Sodium Salts and Solvate of Rebamipide: Synthesis, Structure, and Pharmacokinetic Study

Article abstract of DOI:10.1021/acs.cgd.5b01839

Two sodium salts (Na(CH₃CH₂OH) (HReb) (1) and Na₂(H₂O)₄(Reb) (2)), one methanol solvate (H₂Reb·CH₃OH (3)), and one methyl ester (4) of the minimally soluble drug, rebamipide (H₂Reb), used for the treatment of gastric ulcers and gastritis have been synthesized. For the first time the structure of rebamipide has been determined by single crystal X-ray diffraction. Although salts 1 and 2 were prepared under the similar conditions, their structures are different. In 1, rebamipide loses the proton of the carboxyl group to interact with the sodium ion, but in 2 the drug molecule converts to its prototropic tautomer and then simultaneously loses the protons of the carboxyl and hydroxyl groups to form salt. Control experiments show that reaction temperature is the key factor influencing the formation of salts. Although all methanol was used in the synthesis of 3 and 4, in 3 methanol acts as solvent involved in the lattice, while in 4 it reacts with rebamipide to form ester. By analyzing the mass spectra of the reaction solution, we speculate that the crystallization of 3 and 4 is controlled by the products solubility. Dissolution studies indicate that both the maximum solubility and dissolution rate of 1-4 in simulated succus gastricus are improved. Furthermore, pharmacokinetic behavior of compounds 1-4 was investigated in rats and the results indicate that the bioavailability of 1, 3, and 4 upon oral administration is enhanced compared to that of API.

Full text of DOI:10.1021/acs.cgd.5b01839

Article
pubs.acs.org/crystal
Sodium Salts and Solvate of Rebamipide: Synthesis, Structure, and
Pharmacokinetic Study
†
,∥
†,∥
‡
†
†
‡
Yingnan Chi, Chuanrong Liu, Tianming Ren, Xiaoying Wang, Qiuhong Yang, Zhichao Yang,
‡
†
,‡
,†
Yan Yang, Song Yang, Jingkai Gu,* and Changwen Hu*
†Key Laboratory of Cluster Science, Ministry of Education of China, Beijing Key Laboratory of Photoelectronic/Electrophotonic,
School of Chemistry, Beijing Institute of Technology, Beijing 100081, P. R. China
‡
Research Center for Drug Metabolism, Jilin University, Changchun 130012, P. R. China
*
S Supporting Information
ABSTRACT: Two sodium salts (Na(CH CH OH) (HReb)
3
2
(
(
1) and Na (H O) (Reb) (2)), one methanol solvate
2 2 4
H Reb·CH OH (3)), and one methyl ester (4) of the
2
3
minimally soluble drug, rebamipide (H Reb), used for the
2
treatment of gastric ulcers and gastritis have been synthesized.
For the first time the structure of rebamipide has been
determined by single crystal X-ray diffraction. Although salts
1
and 2 were prepared under the similar conditions, their
structures are different. In 1, rebamipide loses the proton of the
carboxyl group to interact with the sodium ion, but in 2 the
drug molecule converts to its prototropic tautomer and then
simultaneously loses the protons of the carboxyl and hydroxyl groups to form salt. Control experiments show that reaction
temperature is the key factor influencing the formation of salts. Although all methanol was used in the synthesis of 3 and 4, in 3
methanol acts as solvent involved in the lattice, while in 4 it reacts with rebamipide to form ester. By analyzing the mass spectra of
the reaction solution, we speculate that the crystallization of 3 and 4 is controlled by the product’s solubility. Dissolution studies
indicate that both the maximum solubility and dissolution rate of 1−4 in simulated succus gastricus are improved. Furthermore,
pharmacokinetic behavior of compounds 1−4 was investigated in rats and the results indicate that the bioavailability of 1, 3, and 4
upon oral administration is enhanced compared to that of API.
INTRODUCTION
common organic solvents and its solubility in water is approxi7-
mately 0.0001 and 0.013% (w/v) at pH 3 and 7, respectively.
As a result, the absorption of this drug is poor with the absolute
bioavailability of about 10%.
