Studies on the synthesis, structural characterization, Hirshfeld analysis and stability of apovincamine (API) and its co-crystal (terephthalic acid: Apovincamine = 1:2)

Article abstract of DOI:10.1016/j.molstruc.2015.05.014

Apovincamine and the corresponding co-crystal have been synthesized and characterized by single crystal XRD, FT-IR, UV, TGA/DSG. Components of the crystalline phase have also been investigated in terms of their corresponding Hirshfeld surface. In the crystal lattice, a three-dimensional hydrogen-bonded network is observed in the co-crystal, including the formation of a two-dimensional molecular scaffold. Hirshfeld surfaces and fingerprint plots indicate that the structures are stabilized by H???H, C-H???π, C-H???O and O-H???N intermolecular interactions. In this work, the co-crystal strategy has been applied successfully to apovincamine. One important parameter, i.e. solubility in water, has also been significantly improved, which proves to be an important factor for bioavailability. Furthermore, DSC/TGA analysis indicates that the co-crystal maintains its crystallinity up to 200 °C, suggesting a higher stability of the co-crystal compared to apovincamine.

Full text of DOI:10.1016/j.molstruc.2015.05.014

Journal of Molecular Structure 1097 (2015) 87–97
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Studies on the synthesis, structural characterization, Hirshfeld analysis
and stability of apovincamine (API) and its co-crystal (terephthalic acid:
Apovincamine = 1:2)
⇑
Yu-heng Ma, Shu-wang Ge, Wei Wang, Bai-wang Sun
School of Chemistry and Chemical Engineering, Southeast University, Nanjing 210096, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
The hydrogen bonds between host
and guest molecules have been
analyzed.
ꢀ
ꢀ
ꢀ
The two apovincamine molecules are
unsymmetrical in co-crystal.
The configuration of apovincamine in
co-crystal changed.
The co-crystal strategy was
successfully applied, which have
better physical property in stability
and solubility.
a r t i c l e i n f o
a b s t r a c t
Article history:
Apovincamine and the corresponding co-crystal have been synthesized and characterized by single
crystal XRD, FT-IR, UV, TGA/DSG. Components of the crystalline phase have also been investigated in
Received 26 November 2014
Received in revised form 30 April 2015
Accepted 5 May 2015
terms of their corresponding Hirshfeld surface. In the crystal lattice,
a three-dimensional
hydrogen-bonded network is observed in the co-crystal, including the formation of a two-dimensional
molecular scaffold. Hirshfeld surfaces and fingerprint plots indicate that the structures are stabilized
Available online 15 May 2015
by Hꢁ ꢁ ꢁH, CꢂHꢁ ꢁ ꢁ , C–Hꢁ ꢁ ꢁO and O–Hꢁ ꢁ ꢁN intermolecular interactions. In this work, the co-crystal strategy
p
Keywords:
Co-crystal
Apovincamine
Hirshfeld surface
Hydrogen bond
Stability
has been applied successfully to apovincamine. One important parameter, i.e. solubility in water, has also
been significantly improved, which proves to be an important factor for bioavailability. Furthermore,
DSC/TGA analysis indicates that the co-crystal maintains its crystallinity up to 200 °C, suggesting a higher
stability of the co-crystal compared to apovincamine.
Ó 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://
creativecommons.org/licenses/by/4.0/).
Solubility
Introduction
context have been implemented: Gilli et al. [3] predicted structural
parameters of salts using the strength of donor–acceptor hydrogen
In the crystalline solid state, the difference between a salt and a
co-crystal is rather subtle [1,2]. This is most notably due to the
unambiguous location of the hydrogen atom. Although subject to
debate, generally accepted literature definitions in a broader
bonds with DpKa > 3, whereas co-crystals where observed with
D
pKa < ꢂ3. However, additional factors including the isotopic nat-
ure of the hydrogen, the crystal environment, temperature, and
pressure are all influencing factors affecting the control of the pro-
ton location in the solid-state.
Traditionally focusing on salt formation [4], the formation of
co-crystals has recently been developed as a potential alternative
⇑
Corresponding author. Tel./fax: +86 25 52090614.
E-mail address: chmsunbw@seu.edu.cn (B.-w. Sun).
http://dx.doi.org/10.1016/j.molstruc.2015.05.014
022-2860/Ó 2015 The Authors. Published by Elsevier B.V.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
0

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Y.-h. Ma et al. / Journal of Molecular Structure 1097 (2015) 87–97
N
H
H
H
N
N
N
N
N
N
OH
O
O
N
H
O
O
O
O
O
apovincamine
vincomone
vincadifformine
vincamine
toxicity LD50= 80mg/Kg
LD
=
42 g/Kg
m
50
increase blood flow:88% in cats
increase blood flow:68% in cats
Scheme 1. Structures of alkaloids extracted from Vinca minor Linne.
