Cyclomaltoheptaose mixed esters of anti-inflammatory drugs and short-chain fatty acids and study of their enzymatic hydrolysis in vitro

Article abstract of DOI:10.1016/j.carres.2008.10.001

In an effort to enhance the drug-loading capacity of cyclomaltoheptaose (β-cyclodextrin, βCD) and to combine the function of anti-inflammatory drugs with short-chain fatty acids (SCFA), ternary esters incorporating seven copies of an anti-inflammatory dru

Full text of DOI:10.1016/j.carres.2008.10.001

Carbohydrate Research 344 (2009) 526–530
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Note
Cyclomaltoheptaose mixed esters of anti-inflammatory drugs and
short-chain fatty acids and study of their enzymatic hydrolysis in vitro
c
Feng Cao a,b, Yong Ren b, , Weiyi Hua
*
a Department of Pharmaceutics, China Pharmaceutical University, Nanjing 210009, PR China
b Jiangsu Key Laboratory for Supramolecular Medicinal Materials and Applications, Nanjing Normal University, Nanjing 210046, PR China
c Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
In an effort to enhance the drug-loading capacity of cyclomaltoheptaose (b-cyclodextrin, bCD) and to
combine the function of anti-inflammatory drugs with short-chain fatty acids (SCFA), ternary esters
incorporating seven copies of an anti-inflammatory drug and 14 copies of a SCFA onto a b-cyclodextrin
core were designed and prepared. Acetic, propionic, or butyric esters were introduced at secondary OH
groups, and ibuprofen, flurbiprofen, or felbinac was attached to primary OH groups through ester bonds.
Heptakis[2,3-di-O-butanoyl-6-O-2-(biphenyl-4-yl)-ethanoyl]-cyclomaltoheptaose was very stable in
Received 30 May 2008
Received in revised form 21 September
2008
Accepted 1 October 2008
Available online 8 October 2008
aqueous and esterase solution. It was hydrolyzed by
The total released amount of biphenyl acetic acid was 38% after 24 h when the esterase was added after
the -amylase hydrolysis. The present results suggest that these nine bCD conjugates may release the
anti-inflammatory drug in the colonic contents.
a-amylase (4 units/mL) with t1/2 value of 18 h.
Keywords:
b-Cyclodextrin esters
Anti-inflammatory drug esters
Colon-specific drug delivery
Enzymatic hydrolysis
Biphenyl acetic acid
a
Ó 2009 Published by Elsevier Ltd.
Oral colon-specific drugs are especially valuable in the treatment
of colon diseases.1 Recently, azo-compounds, glycoside, glucuro-
nide, dextran, and polypeptide conjugates have been prepared for
oral colon-specific delivery.2 Conjugates of cyclomaltooligosaccha-
rides (cyclodextrins, CDs) with biphenyl acetic acid,3,4 predniso-
lone,5 and 5-aminosalicylic acid6 have also been prepared for the
same purpose.
Short-chain fatty acids (SCFAs) play an important physiological
role in the maintenance of the health and integrity of the colonic
epithelium. However, oral administration is difficult because the
SCFAs are absorbed or metabolized in the upper intestinal tract.
Hirayama et al.7 prepared a bCD conjugate in an attempt to con-
struct a colon delivery system for SCFAs.
All the CD conjugates reported involved monosubstitution at
C-6 of the cyclodextrin core. One aim of this work was to enhance
the loading capacity of CDs for drugs by per-modification at the
primary and secondary hydroxyl groups. The other was to synthe-
size anti-inflammatory drugs and SCFAs conjugated CD prodrugs,
which may improve therapy of the colon.
These conjugates were prepared by modifying the method of
Berberan-Santos et al.8 The process is shown in Scheme 1. Starting
from per(6-deoxy-6-iodo)-bCD,9 the secondary OH groups were
acylated using acyl anhydrides and pyridine in the presence of a
catalytic amount of DMAP to give the peracylated derivatives in
23–76.1% yield. Then, treatment of these acylated derivatives with
the sodium salt of the selected anti-inflammatory drug in anhy-
drous DMF yielded the desired bCD conjugates. Difficulties in most
of our syntheses arose in the purification of the final products (col-
umn chromatography, TLC, and crystallization). Structures of the
nine new compounds were confirmed by 1H NMR, FABMS, and ele-
mental analysis.
