Impact of the dissolution profile of the cilostazol cocrystal with supersaturation on the oral bioavailability

Article abstract of DOI:10.1021/acs.cgd.6b01425

The objectives of this study are to enhance the oral bioavailability of cilostazol (CLZ), which is a poorly soluble compound, by cocrystallization and to evaluate the correlation between the calculated solubility of the cocrystal by the solubility product (K_sp) and the complexation constant (K₁₁) and the performance of the cocrystal. Cocrystals of CLZ with 4-hydroxybenzoic acid (4HBA), 2,4-dihydroxybenzoic acid (2,4DHBA), and 2,5-dihydroxybenzoic acid (2,5DHBA) were prepared. Stoichiometric 1:1 structures were formed in the crystal packing of the three cocrystals according to single crystal X-ray diffraction. The calculated solubilities of the CLZ-4HBA cocrystal, CLZ-2,4DHBA cocrystal, and CLZ-2,5DHBA cocrystal were 9.5-fold, 14.5-fold, and 34.3-fold higher than that of CLZ, respectively. Interestingly, the supersaturated dissolution profile in the nonsink condition was inversely correlated with the calculated solubility of the cocrystals, and the CLZ-4HBA cocrystal, which mildly enhanced the solubility compared to the other cocrystals, effectively prolonged the supersaturation. The in vivo performance correlated with the in vitro dissolution profile, and the bioavailability of the CLZ-4HBA cocrystal in beagles was also significantly enhanced even when compared to the amorphous solid dispersion. The cocrystallization of CLZ could be an effective means to enhance the bioavailability, but excessive solubility enhancement was not preferable for the CLZ cocrystal.

Full text of DOI:10.1021/acs.cgd.6b01425

Article
Crystal Growth &
Subscriber access provided by UB
Design is published
+
Fachbibliothek Chemie | (FU-
by the American
Bibliothekssystem)
Chemical Society.
1
155 Sixteenth Street
N.W., Washington, DC
0036
2
Published by American
Chemical Society.
Copyright © American

Impact of the
DissolutioCr
n
ysta
P
l G
r
ro
owthfile
&
Subscriber access provided by UB
Design is published
+
Fachbibliothek Chemie | (FU-
by the American
Bibliothekssystem)
Chemical Society.
1
155 Sixteenth Street
N.W., Washington, DC
0036
2
Published by American
Chemical Society.
Copyright © American

of the Cilostazol
Cocryst
C
a
rys
l
tal
withth &
Gro
w
Subscriber access provided by UB
Design is published
+
Fachbibliothek Chemie | (FU-
by the American
Chemical Society.
Bibliothekssystem)
1
155 Sixteenth Street
N.W., Washington, DC
0036
2
Published by American
Chemical Society.
Copyright © American

Supersaturation
Crystal Growth &
Subscriber access provided by UB
Design is published
+
Fachbibliothek Chemie | (FU-
by the American
Bibliothekssystem)
Chemical Society.
1
155 Sixteenth Street
N.W., Washington, DC
0036
2
Published by American
Chemical Society.
Copyright © American

on the Oral
Bioavai
C
l
r
a
yst
bilityth &
al
G
ro
w
Subscriber access provided by UB
Design is published
+
Fachbibliothek Chemie | (FU-
by the American
Chemical Society.
Bibliothekssystem)
1
155 Sixteenth Street
N.W., Washington, DC
0036
2
Published by American
Chemical Society.
Copyright © American

Motoyasu Yoshimura,
Masateru Miyake, Tatsuya
Crystal Growth &
Subscriber access provided by UB
Design is published
+
Fachbibliothek Chemie | (FU-
by the American
Bibliothekssystem)
Chemical Society.
1
155 Sixteenth Street
N.W., Washington, DC
0036
2
Published by American
Chemical Society.
Copyright © American

