Novel solid-solution of the antiretroviral drugs lamivudine and

9 hours ago - The crystalline structures of two members of the solid-solution were determined showing a non-uniform distribution of the solute among t...
2 downloads 6 Views 1MB Size
Subscriber access provided by Warwick University Library

Novel solid-solution of the antiretroviral drugs lamivudine and emtricitabine Jéssica de Castro Fonseca, Juan Carlos Tenorio Clavijo, Natalia Alvarez, Javier Ellena, and Alejandro Pedro Ayala Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.8b00164 • Publication Date (Web): 23 Apr 2018 Downloaded from http://pubs.acs.org on April 24, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26 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

Crystal Growth & Design

1

Novel solid-solution of the antiretroviral drugs lamivudine and emtricitabine Jéssica de Castro Fonseca1, Juan Carlos Tenorio Clavijo², Natalia Alvarez ³, Javier Ellena2 and Alejandro Pedro Ayala5*

¹ Departamento de Farmácia, Universidade Federal do Ceará, Fortaleza, CE, Brazil ² Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil ³ Facultad de Química, Universidad de la República, Montevideo, Uruguay 5

Departamento de Física, Universidade Federal do Ceará, Fortaleza, CE, Brazil

* Correspoding author: Alejandro Pedro Ayala, Departamento de Física, Universidade Federal do Ceará, Po. Box 6030, 60.455-970, Fortaleza (CE) Brazil. E-mail: [email protected]

ABSTRACT Solid solutions could represent a viable alternative to better understand and control structure-property relationships of drugs, in order to optimize their properties for practical applications. Lamivudine (3TC) and emtricitabine (FTC) are nucleoside analogue reverse transcriptase inhibitor antiretroviral drugs, which have similar molecular structures, differing by a single fluorine atom, which is only present in the FTC molecule. Due to these similarities in structure and molecular resemblances, lamivudine and emtricitabine are good candidates for producing a solid solution with physicochemical properties controlled by the stoichiometry. Following this hypothesis, the formation of a nonconventional solid-solution was verified, whose crystalline

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

Page 2 of 26

2

structure is not defined by any of the individual constituents but by the one of the lamivudine hydrate with emtricitabine as a solute. The crystalline structures of two members of the solid-solution were determined showing a non-uniform distribution of the solute among the independent molecules of the asymmetric unit of the lamivudine hydrate structure. Thermal analysis investigations confirmed that physicochemical properties could be controlled through the variation of the emtricitabine content.

Keywords: solid solution, mixed crystal, lamivudine, emtricitabine

ACS Paragon Plus Environment

Page 3 of 26 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

Crystal Growth & Design

3

1. INTRODUCTION The crystal engineering research field is well-defined as the study of the modelling, design, synthesis and application of crystalline solids with predefined and desired aggregation of molecules and ions 1, enabling the design of solid materials with tailored properties 2. Depending on the solid-state form, differences in several physicochemical properties, such as morphology, density, stability, melting point, solubility and even color, may be observed. Properties like solubility, hardness and compressibility could affect the stability, bioavailability and processability of the active pharmaceutical ingredient (API) 3. Homogeneous multicomponent solids (solid solutions)

4, 5

could

represent a viable alternative that could provide a better understanding and controlling of the structure-property relationships in drugs, in order to optimize their physicochemical properties for practical applications. These phases consist of different molecular constituents randomly occupying equivalent crystallographic sites; and, more importantly, the stoichiometry of the solid solutions allow changes in the concentration of each component without limitation to integer values 6. Those changes in composition are often accompanied by a continuous change in some physical and/or chemical properties (e.g., density, solubility, stability, reactivity) 4. One of the main requirements for solid solution formation is based on geometrical approaches, which consider the shape of the host and guest molecules; the packing density in the mixed crystal, as well as, the symmetry of the crystalline structure of the pure components. According to Kitaigorodsky

4

, solid solutions occur between

isomorphic materials, which means the same space group and unit cell dimensions and/or substantially the same type and position of atoms or functional groups; or isostructural materials, having the same structure but not necessarily the same size or chemical composition in the unit cell. In the case of organic solid solutions, the

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

Page 4 of 26

4

similarity between the molecular forms is not only required, but the foremost and necessary condition for its formation 4. The vast land of mixed crystals includes several applications in engineering organic field 7 and pharmacological field8, 9, among others. Lamivudine (3TC) and emtricitabine (FTC) (Figure 1) are nucleoside analogue reverse transcriptase inhibitor antiretroviral drugs, use for HIV-positive and hepatitis Bpositive patients 10. The crystalline structure of several polymorphs and multicomponent forms of 3TC and one form of FCT were already reported 11-17. However, from the point of view of this study, just pure and hydrated forms are relevant. Thus, 3TC can be found as a pure form, with a single 3TC molecule per asymmetric unit; a hydrate, whose asymmetric unit contains one water and five 3TC molecules; and a hemihydrate, presenting four lamivudine and two water molecules per asymmetric unit

11, 18

. Despite

the fact that FTC has an extremely similar molecular structure to 3TC, differing by a single fluorine atom at the C5 position of the aromatic ring (Figure 1), just one crystalline structure of the former was reported 17.