To gain insight into the stability of the conjugates, the hydroly-
sis of 12 in aqueous solution at various pHs was analyzed in detail
as an example. Figure 1 indicates that 12 was hydrolyzed according
to first-order kinetics, and the half-lives of 12 were 25.1, 626.9, and
125.4 days at pH 1.0, 6.8, and 7.4, respectively. The half-lives of 12
are much higher as compared to the monosubstituted b-cyclodex-
trin biphenylacetic acid conjugate.4
Sugar-degrading and ester-hydrolyzing enzymes were applied
to gain insight into the hydrolysis mechanism of the conjugates tak-
ing 12 as a model. Esterase from porcine liver was used as a model
ester-hydrolyzing enzyme. As shown inFigure 2A, neither apprecia-
ble hydrolysis of 12 nor release of biphenyl acetic acid was observed
for 46 h. These results indicate that 12 is not susceptible to the
esterase-catalyzed hydrolysis. Aspergillus oryzae
a-amylase was
chosen as the model of sugar-degrading enzyme.10,11 As shown in
Figure 2B, the conjugate was hydrolyzed by the enzyme according
to a first-order kinetics, with a t1/2 value of 18 h at enzyme concen-
tration of 4 units/mL. The hydrolysis of the conjugate 12 was slower
* Corresponding author. Tel./fax: +86 25 8589 1591.
E-mail address: renyong@njnu.edu.cn (Y. Ren).
0008-6215/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.carres.2008.10.001

F. Cao et al. / Carbohydrate Research 344 (2009) 526–530
527
I
I
O
O
i
ii
R1OR1
R2Na
R1O
HO
OH
OR1
O
7
O
7
R1=CH3CO
1
2
3
CH3CH2CO
CH3CH2CH2CO
OR2
O
R1O
OR1
O
7
CH3
CH3
H3C
H3C
R2=
CH2COO
CH
C
H
COO
CH2
C
H
COO
F
R1=CH3CO
CH3CH2CO
CH3CH2CH2CO
4
5
6
9
7
8
10
11
12
Scheme 1. Sythesis of cyclomaltoheptaose mixed esters of anti-inflammatory drugs and short-chain fatty acids. Reagents and conditions: (i) DMAP, pyridine, rt, 48 h; (ii)
DMF, 100 °C, 96 h.
Figure 1. Time courses of disappearance of the conjugate IV3 (h, 8.0 ꢀ 10ꢁ5 M) and appearance of biphenyl acetic acid (4) in the aqueous solutions at 37 °C: (A)
hydrochloride acid solution (0.1 M); (B) phosphate buffers (pH 6.8, I = 0.2); (C) phosphate buffers (pH 7.4, I = 0.2).
Figure 2. Time courses of disappearance of the conjugate IV3 (h, 8.0 ꢀ 10ꢁ5 M) and appearance of biphenyl acetic acid (4) in the presence of enzymes at 37 °C: (A) with the
a-amylase (4 units/mL) in acetate buffer (pH 5.5); (B) with the carboxylic esterase (40 units/mL) in HEPES buffer (pH 7.4); (C) the esterase (40 units/mL) was added 12 h after
the a-amylase (4 units/mL) hydrolysis. Key: (a) addition of the a-amylase; (b) addition of the esterase. Each point represents the mean SD of three experiments.
