Development of a water-soluble matrix metalloproteinase inhibitor as an intra-arterial infusion drug for prevention of restenosis after angioplasty

Article abstract of DOI:10.1021/jm020356g

To prevent restenosis after percutaneous transluminal coronary angioplasty (PTCA) and/or stenting of atherosclerotic stenosed arteries, we designed and developed two water-soluble matrix metalloproteinases (MMPs) inhibitors. The first inhibitor was monome

Full text of DOI:10.1021/jm020356g

J . Med. Chem. 2003, 46, 3497-3501
3497
Develop m en t of a Wa ter -Solu ble Ma tr ix Meta llop r otein a se In h ibitor a s a n
In tr a -a r ter ia l In fu sion Dr u g for P r even tion of Resten osis a fter An giop la sty
Takeshi Masuda and Yasuhide Nakayama*
Department of Bioengineering, National Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita,
Osaka 565-8565, J apan
Received August 19, 2002
To prevent restenosis after percutaneous transluminal coronary angioplasty (PTCA) and/or
stenting of atherosclerotic stenosed arteries, we designed and developed two water-soluble
matrix metalloproteinases (MMPs) inhibitors. The first inhibitor was monomeric in type and
was chemically synthesized by succinylation of the synthetic MMP inhibitor, N-hydroxy-5-
hydroxy-2(S)-methyl-4(S)-(4-phenoxybenzoyl)aminopentanamide (ONO-M11-335). The second
inhibitor was polymeric and was a radical copolymer of the vinyl derivative of ONO-M11-335
and a water-soluble monomer, N,N-dimethylacrylamide (DMAAm). For the second inhibitor,
NMR analyses and UV-vis spectra measurements showed that the content of the ONO-M11-
335 unit in the copolymers (Mn; ca. 10 000 and 20 000 by GPC measurements) was about 8 per
molecule. The MMP inhibitors were all highly soluble in water, even under neutral pH. The
succinylated derivative markedly inhibited MMP-2, MMP-9, and MMP-12 in vitro, as did ONO-
M11-335. In contrast the copolymers, which can maintain effective plasma levels for extended
periods by prevention of hepatic uptake, showed a ca. 100-fold reduced inhibition activity. Such
water-soluble MMP inhibitors, developed in this study, may potentially be useful as an intra-
arterial infusion drug for vascular injury.
In tr od u ction
Percutaneous transluminal angioplasty in coronary
and peripheral arteries (PTCA or PTA) has been widely
used for clinical treatment of atheroscleotic stenosis
using either balloon catheters or metallic stents. How-
ever, it has been reported that around 30-50% of
restenosis cases have occurred few months after balloon
angioplasty. Such a phenomenon remains an unsolved
problem. One of the major causes of the restenosis was
excessive tissue ingrowth (intimal hyperplasia), result-
ing from migration and proliferation of smooth muscle
cells (SMC), and is triggered by degradation of the
extracellular matrix by SMC derived MMP. MMP
inhibitors therefore offer potential as a therapeutic tool
for prevention of restenosis, and to this end, chemically
synthesized MMP inhibitors have been widely developed
over the past few decades.7 X-ray crystallographic
analyses of a single crystal of the MMP-inhibitor
complex has been revealed together with determination
of the enzyme’s active site and the interaction mecha-
nism between inhibitor and MMP.8 Such information
has been instrumental in the design of synthetic MMP
inhibitors.9 A number of MMP inhibitors, with varying
efficiency, have been described which are involved in
collagen peptidomimetics and nonpeptidomimetics.10
These have been further classified according to the
variety of their coordination groups binding to the zinc
atom localized on the enzyme active site such as
hydroxamate, phosphinate, thiol, and sulfodiimine.
However, since most of these MMP inhibitors show
extremely poor water solubility they are not maintained
at high levels in the plasma, which is not suitable for
an intra-arterial infusion drug.
