Discovery of 2-arylthiazolidine-4-carboxylic acid amides as a new class of cytotoxic agents for prostate cancer

Article abstract of DOI:10.1021/jm049208b

To improve the selectivity and antiproliferative activity of previously reported serine amide phosphates (SAPs), we designed a new series of 4-thiazolidinone amides, in which the 4-thiazolidinone moiety was introduced as a phosphate mimic. However, these 4-thiazolidinone derivatives demonstrated less cytotoxicity in prostate cancer cells despite improved selectivity over RH7777 cells. To further optimize the thiazolidinone analogues in terms of cytotoxicity and selectivity, we made closely related structural modifications, which led us to the discovery of a new class of 2-arylthiazolidine-4-carboxylic acid amides. These compounds were potent cytotoxic agents with IC₅₀ values in the low micromolar concentration range and demonstrated enhanced selectivity in receptor-negative cells compared to SAPs and 4-thiazolidinone amides.

Full text of DOI:10.1021/jm049208b

2584
J. Med. Chem. 2005, 48, 2584-2588
Discovery of 2-Arylthiazolidine-4-carboxylic Acid Amides as a New Class of
Cytotoxic Agents for Prostate Cancer†
Veeresa Gududuru,‡ Eunju Hurh,§ James T. Dalton,§ and Duane D. Miller*,‡
Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center,
Memphis, Tennessee 38163, and Division of Pharmaceutics, College of Pharmacy, The Ohio State University,
Columbus, Ohio 43210
Received September 30, 2004
To improve the selectivity and antiproliferative activity of previously reported serine amide
phosphates (SAPs), we designed a new series of 4-thiazolidinone amides, in which the
4-thiazolidinone moiety was introduced as a phosphate mimic. However, these 4-thiazolidinone
derivatives demonstrated less cytotoxicity in prostate cancer cells despite improved selectivity
over RH7777 cells. To further optimize the thiazolidinone analogues in terms of cytotoxicity
and selectivity, we made closely related structural modifications, which led us to the discovery
of a new class of 2-arylthiazolidine-4-carboxylic acid amides. These compounds were potent
cytotoxic agents with IC50 values in the low micromolar concentration range and demonstrated
enhanced selectivity in receptor-negative cells compared to SAPs and 4-thiazolidinone amides.
Introduction
One promising drug development strategy for prostate
cancer involves identifying and testing agents that
interfere with growth factors and other molecules
involved in the cancer cell’s signaling pathways. G-
protein-coupled receptors (GPCRs) are a family of
membrane-bound proteins that are involved in the
proliferation and survival of prostate cancer cells initi-
ated by binding of lysophospholipids (LPLs).1-4 The
importance of G protein-dependent pathways in the
regulation of growth and metastasis in vivo is cor-
Figure 1.
roborated by the observation that the growth of androgen-
independent prostate cancer cells in mice is attenuated
by treatment with pertussis toxin, an inhibitor of Gi/o
proteins.5 Lysophosphatidic acid (LPA) and sphingosine
1-phosphate (S1P) are lipid mediators generated via the
regulated breakdown of membrane phospholipids that
are known to stimulate GPCR-signaling.
LPLs bind to GPCRs encoded by the Edg gene family,
collectively referred to as LPL receptors, to exert diverse
biological effects. Lysophosphatidic acid (LPA) stimu-
lates phospholipase D activity and PC-3 prostate cell
proliferation.6 Further, prior studies have shown that
LPA is mitogenic in prostate cancer cells and that PC-3
and DU-145 cells express LPA1, LPA2, and LPA3 recep-
tors.7 Advanced prostate cancers express LPL receptors
and depend on phosphatidylinositol 3-kinase (PI3K)
signaling for growth and progression to androgen inde-
pendence.2 Thus, these pathways are widely viewed as
one of the most promising new approaches to cancer
therapy8 and provide an especially novel approach to
the treatment of advanced, androgen-refractory prostate
cancer. Despite the promise of this approach, there are
no clinically available therapies that selectively exploit
or inhibit LPA or PI3K signaling.