than b-cyclodextrin (t1/2 = 2.5 h calculated under the same condi-
tion7,12). Figure 2C shows the hydrolysis behavior of 12 when the
(Fig. 2C). However, biphenyl acetic acid was rapidly released when
the esterase was added after the -amylase treatment (38% after
a
carboxylic esterase was added 12 h after the
hydrolysis. -Amylase hydrolyzed 12 to small saccharide conju-
gates,12,13 while no release of biphenyl acetic acid was observed
a
-amylase-catalyzed
24 h of the esterase treatment). These results indicate that the ester
linkage of the small saccharide conjugates is easily cleaved by the
esterase. Moreover, colon transit time may last up to 78 h,14 which
a

528
F. Cao et al. / Carbohydrate Research 344 (2009) 526–530
is likely to increase the time available for biphenyl acetic acid re-
lease from 12. These studies suggest that both sugar-degrading
and ester-hydrolyzing enzymes are required for the release of
biphenyl acetic acid from its bCD conjugate, which is in agreement
with the results of Hirayama et al.7
On another hand, the release of biphenyl acetic acid from 12
was slower than that of butyric acid from the monosubstituted bu-
tyric acid–bCD ester conjugate (55% n-butyric acid was released in
the same conditions).7 It is probable that the esterase hydrolyzed
some butyric acid esters at a secondary position of 12. However,
butyric acid does not exhibit properties for direct detection,15
and it was not quantitatively detected by HPLC in our experiments.
Other possible explanation for the observed drug-release discrep-
ancy could not be excluded. For example, the aqueous solubility
of the persubstituted bCD ester conjugate 12 is lower than that
of the monosubstituted n-butyric acid-bCD ester conjugate, affect-
ing the drug release from the conjugate.
1.4. Heptakis(2,3-di-O-butanoyl-6-deoxy-6-
iodo)cyclomaltoheptaose (3)
A similar experiment as for 1 was carried out with butyric anhy-
dride. After the reaction was stopped, the reaction mixture was
poured into 100 mL of ice and water. The insoluble material was
filtered, and was purified by silica gel chromatography with 3:1
petroleum ether–acetone. Compound 3 was obtained as a white
solid (0.75 g, 23%); mp 110–112 °C; 1H NMR (500 MHz, CDCl3): d
5.33–5.40 (m, 7H, H-3), 5.19 (m, 7H, J 3.9 Hz, H-1), 4.83–4.86 (m,
7H, H-2), 3.71–3.86 (m, 14H, H-4, H-5), 3.60–3.63 (m, 14H, H-6,
H-60), 2.17–2.41 (m, 28H, CH2, CH2), 1.61–1.66 (m, 28H, CH2,
CH2), 0.92–0.99 (m, 42H, CH3, CH3); 13C NMR (500 MHz, CDCl3):
d 172.73, 171.32 (C@O,C@O), 96.23 (C1), 80.30 (C4), 76.96 (C2),
76.71 (C3), 69.76 (C5), 35.42 (CH2), 17.80 (CH2), 13.24 (CH3), 8.18
(C6); FABMS: calcd for C98H151I7NO42 [M+NH4]+: 2902.3, found:
m/z 2902.4.
1.5. General procedure for the preparation of heptakis(2,3-di-
O-acetyl-6-O-acyl)cyclomaltoheptaose derivatives of ibuprofen,
flurbiprofen, and felbinac (4–6)
1. Experimental
1.1. General methods
A mixture of 1 (0.24 mmol) and the respective sodium salts of
ibuprofen, flurbiprofen, or felbinac (3.08 mmol) in 15 mL anhyd
DMF was heated for 96 h at 100 °C. After cooling to room temper-
ature, the reaction mixture was poured into 80 mL of ice and water.
The insoluble material was filtered, and was purified twice by silica
gel chromatography (1:1 petroleum ether–EtOAc or 3:1 ether–ace-
tone or 1.5:1 petroleum ether–EtOAc).
bCD was purchased from Sinopharm Chemical Reagent Co. (Chi-
na). Ibuprofen and Felbinac were purchased from Hubei Biocause
Pharmaceutical Co., Ltd (China). Flurbiprofen was purchased from
Shanghai Sunve Pharmaceutical Co., Ltd (China). A. oryzae a-amy-
lase (EC 3.2.1.1, MW 53,000) and esterase (EC 3.1.1.1, MW
168,000) were bought from Sigma Chemicals Co. (St. Louis, MO,
USA). Other chemicals and solvents were of analytical grade, and
deionized, doubly distilled water was used throughout the study.
