Novel hyaluronic acid-methotrexate conjugates for osteoarthritis treatment

Article abstract of DOI:10.1016/j.bmc.2009.04.063

Hyaluronic acid (HA) provides synovial fluid viscoelasticity and has a lubricating effect. Injections of HA preparations into the knee joint are widely used as osteoarthritis therapy. The current HA products reduce pain but do not fully control inflammation. Oral methotrexate (MTX) has anti-inflammatory efficacy but is associated with severe adverse events. Based on the rationale that a conjugation of HA and MTX would combine the efficacy of the two clinically evaluated agents and avoid the risks of MTX alone, we designed HA-MTX conjugates in which the MTX connects with the HA through peptides susceptible to cleavage by lysosomal enzymes. Intra-articular injection of our HA-MTX conjugate (conjugate 4) produced a significant reduction of the knee swelling in antigen-induced arthritis rat, whereas free MTX, HA or a mixture of HA and MTX showed no or marginal effects on the model. The efficacy of conjugate 4 was almost the same as that of MTX oral treatment. Conjugate 4 has potential as a compound for the treatment of osteoarthritis.

Full text of DOI:10.1016/j.bmc.2009.04.063

Bioorganic & Medicinal Chemistry 17 (2009) 4647–4656
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel hyaluronic acid–methotrexate conjugates for osteoarthritis treatment
a
a
a
a
a
Akie Homma a, , Haruhiko Sato , Akira Okamachi , Takashi Emura , Takenori Ishizawa , Tatsuya Kato ,
Tetsu Matsuura a, Shigeo Sato a, Tatsuya Tamura a, Yoshinobu Higuchi a, Tomoyuki Watanabe a,
Hidetomo Kitamura a, Kentaro Asanuma a, Tadao Yamazaki a, Masahisa Ikemi b, Hironoshin Kitagawa b,
Tadashi Morikawa b, Hitoshi Ikeya b, Kazuaki Maeda b, Koichi Takahashi b, Kenji Nohmi b, Noriyuki Izutani b,
Makoto Kanda b, Ryochi Suzuki b
*
a Research Division, Chugai Pharmaceutical Co., Ltd, 1-135, Komakado, Gotemba, Shizuoka 412-8513, Japan
b Research Center, Denki Kagaku Kogyo K. K, 3-5-1 Asahimachi, Machida, Tokyo 194-8560, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Hyaluronic acid (HA) provides synovial fluid viscoelasticity and has a lubricating effect. Injections of HA
preparations into the knee joint are widely used as osteoarthritis therapy. The current HA products
reduce pain but do not fully control inflammation. Oral methotrexate (MTX) has anti-inflammatory effi-
cacy but is associated with severe adverse events. Based on the rationale that a conjugation of HA and
MTX would combine the efficacy of the two clinically evaluated agents and avoid the risks of MTX alone,
we designed HA–MTX conjugates in which the MTX connects with the HA through peptides susceptible
to cleavage by lysosomal enzymes. Intra-articular injection of our HA–MTX conjugate (conjugate 4) pro-
duced a significant reduction of the knee swelling in antigen-induced arthritis rat, whereas free MTX, HA
or a mixture of HA and MTX showed no or marginal effects on the model. The efficacy of conjugate 4 was
almost the same as that of MTX oral treatment. Conjugate 4 has potential as a compound for the treat-
ment of osteoarthritis.
Received 5 March 2009
Revised 27 April 2009
Accepted 28 April 2009
Available online 3 May 2009
Keywords:
Hyaluronic acid
Methotrexate
Osteoarthritis
Targeting drug delivery system
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
an effective carrier that would deliver MTX to the synovium and
lower the systemic side effects is desirable.
Osteoarthritis (OA) is characterized by degeneration of articular
cartilage1–4 and is commonly described as a noninflammatory dis-
ease. However, there is growing evidence suggesting that synovial
inflammation is reflected in many of the signs and symptoms of
the disease.5,6 It is therefore anticipated that inhibition of the
inflammatory component of OA may provide effective therapeutics
for the treatment of OA.7,8
Oral methotrexate (MTX, Fig. 1) provides a very effective treat-
ment to control the synovial inflammation of rheumatoid arthritis
(RA).9,10 Several lines of evidence have suggested that some path-
ological changes observed in the synovial inflammation of RA are
also observed in that of OA.6,11 It seems likely that MTX would
be effective in altering at least some aspects of the OA pathogenic
process. However, MTX is frequently associated with adverse
events such as pneumonitis, liver fibrosis and myelosuppression
that limit the application of the drug to the other arthritic diseases
such as OA.12,13 Intra-articular injection of MTX is a possible alter-
native route to reduce adverse effects while maintaining the anti-
proliferative and anti-inflammatory effects of the drug. Therefore,
Hyaluronic acid (HA) is an endogenous polysaccharide providing
synovial fluid viscoelasticity and a lubricant effect.14–16 Intra-articu-
lar HA is widely used as a symptom-modifying treatment for OA of
the knee.1,4,7,17–24 Intra-articularly injected HA is distributed in the
synovium, and synovial cells have a mechanism for incorporating
HA through the cell surface receptors such as CD4422,25,26 for HA. It
has been reported that HA can be used as a carrier when coupled
with various agents.27–39 Those conjugates are internalized into
the cell through cell surface receptors, followed by intracellular re-
lease of the active drug.40 In this study, we designed HA–MTX conju-
gates based on the rationale that a conjugation of HA and MTX would
combine the efficacy of two clinically evaluated agents and avoid the
risks of MTX. Current HA products reduce pain but do not fully con-
trol inflammation. We anticipate that HA–MTX conjugates would be
H2N
N
N
N
N
N
O
H
N
NH2
OH
OH
O
O
* Corresponding author. Tel.: +81 550 87 8643.
E-mail address: honmaake@chugai-pharm.co.jp (A. Homma).
Figure 1. Methotrexate (MTX).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.063

4648
A. Homma et al. / Bioorg. Med. Chem. 17 (2009) 4647–4656
a safe and more efficacious intra-articular injection alternative to
current HA products.
Herein we report the design of new HA–MTX conjugates. The
conjugates demonstrated antiproliferative and anti-inflammatory
efficacy in vitro and in vivo suggesting their capability to be a pro-
totype for a future OA drug.
the coupling reaction between 18 and HA, and so the ratio of isomers
was determined again by NMR analysis after conjugation. Similar
equilibrium of migration was observed in the case of conjugate 4.
The pteridine part of 23 shown in Scheme 3 was pre-synthesized be-
cause of ease of use. The MTX binding ratio was calculated by the
dividing the concentration of the MTX by the concentration of the
disaccharide unit (GlcU-GlcNAC). These concentrations were ob-
tained by gel permeation chromatography (GPC) analysis of the con-
jugates. GPC analysis determines both the binding ratio and the
molecular weight (MW) of the conjugates at the same time and thus
we demonstrated that the HA–MTX conjugates prepared have a MW
as high as HA (ꢀ2000 kDa). Actually, we conducted a 1H NMR anal-
ysis and identified the acetyl of HA and the 7-position proton of the
pteridine ring; however, the integration ratio was not robust be-
cause the HA peaks tended to be broad, and the respective pteridine
7-position proton peaks of the free MTX and the HA–MTX conjugate
did not separate well. For the reaction of sodium hyaluronate with
compounds 10a, 10b, 18 and 25, tris[2-(2-methoxyethoxy)ethyl]-
amine was used to carry out the reaction under non-acidic
conditions. This amine was also expected to contribute to the
homogenization in the reaction as a phase transfer catalyst.52 As a
matter of fact, the reaction mixture of the water–THF solution ap-
peared homogeneous and elastic gel formed was easy to stir; the de-
sired conjugates were obtained by basic hydrolysis of the methyl
ester of the Glu of the MTX. When equivalents of 0.05–0.1 of com-
pound 25 were used, the MTX binding ratio of 4 became 0.5–3%. Con-
jugates 1, 2 and 3 were synthesized the same as 4. During the
coupling step, the MW of the conjugates tended to decrease. HA with
a high MW (ꢀ2000 kDa) is known to be the preferred analgesic for
knee joints of OA patients.1,7,17–22 Therefore, it is critical that the
HA–MTX conjugate maintains a MW as high as that of HA. To meet
the requirement, the conjugation was optimized by using 1:1
water–THF solvent and 3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzo-
triazine (HOOBt); the reactions were carried out at 5 °C for 20 h. The
MTX binding ratios for conjugates 1, 2, 3 and 4 were, respectively,
3.1%, 2.7%, 1.3% and 1.2% with MWs of 1610, 1610, 1850 and
1780 kDa.
2. Design of conjugates
The template structure of the conjugates is shown in Figure 2.
Several studies have suggested that conjugates of MTX coupled
to drug carriers appear to increase therapeutic efficacy and reduce
the side effects of MTX.12,13,41–48 However, use of HA as a drug car-
rier to target MTX to the joints of OA patients has not been re-
ported. Due to the properties of HA, HA–MTX conjugates can
preferably accumulate in the synovium and be internalized into
synovial cells through cell surface HA receptors. MTX acts inside
the cell, thus the release of a pharmacologically active form of
MTX from HA is important. We therefore introduced a lysosomal
enzyme-sensitive peptide chain into the HA–MTX conjugate. We
also introduced linkers as well as peptides to the conjugates to
avoid a potential steric barrier to the proteases by the HA back-
bone. In the present study, we selected 4,7,10-trioxa-1,13-tridec-
anediamine (PEG13) as the linker, which is considered to be of
sufficient length so as not to be a steric hindrance. To prove the
concept, we synthesized three different types of HA–MTX conju-
gates. Type 1 (conjugates 1 and 2) has no peptide. Type 2 (conju-
gate 3, Gly-Phe-Leu-Gly49) and type 3 (conjugate 4, Asn-Phe-
Phe50) have peptide chains known to be susceptible to cleavage
by lysosomal enzymes.
