Novel and orally active 5-(1,3,4-oxadiazol-2-yl)pyrimidine derivatives as selective FLT3 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.09.031

5-(1,3,4-Oxadiazol-2-yl)pyrimidine derivative 1 was identified as a new class of FLT3 inhibitor from our compound library. With the aim of enhancement of antitumor activity of 2 prepared by minor modification of1, structure optimization of side chains at the 2-, 4-, and 5-positions of the pyrimidine ring of 2 was performed to improve the metabolic stability. Introduction of polar substituents on the 1,3,4-oxadiazolyl group contributed to a significant increase in the metabolic stability. As a result, a series of compounds showed increased efficacy against MOLM-13 xenograft model in mice by oral administration.

Full text of DOI:10.1016/j.bmcl.2008.09.031

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5472–5477
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel and orally active 5-(1,3,4-oxadiazol-2-yl)pyrimidine derivatives
as selective FLT3 inhibitors
a
a
a
a
Hiroshi Ishida a, , Shoichi Isami , Tsutomu Matsumura , Hiroshi Umehara , Yoshinori Yamashita ,
*
Jiro Kajita a, Eiichi Fuse a, Hitoshi Kiyoi b, Tomoki Naoe c, Shiro Akinaga a, Yukimasa Shiotsu a, Hitoshi Arai a
a Pharmaceutical Research Center, Kyowa Hakko Kogyo Co., Ltd, 1188 Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8731, Japan
b Department of Infectious Diseases, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8560, Japan
c Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8560, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
5-(1,3,4-Oxadiazol-2-yl)pyrimidine derivative 1 was identified as a new class of FLT3 inhibitor from our
compound library. With the aim of enhancement of antitumor activity of 2 prepared by minor modifica-
tion of1, structure optimization of side chains at the 2-, 4-, and 5-positions of the pyrimidine ring of 2 was
performed to improve the metabolic stability. Introduction of polar substituents on the 1,3,4-oxadiazolyl
group contributed to a significant increase in the metabolic stability. As a result, a series of compounds
showed increased efficacy against MOLM-13 xenograft model in mice by oral administration.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 10 April 2008
Revised 26 August 2008
Accepted 6 September 2008
Available online 11 September 2008
Keywords:
FLT3 inhibitor
5-(1,3,4-Oxadiazol-2-yl)pyrimidine
FLT3 (FMS-like receptor tyrosine kinase 3) also referred to as fe-
tal liver kinase-2 (FLK-2) or stem cell kinase 1 (STK-1) is a class III
receptor tyrosine kinase together with KIT, FMS, and platelet-de-
rived growth factor receptor.1,2 In 1996, FLT3/ITD (Internal Tandem
Duplication) mutation was found in AML (Acute Myeloid Leuke-
mia) cells. This mutation is formed by the duplication of juxta-
membrane domain-coding sequence in
a direct head-to-tail
orientation.3 The ITD mutation occurs in 15–35% of AML patients
and promotes ligand-independent dimerization, auto-phosphory-
lation, and constitutive activation of the FLT3 receptor.4 Patients
carrying the ITD mutations are found to have an increased inci-
dence of leukocytosis and a decreased overall survival when com-
pared with patients without ITD mutations.5 Therefore, inhibition
of activated and mutated FLT3 kinase is effective strategy for the
treatment of AML. To date, several FLT3 inhibitors have been sub-
jected to clinical trials in several hematopoietic malignancies
including AML.6–9
In the course of our discovery study for FLT3 inhibitors, the 5-
(1,3,4-oxadiazol-2-yl)pyrimidine derivative 1 was identified as a
primary hit compound via high-throughput screening of our com-
pound library. In our preliminary SAR investigations, we found the
compound 2 which showed about 70-fold more potent FLT3 inhib-
itory activity than 1 (Fig. 1).
Figure 1. FLT3 inhibitors identified via high-throughput screening (1) and
subsequent analog screening (2).
modest antitumor activity against MOLM-13 xenograft model in
SCID mice. However, 2 was found metabolically unstable in both
human and mice liver microsomes, and oxidative reaction of the
n-propylamino group at the 4-position and the 4-(2-amino-
ethyl)pyridyl group at the 2-position were shown to be major met-
abolic pathways from the analysis of metabolite profiles. Thus,
further structure optimization to acquire metabolic stability was
required to enhance in vivo activity, and we started the structure
optimization study of 2. We herein report the synthesis and evalu-
ation of novel 5-(1,3,4-oxadiazol-2-yl)pyrimidine derivatives as
FLT3 inhibitors.