Kawato, Masahiko Bando,
Masafumi Toda, Yusuke
Crystal Growth &
Subscriber access provided by UB
Design is published
+
Fachbibliothek Chemie | (FU-
by the American
Bibliothekssystem)
Chemical Society.
1
155 Sixteenth Street
N.W., Washington, DC
0036
2
Published by American
Chemical Society.
Copyright © American

Kato, Toshiro Fukami,
and Tetsuya Ozeki
Crystal Growth &
Subscriber access provided by UB
Design is published
+
Fachbibliothek Chemie | (FU-
by the American
Bibliothekssystem)
Chemical Society.
1
155 Sixteenth Street
N.W., Washington, DC
0036
2
Published by American
Chemical Society.
Copyright © American

Cryst. Growth Des., Just Accepted
Manuscript • Publication
Date (Web): 2C2rysDtael Gcr2ow0t1h6&
Subscriber access provided by UB
Design is published
+
Fachbibliothek Chemie | (FU-
by the American
Bibliothekssystem)
Chemical Society.
1
155 Sixteenth Street
N.W., Washington, DC
0036
2
Published by American
Chemical Society.
Copyright © American

Downloaded from
http://pubs.acs.org on
DecemberCr2y3st,al2G0r1ow6th &
Subscriber access provided by UB
Design is published
+
Fachbibliothek Chemie | (FU-
by the American
Bibliothekssystem)
Chemical Society.
1
155 Sixteenth Street
N.W., Washington, DC
0036
2
Published by American
Chemical Society.
Copyright © American

Just Accepted
Crystal Growth &
Subscriber access provided by UB
Design is published
+
Fachbibliothek Chemie | (FU-
by the American
Bibliothekssystem)
Chemical Society.
1
155 Sixteenth Street
N.W., Washington, DC
0036
2
Published by American
Chemical Society.
Copyright © American

“Just Accepted” manuscripts have been
online prior to technical editing, formattin
Crystal Growth &
Subscriber access provided by UB
Design is published
+
Fachbibliothek Chemie | (FU-
by the American
Bibliothekssystem)
Chemical Society.
1
155 Sixteenth Street
N.W., Washington, DC
0036
2
Published by American
Chemical Society.
Copyright © American

Society provides “Just Accepted” as
dissemination of scientific material as so
Crystal Growth &
appeSaurbsincrifbuelrl ainccPesDsFprfoovridmeadtbaycUcBompanie
Design is published
+
Fachbibliothek Chemie | (FU-
by the American
Bibliothekssystem)
Chemical Society.
1
155 Sixteenth Street
N.W., Washington, DC
0036
2
Published by American
Chemical Society.
Copyright © American

fully peer reviewed, but should not be co
readers and citable by the Digital Object
Crystal Growth &
to auStuhbosrcsri.beTrhaeccreesfsorpero,vtidheed b“JyuUsBt Accepte
Design is published
+
Fachbibliothek Chemie | (FU-
by the American
Bibliothekssystem)
Chemical Society.
1
155 Sixteenth Street
N.W., Washington, DC
0036
2
Published by American
Chemical Society.
Copyright © American

in the journal. After a manuscript is tech
Accepted” Web site and published as an
Crystal Growth &
chanSguebsscrtioberthaeccemssapnruosvicdreidptbyteUxBt and/or
Design is published
+
Fachbibliothek Chemie | (FU-
by the American
Bibliothekssystem)
Chemical Society.
1
155 Sixteenth Street
N.W., Washington, DC
0036
2
Published by American
Chemical Society.
Copyright © American

and ethical guidelines that apply to the
or consequences arising from the use o
Crystal Growth &
Subscriber access provided by UB
Design is published
+
Fachbibliothek Chemie | (FU-
by the American
Bibliothekssystem)
Chemical Society.
1
155 Sixteenth Street
N.W., Washington, DC
0036
2
Published by American
Chemical Society.
Copyright © American