Matrix metalloproteinases (MMPs) are classified into
a series of the proteinase family and contain over 20
species of zinc-dependent endopeptidases.1 The MMPs
facilitate the degradation and remodeling of the extra-
cellular matrix (ECM), including the interstitial and
basement membranes, fibronectin, elastin, and laminin.
The physiological role of the MMPs is to degrade ECM
components, which promote connective tissue remodel-
ing processes and which are expressed during the
morphogenetic stage of embryonic development and
differentiation, bone resorption, and tissue repair. The
MMPs are also associated with various oncologic or
pathological processes such as tumor invasion, rheu-
matoid joint destruction, uterine adenomyosis, and
cardiovascular disease.2
In normal tissue, expression of MMP is regulated at
the gene transcription level by extracellular stimulators3
such as cytokines, growth factors, and cell or matrix-
cell interactions. Once synthesized, they are secreted
from cells as pre-proenzymes which can be activated by
serine proteinases or other activated MMPs in the
extracellular space.4 In a defined activation mechanism,
gelatinase A (MMP-2) is activated by interacting with
a membrane type-1 MMP (MT1-MMP) that is activated
preliminary by furin and by anchorage to the cell
membrane surface.5 The activity of MMP is inhibited
by a natural tissue inhibitor of the metalloproteinase
(TIMP), which is also secreted by transcriptional regu-
lation and inhibits MMP activity by binding to the
catalytic domain of the activated MMP.6
In this study, we molecularly designed and developed
two types of water-soluble MMP inhibitors by either
derivatization of the carboxyl group or copolymerization
* To whom correspondence should be addressed. Tel: (+81) 6-6833-
5012 (ext 2434). Fax: (+81) 6-6872-8090. E-mail: nakayama@
ri.ncvc.go.jp.
10.1021/jm020356g CCC: $25.00 © 2003 American Chemical Society
Published on Web 07/03/2003

3498 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16
Masuda and Nakayama
Sch em e 1. Synthetic Scheme of Water-Soluble Matrix Metalloproteinase Inhibitors
Ta ble 1. Preparation of Poly(AEMA-ONO-co-DMAAm)
degree of derivatization
of MB unit (mol %)
feed of MB
(mol %)
yield
(%)
no. of MB
1H NMRa
UVb
Mn (×104)
Mw/Mn
unitsc/molecule
PA
PB
5
10
70
81
5.4
9.8
6.0
9.1
1.8
1.1
2.81
3.73
7.8
7.4
a
b
Calculated from integration value ratio of -CH3 protons of DMAAm vs aromatic ring protons of AEMA-ONO. All analytical samples
are prepared as 0.2 mg/mL of EtOH solution and molarity of ONO-M11-335 contained in sample solution was estimated by the molar
extinction coefficient of MB at 253 nm. The λmax was not different from monomer and copolymers. c Calculated from 1H NMR.
with a water-soluble monomer to enable water-solubility
of the synthetic MMP inhibitor ONO-M11-335. As for
the copolymers, controlled drug delivery by hydrolysis
in vivo was predicted. The potential abilities of the MMP
inhibitors for clinical applications in intra-vascular
surgery including PTCA and stenting are discussed.
formation between MA and 2-aminoethyl methacrylate
using DCC-HOBt as the coupling reagent. The amide
bond procedure proceeded quantitatively with little side
reactions or formation of byproducts resulting from the
hydroxamate group. Thereafter, the ONO-M11-335 ap-
pending vinyl monomer (MB) was polymerized with
DMAAm, in the presence of AIBN, as a conventional
radical polymerization initiator (Scheme 1). Table 1
summarizes the composition of the obtained copolymers.