Compound 2 showed not only FLT3 inhibitory activity but anti-
proliferative activity against MOLM-13 cells, a human AML cell line
expressing ITD activating mutation.10 This compound also showed
The synthesis of derivatives with modified side chains at the 2-
and 4-positions is outlined in Scheme 1. Functionalization of the 4-
position of a commercially available ethyl 4-chloro-2-methylthio-
pyrimidine-5-carboxylate 3 followed by hydrolysis of the ester
group and condensation with hydrazine monohydrate gave the
pyrimidinecarboxylic acid hydrazide 4. Cyclization reaction11 of 5
* Corresponding author. Tel.: +81 55 989 2025; fax: +81 55 986 7430.
E-mail address: hiroshi.ishida@kyowa.co.jp (H. Ishida).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.031

H. Ishida et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5472–5477
5473
Scheme 1. Reagents and conditions: (a) R1ONa (for X = O) or R1NH2 (for X = NH), Et3N, THF; (b) 2.0 mol/L NaOH aq, EtOH; (c) i—CDI, THF; ii—NH2NH2ÁH2O, THF; (d)
cyclopropanecarbonyl chloride, CHCl3, satd NaHCO3 aq, 0 °C; (e) PPh3, CCl4, Et3N, CH3CN; (f) i—mCPBA, CH2Cl2; ii—R2R3NH, Et3N, THF; (g) PPh3, DEAD, 2-
(trimethylsilyl)ethanol, toluene; (h) 1.0 mol/L TBAF in THF; (i) POCl3, 60 °C.
obtained by acylation of the hydrazide moiety in 4 provided the
5-(1,3,4-oxadiazol-2-yl)pyrimidine derivatives 6. Subsequent
substitution reaction at the 2-position of 6 afforded the desired
compounds 7 (Method A). Alternatively, to aim for more efficient
optimization at the 4-position by introducing the side chain in
the latter stage of the reaction route, another method was exam-
ined (Method B). Introduction of a (2-trimethylsilyl)ethoxy group12
to the 4-position of 8 prepared by the reported procedure13 was
performed using Mitsunobu reaction, followed by conversion to
12 according to Method A. Then, removal14 of the (2-trimethyl-
silyl)ethyl moiety and treatment with POCl3 yielded 14, which
was converted to 7 via formation of 6.
Our SAR study was started on the 4-position. These results are
summarized in Table 1. Shortening the carbon chain (7a) or intro-
duction of a polar group (7b) resulted in a significant increase in
the metabolic stability. However, their FLT3 kinase inhibitory
activity and anti-proliferative activity against MOLM-13 were less
potent compared to that of 2. A similar tendency was also observed
in the ethoxy analog 7i. Anti-proliferative activity of the anilino
analogs (7d–7h) was maintained. Among them, a 4-hydroxyanilino
analog 7g showed the most potent FLT3 inhibitory activity as well
as anti-proliferative activity against MOLM-13 and good stability
in human microsomes. However, compound 7g was not expected
to show in vivo activity, since 7g was not detected in plasma after
oral administration in mice (data not shown). From these results,
we chose n-propylamino, anilino, and methylamino groups as
desirable substituents for further optimization study because 2,
7a, and 7d show either potent anti-proliferative activity against
MOLM-13 (2, 7d) or good metabolic stability (7a).
With the results in mind, SAR investigations on the 2-position
were also examined for structural tolerability of the pyridyl moiety
with the anilino group at the 4-position, which was identified as
potent anti-proliferative activity in Table 1. These results are sum-
marized in Table 2. The compounds were evaluated for anti-prolif-
erative activity against MOLM-13 cells preferentially to identify
more active compound against tumor cells. In contrast to the 4-po-
sition, structure tolerance for anti-proliferative activity in the 2-
position was quite limited. Replacement with pyridyl N-oxide
(7j), pyrimidyl (7k), and 4-aminophenyl (7q) analogs improved
metabolic stability, but significantly lowered anti-proliferative
activity. As mentioned above, oxidative reaction of the 4-(2-amino-
ethyl)pyridyl moiety at the 2-position of 2 was observed in the
metabolite analysis. Introduction of a methyl group to aim for
blocking the oxidative reaction of the nitrogen atom, however,

5474
H. Ishida et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5472–5477
Table 1
Table 2
SAR for the C-4 modified analogs
SAR for the C-2 and C-4 modified analogs
Compound XR1
FLT3IC50
(nmol/L)
MOLM-13 GI50
(nmol/L)
HLM CLc (h/
L/kg)
a
b
Compound NR2R3
MOLM-13 GI50 (nmol/L) HLM CLb (h/L/kg)
a
2
4.9 1.0
35 10
63 10
36 3.4
139 14
123 7.7
13
7j
3610 657
775 205
113 6.6
999 254
>10,000
0.2
4.4
2.5
n.t.c
n.t.
