Synthesis of water-soluble polymeric prodrugs possessing 4-methylcatechol derivatives by mechanochemical solid-state copolymerization and nature of drug release.

Article abstract of DOI:10.1248/cpb.50.1434

In this study we synthesized the water-soluble polymeric prodrugs possessing a 4-methylcatechol (4MC) derivative as a side chain by mechanochemical solid-state copolymerization. 1-benzoyl-4-methylcatechol (Bz4MC) was selected as a model compound of 4MC, and its methacryloyl derivative (1) was synthesized. 6-O-methacryloyl-D-galactose (2) was also prepared as a water-soluble monomer. The mechanochemical solid-state copolymerization of 1 and 2 was carried out to obtain the water-soluble polymeric prodrug possessing the Bz4MC as a side chain. The mechanochemical copolymerization of 1 and 2 proceeded to completion, and the polymeric prodrug produced possessed a narrow molecular weight distribution. Three kinds of polymeric prodrugs, whose compositions were different from one another, were hydrolyzed in vitro. The hydrolysis of these polymeric prodrugs proceeded to completion. The rate constants of hydrolysis decreased with increasing the mole fraction of 1 in polymeric prodrug. It was suggested that the rate constant of hydrolysis could be controlled by the composition, the mole fraction of 1 in the polymeric prodrug.

Full text of DOI:10.1248/cpb.50.1434

1434
Chem. Pharm. Bull. 50(11) 1434—1438 (2002)
Vol. 50, No. 11
Synthesis of Water-Soluble Polymeric Prodrugs Possessing
4-Methylcatechol Derivatives by Mechanochemical Solid-State
Copolymerization and Nature of Drug Release
b
Shin-ichi KONDO, ,a Yasushi SASAI,a Masayuki KUZUYA,a and Shoei FURUKAWA
*
a Laboratory of Pharmaceutical Physical Chemistry, Gifu Pharmaceutical University; and b Laboratory of Molecular
Biology, Gifu Pharmaceutical University; 5–6–1 Mitahora-Higashi, Gifu 502–8585, Japan.
Received April 15, 2002; accepted September 6, 2002
In this study we synthesized the water-soluble polymeric prodrugs possessing a 4-methylcatechol (4MC) de-
rivative as a side chain by mechanochemical solid-state copolymerization. 1-Benzoyl-4-methylcatechol (Bz4MC)
was selected as a model compound of 4MC, and its methacryloyl derivative (1) was synthesized. 6-O-Methacry-
loyl-D-galactose (2) was also prepared as a water-soluble monomer. The mechanochemical solid-state copolymer-
ization of 1 and 2 was carried out to obtain the water-soluble polymeric prodrug possessing the Bz4MC as a side
chain. The mechanochemical copolymerization of 1 and 2 proceeded to completion, and the polymeric prodrug
produced possessed a narrow molecular weight distribution. Three kinds of polymeric prodrugs, whose composi-
tions were different from one another, were hydrolyzed in vitro. The hydrolysis of these polymeric prodrugs pro-
ceeded to completion. The rate constants of hydrolysis decreased with increasing the mole fraction of 1 in poly-
meric prodrug. It was suggested that the rate constant of hydrolysis could be controlled by the composition, the
mole fraction of 1 in the polymeric prodrug.
Key words mechanochemical polymerization; polymeric prodrug; nerve growth factor (NGF); 4-methylcatechol; sustained re-
lease
Although evidence indicates that nerve growth factor properties observed in such polymers is that the resulting
(NGF) may have promise as a therapeutic agent for neurode- polymeric prodrugs are of very low heterogeneity (narrow
generative diseases like Alzheimer’s,1,2) its potential use in molecular weight distribution), which is of great value in phar-
such a central nervous system (CNS) disorder is prevented maceuticals for highly functionalized polymeric prodrugs.22)
by its inability to cross the blood-brain barrier (BBB). A va- It was also shown that the resulting copolymer under the ap-
riety of low molecular weight compounds has been found as propriate experimental conditions was homogeneous in com-
a NGF stimulator.3—9) Among them, 4-methylcatechol (4MC) position (ideal copolymerization).26) Therefore, the present
is a potent NGF stimulator both in vivo10,11) and in vitro.12—14) reactions seem to be applicable to a wide variety of vinyl
4MC, however, could not penetrate into the brain for lacking monomers of an important bioactive compound with differ-
sufficient lipophilicity. Several dihydropyridine derivatives of ent physicochemical properties and provide a novel and
4MC were synthesized as a potential brain selective targetry simple methodology for syntheses of polymeric prodrugs
forms by Kourounakis et al.15) to increase the lipophilicity through a totally dry process.
and the BBB permeability of 4MC.
