Investigation of amino acid conjugates of (S)-1-[1-(4-aminobenzoyl)-2,3- dihydro-1H-indol-6-sulfonyl]-4-phenyl-imidazolidin-2-one (DW2282) as water soluble anticancer prodrugs

Article abstract of DOI:10.1016/j.ejmech.2014.04.048

The amino acid-conjugates (1a-k) with eleven amino acids attached to primary amine of (S)-1-[1-(4-aminobenzoyl)-2,3-dihydro-1H-indol-6-sulfonyl]-4- phenyl-imidazolidin-2-one (DW2282, 1) were prepared and studied for their prodrug characteristics and anti-

Full text of DOI:10.1016/j.ejmech.2014.04.048

European Journal of Medicinal Chemistry 80 (2014) 439e446
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Investigation of amino acid conjugates of (S)-1-[1-(4-aminobenzoyl)-
2,3-dihydro-1H-indol-6-sulfonyl]-4-phenyl-imidazolidin-2-one
(DW2282) as water soluble anticancer prodrugs
*
Ki-Cheul Lee, Eeda Venkateswararao, Vinay Kumar Sharma, Sang-Hun Jung
College of Pharmacy and Institute of Drug Research and Development, Chungnam National University, Daejeon 305-764, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
The amino acid-conjugates (1aek) with eleven amino acids attached to primary amine of (S)-1-[1-(4-
aminobenzoyl)-2,3-dihydro-1H-indol-6-sulfonyl]-4-phenyl-imidazolidin-2-one (DW2282, 1) were pre-
pared and studied for their prodrug characteristics and anti-cancer activity against SW620 cell line. All
the amino acid derivatives showed not only improved water solubility but also displayed potent anti-
Received 22 July 2013
Received in revised form
14 April 2014
Accepted 15 April 2014
Available online 18 April 2014
cancer activity in vitro. Among these amino acid-conjugates the compounds, DW2282-
L-Ala (1b),
DW2282- -Phe (1e), DW2282- -Leu (1g) and DW2282- -Met (1h) showed good reconversion within 8 h
L
L
L
(104.76%, 84.03%, 95.02% and 78.34%, respectively) to the parent drug in human plasma. In addition, the
compounds 1e, 1g and 1j also showed good bioavailability profile along with potent in vivo anticancer
activity.
Keywords:
Prodrug
Amino acid conjugates
Imidazolidinone
Anti-cancer
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
soluble and chemically stable prodrugs that target peptidase en-
zymes for cleavage to the parent drug in vivo [18,19]. Another water
The drug delivery system such as prodrug is an important aspect
of drug discovery and development. A prodrug serves as a precur-
sor to the anticipated drug [1] and also helps in improving its ab-
sorption, distribution, metabolism and excretion or in other words
improves its physicochemical and pharmacokinetic properties
[2,3]. This system is effective especially in case of the drugs which
are unable to perform in vivo despite proven in vitro activity.
Since more than a decade, among the prodrug and promoieties,
amino acid conjugates are one of the crucial ones, having improved
aqueous solubility of various drugs containing alcohol and amine
functional groups [4e12]. The presence of ionized carboxylate
anion or ammonium cations is the main reason for this improved
solubility. Several amino acid ester prodrugs have been reported
and investigated by the researchers as water soluble derivatives for
oral administration [13e16]. In addition, these conjugates are not
only proven for structural diversity and chemical stability but can
also endure the rapid and quantitative bio-reversion to parent drug
by the action of enzymes such as esterases or peptidases [17].
The amino acid conjugates with amide linkage of dapsone, a
primary aromatic amine, have been synthesized as highly water-
soluble prodrug of an anti-tumor agent 3-[(3-amino-4-methoxy)
phenyl]-2-(3,4,5-trimethoxyphenyl)cyclopent-2-ene-1-one [20], a
novel analog of combretastatin A-4 (CA-4), has also been reported.
It was observed that all the prodrug analogs improve the water
solubility as well as anti-tumor activity without exhibiting signifi-
cant toxicities in mice [20]. These studies are proven examples of
amino acid conjugates as worthy carrier of drug in vivo and have
potential role in new drug discovery system.
As we previously discovered a novel and promising anticancer
candidate,
(S)-1-[1-(4-aminobenzoyl)-2,3-dihydro-1H-indol-6-
sulfonyl]-4-phenyl-imidazolidin-2-one (DW2282, Fig. 1) [21e25]
which not only showed the broad and potent growth inhibitory
activity against various cancer cell lines but also proved its worth in
the in vivo test, by exhibiting approximately 45%, 42%, and 94%
suppression of tumor growth against murine colon adenocarci-
noma (colon 26), Lewis lung carcinoma (3LL), and human colon
cancer (SW620) at a dose of 65 mg/kg (p.o., q2d ꢀ 5), respectively
[26]. One of our previous study on the mechanism of action of
arylsulfonyl-4,5-dihydroimidazolones proved them as tubulin in-
hibitors [25]. These compounds were not only the potent inhibitors
of tubulin polymerization but also maintained activity against
multi-drug resistant tumor cell lines, which implies that they are
not a substrate for p-glycoprotein mediated transport like taxanes
and vinca alkaloids derivatives. Despite the therapeutic potential of
* Corresponding author.
E-mail address: jungshh@cnu.ac.kr (S.-H. Jung).
http://dx.doi.org/10.1016/j.ejmech.2014.04.048
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

440
K.-C. Lee et al. / European Journal of Medicinal Chemistry 80 (2014) 439e446
Fig. 1. Structure of DW2282 (1) and designed prodrugs (1aek).
DW2282, in preclinical trial on beagle dog, the severe gastrointes-
tinal (GI) toxicity was observed. The biopsy revealed considerable
accumulation of drug on the intestinal wall after five day oral
administration, which might be resulted due to its poor water
solubility (0.0235 mg/mL) (Data not published). Hence, its solubi-
lity was considered as a critical parameter amenable to manipula-
tion via a prodrug strategy. As we have discussed above, the
presence of amino acid conjugates with parent drug improves its
aqueous solubility and delivery to the target site. Bearing this in
mind, we designed and synthesized amino acid conjugates of
DW2282. The chemical structure of parent drug DW2282 has two
possible linkable sites. One is the amide nitrogen (Site 1 in Fig. 1) of
heterocyclic imidazolidin-2-one and the other one is a basic
aromatic amine (Site 2 in Fig. 1) attached at indoline moiety. Our
preliminary studies clearly indicated that for the synthesis of pro-
drug, the primary amine is more suitable to collaborate with amino
acid conjugates. Thus, amino acid conjugates of DW2282 with
various amino acids attached to primary amine were designed,
synthesized and evaluated for their aqueous solubility, cytotoxicity
against SW620 human colon cancer cell, reconversion test as well
as the anti-tumor activity.