In a previous contribution from our laboratory,9 we
showed that effective cytotoxic agents were obtained,
by replacing the glycerol backbone in LPA with serine
amide. However, the most potent compounds in that
series of derivatives were nonselective and potently
killed both prostate cancer and control cell lines. To
improve the selectivity and enhance the pharmaco-
kinetic and antiproliferative properties, 2-aryl-4-oxo-
thiazolidine amides with general structure III (Figure
1) were designed, utilizing 4-thiazolidinone moiety as
a biomimetic replacement for the phosphate group.10
This strategic modification showed that the 2-aryl-
thiazolidinone moiety is indeed quite beneficial for
obtaining a new set of antiproliferative compounds with
improved selectivity, but resulted in decreased potency
compared to serine amide phosphates.9 To further
optimize the structural characteristics of these com-
pounds to selectively elicit antiproliferative activity, we
made closely related, minor modifications to 2-aryl-4-
oxo-thiazolidine amides as shown in Figure 1. Our
current work highlights synthesis, structure-activity
relationship (SAR) studies, and biological evaluation of
2-arylthiazolidine-4-carboxylic acid amides (ATCAAs)
for prostate cancer.
* Corresponding author. 847 Monroe Ave., Rm. 227C, Memphis,
TN 38163. Phone: +1-901-448-6026. Fax: +1-901-448-3446. E-mail:
dmiller@utmem.edu.
† Part of this work was presented at 227th ACS National Meeting,
Anaheim, CA, March 28-April 1, 2004 and at 95th AACR Annual
Meeting, Orlando, FL, March 27-March 31, 2004.
‡ University of Tennessee Health Science Center.
§ The Ohio State University.
10.1021/jm049208b CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/22/2005

2-Arylthiazolidine-4-carboxylic Acid Amides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2585
Table 1. Structures and Physical Data of Synthesized Compounds
intermediate 2a-v R
phenyl
n-dodecyl
cyclohexyl
benzyl
compd
R
R1
R2
mp (°C) yield (%)
formula
anal.
3‚HCl
4‚HCl
5‚HCl
6‚HCl
7
8
9
phenyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
C7H15
ND
95
80
83
70
78
69
75
81
65
63
60
75
69
70
83
70
63
73
77
60
70
70
80
78
73
70
95
90
C17H27ClN2OS
C24H41ClN2OS
C28H49ClN2OS
C29H51ClN2OS
C34H68N2OS
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
phenyl
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
14H29
18H37
19H39
18H37
18H37
18H37
18H37
18H37
18H37
18H37
18H37
18H37
18H37
18H37
18H37
18H37
18H37
phenyl
phenyl
n-dodecyl
cyclohexyl
benzyl
3-indolyl
3-pyridinyl
93
85
3-indolyl
3-pyridinyl
3-furanyl
86
60
C28H54N2OS
80
C29H50N2OS
4-dimethyl amino phenyl 10
125
94
C30H49N3OS
3-hydroxyphenyl
4-methoxyphenyl
3,4-dimethoxyphenyl
3,4,5-trimethoxyphenyl
4-acetylamino phenyl
4-fluorophenyl
11
C27H47N3OS
12‚HCl 3-furanyl
99
C26H47ClN2O2S
C30H53N3OS
13
14
4-dimethylaminophenyl
3-hydroxyphenyl
75
50
C28H48N2O2S
C29H51ClN2O2S
C30H53ClN2O3S
C31H55ClN2O4S
C30H52ClN3O2S
C28H47FN2OS
C28H47BrN2OS
C28H47N3O3S
C29H47N3OS
C28H46F2N2OS
C28H46Cl2N2OS
C28H46BrFN2OS C, H, N
C29H50N2OS
C34H53ClN2OS
C30H50N2O2S
C29H50N2O3S2
15‚HCl 4-methoxyphenyl
95
16‚HCl 3,4-dimethoxyphenyl
17‚HCl 3,4,5-trimethoxyphenyl
18‚HCl 4-acetylaminophenyl
103
115
170
65
4-bromophenyl
4-nitrophenyl
4-cyanophenyl
19
20
21
22
23
24
25
26
4-fluorophenyl
3,5-difluorophenyl
2,6-dichlorophenyl
3-bromo-4-fluorophenyl
4-methylphenyl
biphenyl
4-bromophenyl
81
115
90
113
49
4-nitrophenyl
C18H37
4-cyanophenyl
C
C
C
C
C
C
18H37
18H37
18H37
18H37
18H37
18H37
3,5-difluorophenyl
2,6-dichlorophenyl
3-bromo-4-fluorophenyl
4-methylphenyl
100
120
130
90
C, H, N
C, H, N
C, H, N
C, H, N
27‚HCl biphenyl
28
29
phenyl
phenyl
COCH3
SO2CH3 C18H37
C18H37
55
Scheme 1a
Scheme 2a
a Reagents and conditions: (a) RCHO, EtOH; (b) CH3(CH2)nNH2,
EDC, HOBt, CH2Cl2.
