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PAPER

Avoiding irreversible 5-fluorocytosine hydration

via supramolecular synthesis of pharmaceutical
Cite this: New J. Chem., 2018,
42, 14994 cocrystals†
a
Matheus S. Souza, Luan F. Diniz, a Lautaro Vogt, a
Paulo S. Carvalho Jr., a
ab
Richard F. D’vries and Javier Ellena *a

Received 30th May 2018,
Accepted 26th July 2018
DOI: 10.1039/c8nj02647e
rsc.li/njc
The antimetabolite 5-fluorocytosine (5-FC) was used to form pharmaceutical cocrystals in order to modulate
its poor physicochemical stability in humid environments, which leads to the irreversible incorporation of a
water molecule at the structural level under storage conditions. The anhydrous form of 5-FC is a well-known
fluorinated analog of cytosine with antifungal activity and it has become one of the most used medications
for cancer treatment via the gene therapy approach. In this study, novel 5-FC cocrystals were obtained from
the reaction of 5-FC with three nontoxic coformers: caffeine (CAF), p-aminobenzoic acid (PABA) and caprylic
acid (CA). These cocrystals, namely 5FC–CAF, 5FC–PABA and 5FC–CA, were characterized by single-crystal
and powder X-ray diffraction (SCXRD and PXRD), spectroscopic (FT-IR and FT-Raman) and thermal
(thermogravimetric analysis, differential scanning calorimetry, and hot-stage microscopy) techniques. The
physical stabilities of 5-FC and its cocrystals were evaluated in environments with high relative humidity
and the equilibrium solubility was measured in a pH 1.2 buffer medium. These studies show that the
prodrug 5-FC is able to form different homo and heterosynthons that lead to cocrystal formation.
Additionally, the solubility profiles of the novel multicomponent solid forms were found to be similar to
the API raw material, a BCS class I drug, exhibiting a high solubility profile. The hydration stabilities of 5-FC
and its cocrystals were evaluated in humid environments to confirm the irreversible hydration of 5-FC in
contrast with the absence of phase transitions in its cocrystal forms. In this way all 5-FC cocrystals
reported herein maintained to a large degree the API solubility and do not undergo the hydration process
or phase transition under extreme storage conditions, being more stable than the parent 5-FC.

a
Instituto de Fı́sica de São Carlos, Universidade de São Paulo, CP 369, 13.560-970 – São Carlos, SP, Brazil. E-mail: javiere@ifsc.usp.br;
Tel: +55 (016)3373-8096/3373-9876
b
Facultad de Ciencias Básicas, Universidad Santiago de Cali, Calle 5 # 62-00, Cali, Valle del Cauca, Colombia
† Electronic supplementary information (ESI) available: Fig. S1: experimental powder X-ray diffraction patterns of 5-FC and its cocrystals that remain non-solubilized after
the solubility test; Fig. S2: absorption spectra of 5-FC 0.0055 mg mL1, caffeine 0.0086 mg mL1, p-aminobenzoic acid 0.0065 mg mL1; and the cocrystals 5FC–CAF
0.016 mg mL1, 5FC–PABA 0.013 mg mL1 and 5FC–CA 0.016 mg mL1 in buffer pH = 1.2; Fig. S3: heterosynthons observed in the 5-FC cocrystals; Fig. S4: homosynthons
observed in the 5-FC cocrystals; Fig. S5(a) and (c): microscope images of crystalline phases with single-crystals of different habits obtained directly from the SES method for
5FC–CAF and 5FC–PABA cocrystals, respectively, (b) and (d): grinding products recrystallized through SES as a unique crystalline phase; Fig. S6: simulated and
experimental powder X-ray diffraction patterns of 5-FC cocrystals. All diffractograms are in good agreement indicating that the samples present high purity and
representativeness; Fig. S7: experimental powder X-ray diffraction patterns of 5-FC cocrystals obtained from solvent-drop grinding (SDG) compared to simulated ones. Only
the diffractograms of 5FC–PABA are not in good agreement which suggest a possible physical mixture between the starting materials; Fig. S8: TGA and DSC curves of the
5-FC cocrystals; Fig. S9: DSC curves of: (i) the raw material 5-fluorocytosine (5-FC anhydrous); (ii) 5-FC after one week in an environment with high relative humidity (5-FC
monohydrate) and (iii) the two solid coformers, namely: CAF (caffeine) and PABA (p-aminobenzoic acid); Fig. S10: FT-Raman spectra of 5-FC and its cocrystals; Fig. S11:
powder X-ray diffraction patterns of 5-FC confirming that this API undergoes a phase transition from the anhydrous to the monohydrate form when exposed to humid
environments; Fig. S12: 3D surfaces generated through a script in Python from the 5-FC monohydrate pattern overlap collected during a VT-PXRD experiment; Fig. S13: the
DSC curve of the resulting material after VT-PXRD; Fig. S14: powder X-ray diffraction patterns of 5-FC after the solubility study (upper) and different solid forms of 5-FC
anhydrous and hydrated reported in the literature; Fig. S15: DSC profiles of 5-FC before (black) and after (purple) the solubility study; Fig. S16: TGA profiles of 5-FC
monohydrate (blue) and 5-FC anhydrous (purple) after the solubility study; Fig. S17: PXRD patterns of 5-FC (anhydrous, monohydrate, hydrate and hemihydrate) structures
confirming that the 5-FC monohydrate sample gives rise under heating to a totally different crystalline phase that does not correspond to any other similar crystal structure
deposited in the Cambridge Structure Database; Table S1: composition of the solution used for preparing the calibration curves and solubility determinations; Table S2:
standard solutions of 5-FC, caffeine and p-aminobenzoic acid used to construct the calibration curves; Table S3: regression coefficients for the calibration curves of 5-FC
and the cocrystal formers in buffer media, pH = 1.2; Table S4: pH values measured after solubility; Table S5: geometric parameters of the intermolecular interactions for
5-FC organic cocrystals; Table S6: main FT-IR and FT-Raman bands (cm1) for 5-FC and 5-FC cocrystals; and Table S7: solubility values of the four compounds. CCDC
1817618, 1817630 and 1817635. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c8nj02647e