3. Synthesis
The synthesis of conjugates 1–4 is described in Schemes 1–3. To
avoid epimerization of amino acids, the peptide was elongated to-
ward the N-terminal.51 In the case of 1 and 2 without a peptide,
the control of the
3 contained a mixture of
mixture of (also 3/1) because of the equilibriumof the migration.
a
- or
c
a/c
- connection was almost perfect; however,
of 3/1. Compound 18 was obtained as a
4. Antiproliferative effect on human synovial fibroblasts
a/c
stimulated by TNF-
a
The isomers were detectable and separable by HPLC. However, pure
compound 18 was not obtained because of re-migration while
removing the solvents. The equilibrium may have occurred during
Effects of the HA–MTX conjugates with or without peptide
chains on cell proliferation were examined using human
MTX
HA
O
γ
Peptide chain
Linker
H2N
N
N
N
O
OH
N
N
O
O
NH2
O
HO
N
H
OH
O
O
HO
O
O
NHAc
OH
n
α
H2N
N
N
N
O
OH
γ
O
H
N
N
N
H
N
H2N
N
N
N
O
O
NH2
O
N
N
O
N
O
O
O
α
O
H
NH2
OH
N
N HA
H
O
O
N HA
H
H
conjugate 1
conjugate 2
H2N
N
N
O
OH
H2N
N
N
HO
O
NH2
O
N
N
N
N
H
H
N
N
N
N
O
NH2
O
NH2
O
N
H
N
H
N
H
N
H
N
O
N
H
N
H
N
N
O
O
H
O
O
O
O
O
O
O
O
O
O
O
H
H
Gly-Phe-Leu-Gly
Asn-Phe-Phe
N HA
H
N HA
H
conjugate 3
conjugate 4
Figure 2. Drug design and structures of conjugates 1–4.

A. Homma et al. / Bioorg. Med. Chem. 17 (2009) 4647–4656
4649
a
b
O
O
O
H2N
O
O
N
H
H2N
O
O
NH2
O
5
H
O
O
O
O
N
= R1
OMe
O
O
O
O
O
O
N
H
O
O
O
O
N
H
H2N
N
N
N
c, e
c, d
N
O
Z
6a
N
N
H2N
N
N
N
R Boc
N
NH2
Z
O
.
Br HBr
(BPT.HBr)
R Boc
N
= R2
O
O
H
N
OH
8a; R = R1
8b; R = R2
NH2
O
9a; R = R1
9b; R = R2
7
N
H
N
H
O
O
OMe
6b
H2N
H2N
N
N
N
N
N
H
O
O
NH2
N
OMe
f
O
1
O
O
O
O
N
H
O
O
O
NH2
NH2
10a
N
N
N
g, h
N
N
H
N
NH2
N
H
O
2
O
10b
OMe
Scheme 1. Reagents and conditions: (a) Boc2O, THF, À5 °C; (b) Z-Glu(OMe)-OH or Z-Glu(OH)-OMe, EDC–HOBt, DMF, 0 °C to rt; (c) H2, Pd/C, methanol, rt; (d) (4-
benzyloxycarbonylmethylamino)benzoic acid (7), EDC–HOBt, DMF, 0 °C to rt; (e) BPTÁHBr, DMF, 65 °C; (f) TFA, 0 °C; (g) sodium hyaluronate, tris[2-(2-methoxyeth-
oxy)ethyl]amine, EDC–HOOBt, THF–H2O, 5 °C; (h) NaOH aq, 5 °C.
a
b, c
b, d
Leu-Gly
HN PEG
Phe-Leu-Gly HN
Z
Gly HN PEG
Boc
PEG
Z
Z
Boc
Boc
5
N
H
N
H
N
H
11
12
13
O
H
N
Z
PEG
b, e
b, f
Gly-Phe-Leu-Gly HN
Boc
N
b, g
PEG
Gly-Phe-Leu-Gly HN
Boc
Z
N
H
H
14
15
N
O
OMe
Z
7
OH
O
H2N
N
N
N
N
N
N
Z
O
H
b, h
O
H
N
N
NH2
PEG
Gly-Phe-Leu-Gly HN
Boc
Gly-Phe-Leu-Gly HN PEG
Boc
N
H
O
N
H
O
16
17
O
OMe
O
OMe
H2N
N
N
O
N
N
O
O
= PEG
N
j, k
O
H
N
i
NH2
O
3
Gly-Phe-Leu-Gly HN
O
O
NH2
O
18
O
OMe
Scheme 2. Reagents and conditions: (a) Z-Gly-OH, EDC–HOSu, DMF, 0 °C to rt; (b) H2, Pd/C, methanol, rt; (c) Z-Leu-ONp, Et3N, DMF, rt; (d) Z-Phe-OH, EDC–HOBt, DMF, 0 °C to
rt; (e) Z-Gly-OH, EDC–HOBt, DMF, 0 °C to rt; (f) Z-Glu(OMe)-OH, EDC–HOBt, DMF, 0 °C to rt; (g) (4-benzyloxycarbonylmethylamino)benzoic acid (7), EDC–HOBt, DMF, 0 °C to
rt; (h) BPTÁHBr, DMF, 65 °C; (i) TFA, 0 °C; (j) sodium hyaluronate, tris[2-(2-methoxyethoxy)ethyl]amine, EDC–HOOBt, THF–H2O, 5 °C; (k) NaOH aq, 5 °C.
fibroblast-like synoviocytes (HFLS) stimulated by TNF-
a
. As shown
as a biologically active form inside the cells. We have not estab-
lished a method for analyzing the metabolism of HA–MTX conju-
gates so we prepared MTX analogs with the peptides of
conjugates 3 and 4, MTX-Gly-Phe-Leu-Gly and MTX-Asn-Phe-Phe,
and examined whether they were cleaved by cathepsins. MTX-
monopeptides are known to have activity42–46,53 or be further
cleaved to MTX by a peptidase such as a carboxypeptidase and,
in our preliminary studies, both Gly-Phe-Leu-Gly and Asn-Phe-
Phe were cleaved by cathepsins B, D and L to produce MTX,
MTX-Gly or MTX-Asn. These results suggest that conjugates 3
and 4 are likely to be cleaved by cathepsins and release biologically
in Table 1, conjugates lacking a peptide chain (1 and 2) showed no
suppressive effects. In contrast, conjugates 3 (Gly-Phe-Leu-Gly)
and 4 (Asn-Phe-Phe) clearly inhibited proliferation of HFLS, indi-
cating that the peptide chains are required for the inhibitory effect
of HA–MTX conjugates on HFLS proliferation. As mentioned above,
synovial cells have a mechanism for incorporating HA through cell
surface receptors for HA such as CD44. HA–MTX conjugates can
also be incorporated into the synovial cells in a similar manner.
It is therefore likely that the HA–MTX conjugates were cleaved
by lysosomal enzymes such as cathepsins and the MTX released

4650
A. Homma et al. / Bioorg. Med. Chem. 17 (2009) 4647–4656
a
b, a
b, c
Boc
Boc
Boc
PEG
PEG
PEG
Z Phe N
H
Z Phe-Phe N
H
Z Asn-Phe-Phe
N
H
N
H
N
H
N
H
5
19
20
21
H2N
N
N
N
H
N
O
N
N
Boc
PEG
H
N
b, d
b, e
O
Z
Asn-Phe-Phe
N
H
N
NH2
PEG Boc
H
Asn-Phe-Phe
OMe
N
H
N
H
O
H2N
N
N
N
O
OMe
22
24
N
N
O
23
NH2
OH
O
H2N
N
N
N
O
N
N
O
O
= PEG
H
N
O
g, h
f
NH2
O
4
Asn-Phe-Phe
OMe
N
H
O
O
NH2
O
O
25
Scheme 3. Reagents and conditions: (a) Z-Phe-OH, EDC–HOBt, DMF, 0 °C to rt; (b) H2, Pd/C, methanol, rt; (c) Z-Asn-OH, EDC–HOBt, DMF, 0 °C to rt; (d) Z-Glu(OMe)-OH, EDC–
HOBt, DMF, 0 °C to rt; (e) 23, EDC–HOBt, DMF, rt; (f) TFA, 0 °C; (g) sodium hyaluronate, tris[2-(2-methoxyethoxy)ethyl]amine, EDC–HOOBt, THF–H2O, 5 °C; (h) NaOH aq, 5 °C.
Table 1
Effects of the peptide chain of HA–MTX conjugates on inhibition of human synovial
fibroblasts
Vehicle (n=6)
A
HA (n=7)
2.0
Inhibition of cell proliferation IC50, l
mol/L (a)
3 (n=7)
4 (n=7)
Compounds
HA
1
2
3
4
—
(>1.0)
(1.0)
(1.0)
(0.01)
(0.01)
—
1.5
1.0
0.5
0.0
-0.5
>77
>67
0.30
0.35
0.05
MTX
*
*
*
Data represent IC50 values of the compounds converted to equivalent concentra-
tions of MTX.
2
4
6
8
10 12 14 16 18 20
Days after challenge
0
a
Equivalent to the concentration of HA (mg/mL).