Recently, solid state forms, referring to polymorphs, solvates
■
Rebamipide, an amino acid derivative of 2-(1H)-quinolinone,
is a therapeutic agent for gastric ulcers and chronic gastritis.
It was first synthesized by Otsuka Pharmaceutical Company, Ltd.
8
1
in 1985 and subsequently marketed with the trade name of
(
or hydrates), salts, cocrystals, or amorphous forms, play an
Mucosta in 1990. The molecular structure of rebamipide is
shown in Scheme 1. This drug can stimulate the generation of
9
−12
important role in the pharmaceutical field.
A lot of studies
demonstrate that many physical and chemical properties of an
1
3,14
active pharmaceutical ingredient (API), such as stability,
Scheme 1. Molecular Structure of Rebamipide
15−22
23−25
solubility,
and bioavailability,
are influenced by the
selection and optimization of solid state forms. The reported
2
6
solid state forms of rebamipide include one monohydrate,
27
28
two cocrystals, and several salts. Forming salt is the classic
method for improving solubility and dissolution rate of acidic/
25,29−31
basic APIs.
Although choline, arginine, lysine, trometh-
amine, sodium, calcium, zinc, and magnesium salts of rebamipide
were synthesized in patent, no structural characterization was
28
provided. With the existence of two acidic functional groups in
rebamipide, a carboxylic acid and a N-heterocycle, it is not easy to
predict the product when reacting with alkaline agents. A careful
endogenous prostaglandins in the gastric mucosa, thereby
facilitating and accelerating ulcer healing. In addition, recent
2
clinical studies display that rebamipide can effectively treat
3
−5
stomatitis, colitis, and dry eye syndrome.
According to the
Biopharmaceutics Classification System (BCS), rebamipide
Received: December 30, 2015
Revised: April 14, 2016
belongs to the Class IV drugs, which have low solubility and
6
low permeability. Rebamipide is nearly insoluble in most
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.cgd.5b01839
Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design
Article
a
Table 1. Crystallographic Data for Compounds 1−4
1
2
3
4
formula
formula weight
crystal system
space group
a (Å)
C H ClN NaO
C H ClN Na O
C H ClN O
C H ClN O
20 17 2 4
2
1
20
2
5
19 21
2
2
8
20 19
2
5
438.83
monoclinic
P21/c
486.81
402.82
384.81
orthorhombic
Pna2(1)
34.551(3)
5.0944(4)
12.0785(11)
90
monoclinic
C2/c
monoclinic
P21c
6.014(2)
23.279(9)
15.076(6)
90
23.030(2)
19.7573(15)
5.0379(3)
19.5600(12)
90
b (Å)
19.1331(18)
9.6060(9)
90
c (Å)
α (deg)
β (deg)
γ (deg)
Z
99.969(7)
90
90
106.358(2)
90
109.887(7)
90
90
4
4
8
4
3
volume (Å )
2078.9(14)
1.402
2126.0(3)
1.521
4061.4(7)
1.318
1830.8(2)
1.396
Dcalc (g·cm−3)
F (000)
912
1008
1680
800
refns. collected
unique reflns.
Rint
10139
3637
9761
10191
3572
8493
2869
3382
0.0447
0.0634
0.2021
0.1092
0.2620
0.0708
0.0470
0.1020
0.0581
0.1056
1.060
0.0599
0.0612
0.1437
0.1261
0.1618
1.044
0.0258
0.0651
0.1702
0.0898
0.1877
1.065
R1[I > 2σ(I)]
wR [I > 2σ(I)]
2
R1(all data)
wR (all data)
2
GOF on F2
1.052
a
2
o
2
c
2
2 2 1/2
o
R = ∑| |F | − |F | |/∑|F |; wR = ∑[w(F − F ) ]/∑[w(F ) ]
1
o
c
o
2
literature and Cambridge Structural Database (CSD) search
indicates that although rebamipide has been widely investigated
since 1985, so far no crystal structure of rebamipide or its salt,
hydrate, solvate, or cocrystal is reported. However, unambiguous
structural characterization is necessary in the development of
a drug, which is helpful to build the structure−property relation-
ship and guide the controllable synthesis.
and transferred to the electrospray source via an autosampler with a flow
rate of 0.2 mL/min.