Chemistry
a Reagents and conditions: (a) Ac2O, pyridine; (b) CH3SO2Cl,
pyridine.
Compounds described in this study were prepared
following straightforward chemistry. Reaction of L-
cysteine with various aldehydes under reported condi-
tions11 gave corresponding acids, which were isolated
as diastereomeric mixtures. These mixtures were used
directly for the formation of corresponding amides by
reacting with appropriate alkylamines using EDC/HOBt
as shown in Scheme 1. All compounds thus prepared
were characterized as diastereomeric mixtures (Table
1). N-Acyl and N-sulfonyl derivatives (28 and 29) were
synthesized from 5 by standard procedures (Scheme 2).
The synthesis of thiazole derivative 34 was accom-
plished starting from cysteine methyl ester (30) as
shown in Scheme 3. The structures of the synthesized
compounds and the yields of the syntheses are presented
in Table 1.
Scheme 3a
a Reagents and conditions: (a) NaHCO3, EtOH, H2O; (b) NBS,
CCl4; (c) NaOH, MeOH; (d) C18H37NH2, EDC, HOBt, CH2Cl2.
Results and Discussion
The ability of 2-aryl-thiazolidine derivatives (AT-
CAAs) to inhibit the growth of five human prostate
cancer cell lines (DU-145, PC-3, LNCaP, PPC-1, and
TSU-Pr1) was assessed using the sulforhodamine B
(SRB) assay.9 We also included a control cell line
(RH7777) that does not express LPL receptors,12 to
understand whether the antiproliferative activity of
these derivatives was mediated through inhibition of
LPL receptors. We first examined LPL receptor expres-

2586 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Table 2. LPL Receptor mRNA Expression
Gududuru et al.
expression level relative to â-actin
LPL receptor
old name
RH7777
DU145
PC-3
LNCaP
PPC-1
TSU-Pr1
LPA1
LPA2
LPA3
EDG-2
EDG-4
EDG-7
UDa
UD
UD
0
2.16
0.33
0.07
2.56
2.53
0.43
0.27
3.23
UD
2.29
0.41
0.15
2.85
2.13
0.19
UD
0.32
0.28
0.60
sum LPA1-3
a UD: under detection limit.
Table 3. Antiproliferative Effects of Compounds 3-29 and 34
2.32
IC50 (µM)
compd
3‚HCl
4‚HCl
5‚HCl
6‚HCl
7
8
9
10
11
RH7777a
DU-145b
PC-3b
LNCaPb
PPC-1b
TSU-Pr1b
52.2
3.4
44.9
2.4
5.4
>20
8.9
38.5
3.0
7.8
12.4
1.4
2.1
34.7
1.3
2.0
16.8
13.0
9.3
4.0
1.3
2.9
1.1
1.1
1.2
1.5
28.0
2.0
5.0
25.6
NAc
∼20
>20
>20
>20
10.5
10.4
>20
31.0
>20
10.3
11.4
21.1
17.4
>20
∼20
>20
>20
>20
∼20
>20
>20
>20
>20
>20
NDd
NDd
NAc
15.0
>20
16.4
11.5
9.2
13.6
11.9
12.8
4.4
>20
10.7
>20
11.2
4.4
>20
15.3
8.9
2.1
7.5
3.6
7.8
12‚HCl
13
14
6.6
8.1
1.7
4.2
5.3
6.0
1.6
3.0
5.7
6.7
1.7
4.0
15‚HCl
16‚HCl
17‚HCl
18‚HCl
19
8.7
∼20
2.1
NDd
2.4
4.5
5.2
0.85
0.58
3.9
4.0
0.82
1.3
1.9
5.1
8.4
5.9
0.48
0.55
2.1
2.4
0.94
5.4
3.1
5.6
5.7
6.8
20
13.8
15.3
>20
>20
>20
11.3
10.5
>20
∼20
>20
>20
11.9
2.7e
17.3
∼ 20
>20
>20
>20
13.5
12.8
>20
∼20
>20
>20
12.0
3.4e
3.7
15.3
5.0
10.6
17.1
4.7
1.9
>20
12.6
>20
>20
6.4
3.4e
18.3
15.9
>20
>20
∼20
14.0
8.0
21
22
23
11.2
13.1
3.0
1.9
>20
16.1
>20
>20
4.9
2.0e
24
25
26
27‚HCl
28
>20
>20
>20
>20
3.6
29
34
5-FU
paclitaxel
2.1e
a Control cell line. b Prostate cancer cell lines. c No activity. d Not determined. e IC50 in nM.