*
P<0.05 vs HA
active MTX analogs inside the cell. Further studies are required to
clarify the mechanism of conjugates 3 and 4 in synovial cells.
B
Vehicle (PBS) (n=6)
MTX (n=6)
2.0
1.5
1.0
0.5
0.0
5. Anti-inflammatory effect in rat antigen-induced arthritis
The in vivo anti-inflammatory effects of the HA–MTX conju-
gates were evaluated in antigen-induced arthritis rats. Arthritis
was induced in one knee joint by intra-articular injection of meth-
ylated bovine serum albumin (mBSA).54–56 Conjugates 3, 4, HA
(at a dose of 0.3 mg as HA) and vehicle (saline) were intra-articu-
larly injected once a week from 7 days before inducing arthritis.
MTX, at the dose of 0.5 mg/kg and vehicle (PBS) were administered
orally 5 times a week from 7 days before inducing arthritis. Conju-
gate 4 showed significant reduction of knee swelling but HA did
not (Fig. 3A). Conjugate 3 having a similar MTX binding ratio and
MW of HA showed only tendency to inhibit knee swelling, suggest-
ing that cleavage of the conjugate in vivo depends on the peptide
composition. In quantifying the AUC (in this case, joint swelling
over time), the efficacy of conjugate 4 was almost the same as
the oral treatment of MTX (Fig. 3C). The weekly dosage of MTX
administered was ꢀ10 nmol for conjugate 4 and 1500 nmol for oral
MTX.
In the comparison of the efficacy of intra-articular injection of
HA–MTX conjugate 4 with free MTX, HA, and a mixture of HA
and MTX in antigen-induced arthritis rats, conjugate 4 showed a
significant reduction in knee swelling, whereas free MTX did not
(Fig. 4). Free HA and HA + MTX showed only marginal suppression
of joint swelling. These results are consistent with our theory that
intra-articularly-injected free MTX or a mixture of HA and MTX
disappear from the joint cavity, but HA–MTX conjugates remain
much longer.
*
*
*
*
*
*
0
2
4
6
8
10 12 14 16 18 20
Days after challenge
-0.5
*
P<0.05 vs PBS
P<0.05
P<0.05
P<0.005
C
35
30
25
20
15
10
5
0
Vehicle HA
3
4
PBS MTX
p.o.
i.a.
Figure 3. Effects of HA, conjugates 3, 4 and MTX on knee swelling in rats with
antigen-induced arthritis. Knee swelling, the difference in width between the right
and left knee was measured from days 0 to 20 (A and B) and the AUC for knee
swelling (C) calculated. Results are expressed as mean SE or mean + SE.

A. Homma et al. / Bioorg. Med. Chem. 17 (2009) 4647–4656
4651
gaku Kogyo, K. K. All solvents were purchased from Kanto Chemical
Co., Inc. 4-Methylaminobenzoic acid was purchased from Tokyo
Chemical Industry Co., Ltd. 6-Bromomethyl-pteridine-2,4-diamine
trihydrobromide (BPTÁHBr) was purchased from Ube Industries,
Ltd. All other compounds were of the best available commercial
grade. 1H NMR spectra were recorded on a JEOL ECP 270 or
500 MHz or a Mercury 300 MHz instrument in CDCl3, DMSO-d6 or
deuterium oxide solutions. Low-resolution mass spectra were
determined on LC/PDA/MS (HP 1100/TSP UV 6000/LCQ Classic,
LC–MS) system using Cadenza CD-C18 (3.0 mm I.D. Â 20 mm) col-
umn at 35 °C. k was 190–400 nm total area with retention times
evaluated in minutes. The solvents ran as follows: (A) 0.05% TFA,
H2O, (B) 0.05% TFA, MeCN, gradient (A/B): 95/5–0/100 (9.5 min), 0/
100 (2.5 min), flow rate: 1.0 mL/min. The binding ratio of MTX and
the MW of HA–MTX conjugates were determined by GPC on an
LC/PDA/RI (Waters Alliance 2695/2996/2414) system using Shodex
OHpak SB-806 HQ (8.0 mm I.D. Â 300 mm) column at 40 °C. RI was
analyzed at 35 °C. k was detected at 259 nm. The solvent ran as fol-
lows: 50 mM sodium phosphate (pH 6.0), Flow rate: 0.3 mL/min,
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
A
Vehicle (n=10)
MTX (n=10)
HA (n=10)
HA+MTX (n=11)
4 (n=11)
*
*
*
*
0
2
4
6
8
10
12
14
Days after challenge
*
P<0.05 vs HA
P<0.0005
P<0.005
B
P<0.05
P<0.05
35
30
25
20
15
10
injection volume: 100 lL.
7.1.1. 4-[(2,4-Diaminopteridin-6-ylmethyl)-methylamino]benzoic
acid (23)
7.1.1.1. 4-Methylaminobenzoic acid methyl ester.57
A dry
methanol (500 mL) solution of 10.28 g (68.0 mmol) of 4-methyla-
minobenzoic acid was cooled by ice bath. Then 7.4 ml
(102.0 mmol) of SOCl2 was added. After 5 min in an ice bath, the
reaction mixture was refluxed for 3 h. The reaction mixture was
then quenched with saturated NaHCO3 at 0 °C and extracted with
ethyl acetate. The organic layer was washed with brine, dried with
anhydrous sodium sulfate, filtered and evaporated. The product
was purified by silica gel column chromatography (eluent; chloro-
form/methanol, 100/5) to give the titled ester as a white solid
(10.96 g, 98%). 1H NMR (270 MHz, CDCl3) d: 2.88 (d, 3H,
J = 4.9 Hz), 3.85 (s, 3H), 4.16 (s, 1H), 6.55 (d, 2H, J = 8.9 Hz), 7.87
(d, 2H, J = 8.9 Hz).
5
0
Vehicle MTX
HA HA+MTX
4
Figure 4. Effects of HA, MTX, HA + MTX and conjugate 4 on knee swelling in rats
with antigen-induced arthritis. Knee swelling (the difference in width between the
right and left knee) was measured from days 0 to 14 (A), and the AUC for knee
swelling was calculated (B). Results are expressed as mean SE or mean + SE.
7.1.1.2. 4-[(2,4-Diaminopteridin-6-ylmethyl)-methylamino]ben-
zoic acid methyl ester.58–60 4-Methylaminobenzoic acid methyl es-
ter (10.96 g, 66.3 mmol) and BPTÁHBr (22.29 g, 66.3 mmol) were
dissolved in 200 mL of N,N-dimethylacetamide (DMA). After being
kept at 60–70 °C for 1 day, the reaction mixture was cooled to 0 °C,
and ethyl acetate and saturated NaHCO3 were added. The precipitate
was filtered and dried at 30 °C for 1 day. The titled ester (21.54 g,
63.5 mmol, 96%) was obtained as a yellow solid. 1H NMR (270 MHz,
DMSO-d6) d: 3.25 (s, 3H), 3.74 (s, 3H), 4.84 (s, 2H), 6.85 (d, 2H,
J = 8.9 Hz), 7.30 (br, 2H), 7.75 (d, 2H, J = 8.9 Hz), 8.21 (br, 1H), 8.43
(br, 1H), 8.66 (s, 1H). LC–MS m/z: 340.1 (M+H)+.
6. Conclusion
We synthesized novel HA–MTX conjugates with a peptide chain
and linker and examined the potential of these compounds have as
anti-arthritic drugs using both in vitro and in vivo assay systems.
One HA–MTX conjugate (4) was found to have both antiprolifera-
tive and anti-inflammatory effects. The antiproliferative effect of
the conjugates on HFLS in comparison with non-peptide conju-
gates indicates that enzymatic cleavage of the peptide is requisite
for exerting activity. In antigen-induced arthritis rats, intra-articu-
larly-injected conjugate 4 suppressed knee swelling as potently as
does oral MTX. However, intra-articularly injected free MTX and
the mixture of HA and MTX showed little or no effect in the anti-
gen-induced rats, probably due to rapid clearance from the joint
cavity. The dose of the MTX of conjugate 4 injected was approxi-
mately one hundred fiftieth the amount of an oral dose of MTX. Ta-
ken together, these results suggest that conjugate 4 is a promising
lead compound for the development of safe and effective future OA
drugs.
7.1.1.3. 4-[(2,4-Diaminopteridin-6-ylmethyl)-methylamino]ben-
zoic acid (23).61 16.54 g (48.7 mmol) of 4-[(2,4-diaminopteridin-6-
ylmethyl)methylamino]-benzoic acid methyl ester was dissolved in
300 mL of DMSO and aqueous NaOH (5.85 g, 164.2 mmol of NaOH
dissolved in 70 mL of water) was added to the solution. After stirring
for 3.5 h, 400 mL of water was added to the reaction mixture. The
solution was cooled to 0 °C and neutralized to pH 5 with a 10% acetic
acidsolution. Theprecipitate was filteredanddriedat30 °C. The titled
compound(12.1 g,37.2 mmol, 76%)wasobtainedasayellowsolid.1H
NMR (270 MHz, DMSO-d6) d: 3.22 (s, 3H), 4.78 (s, 2H), 6.82 (d, 2H,
J = 8.6 Hz), 7.43 (br, 1H), 7.70 (br, 1H), 7.73 (d, 2H, J = 8.6 Hz), 8.58
(s, 1H). LC–MS m/z: 326.1 (M+H)+.