14994 | New J. Chem., 2018, 42, 14994--15005 This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2018
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Introduction
The concepts of crystal engineering remain an established way
to develop and formulate improved pharmaceuticals.1–3 The
design and synthesis of multicomponent crystals4 is increasing,
allowing us to rearrange the supramolecular assembly and
modulate the physicochemical properties of pharmaceuticals.
This occurs based on the inclusion of chosen molecule(s) within
the API crystal lattice without the need to establish or break
covalent bonds5 – while preserving the inherent activity of the
parent drugs.6 In the case of drug-coformer cocrystals (DCC) these
guest molecules must belong to the GRAS (Generally Recognized
as Safe) list.7 Crystal engineering methodologies become necessary
since not all marketed drugs achieved the parameters required to
be a safe and effective solid medicine. The advantages of obtaining
a homogeneous crystalline entity stabilized by noncovalent inter-
actions such as hydrogen bonds, ionic and p–p interactions, and
van der Waals forces8 are linked to the fine control of important
pharmaceutical questions such as manufacturability, storage and
final clinical performance.9–16
5-Fluorocytosine (4-amino-5-fluoro-1,2-dihydropyrimidin-
2-one, 5-FC, Scheme 1) is a high-dose prodrug17 which is
established at the forefront of the treatment of various invasive
infections caused mainly by the fungal biological species
Candida spp. and Cryptococcus neoformans.18,19 In the last
few years, renowned researchers have dedicated themselves to
obtain the Cytosine Deaminase (CD) enzyme in mammalian
tumor cells to study the intracellular route of 5-FC, its conver-
sion to 5-FU (5-fluoro-2,4-(1H,3H)-pyrimidinedione, Scheme 1)
APIs and its influence in the inhibition of uncontrolled replication
Scheme 1 Chemical structures of the drugs 5-fluorocytosine (5-FC), 5-fluorouracil (5-FU) and the GRAS coformers caffeine (CAF), p-aminobenzoic
acid (PABA) and caprylic acid (CA).
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2018 New J. Chem., 2018, 42, 14994--15005 | 14995
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of genetic material that triggers the development of diseases such
as fungal or some cancers. In this way great effort has been
devoted to the development of new strategies to treat these
diseases.20,21 Under this global scenario, the 5-FC API emerged
as a promising prodrug for cancer treatment via the gene therapy
approach,22–29 exhibiting high solubility and permeability pro-
files – making it belong to Biopharmaceutics Classification
System (BCS) class I.6,30 The anhydrous form of 5-FC is the thermo-
dynamically most stable phase at room temperature but when
exposed to higher relative humidity, the physical stability becomes
very questionable and the conversion to the monohydrate form
happens.31 Such a hydration event leads to serious problems during
processing and storage, making 5-FC tableting not trivial. Generally,
the API’s physical instability17 could affect a range of factors that are
important in view of successful formulation, quality, safety and
efficacy of pharmaceuticals such as solubility, permeability, dissolu-
tion rate, bioavailability and extended ‘‘Shelf Life’’.32
In order to expand the range of new 5-FC solid forms, an
extensive number of structures have been reported in the last
few years.33–36 Recently, our group synthesized and depicted six
5-FC cocrystals using as coformers adipic, succinic, terephtalic,
benzoic and malic acids as well as the antineoplastic drug
5-FU.35 Following this approach, herein we report a standardized
protocol for supramolecular synthesis and the main physical and
chemical properties of three novel cocrystals obtained from reac-
tion of 5-FC with the coformers caffeine (CAF), p-aminobenzoic
acid (PABA) and caprylic acid (CA) (Scheme 1).
Initially, the samples were prepared by slow evaporation of
solvent (SES) or manual mechanochemistry (see the Experi-
mental section), but in an attempt to develop more suitable
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Table 1 Comparison between hydrothermal (SES) and automatic mechanochemistry (SDG) techniques for 5FC–CAF cocrystal supramolecular synthesis

Method SES SDG

1
MW 5-FC 129.09 g mol 129.09 g mol1
MW CAF 194.19 g mol1 194.19 g mol1
Initial mass (5-FC) 5 mg 25 mg
n n=1 n=1
Formula used for CAF mass calculation [(5-FC mass/MW 5-FC)  n  MW CAF] [(5-FC mass/MW 5-FC)  n  MW CAF]
Initial mass (CAF) 7.5 mg 37.6 mg
Total mass used 12.5 mg 62.6 mg
Amount of solvent used 1.5 mL (1500 mL) 0.025 mL (25 mL)
Duration of the experiment 3 to 5 days 1 hour
Productivity 62.6/12.5 = B5 times more
Solvent use 1500/25 = 60 times less
SES: slow evaporation of solvent; SDG: solvent-drop grinding; MW: molecular weight; 5-FC: 5-fluorocytosine; CAF: caffeine and n: mol number.

