Article

Cite This: Cryst. Growth Des. 2018, 18, 5202−5209 pubs.acs.org/crystal

Mechanochemical Synthesis of a Multicomponent Solid Form:

The Case of 5‑Fluorocytosine Isoniazid Codrug
Matheus S. Souza,† Luan F. Diniz,† Lautaro Vogt,† Paulo S. Carvalho Jr.,† Richard F. D’vries,†,‡
and Javier Ellena*,†
†
Instituto de Física de São Carlos, Universidade de São Paulo, 13.560-970 São Carlos, SP, Brazil
‡
Facultad de Ciencias Básicas, Universidad Santiago de Cali, Calle 5 # 62-00, Cali, Valle del Cauca, Colombia
*
S Supporting Information
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ABSTRACT: Mechanochemistry synthesis was applied to the supra-

molecular synthesis and green scale-up production of a 1:1 drug−drug
Downloaded via UNIV OF SAO PAULO on September 5, 2018 at 14:46:39 (UTC).

cocrystal involving the antimetabolite prodrug 5-Fluorocytosine (5-FC)

and the tuberculostatic drug Isoniazid (INH), namely, 5FC-INH. Crys-
talline material, also obtained by traditional slow evaporation methods,
was analyzed by single-crystal X-ray diffraction (XRD). The crystal
packing is stabilized by a classical N−H···N hydrogen-bond interaction
between the amine moiety of 5-FC and the INH pyridine nitrogen.
IR and Raman data provided spectroscopic evidence about the hydrogen
atom positions, thereby confirming the neutral nature of the cocrystal.
Furthermore, 5FC-INH codrug was also evaluated by a range of ana-
lytical techniques such as powder XRD and thermal (thermogravimetric
analysis, differential scanning calorimetry, hot stage microscopy) ana-
lyses. A physical stability study was performed in high relative humidity
conditions to verify possible 5-FC solid-state hydration and/or INH degradation. The equilibrium solubility of this codrug was
compared to the anhydrous 5-FC and INH raw materials, in pH 1.2 buffer media, and it was found to be similar to that of 5-FC,
a biopharmaceutics classification system class I drug. The results show that the cocrystal has superior phase stability properties
against moisture when compared to the starting pharmaceutical ingredients, so it could be considered as a potential candidate
for the treatment of concomitant fungal infections, tuberculosis, and cancer.

1. INTRODUCTION solid-state hydration phenomena induced by atmosphere con-

A particular type of crystal form is represented by multidrug
cocrystals (MDC) or drug−drug cocrystals (DDC, or codrug),
where the structure must be composed by at least two different
active pharmaceutical ingredient (API) molecules fully dis-
sociable and in a stoichiometric ratio within the same crystal
lattice.1−4 In the literature, discussion about DDC is quite
scarce because of the complexity in their rational design and
synthetic procurement. Indeed, the raw materials are thor-
oughly selected among the APIs that will be possibly coadmin-
istered in a specific therapy, rather than being chosen on the
principles of crystal engineering.5 The exploration of solid forms
should be a routine practice during the drug development
process; however, for some marketed substances, research &
development on their active ingredients has not been compre-
hensively performed, as is the case of 5-Fluorocytosine (5-FC,
Scheme 1a) and Isoniazid (INH, Scheme 1a).6−11
Anhydrous 5-FC (4-amino-5-f luoro-1,2-dihydropyrimidin-2-
one) is a high-dose antimetabolite nontoxic prodrug that is in
the forefront on the antifungal treatment against Candida spp.
and Cryptococcus neoformans by the inhibition of the enzyme
thymidylate synthase.9,12 Furthermore, it has boosted studies
on 5-FC on cancer treatment.13 Apart from these features, On
the one hand, one of the main concerns about the 5-FC is a
ditions when exposed to high relative humidity (RH), leading
variable pharmacokinetic profile.14 On the other hand, INH
(pyridine-4-carbohydrazide) is a bacteriostatic drug that is stable
over long time periods at ambient as well as in accelerated
stability conditions. However, some studies show that INH
undergoes a degradation process due to drug−drug interactions
in the fixed-dose combination tablet used in the first phase of
tuberculosis (TB) treatment. The limited stability of the afore-
mentioned APIs has spurred the design of novel solid forms,
such as drug-coformer cocrystals (DCC)7−9,11,15−17 and codrugs,
to enhance its therapeutic behavior and solve its stability issue.
In the literature there are cases of coexistence of invasive
fungal infections (IFIs) and the bacteria that cause TB
(Mycobacterium tuberculosis), mainly in immunocompromised
patients.18,19 This type of coinfection represents a growing
threat with high morbidity and mortality and has emerged
due to the high use of steroids and broad-spectrum antibiotics.
Other studies have addressed a correlation between TB and
lung cancer (LC).20 Some researchers have suggested that
Received: April 27, 2018
Revised: June 14, 2018
Published: July 12, 2018