Page 1 o fC 3r 7y stal Growth & Design
1
2
3
4
5
6
7
8
cilostazol (CLZ)
ACS Paragon Plus Environment
9 4
-hydroxy
2,4-dihydroxy
benzoic acid
(2,4DHBA)
2,5-dihydroxy
benzoic acid
(2,5DHBA)
1
1
1
b0 enzoic acid
1 (4HBA)
2

(
a-1) CLZ-4HBA cocrystal
Crystal Growth & Desig Pn age 2 of 37
(a-2) 4HBA
1
2
(
b-1) CLZ-2,4DHBA cocrystal
3
4
(
b-2) 2,4DHBA
5
6
7
(c-1) CLZ-2,5DHBA cocrystal
8
9
(
c-2) 2,5DHBA
1
0
1
1
(
1d2) CLZ
13
1
4
(
e) Amorphous solid dispersion
1
5
6
7
8
ACS Paragon Plus Environment
1
1
0
5
10
15
20
25
30
1
2 theta. (deg.)

Page 3 of 3 7C rystal Growth & Design
CLZ-4HBA cocrystal
0
CLZ-2,4DHBA cocrystal
1
-
-
-
10
20
30
40
50
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
CLZ-2,5DHBA cocrystal
Amorphous solid dispersion
x10
0
1
CLZ
-
2
3
4
5
6
ACS Paragon Plus Environment
-
8
0
100
120
140
160
180
Temperature (ºC)

Crystal Growth & Desig nP age 4 of 37
a
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
b
0
1
2
3
4
5
6
7
8
9
0 c
1
2
3
4
5
ACS Paragon Plus Environment
6

0.020
y = 0.0836x + 0.0012
Page 5 of 3 7C rystal Growth & Design
R² = 0.9999
0.015
y = 0.4668x + 0.0028
R² = 0.9781
1
2
3 0.010
4
y = 0.0357x + 0.001
R² = 0.9999
5
6 0.005
CLZ-4HBA cocrystal
7
8
CLZ-2,4DHBA cocrystal
CLZ-2,5DHBA cocrystal
ACS Paragon Plus Environment
9 0.000
1
1
0
1
0.0
0.1
0.2
/[coformer] (mM )
0.3
0.4
-
1
1

1.0
0.8
0.6
Crystal Growth & Desig nP age 6 of 37
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
SCC-2,5DHBA
0.4
SCC-2,4DHBA
SCC-4HBA
0
0
.2
1
2
3
CLZ
ACS Paragon Plus Environment
0
.0
4
5
6
0.0
0.2
0.4
0.6
0.8
1.0
Concentration of coformer (mM)

P
1
age 7 of 3 7C rystal Growth & Design
0
0
CLZ-4HBA cocrystal
CLZ-2,4DHBA cocrystal
CLZ-2,5DHBA cocrystal
Amorphous solid dispersion
CLZ hammer-milled
a
8
6
4
0
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
0
0
20
0
0
0
10
20
30
40
50
60
1
2
140
Time (min)
3
b
1
20
4
5
1
00
6
7
8
80
6
0
9
040
1
2
2
3
4
5
6
0
ACS Paragon Plus Environment
0
0
10
20
30
40
50
60
Time (min)

Crystal Growth & Desig nP age 8 of 37
CLZ-4HBA cocrystal
2500
CLZ-2,4DHBA cocrystal
CLZ-2,5DHBA cocrystal
Amorphous solid dispersion
CLZ hammer-milled
1
2
3
4
5
2000
1500
6 1000
7
8
5
00
9
1
1
1
1
0
1
2
3
ACS Paragon Plus Environment
0
0
2
4
6
8
Time (hr)

