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1. Synthesis, characterization, solubility and stability studies of hydrate
cocrystal of antitubercular Isoniazid with antioxidant and anti-
bacterial Protocatechuic acid
Syed Muddassir Ali Mashhadi a, *
, Uzma Yunus a
, Moazzam Hussain Bhatti a
,
Imtiaz Ahmed a
, Muhammad Nawaz Tahir b
a
Allama Iqbal Open University, Department of Chemistry, Islamabad, Pakistan
b
University of Sargodha, Department of Physics, Sargodha, Pakistan
a r t i c l e i n f o
Article history:
Received 29 June 2015
Received in revised form
16 March 2016
Accepted 16 March 2016
Available online 19 March 2016
Keywords:
Hydrate cocrystal
Isoniazid
Anti-oxidants
Protocatechuic acid
Solubility
Stability
a b s t r a c t
Isoniazid is an important component used in “triple therapy” to combat tuberculosis. It has reduced
Tabletting formulations stability. Anti-oxidants are obligatory to counter oxidative stress, pulmonary
inflammation, and free radical burst from macrophages caused in tuberculosis and other diseases. In the
present study a hydrate cocrystal of Isoniazid with anti-oxidant and anti-inflammatory and anti-bacterial
Protocatechuic acid (3,4-dihydroxybenzoic acid) in 1:1 is reported. This Cocrystal may have improved
tabletting stability and anti-oxidant properties. Cocrystal structure analysis confirmed the existence of
pyridine-carboxylic acid synthon in the Cocrystal. Other synthons of different graph sets involving
NeH$$$O and OeH$$$N bonds are formed between hydrazide group of isoniazid and coformer. Solubility
studies revealed that cocrystal is less soluble as compared to isoniazid in buffer at pH 7.4 at 22
C while
stability studies at 80
C for 24 h period disclosed the fact that cocrystal has higher stability than that of
isoniazid.
© 2016 Elsevier B.V. All rights reserved.
1. Introduction
Crystal engineering [1] has discovered a relatively new solid
form of Active Pharmaceutical Ingredient (API) known as Phar-
maceutical cocrystal. Crystal engineering falls into the domain of
Supramolecular Chemistry [2], popularized when the Nobel Prize of
the year 1987 in chemistry was awarded to the founders of this
field. Cocrystals are “a stoichiometric multi component crystal in
which all its components are neutral and solid under ambient
conditions when in pure form” [3], and can be formatted through
several types of interactions, including hydrogen bonding, pi-
stacking, and van der Waals forces.
Pharmaceutical cocrystals provides a way to alter the physico-
chemical properties of API [4]. Pharmaceutical dual drug cocrystals
may have properties like retained molecular structure with
improved bioavailability, increased resistance to hydrate formation
and improved compaction properties for tablet formulation of two
APIs into one dose.
Study of synthons and creation of hydrogen bonds between
synthons is the core of the field of cocrystallization. Hydrogen bond
is energetic, directional [5,6] and is utilized in synthetic schemes to
create specific assemblies [7e11]. Carboxylic acid - pyridine
hydrogen bond in the formulation of cocrystals is an established
fact due to strong donor and strong acceptor functionality of the
carboxylic and pyridine functional groups respectively. This is in
accordance to Etter's rules for the formation of hydrogen bonds
[12,13].
Isoniazid (Antitubercular drug) is the popular name of
pyridine-carboxylic acid hydrazide or isonicotinic acid hydrazide
(INH). It is odorless white crystalline powder with molecular
weight 137.14 g/mol having empirical formula of C5H5N3O(Fig.1a).
It is the primary constituent of “triple therapy” used to effectively
treat tuberculosis as it is highly active against Mycobacterium
tuberculosis since 1952.
Crystalline isoniazid is stable for long time while its Tablet for-
mulations undergo oxidative degradation under high temperature
and humid climatic conditions (40 C, 75% RH) [14]. Exposure to
light and presence of other drugs (pyrazinamide, ethambutol) be-
ing used in combination therapy also enhance isoniazid tablet's* Corresponding author.
E-mail address: muddassir_bakie@yahoo.com (S.M.A. Mashhadi).
