Drug Repurposing of the Antiviral Drug Acyclovir: New Pharmaceutical Salts

: Drug repurposing is becoming interesting in terms of offering advantages over the traditional drug development, once drug discovery is a costly, time-consuming, and highly risky process. In particular, with the coronavirus disease (COVID-19) declared by World Health Organization as a global pandemic, there has emerged a considerable need to develop therapeutic agents capable of preventing viral outbreaks. Concomitantly, well-known and long-used drugs such as acyclovir (Acv) have been tested against COVID-19. Acv is a guanosine analogue that acts as an antiviral drug, commonly used to treat herpes simplex virus (HSV), genital herpes, and varicella zoster virus (VZV). Acv showed to inhibit viral proteases, multiple viral genes expression, and RNA-Dependent RNA Polymerase, helping to recover COVID-19 patients. However, ACV is a BCS class III/IV drug, with low permeability and/or slight water solubility (concentration-dependent). Given the repurposing eligibility of Acv, in this work, two new salts of this drug are presented (nitrate and sulfate), with the aim of improving its pharmacokinetic properties. The new salts were evaluated by X-ray diffraction, and thermal and spectroscopic analyses. A third salt, a chloride one, was also characterized and used for comparison.


Introduction
Active Pharmaceutical Ingredients (APIs) are usually available in a crystalline or amorphous state, spread through several dosage forms such as capsules, powders, suspensions, or gels, and are largely orally administered as pills or tablets, which represent the most convenient, compact, and economical way to deliver them to the population [1,2].In particular, crystalline APIs are more attractive than non-crystalline APIs (amorphous), due to their thermodynamic stability, purity, and manufacturability.However, in the crystalline state, APIs may exist in several different solid forms, such as salts, co-crystals, hydrates, and solvates.Beyond exhibiting polymorphism, each of them presenting specific properties (melting point, solubility, hygroscopicity, etc.) that can affect the quality and safety of a medicine [3].
More than 40% of commercially available APIs exhibit bioavailability issues.The development of new crystalline solid forms of APIs could be an effective strategy to improve their pharmacokinetic and pharmacodynamic properties [4,5].Scientists worldwide have noticed that modifications in the properties of APIs promote, in some cases, secondary health effects, thus opening doors to a new area inside the pharmacy field: Drug Repurposing (DR).In this sense, DR has become a good strategy in terms of offering advantages over traditional drug development, as it is a costly, time-consuming, and risky process [6].Recently, given the pandemic caused by the new coronavirus, SARS-CoV-2, termed by the World Health Organization as coronavirus disease 2019 (COVID-19), a huge need for the development of therapeutic agents capable of preventing virus outbreak emerged.In this scenario, DR gained force and several APIs, especially antivirals, were investigated in an attempt to be used in the treatment of COVID-19 [6,7].
phosphate by cellular enzymes, thus inhibiting viral DNA polymerase by acting as an analogue to deoxyguanosine triphosphate (dGTP) [11].The tests involving Acv showed evidence for promoting some effect against COVID-19 infection [12][13][14][15].However, despite being used for many years, this drug has few crystalline solid forms reported [15][16][17][18].Acv is commercialized as acyclovir sesquihydrate, a solid form known since 1980s that shows to be more stable than the others polymorphs, as well as the anhydrous forms [19], which exhibit solubility/permeability issues [20].These facts place this drug as a BCS class III or IV API, depending on the mg/tablet (drug concentration) [21,22].
Since pharmaceutical cocrystals [21] and salts [22,23] are interesting choices to improve the physico-chemical properties, and the last ones can be synthetized by acid-base reactions [24][25][26], in this work we report two new Acv salts (nitrate and sulfate), aiming to improve its pharmacokinetics properties.These new salts were evaluated by single crystal X-ray diffraction (SCXRD), differential scanning calorimetry (DSC), Fourier transform infrared (FTIR), Raman spectroscopy, Thermogravimetric analysis (TGA) and Hirshfeld surface analysis (HS) and Full Interaction Maps (FIM).In addition, acyclovir hydrochloride [22], whose X-ray powder diffraction study has already been reported in the literature, was studied by SCXRD for the first time and included for comparison reasons [4,24,25].Acv has as the major mechanism of action and its conversion to acyclovir monophosphate by virally encoded thymidine kinase and its subsequent conversion to acyclovir triphosphate by cellular enzymes, thus inhibiting viral DNA polymerase by acting as an analogue to deoxyguanosine triphosphate (dGTP) [11].The tests involving Acv showed evidence for promoting some effect against COVID-19 infection [12][13][14][15].However, despite being used for many years, this drug has few crystalline solid forms reported [15][16][17][18].Acv is commercialized as acyclovir sesquihydrate, a solid form known since 1980s that shows to be more stable than the others polymorphs, as well as the anhydrous forms [19], which exhibit solubility/permeability issues [20].These facts place this drug as a BCS class III or IV API, depending on the mg/tablet (drug concentration) [21,22].
Since pharmaceutical cocrystals [21] and salts [22,23] are interesting choices to improve the physico-chemical properties, and the last ones can be synthetized by acid-base reactions [24][25][26], in this work we report two new Acv salts (nitrate and sulfate), aiming to improve its pharmacokinetics properties.These new salts were evaluated by single crystal X-ray diffraction (SCXRD), differential scanning calorimetry (DSC), Fourier transform infrared (FTIR), Raman spectroscopy, Thermogravimetric analysis (TGA) and Hirshfeld surface analysis (HS) and Full Interaction Maps (FIM).In addition, acyclovir hydrochloride [22], whose X-ray powder diffraction study has already been reported in the literature, was studied by SCXRD for the first time and included for comparison reasons [4,24,25].