O
O
H
O
N
N
N
H
O
N
H
O
H
O
N
H
O
N
O
O
co-crystal
apovincamine
O
Scheme 2. The main hydrogen bonding synthons identified in the co-crystal.
Table 1
Crystal data and structure refinement for apovincamine and its co-crystal.
Practical applications of indole alkaloids have been employed
for many years and more than 40 compound families have been
isolated from plants ever since. However, the mechanism of active
compounds reflected in a variety of protective and/or cognitive
effects is rather complex. In the field of indole alkaloids, several
target compounds [10,11] contain an eburnane skeleton, with
examples including vincamine, apovincamine, vincomone and vin-
cadifformine (Scheme 1). The latter can be extracted from Vinca
minor Linne and used in a variety of medical applications, e.g. as
a pharmaceutical against cerebrovascular disease [12].
In recent years much scientific attention has been devoted to
the study of synthetic routes and biological activities [13] of
vincamine derivatives in an effort to illustrate the further
mechanisms of action and to find improved lead compounds with
vasodilatory activity on cerebal blood vessels. Apovincamine
has been demonstrated to exhibit particularly interesting
Apovincamine Co-crystal
Empirical formula
Formula weight
Wavelength(A)
Crystal system
Space group
a (Å)
C
21
H
24
N
2
O
2
50 54 4 8
C H N O
336.42
0.71073
838.97
0.71073
Triclinic
P 1
7.2450(14)
11.289(2)
14.240(3)
80.07(3)
78.00(3)
71.85(3)
1075.3(4)
1
Monoclinic
P 21
9.2278(18)
9.1475(18)
10.619(2)
90
98.04(3)
90
887.5(3)
2
b (Å)
c (Å)
a
(°)
b (°)
(°)
c
3
V (Å )
Z
T/K
293(2)
1.259
293(2)
1.296
Density (calculated) (g/
cm )
3
Absorption coefficient
0.081
0.088
ꢂ1
(
mm
)
h, k, l (min, max)
(ꢂ11,11), (ꢂ11,11),
(0,8), (ꢂ12,13),
(ꢂ16,17)
565
(
ꢂ13,13)
Parameters
228
F(000)
360
446
Goodness-of-fit on F2
0.993
1.039
Final R indices [I>2
r
(I)]
R1 = 0.0815
R1 = 0.0713
CCDC
xR2 = 0.1201
xR2 = 0.1925
1021467
1021468
with some of the produced co-crystals shown to display unique
properties. In general, pharmaceutical co-crystals [5] are defined
as multi-component crystalline structures composed of an
active pharmaceutical ingredient (API) and co-crystal forming
components that are solid at ambient conditions and bound
via non-covalent interactions [6]. Recently, pharmaceutical
co-crystals have gained a lot of attention due to their ability to
tailor physiochemical properties, particularly solubility, dissolu-
tion, stability and bioavailability. With increasing popularity, the
technique of co-crystallization offers the ability to design new
supramolecular scaffolds with desired functional properties [7–9].
Fig. 1. The overlaid UV spectra of apovincamine.

Y.-h. Ma et al. / Journal of Molecular Structure 1097 (2015) 87–97
89
1
Fig. 2. Molecular structure of apovincamine, intramolecular hydrogen bonds are in dashed lines and supramolecular synthons around the S
1
(7) unit (a); crystal packing
0
stabilized by an obvious C–Hꢁ ꢁ ꢁ
p intermolecular interactions were observed with distance of 3.568 ÅA (b); a two-dimensional network viewed from a-axis (c); the layer
structure viewed from a-axis with the width of 12.001 ÅA (d).
0
characteristics with an activity approximately twice that of
vincamine and a hypotensive activity remaining mostly unchanged
crystal structures of the co-crystals (Scheme 2). The newly
synthesized apovincamine and its co-crystal were screened for
solubility and stability; experimental details of these studies
follow in subsequent sections.
[
14]. It has already been explained in great detail why the extracts
show superior effects over pure vincamine.
Typically, alkaloids with widespread applications include vin-
camine and its derivatives. However, a general disadvantage is that
these compounds show poor water solubility. The purpose of this
work is to apply the co-crystal strategy to apovincamine in an
effort to develop co-crystals with antioxidant features, good
solubility [15] and efficient bioavailability, a feature critical to
pharmaceutical applications in the treatment and/or prevention
of diseases. Furthermore, to study the interaction of apovincamine
with terephthalic acid, we report the synthesis and corresponding
Experimental section
General
The corresponding starting material and all reagents were
obtained from commercial sources and were used without further
purification. Infrared spectra were recorded on a SHIMADZU IR

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Y.-h. Ma et al. / Journal of Molecular Structure 1097 (2015) 87–97
Table 2
Geometrical parameters for the hydrogen bonds and C–Hꢁ ꢁ ꢁp interactions in apovincamine and its co-crystal.