In order to gain a fundamental insight to develop a poly-
Major advancements have been made in the understanding meric prodrug that can passively be delivered to the disease
of Alzheimer’s disease. It has been suggested that the in- parts of brain by the peripheral administration and slowly
tegrity of BBB may be compromised in Alzheimer’s dis- release 4MC derivatives, we synthesized the polymeric
ease.16,17) Immunohistological studies have demonstrated the prodrugs possessing 4MC derivatives as a side chain by
presence of serum proteins in the cerebrovascular amyloid in mechanochemical solid-state polymerization. Thus, 1-ben-
Alzheimer’s patients suggesting abnormal BBB permeabil- zoyl-4-methylcatechol (Bz4MC) was selected as a model
ity.18—20) Therefore, it is expected that the polymeric pro- compound of 4MC derivatives. The mechanochemical solid-
drugs, which possess 4MC or its derivative as a side chain, state polymerization of methacryloyl derivatives of Bz4MC
can passively be delivered into the diseased part of the brain and water-soluble monomers, 6-O-methacryloyl-D-galactose,
in Alzheimer’s patients. It is also known that 4MC is easily was carried out to obtain water-soluble polymeric prodrugs.
oxidized in vivo. The polymeric prodrugs possessing 4MC The nature of drug release from the polymeric prodrugs
would be useful in terms of the stabilization and sustained re- formed was examined in vitro.
lease of 4MC.
Experimental
We have reported the synthesis and nature of drug release
Ultraviolet spectral measurements were performed with a Shimadzu
of novel polymeric prodrugs prepared by mechanochemical
Recording Spectrophotometer UV-3100 using 1-cm quartz cells. 1H-NMR
solid-state polymerization.21—33) Several important conclu-
spectra were run on a JEOL JNM-GX270 FT-NMR spectrometer using
sions were reached from a series of such studies. The
monomers prepared on the basis of the structural criteria de-
rived from quantum chemical considerations underwent
facile mechanochemical solid-state polymerizations to give
corresponding polymeric prodrugs essentially quantita-
tively.21,22,25) Thus, this method eliminates the need for any
tetramethylsilane as an internal or external standard. Reading of pH was car-
ried out on a TOA pH meter HM-16S at room temperature. Melting points
were measured on a capillary melting-point apparatus and uncorrected. IR
spectra were obtained on a Perkin Elmer 1650 FT-IR spectrometer. MS spec-
tra were obtained on a JEOL JMS-SX102A mass spectrometer.
Materials Acrylamide supplied by Tokyo Kasei Kogyo Co., Ltd.
(Japan) was commercially available. It was purified by recrystallization from
work-up of the reaction mixture. One of the most striking benzene and dried in vacuo. 2-Methacryloyloxyehtyl isocyanate, Bz4MC
∗ To whom correspondence should be addressed. e-mail: skondo@gifu-pu.ac.jp © 2002 Pharmaceutical Society of Japan

November 2002
1435
which the mole fraction of 1 was 0.05, 0.1, 0.2, 0.3 and 0.4, were used. The
polymeric prodrug powder (5.0 mg) was added to distilled water (10 ml).
The solution containing the polymeric prodrug powder was shaken for
15 min at room temperature. It was observed whether the polymeric prodrug
powder dissolved or not.
Molecular Weight Measurement The molecular weight of polymeric
prodrugs was measured with a gel permeation chromatograph (GPC; Shi-
madzu LC-6A) equipped with a refractive index detector (Shimadzu, RID-
6A), gel column (Shodex, KD-800M and KD-80M), and a data analyzer
(Shimadzu, Chromatopac C-R4A), under the following conditions: elution
solvent, DMF containing 10 mmol of LiBr; flow rate, 0.7 ml/min; column
temperature, 40 °C. Calibration was carried out with a standard specimen of
poly(ethylene oxide).