Supramolecular Synthesis
Acv salts were obtained by suspending 22.5 mg (0.1 mmol) of the API in 1 mL of alcohol for 5 min, after that, 0.2 mL of a 1.0 M solution of the respective inorganic acid (1:1 molar ratio) was added to the suspension, and the reaction was stirred for another 15 min.The solutions were cooled to room temperature and left to slowly evaporate.The yield of the salts synthesis is about 78%, 82% and 74% for the HAcv•HSO 4 , HAcv•Cl and HAcv•NO 3 , respectively.

Fourier Transform Infrared
FT-IR spectra were collected using a Bomem Michelson FT MB-102 spectrometer in the 4000−400 cm −1 range.To record the IR spectra, 1 mg of each compound in 100 mg of KBr was used to prepare the pellets.

Raman Spectroscopy
Raman spectra were collected using a Horiba LabRAM HR Evolution UV-VIS-NIR spectrometer (Palaiseau, Saclay Campus, France) and measured in the 200−4000 cm −1 range with a 532 nm diode laser and a Horiba Synphony CCD detector (Palaiseau, Saclay Campus, France).

Single Crystal X-ray Diffraction (SCXRD)
SCXRD measurements were carried out on a Rigaku XtaLAB Synergy-S Dualflex diffractometer equipped with a HyPix-6000HE hybrid photon-counting detector using Cu-Kα radiation (λ = 1.54184Å).The crystals were kept at 100 K during data collection.Cell refinement, data collection, data reduction, and absorption correction were performed using CrysAlisPro [27]. he intrinsic phasing method was employed to solve the structures using the SHELXT-2018/2 solution program [28], while the refinement was performed using least-squares methods in the SHELXL-2019/2 program [29] both within Olex2 software [28]; non-hydrogen atoms were located in subsequent Fourier-difference map analyses and refined anisotropically.The hydrogen atoms positions were located in idealized positions and refined with isotropic displacement parameters using the riding model [Uiso(h) = 1.2 Ueq(N, C) or 1.5 Ueq(O)] [29].The Olex2 software [30] was also used to generate graphical illustrations.The data collection and refinement parameters of the salts are listed in Table 1.