2. Chemistry
Target compounds 1aek were prepared using the synthetic
pathway shown in Scheme 1. Condensation of DW2282 (1) with
Scheme 1. Synthesis of prodrugs 1aek. Reagents and conditions; (a) protected amino acid (N-Boc-Gly for 2a, N-Boc-L-Ala for 2b, N-Boc-L-Pro for 2c, N-Boc-L-Val for 2d, N-Boc-L-Phe
for 2e, N-Boc-L-Ile for 2f, N-Boc-L-Leu for 2g, N-Boc-L-Met for 2h N-Boc-S-trityl-L-Cysteine for 2i, N-Boc-L-Asp(O-Bzl)-OH for 2j, N-Fmoc-O-tert-Bu-L-Ser for 2k) DCC, HOBt/H2O, DMF;
(b) TFA, CH2Cl2 (for 1aeh), TFA, Triethylsilane, CH2Cl2 for 1i, Pd/C in MeOH then TFA, CH2Cl2 (for 1j), Piperidine, CH2Cl2 then 4N-HCledioxane, reflux (for 1k).

K.-C. Lee et al. / European Journal of Medicinal Chemistry 80 (2014) 439e446
441
Table 2
protected amino acids was carried out in the presence of dicyclo-
hexylcarbodiimide (DCC) and 1-hydroxybenzotriazole hydrate
(HOBt$H2O) as coupling reagents. Deprotection of tert-butylox-
ylcarbonyl (Boc) group of 2aeh was performed by the treatment
with trifluoroacetic acid (TFA) in dichloromethane to obtain the TFA
salt of 1aeh. Compound 1i was prepared by the deprotection of 2i
treated with TFA at room temperature until the color of reaction
turned into brown and then treated with triethylsilane until the
reaction is colorless. To obtain the compound 1j the protected
compound 2j was first deprotected by the treatment of TFA in
dichloromethane to remove the Boc group, followed by hydro-
genolysis of the benzyl ester group using hydrogen gas and Pd/C.
The hydrochloride salt of compound 1k was obtained by the
deprotection of Fmoc group of 2k using piperidine followed by
refluxing in 4 N HCledioxane. All final products were obtained in
sufficient purity after purification through a silica gel column
eluting with 5e10% MeOH in CH2Cl2 and/or recrystallization from
appropriate solvents.
The in vitro anticancer activity of prodrugs (1aek) against human cancer cell
line (SW620).
Compound Cytotoxicitya (IC50b mM) Compound
Cytotoxicitya (IC50b mM)
1a (Gly)
1b ( -Ala)
-Pro)
0.068
0.020
0.648
0.038
0.024
0.095
1g (
1h (
L
-Leu)
0.198
0.095
0.415
0.383
0.992
L
L
-Met)
-Cys)
-Asp)
1c (
L
1i (
1j (
L
L
1d (
L
-Val)
-Phe)
-Ile)
1e (
L
1k (L-Ser)
1f (
L
1 (DW2282) 0.057
a
Cancer cell line; SW620 (Human colorectal adenocarcinoma cell).
All experiments were performed at least in triplicate using the MTT assay
b
[28,29] according to the previously reported procedure with a minor modification.
3.3. In vitro reconversion test of 1aek to 1 in human plasma
In order to predict the rate of reconversion from 1aek to 1 in
body, the reconversion of amino acid conjugates 1aek to 1 in hu-
man plasma in 24 h were measured as shown in Fig. 2. The
maximum amounts of conversion of 1aek to 1 were obtained in 8 h
and were not changed much after that (Table 3). Compounds 1b
(104.76%, T1/2 ¼ 8.0 min), 1e (84.03%, T1/2 ¼ 135.5 min), 1g (95.02%,
T1/2 ¼ 50.1 min) and 1h (78.34%, T1/2 ¼ 154.3 min) are highly
susceptible to hydrolytic conversion to 1 in human plasma.
These results suggest that these analogs might be considered as
prodrugs.
3. Results and discussion
3.1. Water solubility
The solubility of all the amino acid conjugates 1aek was
measured in distilled water at 25 ꢁC as reported earlier [27]. The
results showed that all the amino acid conjugates have much better
solubility in water compared to 1 (Table 1). Especially, compounds
3.4. Pharmacokinetic studies
1c (
L
-Pro, 115 times), 1d (
L
-Val, 64 times), 1g (
L
-Leu, 65 times), 1h
Compounds for pharmacokinetic study in Balb/c mice were
selected based on relatively two acceptable parameters among
water solubility, cytotoxicity and reconversion rate. Thus com-
pounds 1deg were selected and additionally 1j was also selected
due to its carboxylic acid moiety, even though these properties
were not much improved. Since compounds were expected to form
compound 1 in vivo system, the blood concentration was measured
by HPLC after oral administration of 70 mg/kg in 1% CMC solution.
The pharmacokinetic results are shown in Table 4 and Fig. 3. The
(L-Met, 90 times) and 1k (
L-Ser, 50 times) had remarkably increased
water solubility compared to 1. Conversely, the measurement of
solubility is not correlated to hydrophilic properties of side chain of
amino acids.
3.2. In vitro anti-cancer activity
In the next set of experiment growth inhibitory activity of 1aek
against human colorectal adenocarcinoma cell (SW620) line using
MTT assay [28,29] was measured (Table 2). Interestingly, com-
data clearly indicated that 1e (AUC, 344.33
mg min/mL), 1g (AUC,
379.05 g min/mL) and 1j (AUC, 672.23 g min/mL) exhibit good
m
m
exposure by the oral route. The pharmacokinetic profiles of 1e and
1g are well correlated to their reconversion rate (1e: 84.03% and 1g:
95.02%) in human plasma. However, good pharmacokinetic profile
of 1j was unexpected since its reconversion rate was very poor in
human plasma. This result obviously indicates species different
pounds 1b (IC50 ¼ 0.020
(IC50 ¼ 0.024 M) were three times more active than 1 while
M), 1f (IC50 ¼ 0.095 M) and 1h
M) showed comparable activity to
M). On the other hand compounds 1c
M), 1i (IC50 ¼ 0.415 M), 1j
M) showed lesser activity
mM), 1d (IC50 ¼ 0.038
mM) and 1e
m
compounds 1a (IC50 ¼ 0.068
m
m
(IC50
(IC50
¼
¼
0.095
0.057
m
m
1
metabolism. Compounds 1d (AUC, 19.95
mg min/mL) and 1f (AUC,
(IC50 ¼ 0.648
mM), 1g (IC50 ¼ 0.198
m
m
44.24 g min/mL) have very poor pharmacokinetic profiles as ex-
m
(IC50 ¼ 0.383
mM) and 1k (IC50 ¼ 0.992
m
pected in the reconversion test. Therefore, the compounds 1e, 1g
and 1j were subjected for animal study.
than 1. In general, amino acid conjugates with hydrophobic side
chain increase the activity, with exception of proline (1c) and
leucine (1g) conjugates whereas amino acid conjugates (1i, 1j, 1k)
with hydrophilic side chain decrease the activity.