© 2018 American Chemical Society 5202 DOI: 10.1021/acs.cgd.8b00647

Cryst. Growth Des. 2018, 18, 5202−5209
Crystal Growth & Design
Scheme 1. (a) Chemical Structure of the Drugs
5-Fluorocytosine and Isoniazid; (b) Heterosynthon
Presented in 5FC-INH Novel Codrug
inflammation and pulmonary fibrosis caused by TB may be
responsible for inducing genetic damage and, consequently,
causing a greater propensity to trigger an LC.21
To expand the range of new solid forms comprising two
APIs, some structures have been reported in the past few
years.4 Recently, Lamivudine (β-L-2′,3′-dideoxy-3′-thiacytidine;
3TC) and Emtricitabine, antiretroviral (anti-HIV) drugs, were
used by members of our group in collaboration for producing
a solid solution.22 In addition to this, a 5-FC cocrystal with
the antineoplastic drug 5-Fluorouracil (5-FU) was also reported
aiming its potential future application in cancer therapy.8 Other
notable examples are 3TC and Zidovudine (anti-HIV drugs,
commercialized as Combivir),23 Theophylline (antiasthmatic)
with 5-FU24 or Barbital (sleeping aid),25 Lamotrigine and
Phenobarbital (anticonvulsants),26 among a few other research
articles27−29 and patents.30,31 Following this approach, herein
we report a standardized protocol for supramolecular synthesis
as well as the main physical and chemical properties of a
codrug obtained from the reaction of the prodrug 5-FC with
the anti-TB drug INH (Scheme 1b). Initially, the sample was
prepared by slow evaporation of solvent (SES; see Section 2.2).
Afterward, we develop a scale-up method based on the principles
of green chemistry32 involving the solvent-drop grinding (SDG)
method.5,33−36
2. EXPERIMENTAL SECTION
2.1. Materials. 5-FC anhydrous and INH samples were purchased
from Sigma-Aldrich and used without any further purification. The
ultrapure deionized water used in this experiment was obtained from a
Milli-Q System (18.2 mΩ cm), while other solvents used like isopropyl
alcohol were high-performance liquid chromatography (HPLC) grade
and purchased from Acros Organics.
2.2. Experimental Design and Supramolecular Cocrystal
Synthesis. To obtain the optimal reaction conditions several
protocols involving different stoichiometric ratios of 5-FC/INH
5203
Article
were designed (1:0.5, 1:1, and 1:2). Also, different solvents were
tested in the cocrystal formation search. The proper selection of solvents
or mixed solvents was considered an important cocrystallization step in
the protocol development. SES methods were preferred for screening
homogeneous single crystals suitable for structural characterization. The
SDG mechanochemical method was employed aiming the possibility of
a large-scale codrug production under the green chemistry principles.
5-Fluorocytosine Isoniazid Codrug. 5-FC (5.00 mg, 0.039 mmol)
was dissolved in a mixture of isopropyl alcohol/Milli-Q water (1:1, v/v)
and stirred at 100 °C. To this solution was added 5.31 mg (0.039 mmol)
of INH, in a 1:1 (drug/drug) molar ratio, and this system was stirred
using a temperature-controlled magnetic stirrer at 100 °C until com-
plete dissolution of the API. Then, it was allowed to cool slowly to
room temperature (∼25 °C) and covered with Parafilm for slow
evaporation of the solvent. Colorless prismatic crystals were obtained
after 3−5 d. Additionally, the same 5FC-INH phase was also obtained
by SDG method using an oscillatory ball mill Mixer Mill MM400
RETSCH. This experiment was performed by the addition of 50 mg
of 5-FC (0.39 mmol), 53 mg of INH (0.39 mmol), and 25 μL of
isopropyl alcohol/Milli-Q water (1:1, v/v) mixture. A powder sample
was placed in 1.5 mL volume stainless steel milling jar containing two
7 mm diameter stainless steel balls. The final optimized condition
to obtain this new solid form was achieved by milling the system at
25 Hz for 60 min at room temperature. External temperature of the
grinding jar did not exceed 25 °C.
2.3. Single-Crystal X-ray Diffraction. The crystallographic data
for the codrug 5FC-INH were collected at room temperature (293 ±
2 K) on an Agilent Super Nova diffractometer with CCD detector
system equipped with a Mo source (λ = 0.710 73 Å). Data integration,
cell determination, and final parameters were obtained using the
software CrysAlisPro.37 Using Olex2,38 the structure was solved by
direct methods, and the model obtained was refined by full-matrix
least-squares on F2 (SHELXT).39 All the hydrogen atoms were placed
in calculated positions and refined with fixed individual displacement
parameters [Uiso(H) = 1.2Ueq or 1.5Ueq] according to the riding
model. Molecular representations, tables, and pictures were generated
by MERCURY 3.1040 and Olex2.38
2.4. Powder X-ray Diffraction. The milled sample was analyzed
by powder X-ray diffraction (PXRD) at 25 °C using a Rigaku RU200B
Rotaflex diffractometer, in Bragg−Brentano reflective geometry, with Cu
Kα radiation (λ = 1.54 Å) from a voltage of 40 kV, current of 60 mA,
and Ni filter. The raw materials and the novel codrug were scanned
from 5 to 50° (2θ) with a step width of 0.02° θ min−1 and a constant
counting time of 3 s per step, providing unique structural information
about the crystallinity degree of the samples.
2.5. Hot-Stage Polarized Optical Microscopy. Polarized
microscopy study was performed on 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. The
single 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 sample was heated at a ramp rate of 10 °C min−1 until the
beginning of the degradation.
2.6. Thermal Analysis. Thermogravimetric analysis (TGA) was
performed using a Shimadzu TGA-50 thermobalance. An amount of
∼5.0 mg ± 0.001 mg of sample was placed in Al2O3 crucible and
heated at 10 °C min−1 under a N2 atmosphere (50 mL min−1)
between 50 and 500 °C. The differential scanning calorimetry (DSC)
data acquisition was performed on a Shimadzu DSC-60 calorimeter
according to the previously acquired TGA data, that is, until the degra-
dation temperature of the compound. The sample (2.0 mg ± 0.01 mg)
was heated from 50 to 350 °C at a 10 °C min−1 rate in a crimped sealed
aluminum pan. Nitrogen was used as purge gas under a 50 mL min−1
flow. The data were processed using the Shimadzu TA-60 thermal
data analysis software (version 2.2).
2.7. Vibrational Spectra. Fourier transform infrared (FT-IR)
spectra were recorded on an Alpha Bruker FT-IR spectrophotometer,
using KBr pellets, in the range of 3600−600 cm−1, with an average of
64 scans and 2 cm−1 of spectral resolution. Fourier transform Raman
(FT-Raman) spectroscopy was performed using a Bruker RFS 100
DOI: 10.1021/acs.cgd.8b00647
Cryst. Growth Des. 2018, 18, 5202−5209
Crystal Growth & Design Article

