Selective cytotoxicity of azatyrosinamides against ras-transformed NIH 3T3 cells

Article abstract of DOI:10.1016/j.bmcl.2005.06.048

This study aims to develop novel azatyrosinamide compounds structurally modified from ras-specific antioncogenic azatyrosine. Analogues 4-15 were prepared and their inhibition on the growth of wild-type and ras-transformed NIH 3T3 cell lines was compared. Compound 12 was found to be the most active with IC₅₀ 16.5 ± 2.2 μM which is 458-fold more potent than that of azatyrosine. The selective toxicity, defined as IC _{50 wild-type}/IC_{50 ras-transformed} for this compound was 138.5.

Full text of DOI:10.1016/j.bmcl.2005.06.048

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4272–4274
Selective cytotoxicity of azatyrosinamides against
ras-transformed NIH 3T3 cells
H. P. Wang,a,* T. L. Hwang,a On Lee,b Y. J. Tseng,c C. Y. Shud and S. J. Leed
aGraduate Institute of Natural Products, Chang Gung University College of Medicine, 259 Wen-Hwa 1st Road,
Kwei-Shan, Tao-Yuan 333, Taiwan
bBiomedical Engineering Center, No. 321 Kuangfu Road, Section 2, Hsinchu County, Taiwan
cDepartment of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago,
833 South Wood Street, Chicago, IL 60612, USA
dGraduate Institute of Biochemistry, National Taiwan University College of Medicine, No. 1,
Jen-ai Road, Section 1, Taipei 100, Taiwan
Received 28 April 2005; revised 21 June 2005; accepted 21 June 2005
Available online 21 July 2005
Abstract—This study aims to develop novel azatyrosinamide compounds structurally modified from ras-specific antioncogenic
azatyrosine. Analogues 4–15 were prepared and their inhibition on the growth of wild-type and ras-transformed NIH 3T3 cell
lines was compared. Compound 12 was found to be the most active with IC50 16.5 2.2 lM which is 458-fold more potent than
that of azatyrosine. The selective toxicity, defined as IC50 wild-type/IC50 ras-transformed for this compound was 138.5.
Ó 2005 Elsevier Ltd. All rights reserved.
Small GTPases of the Ras superfamily are crucial com-
ponents of signal transduction pathways leading from
cell-surface receptors to the control of cell proliferation,
differentiation, or death. The mammalian genome con-
tains three ras genes that encode highly related proteins
of 21 kDa termed H-Ras, N-Ras, and K-Ras with its
two variants, K-Ras4A and K-Ras4B, generated from
two alternative fourth exons. The three ras genes are
concurrently expressed in most mouse and human tis-
sues.1 Uncontrolled Ras-signaling has been implicated
in the development of human cancer.2 Signaling trans-
mitted through Ras results in activation of several
downstream effectors, with the Raf/ERK1/ERK2 and
the phosphatidylinositol 3-kinase/AKT pathways play-
ing a crucial role in modulation of the ras target genes
which in turn regulate cell functions.3,4 Azatyrosine
(L-b-(5-hydroxy-2-pyridyl)alanine), an antibiotic from
Streptomyces chibanensis,5 was first reported by Shin-
do-Okada et al. to suppress the growth of NIH 3T3 cells
transformed by c-Ha-ras, c-Ki-ras, N-ras, or c-raf onco-
gene while having no effect on the growth of wild-type
cells.6 Those azatyrosine-treated transformed cells that
survived reverted to an apparently normal phenotype
and the normal appearance and growth characteristics
of the cells persisted for months after removal of the
compound.7,8 Some of the revertant clone demonstrated
complete loss of tumorigenicity in nude mice.6 Studies
also proved that this compound is involved in the regu-
lation of other oncogenic cell growth.9–12 The high
reversion efficiency toward oncogenic transformed cells
combined with low toxicity to normal cells suggested
azatyrosine to be a new lead for developing anticancer
agents.
However, the potential of azatyrosine in cancer treat-
ment is limited due to its potency. Relatively high con-
centrations (1–2 mM)10 are necessary for the activity.
We believe that this is partially due to the low intracel-
lular availability of azatyrosine. The intracellular avail-
ability is limited not only by the number and the
turnover rate of the tyrosine amino acid transporter,
but also by the fact that azatyrosine must compete with
tyrosine for access to the transporter. Structural modifi-
cation of azatyrosine such that it is no longer dependent
upon the membrane-associated amino acid transporter
therefore becomes a rational approach to circumvent
this limitation. This report describes the synthesis of a
series of ester and amide derivatives of azatyrosine.
The in vitro inhibitory activities of the analogues were
determined. Selective toxicity in terms of inhibition on
*
Corresponding author. Tel.: 88632118506; fax: +88632118506;
e-mail: hpw@mail.cgu.edu.tw
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.06.048