3.5. Anticancer activity
Such significant bioavailability of compounds 1e, 1g and 1j led
us to investigate their antitumor activities in vivo against human
colon carcinoma (SW620) xenograft tumor models in mice
(Table 5) [30]. Compound 1e (Dose: 70 mg/kg, b.i.d., 5 days, p.o.)
showed 99% TGI against SW620 xenograft tumor models in mice
with 9.5% decrease in body weight. Compound 1g (Dose: 70 mg/kg,
b.i.d., 5 days, p.o.) showed 86% TGI against SW620 xenograft tumor
models in mice with 10.5% decrease in body weight. Although there
are some body weight changes, antitumor activities of these com-
pounds are remarkable at this single dose experiment. However,
compound 1j (70 mg/kg, b.i.d., 5 days, p.o.) showed 63% TGI with
markedly decrease in body weight as shown in Table 5, which in-
dicates significant toxicity.
Table 1
Water solubility of prodrugs 1aek.
Compound
Solubility (mg/mL)a
Compound
1g ( -Leu)
Solubility (mg/mL)a
1a (Gly)
0.064
0.214
2.758
1.541
0.865
0.953
L
1.620
2.176
0.353
0.732
1.201
0.024b
1b (
L-Ala)
1h (
L
-Met)
-Cys)
-Asp)
1c (L-Pro)
1i (
1j (
L
1d (
L
-Val)
-Phe)
-Ile)
L
1e (
L
1k (L-Ser)
1f (
L
1 (DW2282)
a
Solubility was measured in distilled water at 25 ꢁC as reported previously [27].
Solubility values are taken as a mean from 3 experiments.
b

442
K.-C. Lee et al. / European Journal of Medicinal Chemistry 80 (2014) 439e446
Fig. 2. Reconversion curve in human plasma of prodrugs (1aek) to 1 (DW2282).
4. Conclusion
measured on an Electrothermal melting point apparatus. IR spectra
were obtained on KBr disks using a JASCO Report 100 spectro-
photometer. 1H NMR and 13C NMR spectra were recorded on JEOL,
JNM-AL-400 (Alice) 400 FT-NMR spectrometer and the chemical
For finding a possible prodrug candidate of 1, its eleven amino
acid conjugates were synthesized and tested for their water solu-
bility, reconversion rate in human plasma, pharmacokinetic profile
and in vitro and in vivo anticancer activity.
The results indicated improved solubility by all the amino acid
derivatives. Amino acid conjugation to 1 varied the activity of these
analogs. In general, amino acid conjugates with hydrophobic side
chain increase the in vitro activity with exception of proline (1c)
shift (d) were reported in parts per million downfield from tetra-
methylsilane (TMS) and from solvent references. High resolution
mass spectra (HRMS) were measured by using Shimadzu LCMS-IT-
TOF spectrometer. Chromatographic separations were performed
on silica gel columns by flash (Kieselgel 40, 0.040e0.063 mm;
Merck) or gravity column (Kieselgel 60, 0.063e0.200 mm; Merck).
The reactions were monitored by thin-layer chromatography (TLC)
on Merck glass silica gel (Kieselgel 60 F254) plates that were visu-
alized under a UV lamp. HPLC was performed using a Shimadzu
liquid chromatograph model Class-vp version 6.12, equipped with a
SPD-10A UVevis detector (Shimadzu). The HPLC analysis was per-
formed using a Symmetry TMÒ C-18 column (3.9 ꢀ 150 mm, Part
No. WAT054205, Waters, Milford, MA). The elution condition was a
linear gradient of 31e95% acetonitrile in 0.1% TFA for 30 min at a
flow rate of 1 mL/min with UV detection at 254 nm. All solvents for
and leucine (1g) conjugates. Compounds 1b (
-Leu) and 1h ( -Met) showed good reconversion rate in human
plasma. The pharmacokinetic study obviously indicated that 1e
(AUC, 344.33 g min/mL), 1g (AUC, 379.05 g min/mL) and 1j (AUC,
672.23 g min/mL) exhibited good exposure by the oral route in
L-Ala), 1e (L-Phe), 1g
(L
L
m
m
m
mice as compound 1. Compounds 1e (99% TGI) and 1g (86% TGI)
showed remarkable tumor growth inhibition against SW620
xenograft tumor model in mice at single dose experiment (Dose:
70 mg/kg, b.i.d., 5 days, p.o.). Therefore, compounds 1e and 1g can
be considered as potential prodrug candidates.
HPLC were filtered through a 0.45 mm membrane filter (Waters,
USA).
5. Experimental
5.1.1. Synthesis of amino acid prodrugs 2aek
5.1. Chemistry
5.1.1.1. DW2282-N-Boc-Gly (2a). 1-Hydroxybenzotriazole hydrate
(HOBt$H2O) (0.436 g, 3.23 mmol) and N,N’-Dicyclohex-
ylcarbodiimide (DCC) (0.532 g, 2.58 mmol) were added to solution
All reagents and solvents were dried prior to use according to
the standard methods. Commercial reagents were used without
further purification unless otherwise stated. Melting points were
Table 4
Pharmacokinetic Parameters for prodrug (1deg and 1j) tested in vivo.
Table 3
PK parameters
1d (L-Val) 1e (L-Phe) 1f (L-Ile) 1g (L-Leu) 1j (L-Asp)
Reconversion rate of prodrugs (1aek) to 1 (DW2282) in human plasma.
Cmax
(
m
g/mL)a
0.95
60.00
X
6.61
30.00
69.94
0.83
120.00
X
6.24
30.00
60.00
9.09
60.00
32.81
Compound Reconversion Half life Compound Reconversion Half life
Tmax (min)b
ratea (%)
(min)
ratea (%)
(min)
T1/2 (min)c
1a (Gly)
1b ( -Ala)
-Pro)
37.56
104.76
5.74
1.59
84.03
13.84
e
1g (
1h (
L
-Leu)
95.02
78.34
4.09
10.00
15.09
50.1
154.3
e
AUC0e240
(
m
g min/ml)d 19.95
344.33
412.94
44.25
X
379.05
426.66
672.23
680.27
L
8.0
e
L
-Met)
-Cys)
-Asp)
AUC0eN
(
mg min/ml)
X
1c (
L
1i (
1j (
L
L
a
Cmax is a maximum blood concentration of DW2282.