Thermal Analysis
Thermogravimetric analysis was performed using a Shimadzu TGA-50 thermobalance instrument.Approximately 4.0 mg of each sample was used for the measurements in an alumina crucible and heated at 10 • C min −1 under N2 atmosphere (50 mL min −1 ).DSC data were acquired according to previous TGA data, that is, up to the degradation temperature of each compound.These experiments were performed using a Shimadzu DSC-60 calorimeter.The samples were heated at a rate of 10 • C min −1 with a crimped sealed aluminum pan using nitrogen as the purge gas (50 mL min −1 ).The data were processed using Shimadzu TA-60 thermal data analysis software.

Hirshfeld Surface
The Hirshfeld surfaces and two-dimensional fingerprint plots for the salts were obtained from the crystallographic information files (CIFs) generated by SCXRD analyses using the CrystalExplorer 17.5 program package [31].The d norm surfaces were mapped over the color scale from −0.7 (red) to 1.3 (blue), and the shape index surfaces were obtained in the range of −1.0 (red) to 1.0 (blue).The bidimensional fingerprint plots were generated with the combination of the d i and d e distances, in the scale of 0.4 to 3.0 Å [31,32].

Full Interaction Maps
This analysis is useful to explore the intermolecular interactions in solid state and were calculated using the Mercury program [33] based on the molecular interactions library in the Cambridge Structural Database (CSD).

H Nuclear Magnetic Resonance
The 1 H Nuclear Magnetic Resonance (NMR) spectra of the studied compounds were collected on a 9.4 T Bruker Avance III (400 MHz) spectrometer using DMSO-d 6 as solvent.

Equilbirum Solubility Studies
The aqueous solubility of the commercial Acv and its salts was investigated at room temperature using a UV-Vis 1800 Shimadzu spectrophotometer.Saturated solutions of the compounds were prepared, suspending each compound in deionized water (Milli-Q water) and left to stir for 24 h.After 24 h, the solutions were filtered using a 0.45 µm filter, diluted, and measured in the 200-600 nm range.The concentrations of the compounds were determined in comparison with calibration curves constructed using the absorbance values at λ = 251 nm for the standard solution prepared with concentrations ranging from 0.025 to 0.2 mg mL −1 .

Single Crystal X-ray Diffraction (SCXRD)
Acyclovir is a guanine derivative and possess amphoteric characteristic, acting as weak acid (pk a between 2.16 and 2.27) or base (pK a between 9.04 and 9.25), depending on the system which is present.Three different salts were synthesized, considering the basicity of the purine group, using the following inorganic acids: hydrochloric, nitric, and sulfuric.The asymmetric unit of the salts and the geometric parameters of the hydrogen bonds are shown in Figure 2 and Table 1, respectively, while the hydrogen bonds and bond lengths of Acv and its salts are available in Tables S1 and S2.

Equilbirum Solubility Studies
The aqueous solubility of the commercial Acv and its salts was investigated at room temperature using a UV-Vis 1800 Shimadzu spectrophotometer.Saturated solutions of the compounds were prepared, suspending each compound in deionized water (Milli-Q water) and left to stir for 24 h.After 24 h, the solutions were filtered using a 0.45 μm filter, diluted, and measured in the 200-600 nm range.The concentrations of the compounds were determined in comparison with calibration curves constructed using the absorbance values at λ = 251 nm for the standard solution prepared with concentrations ranging from 0.025 to 0.2 mg mL -1 .