22
7a
7b
1.2
1.2
7k
7l
7c
7d
7e
515 146
47 7.4
588 235
40 6.3
40 8 7
n.t.d
19
7m
7n
7o
7p
7q
134 31
13
137 10
44 6.7
7f
106 30
4.8 1.2
34 17
80 14
23 1.8
11
7g
7h
7i
3.2 0.34.8
10 1.5
5.1
4.1
16
287 33
263 139
1.4
GI50 values are SEM means of three experiments.
a
See Ref. 16 for assay details.
See Ref. 17 for assay details.
n.t., not tested.
IC50 and GI50 values are SEM means of three experiments.
b
a
See Ref. 15 for assay details.
See Ref. 16 for assay details.
See Ref. 17 for assay details.
c
b
c
d
n.t., not tested.
to the above method, which underwent further deprotection of the
hydroxy group to give 25. Conversion of a 4-hydroxy group of the
pyrimidine ring to mesylates and substitution reaction18 with
amines provided the desired compounds 23 successfully.
did not improve metabolic stability (7o). 2-Methoxypyridyl (7m)
and 2-cyanopyridyl (7n) analogs also showed diminished activity.
Only 2-hydroxypyridyl (7l) and 4-hydroxyphenyl (7p) analogs sat-
isfied both criteria of anti-proliferative activity and microsomal
stability. However, compounds 7l and 7p were not detected in
plasma after oral administration in mice likewise 7g (data not
shown). From the results, we estimated the 4-(2-aminoethyl)pyri-
dyl group was quite important to show in vivo activity.
Through these unsatisfied results, we decided to examine mod-
ification of the side chain on the 1,3,4-oxadiazolyl group at the 5-
position, since SAR investigations on the position were carried out
to a lesser extent. Thus, we planned to reduce their lipophilicity
through introduction of polar substituents at the end of the 5-posi-
tion to improve metabolic stability. To incorporate the above design
into the structure, we developed new synthetic routes as shown in
Scheme 2. Treatment of compounds 4 used as starting materials
with acetoxyacetyl chloride afforded the corresponding 15 fol-
lowed by the cyclization reaction to give 17. Compounds 19 were
obtained by introduction of the side chain at the 2-position of 17.
Hydrolysis of the acetyl group of 19 gave 21 followed by conversion
to mesylates and subsequent substitution reaction with various
amines provided 23. Synthesis of 23 with XR1 = OH could be alter-
natively achieved. Compounds 24 were prepared from 10 according
These compounds were evaluated for anti-proliferative activity
against MOLM-13 cells. Table 3 summarizes the SAR studies for the
substituted 1,3,4-oxadiazolyl group. We identified the 5-position
with extensive structural tolerability. Polar substituents on the
side chain of the 1,3,4-oxadiazolyl group were well tolerated for
anti-proliferative activity against MOLM-13 cells. It is also note-
worthy that the microsomal intrinsic clearance values of these
compounds varied depending on the side chains. The distinct
improvement of metabolic stability by the introduction of dimeth-
ylamino (23a), morpholino (23b), and 4-acetylpiperazinyl (23d)
groups was not observed. Meanwhile, the introduction of hydroxy
(21a), 4-methylpiperazinyl (23c), piperazinyl (23f), and 2-(dimeth-
ylamino)ethylamino (23i) groups increased metabolic stability.
Above all, compound23f gave a best result and substituted or
unsubstituted piperazinyl groups were shown to be suitable for
the improvement of metabolic stability. We therefore tried to ex-
plore a combination of the side chains at the 4- and 5-positions
which showed both potent activity and good metabolic stability.