Tmax: Time when the blood concentration of DW2282 is maximum.
T1/2 is half life time.
1d (
L
-Val)
-Phe)
-Ile)
e
e
b
c
1e (
L
135.5
e
1k (
L
-Ser)
e
1f (
L
d
AUC: 0 / 240 is a integral value of area under the concentrationetime curve
a
Reconversion rate: measured at 8 h after initiation of reaction.
from 0 min to 240 min.

K.-C. Lee et al. / European Journal of Medicinal Chemistry 80 (2014) 439e446
443
Table 5
5.1.1.4. DW 2282-N-Boc-
L
-Val (2d). The compound (2d) was pre-
Antitumor activity of prodrugs (1e, 1g and 1j).
pared by the same procedure described for DW2282-N-Boc-Gly
(2a) using N-Boc- -Val instead of N-Boc-Gly.
Yield: 23.8%; Rf 0.45 (3:1 ethyl acetate/hexane); 1H NMR (CDCl3-
L
Compound
Schedule
% TGI
Body weight
change (%)
d6)
d
1.03 (q, J ¼ 9.2, 6.8 Hz, 6H), 1.36 (s, 9H), 2.11 (m, 1H), 3.14 (m,
1e
1g
1j
1
70 mg/kg, b.i.d., 5 days, p.o.
70 mg/kg, b.i.d., 5 days, p.o.
70 mg/kg, b.i.d., 5 days, p.o.
70 mg/kg, 1 time/2days, 3 times, p.o.
99
86
63
86
ꢂ9.5
ꢂ10.5
ꢂ28.6
1.5
2H), 3.80 (m, 1H), 4.07e4.17 (m, 3H), 4.31 (t, J ¼ 8.8 Hz, 1H), 4.80 (t,
J ¼ 8.4 Hz, 1H), 5.28 (d, J ¼ 8.8 Hz, 1H), 7.26e7.56 (m, 12H), 7.86 (m,
2H), 9.23 (s, 1H).
5.1.1.5. DW 2282-N-Boc-
pared by the same procedure described for DW2282-N-Boc-Gly
(2a) using N-Boc- -Phe instead of N-Boc-Gly.
Yield: 46.0%; Rf 0.24 (2:1 ethyl acetate/hexane); 1H NMR
(DMSO-d6) 1.32 (s, 9H), 2.86e3.05 (m, 2H), 3.15 (m, 2H), 3.51 (m,
1H), 4.14 (m, 3H), 4.28e4.30 (m, 2H), 4.80 (m, 1H), 5.57 (d,
J ¼ 8.0 Hz, 1H), 7.20e7.40 (m, 13H), 7.61e7.80 (m, 6H), 10.35 (s, 1H).
L
-Phe (2e). The compound (2e) was pre-
of DW2282 (1, 1.0 g, 2.16 mmol) in anhydrous dimethylformamide
(DMF) (30 mL) at 0 ꢁC. The reaction mixture was kept under
continuous stirring for 15 min. N-Boc-Gly (0.452 g, 2.58 mmol) in
the solid form was the added at once, and the reaction mixture was
stirred for additional 24 h while it was gradually allowed to reach
ambient temperature. The solvent was then removed under
reduced pressure. The crude product was dissolved in ethyl acetate
(50 mL), washed with water (50 mL), dried (Na2SO4) and evapo-
rated. Chromatography of crude product on silica gel (hexane:ethyl
acetate ¼ 1:1 / hexane:ethyl acetate:methanol ¼ 10:10:1) gave
DW2282-N-Boc-Gly (2a) as a white solid.
L
d
5.1.1.6. DW 2282-N-Boc-
by the same procedure described for DW2282-N-Boc-Gly (2a) using
N-Boc- -Ile instead of N-Boc-Gly.
Yield: 28.3%; Rf 0.35 (3:1 ethyl acetate/hexane); 1H NMR
(DMSO-d6) 0.80e0.88 (m, 6H), 1.23 (m, 1H), 1.39 (s, 9H), 1.62 (m,
L
-Ile (2f). The compound (2f) was prepared
L
Yield: 0.635 g (47.7%); Rf 0.43 (10:10:1 ethyl acetate/hexane/
d
methanol); 1H NMR (DMSO-d6)
3.49 (m, 1H), 3.76 (d, J ¼ 6.0 Hz, 2H), 4.13 (m, 2H), 4.27 (m. 1H), 4.76
(t, J ¼ 7.6 Hz, 1H), 7.20e7.40 (m, 5H), 7.59e7.61 (m, 2H), 7.69e7.78
(m, 5H), 8.21 (s, 1H).
d
1.39 (s, 9H), 3.15 (t, J ¼ 8.0 Hz, 2H),
1H), 1.72 (m, 1H), 3.16 (m, 2H), 3.50 (m, 2H), 3.97 (m, 1H), 4.15 (m,
2H), 4.28 (t, J ¼ 8.8 Hz,1H), 4.80 (t, J ¼ 7.2 Hz,1H), 5.57 (d, J ¼ 8.0 Hz,
1H), 7.22e7.40 (m, 5H), 7.59e7.80 (m, 6H), 10.29 (s, 1H).
5.1.1.7. DW 2282-N-Boc-
pared by the same procedure described for DW2282-N-Boc-Gly
using N-Boc- -Leu instead of N-Boc-Gly.
Yield: 60.1%; Rf 0.44 (2:1 ethyl acetate/hexane); 1H NMR (CDCl3-
0.95 (m, 6H), 1.36 (s, 9H), 1.69 (m, 3H), 3.17 (m, 2H), 3.51 (m,
L
-Leu (2g). The compound (2g) was pre-
5.1.1.2. DW2282-N-Boc-
by the same procedure described for DW2282-N-Boc-Gly (2a) using
N-Boc- -Ala instead of N-Boc-Gly.
Yield: 40.1%; Rf 0.34 (3:1 ethyl acetate/hexane); 1H NMR (,
DMSO-d6)
(m, 1H), 4.10e4.19 (m, 3H), 4.25 (m, 1H), 4.79 (t, J ¼ 7.4 Hz, 1H), 5.77
(s, 1H), 6.58 (d, J ¼ 8.4 Hz, 1H) 7.22e7.38 (m, 7H), 7.59e7.61 (m, 2H),
7.69e7.78 (m, 3H), 8.21 (s, 1H), 10.21 (s, 1H).
