Novel thienopyridine derivatives as specific anti-hepatocellular carcinoma (HCC) agents: Synthesis, preliminary structure-activity relationships, and in vitro biological evaluation

Article abstract of DOI:10.1016/j.bmcl.2010.08.088

Novel thienopyridine derivatives 1b-1r were synthesized, based on a hit compound 1a that was found in a previous cell-based screening of anticancer drugs. Compounds 1a-1r have the following features: (1) their anticancer activity in vitro was first reported by our group. (2) The most potent analog 1g possesses hepatocellular carcinoma (HCC)-specific anticancer activity. It can specifically inhibit the proliferation of the human hepatoma HepG2 cells with an IC₅₀ value of 0.016 μM (compared with doxorubicin as a positive control, whose IC₅₀ was 0.37 μM). It is inactive toward a panel of five different types of human cancer cell lines. (3) Compound 1g remarkably induces G₀/G₁ arrest and apoptosis in HepG2 cells in vitro at low micromolar concentrations. These results, especially the HCC-specific anticancer activity of 1g, suggest their potential in targeted chemotherapy for HCC.

Full text of DOI:10.1016/j.bmcl.2010.08.088

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6282–6285
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel thienopyridine derivatives as specific anti-hepatocellular carcinoma
(HCC) agents: Synthesis, preliminary structure–activity relationships,
and in vitro biological evaluation
Xiu-Xiu Zeng a,b, , Ren-Lin Zheng a, , Tian Zhou a, , Hai-Yun He a, Ji-Yan Liu c, Yu Zheng a, Ai-Ping Tong a,
Ming-Li Xiang a, Xiang-Rong Song a, Sheng-Yong Yang a, Luo-Ting Yu a, Yu-Quan Wei a, Ying-Lan Zhao a,
,
⇑
Li Yang a,
⇑
a State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medicinal School, Sichuan University, Chengdu 610041, Sichuan, China
b Department of Pharmaceutical and Bioengineering, School of Chemical Engineering, Sichuan University, Chengdu 610065, Sichuan, China
c Department of Medical Oncology, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medicinal School, Sichuan University,
Chengdu 610041, Sichuan, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 March 2010
Revised 27 July 2010
Accepted 18 August 2010
Available online 21 August 2010
Novel thienopyridine derivatives 1b–1r were synthesized, based on a hit compound 1a that was found in
a previous cell-based screening of anticancer drugs. Compounds 1a–1r have the following features: (1)
their anticancer activity in vitro was first reported by our group. (2) The most potent analog 1g possesses
hepatocellular carcinoma (HCC)-specific anticancer activity. It can specifically inhibit the proliferation of
the human hepatoma HepG2 cells with an IC50 value of 0.016
lM (compared with doxorubicin as a posi-
tive control, whose IC50 was 0.37 M). It is inactive toward a panel of five different types of human cancer
l
Keywords:
cell lines. (3) Compound 1g remarkably induces G0/G1 arrest and apoptosis in HepG2 cells in vitro at low
micromolar concentrations. These results, especially the HCC-specific anticancer activity of 1g, suggest
their potential in targeted chemotherapy for HCC.
Hepatocellular carcinoma (HCC)
HCC-specific anticancer activity
Thienopyridine
Structure–activity relationships (SARs)
G0/G1 cell cycle arrest
Ó 2010 Elsevier Ltd. All rights reserved.
Hepatocellular carcinoma (HCC) is one of the most common
causes of cancer death and its incidence is increasing worldwide.1
Liver transplantation is considered to be the only curative therapy
at present but a majority (>80%) of patients with advanced and
unresectable HCC are not suitable for transplantation or surgical
resection.2 Chemotherapy is one of the commonly used treatment
options, especially for patients with unresectable tumors. How-
ever, the use of conventional cytotoxic drugs, including doxorubi-
cin, cisplatin, fluorouracil, etc., has not shown any improvement
in survival, and severe adverse effects were observed frequently
in patients with these treatments.3 Thus, there is an urgent need
to develop targeted chemotherapeutic agents for HCC. The success
of sorafenib, which was found to prolong significantly the survival
of patients with advanced HCC,4 suggested that small-molecule
targeted chemotherapy is a promising strategy to combat this
cancer.5
a previous cell-based screening of anticancer drugs,7 we found a
3-amino-thieno[2.3-b]pyridine derivative, 1a (Fig. 1) that can inhi-
bit efficiently the growth of human hepatocellular carcinoma
HepG2 cells, and induce their apoptosis in vitro.