Synthesis of Na(CH CH OH) (HReb) (1). Rebamipide (100 mg,
.27 mmol) was dissolved in 15 mL ethanol and then stirred for 2 h.
3
2
0
To this was added 0.075 mL NaOH (5 M) with vigorously stirring.
The resulting solution was filtered and allowed for slow evaporation
under ambient conditions. Colorless block single crystals of 1 suitable
for single X-ray diffraction were obtained and dried under ambient
conditions (yield of 95 mg, ∼80% based on rebamipide).
Herein, for the first time we determine the crystal structures
of two rebamipide sodium salts, namely Na(CH CH OH)
Synthesis of Na (H O) (Reb) (2). Compound 2 was prepared using
3
2
2
2
4
(
HReb) (1) and Na (H O) (Reb) (2), and one methanol
a procedure similar to that for 1. The only difference is that the mixture
of rebamipide and sodium hydroxide solution was heated at 50 °C for
2
2
4
solvate, H Reb·CH OH (3) (H Reb = rebamipide). Moreover,
2
3
2
3
0 min, then filtered and allowed for crystallization. After 1 day, colorless
during the screening of solid state form, the single crystal of
rebamipide methyl ester (4) has been fortunately obtained. In
addition, compounds 1−4 have been characterized by powder
X-ray diffraction, Fourier transform-infrared spectroscopy
needle-like single crystals of 2 were isolated and dried at ambient
conditions (yield of 98 mg, ∼75% based on rebamipide).
Synthesis of H Reb·CH OH (3). Rebamipide (45 mg, 0.12 mmol)
2
3
was dissolved in 15 mL methanol and stirred for 2 h. The solution was
then transferred to a Teflon-lined autoclave (15 mL) and kept at 120 °C
for 48 h. After the mixture was slowly cooled to room temperature,
the resulting solution was filtered and slowly evaporated at ambient
conditions. After 2 days, colorless needle-shaped crystals of 3 were
harvested (yield of 40 mg, ∼82% based on rebamipide) and one single
crystal was chosen for X-ray analysis.
(
FT-IR), thermogravimetry, and differential scanning calorimetry.
It is notable that both the maximum solubility and bioavailability
of 1, 3, and 4 are enhanced compared to those of API.
EXPERIMENTAL SECTION
■
Materials and General Methods. Rebamipide (purity >98%) was
provided by Research Center for Drug Metabolism of Jilin University
and used without further purification. The other chemicals were reagent
grade, purchased from commercial sources, and used without further
purification. IR spectra were obtained (as KBr-pressed pellets) using a
Synthesis of Rebamipide Methyl Ester (4). Compound 4 was
synthesized using a procedure similar to that of 3. The only difference is
that the API was dissolved in the methanol-H O (v:v = 3:1) solution.
2
The obtained solution was heated at 120 °C in a Teflon-lined autoclave
(15 mL) for 48 h, and then the resulting solution was allowed to
evaporate at ambient conditions. Colorless block crystals of 4 were
−1
Nicolet 170SXFT/IR spectrometer in the range of 500−4000 cm .
Powder X-ray diffraction (PXRD) data were collected on a Bruker
instrument equipped with graphite-monochromatized Cu Kα radia-
tion (λ = 0.154060 nm, scan speed = 8°/min, 2θ = 5−35°, stepsize =
isolated after about 7 days (yield 35 mg, ∼75% based on rebamipide).
1
H NMR (DMSO-d , 400 MHz) δ (ppm) 11.66 (1H, s, imino group),
6
9.03 (1H, d, imino group), 7.21−7.85 (8H, m, phenyl), 6.44 (1H, s,
alkenyl), 4.35 (1H, t, methine), 3.69 (3H, s, methyl), 2.06 (2H, s,
methylene). (Supporting Information, Figure S1)
Single Crystal X-ray Diffraction. The single crystal data for
compounds 1−4 were collected on a Bruker-AXS CCD diffractometer
equipped with a Mo Kα radiation source (λ = 0.07103 Å) at 298 K. The
absorption corrections were performed with the SADABS program.