L
-Ala (2b). The compound 2b was prepared
L
L
d1)
d
1H), 3.96 (m, 1H), 4.09e4.32 (m, 3H), 4.80 (m, 1H), 5.23 (m, 1H),
7.21e7.86 (m, 12H).
d
1.26 (d, J ¼ 7.2 Hz, 3H), 1.38 (s, 9H), 3.15 (m, 2H), 3.49
5.1.1.8. DW 2282-N-Boc-
pared by the same procedure described for DW2282-N-Boc-Gly
(2a) using N-Boc- -Met instead of N-Boc-Gly.
Yield: 41.6%; Rf 0.35 (3:1 ethyl acetate/hexane); 1H NMR (CDCl3-
1.35 (s, 9H), 1.87e2.10 (m, 2H), 2.12 (s, 3H), 3.20 (m, 2H), 3.67
(m, 2H), 4.29 (m, 3H), 4.80 (m,1H), 5.55 (m,1H), 7.27e7.85 (m,12H).
L
-Met (2h). The compound (2h) was pre-
L
5.1.1.3. DW 2282-N-Boc-
by the same procedure described for DW2282-N-Boc-Gly (2a) using
N-Boc- -Pro instead of N-Boc-Gly.
Yield: 36.5%; Rf 0.40 (10:10:1 ethyl acetate/hexane/methanol);
1H NMR (DMSO-d6)
1.39 (s, 9H), 1.79e1.89 (m, 3H), 2.20 (m, 1H),
L
-Pro (2c). Compound (2c) was prepared
d1)
d
L
5.1.1.9. DW2282-N-Boc-S-trityl-
prepared by the same procedure described for DW2282-N-Boc-Gly
(2a) using N-Boc-S-trityl- -Cys instead of N-Boc-Gly.
Yield: 46.3%; Rf 0.65 (1:1 ethyl acetate/hexane); 1H NMR
(DMSO-d6) 1.38 (s, 9H), 2.62 (m, 1H), 3.13 (m, 2H), 3.67 (m, 1H),
4.12e4.18 (m, 3H), 4.25 (m, 1H), 4.75 (m, 1H), 7.18e8.33 (m, 28H).
L
-Cys (2i). The compound (2i) was
d
3.15 (m, 2H), 3.49 (m, 1H), 4.11e4.29 (m, 4H), 4.79 (t, J ¼ 7.4 Hz, 1H),
7.22e7.38 (m, 6H), 7.60e7.62 (m, 2H), 7.71e7.79 (m, 4H), 8.21 (s,
1H), 10.26 (s, 1H).
L
d
5.1.1.10. DW2282-N-Boc-
prepared by the same procedure described for DW2282-N-Boc-Gly
(2a) using N-Boc- -Asp(OBzl)-OH instead of N-Boc-Gly.
Yield: 57.7%; Rf 0.50 (2:1 ethyl acetate/hexane); 1H NMR (CDCl3-
1.48 (s, 9H), 2.80e3.10 (m, 2H), 3.17 (m, 2H), 3.44 (m, 1H), 3.70
L
-Asp(OBzl) (2j). The compound (2j) was
L
d6)
d
(q, J ¼ 9.2, 7.2 Hz, 1H), 4.17 (t, J ¼ 8.4 Hz, 2H), 4.30 (t, J ¼ 8.8 Hz, 1H),
4.78 (t, J ¼ 7.6 Hz, 1H), 5.18 (d, J ¼ 5.2 Hz, 2H), 5.90 (m, 1H), 7.22e
7.37 (m, 10H), 7.54e7.57 (m, 4H), 7.60e7.92 (m, 4H).
5.1.1.11. DW2282-N-Fmoc-O-tert-Bu-
was prepared by the same procedure described for DW2282-N-
Boc-Gly (2a) using N-Fmoc-O-tert-Bu- -Ser instead of N-Boc-Gly.
Yield: 38.0%; Rf 0.31 (3:1 ethyl acetate/hexane); 1H NMR (CDCl3-
L
-Ser (2k). The compound (2k)
L
d6)
d
1.28 (s, 9H), 3.19 (t, J ¼ 8.4 Hz, 2H), 3.49 (t, J ¼ 8.8 Hz, 1H), 3.69
(q, J ¼ 9.6, 6.8 Hz, 1H), 4.16e4.46 (m, 8H), 4.78 (t, J ¼ 8.0 Hz, 1H),
Fig. 3. The blood concentrationetime curve of DW2282 (1) in the female Balb/c mice
soon after the administration of prodrugs (1deg and 1j), respectively.
7.20e7.92 (m, 22H).

444
K.-C. Lee et al. / European Journal of Medicinal Chemistry 80 (2014) 439e446
5.1.2. Synthesis of TFA salt of amino acid prodrugs 1aek
J ¼ 9.2 Hz, 1H), 4.79 (q, J ¼ 8.8, 6.0 Hz, 1H), 7.20e7.22 (m, 2H), 7.29e
7.36 (m, 9H), 7.60e7.84 (m, 6H); 13C NMR (DMSO-d6)
. 174.0, 169.1,
168.5, 164.9, 158.5, 154.7, 147.5, 140.9,140.3,135.3, 134.7, 134.1, 131.7,
130.2, 129.6, 128.9, 128.7, 128.4, 128.1, 127.8, 126.0, 124.6, 119.2,
118.8, 54.4, 52.3, 51.5, 48.9, 37.0, 27.4. HRMS Calcd for
5.1.2.1. DW2282-Gly.TFA (1a). DW2282-N-Boc-Gly (0.5g, 0.8 mmol)
was dissolved in TFA/CH2Cl2 (20 mL þ 40 mL) and stirred at room
temperature for 3 h. After removal of solvent, chromatography of
crude product on silica gel (dichloromethane:methanol ¼ 10:1)
gave DW2282-Gly (1a) as a white solid.
d
C35H32F3N5O7S m/z 723.1975, found 723.1971.
Yield: 0.62 g (90.3%); mp 167.0e168.0 ꢁC; Rf 0.22 (10:1
dichloromethane:methanol); FT-IR (cmꢂ1) 3254, 2360, 1723, 1381,
5.1.2.6. DW2282-Ile. TFA (1f). The compound (1f) was prepared by
the same procedure described for DW2282-Gly.TFA (1a) using
DW2282-N-Boc-Ile instead of DW2282-N-Boc-Gly.
