Influence of Impurities on the Solution-Mediated Phase

Transformation of an Active Pharmaceutical Ingredient

Takashi Mukuta,
,
Alfred Y. Lee,

Takeshi Kawakami,

and Allan S. Myerson*

,
Process Chemistry Labs, Astellas Pharma Inc., 160-2, Akahama, Takahagi-shi, Ibaraki,
318-0001 Japan, and Department of Chemical and Environmental Engineering, Illinois
Institute of Technology, Chicago, Illinois 60616
Received October 14, 2004
ABSTRACT: The solution-mediated phase transformation of the metastable A form of an active pharmaceutical
ingredient (1) to the stable B form is investigated in 2-propanol. The transformation behavior (or rate) is quantified
using powder X-ray diffraction. The studies show that the rate of transformation is sensitive to the tailor-made
impurities and that the presence of certain inhibitors reduces the rate of transformation. Concurrently molecular
modeling studies are undertaken to investigate the incorporation of these structurally related impurities into the
crystal lattice, and it is observed that the build-in approach used in morphology predictions for additive-host systems
can be applied to evaluate the extent of impurity incorporation. The build-in approach employs the attachment
energy method in which the host molecules are substituted by impurity molecules, and the relative incorporation
energies are calculated for various crystal faces. The order of the relative incorporation energies of the structurally
similar impurities is identical to the order of the percentages of the amount of impurities incorporated into the
crystal lattice as determined by high performance liquid chromatography (HPLC).
Introduction
Polymorphism is the ability of a chemical entity to
exist in more than one distinct crystalline form as a
result of differences in the packing arrangement and/
or molecular conformation.
1
This phenomenon is often
observed in organic molecular crystals
2
and is of para-
mount importance in the pharmaceutical industry where
different solid forms of the same chemical compound can
exhibit different physical and chemical properties as
well as different solubility and dissolution, which in turn
affects the bioavailability and stability of the drug
substance. Pharmaceutical manufacturers are required
by the Food and Drug Administration to consistently
produce the desired polymorph of a drug.
3-5
Discovery
and characterization of polymorphs are crucial in the
early stages of the development of the drug product, as
unanticipated appearance or disappearance
6
of a poly-
morph can impact the time to market for a drug, or in
the case of ritonavir
7,8
it can result in a withdrawal of
a commercial pharmaceutical product. As a result,
polymorph screening, in which a compound is crystal-
lized in various process conditions under a variety of
crystallization methods (e.g., sublimation, crystalliza-
tion from the melt, vapor diffusion, thermal treatment,
and crystallization from a single solvent or combinations
of solvents), has been particularly important.
9
More
recently, high-throughput crystallization screens have
been developed using a combinatorial approach to
capture crystal form diversity.
10-14
This approach en-
ables a more comprehensive exploration of solid forms
and has been applied to various highly polymorphic
pharmaceutical compounds such as acetaminophen,
15
MK-996,
16
ritonavir,
17
and sertraline HCl.
16,18
It is important to identify the most stable polymorph
as well as to fully understand and control the conditions
to obtain the desired solid form. Numerous methods and
strategies have been used to control polymorphism,
including capillary crystallization,
19-21
laser-induced
nucleation,
22,23
solvent-drop grinding,
24
spray drying,
25
supercritical fluid crystallization,
26
self-assembled mono-
layers,
27,28
surfaces of metastable crystal forms,
29
nano-
porous polymer monoliths and glass matrixes,
30
polymer
heteronuclei,
31
and organic single-crystal surfaces that
direct the selectivity of polymorphs through epitaxial
matching.
32,33
In addition, designer additives have been
shown to inhibit the formation of one polymorph, in turn
promoting the crystallization of another polymorph.
34,35
These additives have also been exploited to engineer
crystal morphology
36,37
and kinetically resolve chiral
molecules.
38,39
Similarly, impurities and synthesis byprod-
ucts can influence the nucleation and growth of poly-
morphs as can be seen in the case of terephthalic acid
where an impurity induced twinning and inhibited a
solid-state transformation, leading to the stabilization
of the metastable form.
40
Recent works have utilized
additives or impurities in manipulating the polymorphic
outcome.
41-45
Structurally related additives or impurities may be
incorporated into the host crystal lattice as crystal faces
are sometimes unable to discriminate between the host
and the additive/impurity molecule.