7. Experimental
7.1. Chemistry
All amino acid derivatives, 1-hydroxybenzotriazole monohy-
drate (HOBtÁH2O) and 1-ethyl-3-(3-dimethylaminopropyl)carbodi-
imide hydrochloride (EDCÁHCl) were purchased from Watanabe
Chemical Industries, Ltd. Hyaluronic acid was supplied by Denki Ka-
7.1.2. N-tert-butoxycarbonyl-4,7,10-trioxa-1,13-tridecandiamine
(Boc-NH-PEG-NH2, 5)62
To a solution of 50 g (0.227 mol) of 4,7,10-trioxa-1,13-tridecan-
diamine in dry THF (80 mL) under an nitrogen atmosphere at a

4652
A. Homma et al. / Bioorg. Med. Chem. 17 (2009) 4647–4656
temperature of –5 °C was added dropwise di-tert-butyl dicarbon-
ate (Boc2O, 14.6 g, 0.07 mol, 0.3 equiv) in dry THF (80 mL). After
1 h, THF was evaporated in vacuo and to the resulting mixture
was added 1 N HCl (to pH 4) at a temperature of 0 °C and extracted
with ethyl acetate (500 mL) to remove the diprotected byproduct.
To the aqueous layer was added a Na2CO3 solution (to pH 10) and
brine and stirred for an hour. Then the solution was extracted with
ethyl acetate and the organic layer was dried over sodium sulfate
and evaporated. The product was purified by silica gel column
chromatography (eluent; chloroform/methanol/17% NH3aq, 100/
10/1), obtaining 5.84 g (0.018 mol, 8.0%) of desired compound 5
as an oil. 1H NMR (300 MHz, CDCl3) d: 1.44 (s, 9H), 1.71–1.80 (m,
4H), 2.06 (br, 2H), 2.83 (t, 2H, J = 6.6 Hz), 3.22 (q, 2H, J = 6.0 Hz),
3.52–3.67 (m, 12H), 5.09 (br, 1H). LC–MS m/z: 321.0 (M+H)+.
of dry DMF. To the solution 4-(benzyloxycarbonylmethylami-
no)benzoic acid (7, 509 mg, 1.78 mmol, 1.20 equiv), HOBtÁH2O
(228 mg, 1.49 mmol, 1.0 equiv) and EDCÁHCl (341 mg, 1.78 mmol,
1.20 equiv) were added at 0 °C. The reaction mixture was warmed
to room temperature, stirred for 18 h, and ethyl acetate added. The
organic layer was washed with saturated NaHCO3 and brine, dried
over sodium sulfate and evaporated. The desired compound 8b
was purified by silica gel column chromatography (eluent; dichlo-
romethane/methanol, 100/5) to afford 1.06 g (1.45 mmol, 97%) as
colorless oil. 1H NMR (270 MHz, CDCl3) d: 1.43 (s, 9H), 1.73–1.82
(m, 4H), 2.06–2.39 (m, 2H), 2.45–2.68 (m, 2H), 3.17–3.31 (m,
2H), 3.35 (s, 3H), 3.36–3.49 (m, 2H), 3.56–3.63 (m, 12H), 3.65 (s,
3H), 4.61 (br, 1H), 5.10 (br, 1H), 5.18 (s, 2H), 7.00 (br, 1H), 7.30–
7.36 (m, 8H), 7.81 (d, 2H, J = 8.4 Hz). LC–MS m/z: 731.3 (M+H)+.
7.1.3. Z-Glu(OMe)-NH-PEG-NH-Boc (6b)63,64
7.1.7. 4-Z-N(Me)C6H4-C(@O)-Glu-OMe(NH-PEG-NH-Boc) (8a)
To a solution of 500 mg (1.56 mmol) of 5 in 5 mL of dry N,N-
Substitution of a
-derived one and the use of 7 (532 mg, 1.86 mmol, 1.20 equiv),
HOBtÁH2O (237 mg, 1.66 mmol, 1.0 equiv) and EDCÁHCl (357 mg,
1.86 mmol, 1.20 equiv) provided -compound 8a (931 mg,
c-derived compound 6a (928 mg, 1.55 mmol)
dimethylformamide (DMF) was added N-
a-carbobenzoxy-
L
-glu-
for a
tamic acid -methyl ester (Z-Glu(OMe)-OH, 461 mg, 1.56 mmol,
c
1.00 equiv), HOBtÁH2O (119 mg, 0.78 mmol, 0.50 equiv) and
EDCÁHCl (329 mg, 1.72 mmol, 1.10 equiv) at 0 °C. After being kept
at 0 °C for 2 h, the reaction mixture was warmed to room temper-
ature. After 3 h ethyl acetate was added. The organic layer was
washed with saturated NaHCO3 and brine, dried over anhydrous
sodium sulfate and evaporated. The product was purified by silica
gel column chromatography (eluent; dichloromethane/methanol,
100/3) to afford 892 mg (1.49 mmol, 95.7%) of the desired com-
pound 6b as colorless oil. 1H NMR (270 MHz, CDCl3) d: 1.43 (s,
9H), 1.72–1.76 (m, 4H), 1.89–2.22 (m, 2H), 2.38–2.57 (m, 2H),
3.18–3.21 (m, 2H), 3.35–3.37 (2H, m), 3.49–3.64 (m, 12H), 3.66
(s, 3H), 4.21 (br, 1H), 5.02 (br, 1H), 5.10 (s, 2H), 5.79 (br, 1H),
6.90 (br, 1H), 7.26–7.34 (m, 5H). LC–MS m/z: 598.3 (M+H)+.
c
1.56 mmol, 99.8%) as a colorless oil. 1H NMR (270 MHz, CDCl3) d:
1.43 (s, 9H), 1.71–1.76 (m, 4H), 2.19–2.40 (m, 4H), 3.16–3.33 (m,
2H), 3.33–3.40 (m, 2H), 3.35 (s, 3H), 3.48–3.60 (m, 12H), 3.76 (s,
3H), 4.65 (br, 1H), 5.00 (br, 1H), 5.18 (s, 2H), 6.72 (br, 1H), 7.30–
7.36 (m, 7H), 7.88 (d, 2H, J = 8.4 Hz), 8.10 (br, 1H). LC–MS m/z:
731.3 (M+H)+.
7.1.8. MTX(OMe)-a
-NH-PEG-NH-Boc (9b)59
1.06 g (1.45 mmol) of compound 8b was dissolved in 10 mL of
methanol. To the mixture, 30 mg of 10% Pd–C was added. After stir-
ring under hydrogen atmosphere for 1 h, the reaction mixture was
filtered and evaporated, and the residue was dissolved in 7 mL of
dry DMA. To the solution, BPTÁHBr (536 mg, 1.60 mmol, 1.10 equiv)
was added and stirred at 65 °C for 9 h. The reaction mixture was
diluted dichloromethane and purified by column chromatography
(aminosilica gel, eluent; dichloromethane/methanol, 100/3–100/
10, 100/2–100/3, 2 times). The desired compound 9b was obtained
611 mg (0.79 mmol, 55%) as a yellow amorphous substance. 1H
NMR (270 MHz, CDCl3) d: 1.42 (s, 9H), 1.73–1.80 (m, 4H), 2.05–
2.70 (m, 4H), 3.16–3.25 (m, 2H), 3.20 (s, 3H), 3.36–3.40 (m, 2H),
3.51–3.59 (m, 12H), 3.64 (s, 3H), 4.62 (br, 1H), 4.76 (s, 2H), 5.15
(br, 3H), 6.76 (d, 2H, J = 8.6 Hz), 6.50–7.20 (br, 4H), 7.72 (d, 2H,
J = 8.6 Hz), 8.66 (s, 1H). LC–MS m/z: 771.4 (M+H)+.
7.1.4. Z-Glu-OMe(NH-PEG-NH-Boc) (6a)
Substitution of N-
a-carbobenzoxy-
L-glutamic acid
a-methyl es-
ter (Z-Glu(OH)-OMe) for Z-Glu(OMe)-OH,
c
-compound (931 mg,
1.56 mmol, 99.8%) was obtained as a colorless oil. 1H NMR
(270 MHz, CDCl3) d: 1.43 (s, 9H), 1.69–1.78 (m, 4H), 1.96–2.23
(m, 4H), 3.19–3.13 (m, 2H), 3.34–3.38 (m, 2H), 3.49–3.61 (m,
12H), 3.74 (s, 3H), 4.34 (br, 1H), 5.00 (br, 1H), 5.01 (s, 2H), 5.94
(br, 1H), 6.50 (br, 1H), 7.26–7.34 (m, 5H). LC–MS m/z: 598.3
(M+H)+.
7.1.5. 4-(Benzyloxycarbonylmethylamino)benzoic acid (7)65
To a solution of 4.0 g (26.5 mmol) of 4-methylaminobenzoic
acid in100 mL of ether was added NaHCO3 solution (10 g,
119 mmol in 65 mL of water) and dropwise of benzyl chlorofor-
mate (6.7 g, 39.3 mmol, 1.5 equiv) at the temperature of 0 °C. The
reaction mixture was stirred at 0 °C for 1.5 h and at room temper-
ature for 2.5 h. Then the solution was cooled to 0 °C and 50 mL of
1 N HCl solution added. The precipitate which formed was filtered
and dried at reduced pressure. The filtrate was dissolved with ethyl
acetate and the organic layer was washed with brine, dried over
sodium sulfate and evaporated. The precipitate and the residue
were suspended in ethyl acetate and hexane. The desired product
(6.37 g, 22.3 mmol, 84%) was obtained by filtration. 1H NMR
(270 MHz, CDCl3) d: 3.38 (s, 3H), 5.20 (s, 2H), 7.34 (m, 5H), 7.39
(d, 2H, J = 8.4 Hz), 8.07 (d, 2H, J = 8.6 Hz). LC–MS m/z: 285.9
(M+H)+.