Figure 1. Molecular structure of 3TC and FTC, numbered following the IUPAC rules.

Examples of organic mixed crystals can be seen in the 2-R-naphthalenes family 19, omeprazole tautomeric forms 9, benzylidenecyclopentane derivatives 7, they generally

ACS Paragon Plus Environment

Page 5 of 26 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

Crystal Growth & Design

5

present isomorphous constituents, nonetheless, several solid solutions of nonisostructural crystals have also been previously reported 20, 21. In this work we report the unprecedented crystalline structure of the solid solution between two APIs: 3TC and FTC. Single-crystal structure determinations combined with thermal analysis assays allowed us to investigate the impact of the solid solution stoichiometry in the physicochemical properties.

2. EXPERIMENTAL SECTION Materials and sample preparation. Lamivudine (3TC) and Emtricitabine (FTC) were gently provided from Nortec. All other chemicals and solvents were commercially available and used without further purification. 3TC and FTC mixtures in 1:1 and 2:1 rations were dissolved under magnetic stirring using the minimum amount of solvent (1:1 methanol:water solution). After filtering, the resulting solutions were allowed to slowly evaporate at low temperature (2ºC ~ 8ºC). Single crystals were harvested before the complete evaporation of the solvent. Other concentration ratios were tested unsuccessfully.

Powder X-ray Diffraction (PXRD). Powder diffraction patterns were recorded on a D8 Advanced Bruker AXS difractometer equipped with a theta/theta goniometer, operating in the Bragg Brentano geometry with a fixed specimen holder, CuKα radiation source and a LynxEye detector. The voltage and electric current applied were 40 kV and 40 mA, respectively. The slit opening for the incident beam on the sample was set at 0.2 mm. Samples were scanned within the scan range in 2θ of 5° to 35° in the continuous scan mode, with a scan rate of 1 deg min-1.

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

Page 6 of 26

6

Single Crystal X–ray Structure Determination. Single-crystal X–ray diffraction data collections (ϕ scans and ω scans with κ offsets) were performed on a Bruker D8 Venture diffractometer equipped with a PHOTON 100 CMOS detector using graphite– monochromated CuKα radiation at room temperature. Data were processed using 22

Bruker APEX 3 package

. The structures were solved by iterative methods

implemented in ShelXT 23 and refined with ShelXL 24 using the Olex2 program 25. The programs MERCURY

26

, ORTEP–3

27

and VEGA28 were used to prepare the

crystallographic information files (CIF) and artwork representations for publication. The fluorine occupation was refined with no constrains. No significant correlations between the thermal ellipsoid and the fluorine occupation factors were observed. Hirshfeld surfaces and the corresponding fingerprint maps were calculated with the program Crystal Explorer

29

. The CIFs files are deposited in the Cambridge Structural

Data Base under the codes 1541526 (3TCH:56) and 1541527 (3TCH:43).

Thermal analysis. Thermogravimetric (TG) and Differential Scanning Calorimetry (DSC) curves were obtained using Simultaneous Thermal Analysis equipment (Jupiter STA 449, Netzch). Approximately 5 mg of the sample were placed in sealed aluminum crucibles with pierced lids. Measurements were made from room temperature up to 200 °C using 5 °C/min heating rate. The experiment was carried out under a constant flow of nitrogen (70mL/min). A polarized Leica microscope (DM2500P) coupled with a hotstage Linkam (FTIR600) was used to perform Hot Stage Microscopy assays. Images were recorded with a QICAM (Fast1394) camera and processed with the Linksys32 software.

ACS Paragon Plus Environment

Page 7 of 26 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

Crystal Growth & Design

7

High Performance Liquid Chromatography. HPLC was performed using a Varian equipment with two Dynamax Rainin SD-200 pumps, UV/VIS detector, diode Prostar 335 grid. The mobile phase was a solution of phosphate buffer and acetonitrile (95:5), under pH 3,0; the column Kinetex EVO 5 µm C18 100A, 150x4,6mm was used on a LC Workstation v 6.41 system. The 20µL injection were performed in triplicates to increase precision of the results. A protocol for determination 3TC and FTC independently was developed (see Supporting information) based on the British Pharmacopoeia

30

and

Indian Pharmacopoeia 31.