Single Crystal X-ray Diffraction (SCXRD)
Acyclovir is a guanine derivative and possess amphoteric characteristic, acting as weak acid (pka between 2.16 and 2.27) or base (pKa between 9.04 and 9.25), depending on the system which is present.Three different salts were synthesized, considering the basicity of the purine group, using the following inorganic acids: hydrochloric, nitric, and sulfuric.The asymmetric unit of the salts and the geometric parameters of the hydrogen bonds are shown in Figure 2 and Table 1, respectively, while the hydrogen bonds and bond lengths of Acv and its salts are available in Tables S1 and S2  The formation of the Acv cation occurs through protonation of the N5 atom of the imidazole ring due of the significant differences in the pKa values between Acv and the inorganic acids (ΔpKa > 3) [34,35].Fourier map analyses and vibrational spectroscopy studies confirmed the presence of hydrogen in N5.The three salts are composed of one protonated ACV molecule and one counterion: hydrogen sulfate, nitrate, or chloride for HAcv•HSO4, HAcv•NO3 and HAcv•Cl, respectively.The formation of the Acv cation occurs through protonation of the N5 atom of the imidazole ring due of the significant differences in the pKa values between Acv and the inorganic acids (∆pK a > 3) [34,35].Fourier map analyses and vibrational spectroscopy studies confirmed the presence of hydrogen in N5.The three salts are composed of one protonated ACV molecule and one counterion: hydrogen sulfate, nitrate, or chloride for HAcv•HSO 4 , HAcv•NO 3 and HAcv•Cl, respectively.
The Acv molecule presents conformational flexibility around the (2-hydroxyethoxy)methyl group attached to N4, where the orientation of this group influences the organization of the Acv molecules on the crystal lattice.Figure 3 shows the overlay of the Acv molecular conformations observed in the structure of the salts, as well as the free Acv (CCDC code MECWIC) [36] The Acv molecule presents conformational flexibility around the (2-hydroxyethoxy)methyl group attached to N4, where the orientation of this group influences the organization of the Acv molecules on the crystal lattice.Figure 3 shows the overlay of the Acv molecular conformations observed in the structure of the salts, as well as the free Acv (CCDC code MECWIC) [36].It can be noticed that the Acv molecule in HAcv•NO3 has the most different conformation for the (2-hydroxyethoxy)methyl group, which is positioned in the opposite direction when compared to Acv and the other salts.This conformational difference involves the δ angle and is a consequence of the difference in the intermolecular interactions between Acv and the anions.The conformational analyses the structures shows that the N5-C5 bond length is stable along the three salts,  The HAcv•HSO4 salt crystallizes in the triclinic P-1 space group, with one HAcv + cation and one hydrogen sulfate anion per asymmetric unit.The presence of only one O-H group in the HSO4 − counterion is evidenced by the S1-O5 bond length, which has a single bond character and is at least 0.11 Å longer than the others S-O bonds [37].Figure 4a  2 (8) motifs (Figure 4c).These ribbons are linked by N5-H5• • • O3 interactions.In this structure the hydrogen sulfate anions participate in HSO 4 − • • • π interactions with the acyclovir rings with distance 2.969 (12) Å and 3.083 (12) Å as reported previously [38].
The asymmetric unit of the HAcv•NO 3 salt is composed of one HAcv + cation and one nitrate anion and crystallizes in the monoclinic P2 1 /c space group with a twinned structure along the 2 (9) graph set).In addition, the interactions between the HAcv + and NO 3 − ions form synthons with R 3 3 (10) and R 2 3 (9) graph sets (Figure 5c).

Hirshfeld Surfaces Analysis
Hirshfeld surfaces (HS) analysis and the 2D-fingerprint plots (FP) allow the investigation of intermolecular interactions in crystal structures.Normalizing the distances from the HS to the nearest atom inside (di) and outside (de) the surface by the van der Waals radii of the atoms gives the origin to the dnorm HS, which shows the contacts in the crystal structure of a compound.The dnorm surface shows regions in red, white, and blue to indicate the contacts with distances shorter and longer than the sum of the vdW radii of the involved atoms, respectively.The shape index (S) is another HS property that helps evaluate the π•••π stacking interactions, which are indicated by pairs of blue and red triangles.The HS mapped on dnorm property are represented in Figure 7, while the FP and shape index surfaces are shown in Figures S1-S4.

Hirshfeld Surfaces Analysis
Hirshfeld surfaces (HS) analysis and the 2D-fingerprint plots (FP) allow the investigation of intermolecular interactions in crystal structures.Normalizing the distances from the HS to the nearest atom inside (d i ) and outside (d e ) the surface by the van der Waals radii of the atoms gives the origin to the d norm HS, which shows the contacts in the crystal structure of a compound.The d norm surface shows regions in red, white, and blue to indicate the contacts with distances shorter and longer than the sum of the vdW radii of the involved atoms, respectively.The shape index (S) is another HS property that helps evaluate the π• • • π stacking interactions, which are indicated by pairs of blue and red triangles.The HS mapped on d norm property are represented in Figure 7, while the FP and shape index surfaces are shown in Figures S1-S4