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
http://dx.doi.org/10.1016/j.molstruc.2016.03.057
0022-2860/© 2016 Elsevier B.V. All rights reserved.
Journal of Molecular Structure 1117 (2016) 17e21

2. degradation [15,16]. It is therefore important to develop stable
formulations of isoniazid.
Isoniazid is very useful supramolecular reagent to synthesize
novel supramolecular structures; this is due to the fact that Pyr-
idine ring and carbohydrazide group of Isoniazid act as hydrogen
bond acceptor for carboxylic acids in form of O and N atoms and
donor in the form of three H atoms respectively. Therefore, INH is
a potential supramolecular reagent to synthesize pharmaceutical
cocrystals. In past cocrystals of isoniazid [17e24] with Carbox-
ylicÀpyridine synthons have been reported. Hydroxy benzoic
acids, Gallic Acid, 4-aminosalicylic acid, dicarboxylic acids, ter-
ephthalic acid, tartaric acid and 2,2-dithiodibenzoic acid have
been synthesized. Moreover, hydrazideÀhydrazide hydrogen
bonds are also present in the pharmaceutical cocrystals of
isoniazid.
Oxidative stress in tuberculosis and some other diseases is
common due to tissue inflammation and free radical burst from
macrophages. These free radicals results in pulmonary inflam-
mation if not countered by anti-oxidants [25e27]. Oxidation re-
actions are the main reasons of degradation of APIs which
decreases the shelf life of pharmaceutical formulations. Anti-
oxidants are required to combat above said oxidative stress and
degradation.
In the present study Hydrate cocrystal of Isoniazid with an
antioxidant, antibacterial Protocatechuic acid (Fig. 1b) is synthe-
sized by slow evaporation method and characterized by Fourier
Transform Infrared spectroscopy (FTIR), Single crystal X-ray
diffraction and Differential Scanning Calorimetry (DSC) studies.
Moreover, the Solubility, Stability and Spectral studies were also
performed to investigate and compare the properties of cocrystal
with that of isoniazid.
2. Experimental section
All the chemicals were used as received from the supplier
without any further purification.
Melting point was studied by using a Gallenkamp (UK) 50 Hz
220/240 V melting point apparatus. The IR spectra were recorded
on Varian 640-IR spectrophotometer.
Single crystal X-ray diffraction data was collected by using
Bruker Kappa APEX II CCD diffractometer equipped with a
graphite monochromator at 296 K. Fine focus of molybdenum Ka
tube was used. Data was collected using APEX2 software, SAINT
for indexing the reflections and determining the unit cell
Fig. 1. (a) Structural formulas of Isoniazid (b) Structural formulas of Protocatechuic
acid.
Table 1
Physical data of Isoniazid and Cocrystals (C-01).
Code Physical appearance Melting point (o
C) Stability Solubility lmax
Isoniazid White crystal 172
C 93.86% 76.30 mg/mL 263 nm
Protocatechuic acid Light brown 221
C 93.40% 12.40 mg/mL 258 nm
C-01 Orange Prism 185
C 94.46% 6.57 mg/mL 252 nm
Table 2
IR spectral data of Cocrystals (C-01).
Functional groups Isoniazid y cmÀ1
Protocatechuic acid C-01 y cmÀ1
Asymmetric eNH2 stretching 3302 e 3248
Aromatic CeH Stretching 3010 3176 e
CeO Stretching 1662 1667 1651
CeN Stretching 1602 e 1612
Aromatic ring Vibration 1492 1528,1465 1526, 1493
Pyridine ring 1411 e 1407
Carboxylic acid OH e 2632 e
Table 3
Single Crystal XRD data of Cocrystals (C-01).
Cocrystal C-01
Empirical formula C26H30N6O12
Formula Weight 618.56
Temperature (K) 293(2)
Wavelength (Ǻ) 0.71073
Crystal System Monoclinic
Space Group P 21
Cell formula unit-Z 1
a (Ǻ) 6.9626(5)
b (Ǻ) 22.664(5)
c (Ǻ) 8.8114(5)
a (º) 90
b(º) 100.124(4)
g(º) 90
Volume (Ǻ3) 1368.8(3)
Absorption coefficient (mmÀ1
) 0.121
R factor (%) 6.26
Table 4
Hydrogen bond distances in Isoniazid-Cocrystals (C-01).