D–HꢁꢁꢁA
D–H (Å)
Hꢁ ꢁ ꢁA (Å)
Dꢁ ꢁ ꢁA (Å)
<D–Hꢁ ꢁ ꢁA (deg)
Symmetry code
Apovincamine
Co-crystal
Inter C14–H14ꢁ ꢁ ꢁO1
O2–H2ꢁ ꢁ ꢁN2
0.93
0.82
0.82
0.93
0.93
0.93
0.97
0.97
2.58
1.80
1.8
2.54
2.45
2.54
2.4
2.3
Hꢁ ꢁ ꢁCg
2.90
2.88
2.70
2.76
2.94
3.100 (5)
2.592(6)
2.603(6)
3.471(3)
2.759(5)
3.464(6)
3.338(4)
3.241(3)
Xꢁ ꢁ ꢁCg
3.568(5)
3.521(3)
3.418(2)
3.514(5)
3.776(4)
116
162
168
175
100
173
164
164
1 + x,y, ꢂ1 + z
x,1 + y,z
O3–H3ꢁ ꢁ ꢁN4
C9–H9ꢁ ꢁ ꢁO5
Inter C14–H14ꢁ ꢁ ꢁO2
C19–H19ꢁ ꢁ ꢁO9
C42–H42Aꢁ ꢁ ꢁO2
C48–H48bꢁ ꢁ ꢁO3
ꢂ1 + x,y,1 + z
1 + x,y,ꢂ1 + z
Symmetry code
C–Hꢁ ꢁ ꢁ
p
X–Hꢁ ꢁ ꢁCg
a
Apovincamine
Co-crystal
C13–H13ꢁ ꢁ ꢁCg(1)
130
127
134
136
146
1ꢂx,1/2 + y, 1ꢂz
1 + x,1 + y,ꢂ1 + z
1 + x,1 + y,ꢂ1 + z
ꢂ1 + x,y,z
b
c
C14–H14ꢁ ꢁ ꢁCg(27)
C28–H28ꢁ ꢁ ꢁCg(23)
b
C49–H49Bꢁ ꢁ ꢁCg(27)
d
C53–H53Aꢁ ꢁ ꢁCg(1)
ꢂ1 + x,y,z
a
b
c
6
6
5
6
-Membered Ring N(1) ? C(3) ? C(15) ? C(5) ? C(11).
-Membered Ring C(20) ? C(21) ? C(50) ? C(40) ? C(52) ? C(38).
-Membered Ring N(1) ? C(2) ? C(27) ? C(21) ? C(20).
d
-Membered Ring N(3) ? C(1) ? C(3) ? C(7) ? C(11) ? C(30).
prestige-21 FTIR-8400S spectrometer in the spectral range
A general experimental procedure for obtaining co-crystal is as
follows: apovincamine (1 mmol, 338 mg) and terephthalic acid
(0.05 mmol, 83 mg) were dissolved in 50 ml methanol; the solu-
tion was stirred for one hour, filtered and the solvent was allowed
to slowly evaporate under ambient conditions to yield colorless
ꢂ1
4
00–4000 cm , with all samples analyzed in potassium bromide
pellets. Differential scanning calorimetry (DSC) and thermo gravi-
metric analysis (TGA) were performed using a Mettler-Toledo
TGA/DSC STARe system at a heating rate of 10 Kmin under a
ꢂ1
3
ꢂ1
dry N
2
atmosphere and at a constant flow (20 cm /min) over a
crystals (Mp: 197.0 °C and IR, KBr pellet, cm : 3421, 2962, 1726,
range from 40 °C to 500 °C. Samples were placed in open
aluminum oxide crucibles, annealed to 1100 °C. The TGA/DSC data
were analyzed using STAR software provided by Mettler-Toledo.
1641, 1457, 1257, 1085, 736, 642, 611, 590, 572, 559, 538, 507,
474, 431).
e
LC–MS analysis was performed on a TSQ Quantum Ultra AM
X-ray crystallographic studies
LC-MS (Thermo Finnigan) with an electrospray ionization source
+
and operated in the positive (ESI ) mode at 350 °C with N
2
as
Single crystal X-ray diffraction data was collected at 293 K using
graphite monochromated Mo K
Rigaku’s SCX mini diffractometer with the
nebulizer. The source temperature was kept at 150 °C and the
capillary voltage was maintained at 5.0 kV.