3. RESULTS AND DISCUSSION The molecular similarity between 3TC and FTC supported the hypothesis of formation of a solid solution. However, from the point of view of their crystalline structures, they exhibit different crystal packing motifs. Crystalline structure of the only anhydrous form of 3TC belongs to the P43212 space group and consists of a highly symmetric H-bond network of N–H···O and O–H···N type, between the cytosinic fragments and the –CH2OH groups in a tetragonal fashion 11. On the other hand, FTC crystallizes in the P21 space group with the molecules forming dimers connected by the classic homosynthon NCN···NCN between cytosinic fragments, which in turn are linked by H-bonds in helical arrangement along the crystallographic b-axis 17. Thus, in principle, the 3TC-FTC system seems to fail to fulfill the conditions for obtaining a solid solution. Despite of that, slow evaporation crystallization experiments following the procedure described in the Experimental Section were conducted and mixed crystals were obtained. Single crystals of two concentrations of 3TC-FTC mixed crystals were isolated and the corresponding crystalline structures determined (Table S1). The studied solid

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

Page 8 of 26

8

solutions are isomorphous and crystallize in the P212121 space group. The asymmetric unit contains five 3TC/FTC independent molecules (Z’=5), as well as one water molecule. A comparison with the reported structures for the single components showed that the mixed crystals are isomorphous to the lamivudine hydrate (3TC:0.2 H2O) reported by Harris et al.

11

, as it can be verified by superimposing the corresponding

asymmetric units (Figure 2a). This superposition gives a very low deviation factor (rmsd = 0.3), which is mainly associated to the positional disorder of the water molecule as it will be discussed in detail later. It is also important to point out, that several attempts to produce the FTC hydrate were unsuccessful suggesting that it might be not as stable as the 3TC one. Based on this observation, hereafter, the lamivudine hydrate (3TCH) will be considered the matrix where a x concentration of FTC is dissolved by substituting 3TC molecules (hereafter, 3TCH:xFTC). Regarding to the molecular conformations, overlapping all conformers of the 3TCH:xFTC structure (Figure 2b) shows that conformers A and B are equivalent, whereas the other ones differ in the relative orientation of the phenyl rings and the hydroxyl-methylene groups.

ACS Paragon Plus Environment

Page 9 of 26 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

Crystal Growth & Design

9

Figure 2. Overlap of the (a) asymmetric units and (b) molecular conformers.

The reported structures are unusual examples of mixed crystals showing two nonisomorphous molecules forming a solid-solution in a solvated form. Recently, a solid solution of the related compounds cytosine/5-flucytosine was also reported as a monohydrate, which has is isomorphous to a 5-flucytosine monohydrate, but the cytosine counterpart was not observed 8, 32. As stated, in the obtained solid solution, there are five molecules in the asymmetric unit where 3TC and FTC are disordered. These conformers (labeled from A to E) have similar conformational parameters differing only in the occupation factor (s.o.f.) of the F atoms of FTC molecules, as they are dissolved into to the 3TCH structure which acts as the host in the solid solution. Naturally, the concentrations of F-atoms depend on the 3TC:FTC stoichiometry, the higher the FTC concentration, the higher the site occupation of F-atoms. However, in spite of the good resolution attained in the

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

Page 10 of 26

10

crystallographic measurements, the structure refinement could not be precise enough to establish without doubt the FTC concentration. The 3TC:FTC ratio in the solid solution was determined directly from the single crystals using the HPLC protocol developed for this purpose (see Supplementary material), which provided great resolution as the peaks associated with both components are clearly isolated (Figure S2). To verify that the studied single crystals were indeed a good representation of the correspondent bulk materials, the samples were also studied by PXRD (Figure 3). Despite the isomorphous character of both solid solutions, the PXRD patterns exhibit a large number of features fingerprinting each composition. HPLC results have confirmed that two well defined concentrations were produced: x=0.56(2) and x=0.43(2), in good agreement with the stoichiometry obtained by refining the F atoms s.o.f. in each structure (x=0.54(2) and 0.40(2)).

Figure 3. Experimental (E) and calculated (C) powder X-ray diffraction patterns of the solid solution: 3TCH:0.56FTC and 3TCH:0.43FTC.

ACS Paragon Plus Environment

Page 11 of 26 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

Crystal Growth & Design

11

The s.o.f of the fluorine atoms were carefully refined for both solid solutions and the corresponding values listed in Table 1. It is a relevant parameter because the occupation of the fluorine sites is the marker for the 3TC molecule substituted by FTC. It is clear that fluorine atoms (FTC molecules) are not uniformly distributed among the five 3TC sites. Conformer C is preferentially occupied by FTC, whereas the remaining sites form two groups A/B and D/E with similar occupations, being the latter the one with smaller s.o.f.

It is important to notice that this distribution is maintained

independently of the FTC concentration, but, as the FTC concentration increases, sites B and C are slightly preferred (∆s.o.f.~0.16) over sites D and E (∆s.o.f.~0.12) and site A (∆s.o.f.~0.09).

