NJC

PAPER

Supramolecular synthesis and characterization of

crystalline solids obtained from the reaction of
Cite this: New J. Chem., 2019,
43, 15924 5-fluorocytosine with nitro compounds†
a
Matheus S. Souza, Luan F. Diniz, a Natalia Alvarez, b
a
Cecı́lia C. P. da Silva and Javier Ellena *a

Received 26th June 2019,
Accepted 12th September 2019
DOI: 10.1039/c9nj03329g
rsc.li/njc
Two novel multicomponent crystal forms of the prodrug 5-fluorocytosine (5-FC), an ionic cocrystal
and a monohydrate salt, with 2,4,6-trinitrophenol (picric acid, PA) and 3,5-dinitrosalicylic acid (DNSA),
respectively, were rationally designed and synthesized. Nitro compounds are used to treat many
diseases, such as cancer, one of the 5-FC targets via gene-directed prodrug therapy. In addition, within
the wide range of applications for nitro compounds is the formation of charge-transfer complexes with
organic molecules. Among all 5-FC crystalline structures depicted, only two contain nitro compounds: a
salt with PA and a dihydrate cocrystal with 5-nitrouracil. Therefore, the two new single translucent pale
gold prism crystals of 5FC-PA and 5FC-DNSA were successfully obtained by slow evaporation from
aqueous solution and characterized by X-ray diffraction (single crystal and powder), Fourier Transform
Infrared (FT-IR), and Raman (FT-Raman) spectroscopies. In both crystal structures, the 5-FC pyrimidine
ring is protonated at the N3 atom and the respective packings are mainly stabilized by N–H  O
H-bonds. For the 5FC-PA ionic cocrystal, the formation of triple H-bonds (two N–H  O and one charge
assisted N–H+  N H-bonds) among the 5-FC molecules was observed. This is consistent with the
expected stability of the duplex structure, which is considered the strongest association between two
species. In addition to the structural study, Hirshfeld surface analysis was carried out based on the single
crystal X-ray diffraction data. Thermal stability was assessed through thermogravimetric analysis (TGA),
differential scanning calorimetry (DSC), and hot-stage microscopy (HSM), indicating that these compounds
are stable up to approximately 200 1C. The results contribute to the diversity of new 5-FC solid forms and
introduce two novel compounds, with possible application in fungal/cancer medicine formulations or to
better understand the behavior of cytosine in DNA/RNA base pairing.

1. Introduction is the key to modulate properties like solubility, bioavailability,

Crystal engineering plays an important role in many pharma-
ceutical industrial processes, given that designing new solid-
state forms of known active pharmaceutical ingredients (APIs)
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;
Fax: +55 (016) 3373-8096/3373-9876
b
Facultad de Quı́mica, Universidad de la República, General Flores 2124,
Montevideo, Uruguay
† Electronic supplementary information (ESI) available: Fig. S1. Calculated and
experimental powder X-ray diffraction patterns of 5-fluorocytosine. The diffrac-
tion patterns are in good agreement indicating that the starting material presents
high crystallinity and purity; Fig. S2. Calculated and experimental (in different
stoichiometries) powder X-ray diffraction patterns of FCPA salt; Table S1. Angular
dimensions of protonated and neutral 5-fluorocytosine and Table S2. Calculated
DpKa values for the reaction of 5-FC with the nitro acids studied here. CCDC
1840645 and 1840646. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c9nj03329g
flow, compressibility, thermal stability, crystallinity, and hygro-
scopicity, among many others.1–4 Aiming to tune the physico-
chemical profile of pharmaceutically active molecules in order
to circumvent problems related to development and manufacturing,
recent studies have examined the solid-state landscape of cytosine
derivatives for the synthesis of new crystal forms, whether for use in
pharmaceuticals or biological sensors/systems, taking into account
that cytosine plays a crucial role in DNA/RNA base pairing, through
several H-bonding patterns.1,5–19 The search for new solid forms has
recently focused on salt20 and cocrystal formation.21,22 In the first
one, the former is restricted to ionizable molecules, while the latter
provides a wider spectrum of options. A particular type of pharma-
ceutical solid form in the cocrystal family is the ionic cocrystals
(ICCs), in which the coformer is a salt. According to Song et al.23 this
subset of compounds can improve the parent compound’s stability
because of interactions between the organic moiety and the ions
from the salt. Moreover, the choice of coformers is fundamental and

