Synthesis and mechanism of action of novel pyrimidinyl pyrazole derivatives possessing antiproliferative activity

Article abstract of DOI:10.1016/S0960-894X(02)00568-1

Pyrimidinyl pyrazole derivatives 1-4, prepared as a new scaffold of an anti-tumor agent, showed antiproliferative activity against human lung cancer cell lines and inhibited tubulin polymerization. Furthermore, it was found that compound 2 bound at the colchicine site on tubulin, but the tubulin binding pattern was different from that of colchicine. Here, we describe the synthesis of the derivatives and the differences of the action mechanism on tubulin polymerization inhibition between compound 2 and colchicine.

Full text of DOI:10.1016/S0960-894X(02)00568-1

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3191–3193
Synthesis and Mechanism of Action of Novel Pyrimidinyl
Pyrazole Derivatives Possessing Antiproliferative Activity
Hitoshi Ohki,a,* Kenji Hirotani,b Hiroyuki Naito,a Satoru Ohsuki,a Megumi Minami,b
Akio Ejima,a Yukiko Koisoc and Yuichi Hashimotoc
aMedicinal Chemistry Research Laboratory, DaiichiPharmaceutical Co. Ltd., 16-13, Kita-kasai1-chome, Edogawa-ku, Tokyo134, Japan
bNew Product Research Laboratories III, Daiichi Pharmaceutical Co. Ltd., 16-13, Kita-kasai 1-chome, Edogawa-ku, Tokyo 134, Japan
cInstitute of Molecular and Cellular Biosciences (IMCB), The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113, Japan
Received 27 May 2002; accepted 17 July 2002
Abstract—Pyrimidinyl pyrazole derivatives 1–4, prepared as a new scaffold of an anti-tumor agent, showed antiproliferative activity
against human lung cancer cell lines and inhibited tubulin polymerization. Furthermore, it was found that compound 2 bound at
the colchicine site on tubulin, but the tubulin binding pattern was different from that of colchicine. Here, we describe the synth-
esis of the derivatives and the differences of the action mechanism on tubulin polymerization inhibition between compound 2 and
colchicine.
# 2002 Elsevier Science Ltd. All rights reserved.
In our previous paper, we reported that pyrimidinyl
pyrazole 1 showed antiproliferative and inhibitory
activities on tubulin polymerization.1,2 As a result of
focusing on further modification of the pyrimidine ring
in order to find better compounds, we obtained new
analogues 2–4 which showed more potent cytotoxicity
in vitro than 1. It has been known that there are two
major binding sites on tubulin, which are called the
colchicine (CLC) and vincristine (VCR) sites for tubulin
polymerization inhibitors.3 The binding site of pyri-
midinyl pyrazole derivatives was investigated by the use
of compound 2. In this paper, we describe the synthesis,
in vitro cytotoxicity, the tubulin polymerization inhibi-
tory activity of compounds 2–4, and the binding site of
compound 2 on tubulin (Fig. 1).
pyrazole rings. Therefore, compound 4 bearing an
amino group on the pyrimidine ring was prepared via
reductive amination of formyl derivative 16 with 9 in
the presence of NaBH3CN, and deprotection of the
benzyl group by the treatment with TFA. To perform
this reductive amination, compound 11 derived from 55
was cyclized with 12 instead of 6 to give 13. The ester of
13 was reduced by diisobutylaluminum hydride
(DIBAL), then successively oxidized with MnO2, and
treated with 14 to give 15. The ester 15 was reduced and
oxidized again to afford the formyl derivative 16, which
was led to 4 through reductive amination with 9.
In vitro cytotoxicity and the tubulin inhibitory activity
of compounds 2–4 were assessed in comparison with 1,
vinblastine (VBL), and CLC as shown in Table 1. The
cytotoxicity (MTT assay6) of 2 bearing a difluoro group
Compounds 2 and 3 having a difluoro group on the phenyl
ring were prepared as described for the synthesis of com-
pound 1 via the Mannich reaction of 7 or 8 with 9.1,4
Compound 10 was treated with trifluoroacetic acid (TFA),
POCl3, and concd NH4OH to give 3 as shown in Scheme 1.
The Mannich reaction of compounds having an amino
group on the pyrimidine ring with 9, however, caused the
cleavage of the C–N bond between the pyrimidine and
*Corresponding author. Tel.: +81-3-3680-0151; fax: +81-3-5696-
8344; e-mail: okihi10g@daiichipharm.co.jp
Figure 1. Structure of pyrimidinyl pyrazole derivatives.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00568-1