1H and 13C NMR spectra were recorded on a Bruker ACF-500 instru-
ment using TMS as an internal standard. Elemental analysis was
performed with an Elementar Vario EL III equipment. FAB mass
spectra were recorded on a PE ESI-TOF mass spectrometer. Melting
points were determined on an X4 micromelting point apparatus,
and were uncorrected. Silica gel (200–300 mesh) for chromatogra-
phy and Silica Gel GF254 for TLC were purchased from Qingdao
Haiyang Chemical Co., Ltd. [Editor’s note: required optical rotations
were not provided due to shortage of products].
1.5.1. Heptakis[2,3-di-O-acetyl-6-O-2-(4-
isobutylphenyl)propionoyl)]cyclomaltoheptaose (4)
From 0.6 g of 1 and 0.71 g of ibuprofen sodium salt; white solid
(0.35 g, 48.2%); mp 110–112 °C; 1H NMR (500 MHz, CDCl3): d 7.18
(d, 14H, J 5.7 Hz, Ar), 7.05–7.09 (m, 14H, Ar), 5.14 (d, 7H, J 8.0 Hz,
H-2), 4.81–4.99, 4.27–4.45 (m, 7H, H-1), 4.66–4.70 (m, 7H, H-5),
4.55–4.57 (m, 7H, H-3), 4.09–4.21 (m, 7H, H-6), 3.84–4.04 (m,
7H, H-60), 3.74–3.77 (m, 7H, CH), 3.25–3.51 (m, 7H, H-4), 2.42–
2.43 (m, 14H, CH2), 1.98–2.42 (m, 42H, CH3, CH3), 1.83–1.85 (m,
7H, CH), 1.48–1.49 (m, 7H, CH3), 0.88–0.89 (d, 42 H, (CH3)2); FAB-
MS: calcd for C161H214NO56 [M+NH4]+: 3059.4, found: m/z 3059.4.
Anal. Calcd for C161H210O56ꢂ2H2O: C, 62.84; H, 7.01. Found: C,
62.45; H, 6.86.
1.2. Heptakis(2,3-di-O-acetyl-6-deoxy-6-
iodo)cyclomaltoheptaose (1)
The preparation was analogous to that described by Baer et
al.,16 except that the purification stage was omitted. To a soln of
per-6-deoxy-iodo-b-cyclodextrin10 (1.91 g, 1.0 mmol) in 11 mL of
dry pyridine were added 9 mL of acetic anhydride and 13.0 mg
(0.11 mmol) of DMAP. The reaction mixture was stirred at room
temperature under N2 for 48 h. The reaction mixture was poured
into 80 mL of ice and water. The insoluble material was filtered
and crystallized from MeOH. Compound 1 (1.89 g, 76.1%) was ob-
1.5.2. Heptakis[2,3-di-O-acetyl-6-O-2-(2-fluoro-4-
biphenylyl)propionoyl]cyclomaltoheptaose (5)
From 0.6 g of 1 and 0.82 g of flurbiprofen sodium salt; white so-
lid (0.15 g, 21%); mp 114–116 °C; 1H NMR (500 MHz, CDCl3): d
7.51–7.62 (m, 14H, Ar0), 7.26–7.42 (m, 28H, Ar), 7.13–7.21 (m,
14H, Ar, Ar0), 5.15–5.17 (m, 7H, H-2), 4.89–4.94, 4.38–4.42 (m,
7H, H-1), 4.73–4.85 (m, 7H, H-5), 4.43–4.61 (m, 7H, H-3), 4.19–
4.31 (m, 7H, H-6), 3.95–4.12 (m, 7H, H-60), 3.78–3.88 (m, 7H,
CH), 3.21–3.41 (m, 7H, H-4), 1.85–2.01 (m, 42H, CH3, CH3), 1.55–
1.60 (m, 21H, CH3); FABMS m/z calcd for C175H179F7NO56
tained as a white solid; mp 179–180 °C [dec]; lit.16 180 °C; [
+82.5 (c 1, CHCl3).
a]
D
[M+NH4]+:
3325.3,
found:
3325.0.