15924 | New J. Chem., 2019, 43, 15924--15934 This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019
Paper
Scheme 1 Chemical structure of the prodrug 5-fluorocytosine and the
coformers picric and 3,5-dinitrosalicylic acids.
motivated mostly by acid–base and electronic properties, where
the goal is to exploit their combined effect giving rise to products
with improved features.24 It is also important to mention that the
synthesis of salts and cocrystals is based on the principles of
Supramolecular Chemistry, once these compounds are formed
and stabilized mainly by non-covalent interactions (H-bonds,
charge-assisted bonds, hydrophobic interactions, van der Waals
forces, p  p stacking, and dipole–dipole bonds) and electrostatic
forces (magnetic forces), without modifying the pharmacophore
group of the API.
5-Fluorocytosine (4-amino-5-fluoro-1,2-dihydropyrimidin-2-
one, 5-FC, Scheme 1) is a synthetic cytosine derivative used
as a fungicide and antineoplastic prodrug, via mammalian cell
gene therapy.25–32 5-FC belongs to the group of the halopyrimidines
due to replacement of hydrogen by a fluorine group.33–35 This
monofluorinated analogue of cytosine is geometrically very similar
to its parent molecule, meeting the steric requirements at enzyme
receptor sites. In addition, the substitution of two carbon atoms in
the phenyl ring for nitrogen increases its nucleophilicity, becoming
prone to interacting with p deficient systems.36,37 Taking these
features of 5-FC into account and given the importance of having
more systems capable of offering insights concerning the DNA/RNA
base pairing, as part of the ongoing interest in studying supra-
molecular architectures of biological proton-transfer compounds,
our group has chosen picric acid (PA)38,39 and 3,5-dinitrosalicylic
acid (DNSA)40 as coformers for 5-FC in the search of new solid
forms (Scheme 1). This choice was made by considering the
strong electron-withdrawing properties addressed to nitro groups
in the phenole aromatic ring that make them potent p-acceptor
moieties and, consequently, place them as suitable options for
synthesis of charge-transfer complexes.41,42 In addition, nitro
compounds, as part of drugs, are long used as antineoplastic,
antibiotic, and antiparasitic agents, which also allows the future
application of the new structures for medicinal purposes.33–35 PA,
although regarded as toxic, similar to many other nitroaromatic
and heteroaromatic compounds, also has application in the treat-
ment of malaria, trichinosis, herpes, smallpox, and as an antiseptic.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019
NJC
Toxicity is indubitably a major concern when dealing with
medicines, but selective toxicity also is the base for using these
nitro compounds for pharmaceutical purposes. DNSA, to the
best of our knowledge, still has no medicinal applications, but
as a salicylic acid derivative, perhaps may find some chemo-
therapy application. On the other hand, DNSA has been used as
a good charge-transfer complex before, and provides one of
the best chemical synthons for the construction of H-bonded
structural motifs.43–45
In this manuscript, new structures have been prepared
considering the protonable pyrimidine N3-atom of the 5-FC mole-
cule (pKa: 3.26) and the possible resultant pyrimidinium  anion
(N3+  A) synthon.8,20,46,47 To simplify the discussion, the following
nomenclature is adopted: FC for neutral 5-FC molecule, FCH for
protonated 5-FC molecule, FCPA for the picrate salt, 2FCPA for
the ionic cocrystal with picric acid, and FCDNS for the mono-
hydrate salt. It is worth mentioning that the crystalline struc-
ture of the FCPA has already been reported in the crystal
structure and hydrogen-bonding patterns in 5-fluorocytosinium
picrate, however the physicochemical properties have not been
reported in Mohana and coworker’s manuscript.15 The obtained
solid forms were characterized by single crystal X-ray diffraction
(SCXRD), powder X-ray diffraction (PXRD), Fourier Transform
Infrared (FT-IR), and Raman (FT-Raman) spectroscopies. Their
thermal stability was assessed using hot-stage microscopy (HSM),
thermogravimetric analysis (TGA) and differential scanning
calorimetry (DSC).
2. Experimental details
Anhydrous 5-FC was purchased from Sigma-Aldrich Brazils
and used for the crystallization batches without any further
purification. Crystalline purity of 5-FC was assessed by PXRD
(Fig. S1, see the ESI†). PA, DNSA, Milli-Q water, acetonitrile and
isopropyl alcohol were used as commercially available.
2.1 Supramolecular synthesis
FCPA. 5-Fluorocytosine picrate salt was obtained by dissolving
5 mg (0.039 mmol) of 5-FC in 2 mL of acetonitrile/water (1 : 1
mixture) at 100 1C and adding an aqueous solution with
equimolar quantity of PA (8.93 mg, 0.039 mmol) under constant
stirring. Experiments carried out with an excess of PA (13.4 mg,
0.585 mmol) led to the formation of the same crystal structure
(Fig. S2, see the ESI†).
2FCPA. Similar to FCPA, for 5-fluorocytosine ionic cocrystals,
5 mg (0.039 mmol) of 5-FC were added to 2 mL of acetonitrile/
water (1 : 1 mixture) at 100 1C. Then, 4.47 mg (0.0195 mmol) of
PA was added to the initial 5-FC solution and stirred well at
100 1C to yield a homogeneous system.
FCDNS. Finally, for 5-fluorocytosine monohydrate salt, 5 mg
(0.039 mmol) of 5-FC were added to 2 mL of isopropyl alcohol/
water (1 : 1 mixture) at 100 1C. Next, 8.8 mg (0.039 mmol) of
DNSA were added to this solution and the system was stirred
using a temperature controlled magnetic stirrer at 100 1C until
complete dissolution of the acid. The resulting solution was
New J. Chem., 2019, 43, 15924--15934 | 15925
NJC Paper

allowed to cool slowly at room temperature and covered with
Parafilms for slow evaporation of the solvent.
It is worth noting that all systems described above were
maintained at room temperature in a controlled crystallization
room at 25 1C. Yellow crystals were obtained in all cases within
3 to 5 days.
2.2 Single crystal X-ray diffraction
Adequate single crystals were selected for X-ray diffraction data
collection at room temperature (293  2 K). In the case of FCPA,
the crystal structure has been previously reported by McMillen
et al.15 Crystallographic data for 2FCPA were collected on an
Enraf-Nonius Kappa-CCD diffractometer (95 mm CCD camera
on k-goniostat) using graphite-monochromated MoKa radiation
(0.71073 Å). The software COLLECT48 and the HKL package (Denzo,
XDisplayF and Scalepack software)49 were used for data acquisition,
indexing, integration and scaling of Bragg-reflections. The structure
was solved by iterative methods using SHELXT50 within Olex2.51
Data collection for FCDNS was carried out on a Bruker APEXII-CCD
diffractometer using graphite-monochromated MoKa radiation
(0.71073 Å), using APEX2 software for strategy planning and data
reduction. The structure was solved using dual space methods
using SHELXD52 within Olex2.51 The obtained structures were
refined by full–matrix least squares on F2 with SHELXL53 within
Olex2.51 Hydrogen atoms were positioned in calculated positions
and refined isotropically constraining their displacement para-
meters to be equal to 1.2 or 1.5 times the corresponding
isotropic displacement parameters of the bonded atom [Uiso(H) =
1.2 Ueq(N, C) or 1.5 Ueq(O)] according to the riding model. The
protonation was confirmed by the location of the proton in the
difference Fourier map as well as by the analysis of the widening of
the C2–N3–C4 5-FC pyrimidinic internal angle.54 In all cases, the
use of spectroscopic techniques, such as FT-IR, was imperative for
confirming the proton transfer.
MERCURY (version 3.10.2)55 and ORTEP-356 software were
used to prepare the graphical representations for publication.
The CIFs of the two novel 5-FC structures were deposited in the
Cambridge Structural Database (CSD)57 under refcodes 1840645
and 1840646.†
2.3 Powder X-ray diffraction (PXRD)
PXRD measurements of the ground samples were performed
on a Rigaku Ultima IV powder diffractometer using CuKa
radiation, l = 1.5418 Å; 40 kV; 30 mA; step scan width of
0.021 in an interval of 5–501 in 2y and 3 s per step.
2.4 Hot-stage polarized optical microscopy (HSM)
Tests were performed on a Leica DM2500P microscope connected
to the Linkam T95-PE hot-stage equipment controlled with
Linksys 32 software (version 1.96). One crystal was placed on
a 13 mm glass coverslip, placed on a 22 mm diameter pure
silver heating block inside the stage. The sample was heated at
a ramp rate of 10 1C min1 up to total sublimation for FCPA
and 2FCPA or melting for FCDNS.
2.5 Thermal analysis
Thermogravimetric analysis was performed using a Shimadzu
TGA-50 thermobalance. Approximately 5.0 mg of each sample
were placed in an alumina crucible and heated at 10 1C min1
under a N2 atmosphere (50 mL min1) from 25 to 320 1C. The
Differential Scanning Calorimetry (DSC) data acquisition was
carried out according to previous TGA data, that is, until the
degradation temperature of each compound. This experiment
was performed on a Shimadzu DSC-60 calorimeter. The samples
(2.0 mg) were heated from 25 to 320 1C with a heating rate of
10 1C min1 in a crimped sealed aluminum pan, using nitrogen
as the purge gas (50 mL min1). Data were processed using
Shimadzu TA-60 thermal data analysis software (version 2.2).