7.1.9. MTX(OMe)-
Substitution of a
for an
-derived one and the use of BPTÁHBr (483 mg, 1.44 mmol,
1.05 equiv) in 10 mL of DMA provided -compound (9a, 622 mg,
c
-NH-PEG-NH-Boc (9a)
c-derived compound (8a, 1.00 g, 1.37 mmol)
a
c
0.81 mmol, 59%) as a yellow amorphous substance. 1H NMR
(270 MHz, CDCl3) d: 1.43 (s, 9H), 1.69–1.76 (m, 4H), 2.09–2.42
(m, 4H), 3.20 (s, 3H), 3.20–3.40 (m, 4H), 3.47–3.60 (m, 12H), 3.75
(s, 3H), 4.67 (br, 1H), 4.76 (s, 2H), 5.16 (br, 3H), 6.77 (d, 2H,
J = 8.9 Hz), 6.60–7.60 (br, 4H), 7.76 (d, 2H, J = 8.9 Hz), 8.66 (s, 1H).
LC–MS m/z: 771.4 (M+H)+.
7.1.10. MTX(OMe)-a-NH-PEG-NH2 (10b)
285 mg (0.37 mmol) of compound 9b was dissolved in 3 mL of
trifluoroacetic acid (TFA) at a temperature of 0 °C. After the reac-
tion mixture was kept at 0 °C for 0.5 h, dichloromethane was added
and evaporated to remove TFA and solvent. The residue was puri-
fied by column chromatography (aminosilica gel, eluent; dichloro-
methane/methanol, 100/5–100/8). The titled compound 10b
(211 mg, 0.31 mmol, 85%) was obtained as yellow oil. 1H NMR
(270 MHz, CDCl3) d: 1.68–1.75 (m, 4H), 2.17–2.41 (m, 4H), 2.77
(t, 2H, J = 6.6 Hz), 3.16 (s, 3H), 3.28–3.40 (m, 2H), 3.46–3.60 (m,
12H), 3.74 (s, 3H), 4.66 (br, 3H), 5.51 (s, 2H), 6.00–6.73 (br, 2H),
7.1.6. 4-Z-N(Me)C6H4-C(@O)-Glu(OMe)-NH-PEG-NH-Boc (8b)
889 mg (1.49 mmol) of compound 6b was dissolved in 10 mL of
methanol. To the mixture, 30 mg of 10% palladium on carbon (Pd–
C) was added. After stirring under hydrogen atmosphere for
80 min, the reaction mixture was filtered and evaporated to dry-
ness at reduced pressure, and the residue was dissolved in 10 mL

A. Homma et al. / Bioorg. Med. Chem. 17 (2009) 4647–4656
4653
6.73 (d, 2H, J = 8.9 Hz), 7.11 (br, 1H), 7.65–7.76 (m, 3H), 8.62 (s,
1H). LC–MS m/z: 671.4 (M+H)+.
3.35–3.40 (m, 2H), 3.49–3.63 (m, 12H), 3.84 (br, 2H), 4.20 (br,
2H), 5.07 (s, 2H & br, 1H), 5.21 (br, 1H), 6.43 (br, 1H), 6.80 (br,
2H), 7.16–7.33 (m, 10H). LC–MS m/z: 772.5 (M+H)+.
7.1.11. MTX(OMe)-
c
c
-NH-PEG-NH2 (10a)
-derived compound (9a, 319 mg, 0.41 mmol)
-compound 10a (238 mg,
Substitution of a
7.1.15. Z-Gly-Phe-Leu-Gly-NH-PEG-NH-Boc (14)
for an
a-derived one provided
c
5.35 g (6.93 mmol) of compound 13 was dissolved in 20 mL of
methanol. To the mixture, 300 mg of 10% Pd–C was added. After stir-
ring under hydrogen atmosphere for 2 h, the reaction mixture was
filtered and evaporated. The residue was dissolved in 20 mL of dry
DMF. To the solution, Z-Gly-OH (1.59 g, 7.62 mmol, 1.10 equiv),
HOBtÁH2O (1.06 g, 6.93 mmol, 1.00 equiv) and EDCÁHCl (1.46 g,
7.62 mmol, 1.10 equiv) were added at the temperature of 0 °C. The
reaction mixture was warmed to room temperature and ethyl ace-
tate was added following stirring for 17 h. After following the same
procedure as described for 11, the residue was obtained. The desired
compound 14 was purified by silica gel column chromatography
(eluent; dichloromethane/methanol, 100/5) to afford 5.61 g
(6.77 mmol, 98%) as an amorphous substance. 1H NMR (270 MHz,
CDCl3) d: 0.89 (d, 6H, J = 6.5 Hz), 1.43 (s, 9H), 1.67–1.87 (m, 7H),
3.10–3.12 (m, 4H), 3.30–3.32 (m, 2H), 3.47–3.59 (m, 12H), 3.79
(br, 2H), 3.87 (br, 2H), 4.42 (br, 1H), 4.65 (br, 1H), 5.02 (s, 2H & br,
1H), 6.19 (br, 1H), 6.90–7.34 (m, 14H). LC–MS m/z: 829.6 (M+H)+,
852.5 (M+Na)+.
0.35 mmol, 85%) as a yellow oil. 1H NMR (270 MHz, CDCl3) d:
1.67–1.80 (m, 4H), 2.06–2.31 (m, 2H), 2.41–2.60 (m, 2H), 2.79 (t,
2H, J = 6.6 Hz), 3.17 (s, 3H), 3.34–3.42 (m, 2H), 3.50–3.61 (m,
12H), 3.62 (s, 3H), 4.65 (br, 1H), 4.73 (s, 2H), 5.50 (s, 2H), 6.00–
6.73 (br, 2H), 6.73 (d, 2H, J = 8.6 Hz), 7.22 (s, 1H), 7.55 (br, 1H),
7.71 (d, 2H, J = 8.9 Hz), 8.63 (s, 1H). LC–MS m/z: 671.4 (M+H)+.
7.1.12. Z-Gly-NH-PEG-NH-Boc (11)
To a solution of 3.50 g (10.9 mmol) of 5 in 30 mL of dry DMF
was added N-a-carbobenzoxy-glycine (Z-Gly-OH, 2.29 g,
10.9 mmol, 1.0 equiv), N-hydroxy succinimide (HOSu) (628 mg,
5.46 mmol, 0.5 equiv) and EDCÁHCl (2.09 g, 10.9 mmol, 1.0 equiv)
at the temperature of 0 °C. The reaction mixture was warmed to
room temperature. After stirring for 17 h, ethyl acetate was added.
The organic layer was washed with 10% citric acid, saturated NaH-
CO3 and brine, dried over sodium sulfate and evaporated. The
product was purified by silica gel column chromatography (eluent;
dichloromethane/methanol, 100/3) to afford 4.76 g (9.30 mmol,
85%) of the desired compound 11 as a colorless oil. 1H NMR
(270 MHz, CDCl3) d: 1.43 (s, 9H), 1.68–1.75 (m, 4H), 3.14–3.19
(m, 2H), 3.37–3.41 (m, 2H), 3.47–3.58 (m, 12H), 3.83–3.85 (m,
2H), 4.96 (br, 1H), 5.13 (s, 2H), 5.82 (br, 1H), 6.98 (br, 1H), 7.32–
7.35 (m, 5H). LC–MS m/z: 512.3 (M+H)+.
7.1.16. Z-Glu(OMe)-Gly-Phe-Leu-Gly-NH-PEG-NH-Boc (15)
1.93 g (2.33 mmol) of compound 14 was dissolved in 10 mL of
methanol. To the mixture, 300 mg of 10% Pd–C was added. After stir-
ring under hydrogen atmosphere for 3 h, the reaction mixture was
filtered and evaporated. The residue was dissolved in 7 mL of dry
DMF. To the solution Z-Glu(OMe)-OH (756 mg, 2.56 mmol,
1.10 equiv), HOBtÁH2O (356 mg, 2.33 mmol, 1.00 equiv) and
EDCÁHCl (491 mg, 2.56 mmol, 1.10 equiv) were added at the temper-
ature of 0 °C. The reaction mixture was warmed to room tempera-
ture and ethyl acetate was added following stirring for 17 h. After
following the same procedure as described for 11, the residue was
obtained. The product 15 was purified by silica gel column chroma-
tography (eluent; dichloromethane/methanol, 100/5) to afford
2.10 g (2.16 mmol, 93%) as an amorphous substance. 1H NMR
(270 MHz, CDCl3) d: 0.89 (d, 6H, J = 5.7 Hz), 1.43 (s, 9H), 1.44–1.82
(m, 7H), 1.90–2.21 (m, 2H), 2.40–2.48 (m, 2H), 3.00–3.38 (m, 6H),
3.47–3.63 (m, 12H), 3.64 (s, 3H), 3.65–4.02 (m, 4H,), 4.25 (br, 1H),
4.50–4.79 (br, 2H), 5.02–5.14 (m, 3H), 6.59 (br, 1H), 7.00 (br, 1H),
7.17–7.32 (m, 13H), 7.82 (br, 1H). LC–MS m/z: 972.9 (M+H)+, 994.6
(M+Na)+.
