11 J?g-1,is significantly different from that of the dihydrate,32.08 J?g-1.

Table 1 Thermal characteristics of the heterosolvate and the dihydrate

The elemental analysis result of the heterosolvate shows as:m(C)=64.28%,m(H)=10.12%,m(N)=3.76%,m(S)=4.30%,m(O)=17.54%.This is similar to the theoretical values,which are:m(C)=64.36%,m(H)=10.13%,m(N)=3.80%,m(S)=4.34%,m(O)=17.37%.The water content in the heterosolvate is (3.73±0.09)%(mass fraction)using the Karl Fischer method.Further,a simplified molar ratio in the heterosolvate is determined as EMTA/water/acetone=1/1.5/0.5 by the result of thermal analysis and composition analysis.According to the simplified molar ratio,the general chemical name of the obtained heterosolvate can be defined as erythromycin thiocyanate sesquihydrate hemiacetonate.Structures with the solvent water and an organic solvent molecule are consequently called ‘hydrate solvates’.Thus,EMTC heterosolvate can also be defined as hydrate solvate.

Fig.2.TG curves of the heterosolvate and the dihydrate of EMTC.

3.2.Powder X-ray diffraction

PXRD was applied to examine the structures of the heterosolvate and the dihydrate.As shown in Fig.3(1) and (4),the diffractograms produced from the heterosolvate and the dihydrate exhibit sharp,distinct peaks,which are indicative to a high degree of crystallinity.It is worth noting that the characteristic peaks of the heterosolvate and the dihydrate are almost located in exactly the same positions,except for the varied intensity of some peaks.This isostructure phenomenon is not unique and similar cases of pharmaceutical polymorph forming compounds can be found in literature,such as pioglitazone HCl form IIvs.pioglitazone base,and erythromycin dihydratevs.erythromycin anhydrate,just to name a few [25–33].The PXRD patterns of the heterosolvates obtained with different evaporation rates in Fig.3(1) and (2) are consistent,indicating that the same crystal form can be obtained under different experimental conditions.In addition,the experimental diffraction peaks match well with the simulated powder patterns based on the single crystal.In Fig.3(4)–(6),the PXRD patterns of dihydrates obtained under different conditions have the same characteristic peaks with that of reported dihydrates in the literature[34](Fig.3(7)),indicating that they have the same crystal form.

3.3.Morphology

Fig.4 shows the SEM micrographs of the two solid forms which differ notably.The heterosolvate could easily be distinguished by its cubic morphology and the dihydrate exhibited a triangular prism habit.This illustrates that the effect of acetone solvent on the shape of heterosolvate crystals in comparison to the dihydrate samples is remarkable.Fig.4(a) and (c) show the SEM images of the heterosolvate under different evaporation conditions,and the crystal morphology does not change much,indicating that the evaporation rate has no effect on the crystal morphology.InFig.5 samples of the heterosolvate was observed under a hot stage microscope.The removal of acetone leads the crystal diaphaneity decline remarkably with the increasing of temperature from 308.15 to 358.15 K.Cracks appeared then on the crystal surfaces.

Fig.3.Powder X-ray diffraction patterns of the heterosolvate with (1) slow evaporation,(2) rapid evaporation,(3) simulated powder patterns and the dihydrate with (4) the original condition,(5) different concentrations of acetic acid,(6) different amounts of water,(7) reported in the literature.

3.4.Raman spectroscopy and FTIR

As shown in Fig.1,EMTC contains many functional groups,-NH2-,-COO-,-CO-,-OH,which possess the ability to form hydrogen bonds with solvent molecules.In the Raman spectrum as depicted in Fig.6,a clear difference between the two solid forms is the strong absorption band at 1705 cm-1which is assigned to the carbonyl stretching vibration.The heterosolvate shows an apparent characteristic Raman peak at this band while the dihydrate doesn’t have such a peak,confirming the acetone molecules form intermolecular hydrogen bonds with the solute.It should be pointed out that a red shift in the range from 1327 to 1221 cm-1is found in the spectrum of the heterosolvate resulting from the reformation of hydrogen bonds network.The Raman spectrum of the dihydrate contains two more -OH stretching bonds at 1088 and 1425 cm-1.However,the corresponding bands at 1088 and 1425 cm-1are missing in the spectrum of the heterosolvates.The 1362 cm-1band in the dihydrate is replaced by a doublet peak at 1356 and 1364 cm-1in the heterosolvate which are assigned to the branching of the methyl group.