Table 1 Crystal data and structure refinement of the novel solid forms 2FCPA and FCDNS

Identification 2FCPA FCDNS

Empirical formula C14H11F2N9O9 C11H10FN5O9
Formula weight 487.32 375.24
Temperature/K 293(2) 293(2)
Crystal system Triclinic Monoclinic
Space group P1% C2
a/Å 8.4380(2) 21.337(3)
b/Å 10.6270(3) 5.4905(9)
c/Å 11.2100(3) 12.445(2)
a/1 76.3720(10) 90
b/1 87.981(2) 103.196(3)
g/1 73.180(2) 90
Volume/Å3 934.53(4) 1419.5(4)
Z 2 4
rcalc mg cm3 1.732 1.756
m/mm1 0.158 0.162
Crystal size/mm 0.18  0.09  0.06 0.22  0.08  0.03
F(000) 496 768
Independent reflections 3568 [R(int) = 0.0301] 4532 [R(int) = 0.0255]
Radiation MoKa MoKa
Data/restraints/parameters 3568/0/307 4532/1/239
Goodness-of-fit on F2 0.894 1.070
Final R indices [I Z 2s (I)] R1 = 0.0497, wR2 = 0.1203 R1 = 0.0306, wR2 = 0.0808
R indices [all data] R1 = 0.0711, wR2 = 0.1279 R1 = 0.0333, wR2 = 0.0832

15926 | New J. Chem., 2019, 43, 15924--15934 This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019
Paper NJC

Table 2 Hydrogen-bonding geometry (Å, 1) for the novel 5-fluorocytosine structures

Interaction DA(Å) HA(Å) D–HA(1) Symmetry code

2FCPA
N3–H3O21 3.552(2) 2.901(2) 133.92(2) x,y,z
N3–H3N3 2.825(2) 1.968(2) 174.50(2) x,y + 2,z + 2
N1–H1O3 2.696(2) 2.018(2) 135.05(2) x + 1,y + 2,z + 1
N1–H1O9 3.173(2) 2.407(2) 148.63(2) x,y,z
N41 0 H41CO21 2.869(2) 2.013(2) 173.58(2) x,y,z
N41–H41AO21 0 2.824(2) 1.967(2) 174.46(2) x + 1,+y,+z  1
N1 0 H1 0 O21 0 2.853(2) 2.003(2) 169.30(2) x + 1,+y,+z
C15–H15O8 3.441(2) 2.890(2) 119.24(2) x + 1,y + 1,z + 2
C15–H15O9 3.799(2) 2.912(2) 159.95(2) x + 1,y + 1,z + 2
N41–H41BO4 3.048(2) 2.191(2) 174.60(2) x + 1,y,z + 1
N41–H41BO5 3.611(2) 2.919(2) 138.82(2) x + 1,y,z + 1
N41–H41BN7 3.747(2) 2.917(2) 162.54(2) x + 1,y,z + 1
C6–H6F51 3.204(2) 2.447(2) 138.55(2) x + 1,y,z + 1
C6 0 H6 0 O5 3.191(2) 2.284(2) 165.04(2) x + 1,+y + 1,+z  1

FCDNS
O1–H2AN2 2.731(4) 1.916(3) 171.76(19) x,y,z
O4–H4CN3 2.819(4) 2.190(5) 148.96(16) x,y,z
N1–H1 O1 2.851(3) 2.128(2) 141.49(19) x,+y  1,+z
N5–H5AO3 2.992(5) 2.180(3) 155.81(21) x + 1,+y  1/2,z + 2
N3–H3BO1 3.218(5) 2.454(4) 143.34(17) x + 1,+y  1/2,z + 2
N3–H3AO5 3.091(4) 2.239(3) 169.46(23) x,+y + 1,+z
N4–H4AN4 3.201(5) 2.396(4) 154.27(26) x,+y  1/2,z
N4–H4BO5 2.998(5) 2.170(4) 162.12(20) x + 1,+y + 1/2,z + 1

2.6 Vibrational spectra 3. Results and discussion

Fourier Transform infrared (FT-IR) spectra were recorded on an
Alpha Bruker FT-IR spectrophotometer as KBr pellets, in the
range of 4000–400 cm1, with an average of 64 scans and 2 cm1
spectral resolution. FT-Raman spectroscopy was performed
using a Bruker RFS 100 instrument with Nd3+/YAG laser
operating at 1064 nm in the near-infrared region and a CCD
detector cooled with liquid nitrogen using a spectral resolution
of 4 cm1.
3.1 Structural description
A summary of crystallographic data and refinement parameters
for 2FCPA and FCDNS is shown in Table 1.
In both structures, FC is protonated at the N3 atom of
the pyrimidine ring, this being evident by the widening of the
C2–N3–C4 pyrimidinic internal angle (Table S1, ESI†), which
assumes a value of 1201 in the FC molecule58 and values close

Fig. 1 Asymmetric unit of FCPA, 2FCPA, and FCDNS.