The molecular weight of the copolymers (P A and P B)
was around 20 000 and 10 000, respectively. The degree
of derivatization of the MB unit in the copolymers was
calculated using two alternative methods. The first
method calculated the value from the ratio of the
integrals of the aromatic ring protons (δ ) 7.0-7.8 ppm)
of the MMP inhibitor unit with the dimethyl amide
protons (δ ) 2.6-3.4 ppm) of the DMAAm unit in the
1H NMR spectra. The second method determined the
value from the UV/vis absorption spectrum of the
copolymers using the absorption coefficient of the MB,
which has a maximum absorption at a wavelength of
253 nm in ethanol (ꢀ ) 16 230). There was good
agreement between the copolymer compositions calcu-
lated from 1H NMR spectra and from UV/vis spectra.
The degree of derivatization of MMP inhibitor unit in
the copolymers was around 5 mol % (P A) and 10 mol %
(P B), respectively. Thus, the number of the inhibitor
unit in the two copolymers was about 8 per molecules.
The succinylated derivative (MA) and two copolymers
Resu lts
P r ep a r a tion of Wa ter -Solu ble MMP In h ibitor s.
Two different molecular designs were used for the
water-solubilization of the synthetic MMP inhibitor
ONO-M11-335 (Scheme 1). The first approach involved
synthesis of a monomeric inhibitor (MA), whereas the
second approach involved synthesis of a polymeric
inhibitor (P A and P B).
The monomeric MMP inhibitor (MA) was synthesized
by derivatization of a succinic acid moiety to the ONO-
M11-335 through an ester bond. The esterification of
ONO-M11-335 was performed by nucleophilic attack of
the 5-OH group of ONO-M11-335 to the carbonyl group
of succinic anhydride in anhydrous pyridine at room
temperature. The unfavorable nucleophilic attack of the
nonprotected OH group of the hydroxamate moiety was
preferentially prevented to minimize production of an
amide derivative being negative to hydroxamic acid
method.
The polymeric inhibitors (P A and P B) were synthe-
sized by copolymerization of DMAAm with a vinyl
derivative of ONO-M11-335 (MB) by an amide bond

Water-Soluble MMP Inhibitor
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16 3499
Ta ble 2. Solubility of MMP Inhibitors in Various Aqueous
Solutions
concerned with structure and function, and the impor-
tance of the vascular ECM for the vascular reconstruc-
tion response to arterial injury, such as balloon and/or
stent angioplasty. ECM degradation, which has a
central role in SMC migration and proliferation during
the early stages of neointimal hyperplasia, depends on
a group of proteases known as MMPs. In mechanical
arterial injury, overexpression of various MMPs, includ-
ing stromelysin (MMP-3), collagenases (MMP-1), and
gelatinases (MMP-2 and MMP-9) occurs during SMC
migration.
solubility (µg/mL)
MMP inhibitor artificial bile buffer (pH 7.4) purified water
ONO-M11-335
432
>1000
>1000
>1000
307
>1000
>1000
>1000
372
398
MA
PA
PB
Ta ble 3. Human Matrix Metalloproteinase Inhibition
Activities
IC50 value (nM)a
Many different types of synthetic MMP inhibitors
have been developed and assessed in clinical trials. For
example marimastat,13 a synthetic hydroxamate type
MMP inhibitor, mimics the structure of collagen at the
site where MMP binds to the zinc ion, which is located
at the active site of the MMP and is held in a stereospe-
cific manner. Marimastat is widely known to inhibit the
activity of MMP-1, -2, -3, -7, and -9 but possesses a
relatively nonspecific selectivity for the MMPs. Other
peptide-mimetic hydroxamic acid type MMP inhibitors
include GM 6001,14 developed by Glycomed, Inc., which
has been studied for its SMC antiproliferation activity
in SMC migration assays and has been found to almost
completely inhibit SMC migration, without reducing the
number of cells available to migrate from the media to
intima. On the other hand, ONO-4817,15 a novel hy-
droxamic acid-based nonpeptide type orally active syn-
thetic MMP inhibitor, was developed in ONO Pharma-
ceutical Co., which shows a broad inhibitory activity
against MMPs except MMP-1 and MMP-7 at nanomolar
concentration levels.