productivity methods based on the principles of green chemistry37
we also establish protocols involving automatic mechano-
chemistry (solvent-drop grinding, SDG) for the production of
the 5FC–CAF cocrystal sample (Table 1).38–43 This technique
provides considerable benefits in terms of economy, efficiency
and environmentally sustainable by reducing total processing
times from days/weeks to hours, and minimizing exposure to
aqueous conditions and also the use of large quantities of
solvent, which reduce or eliminate side-reactions.44 However, in
spite of our efforts it was not possible to obtain the 5FC–PABA
cocrystal by this method. Finally, we also report here the
hydration stability study of the cocrystals as well as the 5-FC
raw material under controlled humidity conditions, and their
solubility profiles at stomach pH.
Experimental details
5-FC anhydrous was purchased from Sigma-Aldrich Brazils and
used without any further purification. All coformers were obtained
from commercial sources and used directly. Milli-Q water and
acetonitrile were used as solvents.
Preparation of cocrystals
Cocrystallization of equimolar amounts of 5-FC and the co-
formers was performed by slow evaporation as well as by
mechanochemical methods. Manual mechanochemistry experi-
ments were carried out using a mortar, a pestle and stoichio-
metric quantities of molecular solvents. 5-FC and the cocrystal
formers CAF and PABA were ground for about 10–15 minutes.
A small amount of solvent (300 mL) was added to the grinding
mixture. After that, solvent evaporation was used to obtain
single crystals for X-ray diffraction experiments. For the
case of the CA coformer the cocrystallization solutions were
made using the SES method from mixture solvents like
acetonitrile–water.
5-Fluorocytosine caffeine cocrystal (5FC–CAF). Suitable
single-crystals were obtained by manual mechanochemistry.
5.00 mg of 5-FC (0.039 mmol) and 7.50 mg of CAF (0.039 mmol)
were ground in a pestle and mortar by adding 300 mL of a hot
acetonitrile/Milli-Q water (3 : 1, v/v, 100 1C) mixture until a
homogeneous mass was formed. The resulting ground material
was dissolved in the same composition hot mixture for its
recrystallization through the slow evaporation method. Colorless
prismatic crystals were obtained within 3–5 days.
5-Fluorocytosine p-aminobenzoic acid cocrystal (5FC–PABA).
Suitable single-crystals were obtained by manual mechano-
chemistry. 5.00 mg of 5-FC (0.039 mmol) and 5.31 mg of PABA
(0.039 mmol) were macerated together in a pestle and mortar
by adding 300 mL of a hot acetonitrile/Milli-Q water (1 : 1, v/v,
100 1C) mixture until a homogeneous system was formed. The
resulting ground material was dissolved in the same hot mixture
for its recrystallization through the slow evaporation method.
Colorless plate crystals were obtained within 3–5 days.
5-Fluorocytosine caprylic acid cocrystal (5FC–CA). These
cocrystals were obtained by dissolving 20 mg of 5-FC (0.155 mmol)
in an acetonitrile/Milli-Q water (5 : 1, v/v, 150 1C) mixture. Next,
to this hot homogeneous solution was added 500 mL of CA. The
system was stirred at 100 1C for approximately 15 minutes.
Then, the crystallization batches were allowed to cool down
slowly until room temperature and covered with Parafilms for
slow evaporation of the solvent. All these systems were main-
tained at 5 1C until the appearance of crystals, which occurs
within 10–15 days.
Additionally, the same 5FC–CAF phase was also obtained
by an automatic mechanochemistry method (SDG) using an
oscillatory mixer ball mill MM400 RETSCH. The sample powder
was placed in a 1.5 mL volume stainless steel milling jar
containing two 7 mm diameter stainless steel balls. The opti-
mized final condition to obtain this new solid form was
achieved by milling the system at a frequency of 25 Hz, at room
temperature, and for 1 hour. This experiment was performed by
the addition of a 50 mg of 5-FC (0.39 mmol), 75 mg of CAF
(0.39 mmol) and only 25 mL of acetonitrile/Milli-Q water (3 : 1, v/v)
mixture.
Single crystal X-ray diffraction (SCXRD)
The crystallographic data for the cocrystals 5FC–CAF and 5FC–
PABA were collected at room temperature (298  2 K) using an
Agilent Super Nova diffractometer equipped with a CCD detec-
tor system and a Mo source (l = 0.71073 Å). Cell determination,
data integration and reduction and final parameters were
obtained using the software CrysAlisPro45 (version 171.38.43b).
Using Olex2,46 the structures were solved by direct methods and
the models obtained were refined by full-matrix least squares