46
This can lead to
severe consequences as incorporated impurities can
alter the physical and chemical properties of the crystals
and quite possibly have toxicological effects. Thus,
control and minimization of the impurity content in
pharmaceutical products are of utmost importance.
Molecular modeling techniques employing the attach-
ment energy method have shown that impurity-modified
crystal habit can be successfully predicted
47-52
and that
relative incorporation energies can be used as an
indicator for the likelihood of impurity incorporation on
crystal surfaces.
51,52
In most cases, the crystallization of polymorphs often
obeys Ostwalds Law of Stages
53
where the kinetically
* To whom correspondence should be addressed. Phone: 312-567-
3163. Fax: 312-567-7018. E-mail: myerson@iit.edu.

Astellas Pharma Inc.

Illinois Institute of Technology.

CRYSTAL
GROWTH
&DESIGN
2005
VOL. 5, NO. 4
1429-1436
10.1021/cg049646j CCC: $30.25 2005 American Chemical Society
Published on Web 05/18/2005
metastable form initially appears followed by its trans-
formation to the more stable polymorph. Understanding
the different factors (e.g., solvent, temperature, and
agitation rate) that affect the conversion between the
forms is essential as these variables can facilitate or
impede the transformation rate. In addition, impurities
in the process can also impact the transformation
behavior as evident in studies in which additives can
stabilize the kinetic crystal form.
40-45
In this work, the influence of four structurally related
impurities on the solution-mediated phase transforma-
tion of an active pharmaceutical ingredient (1) is
investigated. The polymorphic transformation is moni-
tored using powder X-ray diffraction, and the extent of
impurity incorporation is determined by high perfor-
mance liquid chromotography (HPLC) and compared to
relative incorporation energies derived for measuring
the compatibility of impurity with the host crystal
lattice. The four structurally related compounds were
chemically synthesized and chosen as model inhibitors.
Experimental Section
Materials. Cyclohexane and 2-propanol were obtained from
Pharmco Products (Brookfield, CT). Form B of compound 1
was supplied from Astellas Pharma Inc. and used without
further purification. The A form was prepared from cyclohex-
ane at reflux temperature. Form C was crystallized in a
mixture of ethyl acetate and n-heptane. The structurally
related compounds RS1, RS2, RS3, and RS4 as shown in
Figure 1 were synthesized and have been identified by nuclear
magnetic resonance (NMR) and mass spectroscopy (MS).
Solubility and Dissolution. The solubility of each poly-
morph of compound 1 was measured in the temperature range
of 0 to 40 C and determined by high performance liquid
chromatography (HPLC) using a Shimadzu HPLC system
(YMC-GEL ODS column, injection volume was 5 L, flow rate
was 1.0 mL/min, UV detector was set at 210 nm and the mobile
phase consisted of 40% acetonitrile/60% phosphoric acid buffer
pH 5.0). The experimental setup consisted of a 50 mL glass
vessel equipped with an agitator where 200 mg of compound
1 was suspended in 10 mL of 2-propanol at a desired temper-
ature controlled by a thermostat. After 30 min, the solution
was filtered and the concentration of compound 1 in the
supernatant was determined by HPLC. The residual crystal
of compound 1 on the filter was dried, and powder X-ray
diffraction was performed to ensure that the polymorph
transformation did not occur during solubility measurement.
Also, the stable polymorph concentration after the solution-
mediated phase transformation was determined by HPLC in
the absence and the presence of the impurities.
To measure the amount of impurities incorporated in
compound 1, the crystals were dissolved in the mobile phase.
After dissolution, the amount of impurities in the effluent or
the degree of impurity incorporation within the crystal lattice
was determined by HPLC analysis.
Crystallization Studies. The solution-mediated phase
transformation of the metastable A form of compound 1 to the
stable B form was carried out at 30 C. Experiments were
performed in a 100 mL three-neck flask equipped with a
mechanical stirrer (stirring speed of 300 rpm). The tempera-
ture was chosen in to avoid the appearance of form C being
mixed with form B since form C was more stable below 20 C
despite having a long transformation time from form B to form
C. Different concentrations of impurities RS1, RS2, RS3, and
RS4 were mixed with form A and added into 100 mL of
2-propanol. Samples were removed at desired time intervals;
the solid phase was immediately filtered under reduced
pressure, and the physically adsorbed solvent was removed
by drying for 15 h at 30 C. The phase composition in the solid
was examined by powder X-ray diffraction, and the level of
impurities incorporated in the crystal lattice was assessed by
HPLC. Also the effects of seeding of the stable B form on the
rate of transformation were studied. Two different seeding
levels (0.1 and 0.5 wt %) were used in the presence of the most
effective inhibitor. The form B seed crystals were added to the
slurry of the A form at time 0 s and at 30 C.