Table 4 summarizes the SAR studies for the 4-position in combina-
tion with the favorable piperazinyl groups on the side chain of the
5-position, which were identified in Table 3. Reduction of lipophil-

H. Ishida et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5472–5477
5475
Scheme 2. Reagents and conditions: (a) AcOCH2COCl, CHCl3, satd NaHCO3 aq, 0 °C; (b) PPh3, CCl4, Et3N, CH3CN; (c) i—mCPBA, CH2Cl2; ii—4-(2-aminoethyl)pyridine, THF; (d)
2.0 mol/L NaOH aq, MeOH; (e) i—Ms2O, Et3N, CH2Cl2; ii—R4R5NH, THF; (f) 1.0 mol/L TBAF in THF; (g) i—Ms2O, Et3N, CH2Cl2; ii—R1NH2 (for X = NH), Et3N, CH2Cl2.
Table 4
icity at the 4-position (e.g., 23c vs 23j or 23k, 23h vs 23r) was
found also effective for metabolic stabilization, which was already
shown in Table 1. Incorporation of F atom to aim for blocking
SAR for the C-4 and C-5 modified analogs
Table 3
SAR for the C-5 modified analogs
Compound XR1
NR4R5
MOLM-13
HLM CLb clogpc
GI50 (nmol/L) (h/L/kg)
a
23j
23k
23l
262 51
102 5.1
52 4.4
<0.1
2.7
0.91
1.44
2.0
XR4R5
MOLM-13 GI50
(nmol/L)
HLM CLb
(h/L/kg)
clogpc
a
Compound
21a
23a
58 8.6
2.1
0.74
1.61
5.2
73 8.1
11
23m
23n
308 55
49 4.0
0.7
1.5
1.51
1.29
23b
23c
23d
23e
23f
43 4.2
19 3.6
66 4.6
57 4.3
29 1.9
74 5.8
10
1.52
1.97
1.11
1.43
1.51
0.82
4.9
8.2
3.0
1.1
3.0
23o
23p
50 7.7
74 5.8
6.3
2.3
1.37
2.43
23g
23q
23r
99 4.9
94 10
<0.1
0.8
0.93
2.02
23h
23i
43 8.1
78 4.1
6.9
3.0
2.55
1.53
GI50 values are SEM means of three experiments.
GI50 values are SEM means of three experiments.
a
a
See Ref. 16 for assay details.
See Ref. 17 for assay details.
See Ref. 16 for assay details.
See Ref. 17 for assay details.
b
b
c
Calculated by Daylight.19
c
Calculated by Daylight.19

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H. Ishida et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5472–5477
Table 5
Anti-proliferative activity of 5-(1,3,4-oxadiazol-2-yl)pyrimidine derivatives against several cell lines
Compound
MOLM-13 GI50 (nmol/L)
ML-1 GI50 (nmol/L)
FLT3-D
599GI50 (nmol/L)
FLT-D835Y GI50 (nmol/L)
2
36 3.4
56 8.6
19 3.6
29 1.9
102 5.1
94 10
4318 905
5958 1552
>10,000
9108 1045
>10,000
36 9.2
76 22
37 11
74 14
114 13
74 14
52 13
116 38
31 5.9
65 21
63 10
48 19
21a
23c
23f
23k
23r
>10,000
GI50 values are SEM means of three experiments. See Ref. 16 for assay details.
Table 6
indebted to Dr. Tatsuhiro Ogawa for his helpful discussion. We also
thank Ms. Nana Oiwa, Mr. Satoshi Tashiro, Ms. Shinobu Shioya, Ms.
Yoko Tahara, and Ms. Masayo Suzuki for conducting the human li-
ver microsomal stability studies and in vitro cytotoxic assays.
Antitumor activity of 5-(1,3,4-oxadiazol-2-yl)pyrimidine derivatives
Dosea
(mg/kg)
T/Cmin
Regression V/V0
minc (on day)
Mortality
b
Compound
(on day)
Control
2
1.00
—
—
—
—
—
—
0/5
0/5
1/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
50
100
50
50
50
100
200
50
50
100
0.47 (7)
0.34 (7)
0.45 (7)
0.27 (7)
0.25 (10)
0.10 (7)
0.006 (10)
0.24 (7)
0.20 (7)
0.014 (7)
Supplementary data
23a
23c
23f
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.09.031.