H. P. Wang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4272–4274
4273
the growth of wild-type and ras-transformed NIH 3T3
cell lines was also determined.
ison of their cytotoxicity.15 Screening was carried out in
96-well plate and the cells, with a density of 1500–2500
cells/well, were cultured in DulbeccoÕs modified EagleÕs
medium containing 10% fetal bovine serum at 37 °C un-
der an atmosphere of 7.5% CO2 in air. Growth curves
for each cell line were established. The DMSO solution
of each compound was incubated with the cell for 48 h.
The Hansen MTT assay method was modified for
estimating the cell number.16 In brief, the absorption
of generated formazan blue at k570 nm was recorded with
ELISA reader. The number of surviving cells was deter-
mined from a calibration curve derived by correlating
the absorption to the cell number determined from a
hemacytometer. At least three experiments were carried
out for each compound and IC50 was calculated using
Analogues 4–15 of azatyrosine were prepared via the
route depicted in Scheme 1. Condensation of 2 with
113 afforded a malonate derivative (3), which upon
hydrolysis with aqueous 3-N sulfuric acid afforded the
benzyl derivative of azatyrosine 4. Protection of the ami-
no group of 4 with Boc gave analogue 5. Benzylation of
5 followed by the removal of Boc group gave the ester
analogue 6. Amide analogues 7–15 were obtained by
condensation of 5 with a variety of amines.
The synthesized compounds14 were screened on wild-
type and ras-transformed NIH 3T3 cells for the compar-
Scheme 1.
Table 1. Growth inhibition of azatyrosine and its analogues against wild-type and ras-transformed NIH 3T3 cell lines
Compound
R1
R2
IC50 (lM)a
Wild-type
ras-Transformed
SIb
1.42
Azatyrosine
—
—
—
—
—
—
H
H
H
H
H
H
10793.0 471.8
—
—
7554.8 417.5
1000
4
5
—
—
—
—
—
1000–2000
100
6
—
7
H
1716.8 10.4
989.7 707.3
213.7 111.5
410.8 247.3
1926.1 898.7
2284.8 1763
74.8 18.7
148.1 12.5
154.4 3.9
73.1 41.8
34.8 0.8
74.2 15.2
45.8 1.65
104.5 83.8
16.5 2.2
42.0 3.9
59.3 17.8
50.3 1.04
23.5
8
n-Propyl
Allyl
28.4
2.9
9
10
11
12
13
14
15
Propagyl
Cyclopropyl
Cyclohexyl
9.0
18.4
138.5
1.8
˜
–(CH2)5N
˜
–(CH2)4N
–(CH2)2–S–(CH2)2–
2.5
3.1
a Data presented are means SD of 3–5 experiments.
b SI denotes IC50 wild-type/IC50 ras-transformed
.

4274
H. P. Wang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4272–4274
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sigmoidal regression. SI, the selectivity of growth inhibi-
tion of each test compound, was defined as the ratio of
IC50 on wild-type to IC50 on ras-transformed cell lines.
5. Inouye, S.; Shomura, T.; Tsuruoka, T.; Ogawa, Y.;
Watanabe, H.; Yoshida, J.; Niida, T. Chem. Pharm. Bull.
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Nakajima, K.; Nishimura, S. Proc. Natl. Acad. Sci.
U.S.A. 1992, 89, 4879.
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ura, S. Ann. N.Y. Acad. Sci. 1999, 886, 109.
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Arce, C. A. Biochem. J. 2003, 375, 121.
10. Campa, M. J.; Glickman, J. F.; Yamamoto, K.; Chang, K.
J. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 7654.
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The inhibitory activity of compounds 4–15 is summa-
rized in Table 1. The acid analogues 4 and 5 were slight-
ly more active than azatyrosine on inhibiting the growth
of ras-transformed cells. The ester analogues 6 showed
an IC50 about 75-fold lower than that of azatyrosine.
Comparison of the IC50 between azatyrosine and its
amide analogue 7 indicated that high lipophilicity might
increase the intracellular bioavailability for exhibiting
the inhibitory activity. Thus, we further synthesized a
series of amide analogues 8–15. As indicated in Table
1, all the amide analogues showed higher activity than
azatyrosine on both cell lines. Compound 12 exhibited
the highest activity among all the amide analogues test-
ed with IC50 16.5 2.2 lM, a concentration 458-fold
lower than that for azatyrosine. The SI of compound
12 was 138.5, indicating its high selective toxicity on
ras-transformed cells. Although cyclic tertiary amides
(13–15) showed good activity, their SIÕs were unfortu-
nately low.
In conclusion, most of the compounds exhibited an
inhibitory effect on ras-transformed NIH 3T3 cells with
activities more potent than that of azatyrosine. Com-
pound 12 was the most active with the highest selective
toxicity on ras-transformed cells. Whether the activity
came from the compound per se or from azatyrosine,
the potential metabolite, need to be further investigated.
14. All compounds were characterized by 1H NMR, mass and
C, H, N elemental analysis. Spectral data of representative
compounds. 8: mp 117.0–117.5 °C; 1H NMR (CDCl3)
d, ppm: 8.23 (pyridine-a-H, 1H, d, J = 2.6 Hz), 7.37 (Ph,
5H, m), 7.17 (pyridine-c-H, 1H, dd, J = 2.6, 8.6 Hz),
7.10 (pyridine-b-H, 1H, d, J = 8.6 Hz), 6.73 (NH,
1H, br), 6.19 (–CH–CO, 1H, d, J = 6.8 Hz), 5.06
(Ph–CH2–, 2H, s), 4.46 (NH, 1H, br), 3.11
(–CH2CHCONHCH2–, 4H, m), 1.41 (t-Bu, 9H, s), 1.36
(–NHCH2CH2CH3, 2H, hexalet, J = 7.2 Hz), 0.77
(–NHCH2CH2CH3, 3H, t, J = 7.2 Hz); EI-MS (70 eV),
339 (100%), 248, 225, 91; Anal. Calcd for C23H31N3O4: C,
66.81; H, 7.56; N, 10.16. Found: C, 66.43; H, 7.26; N 10.20.
15. NIH 3T3 wild-type and NIH 3T3 val12-ras cells were
kindly supplied by Professor W. P. Wang of the Immu-
nology Department of National Taiwan University Med-
ical College and Dr. M. Campa of Duke University,
respectively.
Acknowledgment
This study was supported by a grant supported by
Standard Pharmaceutical Company, Taiwan (1999).
References and notes
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2. Barbacid, M. Annu. Rev. Biochem. 1987, 56, 779.
16. Hansen, B. M.; Nielson, S. E.; Berg, K. J. Immunol.
Methods 1989, 119, 203.