Full Interaction Maps Analysis
A search in the CSD database only show a few reported Acv solid form crystal structures, and none of them show a further evaluation of the interactions of the functional groups.The display of the Full Interaction Maps (FIMs) created using carbonyl oxygen and alcohol oxygen probes for the Acv, and with an additional charged (NH) nitrogen probe for the salts; Providing an overview of the preferred directionality and the strength of the potential interactions that the functional groups in the molecule can form.
The FIM of the anhydrous Acv form can be observed in Figure S5a.The blue regions (related to the donor probe) indicate a higher propensity of the O1, O2, and N5 atoms (donor region) to donate electron density to the O3, N2, and N1 atoms (acceptor region).  2 summarizes the hydrogen bonds in the studied compounds.

Blue
Red Atoms in bold are related to the region detached.

Thermal Analysis
The thermal behaviour of the Acv salts was studied using a combination of DSC/TGA techniques.The curves are presented in Figure 8

Spectroscopic Analysis
The FT-IR and Raman studies of Acv and its salts were performed to confirm the formation of salts (Figures 9 and 10).FT-IR and Raman spectra of the Acv sample confirmed that the API used in the supramolecular synthesis corresponded to Acv sesquihydrate [19].The salt formation was identified by evaluating the changes in the frequency of some vibrational modes related to the promoted N5 atom from the imidazolium group, as well as the formation of new hydrogen bonds between the API and anions.The band assignments are listed in Table 3.The FT-IR spectra show that ν(N-H) stretching frequencies associated with the imidazolium group are present in HAcv•HSO 4 (3417 cm −1 ), HAcv•Cl (3523 cm −1 ) and HAcv•NO 3 (3425 cm −1 ), but not in Acv (Figure 9).The inorganic anions used as coformers show different FT-IR characteristics, starting with the HSO 4 − and NO 3 − anions, which show ν(SO) and ν(NO 3 − ) bands at 1054 cm −1 and 1384 cm −1 , respectively.The Raman spectra of Acv and its salts present clear differences that can be attributed to the protonation of the imidazolium group and the vibrational modes arising from the inorganic anions (see Figure 10).The HSO 4 − and NO 3 − anions show ν(SO) and ν(NO 3 ) stretching bands at 1020 cm −1 and 1282 cm −1 , respectively.As expected, no bands corresponding to the Cl − anion were observed in the Raman spectrum of HAcv•Cl.A new δ(N-H) band was present in the Raman spectra of the salts HAcv•HSO 4 (3152 cm −1 ), HAcv•Cl (3312 cm −1 ) and HAcv•NO 3 (3321 cm −1 ), which confirms the protonation of the imidazolium ring from Acv.The Raman spectra of Acv and its salts present clear differences that can be attributed to the protonation of the imidazolium group and the vibrational modes arising from the inorganic anions (see Figure 10).The HSO4 -and NO3 -anions show ν(SO) and ν(NO3) stretching bands at 1020 cm −1 and 1282 cm −1 , respectively.As expected, no bands corresponding to the Cl -anion were observed in the Raman spectrum of HAcv•Cl.A new δ(N-H) band was present in the Raman spectra of the salts HAcv•HSO4 (3152 cm −1 ), HAcv•Cl (3312 cm −1 ) and HAcv•NO3 (3321 cm −1 ), which confirms the protonation of the imidazolium ring from Acv.

H NMR Spectroscopy
The 1 H NMR spectra for Acv and its salts were obtained using DMSO-d6 as solvent and reference.The 1 H spectra for each compound and the chemical shifts (δ) are presented in the Supplementary material (Figures S8-S10 and Table S2).
The spectrum of the free Acv shows the signals of the guanine group in the range of 10.70 to 6.50 ppm, appearing as singlets with the integration of 1, 1, and 2 for the -NH-C(O)-(10.64ppm), N=CH-N (7.82 ppm) and -NH2 (6.51 ppm) groups, respectively.The hydrogens atoms from the methylene carbons of the (2-hydroxyethoxy)methyl group appear as signals in 5.35 and 3.47 ppm, with the first one as a singlet (integration of 2) correspondent to the -NCH2Ogroup, while the last one as a multiplet integrating for 4, being a superposing of the signals correspondent to the hydrogens from the ethyl group.These results are comparable to some data reported previously for Acyclovir [39].
The spectra of the salts also showed the signals corresponding to the hydrogens from the N=CH-N, -NH2 and -NCH2O-groups, but in regions with higher chemical shifts, which occurred due to the protonation of the Acv molecule.The signal related to the ethyl group in the free Acv spectrum is seen as two multiplets in the salts spectra, with the