MMP inhibitor
MMP-2
MMP-9
MMP-12
ONO-4817
ONO-M11-335
MA
0.73
0.65
1.10
50
2.10
0.80
2.80
83
0.45
3.50
2.60
120
PAb
PBb
100
200
260
a
b
50% inhibitory concentration. Exact molecular weights of
polymers were not clear; however, their molar concentration was
determined by a net inhibitor ratio involved in each polymers and
number-average molecular weight estimated from GPC.
(P A and P B) were all very soluble in water even under
neutral pH (Table 2).
In h ibition Activity for Hu m a n Ma tr ix Meta llo-
p r otein a se. ONO-M11-335 was a highly potent and
relatively selective MMP inhibitor for MMP-2 and
gelatinase B (MMP-9) compared with MMP-1 or MMP-
7. The IC50 values (50% inhibitory concentration) for
MMP-2, MMP-9, and MMP-12 (metalloelastase) were
0.65, 0.80, and 3.50 nM, respectively (Table 3). MA also
showed high MMP inhibition activities comparable to
ONO-M11-335, with IC50 values for MMP-2, MMP-9,
and MMP-12 of 1.10, 2.80, and 2.60 nM, respectively.
The IC50 values of MMP inhibitor-DMAAm copolymers
for MMP-2, MMP-9, and MMP-12 were also estimated
and were revealed to be 100, 200, and 260 nM for P A
and 50, 83, and 120 nM for P B.
In contrast, our group developed a new percutaneous
transluminal coronary angioplasty catheter with mul-
tiple functions for balloon inflation, local drug delivery,
and coronary perfusion.16-18 Water-soluble drugs can be
infused from a port located distal to the inflated balloon
during continuous blood perfusion via the perfusion
lumen. Recently, local administration of several agents,
including hepatocyte growth factor, docetaxel,17 and a
C-type natriuretic peptide,18 directly to the injured site
has attracted increasing interest as a potential thera-
peutic method for the prevention of restenosis.19 Local
drug delivery may allow a sufficient concentration of the
drug to be achieved at tissue while minimizing systemic
adverse effects. To use in intra-arterial infusion drug,
water solubility is necessary. The synthetic works have
been done for the design of water-soluble. For example,
Scozzafava et al. synthesized water-soluble sulfona-
mides by attaching water-solubilizing moieties such as
pyridine carboximide, carboxypyridinecarboxamide, etc.,
in which carbonic anhydrase inhibitors derivatized with
water-soluble tail structure showed topical activity as
an antiglaucoma drug.20 In this study, we first designed
two alternative approaches for water solubilization of
the MMP inhibitor ONO-M11-335 by applying simple
chemical reactions. As expected, both MMP inhibitors
were highly soluble in water even under neutral pH
(Table 2). The first inhibitor, which is monomeric
succinate derivative (MA), can potentially acquire high
doses at local areas using the above-mentioned multiple
functional balloon catheter. On the other hand, the
second inhibitor, which is copolymer with N,N-dimeth-
ylacrylamide (P A and P B), can potentially acquire an
Discu ssion
Both vascular constructive remodeling and neointimal
hyperplasia are major causes of restenosis in response
to arterial injury after balloon angioplasty. In stenting,
the possibility of constructive remodeling is almost
excluded. It has, however, been reported that 20-30%
of in-stent restenosis cases are observed a few months
after stenting, which may be mainly due to neointimal
hyperplasia, triggered by early SMC migration to the
luminal surface of the artery after angioplasty injury
and followed by SMC proliferation and ECM production.