sion in these cell lines by RT-PCR to validate their use
as in vitro models (Table 2). LPA1 was the predominant
LPL receptor expressed in these cell lines. However,
LNCaP cells did not express this receptor subtype. LPA2
was also expressed in all prostate cancer cell lines
examined. Interestingly, ovarian cancer cells also dem-
onstrated overexpression of LPA2 compared to normal
ovarian epithelial cells.8 PC-3 and LNCaP cells, but not
DU-145 cells, expressed LPA3, consistent with published
data.7 None of the LPL receptors was expressed in
RH7777 cells.
The diastereomeric mixtures of the target compounds
3-29 were used as such to evaluate their in vitro
inhibitory activity against prostate cancer cell lines, and
the results are summarized in Table 3. Paclitaxel and
5-fluorouracil were used as reference drugs for com-
parison. Since preparation of isolated enantiomers was
not easy to achieve, the IC50s were obtained on diaster-
eomeric mixtures in order to select the most promising
compounds. Many of these thiazolidine analogues were
very effective in killing prostate cancer cell lines with
IC50 values as low as 480 nM (Table 3). Examination of
the cytotoxic effects of 3-5 showed that as the chain
length increased from C7 to C18, the potency also
increased. However, a further increase in the alkyl chain
length by one carbon unit (6) caused a significant loss
of activity. Interestingly, the C14 derivative (4) demon-
strated higher potency than 5, but was 8-fold less
selective against the RH7777 cell line. Thus, an alkyl
chain with C18 unit was optimal for maintaining the
potency and selectivity observed in this series of com-
pounds. N-Acyl and N-sulfonyl derivatives (28 and 29)
were significantly less cytotoxic than parent compound
5. Replacement of the phenyl ring with an alkyl or
cyclohexyl group reduced the potency (7 and 8) relative
to the thiazolidine derivative (5). Introduction of a
methylene spacer separating the phenyl ring and the
thiazolidine ring furnished a compound 9, which was
less active than the parent compound 5.
Replacements of the phenyl ring with a heterocycle,
such as an indole, pyridine, or furan ring was investi-
gated by synthesizing analogues 10-12. The furanyl
derivative 12 showed equivalent cytotoxicity as 5, but
was 3-fold less selective against RH7777 cells.
The cytotoxicity data of compounds 13-27 provides
a summary of a broad survey of phenyl ring-substituted
analogues. Examination of the IC50 values of these
analogues demonstrates a greater tolerance for diverse
substituents in the phenyl ring. In general, the most
potent analogues possessed electron-donating substit-

2-Arylthiazolidine-4-carboxylic Acid Amides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2587
Table 4. Thiazolidine Amide-Induced Apoptosis
most potent and selective cytotoxic agents with an IC50
of 0.55 µM and 38-fold selectivity in PPC-1 cells.
Further, the ability of these analogues to induce apop-
tosis in LNCaP and PC-3 cells provides an important
clue to understand their mechanism of action, and
suggests that they may have therapeutic utility in the
treatment of prostate or ovarian cancer. All compounds
discussed in this report have been prepared and tested
as diastereomeric mixtures. Future efforts shall be
aimed at synthesis and evaluation of pure individual
stereoisomers of the most promising thiazolidines dis-
cussed above.
compd for 72 h
PC-3
LNCaP
RH7777
4
2 µM
1.8
18.7
54.0
1.4
14.1
75.4
80.7
4.5
45.2
37.1
26.1
2.6
3.2
5 µM
10 µM
2 µM
5 µM
10 µM
20 µM
2.5
5
NDa
2.3
3.4
12.7
a ND: not determined.
uents, as exemplified by comparison of 13 and 16-18,
relative to 5. Compound 18 was one of the most active
compounds with an IC50 of 0.55 µM and was 38-fold
more selective in PPC-1 cells compared to RH7777 cells.