H NMR Spectroscopy
The 1 H NMR spectra for Acv and its salts were obtained using DMSO-d 6 as solvent and reference.The 1 H spectra for each compound and the chemical shifts (δ) are presented in the Supplementary material (Figures S8-S10 and Table S2).
The spectrum of the free Acv shows the signals of the guanine group in the range of 10.70 to 6.50 ppm, appearing as singlets with the integration of 1, 1, and 2 for the -NH-C(O)-(10.64ppm), N=CH-N (7.82 ppm) and -NH 2 (6.51 ppm) groups, respectively.The hydrogens atoms from the methylene carbons of the (2-hydroxyethoxy)methyl group appear as signals in 5.35 and 3.47 ppm, with the first one as a singlet (integration of 2) correspondent to the -NCH 2 O-group, while the last one as a multiplet integrating for 4, being a superposing of the signals correspondent to the hydrogens from the ethyl group.These results are comparable to some data reported previously for Acyclovir [39].

Figure 2 .
Figure 2. ORTEP type illustration of the asymmetric unit of (a) Acv hydrogen sulfate, (b) Acv nitrate and (c) Acv hydrochloride with crystallographic labelling.Thermal ellipsoids represented at 50% probability level.

Figure 2 .
Figure 2. ORTEP type illustration of the asymmetric unit of (a) Acv hydrogen sulfate, (b) Acv nitrate and (c) Acv hydrochloride with crystallographic labelling.Thermal ellipsoids represented at 50% probability level.
. It can be noticed that the Acv molecule in HAcv•NO 3 has the most different conformation for the (2-hydroxyethoxy)methyl group, which is positioned in the opposite direction when compared to Acv and the other salts.This conformational difference involves the δ angle and is a consequence of the difference in the intermolecular interactions between Acv and the anions.The conformational analyses the structures shows that the N5-C5 bond length is stable along the three salts, meanwhile the N5-C3 bond length shows different values: 1.391 (3) Å in Acv and 1.383 (2) Å, 1.385 (2) Å and 1.386 (5) Å in HAcv•HSO 4 , HAcv•Cl and Hacv•NO 3 , respectively.In contrast, the C3-N5-C5 P5 bond angles show small differences: 107.970 • (11), 108.221 • (14) and 108.669 • (3) for HAcv•HSO 4 , HAcv•Cl and HAcv•NO 3 , respectively.However, no other substantial changes in the imidazole ring were verified due to the protonation.

Figure 3 .
Figure 3. Overlay of the Acv molecule with the HAcv + cations obtained by superposing of pyrimidine rings.Hydrogen atoms were omitted for clarity.
,b show the crystal packing of HAcv•HSO4 with the representation of the N-H•••O, N-H•••N and O-H•••O intermolecular interactions.

Figure 3 . 17 Figure 4 . 2 Figure 4 .
Figure 3. Overlay of the Acv molecule with the HAcv + cations obtained by superposing of pyrimidine rings.Hydrogen atoms were omitted for clarity.The HAcv•HSO 4 salt crystallizes in the triclinic P-1 space group, with one HAcv + cation and one hydrogen sulfate anion per asymmetric unit.The presence of only one O-H group in the HSO 4 − counterion is evidenced by the S1-O5 bond length, which has a single bond character and is at least 0.11 Å longer than the others S-O bonds[37].Figure4a,b show the crystal packing of HAcv•HSO 4 with the representation of the N-H• • • O, N-H• • • N and O-H• • • O intermolecular interactions.Crystals 2023, 12, x FOR PEER REVIEW 7 of 17 [001] direction.The crystal structure of HAcv•NO 3 is stabilized by classical O-H• • • O and N-H• • • O hydrogen bonds (Figure 5) that form a bi-dimensional supramolecular network along the [100] plane.Dimeric arrangements are formed with the association of two HAcv + cations through the N1-H1• • • O3 hydrogen bond as well as π• • • π stacking interaction (3.624 Å) involving the pyrimidine rings (Figure 5b).The dimers are connected through the N2-H2a• • • O1 as well as the bifurcated N2-H2b• • • O1 • • • H5 - N5 interactions (C 1