Table 1: Aspects of Substitutional Replacement in the 3TCH:xFTC solid slution. Unit Cells a (Å) b (Å) c (Å) 3

Volume (Å )

3TCH:0.56FTC

3TCH:0.43FTC

3TCH (Form I)

10.5233(3) 14.4708(4)

10.8415(3) 14.3550(4)

10.427(2) 14.327(3)

34.7146(10)

34.2245(9)

34.851(7)

5286.4(3)

5326.4(3)

5206.3(2)

Solid Solutions x

0.54(2) s.o.f

s.o.f

∆sof

F5A

0.593(8)

0.497(12)

0.096(14)

F5B

0.676(8)

0.505(13)

0.171(15)

F5C

0.724(10)

0.565(13)

0.159(16)

F5D F5E

0.346(9) 0.351(9)

0.222(13) 0.228(12)

0.124(16) 0.123(15)

0.40(3)

The crystal packing of the solid solutions show a complex network of intermolecular interactions (Table S2), stabilized by the formation of molecular columns constituted by B conformers and water molecules (B-columns, Figure 4). The water molecule adopts the most common environment according to the Gillon et al.

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

Page 12 of 26

12

classification 33. B-columns are the result of the helical distribution of the B-conformers linked by classic hydrogens bonds involving water molecules through N4B– H41B···O2B, O1W–H1WA···O2B, O1W–H1WB···N3B and N4B–H42B···O1W interactions, around the 21-screw axis along the crystallographic a-axis. Even though the water molecules contribute to stabilization of the B-columns, they are located in a 41 Å3 cavity, which offers some occupational flexibility. Thus, two different organizations were observed, the water molecule is at a well-defined position in 3TCH:0.56FTC, whereas it is disordered among three sites in 3TCH:0.43FTC. Differently to the behavior of other continuous solid solutions

6, 34

, a well defined

variation of the lattice parameters was not observed in the case of 3TCH:xFTC, as it can be noticed in Table 1. This effect is consequence of the non-uniform distribution of the guest molecules, as well as, the possibility of water being disordered. It is clear that the FTC increases the lattice volume due to the replacement of one hydrogen by fluorine, but the concentration dependence is not linear. For example, the a lattice parameter is bigger for x=0.43 than 0.56, probably due to the disorder of the water molecule linking the B-conformers along the [100] direction (Figure 4a). On the other hand, c exhibits an opposite behavior, whereas b continuously increases following the FTC concentration. Thus, the crystalline structure of more members of the 3TCH:xFTC solid solution needs to be determined in order to develop a reliable model for the concentration dependence of the lattice parameters.

ACS Paragon Plus Environment

Page 13 of 26 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

Crystal Growth & Design

13

Figure 4. (a) B-columns along the a-axis. (b) bc-projection of the B-D supramolecular structure.

B-columns are linked to D-conformers through O5’B–H5’B···O2D and N4D– H42D···O5’B hydrogen bonds. Based on these interactions, B and D conformers form a 10 member ring stacked along the [100] direction (Figure 4). The remaining conformers are placed in the interstices of this supramolecular structure. A dimer formed by A and E conformers (A=E dimer) can be easily identified, determined by base-pairing through N–H···O interactions of the cytosine fragments (homodimers), commonly found in the literature (Figure 5a)

35, 36

. This homodimer forms a R22(8) motif through the N4A–

H41A···N3E and N4E–H41E···N3A H-bonds. It is also interesting to point out that the A=E dimer is similar to the one observed in the FTC crystalline structure 17 , and could be considered as the base motif for the dissolution of this molecule into the 3TCH

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

Page 14 of 26

14

structure. In addition, the hydroxyl-methylene groups of the C-conformers are also bonded to the A=E dimers, acting as H bonds donor/acceptor simultaneously through O5’C–H5’C···O2E and N4A–H42A···O5’C interactions giving rise to a ܴଷଷ (8) motif (Figure 5a). The A=E-C trimer are placed in the B-D supramolecular structure cavities linked by classical H-bonds just to the D conformers. The A=E dimer is connected to two D conformers: one at the cytosine group (N4D–H41D···O2A and N4E–H42E···N3D) and other at the hydroxyl-methylene group (O5’A–H5’A···O5’D). On the other hand, the cytosine moiety of the C-conformers is also linked to the hydroxyl group of a D conformer (O5’D–H5’D···N3C). In addition to the described interactions, several Hbonds listed in Table S2 allow the helical stacking of the A=E-C trimers along the baxis. Figure 5d shows the HS of conformed D mapped with the shape index exhibiting the complementary blue and red spots with tie shape around the centroids of the cytosinic fragments fingerprinting a π···π interaction 37, 38. This interaction disposes the cytosine fragments of the D and E conformers in a face-to-face stacking (Table S2)

ACS Paragon Plus Environment

Page 15 of 26 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

Crystal Growth & Design

15

Figure 5. Selected motifs related to the intermolecular interactions of the 3TCH-xFTC solid solution.