Page 9 of 37
Crystal Growth & Design
1
2
3
4
5
6
7
8
9
Impact of the Dissolution Profile of the Cilostazol
Cocrystal with Supersaturation on the Oral
Bioavailability
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
*,†,§
†
‡
‡
Motoyasu Yoshimura , Masateru Miyake , Tatsuya Kawato , Masahiko Bando , Masafumi
†
†
#
§
Toda , Yusuke Kato , Toshiro Fukami , and Tetsuya Ozeki
†
‡
Formulation Research Institute and Medicinal Chemistry Research Laboratories, Otsuka
§
Pharmaceutical Co., Ltd., Tokushima 771ꢀ0182, Drug Delivery and Pharmaceutics, Graduate
School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi 467ꢀ8603, and
#
Department of Molecular Pharmaceuticals, Meiji Pharmaceutical University, Kiyose, Tokyo
204ꢀ8588, Japan.
*
Yoshimura.Motoyasu@otsuka.jp
ABSTRACT: The objectives of this study are to enhance the oral bioavailability of cilostazol
(CLZ), which is a poorlyꢀsoluble compound, by cocrystallization and to evaluate the correlation
between the calculated solubility of the cocrystal by the solubility product (K ) and the
sp
complexation constant (K ) and the performance of the cocrystal. Cocrystals of CLZ with 4ꢀ
11
hydroxybenzoic acid (4HBA), 2,4ꢀdihydroxybenzoic acid (2,4DHBA), and 2,5ꢀ
dihydroxybenzoic acid (2,5DHBA) were prepared. Stoichiometric 1:1 structures were formed in
the crystal packing of the three cocrystals according to single crystal Xꢀray diffraction. The
calculated solubilities of the CLZꢀ4HBA cocrystal, CLZꢀ2,4DHBA cocrystal, and CLZꢀ
ACS Paragon Plus Environment
1

Crystal Growth & Design
Page 10 of 37
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
,5DHBA cocrystal were 9.5ꢀfold, 14.5ꢀfold, and 34.3ꢀfold higher than that of CLZ, respectively.
Interestingly, the supersaturated dissolution profile in the nonꢀsink condition was inverselyꢀ
correlated with the calculated solubility of the cocrystals, and the CLZꢀ4HBA cocrystal, which
mildly enhanced the solubility compared to the other cocrystals, effectively prolonged the
supersaturation. The in vivo performance correlated with the in vitro dissolution profile, and the
bioavailability of the CLZꢀ4HBA cocrystal in beagles was also significantly enhanced even
when compared to the amorphous solid dispersion. The cocrystallization of CLZ could be an
effective means to enhance the bioavailability, but excessive solubility enhancement was not
preferable for the CLZ cocrystal.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
INTRODUCTION
Recently, the number of poorly soluble drugs has been increasing in the market because
hydrophobic substituents tend to be introduced to pharmaceutical compounds in order to enhance
pharmacodinamic activity. The Biopharmaceutics Classification System (BCS) is proposed in
1
order to categorize the drug type based on solubility and permeability. According to a previous
report, approximately 40% of the market drugs and 80~90% of the R&D pipelines are poorly
soluble drugs (BCS class II or class IV), and these compounds are likely to fail due to the poor
2
solubility. Therefore, solubility enhancement of poorly soluble drugs is very important for
pharmaceutical development, and various formulation techniques (i.e., micronized or nanonized
techniques, amorphous solid dispersions, salts, cocrystals, and lipidꢀbased formulations) have
been studied in order to improve solubility properties. It is important to select the suitable
formulation type for solubility improvement based on the physicochemical properties of the
compound. In the case of a highlyꢀlipophilic compound, the bioavailability of lipidꢀbased
ACS Paragon Plus Environment
2