Although 3-amino-thieno[2.3-b]pyridine derivatives demon-
strate various biological activities, such as inhibition of IjB ki-
nase-b (IKK-b),8 modulation of muscarinic acetylcholine receptors
(mAChRs),9 agonist activity towards luteinizing hormone (LH)
receptor,10 promotion of bone formation,11 etc., their antitumor
activity was seldom reported.12 To the best of our knowledge,
the antitumor activity of 6-aryl-3-amino-thieno[2,3-b]pyridine
derivatives 1a is being first reported by our group. Here we report
NH2
3
O
Our research group has been interested in the design, screening,
synthesis, and biological evaluation of novel tumor growth inhibi-
tors and apoptosis inducers as potential new anticancer agents.6 In
MeO
2
S
1a
HN
N
6
⇑
Corresponding authors. Tel.: +86 28 85164063; fax: +86 28 85164060 (L.Y.).
E-mail addresses: alancenxb@sina.com (Y.-L. Zhao), yangli@scu.edu.cn (L. Yang).
Cl
These authors contributed equally to this paper.
Figure 1. The structure of hit compound 1a.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.088

X.-X. Zeng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6282–6285
6283
NH2
CN
O
O
c
R
a
b
Ar
N
Ar
N
H
S
Ar
2a-2m
2a: 3-methoxyacetophenone
S
Ar
N
1a-1r
5
3a-3m
4a-4m
5a: 2-chloro-N-(4-chlorobenzyl)acetamide
5b: chloroacetonitrile
5c: ethylchloroacetate
5d: chloroacetamide
Scheme 1. Synthesis of analogs 1a–1r.15 Reagents and conditions: (a) DMF-DMA (2.0 equiv), reflux, 4–24 h, 75–95%; (b) DABCO (0.5 equiv), EtOH, reflux, 2–4 h, 40–97%;
(c) compound 5, 10% aq KOH/DMF, rt, 10–15 min and then 85 °C, 6 h, 50–80%.
the synthesis, the structure–activity relationship (SAR) study, and
preliminary biological evaluation of this novel class of anticancer
agents.
Among them, the most potent analog 1g significantly inhibited
the proliferation of the human hepatoma HepG2 cells at low
nanomolar concentrations (IC50 = 16 nM). With compounds 1a–
1f in hand, we started our SAR studies at the 2-position (R1).
The importance of the unsubstituted formamide group was re-
vealed by the dramatic loss of activity upon replacement with
substituted formamide (1a and 1b), a cyano group (1c), formate
ester (1d), or a carboxylic group (1f). The activity of the analog
As illustrated in Scheme 1, compound 1a13 was prepared readily
through the condensation of 6-aryl-3-cyanopyridine-2-(1H)-thi-
one 4a with compound 2-chloro-N-(4-chlorobenzyl)acetamide
5a, via the Thorpe-Ziegler cyclization.14 The key intermediate 4a
was prepared as follows: readily available acetophenone 2a was
reacted with N,N-dimethylformamide dimethyl acetal (DMF-
DMA) to give enaminone 3a. Subsequent condensation with cya-
nothioacetamide, catalyzed by DABCO, yielded 4a in 80% overall
yield. Using the same synthetic strategy shown in Scheme 1, ana-
logs 1b–1e were prepared through the condensation of 4b with
5a, chloroacetonitrile (5b), ethyl chloroacetate (5c), and chloro-
acetamide (5d), respectively. Compounds 1g–1r were synthesized
in parallel from the corresponding aryl ketones 2 with the same
synthetic sequence of 1a–1e. The only exception was the analog
1f, which was prepared via the hydrolysis of the parent compound
1d under basic conditions. The structures of 1a–1r were fully char-
acterized by 1H NMR, 13C NMR, and ESI-MS analysis.
with unsubstituted amide (1e, IC50 = 0.019 lM) at 2-position
was about 500–5000-fold greater than that of the substituted
amide analogs (1a, 1c, 1d).