On the other hand, thiazolidine analogues (19-25), with
electron-withdrawing substituents demonstrated less
cytotoxicity. Comparison of the potencies of 26 and 27
suggest that substitution of the phenyl ring with a bulky
group reduces the activity. To understand the effect of
unsaturation on potency and selectivity, and to over-
come the problems associated with stereoisomers, we
replaced the central thiazolidine core in 5 with a
thiazole ring. However, thiazole derivative (34) did not
show any activity below 20 µM in both prostate and
RH7777 cells, which suggests that thiazolidine ring with
two chiral centers plays an important role in providing
potency and selectivity.
From the LPL receptor mRNA expression studies
(Table 2), it was evident that these cell lines serve as
an excellent model system to explore the effects of LPL
receptors in prostate cancer cell growth. Given the
structural similarity of SAPs to ceramide (and the
known ability of ceramide to induce apoptosis), we next
determined whether the antiproliferative effects of
thiazolidine analogues were mediated via apoptotic
events. We examined the ability of our analogues to
induce apoptosis in LNCaP, PC-3, and RH7777 cells
using a quantitative sandwich ELISA13 that measures
DNA-histone complex released during apoptosis. The
enrichment factor calculated as ratio of OD405 in
treated and untreated cells provides a quantitative
assessment of the degree of apoptosis induced. Initially,
we used only two compounds (4 and 5) for this study.
Apoptotic activity of analogue 4 was selective in prostate
cancer cells despite nonselective cytotoxicity in RH7777
negative control cells (Table 4). Analogue 5 induced
apoptosis in PC-3 and LNCaP cells, but to a lesser
extent in PC-3 cells perhaps due to lower potency in this
cell line. These data suggests that thiazolidine ana-
logues may act as potent inducers of apoptosis and
selectively kill a variety of prostate cancer cell lines.
Experimental Section
All reagents and solvents used were reagent grade or were
purified by standard methods before use. Moisture-sensitive
reactions were carried under an argon atmosphere. Progress
of the reactions was followed by thin-layer chromatography
(TLC) analysis. Flash column chromatography was carried out
using silica gel (200-425 mesh) supplied by Fisher. Melting
points were measured in open capillary tubes on a Thomas-
Hoover melting point apparatus and are uncorrected. All
compounds were characterized by NMR and MS (ESI). 1H
NMR spectra were recorded on a Varian 300 instrument.
Chemical shifts are reported as δ values relative to Me4Si as
internal standard. Mass spectra were obtained in the electro-
spray (ES) mode using Esquire-LC (Bruker) spectrometer.
Elemental analyses were performed by Atlantic Microlab Inc.
(Norcross, GA).
General Procedure for the Preparation of 2a-v. A
mixture of L-cysteine (1, 0.5 g, 4.12 mmol) and appropriate
aldehyde (4.12 mmol) in ethanol (15 mL) was stirred at room
temperature for 5 h, and the solid separated was collected,
washed with diethyl ether and dried to afford 2a-v.
(2RS,4R)-2-Phenylthiazolidine-4-carboxylic Acid (2a).
Obtained as colorless crystals (0.82 g, 95%). 1H NMR (DMSO-
d6) δ 7.24-7.53 (m, 5H), 5.67 (s, 0.6H), 5.50 (s, 0.4H), 4.22
(dd, J ) 6.9, 4.5 Hz, 0.6H), 3.90 (dd, J ) 8.7, 7.2 Hz, 0.4H),
3.27-3.40 (m, 1H), 3.04-3.16 (m, 1H); MS (ESI) m/z 208
(M - 1).
General Procedure for the Preparation of 3-27. A
mixture of appropriate carboxylic acid (2a-v, 0.3-0.5 g), EDC
(1.25 equiv) and HOBt (1 equiv) in CH2Cl2 (25-50 mL) was
stirred for 10 min. To this solution, appropriate alkylamine
(1 equiv) was added and stirring continued at room temper-
ature for 6-8 h. Reaction mixture was diluted with CH2Cl2
(100-150 mL) and sequentially washed with water, sat.
NaHCO3, and brine and dried over Na2SO4. The solvent was
removed under reduced pressure to yield a crude solid, which
was purified by column chromatography. The purified com-
pounds (3-6, 12, 15-18, and 27) were converted to corre-
sponding hydrochlorides using 2 M HCl/Et2O.