1156; 1H NMR (DMSO-d6)
d
3.16 (m, 2H), 3.48 (q, J ¼ 9.2, 6.0 Hz 1H),
3.83 (s, 2H), 4.12 (t, J ¼ 8.4 Hz, 2H), 4.27 (t, J ¼ 9.2 Hz, 1H), 4.79 (t,
J ¼ 7.2 Hz, 1H), 7.22e7.37 (m, 5H), 7.64e7.79 (m, 6H), 8.16 (s, 3H),
Yield: 62.3%; mp 114.0e115.0 ꢁC; Rf 0.22 (10:1 dichloromethane/
8.21 (s, 1H), 10.73 (s, 1H); 13C NMR (400 MHz, DMSO-d6)
d. 170.7,
methanol); FT-IR (cmꢂ1) 3254, 2359,1724,1667,1381,1156; 1H NMR
169.2, 168.8, 168.5, 158.6, 147.5, 140.9, 140.4, 134.3, 131.5, 131.4,
128.9, 128.1, 127.8, 126.0, 124.3, 117.7, 116.0, 57.8, 51.3, 50.9, 48.9,
27.6. HRMS Calcd for C28H26F3N5O7S m/z 633.1505, found 633.1501.
(CD3OD-d4)
d
1.00 (m, 6H), 1.10 (d, J ¼ 6.8 Hz, 3H), 1.28 (m, 1H), 1.65
(m, 1H), 2.05 (m, 1H), 3.20 (m, 2H), 3.60 (q, J ¼ 9.6, 6.0 Hz, 1H), 3.86
(d, J ¼ 5.6 Hz,1H), 4.20 (t, J ¼ 8.0 Hz, 2H), 4.31 (t, J ¼ 9.6 Hz,1H), 4.79
(q, J ¼ 8.8, 6.0 Hz, 1H), 7.20e7.22 (m, 2H), 7.30e7.37 (m, 3H), 7.63e
5.1.2.2. DW2282-Ala. TFA (1b). The compound (1b) was prepared
by the same procedure described for DW2282-Gly.TFA (1a) using
DW2282-N-Boc-Ala instead of DW2282-N-Boc-Gly.
7.65 (m, 2H), 7.77e7.80 (m, 3H), 7.85 (s, 1H); 13C NMR (DMSO-d6)
d.
174.4, 169.3, 168.6, 168.1, 158.6, 154.7, 147.8, 141.0, 140.3, 134.3, 131.9,
131.6, 128.9, 128.4, 128.1, 127.8, 126.0, 124.6, 119.2, 118.8, 57.6, 52.3,
51.5, 51.1, 36.4, 27.5, 24.0, 14.7, 11.0. HRMS Calcd for C32H34F3N5O7S
m/z 689.2131, found 689.2125.
Yield: 70.6%; mp 238.0e239.0 ꢁC; Rf 0.25 (10:1 dichloro-
methane/methanol); FT-IR (cmꢂ1) 3254, 2358, 1746, 1404, 1156; 1H
NMR (DMSO-d6)
d
1.44 (d, J ¼ 6.8 Hz, 3H), 3.16 (m, 2H), 3.49 (q,
J ¼ 9.2, 6.0 Hz, 1H) 4.01 (m, 1H), 4.12 (t, J ¼ 8.0 Hz, 2H), 4.27 (t,
J ¼ 8.8 Hz, 1H), 4.80 (m, 1H), 7.22e7.24 (m, 2H), 7.30e7.39 (m, 3H),
7.64e7.79 (m, 6H), 8.21 (s, 1H), 10.73 (s, 1H); 13C NMR (400 MHz,
5.1.2.7. DW2282-Leu. TFA (1g). The compound (1g) was prepared
by the same procedure described for DW2282-Gly.TFA (1a) using
DW2282-N-Boc-Leu instead of DW2282-N-Boc-Gly.
DMSO-d6)
d
. 170.5, 169.2, 168.6, 168.3, 157.8, 154.5, 147.5, 140.7,
Yield: 93.0%; mp 142.0e143.0 ꢁC; Rf 0.28 (10:1 dichloro-
140.1, 134.0, 131.5, 131.4, 128.6, 128.4, 128.1, 127.8, 125.8, 124.3,118.8,
115.8, 52.1, 51.3, 50.9, 48.9, 27.2, 16.9. HRMS Calcd for
methane/methanol); FT-IR (cmꢂ1) 3262, 2360, 1724, 1654, 1381,
1156; 1H NMR (DMSO-d6)
d
0.95(d, J ¼ 3.6 Hz, 6H), 1.69 (m, 3H), 3.17
C
29H28F3N5O7S m/z 647.1662, found 647.1656.
(t, J ¼ 8.0 Hz, 2H), 3.51 (q, J ¼ 8.8, 6.4 Hz 1H), 3.96 (m, 1H), 4.13 (t,
J ¼ 8.0 Hz, 2H), 4.28 (t, J ¼ 9.2 Hz, 1H), 4.80 (t, J ¼ 7.2 Hz, 1H), 7.23e
7.25 (m, 2H), 7.31e7.40 (m, 3H), 7.66e7.68 (m, 2H), 7.73e7.81(m,
5.1.2.3. DW2282-Pro. TFA (1c). The compound (1c) was prepared
by the same procedure described for DW2282-Gly (1a) using
DW2282-N-Boc-Pro instead of DW2282-N-Boc-Gly.
4H), 8.28 (s, 2H), 10.76 (s, 1H); 13C NMR (DMSO-d6)
d. 176.6, 169.3,
168.6,162.5,158.2,154.7,147.7, 140.9,140.3,134.2,131.7, 131.6, 128.9,
128.5, 128.1, 127.7, 126.0, 124.5, 119.5, 119.2, 52.3, 51.9, 51.5, 51.2,
35.8, 30.7, 22.6, 21.8. HRMS Calcd for C32H34F3N5O7S m/z 689.2131,
found 689.2126.
Yield: 60.7%; mp 142.0e143.0 ꢁC; Rf 0.28 (10:1 dichloro-
methane/methanol); FT-IR (cmꢂ1) 3105, 2359, 1673, 1381, 1130; 1H
NMR (CD3OD-d4)
d 2.10e2.17(m, 3H), 2.54 (m, 1H), 3.21 (m, 2H),
3.36e3.50 (m, 2H), 3.60 (q, J ¼ 10.0, 6.4 Hz, 1H), 4.20 (t, J ¼ 8.4 Hz,
2H), 4.32 (t, J ¼ 9.6 Hz,1H), 4.44 (m, 1H), 4.80 (m, 1H), 7.20e7.23 (m,
2H), 7.30e7.37 (m, 4H), 7.63e7.65 (m, 2H), 7.76e7.78 (m, 4H) 7.85
5.1.2.8. DW2282-Met. TFA (1h). The compound (1h) was prepared
by the same procedure as described for the preparation of
DW2282-Gly.TFA (1a) using DW2282-N-Boc-Met instead of
DW2282-N-Boc-Gly.