Page 11 of 37
Crystal Growth & Design
1
2
3
4
5
6
7
8
9
formulation was remarkably enhanced compared to conventional formulation or drug
3
ꢀ5
suspension.
It has been reported that the compounds consisting of a dissociable group were
6
ꢀ8
able to form salt, leading to high solubility and oral absorption.
Whereas amorphous solid
the conversion to a stable
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
9ꢀ11
dispersion is a useful formulation for bioavailability enhancement,
crystal and the chemical degradation of the amorphous solid dispersion with high internal energy
1
1ꢀ13
and specific volume were found to be critical.
On the other hand, salts and cocrystals are
1
4
prone to be physicochemically stable. In particular, cocrystals are very useful for undissociated
and poorlyꢀsoluble compounds because it is formed with various coformers without an ionic
8,9,15ꢀ17
bond, and a high potential for enhancing oral absorption has been reported.
1
8
Cilostazol (CLZ) is a synthetic antiplatelet agent with a vasodilating effect. It is a poorly
1
9
soluble compound categorized in BCS class II without dissociable groups, as shown in Figure
Because the area under the serum concentration ꢀ time curve (AUC) of CLZ for a
conventional 50ꢀmg tablet is significantly lower compared to an ethanol solution in human, it has
1.
2
0
been suggested that the poor solubility of CLZ limits bioavailability.
In this research, the cocrystallization of CLZ was investigated in order to improve the poor
solubility and to understand how to enhance the oral absorption. In prior investigation, cocrystal
screening of CLZ was performed by liquid assisted grinding (LAG). As a result, CLZ formed
cocrystals with 4ꢀhydroxybenzoic acid (4HBA), 2,4ꢀdihydroxybenzoic acid (2,4DHBA), and 2,5ꢀ
dihydroxybenzoic acid (2,5DHBA), whose structures are shown in Figure 1. In the experiment,
the cocrystals were newly prepared by the slurry method under adjusted conditions because the
cocrystals prepared by the LAG method may partially contain the amorphous form of CLZ, and
the control of cocrystal particle size was difficult. All coformers forming cocrystals with CLZ
were derivatives of hydroxybenzoic acid, and the substitution site and ratio of the hydroxyl
ACS Paragon Plus Environment
3

Crystal Growth & Design
Page 12 of 37
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
group influenced the solubilities of these coformers (the solubilities of 4HBA, 2,4DHBA, and
2
1,22
2,5DHBA were 4.9, 6.0, and 22 mg/mL at 25 °C, respectively
). As the solubility of the
2
3
cocrystal is proportional to that of the coformer, the unique performances in each cocrystal
were predicted even though these were formed with similar coformers. In addition, it has been
reported that the coformers influenced not only the solubility but also the permeability and the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4,25
oral absorption in vivo.
Generally, a cocrystal forms by weak interactions, such as hydrogen
bonding, between an active pharmaceutical ingredient and a coformer rather than by ionic
1
4
bonding to form a salt crystal; hence, a cocrystal is likely to dissociate more quickly than a salt.
Some of the amorphous solid dispersions or cocrystals cause not only solutionꢀmediated
transformation but also surfaceꢀmediated transformation to the more stable crystal as soon as
26,27
these come in contact with liquids.
Therefore, a formulation design to prevent the conversion
28,29
to a stable crystal or precipitation is important for the enhancement of bioavailability.
Figure 1. Chemical structures of CLZ and coformers.
The purpose of this research is to clarify the correlation between the solubility enhancement of
a cocrystal and the supersaturated profile and to investigate the effect of supersaturation in vitro
ACS Paragon Plus Environment
4