The structures were determined by direct methods and expanded
using Fourier techniques. The non-hydrogen atoms were refined
0
.019762). TG analyses were carried out in nitrogen atmosphere
between 30 and 350 °C at a heating rate of 10 °C/min on a SDT Q600
simultaneous thermal analyzer. Differential scanning calorimetry was
performed on a Q100 DSC module with nitrogen flow rate of
−1 1
5
0 mL·min . H NMR spectra were recorded using a Bruker-400 NMR
spectrometer. Mass spectra were recorded in the positive mode on
an Agilent 6520 Q-TOF LC/MS mass spectrometer. Sample solutions
were made to approximately 10 M in ultrapurified water or methanol
−6
B
DOI: 10.1021/acs.cgd.5b01839
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Article
Table 2. Hydrogen Bonding Interactions in Compounds 1−4
D−H···A
D−H/Å
H···A/Å
D···A/Å
D−H···A/°
symmetry code
compound 1
N1−H1···O1
N2−H2A···O1
O5−H5A···O4
compound 2
0.860
0.860
0.849
2.004
2.338
1.818
2.858
3.102
2.685
172.05
148.23
169.92
−x+2, −y+1, -z+1
x−1, y, z
x−1, y, z
N2−H2A···O2
O5−H5A···O1
O5−H5B ···N1
O6−H6A···N1
O6−H6B···O4
O7−H7A···O8
O7−H7B···N1
O8−H8A···O4
O8−H8B···O2
compound 3
0.860
0.970
0.970
0.850
0.850
0.970
0.970
0.852
0.851
2.333
1.886
2.492
2.078
2.036
1.964
2.262
1.922
1.961
3.175
2.720
3.203
2.920
2.839
2.726
2.871
2.772
2.785
166.11
142.54
130.03
170.47
157.29
133.78
119.81
174.68
162.71
x, y−1, z
−x+1, −y, z−1/2
−x+1, −y+1, z−1/2
x, y−1, z
−x+1, −y, z−1/2
intramolecular
−x+1, −y, z−1/2
−x+1, −y, z−1/2
−x+1, −y+1, z−1/2
N1−H1···O3
N2−H2A···O2
O4−H4B···O5
O5−H5A···O1
O5−H5B···O1
compound 4
0.860
0.860
0.850
0.820
0.850
2.026
2.122
1.782
1.979
2.018
2.883
2.869
2.609
2.701
2.701
174.03
144.90
163.88
146.53
136.78
−x+3/2,y+1/2,−z+1/2
x, −y+1, z−1/2
intramolecular
−x+3/2,y−1/2, −z+1/2
−x+3/2,y−1/2, −z+1/2
N1−H1···O1
N2−H2A···O2
0.860
0.860
1.972
2.007
2.826
2.923
172.10
167.62
x, y−1, z
−x,y+1/2,−z−1/2
anisotropically. The H atoms on C atoms were fixed at the calculated
positions and those on the water molecules were located from the
different Fourier maps. The H atoms were isotropically refined. All
from the eyeball veins at intervals of 0.17, 0.33, 0.5, 0.75, 1, 2, 3, 5,
7, 9, 11, and 24 h from each rat, immediately centrifuged for 5 min
at 13300 rpm at ambient temperature, and stored at −20 °C until
LC−MS/MS analysis. Analysis was performed on a LC−MS/MS system
comprising Agilent 1100 Series HPLC system (Agilent Technologies,
Palo Alto, CA, USA) and a Q-trap 2000 mass spectrometer (Applied
BiosystemsSciex, Ontario, Canada). Chromatographic separation was
achieved using an Agilent ZORBAX 300 SB-C18 column (150 mm ×
3
2
calculations were performed using the SHELXTL-97 program.
CCDC-1439505 (1) 1404896 (2) 1404894 (3) and 1439500 (4) contain
the supplementary crystallographic data for this paper. The crystallo-
graphic details of the compounds are summarized in Table 1. The
hydrogen bonding distances and angles are present in Table 2 and the
selected bond length and angles are given in Table S2.
4
.6 mm I.D., 5 μm particle size) at 40 °C with a mobile phase of formic
acid/water (v/v = 70:30) at 1 mL/min. Analysis was carried out with an
electrospray ionization (ESI) source using positive ion mode. Data were
acquired using Drug and Statistics Software DAS 3.0 (Mathematical
Pharmacology Professional Committee of China, Shanghai, China). The
hydrolysis experiment of compound 4 in vivo has been performed using
the similar method.