Scanning Electron Microscopy. The morphology of each
crystalline form was observed with a scanning electron mi-
croscope (SEM, Hitachi S-800). Samples were sputter coated
with gold before examination to improve conductivity, and
images were acquired at an operating voltage of 5 kV.
Single-Crystal X-ray Structure Determination. Data
were collected using a four axis single-crystal X-ray diffrac-
tometer (Rigaku AFC-7R) at ambient temperature. Measure-
ments were carried out under the following conditions: graph-
ite monochromated CuKR ( ) 0.154178 nm) radiation;
voltage, 40 kV; current, 40mA; and scanning speed, 8/min.
The crystal structures for all three polymorphs were solved
by direct methods with the crystallographic software package,
teXsan.
54
Powder X-ray Diffraction. X-ray powder diffraction was
obtained with a Rigaku Miniflex diffractometer with CuKR
radiation ( ) 0.15418 nm), and measurements were carried
out at a power of 30 kv and 15 mA. Samples were manually
ground into fine powder in a mortar and pestle and packed
on glass slides for analysis. Data were collected from 3 to 30
with a scan rate and step size of 1/min and 0.1, respectively.
Powder X-ray diffraction was utilized to quantify the relative
amounts of forms A and B present in the mixture based on
the differences between the two distinct powder patterns. For
calculation purposes, the area of all the peaks in the scan range
(3 to 30) of both forms were utilized. The conversion to form
B or the content of form A of the sample collected with time
was determined on the basis of the area ratio of the X-ray
peaks of the two crystalline forms. Binary mixtures of both
polymorphic forms in various ratios were prepared in a mortar
and pestle. Figure 2 shows typical powder patterns for a
number of standard mixtures of different compositions (0, 30,
50, 70, and 100 wt % of form A). It can be seen that there are
four unique peaks in the powder pattern for form A (2 ) 6.8,
13.4, 21.6, and 24.4) that can be differentiated from peaks
of pure form B. Thus, the ratio of the area of these four
characteristic peaks for the two forms was chosen for use in
the construction of a calibration curve for determining quan-
titatively the polymorphic composition. The calibration curve
Figure 1. Molecular structures of compound 1 and the
structurally related impurities.
1430 Crystal Growth & Design, Vol. 5, No. 4, 2005 Mukuta et al.
for the extent of phase transformation of form A to form B is
shown in Figure 3.
Molecular Modeling. Relative incorporation or binding
energy calculations, including molecular dynamics (MD) and
mechanics simulations, were carried out with the software
Cerius
2
. The molecular modeling methodology and details of
the procedures for the build-in approach have been described
elsewhere.
47
Briefly, the build-in approach consists of four
main steps. First molecular mechanics (MM) simulations are
performed using a suitable potential function to predict the
crystal morphology to identify the morphologically important
faces. Next, in each symmetry position of the unit cell, the host
molecule is replaced by an impurity molecule. Molecular
mechanics simulations are then carried out again, using the
conjugate gradient method
55
to minimize the energy of the
impurity molecule within the host crystal lattice, in combina-
tion with MD simulations, where external forces on the
molecule are applied and Newtons equations of motions are
solved to compute the new atomic positions. This sequence of
MM and MD simulations is repeated until a global minimum
energy is obtained to ensure that the conformation of the
impurity molecule is adjusted in such a way that it is situated
at its optimum position within the host crystal lattice. In this
work, the DREIDING 2.21
56
force field is used for all molecular
simulations, van der Waals forces are modeled with the
Lennard-Jones 12-6 expression, and hydrogen bonding energy
is approximated using a Lennard-Jones-like 12-10 expression.
Partial atomic charges are calculated with MOPAC using a
modified neglect of diatomic overlap (MNDO) Hamiltonian
approximation,
57
and the Ewald summation technique is
utilized for the summation of long-range van der Waals and
electrostatic interactions under the periodic boundary condi-
tions.