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H. Ohki et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3191–3193
Scheme 1. Reagents and conditions: (a) NH2NH2, K2CO3, EtOH, reflux, 18 h; (b) 6, EtOH, 80 ꢀC, 1 h; (c) 9, (HCHO)n, p-TsOH, EtOH, reflux, 24
h; (d) NaBH4, EtOH, THF, rt, 4 h; (e) p-TsOH, dioxane, THF, reflux, 3 h; (f) TFA, thioanisole, 60 ꢀC, 30 min; (g) POCl3, 60 ꢀC, 2h; (h) concd
NH4OH, THF, 80 ꢀC, sealed, 2h; (i) p-methoxybenzylamine [H2NBzl(OMe)], K2CO3, THF, reflux, 2days; (j) TFA, anisole, rt, 2days; (k) 12,
EtOH, 80 ꢀC, 1 h; (l) DIBAL, CH2Cl2, À78 ꢀC, 4 h; (m) MnO2, dioxane, rt, 24 h; (n) 14, toluene, 80 ꢀC, 15 h; (o) 9, NaBH3CN, AcOH, EtOH, rt, 2
days; (p) TFA, anisole, reflux, 4 days.
was stronger than that of 1 bearing a chloro group; and
compounds 3 and 4 possessing an amino group on the
pyrimidine ring showed more potent cytotoxicity than
2. Introduction of an amino group was considered to
enhance the cytotoxicity. Inhibitory activity of tubulin
polymerization was examined by measurement of the
turbidity of an aqueous solution of porcine brain tubu-
lin.7,8 Among the compounds, 4 showed high tubulin
polymerization inhibitory activity (IC50=3.72 mM),
which was more potent than that of CLC (IC50=29.3
mM); but the activity was lower than that of VBL. There
was not a clear correlation between cytotoxicity and
tubulin polymerization inhibitory activity, but com-
pounds showing strong cytotoxicity tended to show
stronger tubulin polymerization inhibitory activity. The
time-dependent changes in the turbidity of 2 were
shown in Figure 2. Compound 2 inhibited tubulin poly-
merization in a concentration-dependent manner. Fur-
thermore, the inhibitory pattern of 2 on microtubule
assembly was compared with colchicine. After tubulin
was polymerized at 37 ꢀC for 20 min, compound 2 was
added to the assembled microtubulin. As shown in Figure
3, microtubule disassembly was observed at higher
doses, whereas CLC showed no such disassembly
activity (data not shown). A repeated tubulin polymeri-
zation-depolymerization assay (i.e., tubulin was assem-
bled in the presence of CLC at 37 ꢀC for 25 min; then it
was disassembled at 4 ꢀC for 10 min, and reincubated at
37 ꢀC again) indicated that CLC-treated tubulin was
more sensitive toward the polymerization inhibiting
Figure 2. Effect of compound 2 on tubulin polymerization. Micro-
tubule proteins (1.5 mg/mL) were incubated at 37 ꢀC in the absence or
presence (1–10 mg/mL) of the compound, and the turbidity was mon-
itored at 400 nm. The IC50 value of compound 2 was calculated from
the turbidity at 20 min after incubation.
Table 1. Inhibition of tubulin polymerization (IC50) and in vitro
cytotoxic activity (growth inhibition: GI50) of compounds 1–4, vin-
blastine, and colchicine against human cancer cell lines
Compd
Inhibition of
microtubule
assembly (%)
IC50 (mM)
In vitro cytotoxic
activity GI50 (ng/mL)
PC-12HCT116
1
2
3
35.96
9.08
11.05
181.0
62.5
21.6
254.0
70.1
20.8
4
3.7219.9 .9
0.60
29.30
32
10.21.0
31.5
Vinblastine
Colchicine
8.4
Microtubule proteins (1 mg/mL) were incubated at 37 ꢀC in the
absence or presence of the compound, and the turbidity was mon-
itored at 400 nm.8,9 The IC50 values of compounds were calculated
from the turbidity at 20 min after incubation. In vitro cytotoxic activ-
ity (a growth inhibition of 50%: GI50) was calculated according to the
published procedure.7
Figure 3. Microtubule disassembly by compound 2. Microtubule pro-
teins (1.5 mg/mL) were incubated at 37 ꢀC without a compound.
Compound 2 (3–300 mg/mL) was added 20 min later, and the turbidity
was monitored at 400 nm.