Anal.
Calcd
for
1.3. Heptakis(2,3-di-O-propanoyl-6-deoxy-6-
iodo)cyclomaltoheptaose (2)
C175H175F7O56ꢂ2H2O: C, 62.87; H, 5.40. Found: C, 62.55; H, 5.05.
1.5.3. Heptakis[2,3-di-O-acetyl-6-O-2-(biphenyl-4-yl)-
ethanoyl]cyclomaltoheptaose (6)
Similar experiments as for 1 carried out with propionic anhydride
afforded 2 as an amorphous solid (2.23 g, 58.5%); mp 140–142 °C
(MeOH); 1H NMR (500 MHz, CDCl3): d 5.32–5.36 (t, 7H, J 8.58 Hz,
H-3), 5.17 (d, 7H, J 3.2 Hz, H-1), 4.84–4.86 (m, 7H, H-2), 3.71–3.82
(m, 14H, H-4, H-50), 3.60–3.64 (m, 14H, H-6, H-60), 2.17–2.43 (m,
28H, CH2, CH2), 1.08–1.13 (m, 42H, CH3, CH3); 13C NMR (500 MHz,
CDCl3): d 173.59, 172.26 (C@O,C@O), 96.35 (C-1), 80.27 (C4), 76.9
(C2), 76.4 (C3), 69.8 (C5), 27.02 (CH2), 8.56 (CH3), 7.99 (C-6).
From 0.6 g of 1 and 0.72 g of felbinac; white solid (0.20 g, 27%);
mp 131–132 °C; 1H NMR (500 MHz, CDCl3): d 7.44–7.52 (m, 28H,
Ar0), 7.27–7.37 (m, 35H, Ar), 5.21–5.25 (t, 7H, J 8.56 Hz, H-2),
4.94–4.95 (d, 7H, J 3.73 Hz, H-1), 4.65–4.70 (m, 14H, H-3, H-5),
4.29–4.32 (m, 7H, H-6), 4.09–4.11 (d, 7H, J 9.1 Hz, H-60), 3.65–
3.74 (m, 14H, CH2), 3.48–3.52 (t, 7H, J 8.8 Hz, H-4), 2.00 (s, 21H,
CH3), 1.94 (s, 21H, CH3); FABMS: calcd for C168H168NaO56

F. Cao et al. / Carbohydrate Research 344 (2009) 526–530
529
[M+Na]+: 3104.0, found: m/z 3104.0. Anal. Calcd for
1.7.1. Heptakis[(2,3-O-butanoyl-6-O-2-(4-
C168H168O56ꢂ2H2O: C, 65.69; H, 5.56. Found: C, 64.61; H, 5.20.
isobutylphenyl)propionoyl]cyclomaltoheptaose (10)
From 0.6 g of 3 and 0.71 g of ibuprofen sodium salt; white solid
(0.12 g, 17%); mp 73–74 °C; 1H NMR (500 MHz, CDCl3): d 7.18–7.22
(m, 14H, Ar), 7.05–7.07 (m, 14H, Ar), 4.84–5.01 (m, 7H, H-2), 4.81–
4.98, 4.29–4.41 (m, 7H, H-1), 4.68–4.82 (m, 7H, H-5), 4.47–4.52 (m,
7H, H-3), 4.11–4.25 (m, 7H, H-6), 3.89–4.02 (m, 7H, H-60), 3.63–
3.77 (m, 7H, CH), 3.18–3.52 (m, 7H, H-4), 2.39–2.55 (m, 14H,
CH2), 2.21–2.38 (m, 28H, CH2, CH2), 1.84–1.85 (m, 7H, CH), 1.58–
1.72 (m, 28H, CH2, CH2), 1.40–1.60 (m, 7H, CH3), 0.89–0.96 (m,
84H, CH3, CH3, (CH3)2); FABMS: calcd for C189H270NO56
[M+NH4]+: 3452.1, found: m/z 3452.3. Anal. Calcd for
C189H266O56ꢂ3H2O: C, 65.08; H, 7.86. Found: C, 64.98; H, 7.99.