In the final step, attachment energy
58
calculations are
performed with the impurity species in each symmetry position
and the relative incorporation energy for each crystal face is
given by
where E
hkl
b
, E
hkl
sl
, and E
hkl
att
are the incorporation energy of the
host molecule on the {hkl} face, the slice energy, and the
attachment energy of the {hkl} face, respectively. Ki is the ratio
of the lattice energy of the pure crystal to the lattice energy of
the crystal with the impurity in symmetry position i. The
lattice energy is calculated by summing all the atom-atom
interactions between a central molecule and all the surround-
ing molecules in the crystal. E
hkl,i
b
is the incorporation energy
of the impurity molecule on the {hkl} face, while E
hkl,i
sl
and
E
hkl,i
att
are the slice and attachment energy of the {hkl} face
with the impurity in symmetry position i, respectively.
Minimum change in the relative incorporation energy (i.e.,
low b) indicates where the impurities are most likely to
incorporate.
48,49
The energy is a useful measure on the compat-
ibility of the impurity with the host crystal lattice and has
been successful in predicting an impurity-modified crystal
morphology
47-52
and thus used herein to assess the impact of
the impurities on the purity of the crystals.
Results and Discussion
Crystal Structures. Compound 1 exists in three
distinct crystalline forms. Table 1 summarizes the
crystallographic data for each polymorph. All three
crystal structures are rich in hydrogen bonds and are
composed of sheets where it is observed that the crystal
building block or growth unit for each modification is a
centrosymmetric dimer interconnected by symmetrical
NsH NtC interactions. In forms A and B, compound 1
molecules are packed to form the centrosymmetric
aggregate between the hydroxyl hydrogen and the
Figure 2. Powder X-ray diffraction patterns for a number of
mixtures of form A and form B.
Figure 3. Calibration curve for the degree of conversion of
form A to form B using powder X-ray diffraction.
Table 1. Crystallographic Data for Compound 1
Polymorphs
form A form B form C
crystal system triclinic monoclinic triclinic
space group P1h P21/c P1
a () 10.614(2) 10.488(7) 9.5060(8)
b () 13.419(4) 4.811(1) 14.997(1)
c () 5.123(1) 28.263(1) 5.276(1)
R () 90.84(2) 90 98.00(1)
() 95.52(2) 91.23(2) 101.76(1)
() 88.98(3) 90 103.845(8)
cell volume (
3
) 726.1(3) 1425(1) 700.8(2)
Z 2 4 1
Fcalc (g/cm
3
) 1.41 1.436 1.461
temp (K) 293.2 293.2 293.2
radiation CuKR CuKR CuKR
wavelength 1.5418 1.5418 1.5418
R 0.1016 0.0638 0.083
R
w 0.1788 0.1063 0.1312
b ) E
hkl
b
- E
hkl,i
b
) (E
hkl
sl
+ (1/2)E
hkl
att
) - K
i
(E
hkl,i
sl
+
(1/2)E
hkl,i
att
)
Solution-Mediated Phase Transformation Crystal Growth & Design, Vol. 5, No. 4, 2005 1431
trifluoromethyl fluorine atoms (Figure 4a,b). In contrast,
molecules in form C are organized in which an inter-
molecular hydrogen bond is formed between the hy-
droxyl hydrogen and the carbonyl oxygen atom of a
neighboring molecule (Figure 4c). In form B, the fluorine
atoms of the trifluoromethyl group in compound 1 are
disordered and are assigned a site occupancy factor
(SOF) of 0.7/0.3 as determined from refinement.
In all three crystalline structures, there are two
hydrogen bond donors, the amide hydrogen (N-H) and
the hydroxyl hydrogen (O-H), within the molecule, and
there are two hydrogen bond acceptors: for forms A and
B, the cyano nitrogen (CtN) and the trifluoromethyl
fluorine (CF
3
), and for form C the cyano nitrogen (Ct
N) and the carbonyl oxygen (CdO). In addition, these
donors and acceptors are involved in intramolecular
hydrogen bonds with the exception of the trifluorometh-
yl group. The hydrogen bonding interaction between the
amide hydrogen and cyano nitrogen links the growth
unit of each polymorph. Clearly, hydrogen bonding is
an essential feature in the crystal structures of com-
pound 1. The similar hydrogen bonding motifs in forms
A and B suggest that disruptions in the hydrogen
bonding sequence through the incorporation of an
impurity might not only interfere with form A but also
affect form B. On this basis, it might not be possible to
stabilize the metastable A form by inhibiting the stable
B form with an impurity that hinders the hydrogen bond
formation; it would also likely disrupt the structure and
the crystallization process of the metastable phase and
possibly negate the suppression of the transformation
to the more stable polymorph.