0.571 (4)
0.098 (10)
—
1.39 (7)
0.12 (7)
References and notes
23k
23r
1. Rosnet, O.; Schiff, C.; Pebusque, M. J.; Marchetto, S.; Tonnelle, C.; Toiron, Y.;
Birg, F.; Birnbaum, D. Blood 1993, 82, 1110.
2. Lyman, S. D.; James, L.; Zappone, J.; Sleath, P. R.; Beckmann, M. P.; Bird, T.
Oncogene 1993, 8, 815.
3. Kiyoi, H.; Towatari, M.; Yokota, S.; Hamaguchi, M.; Ohno, R.; Saito, H.; Naoe, T.
Leukemia 1998, 12, 1333.
a
Compounds were orally administrated at b.i.d. intervals for 5 days.
Averages of tumor volume ratio in the treated to control mice.
Averages of the relative tumor volume to the initial tumor volume on day 0. See
b
c
Ref. 10 for assay details.
4. Kiyoi, H.; Yanada, M.; Ozeki, K. Int. J. Hematol. 2005, 82, 85.
5. Levis, M.; Small, D. Leukemia 2003, 17, 1738.
6. Stone, R. M.; DeAngelo, D. J.; Klimek, V.; Galinsky, I.; Estey, E.; Nimer, S. D.;
Grandin, W.; Lebwohl, D.; Wang, Y.; Cohen, P.; Fox, E. A.; Neuberg, D.; Clark, J.;
Gilliland, D. G.; Griffin, J. D. Blood 2005, 105, 54.
7. Knapper, S.; Burnett, A. K.; Littlewood, T.; Kell, W. J.; Agrawal, S.; Chopra, R.;
Clark, R.; Levis, M. J.; Small, D. Blood 2006, 108, 3262.
8. Fiedler, W.; Serve, H.; Döhner, H.; Schwittay, M.; Ottmann, O. G.; O’Farrell, A.-
M.; Bello, C. L.; Allred, R.; Manning, W. C.; Cherrington, J. M.; Louie, S. G.; Hong,
W.; Brega, N. M.; Massimini, G.; Scigalla, P.; Berdel, W. E.; Hossfeld, D. K. Blood
2005, 105, 986.
9. DeAngelo, D. J.; Stone, R. M.; Heaney, M. L.; Nimer, S. D.; Paquette, R. L.;
Klisovic, R. B.; Caligiuri, M. A.; Cooper, M. R.; Lecerf, J.-M.; Karol, M. D.; Sheng,
S.; Holford, N.; Curtin, P. T.; Druker, B. J.; Heinrich, M. C. Blood 2006, 108, 3674.
10. Kiyoi, H.; Shiotsu, Y.; Ozeki, K.; Yamaji, S.; Kosugi, H.; Umehara, H.; Shimizu,
M.; Arai, H.; Ishii, K.; Akinaga, S.; Naoe, T. Clin. Cancer Res. 2007, 13, 4575.
11. (a) Brown, P.; Best, D. J.; Broom, N. J. P.; Cassels, R.; O’Hanlon, P. J.; Mitchell, T.
J.; Osborne, N. F.; Wilson, J. M. J. Med. Chem. 1997, 40, 2563; (b) Rajapakse, H.
A.; Zhu, H.; Young, M. B.; Mott, B. T. Tetrahedron Lett. 2006, 47, 4827.
12. Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis; John Wiley
and Sons: New York, 1991. chapter 2, p 41 and references cited therein.
13. Landreau, C.; Deniaud, D.; Reliquet, A.; Reliquet, F.; Meslin, J. C. J. Heterocycl.
Chem. 2001, 38, 93.
14. Gerstenberger, B. S.; Konoperski, J. P. J. Org. Chem. 2005, 70, 1467.
15. FLT3 kinase assays. To evaluate the kinase inhibitory activities against FLT3 by
ELISA, GST-tagged FLT3 cytoplasmic domain and biotinylated poly-(Glu/Tyr
4:1) substrate (CIS bio International) bound to the surface of 96-well assay
plates were used. Phosphorylated substrate was bound to anti-
phosphotyrosine antibody conjugated to europium. The bound antibody was
measured using ARVO (Perkin-Elmer) at excitation/emission of 340/615 nm.
metabolism (23f vs 23q) at the 4-position was also effective; how-
ever, cycloalkyl moieties (23l and 23o) exhibited relatively low
metabolic stability. Introduction of an alkoxy unit (23m) resulted
in a substantial reduction in activity likewise for 7i.