Dissolution and Solubility. The absorbance of a known
concentration of rebamipide was measured at the given λmax (228 nm)
using a TU-1901 UV−vis spectrometer. The absorbance values were
plotted against several known concentrations to prepare the concentra-
2
tion vs intensity calibration curve (R = 0.9996). From the slopes of the
calibration curves, the molar extinction coefficients of compounds 1−4
were calculated. The rebamipide derivatives were milled to powders
and sieved using standard mesh sieves to provide samples with the
approximate particle size distribution of 75−150 μm. In order to
determine the equilibrium solubility, an excess amount of compound
was added to 10 mL of simulated gastric fluid media (HCl solution with
pH = 1.2). The supersaturated solution was stirred (50 rpm) at room
temperature. The suspension was filtered through a 0.22 μm nylon filter
after 24 h. The filtered aliquots were diluted and the absorbance was
RESULTS AND DISCUSSION
■
Crystal Structure of Na(CH CH OH) (HReb) (1). When
3
2
reacting with NaOH solution, rebamipide loses the proton of
the carboxylic acid group to form salt 1. As shown in Figure 1,
+
the asymmetric unit of salt 1 consists of one Na , one ethanol
measured at the given λmax
.
In the dissolution experiment, 50 mL of HCl solution (pH = 1.2) was
added to a 100 mL flask containing excess powder samples, and the
resulting mixture was stirred at 25 °C and 500 rpm. The solution was
withdrawn from the flask at regular intervals and filtered through a
0.22 μm nylon filter. The concentration of the aliquots was determined
with appropriate dilutions measured with UV−vis spectrophotometry.
Each experiment was conducted three times and the mean value in each
case was calculated.
Pharmacokinetic Study (PK). The pharmacokinetics of com-
pounds 1−4 and original API were conducted using Wistar rats. After
an overnight fast, 15 healthy rats (7 months, weighing 220−260 g) were
randomly divided into five groups before dosing. Pure powder samples
of rebamipide API and compounds 1−4 as oral formulations were
administered to rats in different groups by gastric perfusion using a
dry powder insufflator at a single dose of 12 mg/kg for each group.
Subsequently, 0.5 mL aliquots of whole blood samples were collected
Figure 1. An asymmetric unit of salt 1.
C
DOI: 10.1021/acs.cgd.5b01839
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Article
Figure 2. (a) 1D chain in salt 1. (b) 2D supermolecular network formed by hydrogen bonding interactions of N1−H···O3. H atoms are omitted for the
sake of clarity and green dashed lines represent the hydrogen bonding interactions.
−
+
2−
molecule, and one [HReb] . In the rebamipide anion, the
two Na , four coordinated water molecules, and one [Reb]
dihedral angle between quinolinone and chlorobenzene ring is
in the asymmetric unit of 2. Compared with 1, two obvious
differences are found in this case. First, prototropic tautomerism
is observed in the quinolinone part and as a result the amide
form transforms to an imidic acid form (Scheme 2). Second,
+
78.87°. The Na ion exhibits a distorted tetrahedral geometry
and coordinates with two oxygen atoms of carboxyl groups
O3, O4) from different drug molecules, one oxygen atom of
acylamino group (O2), and one oxygen atom of ethanol (O5)
(
+
(
Figure 2a). Two Na cations link two adjacent rebamipide ions
Scheme 2. Equilibrium between Two Tautomers
to form an 8-membered ring. These 8-membered rings are
connected by the Na1−O2 coordination bonds into a 1D chain
along a-axis (Figure 2a). In this case, Na−O distances are in the
range of 2.290−2.343 Å. In addition, N1−H···O1 hydrogen
bond interactions extend the 1D chain into a 2D layer on the
ac-plane (Figure 2b).
Crystal Structure of Na (H O) (Reb) (2). When rebami-
2
2
4
pide reacts with NaOH aqueous under heating conditions,
another sodium salt 2 is isolated. As shown in Figure 3, there are
rebamipide loses two protons: one from a carboxyl group and the
other from a hydroxyl group, to coordinate with sodium cations.