The B-conformers are involved in the formation of B-columns with the water molecules, and the substitutional replacement leads to a slight shortening of the intermolecular bond distances (see Table S2) when comparing to those of the 3TCH. Furthermore, A and C conformers, located into the voids formed by the distribution of the B-columns, also shows a slight shortening of the bonds distances. It is worth mentioning that the positioning of these conformers leads to a lower number of intermolecular interactions, leaving them freely along with the B conformers to carry out the substitutional replacement, since in these positions the substitution of 3TC by FTC molecules leads to a slight stabilization of the structure, at least from the intermolecular interactions standpoint. On the contrary, D and E are responsible for linking the B-columns and placed in their interstices. These conformers are involved in a complex intermolecular interaction network associating all the other conformers and

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

Page 16 of 26

16

even contributing with π-stacking between them. Therefore, these features of the supramolecular role of conformers D and E do not favor the substitutional replacement in these positions, leaving the 3TC molecules firmly bonded in these positions, which would be in agreement with the low s.o.f values of F-atoms found for these conformers. A comparative analysis of the HS, especially of the contact maps (fingerprints), shows subtle differences among the conformers of the solid solutions. Figure 6 shows the fingerprints of all the conformers in 3TCH:0.56FTC, highlighting selected intermolecular interactions. Furthermore, the percentage contribution of the main interactions is also presented in this figure. In all cases, there are notorious sharp spikes corresponding to the O···H and N···H contacts, which are involved in the H-bond networks. The two O···H sharp spikes are observed for all conformers, but just one N···H can be identified in conformers B and C, due to the absence of XH···N intermolecular contacts. In all cases the biggest contribution is due to the H···H contacts, which present the highest percentages over the surface, probably due to the closely packing that results from the proximity of the molecules in theses crystalline arrangements. These contacts are characterized by a sharp peak in the diagonal of the fingerprint plot (around de= di= 1.1 Å). The only exception being the D-conformers that do not present any clearly H···H contact peak. In this case the most intense peaks correspond to the two O···H and N···H contacts related to the strong O–H···O, O– H···N and N–H···N H-bonds. This effect is related to the fact that the D-conformers are the linkers of the B-columns, being precisely the O···H and N···H contacts the main intermolecular interactions for these conformers.

ACS Paragon Plus Environment

Page 17 of 26 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

Crystal Growth & Design

17

Figure 6. – Fingerprint plots of the conformers in the 3TCH:0.56FTC structure. (bottom-right) Percentage contributions to the Hirshfeld surface area for selected close intermolecular contacts for the different conformers.

An interesting type of intermolecular interaction arises as a result of the substitutional replacement of FTC molecules into the solid solutions. The unusual C– F···F–C interactions, called the organic fluorine, have been extensively studied even by accurate charge density analysis

39

. In order to emphasize these interactions, HS

surfaces were calculated considering the hypothetical structure of the FTC hydrate based on the 3TCH:0.56FTC where all fluorine sites are fully occupied. The normalized HS reveals information about the F···F. In the same way, the curved surface depicts the deformation contours around the contact region. The fingerprint plots show that B, D and E conformers have a slight contribution from F···F contacts (1.6, 0.5 and 2.1% of the corresponding total contacts, respectively). According to Reichenbächer et al. 39, the

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

Page 18 of 26

18

low fluorine polarizability leads to a weak attractive interatomic dispersion forces and, consequently, the F···F interactions are rarely observed. The 3TCH:xFTC solid solution does not present any special feature suggesting that the F···F interactions interaction could be favored over conventional intermolecular interactions. The shorter F···F distance is observed between F5B and F5E (2.803(8) Å), being just below sum of the van der Waals radii. If F···F interactions are favored, both sites should have a high FTC occupation. However, that is not the case, because B conformers are in the set of FTC preferentially occupied sites, but E sites always exhibit a low FTC concentration, suggesting a competitive occupation of these sites. The geometry of the C5BF5B···F5EC5E interaction could be classified as Type I (θ1=126.5o and θ2=115.2o) according to Ramasubbu et al.40. This type of interactions does not involve the polarization of the halogen atoms, but there are originated in the close packing and does not form stabilizing interactions

39

. Notice that, the other two sites with a high FTC

concentration do not exhibit any F···F interaction in the HS fingerprints. The remaining site (D conformer) could form F···F interactions with the E-conformer, but it also exhibits low fluorine s.o.f. These results shows that the FTC site occupation is determined by the environment of each conformer, similarly to what was observed by Mínguez Espallargas et al.41 Is interesting to point out here that the low affinity of the F···F interactions in this compound could also explain the unsuccessful search for the isomorphous FTC hydrate. As verified in the hypothetical structure used for the HS calculations, the F5B···F5E interaction cannot be avoided if all the 3TC molecules are substitute by FTC. In fact, the combined site occupation of B and E sites (s.o.f.B+s.o.f.E) raises from ~0.7 to ~1 when x increases from 0.40 to 0.54. In the later, each asymmetric unit has either B or E sites occupied by FTC. As higher FTC concentrations should enforce F5B···F5E

ACS Paragon Plus Environment

Page 19 of 26 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

Crystal Growth & Design

19

interactions, the solubility limit could be determined by the condition s.o.f.B+s.o.f.E ~ 1. Thus, a continuous solid solution covering the whole range of concentrations could be observed