a
radiation (k = 0.071073 nm) on
-scan technique. The
x
lattice parameters were integrated using vector analysis and
refined from the diffraction matrix. Absorption corrections were
carried out using the Bruker SADABS program with multi-scan
method. A summary of crystallographic data, data collection, and
refinement parameters is summarized in Table 1. The structural
determination was carried out using the SHELX package. The
structures were solved by direct method, with refinements being
Preparation of apovincamine and its corresponding co-crystal [16,17]
A mixture of vincamine (10 g, 28.2 mmol), p-toluenesulfonic
acid monohydrate (10.7 g, 56.3 mmol) in 100 ml of toluene was
refluxed for 2 h. During this period, any water formed in the reac-
tion was removed by azeotropic distillation using a Marcusson
trap. The progress of the reaction was monitored by thin layer
chromatography (TLC). When the starting materials could no
longer be detected, the solution was cooled to room temperature
and evaporated in vacuo. The dry residue was dissolved in water
and the pH was adjusted to 8 with 5% aqueous sodium hydroxide
solution. After one hour, the separated material was filtered,
washed with 5 ml of ethanol, and dried in vacuo to obtain apovin-
2
carried out by full-matrix least-square on |F| using the
SHELXL-97 program [18,19]. Reliability factors were defined as
R
R
1
=
= [
R
R
w
(|F
0
|ꢂ|F
c
|)/
) /w(F
R
|F
0
|
and the function minimized was
2
2 2
4 1/2
w
w
(F
0
ꢂF
c
0
) ] , whereas in the least-squares calcula-
tion the unit weight was used. All non-hydrogen atoms were
refined anisotropically and hydrogen atoms were inserted at their
calculated positions and fixed at their positions. CCDC [20] refer-
ence numbers 1021467 and 1021468 contain the supplementary
crystallographic data reported in this paper as CIF files. These data
sets can be obtained free of charge from The Cambridge
camine (8.6 g, 90%) as a white solid. 1H NMR (500 MHz, CDCl
d1.00 (m, 4H), 1.39 (m, 1H), 1.50 (d, J = 15 Hz 1H), 1.72 (m, 1H),
3
):
1
3
.87 (m, 2H), 2.50 (m, 1H), 2.60 (t, J = 10 Hz 2H), 2.99 (m, 1H),
.23 (m, 1H), 3.34 (dd, J = 5 Hz 1H), 3.95 (s, 1H), 4.15 (s, 1H), 6.13
1
3
Crystallographic
data_request/cif.
Data
Centre
via
www.ccdc.cam.ac.uk/
(
s, 1H), 7.11 (m, 1H), 7.14 (m, 1H), 7.21(m, 1H), 7.45(m, 1H);
C
3
NMR (500 MHz, CDCl ): d8.692, 16.360, 20.285, 27.307, 28.615,
3
1
1
7.746, 44.922, 51.491, 52.465, 55.772, 76.751, 77.006, 77.260,
08.699, 112.414, 118.239, 120.259, 121.958, 128.144, 128.202
Hirshfeld surface calculations
29.064, 134.107, 163.854; MS (ESI): required for C21
H
26
N
2
O
2
ꢂ
[
M] ,337.4; found 337.2.
Calculations of Molecular Hirshfeld surfaces [21,22] were per-
formed using the CrystalExplorer program. When the CIF files were
entered into the CrystalExplorer program, all bond lengths to
hydrogen were automatically modified to typical standard neutron
values [23] (C–H = 1.083 Å, N–H = 1.009 Å and O–H = 0.983 Å). All
Hirshfeld surfaces were generated using a standard (high) surface
resolution [24]. For a given crystal structure and a set of spherical
atomic electron densities, the Hirshfeld surface is unique. The dnorm
The experimental procedure to obtain single crystals of apovin-
camine is as follows: apovincamine (1 mmol, 338 mg) was dis-
solved in 50 ml acetone; the solution was stirred for 1 h, filtered
and the solvent was allowed to slowly evaporate under ambient
conditions, yielding colorless crystals (Mp: 165.5 °C and IR, KBr
ꢂ1
pellet, cm : 3428, 2933, 1727, 1631, 1454, 1280, 1205, 1080,
8
33, 744, 578, 532, 503, 468, 431).

Y.-h. Ma et al. / Journal of Molecular Structure 1097 (2015) 87–97
91
Fig. 3. Molecular structure of co-crystal, the thermal ellipsoid plot at 30% probability and hydrogen bonds between apovincamine molecules and terephthalic acid molecules
a); hydrogen bonds formed by two apovincamine molecules and supramolecular synthons around the R2
(10) dimeric unit (b); an infinite 1D chain motif of a layer viewed
from a-axis (c); crystal packing stabilized by an obvious C–Hꢁ ꢁ ꢁ intermolecular interactions were observed with distance of 3.514 ÅA , 3.521 ÅA , 3.418 ÅA and 3.776 ÅA (d).