instrument with Nd3+/YAG laser operating at 1064 nm in the near- Table 1. Crystal Data and Structure Refinement of the
infrared and a CCD detector cooled with liquid nitrogen using a 5FC-INH Codrug
spectral resolution of 4 cm−1.
2.8. Stability Study in Relative Humidity. Powder samples of identification code 5FC-INH
the anhydrous 5-FC and 5FC-INH codrug (30 mg) were stored in a empirical formula C10H11FN6O2
chamber containing water (100 % RH; 25 °C). Further the milled molecular weight 266.25
materials were subjected to PXRD measurements to monitor possible temperature, K 293(2)
phase transitions. crystal system triclinic
2.9. Spectrophotometric Measurements and Calibration
space group P1̅
Curves. A UV-1800 Shimadzu spectrophotometer was used to
determine the absorbance of standard solutions of the raw precursors a, Å 3.7464(3)
of the cocrystal and construct pattern curves. These curves were used b, Å 9.6648(6)
to determine the unknown concentration of solutions of 5-FC, INH, c, Å 16.3882(13)
or 5FC-INH. The spectra were built in the range from 200 to 400 nm α, deg 76.142(6)
using 1 cm quartz cuvettes in the medium scan speed at a 1.0 nm data β, deg 88.948(6)
interval and 1 nm bandwidth. Standard stock solutions of 5-FC and γ, deg 82.040(5)
INH were prepared separately at pH 1.2 hydrochloric buffer (Table S1, volume, Å3 570.49(7)
see Supporting Information). On the one hand, 5-FC standard solutions Z/Z′ 2/1
were prepared dissolving 10.00 mg of the raw material in 1.5 mL of
ρcalc, g/cm3 1.550
buffer into a 10 mL beaker (24 h agitation), and after that a con-
formation of dissolutions from 0.006 mg mL−1 to 0.024 mg mL−1. μ, mm−1 0.125
On the other hand, INH solutions were prepared dissolving 20.00 mg F(000) 276.0
of the solid drug in 4 mL of buffer into a 50 mL volumetric flask. After reflections collected 9602
complete dissolution (24 h agitation process) different concentration independent reflections 2495
points were made by appropriate dilutions in concentrations ranging unique reflections 2092
from 0.004 to 0.01 mg mL−1 (Table S2). UV−Vis spectra of these data/restraints/parameters 2495/0/173
solutions were used to build individual calibration curves of the drugs R1 [I ≥ 2σ(I)] 0.0457
(Table S3). wR2 [all data] 0.1233
2.10. Equilibrium Solubility Studies. Equilibrium solubility of
goodness-of-fitness on F2 1.069
5-FC, INH, and its codrug were determined by the shake-flask
X-ray diffractometer Agilent Super Nova
method41 at 25 °C in pH 1.2 buffer media. Saturated solutions of the
compounds were prepared stirring an excess amount of 5-FC, INH,
and 5FC-INH, enough to reach saturation, into 2 mL of the disso- parameters are summarized in Table 1. The main intermo-
lution media for a 48 h period. These solutions were prepared in lecular interactions and geometric parameters are listed in
triplicate according to the method outlined in the biopharmaceutics Table 2. The ORTEP43 type diagram of the asymmetric unit
classification system (BCS) guidance.42 After 48 h of sedimentation, the
solutions were filtered through a 0.45 mm poly(tetrafluoroethylene) Table 2. Geometric Parameters of the Hydrogen Bonds for
(PTFE) hydrophilic filter (Millipore). The solid sediment identities 5FC-INH