Solubilities. The solubility curve of each form of
compound 1 in 2-propanol in the temperature range of
0 to 40 C is shown in Figure 5. The solubilities show
that form B and form C, and form A and form C are
enantiotropic with a transition temperature at 20.1 and
35.2 C, respectively. Below these crossover tempera-
tures, form C is the most stable (i.e., lowest solubility)
with respect to the other form, whereas above these
temperatures, the other polymorph is more stable. In
contrast, the A and B form is a monotropic pair where
form A is metastable relative to the B form. The
solution-mediated transformation studies of form A to
form B are carried out at 30 C to avoid the appearance
of form C being mixed with form B since it has the
lowest free energy (most stable phase) below 20 C
despite the fact that the transformation rate from form
B to form C is slow.
Solution-Mediated Transformation. The solution-
mediated phase transformation of compound 1 com-
prises three main steps: dissolution of the metastable
phase, form A, nucleation of the stable phase, form B,
and crystal growth of the stable form. The morphology
of each form is shown in Figure 6. Both forms exhibit a
platelike morphology, and thus it is difficult to identify
the polymorph by the shape of the crystal or monitor
the phase transformation by microscopy. Powder X-ray
diffraction is employed to assess the rate of transforma-
tion; polymorphic fractions are measured based on the
differences between the powder patterns of each form
(Figure 2). Characteristic peaks for both polymorphs are
used to construct the calibration curve to quantitatively
measure the composition change of the two polymorphs
in the slurry (Figure 3). At certain time intervals,
samples of the crystal slurry are removed and the
conversion of form A to form B is monitored. Figure 7
Figure 4. Crystal packing of compound 1 polymorphs: (a)
form A viewed along the c-axis; (b) form B viewed down the
b-axis; and (c) form C viewed parallel to the c-axis. Hydrogen
bonds are represented by the aqua dashed lines. The circled
areas indicate the growth units of each form.
Figure 5. Solubility of the three polymorphs of compound 1
in 2-propanol.
1432 Crystal Growth & Design, Vol. 5, No. 4, 2005 Mukuta et al.
shows the transformation behavior of compound 1 in
2-propanol. In the first three experiments, the stirring
speed is 300 rpm and initial transformation of form A
occurs after the first hour. However, when the degree
of agitation is reduced to 150 rpm, the time elapsed for
the initial appearance of form B increases. Increasing
agitation rate increases the crystallization kinetics (or
the amount of secondary nucleation) of the stable phase,
thus increasing the surface area of this phase and hence
the transformation rate.
The influence of structurally related impurities on the
transformation rate is shown in Figure 8. Impurities
RS1 and RS4 slightly retard the transformation rate,
while with the addition of RS2 and RS3 transformation
of the metastable A form to the stable form is hindered,
particularly RS2, where a trace amount of form B is
observed after 30 h. Overall, the transformation behav-
ior in the presence of RS1 and RS4 is very similar to
the rate of the pure solution. To understand the effect
of the doping level on the rate of transformation, two
different impurity loadings (0.1 and 0.5 w/w%) for the
two best inhibitors (RS2 and RS3) were added to the
solution, and the results are shown in Figure 9. The
stabilization of the metastable modification as reflected
in a decrease in the transformation rate is sensitive to
the doping level. High impurity concentrations (e.g., 0.5
w/w%) of RS2 and RS3 suppress the transformation to
a greater extent than at low concentration consistent
with the notion at low doping levels the nucleation rate
and growth rate coefficients for the stable polymorph
is similar to those in the absence of the impurities,
whereas at high loadings the nucleation rate and growth
rate coefficients decreases, resulting in an increase in
transformation time.
41
The influence of seeding with form B in combination
with the addition of the inhibitor RS2 is shown in Figure
10. At low doping level and seeding, the transformation
behavior resembles very closely to that in the absence
of the impurity as conversion to the stable modification
is completed after 2 h. For high impurity loading and
seeding with form B, small fractions of the stable B
form, as detected from the powder X-ray patterns, are
observed after 6 h resulting in a 5-fold decrease in the
initial appearance of the stable modification when
compared to same doping level without seeding. In
Figure 6. SEM images (500) of form A (left) and form B
(right) of compound 1.
Figure 7. Transformation behavior of form A to form B at
30 C in 2-propanol.
Figure 8. Influence of impurities on the transformation
behavior of compound 1 at 30 C.
Figure 9. Effect of impurity concentration on the transforma-
tion behavior of compound 1 at 30 C.