After identifying the most potent 2-position substituent, we
next turned to examining the SAR at the 6-position (R2). As shown
in Table 1, introduction of a meta-methoxy substituent on the phe-
nyl ring (1g) in the R2 group was well tolerated, whereas the ortho-
substituted analog (1h) led to almost total loss of activity. The
slightly reduced potency of 3,4-disubstituted analogs (1i and 1j)
suggested that the modification at the 3,4-position of the phenyl
ring does not compromise activity. Introduction of the electron-
withdrawing fluoro group to the phenyl ring at the ortho-, meta-,
and para-positions (1k–1m) and the strongly electron-withdraw-
ing trifluoromethyl group (1n) were also not deleterious to the
activity of the parent compound. However, the introduction of a
bulky phenyl ring (1o) in the R2 had a detrimental effect on the po-
tency: that is, a fivefold decrease in potency, compared with that of
the parent compound (1a). Replacement of the phenyl ring in R2
with heteroaryl rings, such as pyridine (1p), thiazole (1q), and fur-
an (1r) groups yielded compounds with IC50 values in the low
micromolar range, but showed no clear SAR trends, and a moderate
decrease in potency was observed compared with 1g. It is notewor-
As shown in Table 1, 17 analogs of 1a were synthesized to sur-
vey the SAR of the 2- and 6-positions on the thieno[2,3-b]pyridine
scaffold by evaluating the cell growth inhibitory activity in hu-
man hepatocellular carcinoma HepG2 cells, using the MTT assay.
Table 1
Cytotoxicity data for 1a–1r
NH2
R1
thy that the most potent analog 1g (IC50 = 0.016
nearly 20-fold improvement in inhibitory activity against HepG2
cells over that of doxorubicin as a positive control (IC50 = 0.37 M).
lM) exhibited a
R2
N
S
a
Compound
R1
R2
HepG2 IC50
(
lM)
l
To further study the cytotoxic profile, the hit compound 1a and
the most potent analog 1g were selected for further evaluation for
inhibitory activity against a panel of five different types of human
cancer cell lines: breast cancer cell line MCF-7, colorectal cancer
cell line HCT-116, lung cancer cell line A549, prostate cancer cell
line PC-3, and epithelial cancer cell line A431. Interestingly, com-
pound 1a showed broad-spectrum antiproliferative activities
whereas compound 1g had a specific cytotoxic effect against
HepG2 cells over the other five common types of human cancer cell
lines in vitro (Table 2). In addition, compound 1g also had signifi-
cant cytotoxicity against human hepatoma Bel-7402 and SMMC-
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
N-(4-Cl–Bn)NHCO–
N-(4-Cl–Bn)NHCO–
–CN
–CO2Et
–CONH2
–COOH
–CONH2
–CONH2
–CONH2
–CONH2
–CONH2
–CONH2
–CONH2
–CONH2
–CONH2
–CONH2
–CONH2
–CONH2
—
3-MeO–Ph–
4-MeO–Ph–
4-MeO–Ph–
4-MeO–Ph–
4-MeO–Ph–
4-MeO–Ph–
3-MeO–Ph–
2-MeO–Ph–
3,4-Di MeO–Ph–
3,4-Di Cl–Ph–
2-F–Ph–
3-F–Ph–
4-F–Ph–
3,5-Di CF3–Ph–
Biphenyl
3-Pyridyl
9.4
>100
68
>100
0.019
>100
0.016
>100
0.72
0.29
0.70
0.63
1.89
6.32
44.7
4.76
1.30
1.97
0.37
7721 cells, with IC50 values <10 lM.
These results suggested that the lead compound 1g might have
different mechanisms of action compared with the parent
compound 1a and its potential in targeted chemotherapy for
HCC. To the best of our knowledge, there has been no reported in-
stance of small molecules with HCC-specific anticancer activity.