34, 42

when all the Kitaigorodsky’s requirements are fulfilled. On the other

hand, if one of the limit cases is not stable, a set of interactions should be driving the instability as the solute contents increases and a solubility limit could be expected. In the case of the solid solution reported here, the F5B···F5E interactions seems to be the main instability point as it becomes shorter as the FTC content increases. Unfortunately, the determination of the FTC concentration limit was not possible on the basis of the current results. The main goal of producing solid solutions is to fine tune key properties by controlling the stoichiometry. As an example of that, the thermal behavior of solid solutions was investigated. DSC curves of the 3TCH:xFTC samples show two overlaped endothermic peaks (Figure 7a). The temperature onsets are Tonset1 = 107.1oC and Tonset2 = 119.2oC and Tonset1 = 112.3oC and Tonset2 = 124.9oC for x=0.56 and x=0.43, respectively. These values agree very well with the DSC curve of 3TCH reported by Harris et al.11, which exibits an event at 119.9 oC clearly associated to a melting followed by recrystallization. Thus, the first peak can be associated to the melting of the hydrated form confirmed by the mass loss oberved in the TG curve (Figure S6). The mass loss at these events was about 1-1.4%, in good agreement with the theoretical value (~1.5%), confirming the lost of the water molecule. The second event is related to the melting point of the recrystallized sample. This assumption was confirmed by hotstage microscopy images (Figure 7b). Finally, the concentration dependence of the melting point of the 3TCH:xFTC solid solutions is presented in Figure 7c showing a linear dependence between this physical property and FTC concentration.

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

Page 20 of 26

20

Figure 7. (a) Differential scanning calorimetry curves of the 3TCH:xFTC solid solution and the raw materials. (b) Hot stage microscopy images of the 3TCH:0.43FTC crystal. (c) Melting point of the solid solution as a function of the FTC concentration.

CONCLUSION A new organic solid solution was obtained using simple execution techniques, supported by the possibility of modulating the concentration of drugs inside the crystalline structure. The 3TCH:xFTC mixed crystals behave as a dynamic structure, based on the 3TC hydrate structure which acted as a template for solid solution formation. The high structural affinity and similarity between the molecules of both APIs is evidently the main cause for the substitutional replacement, as suggested by Kitaigorosdky in his experimental approach about formation of organic solid solutions. Both APIs (3TC and FTC) have similar solubilities, which is also an important feature at the moment of growing the mixed crystal 4. However, the isomorphism condition

ACS Paragon Plus Environment

Page 21 of 26 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

Crystal Growth & Design

21

between the solid solution constituents was not satisfied, since the FTC hydrate seems to be instable. The substitutional replacement of 3TC by FTC is not uniform among the nonequivalent molecules of the asymmetric unit, giving rise to slight variations in the cell dimensions. This effect is also related to the disorder of the water molecules within the B-columns. The site selectivity of the FTC molecules seems to be influenced by the raising of F···F interactions. The crystal packing of the 3TCH structure just allows non attractive type I halogen-halogen interactions 40. In fact, it was verified that this is the preferred configuration for F···F interactions 43, but it needs to be notice that the bond length should be in almost all the cases above the sum of the van der Waals radii 39. For the 3TCH:xFTC solid solution, in the case of a full FTC substitution, the shorter F···F distance should be just below the sum of the van der Waals radii, giving rise to a steric hindrance and, consequently, becoming the main driven force of instability of the elusive FTC hydrate. The final goal of developing mixed crystals is the control of physicochemical properties. It was successfully proven that the melting point of the 3TCH:xFTC solid solution can be tuned by the FTC concentration. However, the solubility limit, and consequently, the melting point range will be subject of further studies.

ACKNOWLEDGMENTS

We gratefully acknowledge the financial support of FUNCAP-PPSUS, CAPES, FAPESP and CNPq.

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

Page 22 of 26

22

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: Crystallographic data including a list of the main interactions. Scanning electron microscopy images of representative crystals of the solid solution. Description of the HPLC methodology. Full x-ray powder patterns of the solid solution and raw materials. Representative thermogravimetry and DSC curves. Additional Hirshfeld surfaces and fingerprints.

Accession codes. CCDC 1541526-1541527 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, Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033