Method of Hydrolysis The hydrolysis of polymeric prodrugs (20—
25 mg) was conducted in the mixture of pH 7.4 phosphate buffer (50 mM
KH2PO4 and 40 mM NaOH) (10 ml) and CH3CN (10 ml) at 37ꢂ0.2 °C. The
pH of the solution unchanged before and after hydrolysis. Released 4MC
and Bz4MC were periodically assayed by using a HPLC procedure. HPLC
was performed with a Shodex Asahipak ODP-50 (4.6 i.d.ꢃ250 mm) and
ODP-50G (4.6 i.d.ꢃ10 mm), and eluted with a mixture of pH 7.4 phosphate
buffer–CH3CN (1 : 1) at a flow rate of 0.5 ml/min.
and 1,2:3,4-di-O-isopropylidene-a-D-galactopyranose were synthesized ac-
cording to the literatures,31,34,35) respectively.
1-Benzoyl-2-methacryloyloxyethylcarbamoyl-4-methylcatechol (1) 2-
Methacryloyloxyethylisocyanate (0.40 g, 2.6 mmol) was added to a solution
of Bz4MC (0.50 g, 2.2 mmol) and triethylamine (0.26 g, 2.6 mmol) in dry
acetonitrile (15 ml). The reaction mixture was stirred at room temperature
overnight. The reaction mixture was filtered and evaporated in vacuo. The
residue was dissolved in CHCl3 (30 ml). The CHCl3 solution was washed
fully with H2O, dried over MgSO4, and evaporated in vacuo. The residue
was chromatographed over silica gel (Wakogel C300) in CHCl3 as an eluent.
The eluted product was further recrystallized from cyclohexane and cyclo-
hexanone (1 : 1) to yield 0.20 g (24%) of 1: mp 69—70 °C. IR (KBr) cmꢀ1
:
3315 (–CONH–), 1741, 1708 (–COO–), 1637 (vinyl group). 1H-NMR
(CDCl3) d: 1.88 (3H, s, –CH3 of methacryloyl group), 2.37 (3H, s, –CH3 of
4-methylcatechol), 3.42—3.50 (2H, m, –CH2OCO–), 4.10—4.18 (2H, m,
–NHCH2–), 5.54 (1H, s, vinyl group), 6.03 (1H, s, vinyl group), 7.05—7.17
(3H, m, aromatic ring of 4-methylcatechol), 7.45—7.67 (3H, m, meta- and
para-H of benzoyl group), 8.16—8.18 (2H, m, ortho-H of benzoyl group).
UV lmax (EtOH) nm (e): 230.9 (17700). EI-MS m/z: 383 (Mꢁ). Anal. Calcd
for C21H21NO6: C, 65.79; H, 5.52; N, 3.65. Found: C, 65.65; H, 5.59; N,
3.67.
6-O-Methacryloyl-D-galactose (2) Methacryloyl chloride (1.6 g,
15.4 mmol) was added to a solution of 1,2:3,4-di-O-isopropylidene-a-D-
galactopyranose (4.0 g, 15.4 mmol) and triethylamine (1.6 g, 15.4 mmol) in
dry acetonitrile (60 ml). The reaction mixture was stirred at room tempera-
ture overnight. The reaction mixture was filtered and evaporated in vacuo.
The residue was dissolved in CHCl3 (300 ml). The CHCl3 solution was
washed with H2O, dried over MgSO4, and evaporated in vacuo to obtain an
oily substance. This oily substance (3.0 g) and 4-methoxyphenol (0.15 g,
1.2 mmol) were dissolved in formic acid (23.75 ml) and then water (5.9 ml)
was added to the solution. The reaction mixture was stirred at 20 °C for 20 h.
Water (30 ml) was added to the reaction mixture. This solution was evapo-
rated in vacuo and EtOH (42 ml) was added to the residue. The EtOH solu-
tion was filtered and evaporated in vacuo. The residue was added to 10 times
the ether. The precipitate was collected and recrystallized from EtOH to
yield 0.80 g (38%) of 2: mp 85—87 °C. IR (KBr) cmꢀ1: 3350 (–OH), 1700
(–COO–), 1640 (vinyl group). 1H-NMR (CDCl3) d: 1.88 (3H, s, –CH3),
2.55—3.69 (4H, br, –OH), 4.14 (2H, m, –CH2–), 4.37—4.94 (4H, m, 2, 3, 4,
and 5-position of galactose), 5.69 (1H, s, vinyl group), 6.03 (1H, s, vinyl
group), 6.23 (1H, m, 1-position of galactose). EI-MS m/z: 248 (Mꢁ). Anal.