17 Figure 7 .
Figure 7. HS of (a) Acv hydrogen sulfate, (b) Acv nitrate and (c) Acv hydrochloride mapped in dnorm.The red spots observed in the dnorm HS of the salts indicate that N-H•••O hydrogen bonds stabilize the three structures.The surfaces also show the presence of O-H•••N hydrogen bonds in the HAcv•HSO4 and HAcv•NO3 structures, due to the presence of oxygen atoms in the counterions as well as N-H•••N interaction in HAcv•HSO4 and N-H•••Cl and O-H•••Cl interactions in HAcv•Cl.Furthermore, it was possible also to observe the presence of non-classical C-H•••O interactions in the structure of the salts.Pairs of red and blue triangles are present on the shape index surface (Figure S4) of HAcv•NO3 and HAcv•Cl, indicating the presence of π•••π interactions in the crystal structure of both salts.The 2D-fingerprint plots of the salts (Figures S1-S3) clearly show the intermolecular contacts present in the structures.The decomposition of these fingerprints allows the quantification of the contribution of each contact to the crystal packing.The FPs of the synthesized compounds, presented in Figure S7, show that the H•••H and O•••H contacts were the major contributors to the crystal packing of these salts.H•••H comprise 28.9%, 27.0% and 36.7% for HAcv•HSO4, HAcv•NO3; and HAcv•Cl, respectively.Meanwhile, the O•••H contacts represented 44.8% (HAcv•HSO4), 49.6% (HAcv•NO3), and 27.2% (HAcv•Cl).The H•••H contribution of HAcv•Cl was higher than the O•••H one due to the absence of oxygen atoms in its anion (Cl -).An opposite situation was observed for the other two salts.The HAcv•NO3 and HAcv•Cl have more C•••C and C•••N contacts contribution than the one observed for HAcv•HSO4.This is due to the presence of the π•••π stacking interactions in the HAcv•NO3 and HAcv•Cl structures, as verified in the shape index surfaces.The O•••H contacts are observed in regions of smaller di and de values in the FPs for the three compounds, an indicative that these interactions are shorter and stronger than the other ones.

17 Figure 8 .
Figure 8. DSC/TGA curves of (a) Acv hydrogen sulfate, (b) Acv hydrochloride and (c) Acv nitrate.According to the TGA data, the thermal degradation of HAcv•HSO4, HAcv•Cl and HAcv•NO3 began at 165 °C, 187 °C and 180 °C, respectively.Thermal stability can be associated with the rupture of the crystal structure upon exposure to the heating process in an inert environment as an experimental condition in the DSC/TGA experiments.The thermal behaviour observed with TGA for Acv salts allows to establish a thermal stability based in the degradation of the compounds in order: HAcv•Cl, HAcv•NO3 and HAcv•HSO4.

Figure 8 .
Figure 8. DSC/TGA curves of (a) Acv hydrogen sulfate, (b) Acv hydrochloride and (c) Acv nitrate.According to the TGA data, the thermal degradation of HAcv•HSO 4 , HAcv•Cl and HAcv•NO 3 began at 165 • C, 187 • C and 180 • C, respectively.Thermal stability can be associated with the rupture of the crystal structure upon exposure to the heating process in an

Table 2 .
Hydrogen bonds by region (Donor and Acceptors).

Table 3 .
Main bands (cm −1 ) for IR and Raman spectra of Acv and its salts.-

Table 3 .
Main bands (cm −1 ) for IR and Raman spectra of Acv and its salts.