Figure 10. Effect of seeding on the transformation rate of
compound 1 at 30 C in 2-propanol.
Solution-Mediated Phase Transformation Crystal Growth & Design, Vol. 5, No. 4, 2005 1433
contrast, seeds that are ground together with a high
level of RS2 impurities reveal that the stabilizing effect
of the impurity is reduced as full conversion to the stable
form is observed after 4 h. The addition of seeds in the
process clearly decreases the transformation time as a
result of secondary nucleation. As the size of the seeds
decreases such as in the case of grinding, higher surface
areas of the seeds are expected, in turn, increasing the
mass transfer and the overall growth rate of the stable
polymorph.
59
Thus, the transformation rate is acceler-
ated and the crystallization to form B is enhanced as
the seeds act as a catalyst during the nucleation process.
The initial appearance of form A and its subsequent
disappearance and conversion to the stable B form
follow Ostwalds Law of Stages concerning the precipi-
tation of the metastable modification followed by its
transformation to the stable form. The driving force for
the solution-mediated transformation is the difference
in the free energy, specifically differences in the solubil-
ity between the stable and the metastable modifications.
Transformation to the less soluble B form occurs at the
expense of the more soluble (or metastable) A form and
the process progresses faster as the solubility difference
between the two forms becomes greater. Full conversion
is obtained when the solution reaches saturation with
respect to the stable polymorph and the metastable
modification is completely dissolved.
The significant retardation effect of the RS2 impurity
is possibly due to its ability to inhibit the nucleation of
form B, in turn, kinetically stabilizing the metastable
phase, as reflected in the increase of the transformation
time. The addition of the impurity most likely reduces
the nucleation rate of form B, perhaps by disrupting and
inhibiting the emerging nucleus. The crystal growth rate
of form B might also be influenced by the impurity but
not to a great extent considering that when small seed
crystals were used in the presence of RS2, the trans-
formation to form B proceeds much more rapidly
compared with the addition of large form B seed crystals
(Figure 10), suggesting that crystal growth of the stable
form in solution ensues despite the impurity. The
prolonged induction period of the stable phase might
also be explained by examining the impact the impuri-
ties have on the solubility of compound 1. Impurities
can influence or alter the solubility of a solute, in turn,
affecting the crystallization process. The higher the
doping level or impurity loading, the more pronounced
the effect becomes. Table 2 shows the effect of the
structurally related impurities on the concentration of
the stable B form of compound 1 in the absence and
presence of the impurities. Although the impurities are
molecularly similar to compound 1, it is believed that
the superior inhibitory impact of RS2 might also be a
result of the increase in the solubility of form B. This
sudden enhancement leads to a smaller driving force
for the transformation process, thus lowering the rate
of transformation and stabilizing the metastable poly-
morph. Differences in the molecular structure of the
impurities lead to a different outcome of the transfor-
mation behavior as each impurity molecule has unique
modes of action and affects the crystallization process
differently. The suppression of the stable B form with
RS2 as determined from powder X-ray diffraction
quantification also reveals that the doping level is
another factor that affects the conversion process and
that a sufficient level of impurities is needed to hinder
the formation of the stable modification.
Impurity Incorporation. Structurally related im-
purities may enter the host crystal lattice and replace
the host molecule at lattice sites as a result of its
molecular compatibility. Relative incorporation energies
are calculated and used to evaluate the extent of the
impurity incorporation. The molecular modeling ap-
proach is a modification of Hartman and Perdoks
classical theory for predicting crystal morphology.