7.1.13. Z-Leu-Gly-NH-PEG-NH-Boc (12)
4.76 g (9.30 mmol) of compound 11 was dissolved in 45 mL of
methanol. To the mixture 250 mg of 10% Pd–C was added. After
stirring under hydrogen atmosphere for 2 h, the reaction mixture
was filtered and evaporated. The residue was dissolved in 30 mL
of dry DMF. To the solution, N-carbobenzoxy-
phenyl ester (Z-Leu-ONp, 3.60 g, 9.30 mmol, 1.0 equiv) and trieth-
ylamine (1.29 mL, 9.30 mmol, 1.0 equiv) were added at
temperature of 0 °C. The reaction mixture was warmed to room
temperature and ethyl acetate was added following stirring for
17 h. After following the same procedure as described for 11, the
residue was obtained. The desired compound 12 was purified by
silica gel column chromatography (eluent; dichloromethane/meth-
anol, 100/5) to afford 5.56 g (8.90 mmol, 96%) as a colorless oil. 1H
NMR (270 MHz, CDCl3) d: 0.94 (d, 6H, J = 5.9 Hz), 1.43 (s, 9H), 1.53–
1.80 (m, 7H), 3.17–3.22 (m, 2H), 3.35–3.40 (m, 2H), 3.48–3.63 (m,
12H), 3.77–4.01 (m ,2H), 4.23 (br, 1H), 5.10 (br, 3H), 5.38 (br, 1H),
6.85 (br, 1H), 7.30–7.34 (m, 6H). LC–MS m/z: 625.6 (M+H)+.
L-leucine-p-nitro-
a
7.1.17. 4-Z-N(Me)C6H4-C(@O)-Glu(OMe)-Gly-Phe-Leu-Gly-NH-
PEG-NH-Boc (16)
2.1 g (2.16 mmol) of compound 15 was dissolved in 10 mL of
methanol. To the mixture, 400 mg of 10% Pd–C was added. After stir-
ring under hydrogen atmosphere for 9 h, to the reaction mixture,
100 mg of Pd–C was added again. After stirring for 4 h, the reaction
mixture was filtered and evaporated. The residue was dissolved in
6 mL of dry DMF. To the solution, 7 (740 mg, 2.59 mmol, 1.20 equiv),
HOBtÁH2O (331 mg, 2.16 mmol, 1.0 equiv) and EDCÁHCl (497 mg,
2.59 mmol, 1.20 equiv) were added at 0 °C. The reaction mixture
was warmed to room temperature and stirred for 18 h, and ethyl
acetate was added. The organic layer was washed with saturated
NaHCO3 and brine, dried over sodium sulfate then n-hexane was
added. The precipitate which formed was filtered and dissolved in
small-scale dichloromethane. The product, 16, was purified by silica
gel column chromatography (eluent; dichloromethane/methanol,
100/5–100/6) to afford 2.02 g (1.83 mmol, 85%) as a white solid. 1H
NMR (270 MHz, CDCl3) d: 0.84–0.91 (m, 6H), 1.41 (s, 9H), 1.59–
1.78 (m, 7H), 2.17–2.21 (m, 2H), 2.41–2.78 (m, 2H), 3.09–3.35 (m,
8H), 3.36 (s, 3H), 3.45–3.60 (m, 14H), 3.70 (s, 3H), 3.94–4.02 (m,
7.1.14. Z-Phe-Leu-Gly-NH-PEG-NH-Boc (13)
5.07 g (8.11 mmol) of compound 12 was dissolved in 30 mL of
methanol. To the mixture, 300 mg of 10% Pd–C was added. After
stirring under hydrogen atmosphere for 2 h, the reaction mixture
was filtered and evaporated. The residue was dissolved in 20 mL
of dry DMF. To the solution, N-carbobenzoxy-L-phenylalanine (Z-
Phe-OH, 2.67 g, 8.93 mmol, 1.10 equiv), HOBtÁH2O (1.24 g,
8.11 mmol, 1.10 equiv) and EDCÁHCl (1.71 g, 8.93 mmol,
1.10 equiv) were added at the temperature of 0 °C. The reaction
mixture was warmed to room temperature and ethyl acetate was
added following stirring for 17 h. After following the same proce-
dure as described for 11, the residue was obtained. The desired
compound was suspended in ethyl acetate–hexane and the prod-
uct (13, 5.87 g, 7.60 mmol, 94%) was obtained by filtration as a
white powder. 1H NMR (270 MHz, CDCl3) d: 0.88 (d, 6H,
J = 4.3 Hz), 1.43 (s, 9H), 1.72–1.81 (m, 7H), 3.07–3.25 (m, 4H),

4654
A. Homma et al. / Bioorg. Med. Chem. 17 (2009) 4647–4656
2H), 4.37–4.63 (br, 3H), 5.06 (br, 1H), 5.18 (s, 2H), 7.02 (br, 1H), 7.21–
7.39 (m, 14H), 7.58 (br, 1H), 7.85 (d, 2H, J = 8.1 Hz), 8.07 (br, 1H), 8.40
(br, 1H). LC–MS m/z: 1127.4 (M+Na)+.
(d, 2H, J = 9.1 Hz, 1,4-disubstituted benzene). LC–MS m/z: 1031.6
(M+H)+. Retention time: 3.46 min.
7.1.20. Z-Phe-NH-PEG-NH-Boc (19)
7.1.18. MTX(OMe)-
a
-Gly-Phe-Leu-Gly-NH-PEG-NH-Boc (17)
To a solution of 5.0 g (15.6 mmol) of 5 in 150 mL of dry DMF was
added Z-Phe-OH (4.9 g, 16.4 mmol, 1.05 equiv) and HOBtÁH2O
(2.51 g, 16.4 mmol, 1.05 equiv). The reaction mixture was stirred
at 0 °C and EDCÁHCl (3.29 g, 17.2 mmol, 1.10 equiv) was added. After
keeping 0 °C for 1 h, the reaction mixture was warmed to room tem-
perature. After 17 h ethyl acetate was added. The organic layer was
washed with 10% citric acid, saturated NaHCO3 and brine, dried over
magnesium sulfate and evaporated. The product was purified by
means of silica gel column chromatography (eluent; dichlorometh-
ane/methanol, 10/1) to afford 9.82 g (quant.) of the desired com-
pound 19. 1H NMR (300 MHz, DMSO-d6) d: 1.37 (s, 9H), 1.54–1.63
(m, 4H), 2.71–3.32 (m, 4H), 3.33–3.39 (m, 6H), 3.45–3.52 (m, 8H),
4.19 (m, 1H), 4.94 (s, 2H), 6.74 (br, 1H), 7.17–7.30 (m, 10H), 7.46
(d, 1H, J = 8.4 Hz), 7.95 (br, 1H). LC–MS m/z: 624.2 (M+Na)+.
2.02 g (1.83 mmol) of compound 16 was dissolved in 8 mL of
methanol. To the mixture, 400 mg of 10% Pd–C was added. After stir-
ring under hydrogen atmosphere for 2 h, the reaction mixture was
filtered and evaporated. The residue was dissolved in 7 mL of dry
DMA. To the solution, BPTÁHBr (676 mg, 2.01 mmol, 110 equiv)
was added and stirred at 65 °C for 19 h. The reaction mixture was di-
luted with dichloromethane and purified by column chromatogra-
phy (aminosilica gel, eluent; dichloromethane/methanol, 100/4–
100/6, 100/3–100/7, 2 times). The desired compound 17 was
obtained 978 mg (0.85 mmol, 47%) as a yellow amorphous sub-
stance. 1H NMR (270 MHz, CDCl3) d: 0.85–0.89 (m, 6H), 1.42 (s, 9H),
1.64–1.78 (m, 7H), 2.09–2.21 (m, 2H), 2.43–2.78 (m, 2H),
3.07–3.32 (m, 8H), 3.27 (s, 3H), 3.45–3.90 (m, 16H), 3.64 (s, 3H),
4.25 (br, 1H), 4.36 (br, 1H), 4.55–4.57 (br, 2H), 6.85 (d, 2H, J = 8.9 Hz),
7.15–7.20 (m, 5H), 7.79 (d, 2H, J = 8.9 Hz), 8.56 (s, 1H). LC–MS m/z:
1145.6 (M+H)+, 1167.6 (M+Na)+.
7.1.21. Z-Phe-Phe-NH-PEG-NH-Boc (20)
15.6 mmol of compound 19 was dissolved in 400 mL of metha-
nol. To the mixture, 0.98 g of 10% Pd–C was added. After stirring
under hydrogen atmosphere for 1.5 h, the reaction mixture was fil-
tered and evaporated, and the residue was dissolved in 160 mL of
dry DMF. To the solution, Z-Phe-OH (5.13 g, 17.1 mmol,
1.10 equiv), HOBtÁH2O (2.62 g, 17.1 mmol, 1.10 equiv) were added.
The reaction mixture was stirred at 0 °C and EDCÁHCl (3.44 g,
17.9 mmol, 1.15 equiv) was added. After keeping 0 °C for 1 h, the
reaction mixture was warmed to room temperature. After 14 h
ethyl acetate was added. After following the same procedure as de-
scribed for 19, the desired compound 20 was obtained 12.1 g
(quant.). 1H NMR (300 MHz, DMSO-d6) d: 1.37 (s, 9H), 1.54–1.63
(m, 4H), 2.67–3.13 (m, 6H), 3.33–3.39 (m, 6H), 3.45–3.50 (m,
8H), 4.24 (br 1H), 4.87 (br, 1H), 4.93 (s, 2H), 6.74 (br, 1H), 7.16–
7.34 (m, 15H), 7.45 (d, 1H, J = 8.7 Hz), 7.88 (br, 1H), 8.12 (d, 1H,
J = 8.1 Hz). LC–MS m/z: 771.3 (M+Na)+.