Atoms DÀH (Aº) H/A (Aº) D/A (Aº) DÀH/A (deg)
Cocrystal C-01
O8eH8/N6 0.932 1.676 2.593 167.12
S.M.A. Mashhadi et al. / Journal of Molecular Structure 1117 (2016) 17e2118

3. parameters. The structure was solved by direct methods and
refined by full-matrix least square calculations using SHELXL-97
software. Structure of the cocrystal was drawn and other calcu-
lations were carried out by using Mercury 3.1 software. Differ-
ential Scanning Calorimetry (DSC) experiments were performed
with Mettler Toledo instrument. The samples (2e5 mg) were
heated in open aluminum pans at a rate of 10 C/min in nitrogen
(flow of 20.0 ml/min).
Molar concentrations of aqueous solutions of cocrystal were
determined by UV/Vis spectrometry using a UV-1700, Shimadzu,
equipped with 1.0 cm quartz cuvettes. 200e600 nm range was
recorded for the sample and absorption for cocrystal solution was
determined. Concentration measurements were performed for
cocrystal and separate linear calibration curve was plotted for
cocrystal.
Solubility studies of Isoniazid and Cocrystal were performed in
duplicate according to method reported by Higuchi and Connors
[28]. In this solubility study, an excess quantity of Isoniazid and its
cocrystal was placed separately in the vials containing 5 ml of
buffer at pH 7.4. The vials were agitated on shaker (150 agitations/
min) for 24 h at room temperature (22 C). The solution in vials was
then filtered through filter paper to obtain the saturated solutions
and then amount of the Isoniazid and its cocrystal dissolved in
buffer at pH 7.4 was analyzed by UV spectrophotometer.
Stability studies of Isoniazid and Cocrystal were performed in
duplicate by placing known amount of Isoniazid and its Cocrystals
in oven at 80 C for 24 h and then amount of the Isoniazid and its
Cocrystals was determined spectrophotometrically.
3. Results and discussion
Cocrystallization of isoniazid with antioxidant Proto-
catechuic acid resulted in cocrystal and was characterized by
Infra-red spectroscopy, single-crystal X-ray diffraction and
thermal analysis. Physical properties of cocrystal are arranged in
Table 1, IR spectral data is given in Table 2. Crystallographic in-
formation is shown in Table 3 and information regarding
hydrogen bonds of cocrystals is arranged in Table 4. Crystal
structure was deposited at the Cambridge Crystallographic Data
Centre. The data have been assigned the deposition numbers,
CCDC 1016095.
3.1. Cocrystal of Isoniazid and Protocatechuic acid (C-01)
3.1.1. Synthesis
Isoniazid (0.137 g, one mmol) and Protocatechuic acid (0.154 g,
one mmol) were dissolved separately in 20 ml of the mixture of
methanol and water (1:1) then mixed together. Solution was
heated to 70 C for 10 min and kept for slow evaporation for 15
days. The orange prism like crystals were isolated by filtration
through filter paper and dried in the air. Physical parameters are
summarized in Table 1.
3.2. Characterization
3.2.1. Analysis of IR spectrum
IR experiment was performed using ATR technique. IR spec-
trum of Isoniazid showed the stretching ofeNH bond in the high
wave number region of the IR spectrum at 3302 cmÀ1
, while the
cocrystal showed a band of weak intensity at 3248 cmÀ1
(Fig. 2).
Another sharp band is present at 3113 cmÀ1
, which was attrib-
uted to the CeH (aromatic) stretching vibrations while isoniazid
showed at 3104 cmÀ1
for the same bond. C]O stretching vi-
bration was present at 1651 cmÀ1
for cocrystal while
Fig. 2. Over lay of IR spectrum of Cocrystal, Isoniazid and Protocatechuic acid.
Fig. 3. XRD of Cocrystal of Isoniazid and Protocatechuic acid (1:1) (a) The hydrogen
bonding between nitrogen of pyridine ring and hydrogen of carboxylic acid group. (b)
The packing and layer structure of motif. (c), (d) Ring motifs.