were checked by PXRD analysis (Figure S1). UV−Vis spectroscopy was
employed to analyze the supernatant concentration of the compounds. d(D···A) d(H···A) ∠D−H···A
The samples were diluted in the pH 1.2 buffer media before we interaction (Å) (Å) (deg) symmetry codes
started measuring. 5-FC, INH, and its codrug showed similar spec- N5−H5···N3 3.027(2) 2.271(2) 147(1) x,y,z
trum that could be resolved with the addition of absorbances (Figure S2). N41−H41A···N5 3.520(2) 2.824(2) 139(1) x,y,z
Solubilities of 5-FC, INH, as well as 5FC-INH codrug were measured N41−H41A···N4 2.963(2) 2.118(2) 167(1) x,y,z
interpolating their maximum absorbance readings to the correspond- C9−H9···O21 3.225(2) 2.606(2) 124(1) x,y,z
ing calibration curves. After the equilibrium solubility measurements,
N1−H1···O21 2.735(2) 1.876(2) 176(1) −x+1,−y+1,−z+2
the pH values in each dissolution medium were determined (Table S4)
N1−H1···N1 3.589(2) 2.992(2) 128(1) −x+1,−y+1,−z+2
using a pH meter QX 1500 Plus Qualxtron.
N41−H41B···N6 2.946(2) 2.123(2) 160(1) x−1,+y−1,+z
3. RESULTS AND DISCUSSION N4−H4A···O2 3.151(2) 2.211(2) 160(1) −x+2,−y+1,−z+1
N4−H4B···O2 3.659(2) 2.735(2) 149(1) x−1,+y,+z
From the structural point of view, 5-FC and INH possess N4−H4B···N4 3.668(2) 2.863(2) 135(2) −x+1,−y+1,−z+1
multiple donor and acceptor hydrogen-bond sites. 5-FC is a N4−H4B···O2 2.965(2) 2.303(2) 120(1) −x+1,−y+1,−z+1
weak basic molecule (pKa = 3.26) that structurally presents two C6−H6···F51 3.331(2) 2.536(2) 143(1) −x,−y,−z+2
donors: NH(pirimidinic ring), NH2(amine group), and two acceptors: C9−H9···N3 3.517(2) 2.746(2) 141(1) x+1,+y,+z
Nring and carbonyl (CO). INH (pKa = 3.50) also is a weak C10−H10···021 3.401(2) 2.814(2) 122(1) x+1,+y,+z
base that contains two main functional groups: hydrazide and C10−H10···F51 3.532(2) 2.986(2) 119(1) x+1,+y+1,+z
pyridine ring. These groups are the bases for the generation of
the already-reported cocrystals of these APIs with generally
recognized as safe (GRAS) coformers containing COOH and (ASU) is shown in Figure 1. Crystalline purity of this
NH2 functional groups.8,11 As part of our ongoing studies in codrug was assessed by PXRD (Figure S3). The compound
the development of stable multicomponent cocrystal forms, we was also obtained as powder pure phase by SDG method (see
rationally designed (applying the ΔpKa approach, i.d. ΔpKa = Figure 2).
[pKa (conjugate acid of the base) − pKa (acid)]) a cocrystal 3.1. Structural Description. 5FC-INH crystallizes in the
involving these two APIs. The difference between the pKa of triclinic space group P1̅ with one molecule of 5-FC and one of
5-FC and INH gives a ΔpKa value within the required range INH in the ASU (Figure 1). 5-FC molecules form an R22(8)
for cocrystal formation (−0.24 = ΔpKa < 0). Suitable single homodimer through N−H···O (2.735(2) Å, 176.6(2)°)
crystals of 5FC-INH were obtained by evaporation methods. H-bonds. Likewise, INH molecules form an R22(10) homo-
Details of the data collection, refinement, and crystallographic synthon via N−H···O (2.965(2) Å, 120.52(2)°) H-bonds
5204 DOI: 10.1021/acs.cgd.8b00647
Cryst. Growth Des. 2018, 18, 5202−5209
Crystal Growth & Design Article