(
2
0
0
0
0
p
values [25] were mapped onto the Hirshfeld surface by employing
a red–blue–white color scheme: red regions represent closer
contacts and a negative dnorm value; blue regions represent longer
contacts and a positive dnorm value; white regions represent the
distance of contacts exactly corresponding to the Van der Waals
separation with a dnorm value of zero. The 3-D dnorm surfaces can
be resolved into 2-D fingerprint plots, analyzing all of the inter-
molecular contacts at the same time to quantitatively summarize
Solubility studies
An Agilent 1260 HPLC system was used to analyze apovin-
camine and its co-crystal, together with a 1260 variable wave-
length detector (VWD). Before this procedure, an Agilent Eclipse
plus-C18 analytical column (4.6 mm ꢃ 150 mm, 3.5
lm particle
sizes) was used as a separation column. For apovincamine, the
UV detector was set to a wavelength of 273 nm in order to avoid
any interference from near ultraviolet radiation. The corresponding
spectrum is shown in Fig. 1.
[
26,27] the nature and type of all intermolecular contacts experi-
enced by the molecules in the crystal.

9
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Y.-h. Ma et al. / Journal of Molecular Structure 1097 (2015) 87–97
Fig. 4. The terephthalic acid induced configuration change (a); apovincamine molecules (type I and type II) have similar configuration.
A mixture of triethylamine 0.5% (pH = 7.5) and methanol
40:60) was used as the mobile phase. The column temperature
was maintained at 25 °C during analysis. Pre-treatment and con-
centration columns were operated at ambient temperature.
Pumps 1 and 2 were used to deliver mobile phase 1 at a flow rate
of 0.40 ml/min and mobile phase 2 at a flow rate of 0.60 ml/min,
For studies on oxidative stress, sample solutions in 3% hydrogen
peroxide were kept at 65 °C for 2 h. After degradation, the acidic
and alkaline solutions were neutralized by adding proper amounts
of sodium hydroxide or hydrochloric acid which were diluted with
(
solvent to yield a concentration of 10 lg per ml.
respectively. The entire solution was filtered using a 0.45 lm
Results and discussion
membrane filter (Millipore Corp., Bedford) and degassed before
performing HPLC analysis. The system was running at a flow rate
of 1 ml/min and the total time was 60 min. The injection volume
Crystal structures
was 10
ll.
Apovincamine crystallizes as colorless cuboid-shaped crystals,
representing the P21 space group of a monoclinic system. A sche-
matic ORTEP-drawing of apovincamine with thermal ellipsoids at
Stability
3
0% probability is shown in Fig. 2a. A strong intramolecular
The degradation studies of apovincamine and the correspond-
ing co-crystal were carried out under acid/base hydrolysis and con-
ditions of oxidative stress according to methods previously
published [28–30]. For acid hydrolysis of drug substances, solu-
tions of apovincamine and its co-crystal were treated with 0.1 M
hydrochloric acid in a water bath at 65 °C for 2 h. Alkaline hydrol-
ysis was conducted using 0.1 M sodium hydroxide at 65 °C for 2 h.
C14–H14ꢁ ꢁ ꢁO1 hydrogen bond (distance of 3.100 Å and angle of
1
1
16°) can be detected forming a cyclic S
1
(7) system. C–Hꢁ ꢁ ꢁ
p inter-
molecular interactions have been observed with the distance of
0
3.568 ÅA (Fig. 2b), exhibiting crucial parameters for the formation
of
two-dimensional network is observed in this crystal lattice and
a network structure. When viewed from the a-axis, a
Table 3
Geometry parameters of selected bond angle and torsion angle in apovincamine and its co-crystal.
Apovincamine
Apovincamine (type II)
Apovincamine (type I)
Bond length(Å)
O2–C6
1.454(6)