S.M.A. Mashhadi et al. / Journal of Molecular Structure 1117 (2016) 17e21 19

4. at1662 cmÀ1
for isoniazid. eC]N stretching was observed at
1609 cmÀ1
for cocrystal and that of isoniazid at 1602 cmÀ1
. Ar-
omatic ring vibrations were attributed at 1526 cmÀ1
, 1493 cmÀ1
for cocrystal and at 1492 cmÀ1
for isoniazid. Pyridine ring of
cocrystal was identified at 1407 cmÀ1
while the same was
identified at 1411 for isoniazid.
3.3. Analysis of XRD result
The crystal structure comes out to be that of a hydrate coc-
rystal with half water molecule as water of crystallization and
crystallized in the monoclinic space group P21. Molecular for-
mula of the cocrystal was determined to be C52H60N12O24 with
molar mass 1237.12 amu. The cocrystal stoichiometry is a
discrete 1:1 adduct having distinct hydrogen bonding between
nitrogen of pyridine ring of isoniazid and hydrogen of carboxylic
group of acid (Fig. 3a) having bond length 1.746 Ao
. The extended
packing of these discreet adducts formed layers (Fig. 3b). These
findings correlated well with the predicted hydrogen bond in-
teractions as found in the structures of the cocrystals which are
previously discovered. Other parameters are summarized in
Table 3.
Protocatechuic acid is hydrogen bonded to N of pyridine ring of
isoniazid through OeH$$$N. The angle between the carboxyl group
plane and the pyridine ring plane is 167.12. One ring motif having a
graph set of R2
2 (6 (Fig. 3c) and another graph set of R2
2 (6) (Fig. 3d)
are present.
3.4. Analysis of result from DSC
DSC experiments were carried out to study the thermal behavior
of the cocrystal in relation to the individual components. Ther-
mogram of Isoniazid showed the endothermic peak at 174.37 C.
While Protocatechuic acid at 206 C DSC results of isoniazid and
Protocatechuic acid cocrystal (Fig. 4) expressed endothermic peak
at 190.73 C which was in close agreement with the measured
melting range in the melting point determination. A TG curve in the
range of 110e120 C suggested the absorbed water molecule in the
cocrystal. The thermal profile of molecular Cocrystal was distinct,
with a different melting transition from either of the individual
components. This indicates the formation of novel molecular
complex.
3.5. Results of solubility, stability and UV spectral studies
The Cocrystal showed 6.573 mg/mL solubility in buffer solu-
tion of pH 7.4 which was very less as compared to that of
Isoniazid with solubility 76.30 mg/mL under the same condi-
tions applied. The Cocrystal was stable up to 94.46% at 80 C for
24 h. Cocrystal exhibited more stability than Isoniazid whose
stability is 93.86%. Spectral studies revealed that the co crystal
had ƛmax at 252 nm and value of Ɛo is 0.0384 in buffer solution of
pH 7.4.
4. Conclusion
In the present study hydrate cocrystal of Isoniazid with Proto-
catechuic acid have been developed and characterized by Infra-red
spectroscopy, Differential Scanning Calorimetry and Single Crystal
XRD. Cocrystal structure analysis reveals that formation of
pyridine-carboxylic acid synthon and pyridine-hydroxyl group
synthon are the key reason of Cocrystal formation. The study also
establishes the fact that Isoniazid is an important API having great
potential for cocrystallization and Protocatechuic acid have great
affinity to act as coformer for isoniazid so their cocrystals can be
developed easily.
Stability study reveals that cocrystal have stability greater than
that of isoniazid under the same study conditions and also shows
that cocrystal have very less solubility as compared to that of
isoniazid.
The study suggested that the cocrystals of Isoniazid with anti-
oxidant hydroxy benzoic may serve better in reducing oxidative
stress in Tuberculosis patients during treatment as well as
increasing tabletting stability and shelf life of Isoniazid tablets by
decreasing oxidation processes due to the presence of anti-oxidants
in the formulation.
Acknowledgment
The authors are grateful to Allama Iqbal Open University,
Fig. 4. DSC thermogram.
S.M.A. Mashhadi et al. / Journal of Molecular Structure 1117 (2016) 17e2120

5. Islamabad, Pakistan and HEC Pakistan for all the funding for this
research.
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