ACS Paragon Plus Environment

Page 23 of 26 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

Crystal Growth & Design

23

REFERENCES (1) Braga, D.; Desiraju, G. R.; Miller, J. S.; Orpen, A. G.; Price, S. L., Innovation in crystal engineering. CrystEngComm 2002, 4, 500-509. (2) Duggirala, N. K.; Perry, M. L.; Almarsson, Ö.; Zaworotko, M. J., Pharmaceutical cocrystals: along the path to improved medicines. Chem. Commun. 2016, 52, 640-655. (3) Shan, N.; Perry, M. L.; Weyna, D. R.; Zaworotko, M. J., Impact of pharmaceutical cocrystals: the effects on drug pharmacokinetics. Expert opin. drug met. 2014, 10, 1255-71. (4) Kitaigorodsky, A. I., Mixed Crystals.; 1984. Berlin, Heidelberg: Springer Berlin Heidelberg. (5) Vippagunta, S. R.; Brittain, H. G.; Grant, D. J., Crystalline solids. Adv. drug delivery reviews 2001, 48, 3-26. (6) LusiI, M.; Vitorica-Yrezabal, I. J.; Zaworotko, M., Expanding the Scope of Molecular Mixed Crystals Enabled by Three Component Solid Solutions. Cryst. Growth Des. 2015, 15, 4098-4103. (7) Theocharis, C. R.; Desiraju, G. R.; Jones, W., The Use of Mixed-Crystals for Engineering Organic Solid-State Reactions Application to Benzylbenzylidenecyclopentanones. Am. Chem. Soc. 1984, 106, 3606-3609. (8) Braun, D. E.; Griesser, U. J., Prediction and experimental validation of solid solutions and isopolymorphs of cytosine/5-flucytosine. CrystEngComm 2017, 19, 35663572. (9) Mishra, M. K.; Ramamurty, U.; Desiraju, G. R., Solid solution hardening of molecular crystals: tautomeric polymorphs of omeprazole. J. Am. Chem. Soc. 2015, 137, 1794-7. (10) De Clercq, E., Antiviral drugs in current clinical use. J. Clin. Virol. 2004, 30, 15133. (11) Harris, R. K.; Yeung, R. R.; Lamont, R. B.; Lancaster, R. W.; Lynn, S. M.; Staniforth, S. E., 'Polymorphism' in a novel anti-viral agent: Lamivudine. J. Chem. Soc. Perk. T 2 1997, 2653-2659. (12) Vasconcelos, A. T.; da Silva, C. C.; Queiroz Júnior, L. H. K.; Santana, M. J.; Ferreira, V. S.; Martins, F. T., Lamivudine as a Nucleoside Template To Engineer DNA-Like Double-Stranded Helices in Crystals. Cryst. Growth Des. 2014, 14, 46914702. (13) Ellena, J.; Bocelli, M. D.; Honorato, S. B.; Ayala, A. P.; Doriguetto, A. C.; Martins, F. T., Base-Paired and Base-Stacked Structures of the Anti-HIV Drug Lamivudine: A Nucleoside DNA-Mimicry with Unprecedented Topology. Cryst. Growth Des. 2012, 12, 5138-5147. (14) Ellena, J.; Paparidis, N.; Martins, F. T., Toward supramolecular architectures of the anti-HIV drug lamivudine: understanding the effect of the inclusion of water in a hydrochloride form. CrystEngComm 2012, 14, 2373-2376. (15) Bhatt, P. M.; Azim, Y.; Thakur, T. S.; Desiraju, G. R., Co-Crystals of the AntiHIV Drugs Lamivudine and Zidovudine. Cryst. Growth Des. 2009, 9, 951-957. (16) Martins, F. T.; Guimaraes, F. F.; Honorato, S. B.; Ayala, A. P.; Ellena, J., Vibrational and thermal analyses of multicomponent crystal forms of the anti-HIV drugs lamivudine and zalcitabine. J. Pharmaceut. Biomed. 2015, 110, 76-82.