blue) are related by R22(7) heterosynthon (highlighted in red in

Figure 1. ORTEP type diagram with atomic numbering scheme
showing 50% of probability ellipsoids for 5FC-INH.
Figure 2. Experimental PXRD pattern of 5FC-INH obtained from
SDG compared to the simulated one from SCXRD of the crystal
obtained by SES method.
(Figure 3a). The 5-FC (colored in green in Figure 3a) and
INH (colored in blue in Figure 3a) homodimers are alternately
arranged into infinite chains along the [121] direction via
N−H···N (2.946(2) Å, 160.0(2)°) H-bonds (Figure 3a).
Along [001] direction, adjacent chains (color in orange and
Figure 3b) formed between 5-FC of one chain and INH from
another one. Interestingly, in a Cambridge Structural Database
(CSD)44 survey we didn’t find any structure of either 5-FC or
INH presenting this R22(7) heterosynthon. Thus, the molecules
are arranged giving rise to planes as shown in Figure 3c. Such
planes are stacked through N−H···O (3.151(2) Å, 160.78(2)°),
C−H···N (3.517(2) Å, 141.0(2)°), and slip-stacked π···π
(centroid distance: 3.7385 Å for INH···INH and 3.746 Å for
5-FC···5-FC) interactions that give to the three-dimensional
(3D) structure (Figure 3c).
3.2. Thermal Analysis. The phase purity of 5FC-INH
codrug was also assessed by a combination of DSC, TGA, and
hot-stage microscopy (HSM) techniques. DSC and TGA curves
of the sample are shown in Figure 4a. The thermograms of pure
5-FC (anhydrous and monohydrate) as well as those from the
INH were included to characterize the starting materials
(see Figure S4, Supporting Information). The DSC curve of
5-FC raw material (anhydrous) shows an endothermic peak at
∼299.62 °C coupled to an exothermic one, both attributed to
the degradation process. The DSC curve of the 5-FC exposed
to highly humid environment (100% RH and 25 °C), in turn,
is very different from the anhydrous form, since it is charac-
terized by one extended and endothermic peak in the 70.19−
131.91 °C range, which corresponds to the dehydration process,
and one endothermic peak at 237.80 °C, related to the melting
process. According to the TGA data, the 5FC-INH codrug is
thermally stable between 50 and 157.45 °C (Figure 4a). After
this temperature it shows a significant weight loss as observed
in the TGA curve. The 5FC-INH DSC curve (Figure 4a) pre-
sents an endothermic peak at 221.0 °C followed by an exo-
thermic one at 224.06 °C, both attributed to the codrug degra-
dation. No traces of 5-FC/INH peaks were observed in the
thermogram, confirming the purity of the sample.