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on F2 (SHELXTL).47,48 The aromatic ring of the PABA coformer
is disordered over two positions with relative occupancies
of 45 : 55%.
For the 5FC–CA compound, the data collection was carried
out at 298  2 K using a Bruker Apex-II CCD diffractometer
using graphite-monochromated MoKa radiation (0.71073 Å).
The SAINT software49 was used for strategy planning and data
reduction. Using Olex2,46 the structure was solved with the
ShelXT47 structure solution program using intrinsic phasing
and refined with the ShelXL48 refinement package using least
squares minimization.
For all structures, the H atoms bonded to N and O atoms
were located from a difference Fourier map and freely refined.
Methyl H atoms were located in a Fourier calculation and refined
as riding atoms, with Uiso(H) = 1.5Ueq(C). The remaining H atoms
bonded to C atoms were geometrically positioned (aromatic C–H =
0.93 Å) and were refined using a riding model, with Uiso(H) =
1.2Ueq(C). Molecular representations, tables and pictures were
generated by MERCURY 3.10.250 and Olex2.46 For the 5FC–CAF and
5FC–PABA cocrystals one patent application was generated and
deposited on November 16, 2017 under the code BR1020170245764
(title of the invention: pharmaceutical cocrystals and their uses). All
CIFs were deposited in the Cambridge Structural Data Base51 under
the codes CCDC 1817618 (5FC–CAF), 1817630 (5FC–PABA) and
1817635 (5FC–CA).
Powder X-ray diffraction (PXRD)
The milled samples were analyzed at room temperature (293 
2 K) using a Rigaku Rotaflex RU200B diffractometer, in the
Bragg–Brentano reflective geometry, with CuKa radiation
(l = 1.54 Å) at 40 kV–60 mA and a Ni filter. The diffractograms
of the 5-FC raw material and the novel cocrystals were acquired
in the 5–501 2y range with a step width of 0.021 and a constant
counting time of 3 s per step. In addition, the 5-FC mono-
hydrate sample was also measured at different temperatures
in the 25–150 1C range with a heating rate of 2.5 1C min1.
Variable temperature powder X-ray diffraction (VT-PXRD)
experiments were performed using a Rigaku Ultima IV powder
diffractometer coupled to a variable temperature control device
using CuKa radiation (l = 1.54 Å) at 40 kV–30 mA with a step
scan width of 0.021 in an interval of 5–501 in 2y and 3 s per step.
The 3D surfaces were generated through a script in Python
from the VT-PXRD pattern overlap. The points of the corres-
ponding angular positions were connected for a better observa-
tion of the diffractogram evolution and, in this case, for the
phase transition display.
Hot-stage polarized optical microscopy
Polarized microscopy studies were performed using a Leica
DM2500P microscope connected to the Linkam T95-PE hot-
stage equipment. Data were visualized with the Linksys 32
software for hot-stage control. Each crystal was placed on an
individual 13 mm glass coverslip, placed on a 22 mm diameter
pure silver heating block inside of the stage. The samples were
heated at a ramp rate of 10 1C min1 until the beginning of the
degradation.
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Optical microscopy
Cocrystal images (5FC–CAF and 5FC–PABA crystallization medium)
were taken using an Olympus SZ61 microscope connected to the
Leica EC3 camera device (zoom: 4.5; magnification: 10).
Thermal analysis
Thermogravimetric analysis (TGA) was performed using a
Shimadzu TGA-50 thermobalance. An amount of approximately
5.0 mg  0.001 mg of each sample was placed in an alumina
crucible and heated at 10 1C min1 under a N2 atmosphere
(50 mL min1). For the samples 5FC–CAF and 5FC–PABA the
temperature range analyzed was from 50 to 500 1C. For the
sample 5FC–CA, however, this range was from 50 to 200 1C,
since at this temperature the sample has already degraded.
Differential Scanning Calorimetry (DSC) data acquisition was
carried out using a Shimadzu DSC-60 calorimeter until the
degradation temperature of each compound. The samples
(2.0 mg  0.01 mg) 5FC–CAF and 5FC–PABA were heated from
50 to 350 1C and 5FC–CA (2.0 mg  0.01 mg) from 50 to 200 1C
with a heating rate of 10 1C min1 in a crimped sealed aluminum
pan. The purge gas was nitrogen under a flow of 50 mL min1.
All the values reported here were processed using the Shimadzu
TA-60 thermal data analysis software (version 2.2).
Vibrational spectra
Fourier transform infrared (FT-IR) spectra were recorded using
an Alpha Bruker FT-IR spectrophotometer, using KBr pellets,
in the range 3600–600 cm1, with an average of 64 scans and a
2 cm1 spectral resolution. FT-Raman spectroscopy was per-
formed using a Bruker RFS 100 instrument with a Nd3+/YAG
laser operating at 1064 nm in the near-infrared and a CCD
detector cooled with liquid nitrogen using a spectral resolution
of 4 cm1.
Spectrophotometric measurements and calibration curves
A UV-1800 Shimadzu spectrophotometer was used to determine
the absorbance of standard solutions of the raw precursors of
the cocrystals and construct pattern curves. These curves were
used to determine the unknown concentration of solutions
of 5-FC, 5FC–CAF, 5FC–PABA or 5FC–CA. The spectra were
measured in the range from 200 to 400 nm using 1 cm quartz
cuvettes at a medium scan speed at a 1.0 nm data interval and
1 nm bandwidth. Standard stock solutions of 5-FC, caffeine and
p-aminobenzoic acid were prepared separately in a pH 1.2
hydrochloric buffer (Table S1, see ESI†). 5-FC standard solu-
tions were prepared by dissolving 10.00 mg of the raw material
in 1.5 mL of buffer in a 10 mL beaker (24 h agitation) and after that
a series of dissolutions from 0.006 mg mL1 to 0.024 mg mL1
were carried out. CAF solutions were prepared by dissolving
10.00 mg of the solid drug in 4 mL of buffer in a 50 mL volumetric
flask. After complete dissolution (24 h agitation) different concen-
tration points were made by appropriate dilutions at concentra-
tions ranging from 0.007 to 0.02 mg mL1. PABA solutions were
prepared by dissolving 15.00 mg of the solid drug in 4 mL
of buffer in a 50 mL volumetric flask. After 24 h agitation,
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different concentration points were prepared by appropriate
dilutions at concentrations ranging from 0.004 to 0.012 mg mL1
(Table S2, see ESI†). UV-vis spectra of these solutions were used to
build calibration curves of the API (5-FC) and the cocrystal formers
(CAF and PABA) individually (Table S3, see ESI†). Caprylic acid
shows a very low spectra signal at the range studied (200–400 nm).
Because of this, the calibration curve of CA was not constructed
and the 5-FC calibration curve was used to determinate the
concentration of 5FC–CA unknown solutions.
Equilibrium solubility studies
Equilibrium solubilities of 5-FC and its cocrystals were deter-
mined by the shake-flask method at 25 1C in a pH 1.2 buffer
medium.52 Saturated solutions of the compounds were prepared
by stirring an excess amount of 5-FC, 5FC–CAF, 5FC–PABA and
5FC–CA, sufficient to reach saturation, into 2 mL of the dissolu-
tion medium for a 48 h period. These solutions were prepared
in triplicate according to the method outlined in the BCS
guidance.53 After 48 h of sedimentation, the solutions were
filtered through a 0.45 mm PTFE hydrophilic filter (Millipores).
The solid sediment identity was checked by PXRD analysis
(Fig. S1, see ESI†). UV-vis spectroscopy was employed to analyze
the supernatant concentration of the compounds. The samples
were diluted in the pH 1.2 buffer medium before we started
measuring. 5-FC and cocrystals showed similar spectra that
could be resolved with the addition of absorbances (Fig. S2, see
ESI†). Solubilities of 5-FC, 5FC–CAF, 5FC–PABA and 5FC–CA
were measured by interpolating their maximum absorbance
readings to the corresponding calibration curves constructed
previously. After the equilibrium solubility measurements, the
pH value in each dissolution medium was determined (Table S4,
see ESI†) using a pH meter, QX 1500 Plus Qualxtron.
Stability study in relative humidity (RH)
Powder samples of the anhydrous 5-FC and 5-FC cocrystals
(30 mg) were stored in a sealed glass desiccator chamber
containing a saturated K2SO4 salt solution (distilled water) for
B100% RH. At any temperature, the concentration of a satu-
rated solution is fixed and does not have to be determined.
Further, the milled materials were subjected to PXRD measure-
ments as a characterization tool to monitor possible phase
transitions.
Results and discussion
5-FC is a weak basic molecule (pKa = 3.26) that structurally
presents two donors: NH(pirimidinic ring) and NH2(amine group), and
two acceptors: Nring and carbonyl (CQO). It cocrystallizes with
coformers containing COOH and NH2 functional groups. By the
DpKa approach, ie. DpKa = pKa (conjugate acid of the base) 
pKa (acid), cocrystals of 5-FC can be obtained using weak acids,
ca. DpKa o 0. Based on this perspective, caffeine (CAF, pKa =
14.00), p-aminobenzoic acid (PABA, pKa = 2.38) and caprylic
acid (CA, pKa = 4.89) were selected for the cocrystallization
experiments. The differences between the pKas of 5-FC and CAF
14998 | New J. Chem., 2018, 42, 14994--15005 This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2018
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and CA coformers give DpKa values within the ones required for
cocrystal formation (DpKa o 0). These values correspond,
respectively, to 10.74 and 1.63. The only exception was
the PABA coformer (DpKa = 0.88), for which the DpKa value fell
into the range from 0 to 3. Within this range, stated as the
continuum region in the literature, a cocrystal or salt can be
equally obtained.54
Fig. S3 and S4 (ESI†) show the main hydrogen bonding
supramolecular hetero and homosynthons, respectively, found
for the 5-FC cocrystals reported here. The cocrystals were
designed considering the trend of 5-FC to associate with amine,
amide and carboxylic functional groups through amine  amine
(heterosynthon I, Fig. S3, ESI†), amide  carboxyl (heterosynthon
II, Fig. S3, ESI†) and carbonyl  carboxyl (heterosynthon III,
Fig. S3, ESI†) classical synthons. Interestingly, the non-classical
C–H  F interactions connects neighbor 5-FC and CAF mole-
cules helping in the stabilization of the heterosynthon I form
by classical N–H  N hydrogen bonds (Fig. S3, ESI†). In a
Cambridge Structural Database (CSD)55 survey none of the
5-FC structures reported to date presented heterosynthon I.
In addition, heterosynthons II and III appeared in only 10%
of the 5-FC structures. In turn, homosynthons I, II and III
(Fig. S4, ESI†) were found in 38%, 22% and 16% of the structures,
respectively.
In all cases, obtaining single crystals was not a straight-
forward step. The crystallization attempts of 5FC–CAF and
5FC–PABA from solution gave very poor-quality crystals and/or
even mixtures of phases (5-FC raw and the respective coformers;
see Fig. S5, ESI†). In order to obtain pure single crystals for the
X-ray diffraction experiments, manual mechanochemistry methods
were applied only for these two cocrystals. For 5FC–CA, however,
suitable single crystals were obtained directly by evaporation
methods. Thus, 1 : 1 5-FC : CAF, 1 : 1 5-FC : PABA and 1 : 1
5-FC : CA cocrystals were all formed. Crystalline purities of
5-FC cocrystal samples were assessed by PXRD (see Fig. S6,
ESI†). Interestingly, cocrystal 5FC–CAF was also obtained as a
powder pure phase by the SDG method (see Fig. S7, ESI†).
The ORTEP56 diagrams of the asymmetric unit (ASU) of 5-FC
cocrystals are shown in Fig. 1. Details of the data collection,
refinement and crystallographic parameters for the novel com-
pounds are summarized in Table 2. The geometric parameters
of H-bonds present in the crystal structures are listed in Table S5
(see ESI†).
Structural description
5FC–CAF. The ASU comprises two 5-FC as well as two CAF
molecules. As expected from the planar conformation of raw
materials, 5FC–CAF crystallizes in the monoclinic P21/c space
group (Z 0 = 2, Z00 = 4, Fig. 1a), forming a layered structure
stabilized by classical (N–H  N) and non-classical (C–H  F)
interactions (Fig. 2a). In 5FC–CAF, the symmetry-independent
5-FC molecules are connected via N–H  O (2.983(4) Å, 169.3(2)1)
and N–H  N (2.761(4) Å, 176.5(2)1) H-bonds to form a dimeric
R22(8) synthon (Fig. 2b). As a result, these homodimers form
zigzag 1D chains (represented by the black arrows in Fig. 2a)
running along the a axis (highlighted in green in Fig. 2a).
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Fig. 1 ORTEP type diagrams showing atomic numbering schemes and 50% probability ellipsoids for: (a) 5FC–CAF, (b) 5FC–PABA and (c) 5FC–CA
compounds.