There are two crystallographically independent Na ions
in 2. Na1 coordinates with one carboxyl oxygen (O3), one
hydroxyl oxygen (O1), and two water molecules (O5, O6) with
Figure 3. An asymmetric unit of salt 2.
D
DOI: 10.1021/acs.cgd.5b01839
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Crystal Growth & Design
Article
Figure 4. (a) 1D chain in salt 2. (b) 2D sheet formed by Na···O weak interactions on bc plane. (c) The view of the 2D sheet along b-axis. H atoms are
omitted for the sake of clarity and green-blue dashed lines represent the weak Na···O interactions.
seesaw-shaped geometry. Na2 adopts a distorted square geometry
bonding with one hydroxyl oxygen (O1) and three water
molecules (O5, O7, O8). The O1 and O5 acting as bridge
link adjacent Na1 and Na2 ions into a 1D chain along c-axis
Figure 6a, a 1D supermolecular chain is generated along b-axis
with the help of N1−H···O3, O4−H···O5, and O5−H···O1
hydrogen bonds. The neighboring 1D chains are further
connected via N2−H···O2 hydrogen bonds into a 2D sheet on
the bc-plane (Figure 6).
(Figure 4a). The weak interaction of Na1···O6 with the distance
of 2.710 Å further assembles 1D chains into a 2D sheet on the
ac-plane (Figure 4b,c). Besides the Na···O weak interactions, a
series of hydrogen bond interactions contribute to the formation
of 2D sheet (Figure S2 and Table 2).
Rebamipide Methyl Ester (4). When rebamipide dissolves
in methanol-H O (Vmethanl:Vwater = 3:1), the crystals of re-
2
bamipide methyl ester 4 were obtained unexpectedly (Figure 7a).
Rebamipide methyl ester as a prodrug has been synthesized
3
3
Crystal Structure of H Reb·CH OH (3). When rebamipide
through the reaction of rebamipide and methyl iodide.
2
3
dissolve in methanol under solvothermal conditions, the crystals
of solvate 3 were obtained. Asymmetric unit of 3 contains one
rebamipide and one methanol molecule (Figure 5). The dihedral
angle between quinolinone and chlorobenzene rings is 62.79°,
which is less than the angles in salts 1 and 2. As shown in
However, in our case 4 is prepared by esterification of rebamipide
with methanol. The dihedral angle between quinolinone and
chlorobenzene rings in 4 (8.65°) is much smaller than those in
1−3. As shown in Figure 7b, the N2−H···O2 hydrogen bonding
interactions assemble the organic molecules into a 1D chain and
E
DOI: 10.1021/acs.cgd.5b01839
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Article
the adding order of starting material, the amount of NaOH,
and reaction temperature were investigated (see Table S1). The
results display that the formation of salt 1 or 2 depends on the
temperature. As shown in Scheme 2, two tautomers, amide form
(
in 1) and imidic acid form (in 2), exist in a chemical equilibrium.
Generally, the extra ratio of the isomers is influenced by
34−37
temperature, solvent, and pH value.
The previous study
shows that high temperature is beneficial for the formation of
38
imidic acid form. Therefore, the sodium salt 2 based on imidic
acid form was isolated under heating conditions, while under
room temperature the sodium salt 1 based on amide from was
generated. Although the prototropic tautomerism is common in
heterocycles, to the best of our knowledge, the imidic acid form
of rebamipide has not been reported. Moreover, during our
experiment even if the API solution was heated in temperature
range of 120−160 °C, we still did not observe the formation of
imidic acid form as neutral molecules. Therefore, we speculate
that the salt forming process is helpful to stabilize the imidic acid
form.
Figure 5. An asymmetric unit of solvate 3.
two adjacent chains are further linked by N1−H···O1 hydrogen
bondings to form a double supermolecular chain along b-axis.
The formation of rebamipide methyl ester is further proved by
1
Compounds 3 and 4 are all crystallized from the rebamipide
solution prepared under solvothermal conditions (120 °C for 48 h).