1.515(6)
1.519(6)
1.480(6)
1.525(6)
1.552(6)
1.566(6)
O6–C53
C25–C19
C7–C41
N4–C36
C36–C47
C34–C25
C25–C48
1.445(9)
1.505(8)
1.509(8)
1.485(8)
1.531(9)
1.560(8)
1.534(7)
O8–C49
C26–C9
1.437(9)
1.534(8)
1.494(8)
1.503(9)
1.504(10)
1.550(9)
1.537(8)
C8–C12
C5–C17
N2–C18
C18–C9
C10–C8
C8–C7
C27–C44
N2–C43
C43–C37
C15–C26
C26–C42
Bond angle (°)
C6–O2–C4
C12–C1–N1
C11–C5–C17
N2–C2–C11
C8–C7–C19
117.1(4)
C53–O6–C31
C19–C35–N3
C1–C7–C41
N4–C12–C1
C25–C48–C46
116.1(5)
C49–O8–C18
C9–C23–N1
C2–C27–C44
N2–C3–C2
114.8(5)
119.8(4)
119.3(5)
109.2(4)
115.8(4)
118.1(5)
120.7(5)
107.8(4)
117.2(5)
119.8(5)
122.1(5)
106.7(4)
115.4(6)
C26–C42–C54
Torsion angle (°)
O2–C4–C1–C12
O1–C4–C1–N1
C11–C2–C8–C12
C2–C8–C12–C1
C8–C12–C1–N1
C12–C1–N1–C11
C17–C20–N2–C2
C2–N2–C18–C9
C10–C8–C7–C19
C12–C8–C7–C19
41.8(6)
33.7(7)
42.8(5)
ꢂ31.1(6)
2.8(7)
12.8(6)
64.0(5)
ꢂ49.6(5)
55.3(6)
ꢂ66.8(5)
O6–C31–C35–C19
O5–C31–C35–N3
C1–C12–C25–C19
C12–C25–C19–C35
C25–C19–C35–N3
C19–C35–N3–C1
C41–C17–N4–C12
C12–N4–C36–C47
C34–C25–C48–C46
C19–C25–C48–C46
ꢂ144.9(5)
ꢂ157.4(6)
45.3(6)
O8–C18–C23–C9
O7–C18–C23–N1
C2–C3–C26–C9
C3–C26–C9–C23
C26–C9–C23–N1
C9–C23–N1–C2
C44–C33–N2–C3
C3–N2–C43–C37
C15–C26–C42–C54
C9–C26–C42–C54
ꢂ144.9(6)
ꢂ157.1(6)
46.3(6)
ꢂ33.5(7)
ꢂ34.3(7)
0.5(8)
0.8(8)
20.1(8)
65.4(7)
53.6(6)
45.8(8)
19.5(8)
66.6(7)
53.3(6)
47.5(8)
ꢂ75.6(8)
ꢂ74.2(8)

Y.-h. Ma et al. / Journal of Molecular Structure 1097 (2015) 87–97
93
Fig. 5. 3-D dnorm surfaces and 2-D fingerprint plots of apovincamine molecule in two crystals.
the width of the layer is 12.001 Å (Fig. 2c and d). Geometrical
parameters for hydrogen bonds present in apovincamine and its
co-crystal are summarized in Table 2.
The co-crystals also form as colorless cuboid-shaped crystals,
representing the P1 space group of a triclinic system, with the
asymmetric unit (ASU) consisting of one terephthalic acid mole-
cule and two apovincamine molecules. A schematic ORTEP draw-
ing of the co-crystal with thermal ellipsoids at 30% probability
was shown in Fig. 3a, in which the terephthalic acid molecule func-
tions as a bridge connecting two apovincamine units through O–
Hꢁ ꢁ ꢁN and C–Hꢁ ꢁ ꢁO hydrogen bonds. The apovincamine molecule
(type I) and the terephthalic acid molecule formed a dimeric unit
2
by R
2
(7) supramolecular heterosynthon through O–Hꢁ ꢁ ꢁN (dis-
tance of 2.592 and angle of 162°) and C–Hꢁ ꢁ ꢁO (distance of 3.338
Å and angle of 164°) hydrogen bond interactions. The apovin-
camine molecule (type II) also formed a dimeric unit by R2
(7)
2

9
4
Y.-h. Ma et al. / Journal of Molecular Structure 1097 (2015) 87–97
Table 4
Summary of the various contact contributions to apovincamine molecules in
Hirshfeld surface area.
Apovincamine
%)
Apovincamine
(type II) (%)
Apovincamine
(type I) (%)
(
H–H
H–O
C–H
N–H
C–O
C–C
67.7
12.5
17.4
2.2
63.2
17.4
15.4
1.6
0.3
1.9
62.5
17.7
17.6
1.5
0.2
0.3
O–O
0.2
0.2
supramolecular heterosynthon through O–Hꢁ ꢁ ꢁN (distance of 2.603
and angle of 168°) and C–Hꢁ ꢁ ꢁO (distance of 3.241 Å and angle of
1
64°) hydrogen bond interactions. Notable is the fact that these
two pairs of C–Hꢁ ꢁ ꢁO and O–Hꢁ ꢁ ꢁN hydrogen bonds are almost
identical and not centrosymmetric.
Further analysis reveals that the terephthalic acid molecule
1
Fig. 7. Solubility of apovincamine and its co-crystal at different temperature.
formed a S
1
(5) ring through C14–H14ꢁ ꢁ ꢁO2 (distance of 2.759
and angle of 100°) hydrogen bond interactions. However, between
C13–H13 and O3, no intramolecular hydrogen bond can be
(
33.63°). Geometry parameters of selected bond angle and torsion
detected. The two apovincamine molecules form a dimeric unit
angle in apovincamine and its co-crystal are provided in Table 3.