(2RS,4R)-2-Phenylthiazolidine-4-carboxylic Acid Hep-
tylamide Hydrochloride (3‚HCl). 1H NMR (DMSO-d6) δ
8.72 (s, 1H), 7.65 (m, 2H), 7.43 (m, 3H), 5.89 (s, 0.6H), 5.84 (s,
0.4H), 4.66 (t, J ) 6.3 Hz, 0.6H), 4.46 (t, J ) 6.9 Hz, 0.4H),
3.55-3.71 (m, 1H), 3.24-3.34 (m, 1H), 3.13 (d, J ) 5.7 Hz,
2H), 1.44 (m, 2H), 1.25 (s, 8H), 0.83 (t, J ) 6.9 Hz, 3H); MS
(ESI) m/z 307.10 (M + 1).
Conclusions
2-Phenylthiazolidine-4-carboxylic Acid Methyl Ester
(31). To a solution of DL-cysteine (3 g, 24.76 mmol) in MeOH
(50 mL) at 0 °C, SOCl2 (2.76 mL, 37.14 mmol) was slowly
added and warmed to room temperature then refluxed for 3
h. The reaction mixture was concentrated in vacuo to yield a
residue. This residue 30 was taken up in aqueous EtOH (1:1,
30 mL), NaHCO3 (2.28 g, 27.23 mmol) was added, and after
10 min, benzaldehyde (2.5 mL, 24.76 mmol) was added and
stirring continued for 3 h. CHCl3 (200 mL) was added to the
reaction mixture, washed with water and brine, and dried (Na2-
SO4), and solvent was removed in vacuo. The crude product
was purified by column chromatography to afford 31 (4.7 g,
85%). 1H NMR (CDCl3) δ 7.51-7.62 (m, 2H), 7.32-7.42 (m,
3H), 5.84 (s, 0.4H), 5.58 (s, 0.6H), 4.24 (t, J ) 6.3 Hz, 0.4H),
2-Aryl-thiazolidine-4-carboxylic acid amides (AT-
CAAs) were obtained by the modification of previously
reported 4-thiazolidinones. We synthesized a number
of ATCAAs and evaluated for their inhibitory activity
toward the growth of human prostate cancer cell lines.
Introduction of ring activating groups on the phenyl ring
resulted in increased potencies for prostate cancer cell
lines and led to discovery of several new anticancer
agents represented by analogues 16, 17, and 18 with
low/sub micromolar cytotoxicity and high selectivity.
From this study, compound 18 emerged as one of the

2588 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Gududuru et al.
4.01 (t, J ) 7.5 Hz, 0.6H), 3.83 (s, 3H), 3.39-3.55 (m, 1H),
3.10-3.26 (m, 1H); MS (ESI) m/z 224 (M + 1).
plate reader (DYNEX Technologies, Chantilly, VA). Percent-
ages of cell survival versus drug concentrations were plotted
and the IC50 (concentration that inhibited cell growth by 50%
of untreated control) values were obtained by nonlinear
regression analysis using WinNonlin (Pharsight Corporation,
Mountain View, CA). 5-Fluorouracil was used as a positive
control to compare potencies of the new compounds.
Apoptosis. A sandwich ELISA (Roche, Mannheim, Ger-
many) utilizing monoclonal antibodies specific for DNA and
histones was used to quantify degree of apoptosis induced by
the analogues after 72 h exposure. This assay measures DNA-
histone complexes (mono- and oligonucleosomes) released into
cytoplasm from the nucleus during apoptosis. RH7777 cells
were employed because of nonspecific cytotoxicity of compound
4 in receptor-negative cells as well as receptor-positive prostate
cancer cells.
2-Phenylthiazole-4-carboxylic Acid Methyl Ester (32).
This compound was synthesized following a reported proce-
dure.14 N-Bromosuccinamide (2.48 g, 13.9 mmol) and benzoyl
peroxide (0.05 g) were added to 31 (1.5 g, 6.7 mmol) dissolved
in CCl4 (70 mL), and the solution was refluxed for 6 h. Solvent
was removed in vacuo, and the crude product was purified by
1
column chromatography to afford 32 (0.71 g, 48%). H NMR
(CDCl3) δ 8.20 (s, 1H), 8.0-8.04 (m, 2H), 7.45-7.50 (m, 3H),
4.0 (s, 3H); MS (ESI) m/z 220 (M+1).