(m, 1H); 13C NMR (DMSO-d6)
d. 176.9, 169.3, 167.5, 162.5, 158.3,
152.8, 147.7, 140.9,140.4,134.3, 131.5, 131.4, 128.9,128.6,128.1, 127.8,
126.0, 124.3, 119.1, 115.7, 61.1, 59.9, 53.8, 45.9, 35.8, 32.2, 30.8, 29.6
HRMS Calcd for C31H30F3N5O7S m/z 673.1818, found 673.1814.
Yield: 75.5%; mp 147.5e148.5 ꢁC; Rf 0.20 (10:1 dichloromethane/
methanol); FT-IR (cmꢂ1) 3254, 2359, 1724, 1665, 1382, 1156; 1H
NMR (CD3OD-d4)
d 1.87e2.10 (m, 2H), 2.08 (s, 3H), 2.62 (m, 2H),
5.1.2.4. DW2282-Val. TFA (1d). The compound (1d) was prepared
by the same procedure described for DW2282-Gly.TFA (1a) using
DW2282-N-Boc-Val instead of DW2282-N-Boc-Gly.
3.20 (m, 2H), 3.60 (m, 2H), 4.20 (t, J ¼ 8.4 Hz, 2H), 4.31 (t, J ¼ 9.6 Hz,
1H), 4.55 (s, 1H), 4.76 (m, 1H), 7.20e7.22 (m, 3H), 7.30e7.34 (m, 3H),
7.60e7.62 (m, 2H), 7.76e7.84 (m, 5H); 13C NMR (DMSO-d6)
d. 178.0,
Yield: 45.1%; mp 184.0e185.0 ꢁC; Rf 0.24 (10:1 dichloromethane/
169.2,168.6, 167.6, 162.4,154.7, 147.7, 140.9,140.3, 134.3,131.8, 131.6,
128.9, 128.5, 128.1, 127.7, 126.0, 124.6, 119.5, 112.9, 52.6, 52.3, 51.5,
51.1, 35.8, 30.7, 28.3, 14.5. HRMS Calcd for C31H32F3N5O7S2 m/z
707.1695, found 707.1691.
methanol); FT-IR (cmꢂ1) 3251, 2357, 1528, 1201; 1H NMR (CD3OD-
d4)
d
1.11 (q, J ¼ 13.6, 6.8 Hz, 6H), 2.30 (m, 1H), 3.21 (m, 2H), 3.60 (q,
J ¼ 9.6, 6.0 Hz, 1H), 3.84 (d, J ¼ 5.6 Hz, 1H), 4.20 (t, J ¼ 8.4 Hz, 2H),
4.31 (t, J ¼ 9.6 Hz, 1H), 4.79 (q, J ¼ 8.8, 5.6 Hz, 1H), 7.20e7.22 (m,
2H), 7.30e7.36 (m, 3H), 7.63e7.65 (m, 2H), 7.77e7.80 (m, 3H), 7.85
5.1.2.9. DW2282-Cys. TFA (1i). DW2282-N-Boc-S-trityl-L-Cys (0.5 g,
(s, 1H); 13C NMR (DMSO-d6)
d. 178.0, 169.2, 168.5, 164.9, 158.5, 154.7,
0.56 mmol) was dissolved in trifluoroacetic acid/dichloromethane
(20mL þ 40 mL) and stirred at room temperature for 3 h, the color
of the solution turned brown. Triethylsilane was added until the
solution became colorless. Stirring was continued for 1 h. After
removal of solvent, the solid residue was washed several times with
n-hexane to remove most of the triphenylmethane. Then, the solid
was dissolved in a minimal amount of methanol and excess of
diethyl ether. The precipitate was collected and washed with dry
diethyl ether to give the compound (1i).
147.5, 141.0, 140.3,135.3, 134.3, 131.7, 128.9,128.4,128.1, 127.8, 126.0,
124.6, 119.2, 118.8, 58.3, 51.5, 51.1, 48.9, 36.1, 30.0, 18.4. HRMS Calcd
for C31H32F3N5O7S m/z 675.1975, found 675.1969.
5.1.2.5. DW2282-Phe. TFA (1e). The compound (1e) was prepared
by the same procedure described for DW2282-Gly.TFA (1a) using
DW2282-N-Boc-Phe instead of DW2282-N-Boc-Gly.
Yield: 75.5%; mp 169.0e170.0 ꢁC; Rf 0.20 (10:1 dichloro-
methane/methanol); FT-IR (cmꢂ1) 3254, 2359, 1745, 1659, 1383,
Yield: 92.6%; mp 174.0e175.0 ꢁC; Rf 0.30 (10:1 dichloromethane/
1156; 1H NMR (CD3OD-d4)
6.0 Hz, 1H), 4.19 (t, J ¼ 8.4 Hz, 2H), 4.24 (t, J ¼ 7.2 Hz, 1H), 4.31 (t,
d
3.15e3.52 (m, 4H), 3.60 (q, J ¼ 9.6,
methanol); FT-IR (cmꢂ1) 3105, 2360, 1673, 1383, 1157; 1H NMR
(DMSO-d6)
d
1.39 (s, 1H), 3.06 (m, 1H), 3.16 (m, 3H), 3.50 (q, J ¼ 8.8,

K.-C. Lee et al. / European Journal of Medicinal Chemistry 80 (2014) 439e446
445
6.8 Hz, 1H), 4.12e4.18 (m, 3H), 4.28 (t, J ¼ 8.8 Hz, 1H), 4.78 (t,
J ¼ 7.8 Hz, 1H), 7.23e7.37 (m, 6H), 7.62e7.80 (m, 7H), 8.20 (s, 1H),
8.43 (s, 1H), 10.78 (s, 1H); 13C NMR (DMSO-d6)
. 177.9, 169.3, 168.5,
5.3. Procedure for in vitro plasma reconversion kinetics studies of
1aek
d
164.9,157.8, 154.7, 147.5, 141.9, 140.8,135.3, 134.2,131.7, 128.8,128.4,
128.1, 127.8, 126.0, 124.6, 119.3, 116.0, 52.3, 51.5, 51.1, 48.9, 27.4, 20.7.
HRMS Calcd for C29H28F3N5O7S2 m/z 679.1382, found 679.1375.
Human plasma was obtained from Sigma (P9523). The stock
solution (1 mg/mL) of compounds 1aek in dimethylsulfoxide
(DMSO) was diluted 10 times with the plasma and the mixture was
maintained at 37 ꢃ 0.5 ꢁC in a Precision shaking water bath. No
plasma protein precipitation was observed at this concentration of
DMSO. The plasma was sampled at selected times (0, 0.5, 1, 2, 4, 8
and 24 h), and then it was later subjected to the sample preparation
method described below and then analyzed with the modified
HPLC assay (Table 3).