This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019 New J. Chem., 2019, 43, 15924--15934 | 15927
NJC
Fig. 2 (a) Hydrogen bond pattern between coplanar FCH–FC units, (b)
nitro–nitro interactions between picrate anions and (c) hydrogen bond
pattern in the structure.
to 1251 in the FCH one, as previously proposed by Clowney et al.
in 1996.54 By considering that the synthesis of these structures
lies in the salt-cocrystal range, where 0 o DpKa o 3 units
(pKa of PA = 0.3835 and pKa of DNSA = 2.18,59 Table S2, ESI†),
then it is possible to agree with the statement of da Silva et al.8
that FC tends to form protonated structures in the continuum.1,7,8
In addition, the complementary shape and bonding feature
of interacting reagents strengthens the formation of periodic
supramolecules.60 The geometric parameters of H-bonds present
in the crystal structures are listed in Table 2. The ORTEP-view56 of
the asymmetric units, including the one of FCPA (reported on the
CSD57 under database identifier ZAPFEE15), are shown in Fig. 1.
3.1.1 5-Fluorocytosine 5-fluorocytosinium picrate, 2FCPA.
Single-crystal XRD analysis reveals that 2FCPA crystallizes in
the triclinic space group (P1% ) with one FCH, one FC and one
PA in the asymmetric unit (Fig. 1). As previously reported for
related FCH salts, a three-point synthon between an FCH and
FC is formed, showing higher stability with respect to the crystalli-
zation of exclusive FCH moieties.61–63 The supramolecular dimer
Fig. 3 (a) Water mediated chain formation between DNSA anions, (b) crystal packing diagram view along the b crystallographic axis and (c) hydrogen
bond pattern of the FCH moiety.
15928 | New J. Chem., 2019, 43, 15924--15934 This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019
Paper
involves a triple hydrogen bond structure, similar to that observed
between the pyrimidinic cytosine and purinic guanine bases in
the deoxyribonucleic acid (DNA).64 In this arrangement there
are two N–H  O (N41 0 H41C  O21 and N41–H41AO21 0 with
D  A distances 2.013(2) and 1.967(2) Å, respectively) and one
N–H  N (N3–H3  N3 0 , D  A 1.968(2) Å) strong hydrogen bonds,
as classified by Desiraju, et al.,65 forming R22(8) bonding patterns.
Adjacent dimers interact through a two-point homosynthon
between the FC moieties involving N–H  O hydrogen bonds
in an R22(8) pattern giving rise to a supramolecular tetramer
arrangement of 2FCH and 2FC molecules along the 21% 1
plane (Fig. 2a).
The picrate anions are positioned in the plane defined by
the supramolecular tetramer discussed above. The FCH inter-
acts with the carbonyl group in the PA anion through a
N1–H1  O3 H-bond (Fig. 2c). On the other hand, the nitro
substituents participate in classical N–H  O (N41–H41B  O4,
N41–H41B  O5, N1–H1  O9), as well as nonclassical, C–H  O
H-bonds (C15–H15  O8, C15–H15  O9). The nitro group
O9–N9O8 is tilted from the 21% 1 plane to enable the participa-
tion in nitro–nitro interactions (O  O distance: 3.167(2) Å,
Fig. 2b). Nitro–nitro interactions can be explained through an
unequal electron distribution along the group, which normally
presents 2.5 lone electron pairs in their resonant forms.66 This
uneven charge distribution can be evidenced by the difference
in the N–O distances N9–O8 1.229(3) Å and N9–O9 1.219(2) Å,
respectively, leading to the tilting of the nitro groups contain-
ing N9 with respect to the plane of the hydrogen network layer
formation in order to position themselves in an antiparallel
fashion where the electron deficient O8 is positioned in front of
the O9 from the contiguous molecule, and vice versa, confirm-
ing the formation of a dipole–dipole interaction.
3.1.2 5-Fluorocytosinium 3,5-dinitrosalicylate monohydrate,
FCDNS. The structure of FCDNS crystallizes on the monoclinic
space group C2. The asymmetric unit contains one FCH, one
DNSA, and one water molecule (Fig. 1). An interesting feature in
the crystal packing of FCDNS is the formation of R22(10) chains,
constituted by intercalated molecules of DNSA and water running
Paper
Fig. 4 Calculated and experimental powder X-ray diffraction patterns of
FCPA, 2FCPA and FCDNS.
parallel to the [001] direction (Fig. 3a). FCH cations occupy
the space between these chains (Fig. 3b) participating in five
contiguous classical N–H  O H-bonds, being two with FCH, two
with DNSA, and one with a water molecule (Fig. 3c). The amide
groups (N1–H1 and N3–H3) in FCH act as monotopic H-bond
donors to the water molecule and to the carbonyl oxygen
atom (O21) of an adjacent FCH, whereas the amine group
(H41A–N41H41B) acts as a ditopic H-bond donor to the
carboxylic oxygen atom of the DNSA. The fifth hydrogen bond
is between the carbonyl oxygen atom (O21), acting as a mono-
topic H-bond acceptor, with the N1–H1 amide group of a
neighbor FCH molecule. In contrast to the structure discussed
above, the lack of a neutral FC moiety in the structure does not
allow the formation of the three-point synthon.
Concerning the FCFCH triple point H-bond, from the 72
crystalline structures reported in the CSD57,67 (version 5.40,
Fig. 5 Intermolecular interaction contributions in the asymmetric unit of 5-fluorocytosine structures: (a) FCH in 2FCPA, (b) FC in 2FCPA and (c) FCH in FCDNS.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019
NJC
September 2019), only 4 exhibit this pattern (refcodes SIJXAN,68
UWANEN,61 WEWZAA,69 and WOJYAX63), 1 exhibits an FCFC
triple H-bond (refcode PANLAS),70 1 is a case where the hydrogen
atom is shared among the FC molecules (refcode SIJXIU),10 and
another 8 structures exhibit FCcoformer triple point H-bonds
(refcodes MECWEY,71 MECWAU,71 MECVUN,71 MECVOH,71
MECWOI,71 MECWUO,72 MECXEZ,72 and MECXID72). From the
4 structures containing FCFCH triple point H-bonds, only the
ones under refcode WEWZAA69 and WOJYAX63 exhibit the same
pattern of interaction resembling the 2FCPA one with the tetramer
formation. However, it is worth mentioning that this is the first
time, at least to the best of our knowledge, that a nitro compound
exhibits the FCFCH triple point H-bond. The advantage of having
this supramolecular architecture occurring with a nitro compound
is that its formation can perhaps be better evaluated, considering
the charge-transfer properties associated with nitro compounds.
Moreover, there are only 2 structures in the CSD57,67 (version 5.40,
September 2019) involving FC and nitro compounds: the picrate
already mentioned (refcode ZAPFEE)15 and a monohydrate
cocrystal with 5-nitrouracil (refcode GATMUL).18 Nevertheless,
these two nitro structures are different from the FCDNS
depicted herein; once in the new structure it can be observed
that the protonated N3 atom of the FCH molecule forms its
H-bond with the oxygen atom of the adjacent FCH one. In the
GATMUL18 and ZAPFEE15 structures, the protonated N3 atom
of FCH interacts only with the coformer. So, perhaps the FCDNS
structure may be more helpful toward studying charge-transfer