The only difference is that the solvent used in 3 is methanol, but
H NMR (Figure S1). Through the comparison of rebamipide
API and its methyl ester, it is clear that in 4 the active hydrogen
of carboxyl group with δ = 13.0 disappears and an additional
that in 4 is methanol-H O with volume ratio of 3:1. In 3, methanol
single peak of −CH with δ = 3.69 is observed. Moreover, the
2
3
as a solvent is involved in the lattice, but in 4, methanol acts as a
reactant to form ester with API molecule. In order to explore the
key factor controlling the formation of 3 and 4, the reaction
solutions of 3 and 4 are characterized by ESI-MS. The analyses of
the positive mode mass spectra display that both API with m/z =
371.09 and methyl ester of rebamipide with m/z = 385. 09 are
detected in solution and in the two cases the main products are all
rebamipide peaks are slightly shifted to higher field relative to
the peaks of API, because of the electron-donating ability of the
methyl group.
The Factors Influencing the Synthesis of 1−4. Although
sodium salts 1 and 2 were all prepared by the reaction of re-
bamipide ethanol solution and NaOH aqueous, they have totally
different structures. In order to explore the controlling factors,
Figure 6. (a) 1D chain formed by hydrogen bonding in 3. (b) 2D supermolecular network on bc plane. H atoms are omitted for the sake of clarity and
green dashed lines represent the hydrogen bonding interactions.
F
DOI: 10.1021/acs.cgd.5b01839
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the equilibrium goes backward and more solvate 3 is obtained.
However, when deionic water was added, the solubility of
ester dramatically decreases, so the ester precipitates instead
(Scheme 3). With the precipitation of 4, the equilibrium moves
forward, followed by the crystallization of 4. Therefore, the
harvesting of 3 or 4 is controlled by the dynamics and the
solubility of API or ester is adjusted by water.
The Powder X-ray Diffraction and Thermal Analyses.
PXRD is proved to be a reliable technique for assessment of solid
forms in pharmaceutical study. As shown in Figure S4, the PXRD
patterns of compounds 1−4 are obviously different from that of
API, proving the formation of new solid forms. Moreover, the
PXRD patterns of the synthesized samples closely match the
simulated ones (Figure S5).
The thermal behaviors of API and compounds 1−4 were
investigated by differential scanning calorimetry (DSC) and
thermogravimetric analysis (TGA). The results are presented in
Figure 8. In the DSC and TGA plots of 1, an initial endotherm at
1
60 °C corresponds to a weight loss of ethanol molecule
(
obsd.10.40%, calc.10.49%), and the endotherm at 318 °C is
attributed to the decomposition of 1. For 2, the TGA curve
shows a weight loss of 14.70% in the 73−97 °C range, which is
consistent with the loss of four water molecules (calc.14.79%).
Then upon further heating, compound 2 decomposes at 267 °C.
The first endothermic peak of 3 at 151 °C is for the loss of
methanol molecular (obsd.7.98%, calc.7.95%) and 3 decomposes
at 273 °C. The melting point of 4 is 269 °C and above this
decomposition begins.
Spectral Analysis. The structure changes of drug molecules
in 1, 2, and 4 are further confirmed by FT-IR spectroscopy.
Generally, the characteristic absorption peak of −COOH group
−
1
−
is at 1720−1700 cm , whereas the ones of −COO group are
at 1650−1550 cm− (strong) and 1400 cm (weak). The IR
spectra of API and compounds 1−4 are shown in Figure S6. The
strong absorption peak at 1720 cm− in API disappears in salts 1
and 2. Instead, new absorption peaks arising from asymmetric
and symmetric vibration of carboxylate group are observed at
1
−1
1
Figure 7. (a) An asymmetric unit of rebamipide methyl ester 4.
(
b) 1D double chain assembled by hydrogen bonding interactions.
esters (Figure S3). Why we do not isolate ester during the
synthesis of 3? We speculate that solubility is the key factor
controlling the crystallization of 3 and 4. In methanol, the ester is
more soluble than API, so during evaporation API combining
with methanol first precipitates to form 3 (Scheme 3). As a result,
−1
−1
1
614 and 1395 cm (for 1) and 1612 and 1392 cm (for 2).
Moreover, the characteristic absorption peaks of ester group at
−1
1
156 and 1088 cm appear in the IR spectrum of 4.