1.6. General procedure for the preparation of heptakis(2,3-O-
propanoyl-6-O-acyl)cyclomaltoheptaose derivatives of
ibuprofen, flurbiprofen, and felbinac (7–9)
A mixture of 0.22 mmol of 2 and 3.08 mmol of the respective
sodium salts of ibuprofen, flurbiprofen, and felbinac in 15 mL an-
hyd DMF was heated for 96 h at 100 °C. After cooling to room tem-
perature, the reaction mixture was poured into 80 mL of ice and
water. The insoluble material was filtered, and was purified by sil-
ica gel chromatography (4:1 petroleum ether–EtOAc or 1:1 petro-
leum ether–EtOAc).
1.7.2. Heptakis[2,3-di-O-butanoyl-6-O-2-(2-fluoro-4-
biphenylyl)-ethanoyl]cyclomaltoheptaose (11)
1.6.1. Heptakis[(2,3-O-propanoyl-6-O-2-(4-isobutylphenyl)-
propionoyl]cyclomaltoheptaose (7)
From 0.6 g of 3 and 0.82 g of flurbiprofen sodium salt; white so-
lid (0.30 g, 39.7%); mp 100–102 °C; 1H NMR (500 MHz, CDCl3): d
7.51–7.60 (m, 14H, Ar0), 7.30–7.45 (m, 28H, Ar), 7.09–7.21 (m,
14H, Ar, Ar0), 5.11–5.24 (m, 7H, H-2), 4.89–4.94, 4.40–4.52 (m,
7H, H-1), 4.79–4.88 (m, 7H, H-5), 4.58–4.67 (m, 7H, H-3), 4.21–
4.31 (m, 7H, H-6), 3.92–4.10 (m, 7H, H-60), 3.77–3.90 (m, 7H,
CH), 3.31–3.54 (m, 7H, H-4), 1.82–2.38 (m, 28H, CH2, CH2), 1.42–
1.71 (m, 49H, CH2, CH2, CH3), 0.82–0.92 (m, 42H, CH3, CH3); FAB-
MS: calcd for C203H235F7NO56 [M+NH4]+: 3718.0. Found: m/z
3718.2. Anal. Calcd for C203F7H231O56: C, 65.90; H, 6.29. Found: C,
65.68; H, 6.30.
From 0.6 g of 2 and 0.71 g of ibuprofen sodium salt; white solid
(0.12 g, 17%); mp 101–102 °C; 1H NMR (500 MHz, CDCl-d3): d
7.16–7.19 (m, 14H, Ar), 7.03–7.09 (m, 14H, Ar), 5.15–5.19 (m, 7H,
H-2), 4.81–4.98, 4.27–4.41 (m, 7H, H-1), 4.85–4.86 (m, 7H, H-5),
4.42–4.59 (m, 7H, H-3), 4.16–4.22 (m, 7H, H-6), 3.91–4.02 (m,
7H, H-60), 3.73–3.77 (m, 7H, CH), 3.20–3.42 (m, 7H, H-4), 2.39–
2.42 (m, 14H, CH2), 2.17–2.39 (m, 28H, CH2, CH2), 1.80–1.86 (m,
7H, CH), 1.41–1.47 (m, 7H, CH3), 1.06–1.13 (m, 42H, CH3, CH3),
0.88–0.89 (d, 42 H, (CH3)2); FABMS: calcd for C175H242NO56
[M+NH4]+: 3353.6, found: m/z 3354.5. Anal. Calcd for
C175H238O56: C, 64.92; H, 7.41. Found: C, 64.60; H, 7.61.