mechanisms related to cytosine base pairing.
3.2 Powder X-ray diffraction analysis
Powder X-ray diffraction is considered a reliable characterization
tool to confirm the crystalline nature as well as the formation of
novel multicomponent crystal forms. Whenever possible, PXRD
analysis should be used to prove if the single crystal chosen for
New J. Chem., 2019, 43, 15924--15934 | 15929
NJC
Fig. 6 Contact percentage contribution on the dnorm surface for FCPA,
2FCPA and FCDNS crystal structures.
the SCXRD data collection is a representative of the entire sample.
The presence of various and defined Bragg peaks in the experi-
mental diffractograms (Fig. 4) confirms the crystalline nature of
the synthetized materials. Moreover, by observing the PXRD
patterns from 51 to 501 (2y), it is possible to conclude that a
unique crystalline phase has been obtained for the FCPA, 2FCPA,
and FCDNS compounds as a result of the cocrystallization
reactions, since the PXRD experimental patterns exhibit a good
match with the calculated ones (generated from SCXRD data).
This finding has been confirmed by indexing the hkl values of the
10 most intense Bragg peaks observed in the experimental
diffractograms. All Bragg peaks selected have hkl values at the
same 2y position compared to the calculated diffractograms,
Fig. 7 (a)–(c) DSC and TGA curves of FCPA, 2FCPA and FCDNS, respectively. (d) Crystal behavior visually checked by HSM of FCPA, 2FCPA and FCDNS
single crystals.
15930 | New J. Chem., 2019, 43, 15924--15934 This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019
Paper
indicating that there is no contribution of crystalline impurities
in the sample.
3.3 Hirshfeld surface analysis
Hirshfeld surface analysis was carried out to evaluate semiquantita-
tively the influence of each type of intermolecular interaction invol-
ving the FC and FCH moieties in the crystal structures of 2FCPA and
FCDNS using the software Crystal Explorer17.73–75 Fig. 5(a)–(c) depicts
dnorm surface for all analyzed structures, where contacts shorter than
the sum of the van der Waals radii are colored in red, similar in
white, and longer contacts in blue. In all cases, it was possible to
confirm that, as expected, the shorter intermolecular contacts are
associated with the classical H-bonds involving the amine groups in
the FC and FCH molecules, i.e. the triple-point H-bonds. Fig. 5a and
b depicts the short-contact interactions of the FC and FCH moieties
on the 2FCPA crystal, where the FC moiety does not participate in
non-classical hydrogen bonding through the F atom.
As can be seen in Fig. 6, the predominant contacts on the
studied crystals are of polar nature, including mostly classical and
non-classical hydrogen bonds. An interesting feature of the 2FCPA
crystal packing is the presence of F  F interactions which might
explain the slight difference in the degradation temperature when
compared to the FCPA sample: DTonset TGA[2FCPA-FCPA] = 224.83 
192.66 1C = 32.17 1C, Fig. 7a and b.
3.4 Thermal analysis
Thermal stability analysis by DSC (straight solid black line),
TGA (straight dashed orange line) and hot-stage microscopy
Paper
were carried out for FCPA, 2FCPA and FCDNS structures as
depicted in Fig. 7.
The DSC curve of FCPA (Fig. 7a) presents three endothermic
peaks (221.39, 242.60 and 260.82 1C) and an exothermic one at
277.26 1C. The peak at 221.39 1C (Tonset = 217.81 1C) was correlated
with the beginning of sample degradation. This event is followed
by the appearance of well-defined peaks that were detected and
also correlated with the process of material degradation – which
could be confirmed by observing the mass decrease (Tonset =
192.66 1C) represented by the TGA thermogram (Fig. 7a). The
DSC curve of the 2FCPA (Fig. 7b) structure exhibited two asso-
ciated peaks: an endothermic at 267.17 1C (Tonset = 246.33 1C) and
an exothermic at 273.58 1C. These events were assigned to the
sample decomposition and were also observed in the TGA curve
(Tonset = 224.83 1C, Fig. 7b). Beyond the endothermic melting peak
at 236.04 1C, an initial endothermic peak at 99.09 1C is observed
in the DSC curve of FCDNS (Fig. 7c). This peak corresponds to the
sample dehydration process. The loss of one water molecule from
the crystalline lattice is also observed in the TGA curve (Fig. 7c) at
B105 1C (Tonset = 93.53 1C). This mass loss corresponds to 4.31%,
which agrees very well with the theoretical value (4.79%). The
additional exothermic DSC peak at 258.70 1C can be associated
with the decomposition of the sample since a gradual mass loss
occurs in the TGA curve right after the melting process.
The interpretation of the DSC/TGA results was successfully
confirmed by HSM experiments. The images (Fig. 7d) confirm
the beginning of FCPA degradation (B220 1C) and 2FCPA crystals
(B225 1C). For the FCDNS monohydrate salt, it is possible to see a
change in the crystal appearance at B110 1C, consistent with the
discussed dehydration process. The images also show the begin-
ning of the crystal melting occurring at around 240 1C. It is worth
mentioning that the sharpness in the DSC peaks can be easily
coordinated with good crystalline nature of the synthesized
material, corroborating that there is no evidence of impurities
in the samples.
3.5 Spectroscopic analysis
Infrared (Fig. 8) and Raman (Fig. 9) spectra of FC and its new solid
forms were analyzed taking into account the structural features
Fig. 8 FT-IR spectra of 5-FC, FCPA, 2FCPA and FCDNS.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019
NJC
observed in the crystal structure. Analysis and band assignments
(Table 3) were performed using spectroscopic data available for
previously reported structures with nitro and carboxylic acids and
related FC compounds found in the literature.43,76–78 Salt and
ionic cocrystal formation were identified evaluating the changes
in the vibrational modes of specific functional groups of the FC
and the nitro compounds used as coformers.58
FT-IR and FT-Raman spectra of 5-FC and its nitro salts show
clear differences that could be, in part, attributed to the
protonation of the pyrimidine ring of FC and to the vibrational
modes arising from the anions which contain NO2 groups.
A strong absorption band appearing at 1284 cm1 in FT-IR and
at 1288 cm1 in FT-Raman characterizes the FC spectra. These
bands were attributed to the pyrimidine CN stretching bonds
and in the salts spectra, these vibrational modes appear
red shifted from 1274 cm1 to 1270 cm1 (FT-IR) due to the
protonation. With respect to the NH2 group, bands assigned to
NH stretching can be observed in the FC FT-IR spectrum from
3375 cm1 to 3140 cm1. In the salts spectra, these bands also
appear shifted due to the H-bond formation.
As expected, for all 5-FC nitro salts, characteristic bands
associated with the anion vibration were observed. The FT-IR
Fig. 9 FT-Raman spectra of FC, FCPA, 2FCPA and FCDNS.
New J. Chem., 2019, 43, 15924--15934 | 15931
NJC Paper