Figure 3. (a) Partial view down [100] direction of 5FC-INH crystal packing highlighting the formation of alternating 5-FC and INH dimer chains
stabilized by N−H···N and N−H···O interactions and forming R22(8) and R22(10) graph sets motifs. Black dotted lines indicate hydrogen bonds.
(b) View along [001] direction: adjacent chains are related by N−H···N R22(7) heterosynthon formed between 5-FC of one chain and INH from
another. (c) 3D arrangement of crystalline 5FC-INH. The 5-FC and INH molecules are arranged giving rise to plane parallel to the (1̅10) plane.
Such planes are stacked through N−H···O, C−H···N, and slip-stacked π···π interactions leading to 3D structure formation.

5205 DOI: 10.1021/acs.cgd.8b00647

Cryst. Growth Des. 2018, 18, 5202−5209
Crystal Growth & Design
Figure 4. (a) TGA and DSC curves of the 5FC-INH codrug.
(b) Crystal behavior as a function of temperature increase visually
checked by HSM of the novel crystalline form.
The thermal behavior of the codrug was also observed in the
HSM experiment (Figure 4b) and was successfully confirmed
by them. The crystal gradually gets dark as the temperature
rises, becoming opaque at ∼160 °C. From this temperature
forward, the samples continue reducing their mass in a degra-
dation process.
3.3. Spectroscopy Analysis. FT-IR and FT-Raman
spectroscopy provides crucial information about vibrational
modes and molecular conformations of the APIs.45,46 For this
purpose, a comparative analysis of 5-FC and INH and their
5FC-INH codrug FT-IR and FT-Raman spectra (Figure S5
and Figure 5) was performed. This study shows differences in
Figure 5. FT-Raman spectra of the raw materials and the 5FC-INH
codrug.
some typical bands of 5-FC and INH when compared to the
bands present in the codrug spectra, which indicates changes in
the hydrogen-bonding patterns.47,48 The spectra interpretation
and band assignments (Table S5) were performed taking into
account the crystallographic study previously described and
using spectroscopic data available from related 5-FC and INH
compounds found in the literature.49−51
5206
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On the one hand, 5-FC molecule exhibits IR and Raman
stretching frequencies at 3375 (amine N−H stretch), 1682, and
1677 cm−1 (amide CO stretches), at 1337 and 1340 cm−1
(pyrimidine ring C−N stretches), and at 1227 and 1236 cm−1
(C−F stretches). On the other hand, the functional groups
present in the INH molecule (hydrazide, amide, and pyridine
ring) exhibit IR and Raman stretching frequencies at 3303 and
3212 cm−1 (primary amine N−H stretches), 3107 (secondary
amine N−H stretch), at 1664, and 1670 cm−1 (amide CO
stretch), and at 1331 and 1334 cm−1 (pyridine ring C−N stretch).
As expected, the main 5-FC and INH stretching frequencies
are observed in the spectra of 5FC-INH. Moreover, these
vibrational modes appear shifted (10−60 cm−1) in the FT-IR
and FT-Raman spectra (Table S5), in agreement with the
cocrystal formation.
3.4. Representativity, Phase Transition, and Stability
Test. PXRD is the most suitable characterization tool to con-
firm the formation of novel crystalline forms.52 The cocrystal
obtained by both SES and SDG methods displays experimental
diffraction patterns in good agreement with the simulated ones,
obtained from single-crystal X-ray diffraction (SCXRD) ana-
lysis (Figures S3 and 2, respectively). This confirms that, in
one hand, the powder sample obtained from SDG is pure and
presents a high degree of crystallinity and, in the other, that the
single crystal used in the SCXRD experiment is representative
of the whole sample.
The PXRD was also used as a tool to verify the physical sta-
bility of the reported cocrystal. Anhydrous 5-FC is converted
to the monohydrate form after one week of exposure to humid
atmosphere, as can be seen in the diffractograms of Figure 6.
Figure 6. PXRD pattern of 5FC-INH codrug showing the same solid
phase after one week in humid environment, which not occurs with
the 5-FC raw material currently marketed.
It was possible to note that the major peaks of the 5-FC pattern