1.7.3. Heptakis[2,3-di-O-butanoyl-6-O-2-(biphenyl-4-yl)-
ethanoyl]cyclomaltoheptaose (12)
1.6.2. Heptakis[2,3-di-O-propanoyl-6-O-2-(2-fluoro-4-
biphenylyl)-propionoyl]cyclomaltoheptaose (8)
From 0.6 g of 3 and 0.72 g of felbinac sodium salt; white solid
(0.13 g, 17%); mp 110–112 °C; 1H NMR (500 MHz, CDCl3): d 7.43–
7.45 (m, 28H, Ar0), 7.26–7.33 (m, 35H, Ar), 5.23–5.26 (m, 7H, H-
2), 4.97–4.98 (m, 7H, H-1), 4.66–4.71 (m, 14H, H-3, H-5), 4.32–
4.34 (m, 7H, H-6), 4.10–4.12 (d, 7H, J 8.55 Hz, H-60), 3.66–3.72
(m, 14H, CH2), 3.50–3.53 (t, 7H, J 8.4 Hz, H-4), 2.01–2.30 (m,
28H, CH2, CH2), 1.531.62 (m, 28H, CH2, CH2), 0.90–0.96 (m, 21H,
CH3), 0.86–0.89 (m, 21H, CH3); FABMS: calcd for C196H228NO56
[M+NH4]+: 3493.9, found: m/z 3493.5; Anal. Calcd for
C196H224O56ꢂH2O: C, 67.38; H, 6.52. Found: C, 67.37; H, 6.29.
From 0.6 g of 2 and 0.82 g of flurbiprofen sodium salt; white so-
lid (0.20 g, 26%); mp 106–108 °C; 1H NMR (500 MHz, CDCl3): d
7.51–7.62 (m, 14H, Ar0), 7.30–7.49 (m, 28H, Ar), 7.13–7.21 (m,
14H, Ar, Ar0), 5.15–5.19 (m, 7H, H-2), 4.88–4.94, 4.50–4.54 (m,
7H, H-1), 4.79–4.85 (m, 7H, H-5), 4.55–4.63 (m, 7H, H-3), 4.19–
4.31 (m, 7H, H-6), 3.92–4.03 (m, 7H, H-60), 3.79–3.90 (m, 7H,
CH), 3.27–3.56 (m, 7H, H-4), 1.82–2.41 (m, 28H, CH2, CH2), 1.55–
1.60 (m, 21H, CH3), 0.89–1.1 (m, 42H, CH3, CH3); FABMS: calcd
for C189H207F7NO56 [M+NH4]+: 3521.6, found: m/z 3521.3. Anal.
Calcd for C189H203F7O56: C, 64.79; H, 5.84. Found: C, 64.86; H, 5.82.
1.8. Analytical methods
1.6.3. Heptakis[2,3-di-O-propanoyl-6-O-2-(biphenyl-4-yl)-
ethanoyl]cyclomaltoheptaose (9)
The HPLC system that consisted of LC-10ADvp pumps, a DGU-
14A online degasser, an SPD-10ADvp detector, a CTO-10AS vp
column oven, and a Class-VP 6.12 software was purchased from
Shimadzu. Normal-phase HPLC conditions for the determination
of 12 were as follows: mobile phase 93.5:6.5 CH2Cl2–MeCN; flow
rate 1.0 mL/min, and detection of wavelength 253 nm. The injec-
From 0.6 g of 2 and 0.72 g of felbinac sodium salt; white solid
(0.15 g, 21%); mp 110–112 °C; 1H NMR (500 MHz, CDCl3): d 7.42–
7.49 (m, 28H, Ar0), 7.26–7.34 (m, 35H, Ar), 5.22–5.25 (t, 7H, J
9.20 Hz, H-2), 4.94–4.95 (d, 7H, J 3.79 Hz, H-1), 4.67–4.94 (m,
14H, H-3, H-5), 4.29–4.32 (m, 7H, H-6), 4.10–4.12 (d, 7H, J
8.60 Hz, H-60), 3.64–3.73 (m, 14H, CH2), 3.49–3.53 (t, 7H, J 9.2
Hz, H-4), 2.10–2.32 (m, 28H, CH2, CH2), 1.03–1.08 (m, 21H, CH3),
1.01–1.02 (m, 21H, CH3); FABMS: calcd for C182H200NO56
[M+NH4]+: 3295.3, found: m/z 3296.2. Anal. Calcd for
C182H196O56ꢂ2H2O: C, 65.93; H, 6.08. Found: C, 65.88; H, 5.96.
tion vol was 20 lL, and the relative retention time was found to
be about 10.0 min at 25 °C. This method was evaluated through
intra-day and inter-day analysis for precision and accuracy. All
the precisions were less than 2.55%, and all the recoveries were
evaluated to be 90.75–97.15%.