Table 3 Principal FT-IR and FT-Raman bands (cm1) for FCPA, 2FCPA and FCDNS

5-FC FCPA 2FCPA FCDNS

IR Raman IR Raman IR Raman IR Raman Assignment
— — — — — — 3440 — n(OH)hydroxyl
3375 — 3332 — 3479, 3232 — 3338 — nas(NH2)11 amine
3320 — — — 3436 — 3286 — ns(NH2)11 amine
3140 — 3170 — 3352, 3120 — 3184 — n(NH)21 amine
3090 3093 3084 3104 3090 3106 3088 — n(CH)aromatic
1678 1678 1689 1687 1687 1699 1687 1689 n(CO)carbonyl
1645 1633 1632 1635 1627 — 1660 1620 n(CQC)
— — — — — — 1539 1535 nas(COO)carboxylate
— — — — — — 1387 1400 ns(COO)carboxylate
1468 1470 1437 1492 1433 1490 1450 1459 d(NH)amine
1284 1288 1270 1274 1273 1267 1274 1284 n(CN)pyrimidine
— — 1560 1552 1540 1560 1581 1558 nas(NO)nitro
— — 1370 1313 1369 1317 1368 1351 ns(NO)nitro
1224 1236 1239 1244 1228 1239 1227 1255 n(C–F)

and FT-Raman spectra show new bands in the 1581–1540 cm1 Author contributions
and 1370–1317 cm1 regions corresponding to the NO
antisymmetric and symmetric stretching modes, respectively. The manuscript was written through contributions of all authors.
Additionally, the vibrational modes arising from hydroxyl and All authors have given approval to the final version of the
carboxylate functional groups present in the DNSA molecule manuscript.
could be observed in the FT-IR and FT-Raman salt spectra
(Fig. 8 and 9, respectively). Conflicts of interest
The authors declare no competing financial interest.