Dissolution Study and Stability. Powder dissolution
profiles of rebamipide API and compounds 1−4 in simulated
succus gastricus (HCl solution with pH = 1.2) are shown in
Figure 9. It is found that both the dissolution rate and apparent
solubilities of 1−4 are higher than those of API. All the
Scheme 3. Equilibrium between Rebamipide and its Methyl
Ester
compounds reach their maximum solubility (S ) within 17 min
max
and Smax is enhanced by 4.05, 4.73, 3.21, and 1.23 times for 1−4,
respectively, compared to that of API (Figure 9a). Then the
solubilities of 1−3 began to decrease and after 36 h the solubility
of each compound was close to that of API (Figure 9b). Similar
17,22,39
phenomena have been reported by Lu T. B. et al.
The
left solid samples of the dissolution experiments were collected
and characterized by PXRD. The PXRD patterns display that
compounds 1−3 transform to API under acid conditions (see
Figure S7) and this transformation might lead to the decrease
of solubility. The rapid dissolution behavior of 1−4 would be
beneficial to their gastrointestinal absorption. The solid forms
of 1 and 2 were found to be stable under ambient conditions
(∼25 °C and 30% relative humidity) for more than three months
(see Supporting Information Figure S8). However, they are
unstable under moisture conditions (40 °C and 75% relative
humidity) as confirmed by PXRD results.
G
DOI: 10.1021/acs.cgd.5b01839
Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design
Article
Figure 9. Dissolution profiles of API and compounds 1−4 in HCl
solution (pH = 1.2) within (a) 35 min and (b) 36 h.
Figure 10. Mean plasma concentration vs time profiles of original API
and compounds 1−4 in rats after oral administration.
rats, which is a classic model to evaluate a drug’s absorption. The
mean plasma concentration vs time plots for rebamipide and
compounds 1−4 after oral administration are shown in Figure 10.
Three important PK parameters, maximal plasma concentration
(Cmax), the time required to reach the Cmax (tmax), and the area
under the curve (AUC), are present in Table 3.
Figure 8. TG (black) and DSC (red) thermograms of compounds 1−4.
Pharmacokinetic Study. The pharmacokinetic study (PK)
of compounds 1−4 and original API was carried out in Wistar
H
DOI: 10.1021/acs.cgd.5b01839
Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design
Article
Table 3. PK Data of the Original API and Compounds 1-4
AUTHOR INFORMATION
■
Corresponding Authors
E-mail: gujk@jlu.edu.cn.
E-mail: cwhu@bit.edu.cn.
drug
Cmax(ng/mL)
tmax(h)
AUC (ng·h/mL)
F (%)
*
*
original API
compound 1
compound 2
compound 3
compound 4
202.5
239.3
53.82
227.2
355.0
3.00
3.00
0.50
6.00
4.00
1694.0
2052.4
948.20
2348.5
3229.8
100.0
121.2
56.00
138.6
190.7
Author Contributions
Y.C. and C.L. contributed equally to this work.
∥
Notes
The authors declare no competing financial interest.
In pharmacology, bioavailability is measured by the AUC
value. Taking the AUC0‑∞ of API as 100%, the relative
bioavailability of 1−4 was calculated as 121.2, 56.00, 138.6, and
ACKNOWLEDGMENTS
■
This work was financially supported by the NSFC (21231002,
21276026, 21173021, 21371024, 81430087), the 111 Project
(B07012), and 973 Program (2014CB932103).
190.7%, respectively. The results indicate that the absorption
of compounds 1, 3, or 4 upon oral administration is better than
that of API. In addition, Cmax value of 4 is increased by 1.8-fold.
Rebamipide as a class IV drug faces two problems: low solubility
and low permeability. For 4, although its solubility is nearly
the same as that of API, its absorption is superior to others
as the permeability of the drug is enhanced by forming ester.
Moreover, the in vivo experiments display that the methyl ester
of rebamipide, 4, rapidly and completely converts to the API
because in the range of 0.17 to 24 h no methyl ester compound
is detected. For 1 and 3, the improvement of bioavailability is
probably due to their good dissolution behavior. As for 2,
although its dissolution behavior is better than others, its
absorption is poorer than others. We speculate that the poor
absorption behavior of 2 might be relative to the formation of
imidic acid tautomer (see Scheme 2) and its low permeability
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