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

Page 24 of 26

24

(17) Van Roey, P.; Pangborn, W. a.; Schinazi, R. F.; Painter, G.; Liotta, D. C., Absolute configuration of the antiviral agent (-)-cis-5-fluoro-1-[2-hydroxymethyl)-1,3oxathiolan-5-yl]cytosine. Antivir. Chem. Chemoth. 1993, 4, 369-375. (18) Bhattacharya, A.; Roy, B. N.; Singh, G. P.; Srivastava, D.; Mukherjee, A. K., Lamivudine hemihydrate. Acta Crystallogr. C 2010, 66, O329-O333. (19) Haget, Y. C., N. B.; Meresse, A.; Bonpunt, L; Michaud,a F.; Negrier, P.; Cuevas-Diarteb, M. A.; Oonk, H. A. J., Isoomorphism and mixed crystals in 2-Rnaphthalenes: evidence of structural subfamilies and prediction of metastable forms. J. Appl. Crystallogr. 1999, 32, 481-488. (20) Schur, E.; Nauha, E.; Lusi, M.; Bernstein, J., Kitaigorodsky Revisited: Polymorphism and Mixed Crystals of Acridine/Phenazine. Chem-Eur. J. 2015, 21, 1735-1742. (21) Zhan, W. H.; Wu, W. J.; Hua, J. L.; Jing, Y. H.; Meng, F. S.; Tian, H., Photovoltaic properties of new cyanine-naphthalimide dyads synthesized by 'Click' chemistry. Tetrahedron Lett. 2007, 48, 2461-2465. (22) Bruker AXS Inc, M., Wisconsin, USA. APEX3, SAINT and SADABS, 2015. (23) Sheldrick, G. M., Crystal structure refinement with SHELXL. Acta Crystallogr. C 2015, 71, 3-8. (24) Sheldrick, G. M., A short history of SHELX. Acta Crystallogr. A 2008, 64, 112122. (25) Dolomanov, O. V.; Bourhis, L. J.; Gildea, R. J.; Howard, J. A. K.; Puschmann, H., OLEX2 : a complete structure solution, refinement and analysis program. J. Appl. Crystallogr. 2009, 42, 339-341. (26) Macrae, C. F.; Edgington, P. R.; McCabe, P.; Pidcock, E.; Shields, G. P.; Taylor, R.; Towler, M.; van de Streek, J., Mercury: visualization and analysis of crystal structures. J. Appl. Crystallogr. 2006, 39, 453-457. (27) Farrugia, L., ORTEP-3 for Windows - a version of ORTEP-III with a Graphical User Interface (GUI). J. Appl. Cryst. 1997, 30, 568. (28) Pedretti, A.; Villa, L.; Vistoli, G., VEGA: a versatile program to convert, handle and visualize molecular structure on Windows-based PCs. J. Mol. Graph. Model. 2002, 21, 47-49. (29) Turner, M. J.; McKinnon, J. J.; Wolff, S. K.; Grimwood, D. J.; Spackman, P. R.; Jayatilaka, D.; Spackman, M. A. CrystalExplorer17 University of Western Australia.: 2017. (30) British Pharmacopoeia Commission. 2012. British pharmacopoeia 2013. London: Stationery Office. (31) India, and Indian Pharmacopoeia Commission. 2010. Indian pharmacopoeia, 2010. Ghaziabad: Indian Pharmacopoeia Commission. (32) Braun, D. E.; Kahlenberg, V.; Griesser, U. J., Experimental and Computational Hydrate Screening: Cytosine, 5-Flucytosine, and Their Solid Solution. Cryst. Growth Des. 2017, 17, 4347-4364. (33) Gillon, A. L.; Feeder, N.; Davey, R. J.; Storey, R., Hydration in Molecular CrystalsA Cambridge Structural Database Analysis. Cryst. Growth Des. 2003, 3, 663673. (34) Oliveira, M. A.; Peterson, M. L.; Klein, D., Continuously Substituted Solid Solutions of Organic Co-Crystals. Cryst. Growth Des. 2008, 8, 4487-4493. (35) Etter, M. C., Encoding and Decoding Hydrogen-Bond Patterns of OrganicCompounds. Accounts Chem. Res. 1990, 23, 120-126.

ACS Paragon Plus Environment

Page 25 of 26 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

Crystal Growth & Design

25

(36) Etter, M. C.; Macdonald, J. C.; Bernstein, J., Graph-Set Analysis of HydrogenBond Patterns in Organic-Crystals. Acta Crystallogr. B 1990, 46, 256-262. (37) Spackman, M. A.; Jayatilaka, D., Hirshfeld surface analysis. CrystEngComm 2009, 11, 19-32. (38) McKinnon, J. J.; Spackman, M. A.; Mitchell, A. S., Novel tools for visualizing and exploring intermolecular interactions in molecular crystals. Acta Cryst. B 2004, 60, 627-668. (39) Reichenbächer, K.; Süss, H. I.; Hulliger, J., Fluorine in crystal engineering--"the little atom that could". Chem. Soc. Rev. 2005, 34, 22-30. (40) Ramasubbu, N.; Parthasarathy, R.; Murray-Rust, P., Angular Preferences of Intermolecular Forces around Halogens Centers: Preferred Directions of Approach of Electrophiles and Nucleophiles around the Carbon-Halogen Bond. J. Am. Chem. Soc. 1986, 108, 4308-4314. (41) Mínguez Espallargas, G.; Van De Streek, J.; Fernandes, P.; Florence, A. J.; Brunelli, M.; Shankland, K.; Brammer, L., Mechanistic insights into a gas-solid reaction in molecular crystals: The role of hydrogen bonding. Angew. Chem. Int. Edit. 2010, 49, 8892-8896. (42) Yamamoto, N.; Taga, T.; Machida, K., Structure of mixed crystals of benzoic acid and p-fluorobenzoic acid, and their energy evaluation by empirical potential functions. Acta Crystallogr. B 1989, 45, 162-167. (43) Nayak, S. K.; Reddy, M. K.; Guru Row, T. N.; Chopra, D., Role of HeteroHalogen (F···X, X = Cl, Br, and I) or Homo-Halogen (X···X, X = F, Cl, Br, and I) Interactions in Substituted Benzanilides. Cryst. Growth Des. 2011, 11, 1578-1596.

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

Page 26 of 26

26

For Table of Contents Use Only

Novel solid-solution of the antiretroviral drugs lamivudine and emtricitabine Jéssica de Castro Fonseca, Juan Carlos Tenorio Clavijo, Natalia Alvarez, Javier Ellena and Alejandro Pedro Ayala

A novel organic solid solution containing the antiretroviral drugs lamivudine and emtricitabine is reported. This structure is not based on those of the individual components, but on the one of a lamivudine hydrate. The solubility range of emtricitabine in the hydrate structure seems to be determined by the raising of F…F interactions.

ACS Paragon Plus Environment