The novel HCC-specificity suggests that 1g might target one or sev-
eral HCC-specific targets. Therefore, further mechanism studies of
1q
1r
2-Thiazolyl
2-Furanyl
—
Doxorubicin
a
The cytotoxicity effects of various compounds on HepG2 cells were determined
by the MTT assay,16 and the results were expressed as the mean IC50 calculated
from three independent experiments.

6284
X.-X. Zeng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6282–6285
Table 2
The anti-proliferation activities of compounds 1a, 1g, and doxorubicin against various cancer cell lines
a
Compound
IC50
(lM)
Bel-7402
SMMC-7721
HepG2
MCF-7
A549
A431
HCT-116
PC-3
1a
1g
13.1
7.4
0.049
7.6
0.90
1.06
6.0
0.016
0.37
9.9
>100
0.75
13.4
>100
9.2
38.3
>100
0.37
35.4
>100
0.07
7.4
>100
0.60
Doxorubicin
a
The cytotoxicity effects of compounds on various cancer cells were determined by the MTT assay, and the results were expressed as the IC50, that were means calculated
from three independent experiments.
Figure 2. Effects of 1a, 1g, and control on the induction of apoptosis and cell cycle distribution. (a) DNA fluorescence histograms of PI-stained HepG2 cells. Cells were treated
with 1.25 lM of 1a and 1g for 36 h, and the cells in the sub-G1 phase were considered as apoptotic cells (n = 5). (b) Effects of control and 1.25 lM of 1a and 1g on cell cycle
distribution for 36 h.
1g may provide insight into novel targets for targeted chemother-
apy for HCC.
1g exhibited an IC50 value of 0.016
cin as a positive control, with an IC50 of 0.37
l
M (compared with doxorubi-
M) against HepG2
l
Flow cytometric analysis was used to identify and measure the
apoptotic cells (sub-G1 cells) and the cell cycle after propidium io-
dide (PI) staining, as described previously17 to further evaluate the
mechanism of the hit compound 1a and the lead compound 1g.
As can be seen in Fig. 2a, a 36 h exposure of HepG2 cells to com-
cells. It was inactive toward a panel of five common types of hu-
man cancer cell lines. These results, especially the HCC-specific
anticancer activity of 1g, suggest their potential in targeted chemo-
therapy for HCC. Further lead optimization and mechanism studies
are worth pursuing.
pounds 1a and 1g (1.25 lM) resulted in a distinct sub-G1 peak that
represents the population of apoptotic cells.18 Moreover, a signifi-
cant accumulation of cells in the G0/G1 phase, accompanied by a
decrease in G2/M and S phase was observed after treatment with
compound 1g, whereas no G0/G1 arrest was observed after treat-
ment with compound 1a (Fig. 2b). The percentage of cells in the
G0/G1 phase was 49.0% for the control, and 51.2% and 76.9% for
compounds 1a and 1g, respectively. These results suggest that
compound 1g could remarkably induce G0/G1 arrest and apoptosis
in the human hepatoma cell line HepG2 in vitro at low micromolar
concentrations.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (NSFC-81000982), National 863 Project
(2006AA03Z356) and the Youth Foundation of Sichuan University
(No. 2008110). We thank the Analytical and Testing Center of
Sichuan University for NMR measurements.
Supplementary data
In conclusion, novel 3-aminothieno [2,3-b]pyridine-2-carbox-
amide derivatives have been synthesized to survey the SAR of sub-
stituents on the thieno[2,3-b]pyridine scaffold. The SAR analysis
indicated that the substituents of the 2- and 6-positions play a cru-
cial role in the antiproliferative activities. The most potent analog
The list of starting materials 2a–2m, experimental details, and
spectroscopic characterization of compounds 3a, 4a, 1a–1f and
1h–1r. Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.2010.08.088.

X.-X. Zeng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6282–6285
6285
10. Straten, N. C. R. V.; Schoonus-Gerritsma, G. G.; Someren, R. G. V.; Draaijer, J.;
Adang, A. E. P.; Timmers, C. M.; Hanssen, R. G. J. M.; Boeckel, C. A. A. V.
ChemBioChem 2002, 10, 1023.
11. Satoru, N.; Akira, N.; Tsuyoshi, S.; Keiji, S., Kiyoshi, O. WO2007043581, 2007.
12. (a) Hayakawa, I.; Shioya, R.; Agatsuma, T.; Furukawa, H.; Sugano, Y. Bioorg. Med.