Therefore, one of the therapeutic strategies for the
prevention of restenosis is the suppression of SMC
migration using immunosuppressive agents such as
FK506 and Rapamycin.11 Both these agents bind to the
cytosolic receptor. For instance, FK506 binds to FKBP12,
which is physically associated with the type I TGF-â
receptor and inhibits the SMC migration by blocking
the growth factor mediated signal transduction path-
ways. Recent reports have shown that stents coated
with these agents possessed sustained release from the
polymer matrix and resulted in remarkably reduced in-
stent restenosis.12
On the other hand, recent studies on the pathology
of restenosis have focused on the role of ECM, which is

3500 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16
Masuda and Nakayama
N-Hydroxybenztriazole (HOBt) was of special grade and
purchased from Peptide Institute Inc. (Osaka, J apan) and used
without further purification. 2-Aminoethyl methacrylate‚HCl
was purchased from Polyscience Inc., (Niles, IL) and other
chemical reagents were commercially obtained from Wako
Pure Chemical Industries, Ltd (Osaka, J apan). Anhydrous
solvents were distilled after drying.
effective plasma level for long periods by prevention of
hepatic uptake in addition to the local administration
activity.
The in vitro enzyme assay showed that the succinyl-
ated MMP inhibitor and the prodrug polymers main-
tained their inhibition activity for the human matrix
metalloproteinases after chemical modification. Com-
paring the MMP inhibition activities with ONO-4817,
which is only slightly soluble in water, the IC50 values
for each of the MMPs were approximately identical. Due
to the high water solubility of these water-soluble MMP
inhibitors, they offer many advantages for drug dose
dependent clinical trials and may suppress intimal
hyperplasia when the dose concentration of the MMP
inhibitors is increased. As for the prodrug polymers,
since the inhibition activities showed relatively good
retention they offer the potential for antirestenosis
therapy. However, the IC50 values for the prodrug
polymers indicated remarkably inferior inhibition ac-
tivities against all the MMPs compared to the mono-
meric MMP inhibitors. Such inactivation is attributed
to the steric hindrance of the MMP inhibitor moiety,
which inhibits other MMP groups from binding to the
enzyme’s active site. P A, the lower MB contents co-
polymer, showed about twice as much activity as P B.
However, copolymerization of non water-soluble syn-
thetic MMP inhibitors, such as ONO-4817, with mono-
mers of excellent water solubility, such as N,N-dimeth-
ylacrylamide, is an advantageous strategy to maintain
drug concentrations at high levels in plasma and is
expected to express prodrug-like controlled drug delivery
functions by hydrolysis in vivo. Further in vitro studies
of SMC migration assays using matrigel, in the presence
of MA, P A, and P B are currently in progress, as are in
vivo inhibition studies of experimental neointimal hy-
perplasia using local drug delivery systems with the
infusion balloon catheter.
Syn th esis: N-Hyd r oxy-5-ca r boxyleth ylca r bon yloxy-
2(S )-m e t h yl-4(S )-(4-p h e n oxyb e n zoyl)a m in op e n t a n a -
m id e (MA). To an anhydrous pyridine solution (5 mL) of ONO-
M11-335 (500 mg, 1.4 mmol) was added dropwise at 0 °C an
anhydrous pyridine solution (5 mL) of succinic anhydride (280
mg, 2.8 mmol), and the mixture was then stirred overnight at
room temperature. The reaction mixture was diluted with
AcOEt (150 mL) and washed with 1 N HCl (50 mL × 2) and
then with 5% NaHCO3 (50 mL × 2). The pH of the aqueous
layer was adjusted to pH 5 with 1 N HCl and then extracted
in AcOEt (50 mL × 2). After drying over MgSO4, the solvent
was removed by vacuum evaporation after filtration. The white
solid was purified by silica gel column chromatography (elu-
ent: chloroform/methanol, from 10/1 to 5/1) to give the desired
product as a white solid: yield 180 mg (28%); FAB-MASS m/n
459.18 [M + H+]; Mr ) 458.47 calcd for C23H26N2O8; 1H NMR
(D2O + Na2CO3) δ 0.98 (d, 3H, J ) 6.9 Hz, -CH3), 1.63-1.96
(m, 2H, -CH(CH3)CH2-), 2.21-2.28 (q, 1H, J ) 7.2 Hz,
-CH(CH3)-), 2.30 (t, 2H, J ) 6.0 Hz, -CH2CH2CO2H), 2.45
(t, 2H, J ) 6.0 Hz, -CH2CH2CO2H), 4.02-4.28 (m, 3H,
-CHCH2O-), 6.96-7.05 (m, 4H, m-H of -COC6H4- and o-H
of -OC6H5), 7.14 (t, 1H, J ) 7.5 Hz, p-H of -OC6H5), 7.33 (t,
2H, J ) 7.5 Hz, m-H of -OC6H5), 7.61 (d, 2H, J ) 7.5 Hz, o-H
of -COC6H4-). Anal. (C23H26N2O8) C, H, N.