2
by R
(
2
(10) supramolecular heterosynthon through C9–H9ꢁ ꢁ ꢁO5
distance of 3.471 Å and angle of 175°) and C19–H19ꢁ ꢁ ꢁO7 (dis-
tance of 3.464 Å and angle of 173°) hydrogen bond interactions
Fig. 3b). In this crystal lattice, a three-dimensional network can
Hirshfeld surface analysis
(
The 3D Hirshfeld surfaces of apovincamine molecules in
apovincamine and its co-crystal are shown in Fig. 5a–c; they
clearly show the influences of co-formers on the intermolecular
interactions of the apovincamine molecule. The large and deep
red spots on the 3D Hirshfeld surfaces indicate the close-contact
interactions, mainly responsible for the corresponding hydrogen
bond contacts. The small red spots on the surfaces represent the
be observed, which proves to be different from the
two-dimensional network in apovincamine. When viewed from
the a-axis, trimeric units display an infinite 1D chain (Fig. 3c);
the 1D chain stacked across from each other and along the bc
plane; C–Hꢁ ꢁ ꢁ
distances of 3.514 ÅA , 3.521 ÅA , 3.418 ÅA and 3.776 ÅA , respectively
Fig. 3d).
Upon overlay of the three-dimensional structures of the apovin-
camine molecules (Fig. 4), e.g. apovincamine-API and the apovin-
camine molecule (type and type II), we found that the
p intermolecular interactions were observed with
0
0
0
0
(
C–Hꢁ ꢁ ꢁ
p interactions. The 2-D fingerprint plots are shown in
Fig. 5d–f, illustrating the breakdown of 2-D fingerprint plots.
With the help of these analysis results, different interactions can
be separated from each other that would commonly overlap in full
fingerprint plots.
I
apovincamine molecules in the co-crystal maintain a similar con-
figuration, not consistent with the apovincamine structure. For
example, the torsion angle (O5–C31–C35–N3) is found to be
ꢂ157.35°, different from the torsion angle in apovincamine
For apovincamine (Fig. 5d), the Hꢁ ꢁ ꢁH interactions, which are
reflected in the middle of scattered points in the 2D fingerprint
plots, exhibit the most significant contribution (67.7%) to the total
Fig. 6. The DSC and TGA profiles of apovincamine and its co-crystal.

Y.-h. Ma et al. / Journal of Molecular Structure 1097 (2015) 87–97
95
Fig. 8. Chromatogram of apovincamine under different: untreated (a); alkaline hydrolysis (b); oxidation (c).
Hirshfeld surfaces. The C–Hꢁ ꢁ ꢁO hydrogen bonding intermolecular
interactions appear as two very short spikes in the 2D fingerprint
plots, contributing 12.5%to the total Hirshfeld surfaces. The C–
Hꢁ ꢁ ꢁ
p interactions also display a relatively large contribution to
the total Hirshfeld surfaces of apovincamine, totaling 17.4%. The
O–Hꢁ ꢁ ꢁN hydrogen bonding interactions did not exhibit significant

9
6
Y.-h. Ma et al. / Journal of Molecular Structure 1097 (2015) 87–97
ꢂ1
contributions in the 2D fingerprint plots and merely comprise of
.2% of the total Hirshfeld surfaces. Apart from these main
197 °C with a melting enthalpy of 50.63 Jg . Another endothermic
ꢂ1
2
peak appears at 298.3 °C, with a melting enthalpy of 265.31 Jg
.
interactions described above, C–C, C–O and O–O interactions were
not observed, see summarized values in Table 4.
The results of the DSC and TGA analysis both indicate that the
co-crystal maintains its relative crystallinity up to 200 °C, suggest-
ing that the co-crystal also exhibits a better stability than
apovincamine.
The two types of apovincamine molecules (Fig. 5e and f) display
structurally similar shapes and therefore show similar intermolec-
ular contacts. For the apovincamine molecule (type II), the Hꢁ ꢁ ꢁH
interactions also reflect the most significant contribution to the
total Hirshfeld surfaces, totaling 63.2%. The O–Hꢁ ꢁ ꢁN hydrogen
bonding interaction display the second largest contribution to
the total Hirshfeld surfaces, slightly larger than that in apovin-
camine, totaling 17.4%. The third largest contribution can be found
Solubility studies
Solubility reflects an important parameter for bioavailability
estimations. High Performance Liquid Chromatography (HPLC) in
general displays major advantages in terms of sensitivity and
specificity and was therefore used to investigate the solubility of
apovincamine and its co-crystal. Prior to solubility measurements,
stock solutions of apovincamine (1 mg/ml) were prepared in
methanol, together with a series of dilutions. The mobile phase
has been adjusted to 0, 0.2 0.4, 0.6, 0.8 mg/ml in an effort to obtain
the corresponding working standards for the preparation of the
standard curve. Then, saturated solutions of apovincamine and
its co-crystal were prepared and measured by HPLC. The results
of the solubility measurements were obtained through the stan-
dard curve.
for the C–Hꢁ ꢁ ꢁ
molecule (type I), the Hꢁ ꢁ ꢁH, O–Hꢁ ꢁ ꢁN and C–Hꢁ ꢁ ꢁ
same order as the apovincamine molecule (type II), resulting in
2.5%, 17.7% and 17.6%. Although the two types of the
apovincamine molecules are very similar, minor differences can
nevertheless be found in the C–Hꢁ ꢁ ꢁ and hydrogen bonds.