were no longer observed after one week giving rise to the char-
acteristic peaks of the monohydrate form. This phase transition
does not occur in the novel codrug, since the main charac-
teristic peaks remain present after the same exposure time to a
highly humid atmosphere.
3.5. Equilibrium Solubility. The solubility study of the
codrug was performed in a buffer mimicking the stomach pH.
These tests show that the new solid form retains solubility
values statistically similar to the ones found for the 5-FC raw
material (Figure 7 and Table S6, see Supporting Information).
PXRD also shows that the crystal structure of the solid obtained
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Crystal Growth & Design
Figure 7. Solubility concentration of 5-FC, INH, and codrug in
pH 1.2 dissolution media.
after the solubility test remains the same as the original codrug
(Figure S1). The same results are obtained for INH. However,
diffractograms before and after the solubility study of 5-FC do
not show the same profile. In this case the remnant solid shows
additional peaks that correspond to other 5-FC polymorphs as
well as different hydrates (Figure S6). When thermal analyses
of these solids are made four important facts are exposed: DSC
contrast between the solids before and after the solubility study
shows an endothermic signal at 52.19 °C (Figure S7) that
corresponds to a phase transition; Figure S7 also shows an
endothermic signal at 104.42 °C that correlates to a water loss;
TGA comparison between 5-FC monohydrate and the 5-FC
solid form obtained after the solubility studies reveals that the
amount of water in the crystal structure is reduced when
compared to the monohydrate (Figure S8); finally, DSC plot
(Figure S7) shows the same signal at ∼300 °C related to the
decomposition of the 5-FC anhydrous. The summary of these
facts seems to indicate that the anhydrous 5-FC solid form
used in the solubility test leads to a different kind of hydrate in
a pH 1.2 buffer solution. The DSC profiles (Figure S7) show
that all these changes are reversible.
As expected, in the pH 1.2 hydrochloric buffer, the INH
solubility is very high due to the interconversion of INH into
its hydrochloride salt. In fact, the hydrochloride salt formation
was confirmed by an increase of the final pH measured after
the experiment, from 1.20 to 4.40 (Table S4).
4. CONCLUSIONS
Solvent-drop grinding was successfully applied as a synthetic
pathway of the codrug involving the antimetabolite prodrug
5-Fluorocytosine and the tuberculostatic drug Isoniazid. This
mechanochemical technique, as well as solvent evaporation
one, led to the 5FC-INH cocrystal formation, the first one being
in agreement with the green chemistry principles.
Crystal structure analysis revealed that the crystal packing
is stabilized by N−H···N and N−H···O H bonds. FT-IR and
FT-Raman spectra confirm the cocrystal formation by the
concomitant appearing of the 5-FC and INH characteristic
bands in the codrug spectra. However, most of these typical
bands appear shifted in both FT-IR and FT-Raman spectra in
agreement with the new intermolecular interactions formation.
The degradation point (∼220 °C) was found to be between
similar ones of the parent APIs (∼170 °C for INH and ∼300 °C
for anhydrous 5-FC). The solubility profile of INH in pH 1.2
buffer corroborates the high value previously reported for this
drug in a wide range of solvents.53,54 The 5-FC solubility, even
5207
Article
when is statistically lower than the INH one, is still high
enough to classify this prodrug as a class I drug8 due to the
considerable solubilization in the stomach at low pH. Within
this context, the new codrug shows a solubility value very close
to the one of the 5-FC raw material, which reinforces the idea
of cocrystallization as an effective tool to resolve the hydration
problem of an API (as the 5-FC in this case) without severely
impairing its solubility.
Furthermore, this work introduces an important multi-API
cocrystal as a promising candidate for (1) concomitant treat-