7.1.19. MTX(OMe)-a/c-Gly-Phe-Leu-Gly-NH-PEG-NH2 (18)
841 mg (0.73 mmol) of compound 17 was dissolved in 7 mL of
TFA at a temperature of 0 °C. After the reaction mixture was kept
at 0 °C for 1 h, dichloromethane was added and evaporated to
remove TFA and solvent. The residue was purified by column chro-
matography (aminosilica gel, eluent; dichloromethane/methanol,
100/10). The product 18 (723 mg, 0.69 mmol, 94%) was a mixture of
a- and c- isomers (3:1). The desired a-isomer was purified by sepa-
rable HPLC but migrated again. The product was obtained as a free
amine through column chromatography (aminosilica gel, eluent;
dichloromethane/methanol, 100/10) as a yellow amorphous sub-
stance. 1H NMR (500 MHz, DMSO-d6) d: 0.50–0.60 (m, 6H, Leu),
1.15–1.39 (m, 2H, Leu, 4H, PEG), 1.71–1.86 (m, 2H, Glu), 1.99–2.08
(br, 1H, Leu), 2.15 (t, 2H, J = 7.2 Hz, Glu), 2.31–2.37 (m, 2H, PEG),
2.64–2.71 (m, 2H, Phe), 2.78–2.87 (m, 2H, PEG), 2.91 (s, 3H, NMe),
3.00–3.23 (m, 12H, PEG), 3.23 (s, 3H, Glu OMe), 3.28–3.48 (m, 4H,
7.1.22. Z-Asn-Phe-Phe-NH-PEG-NH-Boc (21)
Gly), 3.88–3.95 (m, 1H, H
a
Leu), 4.00–4.04 (m, 1H, H
a
Glu), 4.14–
15.6 mmol of compound 20 was dissolved in 280 mL of metha-
nol. To the mixture, 1.0 g of 10% Pd–C was added. After stirring un-
der hydrogen atmosphere for 1.5 h, the reaction mixture was
filtered and evaporated. The residue was dissolved in 160 mL of
4.23 (m, 1H, H Phe), 4.46 (s, 2H, CH2 6-pteridine), 6.22–6.30 (br,
a
2H, NH2), 6.51 (d, 2H, J = 9.6 Hz, 1,4-disubstituted benzene), 6.75–
6.91 (m, 5H, Phe), 7.18–7.28 (m, 1H, NH PEG), 7.18–7.38 (br, 2H,
NH2), 7.44–7.53 (m, 2H, NH Gly), 7.51 (d, 2H, J = 9.6 Hz, 1,4-disubsti-
tuted benzene), 7.64–7.66 (m, 1H, NH Leu), 7.66–7.71 (m, 1H, NH
Phe), 8.14 (br, 1H, NH Glu), 8.12–8.30 (br, 2H, NH2), 8.26 (s, 1H, CH
dry DMF. To the solution, carbobenzyloxy-L-asparagine (Z-Asn-
OH, 4.51 g, 16.9 mmol, 1.08 equiv), HOBtÁH2O (2.59 g, 16.9 mmol,
1.08 equiv) were added. The reaction mixture was stirred at 0 °C
and was added EDCÁHCl (3.40 g, 17.7 mmol, 1.13 equiv). After
being kept at 0 °C for 1 h, the reaction mixture was warmed to
room temperature. After 2 h, ethyl acetate was added. After using
the same procedure as described for 19, the desired compound
21 was obtained 14.8 g (quant.). 1H NMR (300 MHz, DMSO-d6) d:
1.37 (s, 9H), 1.54–1.63 (m, 4H), 2.27–2.57 (m, 2H), 2.74–3.08 (m,
6H), 3.28–3.36 (m, 6H), 3.45–3.51 (m, 8H), 4.29–4.43 (m, 3H),
5.00 (s, 2H), 6.74 (br, 1H), 6.67 (br, 1H), 7.10–7.37 (m, 15H), 7.45
(d, 1H, J = 8.4 Hz), 7.64 (br, 2H), 8.05 (d, 1H, J = 7.8 Hz), 8.18 (d,
1H, J = 8.4 Hz). LC–MS m/z: 863.1 (M+H)+, 885.4 (M+Na)+.
7-pteridine). The ratio was of
MeCN–H2O iso., 30 min., 305–380 nm.). Retention times: 18.6 min.
a/c was decided by HPLC (ODS, 25%
(a-isomer), 11.7 min. (c-isomer). The ratio was 3/1. LC–MS m/z:
1045.7 (M+H)+, 1067.7 (M+Na)+.
For the analysis, the fraction of isomers of free carboxylic
acid was obtained. Separated chemical shifts of
NMR (500 MHz, D2O) d: 0.67–0.75 (m, 6H, side chain Leu),
1.17–1.28 (m, 1H, side chain Leu), 1.36–1.48 (m, 2H, side chain
Leu), 2.00–2.06 (m, 2H, side chain Glu), 2.23–2.32 (m, 2H, side
chain Glu), 2.83–2.91 (m, 2H, side chain Phe), 4.13–4.17 (m,
a
-isomer, 1H
1H,
Ha Leu), 4.20–4.24 (m, 1H, Ha Glu), 6.70 (d, 2H,
J = 9.1 Hz, 1,4-substituted benzene), 7.68 (d, 2H, J = 9.1 Hz, 1,4-
substituted benzene). LC–MS m/z: 1031.5 (M+H)+. Retention
time: 3.57 min.
7.1.23. Z-Glu(OMe)-Asn-Phe-Phe-NH-PEG-NH-Boc (22)
2.62 g (3.04 mmol) of compound 21 was dissolved in 300 mL of
methanol. To the mixture, 0.3 g of 10% Pd–C was added. After stir-
ring under hydrogen atmosphere for 1.5 h, the reaction mixture
was filtered and evaporated, and the residue was dissolved in
50 mL of dry DMF. To the solution, Z-Glu(OMe)-OH (941.3 mg,
3.20 mmol, 1.05 equiv) and HOBtÁH2O (488.2 mg, 3.2 mmol,
1.08 equiv) were added. The reaction mixture was stirred at 0 °C
and EDCÁHCl (640 mg, 3.34 mmol, 1.10 equiv) was added. After
being kept at 0 °C for 1 h, the reaction mixture was warmed to
Separated chemical shifts of c
-isomer, 1H NMR (500 MHz, D2O)
d: 0.62–0.71 (m, 6H, side chain Leu), 1.38–1.54 (m, 3H, side chain
Leu), 1.86–1.93 (m, 1H, side chain Glu), 2.05–2.13 (m, 1H, side
chain Glu), 2.13–2.21 (m, 1H, side chain Glu), 2.23–2.32 (m, 1H,
side chain Glu), 2.70–2.81 (m, 1H, side chain Phe), 2.90–2.98 (m,
1H, side chain Phe), 4.09–4.13 (m, 1H, Ha Leu), 4.29–4.33 (m, 1H,
Ha
Glu), 6.73 (d, 2H, J = 9.1 Hz, 1,4-disubstituted benzene), 7.72

A. Homma et al. / Bioorg. Med. Chem. 17 (2009) 4647–4656
4655
room temperature. After 2 h, water was added. The product was
obtained by filtration and washed with diluted HCl solution, NaH-
CO3 solution and water. Then purified by silica gel column chroma-
tography (eluent; dichloromethane/methanol, 10/1) to afford
2.50 g (2.48 mmol, 82%) of the desired product 22. 1H NMR
(300 MHz, DMSO-d6); d: 1.36 (s, 9H), 1.54–1.63 (m, 4H), 1.67–
1.81 (m, 2H), 2.26–2.60 (m, 2H), 2.68–3.01 (m, 8H), 3.26–3.36
(m, 6H), 3.45–3.51 (m, 8H), 3.57 (s, 3H), 4.02 (br, 1H), 4.32 (br,
2H), 4.53 (br, 1H), 5.01 (dd, 2H, J = 12.3, 20.4 Hz), 6.75 (br, 1H),
7.06–7.49 (m, 19H), 8.16 (br, 2H), 8.25 (d, 1H, J = 7.2 Hz). LC–MS
m/z: 1028.5 (M+Na)+.
sodium chloride (6 g) dissolved in 160 mL of water and 400 mL of
ethanol were added. The precipitate which formed was separated
by centrifugation and dissolved in 500 mL of water. To which so-
dium chloride (15 g) was added before filtration using a 0.45 lm fil-
ter (Stervex HV: Millipore), and then ethanol (1000 mL) was
aseptically added dropwise. The precipitate was filtered and dried
in vacuo. The conjugate was dissolved in 40 mL of phosphate buffer
solution (2 mM sodium phosphate, 154 mM sodium chloride, pH
7.2) to provide a sterile aqueous solution of the HA–MTX conjugate.
The MW was determined by GPC and the binding ratio of MTX was
calculated by measuring the UV absorption (259 nm). The procedure
for calculating the binding ratio of MTX was as follows: The concen-
tration of HA in the conjugate was determined by the RI peak area
relative to the standard HA solution. Next, the HA concentration
was converted to an equivalent concentration of a disaccharide unit
with one carboxyl group. The concentration of MTX in the conjugate
was determined by the UV peak area relative to the standard MTX
solution. The binding ratio of MTX was obtained by dividing the con-
centration of MTX by the concentration of the disaccharide unit.