58
It
requires the substitution of the host molecule with the
impurity molecule and the calculation of attachment
energies for both the pure and the impurity-modified
crystal surfaces. Table 3 shows the relative incorpora-
tion energies for compound 1 doped with the four
structurally related impurities in each crystallographic
position. The position of each impurity within the host
lattice is optimized through a sequence of molecular
mechanic and molecular dynamics simulations. The
likelihood of an impurity incorporating into the crystal
structure of form B of compound 1 can be indicated by
low values of the relative incorporation energies. Crystal
surfaces that have a minimum change in the energy are
where the impurity will most likely to incorporate. Thus
relative incorporation energies can be used to measure
how easily an impurity can replace the host molecule
on a given crystal plane. It can be seen that the impurity
RS1 has the lowest relative incorporation energies
Table 2. Influence of Structurally Related Impurities on
the Concentration (g/L) of Form B after the
Solution-Mediated Phase Transformation at 30 C in
2-Propanol
RS1 RS2 RS3 RS4
0 w/w% (pure) 3.86
0.1 w/w% 3.77 3.98 3.80 3.73
0.5 w/w% 3.82 3.93 3.87 3.80
1.0 w/w% 3.71 4.00 3.77 3.81
Table 3. Relative Incorporation Energies (kcal/mol) for
Various Crystallographic Planes of Compound 1 in the
Presence of Four Structurally Related Impurities
a
crystal faces {hkl} Z RS1 RS2 RS3 RS4
{100} 1 -2.883 -7.405 -4.345 -14.475
2 -2.475 -7.902 -4.050 -14.453
3 -2.419 -7.608 -3.985 -15.987
4 -2.425 -7.891 -2.760 -15.992
{102h} 1 -3.530 -6.868 -4.621 -15.288
2 -3.314 -7.720 -4.458 -15.265
3 -3.304 -7.515 -4.214 -16.797
4 -3.307 -7.711 -2.848 -16.802
{011} 1 -2.874 -5.564 -4.154 -14.068
2 -2.479 -6.898 -4.540 -14.111
3 -2.323 -6.128 -3.941 -14.882
4 -2.567 -6.877 -3.823 -14.999
{110) 1 -3.005 -5.898 -4.012 -12.685
2 -2.401 -5.986 -3.128 -12.148
3 -2.373 -5.791 -4.021 -11.986
4 -2.046 -5.974 -2.526 -12.633
{102} 1 -2.818 -5.270 -4.141 -12.34
2 -2.367 -5.824 -3.679 -12.317
3 -2.340 -5.528 -4.521 -13.878
4 -2.346 -5.812 -2.453 -13.884
{211h} 1 -2.137 -5.664 -3.948 -11.824
2 -1.874 -8.374 -4.081 -11.12
3 -1.864 -5.736 -3.454 -11.548
4 -1.862 -8.367 -2.802 -11.542
a
Z is the symmetry position in the unit cell: (1) Z ) (x, y, z);
(2) Z ) (-x, y + 1/2, -z + 1/2); (3) Z ) (-x, -y, -z); (4) Z ) (x, -y
+ 1/2, z + 1/2).
1434 Crystal Growth & Design, Vol. 5, No. 4, 2005 Mukuta et al.
signifying that it can easily enter the crystal lattice and
substitute for the host molecules in various crystal faces
of form B of compound 1, in particular, the {211h} surface
where there is a minimal change in the energy for each
symmetry position. In contrast, large energy losses for
the RS4 impurity are observed suggesting that segrega-
tion into the compound 1 crystal is least favorable, while
for the impurity RS3, low incorporation energies are
observed in symmetry position 4 for various crystal-
lographic lattice planes. The impurity molecule, RS2,
yields moderate energetic values suggesting that the
uptake may not be extensive as compared with RS1 and
RS3 impurities.
The extent of impurity incorporation in compound 1
crystals for two different concentrations of impurities
as determined by HPLC is shown in Table 4. In
excellent agreement with the relative incorporation
energies, RS1 appears to easily replace the host mol-
ecule in the crystal lattice of the stable phase, form B,
and incorporates more extensively when compared with
the other structurally similar impurities. The order of
the impurities uptake is RS1 > RS3 > RS2 > RS4,
which mirrors the order of the relative incorporation
energies. Although studies have shown that calculations
from the modified attachment energies have been suc-
cessful in the prediction of impurity-modified crystal
habit
47-52
as impurities can affect the individual growth
rate of crystal faces, the effects of impurities on the
morphology of compound 1 are not examined as the
intent of the simulations is to assess the impact of impu-
rities on the purity of the crystals and possibly under-
stand how the incorporation of impurities can be ex-
ploited for the stabilization of a metastable polymorph.
Crystallographic lattice planes are unable to discrimi-
nate between the host and the impurity molecules as a
result of its molecular similarity and compatibility.