ment of fungal and bacteriological infections, (2) concomitant
treatment of fungal and/or cancer in patients undergoing gene-
directed enzyme prodrug therapy, (3) concomitant treatment
of bacteriological infections and/or cancer in patients under-
going gene therapy, and last but not least (4) increase the
physical stability of the raw APIs, avoiding undesirable 5-FC
phase transitions (hydration) and/or INH degradability in
pharmaceuticals containing them in its formulations.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.cgd.8b00647.
Experimental powder X-ray diffraction patterns of 5-FC,
INH, and codrug that remain nonsolubilized after the
solubility test. Absorption spectra of 5-FC 0.0055 mg mL−1,
INH 0.016 mg mL−1, and codrug (5FC-INH) 0.013 mg
mL−1 in buffer pH = 1.2. Simulated and experimental
powder X-ray diffraction patterns of 5FC-INH codrug.
The diffractograms are in a good agreement indicating
that the sample presents high crystallinity and purity.
DSC curves of: (i) the prodrug 5-Fluorocytosine (5-FC
anhydrous), (ii) the 5-FC after one week in environment
with high relative humidity (5-FC monohydrate), and
(iii) the drug Isoniazid (INH). FT-IR spectra of 5-FC
raw, INH raw, and 5FC-INH codrug. Powder X-ray
diffraction patterns of 5-FC after solubility study and
different solid forms of 5-FC anhydrous and hydrated
reported in literature. DSC profiles of 5-FC before and
after solubility study. TGA profiles of 5-FC monohydrate
and 5-FC anhydrous after solubility study. Composition of
the solution used for the preparation of the calibration
curves and solubility determinations. Standard solutions of
5-FC and INH used to construct the calibration curves.
Regression coefficients for the calibration curves of 5-FC
and INH at buffer media, pH = 1.2. pH values measured
after solubility. Main FT-IR and FT-Raman bands (cm−1)
for 5-FC and INH, and the codrug 5FC-INH and
solubility values obtained for the three compounds (PDF)
Accession Codes
CCDC 1817620 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
data_request@ccdc.cam.ac.uk, or by contacting The Cam-
bridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: javiere@ifsc.usp.br. Phone: +55 (016) 3373-8096/
3373-9876.
DOI: 10.1021/acs.cgd.8b00647
Cryst. Growth Des. 2018, 18, 5202−5209
Crystal Growth & Design
ORCID
Paulo S. Carvalho Jr.: 0000-0002-5551-9155
Richard F. D’vries: 0000-0002-3655-1838
Javier Ellena: 0000-0002-0676-3098
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors acknowledge the Brazilian funding agencies
CAPES (M.S.S.), FAPESP (L.F.D. Grant No. 15/25694-0),
and CNPq (J.E. Grant No. 305190/2017-2) for financial
support. L.V. thanks IFSC/USP for being granted a Salazar
Scholarship, which made possible the active collaboration in
this work. The authors would also like to thank Dra. C. C.
Correa, Dr. L. F. C. de Oliveira (Federal Univ. of Juiz de Fora)
for allowing access to the single-crystal X-ray diffraction and
Raman spectroscopy facilities.
■ ABBREVIATIONS
5-FC, 5-Fluorocytosine; INH, Isoniazid; IR, Infrared; PXRD,
powder X-ray diffraction; TGA, thermogravimetric analysis;
DSC, differential scanning calorimetry; HSM, hot-stage
microscopy; RH, relative humidity; BCS, Biopharmaceutics
Classification System; MDC, multidrug cocrystal; DDC, drug−
drug cocrystal; API, active pharmaceutical ingredient; TB,
tuberculosis; DCC, drug-coformer cocrystal; IFIs, invasive
fungal infections; LC, lung cancer; 3TC, β-L-2′, 3′-dideoxy-3′-
thiacytidine; HIV, human immunodeficiency virus; 5-FU,
5-Fluorouracil; SES, slow evaporation of solvent; SDG,
solvent-drop grinding; SCXRD, single-crystal X-ray diffraction;
HPLC, high performance liquid chromatography; CCD,
charge-coupled device; UV−vis, ultraviolet−visible; PTFE,
polytetrafluoroethylene; GRAS, Generally Recognized as Safe;
ORTEP, Oak Ridge Thermal-Ellipsoid Plot; ASU, asymmetric
unit; CSD, Cambridge Structural Database.
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5209 DOI: 10.1021/acs.cgd.8b00647

Cryst. Growth Des. 2018, 18, 5202−5209