The FTIR spectra shown in Fig.7 also reveal the differences in hydrogen bonds at the carbonyl groups between the heterosolvate and the dihydrate,giving rise to distinct changes in the region of 1650–1750 cm-1.In the heterosolvate,there are three peaks at 1734,1705 and 1685 cm-1,while the peaks at 1739,1704 and 1686 cm-1are to be seen in the dihydrate.Moreover,the -NH2-and-OH stretch is found in the 3100–3500 cm-1region.The dihydrate shows an apparent peak at 3385 cm-1,while the heterosolvate shows the peak at 3434 cm-1.The intensity of the characteristic peaks in the heterosolvate become stronger than that in the dihydrate.

Fig.4.SEM images for the (a) heterosolvate of slow evaporation,(b) dihydrate and (c) heterosolvate of rapid evaporation.

Fig.5.Hot stage micrographs taken at different temperatures during the desolvation process of the heterosolvate:(a) 303.15 K,(b) 346.15 K,(c) 350.15 K,(d) 356.15 K.

Fig.6.Raman spectrum of the heterosolvate and the dihydrate.

3.5.Crystal structure analysis of the heterosolvate

Fig.7.FTIR results of the heterosolvate andthe dihydrate of EMTC.

To better illustrate the roles of the solvents playing during the formation of the heterosolvate,the crystal structure of the heterosolvate was determined for the first time by single crystal X-ray diffraction.Relating data of the heterosolvate are summarized in Table 2.The single crystal structure of the heterosolvate shows that the solid form is a monoclinic system with aP21space group,and the unit cell parametersa=1.4367(3) nm,b=1.7682(3) nm,c=1.7937(4) nm,α=90°,β=90.87(3)°,γ=90°,Z=2,Z′=82,V=4.5561(16) nm3.TheRintvalue was 3.62% and the goodness of fit was 1.036.The refinement of the measurement continued until the final deviation factors,R1andwR2,were 3.71% and 9.29%,respectively.As demonstrated in Fig.8,the asymmetric unit is composed of two molecules of independent solutes,three molecules of water and one molecule of acetone.Three water molecules are linked in the middle of two solutes molecules while the acetone molecule is only occupied by one solute molecule.The forces stabilizing the packing of the heterosolvate are the hydrogen bonds in the crystal cell.

Table 2 Crystallographic data of the heterosolvate

Solvent molecules in crystals have essentially four different functions:(a) to participate in hydrogen-bonding networks;(b)to function as space fillers,with no strong interactions between solvent and solute molecules;(c) to work as ligands completing the coordination around a metal ion;(d) to link bridging between polar and nonpolar regions in the crystal [27].In the case of the heterosolvate of EMTC,the water and acetone solvent molecules mainly participate in the hydrogen-bonding networks.The hydrogen bond length between solute and the two types of solvents are similar to each other,around 0.27 to 0.29 nm.Oxygen atoms in water have proton acceptor and donor functions.Thus,they fit in the hydrogen bond schemes of the structure through multihydrogen bonds.Two of them form three hydrogen bonds,and one of them form four hydrogen bonds through O-H-O or O-H-N.Besides that,these three water molecules in the unit are also linked to each other through O-H-O.While the oxygen atoms in acetone molecules only act as proton acceptor and form single-hydrogen bond with one solute molecule.A 2×2 cell was built as depicted in Fig.9.Solute molecules together with the water molecules form layer structure when observed through a axis.Acetone molecules resident near the channel in the crystal lattice.The consequent interactions between acetone molecules and solute molecules are weaker than those between water and solute molecules,which well explains why during the desolvation process of the heterosolvate only acetone is lost instead of water and acetone together.

Fig.8.The asymmetric unit of the EMTC heterosolvate.

Fig.9.A 2×2 cell of the EMTC heterosolvate observed from a axis.

4.Conclusions

A novel heterosolvate of EMTC was introduced for the first time by evaporation crystallization from an acetone solution.It was characterized using TGA/DSC,SEM/HSM,PXRD,Raman and FTIR,and the relevant results were compared with EMTC dihydrate.Many differences could be found between these two solid forms.Through these solid-state characterization technologies,it was also confirmed that both water and acetone solvents were incorporated into the EMTC crystals and intermolecular hydrogen bonds were formed in the heterosolvate.More specifically,it was found that water molecules jointed with solute molecules by strong multihydrogen bonds and formed layer structures together through the single X-ray diffraction data.Acetone molecules only formed single-hydrogen bonds with solute molecules and existed in the channels between the layers.Therefore,acetone solvents are easier to escape from the crystal structure than water molecules during desolvation.In the future,research on the crystallographic data of EMTC dihydrate and the solvent mediated transformation mechanism from EMTC dihydrate to heterosolvate need to be explored.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Supplementary Material

The X-ray crystallographic information file (CIF) is available for structure of the heterosolvate of EMTC.Supplementary data to this article can be found online at https://doi.org/10.1016/j.cjche.2021.04.005.