Thus, the impurities easily incorporate onto the crystal
surface. Once incorporated, the impurity can disrupt the
lattice and the normal hydrogen bonding sequence. In
the case of the RS2 and RS3 impurities, the hydrogen
bonding interactions are maintained intact as the
substitution of either impurity in the crystal lattice is
still involved in the hydrogen bond network through its
cyano nitrogen, amide hydrogen, hydroxyl hydrogen,
and trifluoromethyl fluorine atoms. In contrast, the
hydrogen bond sequence is broken upon substitution of
the impurities RS1 and RS4, specifically the hydrogen
bond donor-acceptor pairing, the hydroxyl hydrogen
atom, and the trifluoromethyl fluorine atom. It would
be expected that since the growth unit for form B is
disrupted, the crystallization of the stable form will be
inhibited; however, this is not observed as the trans-
formation rate for the metastable A form to the more
stable phase is only slightly hindered. With the impurity
RS2, the level of incorporation may have not been
extensive as for RS1 and RS3, but the inhibitory effect
is considerably greater when compared with the other
structurally related impurities. This can be attributed
to its ability to hinder the nucleation and crystal growth
of form B and possibly its influence on the solubility of
the stable modification, form B. The observed increase
of the concentration of form B in the presence of the
RS2 impurity suggests that the impurity can increase
the solubility of form B, which would lead to a reduction
in the driving force for the phase transformation,
namely, the free energy difference between the two
crystalline forms.
Relative incorporation energies derived from the
modified attachment energy calculations enable us to
assess the compatibility of an impurity to incorporate
into the crystal structure. In addition to accurately
simulating crystal habits in the presence of additives
or impurities as reported in other studies,
47-52
the
energetic calculations can be employed to design or
screen additive/impurity molecules to hinder the trans-
formation of a metastable modification to a stable phase
by examining the differences in the level of incorpora-
tion between crystalline forms. An example where this
might be advantageous is in the case of 4-methyl-2-
nitroacetanilide where it has been observed that as the
incorporation efficiency of isomorphic additives in-
creases in the high-energy form, the rate of transforma-
tion decreases, suggesting that a necessary amount of
impurities incorporated is required to stabilize the
metastable phase.
43
Conclusions
The solution-mediated phase transformation of an
active pharmaceutical ingredient is influenced by the
presence of tailor-made impurities as certain structur-
ally related inhibitors, namely RS2, suppressed the
transformation of the metastable A form to the stable
B form, while other impurities have a slight or moderate
effect on the transformation rate. Powder X-ray diffrac-
tion is employed for quantitative measurements of
polymorph composition in the slurry. The driving force
for the transformation is differences in the solubility
between the two forms. The kinetic stabilization of the
metastable phase in the presence of the RS2 impurity
is a result of its ability to disrupt the nucleation and
crystal growth of the stable polymorph and its enhance-
ment in the solubility of form B, which in turn leads to
a reduction in the driving force. The inhibitors are
molecularly similar to compound 1; consequently, the
impurities may be incorporated into the host crystal
lattice. The compatibility of an impurity substituting
for the host molecule in the lattice can be measured by
calculation of the relative incorporation energies. It is
demonstrated that the modified attachment energy
calculations can be an effective indicator on how likely
the impurity molecule will incorporate as the order of
the impurities uptake, as determined by HPLC, mirrors
the relative incorporation energies. Although the level
of incorporation did not correspond to the inhibitory
effect of the impurities, incorporation energies may be
used to stabilize the metastable phase in cases in which
Table 4. Summary of Impurity Incorporation as
Determined by HPLC in Compound 1 for Different
Concentrations of Impurities
0.1 w/w% 0.5 w/w%
crystal filtrate crystal filtrate
RS1 3.89 mg
(95.8%)
0.17 mg
(4.2%)
15.47 mg
(93.7%)
1.04 mg
(6.3%)
RS2 1.14 mg
(40.0%)
1.71 mg
(60.0%)
6.97 mg
(48.9 mg)
7.29 mg
(51.1%)
RS3 3.27 mg
(72.3%)
1.25 mg
(27.7%)
12.41 mg
(68.1%)
5.81 mg
(31.9%)
RS4 0.404 mg
(9.8%)
3.73 mg
(90.2%)
0.369 mg
(2.1%)
17.2 mg
(97.9%)
Solution-Mediated Phase Transformation Crystal Growth & Design, Vol. 5, No. 4, 2005 1435
the transformation rate is retarded by an increase in
the incorporation level of an additive or impurity. This
might be extremely beneficial in cases where the
metastable modification is desired due to its higher
solubility and improved bioavailability. The energetic
calculations can enable molecules to be rationally
designed to inhibit the crystallization of the stable form
and hence stabilize the metastable modification. Also
the modeling approach might reduce the development
time and the labor-intensive batch experiments that are
performed to determine which additives could be effec-
tive inhibitors. Furthermore, relative incorporation
energies can be employed to screen the impact of
impurities on the purity of the crystals and be utilized
to simulate its effects on the crystal morphology.
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