Table 2 Crystal data and structure refinement of the 5-fluorocytosine cocrystals

Identification code 5FC–CAF 5FC–PABA 5FC–CA

Empirical formula C24H28F2N14O6 C11H11FN4O3 C12H20FN3O3
Molecular weight 646.6 266.24 273.31
Temperature/K 298(2) 298(2) 298(2)
Crystal system Monoclinic Monoclinic Triclinic
Space group P21/c P21/c P1%
a/Å 9.1890(6) 4.8695(3) 5.2617(8)
b/Å 22.4128(17) 24.4911(16) 8.7066(14)
c/Å 13.3425(13) 9.8792(6) 15.710(2)
a/1 90 90 81.892(3)
b/1 93.485(8) 97.567(6) 85.884(3)
g/1 90 90 84.460(3)
Volume/Å3 2742.8(4) 1167.92(13) 707.96(19)
Z/Z 0 4/2 4/1 2/1
rcalc/g cm3 1.566 1.514 1.282
m/mm1 0.126 0.124 0.101
F(000) 1344.0 552.0 292.0
Reflections collected 17 471 19 318 24 416
Independent reflections 6188 2671 5518
Unique reflections 3818 2204 3804
Data/restraints/parameters 6188/0/421 2671/36/215 5518/0/174
R1 [I Z 2s(I)] 0.0671 0.0565 0.0560
wR2 [all data] 0.2286 0.1500 0.2134
Goodness-of-fitness on F2 1.036 1.074 1.078