HPLC conditions for the determination of biphenyl acetic acid
were as follows: mobile phase 70:30:0.1 MeOH–water–phosphoric
acid; flow rate 1.0 mL/min, and detection of wavelength 253 nm.
1.7. General procedure for the preparation of heptakis(2,3-O-
butanoyl-6-O-acyl)cyclomaltoheptaose derivatives of
ibuprofen, flurbiprofen, and felbinac (10–12)
The injection vol was 20 lL, and the relative retention time was
found to be about 10.0 min at 25 °C.
A mixture of 0.21 mmol of 3 and 3.08 mmol of the respective
sodium salts of ibuprofen, flurbiprofen, and felbinac in 15 mL an-
hyd DMF was heated for 96 h at 100 °C. After cooling to room tem-
perature, the reaction mixture was poured into 80 mL ice and
water. The insoluble material was filtered and purified by silica
gel chromatography with 3:1 petroleum ether–EtOAc, 2:1 petro-
leum ether–acetone, or 1:1petroleum ether–EtOAc.
1.9. Stability studies in aqueous solution
The hydrolysis was performed at an initial concentration of the
conjugate 12 (8.0 ꢀ 10ꢁ5 M) in HCl soln (0.1 M) and phosphate buf-
fers (pH 6.8, 7.4, I = 0.2) at 37 °C. At fixed intervals, an aliquot
(0.1 mL) was withdrawn and mixed with 4 vol of cold CH2Cl2 at

530
F. Cao et al. / Carbohydrate Research 344 (2009) 526–530
ꢁ20 °C immediately. For the aliquots with pH 6.8 and 7.4, 0.1 mL of
HCl (0.1 M) at ꢁ20 °C was added to acidify the samples before
extraction with CH2Cl2. The organic phase was analyzed for the
conjugate and biphenyl acetic acid by HPLC.
8.0 ꢀ 10ꢁ5 M, respectively, in pH 5.5 acetate buffer containing
0.01 M CaCl2 at 37 °C. After 12 h, the pH of the soln was changed
to 7.4 by the addition of 0.1 M NaOH, and then esterase from por-
cine liver was added to the soln to start the esterase-catalyzed
hydrolysis. The concentration of the esterase was 39 units/mL.
1.10. Hydrolysis studies with a-amylase and esterase
References
The
ried out in 0.2 M acetate buffer (pH 5.5) containing 0.01 M CaCl2
-amylase and the conju-
a-amylase-catalyzed hydrolysis of the conjugate was car-
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at 37 °C. The concentrations of A. oryzae
a
gate were 4 units/mL and 2.0 ꢀ 10ꢁ5 M, respectively. At appropri-
ate intervals, an aliquot (0.1 mL) of the reaction solution was
acidified by the addition of 0.1 mL HCl (1.0 M), and was shaken
with CH2Cl2 (0.4 mL) for 10 min. After centrifugation (8000 rpm,
5 min), the organic phase was analyzed for the residual conjugate
and biphenyl acetic acid by HPLC.
The esterase-catalyzed hydrolysis of the conjugate was carried
out in 0.1 M HEPES/NaOH buffer (pH 7.4) at 37 °C. The concentra-
tions of carboxylic esterase from porcine liver (EC 3.1.1.1), MW
168,000, Sigma, St. Louis, MO) and the conjugate were 40 units/
mL and 8.0 ꢀ 10ꢁ5 M, respectively. The analytical procedure for
the conjugate and biphenyl acetic acid was the same as that al-
ready described.
1.11. Hydrolysis studies combining a-amylase and esterase
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The
ducted under the conditions as above. In brief, the concentrations
of A. oryzae -amylase and the conjugate were 4.0 units/mL and
a-amylase-catalyzed hydrolysis of the conjugate was con-
a