Calcd for C10H16O7·0.5H2O: C, 46.53; H, 6.68. Found: C, 46.75; H, 6.98.
Mechanochemical Solid-State Copolymerization A mixture of 1 and
2 was mechanically fractured under anaerobic conditions (e.g. in nitrogen)
by ball milling (6.0 mmf, 890 mg) in a stainless steel twin-shell blender
(7.8 mmf, 24 mm long) at room temperature for 2 h at 60 Hz according to
the method previously reported.22) Residual oxygen in this system was re-
moved with Model 1000 Oxygen Trap (Chromatography Research Supplies)
coupled with Indicating Oxygen Trap (Chromatography Research Supplies).
The oxygen concentration was monitored with Oxygen analyzer (Toray En-
gineering Co., Ltd., LC 750/PC-120) and kept below 10 ppm. Copolymer-
ization was made on the mixture of 1 and 2 ranging in mole fraction of 1
from 0.05 to 0.5. Mechanochemical copolymerization of 1 and acrylamide
was performed in the same way as described above.
Results and Discussion
Characterization of Polymeric Prodrugs Synthesized
by Mechanochemical Solid-State Copolymerization We
selected Bz4MC as a model compound of 4MC derivatives.
1-Benzoyl-2-methacryloyloxyethylcarbamoyl-4-methylcate-
chol (1) was specially synthesized as a model monomer of
Bz4MC (Fig. 1). 6-O-Methacryloyl-D-galactose (2) was also
prepared as a water-soluble monomer.
We carried out the mechanochemical solid-state copoly-
merization of 1 and 2 to obtain a polymeric prodrug, Poly I.
The mechanochemical copolymerization of 1 and acrylamide
(AAm), which is a conventional water-soluble monomer, was
also carried out, and Poly II was obtained (Fig. 2). The poly-
mer conversion was determined by observing the disappear-
ance of vinyl protons and the appearance of the correspond-
1
ing alkyl protons of the polymer in the H-NMR spectra of
the resultant powders (Chart 1). The mechanochemical solid-
state copolymerization of these monomers proceeded to
completion to give the corresponding polymeric prodrugs.
This eliminates the need for any work-up such as is required
in reactions in the liquid state.
We observed the behavior of dissolution of the polymeric
prodrugs in water. Poly I (0.1) denotes the Poly I in which
the mole fraction of 1 is 0.1. Poly I (0.05), (0.1) and (0.2)
dissolved in water at room temperature. Poly I (0.3) swelled
in water but did not dissolve. Poly I (0.4) and (0.5) were also
Solubility of Polymeric Prodrugs Five kinds of polymeric prodrugs, in
Fig. 1. Structures of Bz4MC, 1-Benzoyl-2-methacryloyloxyethyl-4-methylcatechol (1) and 6-O-Methacryloyl-D-galactose (2)

1436
Vol. 50, No. 11
Fig. 2. Structures of Poly I and II Prepared by Mechanochemical Solid-State Polymerization
Poly I. In the case of Poly I (0.1) the concentration of
Bz4MC continues to increase for ca. 5 h and then tends to
decrease toward x-axis. The similar result was obtained for
Poly I (0.05) and (0.2). The concentration of 4MC increased
with increasing the reaction time. These results suggest that
Bz4MC released was hydrolyzed to produce 4MC. The total
amount of Bz4MC released, area under the curve shown in
Fig. 3, and the maximum concentration of 4MC indicated
that the hydrolysis of a polymeric prodrug proceeded to com-
pletion.
Chart 2 shows the hydrolysis of Poly I and the subsequent
reaction of Bz4MC. The rate constants of hydrolysis of poly-
meric prodrug and Bz4MC are defined as k1 and k2, respec-
tively. Two rate equations are written
ꢀd[P]/dtꢄk1 [P]
(1)
(2)
Chart 1. NMR Spectra of Poly I (0.1)
d[D]/dtꢄk1 [P]ꢀk2 [D]
insoluble in water. On the other hand, Poly II (0.05) dis- where [P]ꢄamount of Bz4MC bonded to the polymeric pro-
solved in water but the other Poly IIs, such as Poly II (0.1), drug per unit volume at time t and [D]ꢄconcentration of free
were insoluble. The molecular weight of Poly I was similar Bz4MC at time t. These equations give
to those of Poly II, and the number average molecular weight
[D]ꢄk1P0(exp(ꢀk1t)ꢀexp(ꢀk2t))/(k1ꢀk2)
(3)
(M ) was ca. 30000. These results suggest that monomer 2 is
ꢀn
superior to AAm in terms of synthesis of water-soluble poly- where P0ꢄtotal amount of Bz4MC in the polymeric prodrugs
meric prodrug.
per unit volume at tꢄ0.