2-Phenylthiazole-4-carboxylic Acid Octadecylamide
(34). To a solution of 32 (0.5 g, 2.28 mmol) in MeOH (10 mL)
at 0 °C, 1 N NaOH (5 mL) was added and stirred for 2 h. To
the reaction mixture, EtOAc (30 mL) was added and acidified
with 1 N HCl. Extracted with EtOAc (3 × 50 mL), combined
extracts were washed with water and brine and dried (Na2-
SO4), and solvent was removed under vacuo to give crude acid
33, which was converted to 34 (0.30 g, 68%), following the
general procedure used as in the case of synthesis of 3-27.
1H NMR (CDCl3) δ 8.10 (s, 1H), 7.96-7.93 (m, 2H), 7.46-7.50
(m, 3H), 3.49 (dd, J ) 13.5, 6.9 Hz, 2H), 1.69 (m, 2H), 1.27 (m,
30H), 0.89 (t, J ) 6.3 Hz, 3H); MS (ESI) m/z 457.60 (M + 1).
Cell Culture. DU-145, PC-3, and LNCaP human prostate
cancer cells, and RH7777 rat hepatoma cells were obtained
from American Type Culture Collection (Manassas, VA). Dr.
Mitchell Steiner at University of Tennessee Health Science
Center kindly provided PPC-1 and TSU-Pr1 cells. Prostate
cancer cells and RH7777 cells were maintained in RPMI 1640
medium and DMEM (Mediatech, Inc., Herndon, VA), respec-
tively, supplemented with 10% fetal bovine serum (Gibco,
Grand Island, NY) in 5% CO2/95% air humidified atmosphere
at 37 °C.
Acknowledgment. This research was supported by
a grant from the Department of Defense (DAMD17-01-
1-083). Pharsight Corporation generously provided Win-
Nonlin software through an Academic License.
Supporting Information Available: 1H NMR (300 MHz)
and MS (ESI) characterization data for compounds 2b-v and
4-29 are available free of charge via the Internet at http://
pubs.acs.org.
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RT-PCR Analysis of LPA Receptor Expression. Total
RNA was extracted using TRIzol reagent (Invitrogen Corp.,
Carlsbad, CA) according to the manufacturer’s instruction. 0.5
µg (LPA1) or 1 µg (LPA2 and LPA3) of total RNA was used to
perform RT-PCR using SuperScript One-Step RT-PCR with
Platinum Taq (Invitrogen Corp., Carlsbad, CA) with 0.2 µM
of primers. The following primer pairs were used: LPA1
forward 5′-GCTCCACACACGGATGAGCAACC-3′, LPA1 re-
verse 5′-GTGGTCATTGCTGTGAACTCCAGC-3′; LPA2 for-
ward 5′-CTGCTCAGCCGCTCCTATTTG-3′, LPA2 reverse 5′-
AGGAGCACCCACAAGTCATCAG-3′; LPA3 forward 5′-CC-
ATAGCAACCTGACCAAAAAGAG-3′, LPA3 reverse 5′-TCCT-
TGTAGGAGTAGATGATGGGG-3′; â-actin forward 5′-GCTC-
GTCGTCGACAACGGCTC-3′, â-actin reverse 5′-CAAACAT-
GATCTGGGTCATCTTCTC-3′. PCR conditions were as fol-
lows: After 2 min denaturation step at 94 °C, samples were
subjected to 34 to 40 cycles at 94 °C for 30 s, 60 °C (LPA1) or
58 °C (LPA2 and LPA3) for 30 s, and 72 °C for 1 min, followed
by an additional elongation step at 72 °C for 7 min. Primers
were selected to span at least one intron of the genomic
sequence to detect genomic DNA contamination. The PCR
products were separated on 1.5% agarose gels, stained with
ethidium bromide, and the band intensity was quantified using
Quantity One Software (Bio-Rad Laboratories, Inc., Hercules,
CA). Expression levels of each receptor subtype in different
cell lines were expressed as ratios compared to â-actin mRNA
level.
Cytotoxicity Assay. For in vitro cytotoxicity screening,
1000 to 5000 cells were plated into each well of 96-well plates
depending on growth rate and exposed to different concentra-
tions of a test compound for 96 h in three to five replicates.
All the compounds were dissolved in dimethyl sulfoxide at 5
to 20 mM and diluted to desired concentrations in complete
culture medium. Cell numbers at the end of the drug treat-
ment were measured by the SRB assay. Briefly, the cells were
fixed with 10% of trichloroacetic acid and stained with 0.4%
SRB, and the absorbances at 540 nm were measured using a
JM049208B