Further analysis of three types of the apovincamine molecules
p
interactions, totaling 15.4%. For the apovincamine
p
followed the
6
p
revealed an interesting phenomenon: the distance of the C–Hꢁ ꢁ ꢁ
p
interactions corresponding to their corresponding Hirshfeld
surface area c0ontributions follow the order apovincamine
0
Using this method, the solubility of apovincamine has been
(
type II, 3.514 ÅA and 15.4%) < apovincamine (API, 3.568 ÅA and
0
determined to be 5.5 mg/ml in CH
of the co-crystal in the same solvent has been determined to be
.8 mg/ml and the solubility of pure apovincamine under the same
3
OH at 30 °C, while the solubility
1
7.4%) < apovincamine (type I, 3.776 ÅA and 17.6%).
9
Differential scanning calorimetry (DSC) and thermogravimeric
analysis (TGA)
conditions has been determined to be 7.8 mg/ml. The solubility of
apovincamine and its co-crystals at different temperature are
shown in Fig. 7. Additionally, we found that through the
co-crystal strategy we could further improve the solubility in
The DSC and TGA curves of terephthalic acid, apovincamine and
its corresponding co-crystal in a nitrogen atmosphere at a heating
3
CH OH by 25%. To find use as a injectable drug and to yield maxi-
ꢂ1
rate of 10 K min
with a temperature range of 40–400 °C are
mum dosage, the solubility of apovincamine in water is a very
important parameter. Neat apovincamine could not be dissolved
in water, whereas the co-crystal exhibits good solubility. The
co-crystal strategy could therefore be an elegant way to solve this
solubility problem in a favorable fashion.
shown in Fig. 6. For terephthalic acid, the DSC curve shows an
intense endothermic peak at 357.7 °C, with a sublimation enthalpy
ꢂ1
of 532.3 Jg . For apovincamine, the melting temperature is
ꢂ1
approximately 165.5 °C, with a melting enthalpy of 77.61 Jg
.
The TGA curve shows a continued weight loss from 180 °C to
ꢂ1
4
00 °C with an endothermic enthalpy of 88.47 Jg . This can be
Stability studies
attributed to the structural decomposition of the apovincamine
molecules. For the co-crystal on the other hand, the TGA curve
shows two endothermic peaks. When the co-crystal begins to dis-
play a weight loss, the DSC curve exhibits an endothermic peak at
Both apovincamine and its co-crystal were not found to be
labile to acid hydrolysis in 0.1 M HCl at 65 °C. The experimental
results obtained provide direct evidence for the hypothesis that
0.1M NaOH
H
H
N
N
N
N
HO
O
O
O
Chemical Formula: C H N O
2
0
22 2 2
Molecular Weight: 322.40
a
H
N
H
3
% H O
2 2
N
H
N
N
N
+
4.06 min
N
O
O
O
=
tR
tR=11.07 min
O
OH
O
O
O
Chemical Formula: C H N O
Chemical Formula: C H N O
2
1
26
2
3
21 24 2
3
Molecular Weight: 354.44
Molecular Weight: 352.43
b
2 2
Scheme 3. The suggested pathway for the degradation in 0.1 M NaOH (a) and 3% H O (b).

Y.-h. Ma et al. / Journal of Molecular Structure 1097 (2015) 87–97
97
the co-crystal strategy does not reduce the stability of apovin-
Appendix A. Supplementary material
camine. However, apovincamine and its co-crystal have been
shown to be highly labile to alkaline hydrolysis: around 36.7%
degradation of apovincamine has been observed in 0.1 M NaOH
at 65 °C and within 2 h. The degradation peaks appeared at tR
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.molstruc.2015.05.
014.
1
.29 min and similar degradation has been observed in the
co-crystal (Fig. 8b). HPLC analysis revealed that the rate of degra-
dation increased with time. The structural characteristics have
been confirmed the LC–MS analyses. The mass of the degradation
product was found to be m/z 323.17 [M + H] which corresponds
to the mass of apovincaminic acid. The presumed degradation
pathway in 0.1 M NaOH is shown in Scheme 3a.
The co-crystal was found to be relatively stable upon exposure
2 2
to oxidative conditions (e.g. 3% H O at 65 °C for 2 h), the presence
of any degradation product (>1/1000) could not be detected by
HPLC. Neat apovincamine on the other hand was found to be prone
to oxidation under these condition, resulting in about 10% degrada-
tion. Two major peaks corresponding to the degradation product
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Acknowledgements
This work has been supported by the prospective joint research
project of Jiangsu province (BY2012193) and the Fundamental
Research Funds for the Central Universities (CXZZ12_0119).