N-Hyd r oxy-5-[2-[N-[2-(isop r op en ylca r bon yloxy)eth yl]-
ca r b a m oyl]et h ylca r b on yloxy]-2(S)-m et h yl-4(S)-(4-p h e-
n oxyben zoyl)a m in op en ta n a m id e (MB). To an anhydrous
DMF solution (5 mL) of MA (778 mg, 1.7 mmol), 2-aminometh-
acrylate‚HCl (566 mg, 3.4 mmol), HOBt (344 mg, 2.55 mmol),
and NEt3 (523 µL, 3.74 mmol) was added dropwise at -5 °C
DCC (421 mg, 2.0 mmol) in anhydrous DMF (5 mL), and the
mixture was stirred for 2 h followed by stirring overnight at
room temperature. The reaction mixture was extracted into
ethyl acetate and washed successively with three 50 mL
portions of 1 N HCl, three 50 mL portions of 5% NaHCO3, and
50 mL of brine, followed the separation of the organic layer,
drying over MgSO4, condensation, and purification by chro-
matography on silica gel (eluent: chloroform/methanol ) 15/
1) to give the desired product as a white solid: yield 536 mg
(55%); FAB-MASS m/ n 570.25 [M + H+]; Mr ) 569.61 calcd
for C29H35N3O9; 1H NMR (DMSO-d6) δ 1.04 (d, 3H, J ) 5.4
Hz, -CH3), 1.60-1.77 (m, 2H, -CH(CH3)CH2-), 1.87 (s, 3H,
-C(CH3)dCH2), 2.23-2.48 (m, 5H, -OCOCH2CH2CONH-
and -CH(CH3)-), 3.31 (m, 2H, -CONHCH2CH2OCO-), 4.07-
4.21 (m, 5H, -CONHCH2CH2OCO- and -CHCH2O-), 5.60
and 6.01 (s, 2H, -C(CH3)dCH2), 6.99-7.06 (t, 4H, J ) 8.1 Hz,
m-H of -COC6H4- and o-H of -OC6H5), 7.17 (t, 1H, J ) 8.1
Hz, p-H of -OC6H5), 7.43 (t, 2H, J ) 8.1 Hz, m-H of -OC6H5),
7.82 (d, 2H, J ) 8.1 Hz, o-H of -COC6H4-). Anal. (C29H35N3O9)
C, H, N.
Exp er im en ta l Section
Gen er a l Meth od s. 1H NMR spectra were recorded in
DMSO-d6 or D2O using tetramethylsilane (0 ppm) as an
internal standard with a 270 MHz NMR spectrometer (J EOL,
GX-270, Tokyo, J apan) at room temperature. UV-vis absorp-
tion spectra were measured on a Pharmaspec UV-1700 spec-
trophotometer (Shimadzu, Kyoto, J apan). Gel permeation
chromatography (GPC) analyses in N,N-dimethylformamide
were carried out with a HPLC-8020 instrument (Tosoh, Tokyo,
J apan) (column: Tosoh TSKgel R-3000 and R-5000). The
columns were calibrated with narrow weight distribution poly-
(ethylene glycol) standards.