By the N–H  N (3.047(4) Å, 169.4(2)1) H-bonds and C–H  F
(3.067(2) Å, 128.64(2)1) intermolecular interactions the CAF
molecules are inserted on both sides of the 5-FC chain in the
ab plane. The dominant driving force in the 3D network
assemblies is the p  p interactions along the [010] direction
connecting the aromatic centers of CAF and 5-FC from adjacent
planes. Furthermore, C–H  O interactions are observed between
these planes with distances of 3.498, 3.422 and 3.514 Å, forming
supramolecular ‘‘roof tile-like’’ layers (Fig. 2c).
5FC–PABA. 5FC–PABA crystallizes in the monoclinic P21/c
space group (Z 0 = 1, Z00 = 2; 1 : 1 5-FC : PABA stoichiometry),
Fig. 1b. The aromatic rings of the PABA molecules show two site
disorder with 45 : 55 occupancies. In the ASU, the 5-FC and
PABA molecules interact by a N–H  O (2.745(2) Å, 169.9(2)1)
H-bond through an acid–pyrimidine heterosynthon (Fig. 3a).
Like other cocrystals, the 5-FC molecules form a robust R22(8)
homodimer (highlighted in blue, Fig. 3a) through N–H  O
(2.772(2) Å, 177.4(2)1) H-bonds (Fig. 3a). Along the [112] direc-
tion, these homodimers connect two PABA molecules through
N–H  O (2.892(2) Å, 157.0(2)1) and N–H  O (2.942(2) Å,
163.1(2)1) H-bonds to form a R24(8) motif (highlighted in light brown,
Fig. 3a) that gives rise to chains (highlighted in green in Fig. 3b). Two
additional PABA molecules (color in orange in Fig. 3c) cross link
parallel chains through N–H  O (2.745(2) Å, 169.9(2)1) H-bonds.

This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2018 New J. Chem., 2018, 42, 14994--15005 | 14999
NJC Paper

Fig. 2 (a) Partial view down the [001] direction of 5FC–CAF crystal packing highlighting the formation of the layers. Black dotted lines indicate hydrogen
bonds. (b) Robust homosynthon formed between 5-FC’s structures. (c) Three-dimensional arrangement of crystalline 5FC–CAF showing the C–H  O
and p  p interactions that give rise to supramolecular ‘‘roof tile-like’’ layers.

Fig. 3 (a) Partial view down the [100] direction of 5FC–PABA crystal packing highlighting the formation of R24(8) and R22(8) graph set motifs via N–H  O
interactions. Black dotted lines indicate hydrogen bonds. (b) Propagation of 5-FC dimer chains attached to two PABA molecules through N–H  O
H-bonds. (c) Three-dimensional arrangement of crystalline 5FC–PABA showing the insertion of two PABA molecules via N–H  O interactions.
As a consequence, two adjacent chains are placed together along the [1% 01] direction.

As a consequence, two adjacent chains are placed together along
the [1% 01] direction (highlighted in red in Fig. 3c). This arrange-
ment is also stabilized by p  p interactions with an inter-
centroid distance of 3.937(2) Å between 5-FC molecules of
adjacent chains.
5FC–CA. The 5FC–CA cocrystal crystallizes in the triclinic P1%
space group with Z0 = 1. The ASU comprises a 5-FC molecule asso-
ciated with a CA one (Fig. 1c) via a O–H  O [2.617(2) Å, 171.49(2)1]
interaction (Fig. 4a). The 5-FC molecules form 1D chains
through the robust amide  carbonyl (highlighted in red, Fig. 4b)
and amide  amine (highlighted in green, Fig. 4b) homosynthons
(N–H  O: 2.792(2) Å, 173.78(2)1 and N–H  N: 2.981(2) Å,
170.58(2)1). Along this 5-FC chain, the CA molecules are con-
nected via the R23(9) synthon [O–H  O: 2.617(2) Å, 171.49(2)1;
N–H  O: 2.792(1) Å, 173.78(2)1 and C–H  O: 3.150(2) Å,
149.83(2)1, highlighted in yellow, Fig. 4b] and the R44(16) one

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Fig. 4 (a) Asymmetric unit of the cocrystal 5FC–CA stabilized by O–H  O interactions. (b) Partial view down the [100] direction of 5FC–CA crystal
packing highlighting the formation of amide  carbonyl and amine  amide homosynthons as well as R23(9) and R44(16) graph set motifs stabilized by
N–H  O, O–H  O and C–H  O interactions. Black dotted lines indicate hydrogen bonds. (c) Stacking of 5FC–CA molecules showing the p  p
interactions between 5-FC molecules of adjacent chains that give rise to a three-dimensional arrangement in which parallel hydrophilic channels
(the spacefill representation colored in light blue) of 5-FC are intercalated by hydrophobic channels of CA.