H. Ohki et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3191–3193
3193
Figure 4. Comparison of compound 2 with colchicine on tubulin. Microtubule proteins were incubated at 37 ꢀC for 25 min in the absence or
presence of compound 2 (*: 0.5, *: 2.5, &: 2 0mg/mL) or colchicines (*: 5, *: 2 0,&: 100 mg/mL). Microtubule proteins were disassembled at 5 ꢀC
for 10 min. Microtubule proteins were incubated at 37 ꢀC again, and the turbidity was monitored at 400 nm (– control).
Table 2. Effect of compound 2 and colchicine on tubulin-[3H]colchi-
cine binding
12and tubulin polymerization inhibition. Although
compound 2 was found to bind at the CLC site, the
pattern of tubulin polymerization inhibitory effect eli-
cited by 2 was different from that of CLC. These results
suggest that our pyrimidinyl pyrazole derivatives could
be novel leading compounds for the development of
MDR-overcoming antitumor agents targeting tubulin.
Compd
Added concentration (mM)
Inhibitory bindinga (%)
2
2
100
200
82
93
Colchicine
Colchicine
100
200
98
100
Tubulin (2 mM) was incubated with [3H]colchicine (2 mM) at 37 ꢀC for
20 min. Then compound 2 (100 or 200 mM) or colchicine (100 or 200
mM) was added at 37 ꢀC.
References and Notes
aThe inhibitory binding was determined by comparing the radio-
activity under the compound-free condition with that in the presence
of the compound on the tubulin-[3H]colchicine binding portion after
the treatment by Sephadex-G25 chromatography.9
1. Naito, H.; Sugimori, M.; Mitsui, I.; Nakamura, Y.; Iwa-
hana, M.; Ishii, M.; Hirotani, K.; Ejima, A. Chem. Pharm.
Bull. 1999, 47, 1679.
2. Iwahana, M.; Ochi, Y.; Ejima, A. Anticancer Res. 2000, 20,
785.
3. Iwasaki, S. Med. Res. Rev. 1993, 13, 183.
4. Ejima, A.; Sugimori, M.; Mitsui, I. Japan Kokai Tokkyo
Koho JP 97 48776, 1997.
activity of CLC, while 2 showed no such a pattern (Fig. 4).
Therefore, it was suggested that our pyrimidinyl
pyrazole derivatives possess characteristics different
from that of CLC.
5. Mitchell, M. B.; Wallbank, P. J. Tetrahedron Lett. 1991, 32,
2273.
Then we examined the binding site of compound 2 on
tubulin. As a result, compound 2 was found to bind at
the CLC site, because compound 2 at 100 and 200 mM
inhibited 82and 93% of the CLC binding competitively
(Table 2).
6. Mitsui, I.; Kumazawa, E.; Hirota, Y.; Aonuma, M.; Sugi-
mori, M.; Ohsuki, S.; Uoto, K.; Ejima, A.; Terasawa, H.;
Sato, K. Jpn. J. Cancer Res. 1995, 86, 776.
7. Shelanski, M. L.; Gaskin, F.; Cantor, C. R. Proc. Natl.
Acad. Sci. U.S.A. 1973, 70, 765.
8. Peyot, V.; Briand, C.; Andreu, J. M. Arch. Biochem. Bio-
phys. 1990, 249, 611.
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1977, 252, 1134.
In conclusion, this new pharmacophore showed potent
cytotoxicity against two tumor cell lines including the P-
gp-associated multi-drug resistant (MDR) cell line PC-