Chem. Lett. 2004, 14, 3411; (b) Claudia, R.; Alexander, L.; Alexander, S.;
Mohammed, D.; Siegfried, L.; Andreas, K.; Jochen, H. WO2006074919, 2006.
13. The sample of 1a for screening was purchased from Specs Company. Because
there is no synthetic literature of compound 1a, we had to synthesize
compound 1a and its analogs.
References and notes
1. Pisani, P.; Parkin, D. M.; Bray, F.; Ferlay, J. Int. J. Cancer 1999, 83, 18.
2. Thomas, M. B.; Zhu, A. X. J. Clin. Oncol. 2005, 23, 2892.
3. Simonetti, R. G.; Liberati, A.; Angiolini, C.; Pagliaro, L. Ann. Oncol. 1997, 8, 117.
4. Keating, G. M.; Santoro, A. Drugs 2009, 69, 223.
5. Spangenberg, H. C.; Thimme, R.; Blum, H. E. Nat. Rev. Gastroenterol. Hepatol.
2009, 6, 423.
6. For representative papers of our groups, see: (a) Zheng, Y. Z.; Zhao, Y. L.; Deng,
X. Q.; Yang, S. Y.; Mao, Y. Q.; Li, Z. G.; Jiang, P. D.; Zhao, X.; Wei, Y. Q. Cancer
Invest. 2009, 27, 286; (b) Jiang, P. D.; Zhao, Y. L.; Shi, W.; Deng, X. Q.; Xie, G.;
Mao, Y. Q.; Li, Z. G.; Zheng, Y. Z.; Yang, S. Y.; Wei, Y. Q. Cell. Physiol. Biochem.
2008, 22, 431; (c) Xie, H. Z.; Li, L. L.; Ren, J. X.; Zou, J.; Yang, L.; Wei, Y. Q.; Yang,
S. Y. Bioorg. Med. Chem. Lett. 2009, 19, 1944; (d) Wang, H. Y.; Cao, Z. X.; Li, L. L.;
Jiang, P. D.; Zhao, Y. L.; Luo, S. D.; Yang, L.; Wei, Y. Q.; Yang, S. Y. Bioorg. Med.
Chem. Lett. 2008, 18, 4972; (e) Luo, Y. F.; Xu, Y. B.; Chen, L. J.; Hu, J.; Peng, C.; Xie,
D. C.; Shi, J. Y.; Huang, W. C.; Xu, G. B.; Peng, M.; Han, J.; Li, R.; Yang, S. Y.; Wei,
Y. Q. Bioorg. Med. Chem. Lett. 2009, 19, 4702; (f) Wei, Y. Q.; Wang, Q. R.; Zhao, X.;
Yang, L.; Tian, L.; Lu, Y.; Kang, B.; Lu, C. J.; Huang, M. J.; Lou, Y. Y.; Xiao, F.; He, Q.
M.; Shu, J. M.; Xie, X. J.; Mao, Y. Q.; Lei, S.; Luo, F.; Zhou, L. Q.; Liu, C. E.; Zhou, H.;
Jiang, Y.; Peng, F.; Yuan, L. P.; Li, Q.; Wu, Y.; Liu, J. Y. Nat. Med. 2000, 6, 1160.
7. Deng, X. Q.; Wang, H. Y.; Zhao, Y. L.; Xiang, M. L.; Jiang, P. D.; Cao, Z. X.; Zheng,
Y. Z.; Luo, S. D.; Yu, L. T.; Wei, Y. Q.; Yang, S. Y. Chem. Biol. Drug. Des. 2008, 71,
533.
8. (a) Wu, J. P.; Fleck, R.; Brickwood, J.; Capolino, A.; Catron, K.; Chen, Z. D.; Cywin,
C.; Emeigh, J.; Foerst, M.; Ginn, J.; Hrapchak, M.; Hickey, E.; Hao, M. H.; Kashem,
M.; Li, J.; Liu, W. M.; Morwick, T.; Nelson, R.; Marshall, D.; Martin, L.; Nemoto,
P.; Potocki, I.; Liuzzi, M.; Peet, G. W.; Scouten, E.; Stefany, D.; Turner, M.;
Weldon, S.; Zimmitti, C.; Spero, D.; Kelly, T. A. Bioorg. Med. Chem. Lett. 2009, 19,
5547; (b) Morwick, T.; Berry, A.; Brickwood, J.; Cardozo, M.; Catron, K.; Turi, M.