[N–H  O: 2.810(2) Å, 125.95(2)1 and O–H  O: 2.617(2) Å,
171.49(2)1, highlighted in grey, Fig. 4b] forming a plane. Such
planes are stacked through slip-stacked p  p interactions
(highlighted in red, Fig. 4c) between adjacent 5-FC molecules
from different planes (centroid–centroid distance: 3.975(2) Å,
Fig. 4c). Parallel hydrophilic channels of 5-FC (the spacefill repre-
sentation colored in light blue) and CA hydrophobic molecules
characterize the crystal packing assembly (Fig. 4c).
Thermal analysis
The thermal analysis and the phase purity of 5-FC cocrystals
were assessed by a combination of DSC, TGA and HSM techni-
ques. DSC and TGA curves are shown in Fig. S8 (see ESI†). For
comparative purposes, the DSC curve of 5-FC (anhydrous and
monohydrate) and those of all solid coformers were also measured
(see Fig. S9, ESI†).
The DSC curve of the 5-FC raw material (anhydrous) is
characterized by an endothermic peak at B299.62 1C coupled
to an exothermic one, both attributed to the degradation
process. The DSC curve of 5-FC exposed to a humid environ-
ment (B100% relative humidity and 25 1C), in turn, is very
different from the anhydrous form, since it is characterized by
one extended and endothermic peak in the 70.19–131.91 1C
range, which corresponds to the dehydration process, and one
endothermic peak at 237.80 1C, related to the melting process.
All the novel phases exhibited lower thermal stability when
compared to the anhydrous 5-FC raw material. The cocrystal
degradation temperatures showed a direct dependence on the
coformer melting point, i.e. a lower degradation temperature
cocrystal was formed by a lower melting coformer. According to
the TGA data, the cocrystals 5FC–CAF and 5FC–PABA are thermally
stable from 50 1C up to about 196.76 1C and 155.75 1C, respectively.
For the 5FC–CA sample, however, this temperature is about
87.49 1C. After these temperatures, the respective material
shows a significant weight loss as can be observed in its corre-
sponding TGA curve.
Moreover, the DSC curve of 5FC–CAF shows three endo-
thermic peaks at 252.28 1C, 272.80 1C and 294.63 1C, followed by
an exothermic one at 306.08 1C. For the 5FC–PABA cocrystal, two
endothermic peaks at 221.93 1C and 288.03 1C were detected.
Finally, for the sample 5FC–CA, the DSC curve exhibits a single
very well defined endothermic peak at 116 1C followed by
undefined and coupled peaks. For all cases, these events were
attributed to the cocrystal degradation since a gradual mass
loss is observed in the corresponding TGA curves.
The thermal behavior of the cocrystals was also observed in
the HSM experiments (Fig. 5) and was successfully confirmed
by them. The crystals gradually get dark as the temperature
rises becoming opaque at B195.0 1C (5FC–CAF), B150.0 1C
(5FC–PABA) and B95 1C (5FC–CA). From these temperatures
forward the samples continue reducing their mass in a degra-
dation process.
Spectroscopy analysis
Fourier transform infrared (FT-IR) and Raman (FT-Raman)
spectroscopy are popular analytical methods that provide
crucial information about vibrational modes of the molecules
as well as information about hydrogen bonds, molecular con-
formations and crystal packing of the API.57,58 For this purpose,
a comparative analysis of 5-FC and its cocrystals’ FT-IR and
FT-Raman spectra (Fig. 6 and Fig. S10, respectively; see ESI†)
was carried out. This study shows slight dislocations of cocrystal
5-FC bands when compared to the bands present in the
5-FC spectra. Generally speaking, peak shifts of about 10 to

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NJC Paper

in the 3500–3400 cm1 and 1750–1670 cm1 ranges which are

related to the hydroxyl and carbonyl stretches of the COOH group,
respectively. These bands are observed in the FT-IR cocrystal spectra
at 3476 cm1 and 1675 cm1 (5FC–PABA); and 3358 cm1 and
1720 cm1 (5FC–CA). The same findings are observed in the 5FC–
PABA and 5FC–CA Raman spectra, where the CQO absorption
band appears at 1703 and 1722 cm1, respectively. Additionally, in
the case of 5FC–CA, new bands associated with the aliphatic chain
vibrations could be observed. The FT-IR and FT-Raman spectra
show characteristic bands in the region 3000–2800 cm1 assigned
to the C–H stretching modes.

Phase transition and stability test

Fig. 5 Crystal behavior as a function of temperature visually checked by
HSM of the forms (a) 5FC–CAF, (b) 5FC–PABA and (c) 5FC–CA.
PXRD as a function of temperature was used to show the
irreversible hydration process of 5-FC (Fig. 7) and to study the
physical stability of the reported cocrystals (Fig. 8). Variable
temperature powder X-ray diffraction (VT-PXRD) is a method
where XRD experiments are performed at different tempera-
tures and it provides information about the solid-state changes
in materials during heating. This direct information is neces-
sary to understand the structure of pharmaceutical solids in the

Fig. 6 FT-IR spectra of 5-FC raw, 5FC–CAF, 5FC–PABA and 5FC–CA.

30 cm1 in the spectra are indicative of changes in hydrogen

bonding patterns.59,60 Fig. 7 Variable temperature PXRD patterns for 5-FC monohydrate.
The spectra interpretation and band assignments (Table S6,
see ESI†) were performed taking into account the crystallo-
graphic study and the spectroscopic data available for related
5-FC compounds.36,61,62 5-FC molecules exhibit IR and Raman
stretching frequencies at 3375 cm1 (amine N–H stretch),
1682 cm1 and 1677 cm1 (amide CQO stretches), 1337 cm1
and 1340 cm1 (pyrimidine ring C–N stretches) and 1227 cm1
and 1236 cm1 (C–F stretches). Some of these stretching frequen-
cies appear shifted (10–30 cm1) in the FT-IR and FT-Raman
spectra (Table S6, see ESI†), indicating cocrystal formation.
According to the literature, amide groups normally display a
characteristic absorption band at about 1700–1600 cm1 related
to the carbonyl stretch. Thus, two absorption bands are observed
in the 5FC–CAF spectra at 1650 cm1 in the FT-IR and 1630 cm1
in the FT-Raman, and are attributed to the carbonyl stretching
modes of caffeine.
As expected, carboxylic p-aminobenzoic and caprylic acids, Fig. 8 PXRD patterns of 5-FC cocrystals showing the maintenance of the
used as coformers, display two characteristic absorption bands crystalline phase after one week in a humid environment.