Page 13 of 37
Crystal Growth & Design
1
2
3
4
5
6
7
8
9
on the absorption in vivo, in addition to the bioavailability enhancement of CLZ by
cocrystallization. To evaluate the supersaturation of cocrystals, kinetic dissolution and
equilibrium solubility measurements were performed. The solubility product K was evaluated
sp
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
as the equilibrium solubility of the CLZ cocrystals. If CLZ forms a stoichiometrically 1:1
cocrystal with a coformer, the solubility product Ksp is calculated by applying a linear
approximation between the inverse of the coformer concentration and CLZ solubility profile to
Equation 1, where K is the binding constant for the complex formation of CLZ/coformer in
1
1
30
solution.
ꢃꢄꢅ
ꢆꢇꢈꢇꢉꢊꢋꢉꢁꢌ
ꢀ
CLZꢁ =
+ ꢍ ꢍ
(1)
ꢂ
ꢎꢎ ꢏꢐ
ꢀ
Since the hydroxybenzoic acid derivatives that form the CLZ cocrystals are acidic compounds,
ionization of the coformer occurs with an increase in pH, and as a consequence, the solubility of
the cocrystal exponentially increases over the pKa of the coformer, although CLZ does not
2
3,31
dissociate over the entire pH range.
The solubility products of CLZ cocrystals were
evaluated in simulated gastric fluid (SGF) because the solubility in acidic pH condition is
especially important for highlyꢀpermeable CLZ to enhance oral absorption. As the ionization of
the coformer can be ignored in the acidic condition, which is lower than the pKa of the coformer,
the intersection of a stoichiometric 1:1 line and the theoretical solubility curve calculated by
23
Equation 1 introducing K and K signifies the solubility of the cocrystal. If the solubility of
sp
11
the cocrystal is higher than that of CLZ, temporary supersaturation can be detected in the kinetic
dissolution. Accordingly, it is likely that the difference of solubility between the CLZ and
cocrystal influences the supersaturated profile. In this report, the correlative relationship
between the calculated solubility by the solubility product and the supersaturation in the kinetic
dissolution of CLZ cocrystal was investigated.
ACS Paragon Plus Environment
5

Crystal Growth & Design
Page 14 of 37
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
EXPERIMENTAL SECTION
Materials. CLZ was synthesized and hammerꢀmilled in Otsuka Pharmaceutical Co., Ltd.
(Tokushima, Japan). 4HBA and 2,5DHBA were purchased from Wako Pure Chemical
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Industries, Ltd. (Osaka), and 2,4DHBA was purchased from Tokyo Kasei Kogyo Co., Ltd.
(Tokyo) as the coformers. Hypromellose, type 2910 grade TCꢀ5E, for the formation of
amorphous solid dispersion of CLZ and the kinetic dissolution was provided by ShinꢀEtsu
Chemical Co., Ltd. (Tokyo). Lactose monohydrate was provided from DFE Pharma Ltd. (Goch,
Germany), and colloidal silicon dioxide was provided from Nippon Aerosil Co., Ltd. (Tokyo).
These excipients were used to preꢀmix with CLZ substances in order to improve the wettability
when kinetic dissolution was performed. SGF was prepared according to USP without any
addition of enzymes. All other regents for the analytical procedures were purchased from Wako
Pure Chemical Industries, Ltd. (Osaka).
Preparation of CLZ Cocrystal. CLZ cocrystals were prepared by the slurry method. The
slurry suspension for CLZꢀ4HBA cocrystal was prepared by the addition of 10 g of CLZ in 40
mL of acetone containing 7.48 g of 4HBA (stoichiometric 1:2 CLZ:4HBA complex addition).
Similarly, the slurry suspension for CLZꢀ2,4DHBA cocrystal was prepared by the addition of 10
g of CLZ in 40 mL of acetone containing 8.34 g of 2,4DHBA (stoichiometric 1:2 CLZ:2,4DHBA
complex addition). These slurry suspensions were stirred at room temperature for 7 days. The
slurry suspension for CLZꢀ2,5DHBA cocrystal was prepared by the addition of 10 g of CLZ in
15 mL of acetone containing 6.67 g of 2,5DHBA (stoichiometric 1:1.6 CLZ:2,5DHBA complex
addition). As the viscosity of the slurry suspension rapidly increased as soon as CLZ was
introduced in acetone with a high concentration of 2,5DHBA, it was severely shaken at 40 °C for
1
day. After that, it was stirred at room temperature for 6 days. These slurry suspensions were
ACS Paragon Plus Environment
6