D.; Emeigh, J.; Homon, C.; Hrapchak, M.; Jacober, S.; Jakes, S.; Kaplita, P.; Kelly,
T. A.; Ksiazek, J.; Liuzzi, M.; Magolda, R.; Mao, C.; Marshall, D.; McNeil, D.;
Prokopowicz, A.; Sarko, C.; Scouten, E.; Sledziona, C.; Sun, S. X.; Watrous, J.; Wu,
J. P.; Cywin, C. L. J. Med. Chem. 2006, 49, 2898.
14. (a) Litvinov, V. P.; Dotsenko, V. V.; Krivokolysko, S. G. Russ. Chem. Bull. 2005, 54,
864; (b) Yakovlev, M. Y.; Kadushkin, A. V.; Granik, V. G. Khim. Farm. Zh. 1996,
30, 36.
15. Typical procedure for the synthesis of 1a–1e and 1g–1r.
Compound 1g was prepared as follows: To a suspension of 4a (1.0 g, 4.2 mmol) in
DMF (6 ml) was added aq KOH solution (1.8 M, 3.0 ml) followed by the addition
of chloroacetamide (0.5 g, 5 mmol) at room temperature. The resulting mixture
was stirred for 10–15 min at room temperature and then heated to 85 °C for 6 h
after the addition of additional aqueous KOH solution (1.8 M, 3.0 ml). After the
reaction mixture was allowed to cool to room temperature, the precipitate was
collected by filtration, and washed with cold ethanol, then recrystallized from
ethanol to give a yellow solid (0.95 g, 75% yield), Mp: 234–236 °C; 1H NMR
(400 MHz, DMSO-d6): d 8.51 (d, J = 8.4 Hz, 1H), 8.06 (d, J = 8.4 Hz, 1H), 7.76 (d,
J = 8.0 Hz, 1H), 7.72 (s, 1H), 7.44 (t, J = 8.0 Hz, 1H), 7.06 (dd, J = 8.0, 2.0 Hz, 1H),
3.87 (s, 3H); 13C NMR (100 MHz, DMSO-d6): d 166.95, 159.72, 158.67, 156.10,
145.51, 139.39, 131.61, 129.92, 125.31, 119.29, 116.36, 115.34, 112.02, 97.47,
55.18; ESI-MS: m/z 334.16 [M+Cl]À.
16. Briefly, cells (2000/well) were seeded in 96-well plates and cultured for 24 h,
followed by treatment with the compounds for 48 h. Ten microliters of 10 mg/
ml MTT was added per well and incubated for another 2.5 h at 37 °C. Then the
supernatant fluid was removed and 150 lL/well DMSO was added for 15–
20 min. The absorbance (OD) of each well was measured at 570 nm, using a
SpectraMAX M5 microplate spectrophotometer (Molecular Devices).
17. Peng, F.; Wei, Y. Q.; Tian, L.; Yang, L.; Zhao, X.; Lu, Y.; Mao, Y. Q.; Kan, B.; Lei, S.;
Wang, G. S.; Jiang, Y.; Wang, Q. R.; Luo, F.; Zou, L. Q.; Liu, J. Y. J. Cancer Res. Clin.
Oncol. 2002, 128, 223.
9. Shirey, J. K.; Xiang, Z.; Orton, D.; Brady, A. E.; Johnson, K. A.; Williams, R.; Ayala,
J. E.; Rodriguez, A. L.; Wess, J.; Weaver, D.; Niswender, C. M.; Conn, P. J. Nat.
Chem. Biol. 2008, 4, 42.
18. Wei, Y. Q.; Zhao, X.; Kariya, Y.; Fukata, H.; Teshigawara, K. Uchida. Cancer Res.
1994, 54, 4952.