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development process of new formulations.63–67 The VT-PXRD
studies of 5-FC monohydrate show no change until B75 1C.
After this temperature the highly defined diffractions peaks of
the starting material start to disappear showing its amorphiza-
tion due to water molecule loss (colored in green, Fig. 7).
Since the water molecules are strongly bound into the crystal
structure (corroborated by the extended peak in the DSC curve,
Fig. S9, ESI†) their loss destabilizes the crystal packing. However,
in the 125 1C to 150 1C range the crystallinity of the sample
reappears but with another identity, that is, it does not correspond
to the anhydrous 5-FC solid phase (colored in red, Fig. S11, see
ESI†). This phase transition could also be observed in the 3D
surfaces (Fig. S12, see ESI†). This information is also supported by
the DSC curve of the resulting material after VT-PXRD (Fig. S13,
see ESI†). The DSC curve shows four endothermic peaks at
88.99 1C, 235.22 1C, 252.11 1C and 265.75 1C. Is interesting to
note that this new anhydrous phase turns back to the mono-
hydrate form after one week of exposure to a highly humid
atmosphere, as can be seen in the PXRD patterns of Fig. S11
(see ESI†). The degrees of crystallinity of cocrystal forms were
reduced after the accelerated stability test (100% RH atmosphere),
but the main characteristic peaks of the calculated patterns
remain present in the experimental ones after the same exposure
time, confirming no phase transition as can be observed in Fig. 8.
Equilibrium solubility
The solubility study of the cocrystals was performed in a buffer
mimicking the stomach pH. These tests show that the new solid
forms retain solubility values statistically similar to the one of the
5-FC raw material (Fig. 9 and Table S7, see ESI†). PXRD also shows
that the crystal structures of the solids obtained after the solubility
test remain the same as the original ones (Fig. S1, see ESI†).
However, diffractograms before and after the solubility study of
5-FC do not show the same results. In this case, the remnant solid
shows additional peaks that correspond to other 5-FC polymorphs
as well as different hydrates (Fig. S14, see ESI†). When thermal
analyses of these solids are performed three important facts are
exposed: (i) the DSC contrast between the solids before and after
the solubility study shows an endothermic signal at 52.19 1C and
another at 104.42 1C (highlighted in gray, Fig. S15, see ESI†) that
Fig. 9 Solubility concentration of 5-FC and the different novel cocrystal
forms in pH 1.2 dissolution media.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2018 New J. Chem., 2018, 42, 14994--15005 | 15003
NJC
might correspond to the phase transition and/or dehydration
process; (ii) the DSC plot (also in Fig. S15, see ESI†) shows the
same signal at B300 1C (highlighted with a red asterisk) corres-
ponding to the decomposition of 5-FC anhydrous and finally,
(iii) the TGA comparison between 5-FC monohydrate and the 5-FC
solid form obtained after the solubility studies (Fig. S16, see ESI†)
reveals that the amount of water in the crystal structure is reduced
when compared to the monohydrate. The summary of these facts
seems to indicate that solid 5-FC anhydrous, in the presence of
the buffer solution pH 1.2, produces a different kind of hydrates
and also suffers a phase transition. The DSC profiles (Fig. S15,
ESI†) show that all these changes are reversible.
Conclusions
Single-crystal X-ray diffraction studies show that 5-FC is able to
form different homo and heterosynthons, which is a notable
feature of N-heterocyclic derivatives, such as pyridines and
pyrimidines. These results corroborate the instability of the
5-FC anhydrous raw material and the efficacy of methodologies
such as rational planning and supramolecular synthesis in the
development of more stable API solid forms. The vibrational
spectroscopic studies were found to be congruent with the
chemical structures of the three novel cocrystals. VT-PXRD
shows that it is not possible to recover anhydrous 5-FC by
heating the monohydrate form since this hydration process is
irreversible and the heating gives rise to a non-reported crystal-
line phase (see Fig. S17, ESI†). Since the hydration process does
not occur in the new cocrystals reported here they can be used
by the pharmaceutical industry as a very attractive strategy to
increase the physical stability of the API, avoiding in this way
the phase transitions in pharmaceuticals containing 5-FC in
their formulations. The solubility profiles of 5-FC cocrystals in
buffer pH 1.2 show solubility values very close to the 5-FC raw
material. This prodrug is classified as a class I drug due to its
considerable solubilization in the stomach at low pH. This fact
strengthens the establishment of cocrystallization as an effec-
tive tool for the stabilization of an API with respect to hydration
without severe impairment of solubility.
Author contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
Conflicts of interest
The authors declare no competing financial interest.
Acknowledgements
The authors acknowledge the Brazilian funding agencies CAPES
(M. S. S.), FAPESP (L. F. D. grant 15/25694-0) and CNPq (J. E.
grant #305190/2017-2) for financial support. The authors would
NJC
also like to thank Dr Charlane C. Correa, and Dr Luiz F. C.
de Oliveira (Federal University of Juiz de Fora) for allowing access
to the Raman spectroscopy (measurements for all cocrystals
reported here) and single-crystal X-ray facilities (data collection
of the cocrystals 5FC–CAF and 5FC–PABA). L. Vogt thanks IFSC/
USP for being granted with a Salazar Scholarship which made
possible the active collaboration in this work. Moreover, the
authors would like to thank Prof. Dr Christian W. Lehmann
(Max-Planck-Institut für Kohlenforschung) for allowing access to
the X-ray diffraction facilities, which made possible the 5FC–CA
data collection. Finally, the authors would like to thank Igor R. dos
Santos (São Carlos Institute of Physics) for generating the PXRD 3D
surfaces through a script in Python.
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