5.1.2.10. DW2282-Asp. TFA (1j).
DW2282-N-Boc-L-Asp(OBzl)-OH (0.5 g, 0.65 mmol) was dissolved
in trifluoroacetic acid/dichloromethane (20 mL þ 40 mL) and stir-
red at room temperature for 3 h. After removal of solvent, the solid
residue was washed several times with diethyl ether. Then, the
solid was dissolved in methanol (30 mL), 10% Pd/C (about 50 mg)
was added, and the mixture was hydrogenated at room tempera-
ture using a hydrogen balloon. After 2 h, the mixture was filtered
through Celite and evaporated under vacuum. Then, the solid was
dissolved in a minimal amount of methanol and excess of diethyl
ether. The precipitate was collected and washed with dry diethyl
ether to get the compound (1j).
Sample Preparation for RPHPLC: Plasma samples (40
mL) were
diluted with a sufficient volume (3 mL) of methylene chloride to
precipitate the plasma proteins as well as to extract the compounds
of interest. After vortexing for 5 min and centrifuging at 3000 ꢀ g
for 10 min, the organic layer was carefully taken out and dried. The
plasma extracts were reconstituted in 160
mL of methanol with
vortexing. After syringe filtration (0.45
mm, Waters), the filtrate was
Yield: 68.8%; mp 164.0e165.0 ꢁC; Rf 0.24 (10:1 dichloro-
methane/methanol); FT-IR (cmꢂ1) 3254, 2360, 1742, 1665, 1383,
analyzed using RPHPLC.
1157; 1H NMR (DMSO-d6)
d 2.82e3.02 (m, 2H), 3.22 (m, 2H), 3.51 (q,
5.4. Procedure for the in vivo anti-tumor activity of 1e, 1g and 1j
J ¼ 9.2, 6.4 Hz, 1H), 4.13 (t, J ¼ 8.4 Hz, 2H), 4.28 (m, 2H), 4.80 (t,
J ¼ 7.6 Hz, 1H), 7.23e7.40 (m, 5H), 7.65e7.95 (m, 6H), 8.28 (s, 1H),
Each syngeneic host mice were intradermally implanted with
SW620 tumor cells on day zero. When tumor volume reached
proper size, thirty animals were distributed into five groups.
Compounds 1e, 1g and 1j (70 mg/kg, b.i.d., 5 days, p.o.) were fed to
the animals from the following day after grouping. Compound 1
(70 mg, every other day, 3 times, p.o.) was used as a positive control.
Control group received vehicle only. Anti-tumor activities were
evaluated by comparing the tumor volume of treated group with
that of control group. Tumor diameter was serially measured with
vernier calipers and tumor volume (V) was calculated as follows:
10.72 (s, 1H); 13C NMR (DMSO-d6)
d. 177.8, 171.2, 169.2, 168.3, 164.7,
157.8, 154.5, 147.5, 140.7, 140.1, 134.0, 131.8, 131.4, 128.6, 128.4, 128.1,
127.8, 126.0, 125.1, 119.1, 118.5, 52.8, 51.3, 49.8, 48.9, 38.9, 35.3.
HRMS Calcd for C30H28F3N5O9S m/z 691.1560, found 691.1553.
5.1.2.11. DW2282-Ser. HCl (1k).
DW2282-N-Fmoc-O-tert-Bu-L-Ser (0.5 g, 0.60 mmol) was dissolved
in piperidine/dichloromethane (20mL þ 80 mL) and stirred at room
temperature for 3 h. After removal of solvent, the mixture was
dissolved in 4-N hydrochloric acid/dioxane (50mL þ 50 mL) and
refluxed for 3 h. After removal of solvent, chromatography of crude
product on silica gel (dichloromethane:methanol ¼ 10:1) gave the
compound (1k) as a white solid.
V ¼ ½ ꢀ a ꢀ b2 [a: long axis (mm), b: short axis (mm)]
Tumor wet weight of treated (Tw) and control (Cw) group was
measured at the day 10 of each experiment and the percentage of
tumor growth inhibition was calculated as follows:
Yield: 36.7%; mp 152.3e153.0 ꢁC; Rf 0.28 (10:1 dichloro-
methane/methanol); FT-IR (cmꢂ1) 3119, 2803, 2358, 1702, 1665,
1382, 1156; 1H NMR (CD3OD-d4)
d
3.21 (m, 2H), 3.60 (q, J ¼ 9.6,
6.0 Hz,1H), 4.01 (m, 2H), 4.10 (m, 1H), 4.20 (t, J ¼ 8.4 Hz, 2H), 4.31 (t,
Inhibition rate (%) ¼ [1 ꢂ (Tw/Cw)] ꢀ 100
J ¼ 9.6 Hz, 1H), 4.80 (m, 1H), 7.20e7.23 (m, 2H), 7.30e7.34 (m, 4H),
7.61e7.64 (m, 2H), 7.70e7.85 (m, 4H); 13C NMR (DMSO-d6)
d. 178.1,
169.0, 168.5, 164.8, 154.5, 148.9, 140.7, 140.3, 134.0, 131.6, 131.4,
128.9, 128.4, 128.1, 127.8, 126.0, 125.1, 119.0, 110.8, 79.9, 76.0, 52.3,
44.3, 32.8, 27.1. HRMS Calcd for C29H28F3N5O8S m/z 663.1611, found
663.1607.
Acknowledgment
This work was supported by Priority Research Centers Program
through the National Research Foundation of Korea (NRF) funded
by the Ministry of Education, Science and Technology (2009-
0093815).
5.2. Procedure for the measurement of water solubility of 1aek
Standard sample solutions of 1 and 1aek were prepared by
dissolving sample 1 and 1aek (1 mg) in HPLC grade methanol
(10 mL) and the absorbance was measured [27]. Saturated solutions
Appendix A. Supplementary data
were prepared by adding 1 and 1aek (1 mg) in water (250
mixture was vortexed for one hour at room temperature and then
filtered using 0.45 m syringe filter. Absorbance of saturated water
mL). The
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.048.
m
solutions of 1 and 1aek was measured. Solubility of 1 and 1aek
was calculated using equation (1).
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C0 ¼ A0 ꢀ C/A
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where
C
¼
concentration of standard solution (mg/mL),
A ¼ absorbance of standard solution, A0 ¼ absorbance of saturated
sample solution, C0 ¼ concentration of water solution of 1 and 1aek
(mg/mL).

446
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