In Vitr o En zym e Assa y. MMPs were extracted and
purified from human normal dermal fibroblast. To an assayed
buffer solution (40 µL) of the purified MMP (5.0 µM), p-
aminophenylmercuric acetate (APMA: 5.0 µL, 10 mM) was
added to activate the enzyme and preincubated at 37 °C for 1
h. A buffer solution (20 mL) of synthetic substrates, (7-
methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(N3-(2,4-dini-
trophenyl)-L-2,3-diaminopropionyl)-Ala-Arg-NH2 (McaPLDG-
LDpaAR)21 (130 µL; final concentration: 13.5 µM) with or
without MMP inhibitor were incubated at 37 °C for 5 min prior
to addition of the preincubated activate enzyme (50 µL/well)
to each solution. The enzymatic assay was performed at 37
°C after 15 min incubation. The enzymatic activities were
represented by an increase in the fluorescent intensity at 393
nm (excitation: 325 nm) per 1 min over the period of incuba-
tion. Inhibition activity, the IC50 values, was determined from
plots of percentage inhibition vs log of inhibitor concentration.
Ma ter ia ls. ONO-M11-335 was kindly supplied from ONO
Pharmaceutical Co., Ltd. (Osaka, J apan) and used as received.
P oly[N,N-d im et h yla cr yla m id e-co-N-h yd r oxy-5-[2-[N-
[2-(isop r op en ylca r bon yloxy)eth yl]ca r ba m oyl]eth ylca r -
b on yloxy]-2(S)-m et h yl-4(S)-(4-p h en oxyb en zoyl)a m in o-
p en ta n a m id e] (P A a n d P B). Two copolymers of MB and
DMAAm were prepared by radical copolymerization. A typical
procedure is as follows. A glass tube containing a mixture of
MB (80 mg, mmol), DMAAm (250 mg, mmol), 2,2′-azobis-
(isobutyronitrile) (AIBN) (4.36 mg, mmol), and DMF (11 mL)
was sealed under reduced pressure after three freeze-pump-
thaw cycles. After shaking at 60 °C for 24 h, the precipitate,
obtained by addition of ether, was separated from the solution
by filtration. Reprecipitation was carried out in a DMF -ether
system three times. The last precipitate was dried under
vacuum. The yield of poly(AEMA-ONO-co-DMAAm) was 230
mg (70%). The molecular weight was estimated by GPC
1
analysis: Mn )18 000; H NMR (in D2O) δ 0.96 (d, 24H, J )
5.4 Hz, -CH(CH3)-), 1.24-1.54 (m, 352H, CH2 and CH3 of

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main chain, -CH(CH3)CH2-), 2.12-2.14 (m, 8H, -CH(CH3)-),
2.31-2.98 (m, 848H, -N(CH3)2 and -OCOCH2CH2CONH-),
3.43-3.58 (m, 24H, -CONHCH2CH2OCO- and -CHCH2O-),
4.00 (m, 16H, -CHCH2O-), 6.95-7.03 (2d, 32H, J ) 8.4 Hz,
m-H of -COC6H4- and o-H of -OC6H5), 7.13 (t, 8H, J ) 8.4
Hz, p-H of -OC6H5), 7.33 (t, 16H, J ) 8.4 Hz, m-H of -OC6H5),
7.63 (d, 16H, J ) 8.4 Hz, o-H of -COC6H4-). The degree of
derivatization of MB unit was 5 mol % by UV spectral
measurement. Copolymer with 10 mol % of MB unit was
synthesized in a similar procedure by changing the monomer
ratio in the feed (data in the text).
Ack n ow led gm en t. We thank ONO Pharmaceutical
Co., Ltd. for a supply of ONO-M11-335 and measure-
ments of MMPI activity. This study was supported in
part by “Research Grants for Human Genome, Tissue
Engineering Food Biotechnology”, “Advanced Medical
Technology”, and “Aging and Health” from the Ministry
of Health, Labour and Welfare of J apan and by a Grant-
in-Aid for Scienific Research (B2-13450302, 2-14030095)
from the Ministry of Education, Science, Sports and
Culture of J apan.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectrum
of P A. This material is available free of charge via the Internet
at http://pubs.acs.org.
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