Table 1 shows the M and heterogeneity (M /M , M is
The theoretical curves (solid curves) shown in Fig. 3 were
ꢀn ꢀw ꢀn ꢀw
weight-average molecular weight) of Poly I. The value in the obtained by fitting iteratively the rate constants, k1 and k2,
parentheses denotes the mole fraction of 1 in Poly I. The with the obtained data of hydrolysis using a nonlinear least-
number average molecular weight was similar to one another. squares method. The values of the rate constants, k1 and k2,
It was also shown that these polymeric prodrugs were of are listed in Table 2. The curve well fitted with the experi-
lower heterogeneity. In general, the polymer prepared by con- mental data. It was shown that Eq. 3 and Chart 1 properly de-
ventional radical-initiated solution polymerization possesses scribed the hydrolysis kinetics.
a broad molecular weight distribution, and its heterogeneity
It was shown in Table 2 that the value of k1 decreased with
is more than 2. The low heterogeneity is very important in increasing the mole fraction of 1 in Poly I. The value of k2
nature due to avoiding the scatter of the behavior in the body was almost equal to one another. We separately examined the
and the rate of drug release of polymeric prodrugs by molec- hydrolysis of Bz4MC under the same condition. The rate
ular weight.
constant of the hydrolysis of Bz4MC was 0.110 hꢀ1 and al-
Nature of Drug Release from Polymeric Prodrugs in most equal to the value of k2 shown in Table 2. As shown in
Vitro Bz4MC is a hydrophobic compound and slightly sol- Table 1, the molecular weight and heterogeneity of Poly I
uble in water. We carried out the hydrolysis of Poly I in the were similar to one another. Therefore, the difference of the
mixture of acetonitrile and pH 7.4 phosphate buffer solution value of k1 can be ascribed to the composition of polymeric
(1 : 1) to prevent the saturation of Bz4MC.
prodrug, the mole fraction of 1. This result suggests that the
Figure 3 shows the progressive changes in the concentra- rate constant of hydrolysis can be controlled by the mole
tion of Bz4MC and 4MC in the course of the hydrolysis of fraction of 1.

November 2002
1437
Fig. 3. Progressive Changes in the Concentration of Bz4MC (ꢀ) and 4MC (ꢁ) in the Course of Hydrolysis of Poly I in a Solution of a pH 7.4 Phosphate
Buffer and Acetonitrile
Chart 2
Table 1. Molecular Weight and Heterogeneity of Poly I
possessed narrow molecular weight distributions (low hetero-
geneity). This feature is of great value for highly functional-
ized polymeric prodrug.
Number average molecular weight
Compound
Heterogeneity
(M /M )
ꢀw ꢀn
(M )
ꢀn
The hydrolysis of polymeric prodrug proceeded to com-
pletion. It was shown that Bz4MC released was further hy-
drolyzed under the present experimental condition to produce
4MC. It can be suggested that the rate constant of hydrolysis
could be controlled by the composition, the mole fraction of
1 in Poly I. The correlationship between the rate constants of
hydrolysis and copolymer structure (e.g. the composition and
molecular weight) will be the subject of a forthcoming paper.
Poly I (0.05)
Poly I (0.1)
Poly I (0.2)
29000
28000
28000
1.16
1.10
1.10
Table 2. Rate Constants of Hydrolysis Derived from Eq. 3
k1 (hꢀ1
)
k2 (hꢀ1
)
Correlation coefficient
Acknowledgments This work is supported, in part, by Health Science
Research Grant (Research on Brain Science) from the Ministry of Health
and Welfare, which is greatly acknowledged.
Poly I (0.05)
Poly I (0.1)
Poly I (0.2)
0.585
0.299
0.140
0.115
0.118
0.116
0.990
0.995
0.983
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