7.1.24. MTX(OMe)-a-Asn-Phe-Phe-NH-PEG-NH-Boc (24)
302 mg (0.30 mmol) of compound 22 was dissolved in 2 mL of
methanol and 6 mL of DMF. To the mixture, 120 mg of 10% Pd–C
was added. After stirring under hydrogen atmosphere for 3 h, the
reaction mixture was filtered and methanol was removed at re-
duced pressure. To the residue solution, compound 23 (117 mg,
0.36 mmol, 1.20 equiv), HOBtÁH2O (46 mg, 0.30 mmol, 1.00 equiv)
and EDCÁHCl (69 mg, 0.36 mmol, 1.20 equiv) were added at the
temperature of 0 °C. After stirring at room temperature for 19 h,
brine was added to the solution. The yellow precipitate which
formed was filtered and washed with water. The precipitate was
diluted with DMF and recrystallized by the addition of methanol
(2 times). The yellow solid was washed with methanol affording
180 mg (0.15 mmol, 51%) of product 20. 1H NMR (270 MHz,
DMSO-d6) d: 1.36 (s, 9H), 1.54–1.61 (m, 4H), 1.89 (m, 2H), 2.31–
2.43 (m, 2H), 2.71–3.38 (m, 19H), 3.45–3.51 (m, 6H), 3.55 (s, 3H),
4.30–4.35 (m, 3H), 4.49 (br, 1H), 4.79 (s, 2H), 6.60–6.83 (m, 5H),
7.03–7.24 (m, 11H), 7.48–7.72 (m, 4H), 7.73 (d, 2H, J = 8.6 Hz),
8.07–8.23 (m, 4H), 8.57 (s, 1H). LC–MS m/z: 1179.4 (M+H)+.
7.1.26.1. MTX-c-NH-PEG-NH-HA
(1). Sodium
hyaluronate
(500 mg, MW: ca. 2300 kDa) was reacted with compound 10a
(0.0625 mmol) following the general procedure, providing an aque-
ous solution of 1.
This aqueous solution was purified to provide a sterile aqueous
solution of 1. The MW and the binding ratio of MTX were 1610 kDa
and 3.1%, respectively. 1H NMR (500 MHz, D2O) d: 1.46 (m), 1.70
(br), 1.95 (br s), 2.21–2.34 (m), 2.87–3.01 (m), 3.20 (br s), 3.29
(br s), 3.36–3.49 (m), 3.63 (br s), 3.75 (br s), 4.25–4.61 (m), 6.90
(d, J = 8.9 Hz), 7.66 (d, J = 8.9 Hz), 8.62 (s).
7.1.26.2. MTX-
a
-NH-PEG-NH-HA
(2). Sodium
hyaluronate
7.1.25. MTX(OMe)-a-Asn-Phe-Phe-NH-PEG-NH2 (25)
(500 mg, MW: ca. 2300 kDa) was reacted with compound 10b
(0.0625 mmol) following the general procedure, providing an aque-
ous solution of 2.
This aqueous solution was purified to provide a sterile aqueous
solution of 2. The MW and the binding ratio of MTX were 1610 kDa
and 2.7%, respectively. 1H NMR (500 MHz, D2O) d: 1.72 (m), 1.95
(br s), 2.31 (m), 3.19 (br s), 3.27 (br s), 3.36–3.54 (m), 3.63 (br s),
3.75 (br s), 4.27–4.58 (m), 6.87 (d, J = 9.0 Hz), 7.69 (d, J = 9.0 Hz),
8.63 (s).
180 mg (0.15 mmol) of compound 24 was dissolved in 2 mL of
TFA at a temperature of 0 °C. After the reaction mixture was kept
at 0 °C for 0.5 h, dichloromethane was added and evaporated to re-
move TFA and solvent. The residue was purified by column chro-
matography (aminosilica gel, eluent; dichloromethane/methanol,
10/1) and suspended in methanol. The titled compound (25,
145 mg, 0.134 mmol, 88%) was obtained as a yellow solid by filtra-
tion and washed with methanol. 1H NMR (270 MHz, DMSO-d6) d:
1.52–1.59 (m, 4H), 1.87–2.02 (m, 2H), 2.32–3.48 (m, 24H), 3.22
(s, 3H), 3.55 (s, 3H), 4.24–4.56 (m, 4H), 4.79 (s, 2H), 6.60 (br s,
2H), 6.81 (d, 2H, J = 8.6 Hz), 7.04–7.75 (m, 17H), 8.07–8.26 (m,
4H), 8.56 (s, 1H). LC–MS m/z: 1079.5 (M+H)+.
7.1.26.3. MTX-ac-GlyPheLeuGly-NH-PEG-NH-HA
(3). Sodium
hyaluronate (500 mg, MW: ca. 2300 kDa) was reacted with com-
pound 18 (0.0625 mmol) following the general procedure, provid-
ing an aqueous solution 3.
This aqueous solution was purified to provide a sterile aqueous
solution of 3. The MW thereof and the binding ratio of MTX were
1850 kDa and 1.3%, respectively. 1H NMR (500 MHz, D2O) d: 0.72
(d), 0.77 (d), 0.81 (d), 1.32 (m), 1.50 (m), 1.67–1.82 (m), 2.01 (br
s), 2.23 (m), 2.33 (m), 2.75–3.03 (m), 3.51 (br s), 3.58 (br s), 3.71
(br s), 3.83 (br s), 4.16–4.28 (m), 4.46 (br s), 4.54 (br s), 6.85 (d),
6.92–7.06 (m), 7.75 (d), 7.78 (d), 8.63 (s), 8.65 (s).
7.1.26. General procedure of conjugation of amines (10a, 10b,
18 and 25) and HA
In what follows water meant extra-pure water and HA was trea-
ted under sterile condition.
A solution of 3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine
(HOOBt) (0.125 mmol) and amine 10a, 10b, 18 or 25 (0.0625 mmol)
in 20 mL of water–THF (1:1) solvent was added to a suspension of
sodium hyaluronate (500 mg, MW: ca. 2300 kDa) in 10 mL of THF
solution. To the mixture, tris[2-(2-methoxyethoxy)ethyl]amine
(0.094 mmol) dissolved in 10 ml of water–THF (1:1) was added, fol-
lowed by stirring at 5 °C. After 30 min, EDCÁHCl (0.125 mmol) dis-
solved in water (10 mL) was added to the mixture, followed by
stirring at 5 °C for 20 h. A 0.09 N NaOH aqueous solution (220 mL)
was added to the reaction mixture, followed by stirring at 5 °C for
3.5 h. 1 N HCl solution (20 mL) was added to the reaction mixture
for neutralization, sodium chloride solution (9 g in 45 mL of water)
was added, followed by dropwise addition of ethanol (600 mL).
The precipitate which formed was separated by centrifugation.
The precipitate was dissolved in water (40 mL) to provide an aque-
ous solution of the HA–MTX conjugate. To the conjugate solution,
The portions given in italics are minor signals. From the signals,
3 was deduced to be a mixture of
a- and c- isomers.
7.1.26.4. MTX- -AsnPhePhe-NH-PEG-NH-HA (4). Sodium hyal-
a
uronate (500 mg, MW: ca. 2300 kDa) was reacted with compound
25 (0.0625 mmol) following the general procedure, providing an
aqueous solution 4.
This aqueous solution was purified to provide a sterile aqueous
solution of 4. The MW and the binding ratio of MTX were 1780 kDa
and 1.2%, respectively. 1H NMR (500 MHz, D2O) d: 1.60 (m), 1.80
(m), 2.02 (br s), 2.34 (m), 2.54 (m), 2.60–3.05 (m), 3.35 (br s),
3.52 (br s), 3.57 (br s), 3.64 (br s), 3.72 (br s), 3.83 (br s), 4.28

4656
A. Homma et al. / Bioorg. Med. Chem. 17 (2009) 4647–4656
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7.80 (d), 8.61 (s), 8.64 (s).
The portions given in italics are minor signals. From these sig-
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7.2. Biology
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7.2.1. In vitro
HFLS (Cell Applications) was seeded at 5000 cells/well on a
96-well plate (Falcon) and cultured for 3 h in Iscove’s modified
Dulbecco’s medium (IMDM) containing 5% FBS and 1X antibiotic–
antimycotic (GIBCO BRL). After cellular attachment, TNF-
binant human TNF- , R & D Systems) (final concentration: 10 ng/
a (recom-
a
mL) and each HA–MTX conjugate at each concentration was added,
followed by cultivation for 5 days. Two days before the end of
culture, 37 kBq/well of [3H]-deoxyuridine was added to the cells
(MORAVEK), followed by determining the uptake quantity (radioac-
tivity) of [3H]-deoxyuridine using a scintillation counter. Cells were
recovered by unsticking them with 0.05% trypsin–0.2% EDTA.
Radioactivity was calculated as a relative value (% of control),
using, as the control, radioactivity in the group of cells cultured
without any added test substance. Since the concentration of a free
carboxyl group is 2.49 Â 10À3 mol/L (1 g/401/L; 401 is the MW of
N-acetylglucosamine + glucuronic acid) for each 1 mg/mL of hyalu-
ronic acid, the MTX concentration in each HA–MTX was calculated
by multiplying the value by the conjugation rate of MTX. (For
1 mg/mL of HA–MTX conjugate with a conjugation rate of MTX
of 1%, the concentration of MTX was 2.49 Â 10À5 mol/L.) The value
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Acknowledgments
The authors thank Professor Kunio Ogasawara for his helpful
suggestions concerning this study. We also thank Ms. Frances Ford
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References and notes
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