4. Conclusions
Two novel solid forms of 5-FC were obtained by reaction with
picric (PA) and dinitrosalicylic (DNSA) acids. Analysis of the
crystal packing revealed that the novel multicomponent forms
are mainly stabilized by N–H  O H-bonds, the N3 atom of
the 5-FC pyrimidine ring being protonated, which was also
confirmed by FT-IR spectra. This protonation was found to be
partial in the ionic cocrystal of FC with PA leading to the
formation of a triple-point H-bond pattern involving the
FCFCH moiety, resembling the cytosine/guanine DNA/RNA
base pairing. In addition, in both FT-IR and FT-Raman spectra
it was possible to verify the appearance of new bands at
1581–1317 cm1 corresponding to the NO vibrational modes.
The shorter intermolecular contacts observed in the Hirshfeld
surfaces were mainly related to the classical hydrogen bonds
involving the amine groups in the FC and FCH molecules.
The degradation process is tuned by the coformer, for the
salt with PA no melting process was evidenced previous to
decomposition, whereas with DNSA, melting precedes degrada-
tion. Thermal behavior suggests that the crystalline solid forms
are stable to temperature variations. Therefore, the scientific
contributions of this study not only introduce valuable insights
regarding the supramolecular interactions of the antifungal
and promising antineoplastic prodrug 5-FC with nitro cofor-
mers, but also extends the family of pyrimidine solid forms
potentially useful for composing new pharmaceutical formula-
tions derived from this API and introducing two new forms
capable of offering better insights concerning DNA/RNA base
pairing systems, as the two chosen nitro compounds are used
to form charge-transfer complexes.
Acknowledgements
The authors acknowledge the Brazilian funding agencies CAPES
(M. S. S., C. C. P. S. and N. A./Finance Code 001), FAPESP (L. F. D.
grant 15/25694-0) and CNPq (J. E. grant #305190/2017-2) for
financial support, as well as Uruguayan PEDECIBA (N. A. research
grant). The authors would also 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
FCDNS salt data collection.
References
1 L. F. Diniz, P. S. Carvalho Jr, C. C. de Melo and J. Ellena,
Cryst. Growth Des., 2017, 17, 2622–2630.
2 L. F. Diniz, M. S. Souza, P. S. Carvalho, C. P. Cecilia,
R. F. D. Vries and J. Ellena, J. Mol. Struct., 2018, 1153, 58–68.
3 H. Li, Z. Zhang, X. Yang, X. Mao, Y. Wang, J. Wang, Y. Peng
and J. Zheng, Chem. Res. Toxicol., 2019, 32, 681–690.
4 M. Crisan, L. Halip, P. Bourosh, S. A. Chicu and Y. Chumakov,
Chem. Cent. J., 2017, 11, 1–10.
5 B. Lou, S. Reddy and C. Calvin, J. Mol. Struct., 2015, 1099,
516–522.
6 M. J. Almarsson and Ö. Zaworoto, Chem. Commun., 2004,
1889–1896.
7 C. C. P. Da Silva, R. D. O. Pepino, C. C. De Melo, J. C. Tenorio
and J. Ellena, Cryst. Growth Des., 2014, 14, 4383–4393.
8 C. C. P. da Silva, R. De Oliveira, J. C. Tenorio, S. B. Honorato,
A. P. Ayala and J. Ellena, Cryst. Growth Des., 2013, 13,
4315–4322.