Among them, several compounds were subjected to evaluation
for anti-proliferative activity against human leukemia cell lines (Ta-
ble 5).20,21 These selected compounds showed significant inhibition
for mutant FLT3-expressing cells in addition to MOLM-13 cells,
while weak or no inhibition for ML1 cells expressing wild-type
FLT3 was observed. Compound 23f also exhibited selective kinase
inhibitory profile against FLT3 kinase as previously described.10
Finally, these antitumor effects were evaluated by oral adminis-
tration to sc MOLM-13 tumor xenograft model in SCID mice (Table
6). All compounds except for 23a revealed more potent activity
than that of 2 without mortality nor body weight loss. Above all,
23f and 23r showed significant antitumor activity including tumor
regression in a dose dependent manner.
In summary, we have demonstrated a novel class of 5-(1,3,4-
oxadiazol-2-yl)pyrimidine derivatives showed anti-proliferative
activity against MOLM-13 cells by SAR investigations to aim for
metabolic stability. Decreased lipophilicity by incorporating polar
group into the 1,3,4-oxadiazolyl group at the 5-position of the
pyrimidine ring was effective to improve metabolic stability. Inter-
estingly, our compounds showed selective anti-proliferative activ-
ity against multiple leukemia cell lines having FLT3/ITD mutation
without inhibition of wild-type FLT3 expressing cells. As a result,
some of them showed potent in vivo antitumor activity with tumor
regression against sc tumor xenograft model in SCID mice. Further
SAR studies of this class of FLT3 inhibitors will be reported else-
where together with detailed biological data.
16. Growth inhibition assays. MOLM-13, ML-1, FLT3/D599, and FLT3/D835Y were
maintained in RPMI1640 with 10% heat-inactivated FBS, 1% penicillin, and 1%
streptomycin. HMC-1 cells were maintained in IMEM with 10% heat-
inactivated FBS, 0.01%
a-thioglycerol, 1% penicillin, and 1% streptomycin.
These cell lines were seeded in 96-well culture plates in appropriate media and
incubated for 72 h in the presence of test compounds. Cell viability was
measured using the cell proliferation reagent WST-1 (Roche) according to the
instructions of the manufacture; see also Ref. 19 and 20.
17. Human liver microsomes (HLM) stability procedure. Human pooled liver
microsomes were purchased from Human and Animal Bridging Research
Organization. Reaction mixtures (n = 1 or 2) containing test compound (0.1 or
1 lmol/L), liver microsomes (0.2 mg protein/mL), nicotinamide adenine
dinucleotide phosphate, reduced form (5 mmol/L), phosphate buffer
(100 mmol/L, pH 7.4), MgCl2 (6 mmol/L), EDTA (0.1 mmol/L), and DMSO (0.01
or 0.001 vol%) or methanol (1 vol%) were incubated at 37 °C for constant time.
Just before and after incubation, aliquots of the reaction mixtures were added to
twofold volume of CH3CN containing internal standard to stop the reaction
(samples for quantitative analysis). After centrifugation, the supernatant was
Acknowledgments
We are grateful to Ms. Miyako Mizuguchi, Ms. Hiroko akamura,
and Ms. Naoko Kondo for their helpful technical assistance. We are

H. Ishida et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5472–5477
5477
analyzed by LC–MS or LC–MS/MS. The values of intrinsic clearance were
calculated from percent remaining of the test compound.
18. Cain, G. A.; Beck, J. P. Heterocycles 2001, 55, 439.
19. Daylight version 4.83; Daylight Chemical Information Systems, Inc.: Mission
Viejo, CA, 2003.
20. Yamamoto, Y.; Kiyoi, H.; Nakano, Y.; Suzuki, R.; Kodera, Y.; Miyawaki, S.; Asou,
N.; Kuriyama, K.; Yagasaki, F.; Shimazaki, C.; Akiyama, H.; Saito, K.; Nishimura,
M.; Motoji, T.; Shinagawa, K.; Takeshita, A.; Saito, H.; Ueda, R.; Ohno, R.; Naoe,
T. Blood 2001, 97, 2434.
21. Kiyoi, H.; Ohno, R.; Ueda, R.; Saito, H.; Naoe, T. Oncogene 2002, 21, 2555.