15932 | New J. Chem., 2019, 43, 15924--15934 This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019
Paper
9 C. C. De Melo, P. De Sousa, C. Jr, L. F. Diniz, R. F. D.
Vries, A. P. Ayala and J. Ellena, CrystEngComm, 2016, 18,
6378–6388.
10 S. Perumalla, V. Pedireddi and C. Sun, Mol. Pharmaceutics,
2013, 10, 2462–2466.
11 S. R. Perumalla and C. C. Sun, CrystEngComm, 2013, 15,
5756–5759.
12 B. Swapna, D. Maddileti and A. Nangia, Cryst. Growth Des.,
2014, 14, 5991–6005.
13 K. Saminathan and K. Sivakumar, Acta Crystallogr., Sect. E:
Struct. Rep. Online, 2007, 63, 354–356.
14 P. Su, X. Song, R. Sun and X. Xu, Acta Crystallogr., Sect. E:
Crystallogr. Commun., 2016, 449, 861–863.
15 M. Mohana, P. T. Muthiah and C. D. McMillen, Acta Crystallogr.,
Sect. E: Crystallogr. Commun., 2017, 73, 361–364.
16 K. Deák, K. Takács-novák, K. Tihanyi and B. Noszál, Med.
Chem., 2006, 2, 385–389.
17 A. Sukhorukov, A. Sukhanova and S. Zlotin, Tetrahedron,
2016, 72, 6191–6281.
18 G. Portalone, Chem. Cent. J., 2011, 5, 51–58.
19 M. Crisan, L. Halip, P. Bourosh, S. A. Chicu and Y. Chumakov,
Chem. Cent. J., 2017, 11, 1–10.
20 M. S. Souza, C. C. P. da Silva, L. R. Almeida, L. F. Diniz,
M. B. Andrade and J. Ellena, J. Mol. Struct., 2018, 1161,
412–423.
21 M. S. Souza, L. F. Diniz, L. Vogt, P. S. Carvalho, R. F. D’Vries
and J. A. Ellena, Cryst. Growth Des., 2018, 18, 5202–5209.
22 M. S. Souza, L. F. Diniz, L. Vogt, P. S. Carvalho, R. F. D’vries
and J. Ellena, New J. Chem., 2018, 42, 14994–15005.
23 L. Song, K. Robeyns and T. Leyssens, Cryst. Growth Des.,
2018, 18, 3215–3221.
24 P. Cerreia Vioglio, M. R. Chierotti and R. Gobetto, Adv. Drug
Delivery Rev., 2017, 117, 86–110.
25 C. J. Springer, I. Niculescu-duvaz and D. H. Kirn, J. Clin.
Invest., 2000, 105, 1161–1167.
26 C. Altaner, Cancer Lett., 2008, 270, 191–201.
27 M. H. Amer, Mol. Cell. Ther., 2014, 2, 1–19.
28 O. Greco and G. U. Dachs, J. Cell. Physiol., 2001, 187,
22–36.
29 B. Yi, S. U. Kim, Y. Kim, H. J. U. N. Lee, M. Cho and K. Choi,
Oncol. Rep., 2012, 27, 1823–1828.
30 A. V. Patterson, Molecules, 2009, 14, 4517–4545.
31 W. Touati, P. Beaune and I. De Waziers, IntechOpen, 2011,
519–540.
32 M. Nyati, Z. Symon, E. Kievit, K. Dornfeld, S. Rynkiewicz,
B. Ross and A. Rehemtulla, Gene Ther., 2002, 9, 844–849.
33 K. Nepali, H. Y. Lee and J. P. Liou, J. Med. Chem., 2019, 62,
2851–2893.
34 D. Olender, J. Żwawiak and L. Zaprutko, Pharmaceuticals,
2018, 11, 1–29.
35 S. Kk, S. Srivastava, T. Alam and R. Raj, Der Pharma Chem.,
2017, 9, 64–75.
36 S. J. Jennifer and P. T. Muthiah, Chem. Cent. J., 2014, 8,
1–22.
37 M. N. Hopkinson, C. Richter, M. Schedler and F. Glorius,
Nature, 2014, 510, 485–496.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019
NJC
38 K. Anitha, S. Athimoolam and S. Natarajan, Acta Crystallogr.,
Sect. C: Cryst. Struct. Commun., 2006, 62, 567–570.
39 K. Anitha, S. Athimoolam and S. Natarajan, Acta Crystallogr.,
Sect. C: Cryst. Struct. Commun., 2006, 62, 426–428.
40 G. L. Miller, Anal. Chem., 1959, 31, 426–428.
41 M. Briget Mary, V. Sasirekha and V. Ramakrishnan, Spectro-
chim. Acta, Part A, 2006, 65, 414–420.
42 M. A. F. Elmosallamy, Anal. Sci., 2004, 20, 285–290.
43 H. T. Varghese, C. Y. Panicker, D. Philip, J. Chowdhury and
M. Ghosh, J. Raman Spectrosc., 2007, 38, 323–331.
44 I. M. Khan, A. Ahmad and M. F. Ullah, Spectrochim. Acta,
Part A, 2013, 102, 82–87.
45 N. Singh, I. M. Khan, A. Ahmad and S. Javed, J. Mol. Liq.,
2014, 191, 142–150.
46 A. Karthikeyan, P. Thomas Muthiah and F. Perdih,
Acta Crystallogr., Sect. E: Struct. Rep. Online, 2014, 70,
328–330.
47 L. Wang, X. Wen, P. Li, J. Wang, P. Yang, H. Zhang and
Z. Deng, CrystEngComm, 2014, 16, 8537–8545.
48 Enraf-Nonius, COLLECT, Nonius BV, Delft, The Netherlands,
1997–2000.
49 Z. Otwinowski and W. Minor, Methods in Enzymology,
Macromolecular Crystallography, Academic Press, 1997.
50 G. M. Sheldrick, Acta Crystallogr., 2014, A, 3–8.
51 O. V. Dolomanov, L. J. Bourhis, R. J. Gildea, J. A. K. Howard
and H. Puschmann, J. Appl. Crystallogr., 2009, 42, 339–341.
52 G. Sheldrick, Acta Crystallogr., Sect. A: Found. Crystallogr.,
2008, 64, 112–122.
53 G. M. Sheldrick, Acta Crystallogr., 2015, C, 3–8.
54 L. Clowney, S. C. Jain, A. R. Srinivasan, J. Westbrook,
W. K. Olson and H. M. Berman, J. Am. Chem. Soc., 1996,
118, 509–518.
55 C. F. Macrae, et al., J. Appl. Crystallogr., 2006, 39, 453–457.
56 L. J. Farrugia, J. Appl. Crystallogr., 2012, 45, 849–854.
57 C. R. Groom, I. J. Bruno, M. P. Lightfoot and S. C. Ward, Acta
Crystallogr., Sect. B: Struct. Sci., Cryst. Eng. Mater., 2016, 72,
171–179.
58 A. Heinz, C. J. Strachan, K. C. Gordon and T. Rades,
J. Pharm. Pharmacol., 2009, 61, 971–988.
59 G. Smith, U. D. Wermuth and P. C. Healy, Acta Crystallogr.,
Sect. E: Struct. Rep. Online, 2004, 60, 2–6.
60 A. Nangia, J. Chem. Sci., 2010, 122, 295–310.
61 E. J. C. de Vries, S. Kantengwa, A. Ayamine and
N. B. Báthori, CrystEngComm, 2016, 18, 7573–7579.
62 G. Portalone and M. Colapietro, J. Chem. Crystallogr., 2007,
37, 141–145.
63 L. Wang, X. Wen, P. Li, J. Wang, P. Yang, H. Zhang and
Z. Deng, CrystEngComm, 2014, 16, 8537–8545.
64 B. Sridhar, J. B. Nanubolu and K. Ravikumar, CrystEngComm,
2012, 14, 7065.
65 G. R. Desiraju and T. Steiner, The Weak Hydrogen Bond
In Structural Chemistry and Biology, International Union of
Crystallography, Monographs on Crystallography, 9, Oxford
University Press, 1999.
66 J. M. A. Robinson, D. Philp, K. D. M. Harris and B. M. Kariuki,
New J. Chem., 2000, 24, 799–806.
New J. Chem., 2019, 43, 15924--15934 | 15933
NJC
67 F. Allen, Acta Crystallogr., Sect. B: Struct. Sci., 2002, 58, 380–388.
68 R. Fischer, H. Görls, R. Suxdorf and M. Westerhausen,
Organometallics, 2019, 38, 498–511. 74
69 G. Portalone and M. Colapietro, J. Chem. Crystallogr., 2016,
37, 141–145.
70 A. T. Hulme and D. A. Tocher, Acta Crystallogr., Sect. E: 75
Struct. Rep. Online, 2005, 61, o2112–o2113.
71 M. Tutughamiarso, G. Wagner and E. Egert, Acta Crystallogr., 76
Sect. B: Struct. Sci., 2012, 68, 431–443.
72 M. Tutughamiarso, Acta Crystallogr., Sect. B: Struct. Sci., 77
2012, 68, 444–452.
73 M. J. Turner, J. J. McKinnon, S. K. Wolff, D. J. Grimwood, 78
P. R. Spackman, D. Jayatilaka and M. A. Spackman,
15934 | New J. Chem., 2019, 43, 15924--15934 This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019
Paper
CrystalExplorer17, University of Western Australia, Nedlands,
Western Australia, 2017.
D. Jayatilaka, S. K. Wolff, D. J. Grimwood, J. J. McKinnon
and M. A. Spackman, Acta Crystallogr., Sect. A: Found.
Crystallogr., 2006, 62, s90.
J. J. McKinnon, M. A. Spackman and A. S. Mitchell, Acta
Crystallogr., Sect. B: Struct. Sci., 2004, 60, 627–668.
I. R. Lewis, N. W. Daniel and P. R. Griffiths, Appl. Spectrosc.,
1997, 51, 1854–1867.
A. Jaworski, M. Szczesniak, K. Kubulat and W. B. Person,
J. Mol. Struct., 1990, 223, 63–92.
Y. Du, Q. Cai, J. Xue, Q. Zhang and D. Qin, Spectrochim. Acta,
Part A, 2017, 178, 251–257.