Synthesis and anti-tumor activities of novel pyrazolo[1,5-a]pyrimidines

Article abstract of DOI:10.1002/ardp.200600098

A series of novel pyrazolo[1,5-a]pyrimidines were designed and synthesized in order to find novel potent anti-tumor compounds. The structures of all the compounds were confirmed by IR, ¹H-NMR, elemental analysis, and MS. Their anti-tumor activities against cancer cell lines were tested by the MTT method in vitro. Compound 19 displayed potent anti-tumor activity.

Full text of DOI:10.1002/ardp.200600098

Arch. Pharm. Chem. Life Sci. 2006, 339, 593–597
J. Li et al.
593
Full Paper
Synthesis and Anti-tumor Activities of Novel
Pyrazolo[1,5-a]pyrimidines
Juan Li, Yan Fang Zhao, Xiang Lin Zhao, Xiao Ye Yuan, and Ping Gong
School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang, P. R. China
A series of novel pyrazolo[1,5-a]pyrimidines were designed and synthesized in order to find
novel potent anti-tumor compounds. The structures of all the compounds were confirmed by IR,
1H-NMR, elemental analysis, and MS. Their anti-tumor activities against cancer cell lines were
tested by the MTT method in vitro. Compound 19 displayed potent anti-tumor activity.
Keywords: Anti-tumor activities / Pyrazolo[1,5-a]pyrimidines / Synthesis /
Received: June 15, 2006; accepted: July 12, 2006
DOI 10.1002/ardp.200600098
Introduction
Cancer is the second leading cause of death in the world.
In the last few years, a large number of compounds, for
instance 4-anilinoquinazolines, 4-anilinophthalazines, 4-
4-anilinopyrozolo[4,3-d]pyrimidines, indolin-2-ones, and
purines were reported as anticancer agents used in
clinics or in clinical trials [1, 2]. 3, 6-Diarylpyrazolo[1,5-
a]pyrimidine 1 was another template reported as anti-
tumor compound, which targeted vascular endothelial
growth factor receptor-2 [3, 4]. This series of compounds
had been hindered by poor physical properties, such as
low solubility and high protein binding. Our chemistry
efforts began with the goal of improving these limita-
tions and we discovered a new series of pyrazolo[1,5-
a]pyrimidine derivatives with anti-tumor activities.
Herein, we replaced the aryl group with a cyano at posi-
tion C-3 (Fig. 1). Since small substitution changes of struc-
tures might affect the biological activity and drug prop-
erties, we were intrigued to determine the effect on anti-
tumor activity resulting from substitution at the C-5 and
C-7 positions of the pyrazolo[1,5-a]pyrimidine core struc-
ture. Thus, various anilines and amino alkoxy groups,
which were extensively used in the design of tyrosine
kinase inhibitors as anticancer agents, were introduced.
Figure 1. Structures of 3-cyano-5,7-disubstituted pyrazolo[1,5-
a]pyrimidine derivatives 8–21.
In this present work, we would like to report the synth-
esis and anti-tumor activities of a series of novel 3-cyano-
5,7-disubstituted pyrazolo[1,5-a]pyrimidine derivatives
represented by the general formula of 8–21 in Fig. 1.
Results and Discussion
We developed an efficient and facile approach to prepare
a variety of pyrazolo[1,5-a]pyrimindine derivatives with
various C-5 and C-7 substituents. As shown in Scheme 1,
the straightforward five-step synthetic route allowed us
to diversify positions C-5 and C-7 of the pyrazolo[1,5-
a]pyrimidine moiety via the key intermediate 5.
The synthesis of 3-amino-4-cyanopyrazole 3 has been
reported by treatment of malononitrile 2 with triethyl
orthoformate and aniline in methanol firstly, and the
intermediate obtained exchanged with hydrazine
hydrate [6]. In our modified approach, intermediate 3
Correspondence: Ping Gong, School of Pharmaceutical Engineering,
Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe Dis-
trict, 110016 Shenyang, Liaoning, P. R. China.
E-mail: gongpinggp@126.com
Fax: +86 24 2388-2925
i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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J. Li et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 593–597
Reagents and conditions: a: HC(OC2H5)3, Ac2O, reflux, 2 h; b: H2NNH26H2O, r.t., 18 h; c: ClCH2COCH2-
COOC2H5, HAc, reflux, 4 h; d: POCl3, Py, 1208C, 1 h; e: R1NH2, iPrOH, 608C, 2 h; f: NaH, R2R3NCH2CH2OH
(7), 408C, 2 h.
Scheme 1. Synthesis route of compounds 8–21.
Table 1. The substituents and anti-tumor effect of compounds 8–14.
Compound
IC50 (lM)
R1
NR2R3
Bel-7402
HT-1080
8
9
10
11
12
13
14
Cisplatin
3,4-dichlorophenyl
3,4-dichlorophenyl
3,4-dichlorophenyl
3-chlorophenyl
3-chlorophenyl
3-chlorophenyl
dimethylamino
diethylamino
4-methylpiperazinyl
dimethylamino
diethylamino
27.2
78.5
26.1
156.8
157.9
143.6
122.0
76.7
22.2
198.6
52.2
113.5
130.3
98.2
pyrrolidinyl
4-methylpiperazinyl
3-chlorophenyl
204.2
63.3
Table 2. The substituents and anti-tumor effect of compounds 15–21.
Compound
IC50 (lM)
R1
NR2R3
Bel-7402
HT-1080
15
16
17
18
19
20
21
Cisplatin
3-fluro-4-bromophenyl
3-fluro-4-bromophenyl
pyrrolidinyl
4-methylpiperazinyl
pyrrolidinyl
4-methylpiperazinyl
piperdinyl
morpholinyl
126.4
59.4
19.7
178.9
13.7
79.8
81.6
75.2
154.7
129.1
44.3
200.0
17.6
46.7
119.5
58.2
3,4-methylenedioxyphenyl
3,4-methylenedioxyphenyl
3,5-di(trifluoromethyl)phenyl
3,5-di(trifluoromethyl)phenyl
3,5-di(trifluoromethyl)phenyl
4-methylpiperazinyl
can be synthesized easily in one pot. Moreover, the yield dine gave 5 [7, 8]. Substitution of 7-Cl in 5 with various
was about 25% higher than that reported [6]. anilines provided the 7-anilinopyrazolo[1,5-a]pyrimidine
Intermediate 3 was then cyclized with ethyl 4-chloroa- 6. Treatment of 6 with various alcohols in basic DMF solu-
ceto-acetate to obtain the intermediate 4. Chlorination of tion generated the desired final products 8–21 (Tables 1
4 with phosphorus oxychloride in the presence of pyri- and 2). The pharmacological anti-tumor activities of com-
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Arch. Pharm. Chem. Life Sci. 2006, 339, 593–597
Novel Pyrazolo[1,5-a]pyrimidines
595
pounds 8–21 are shown in Tables 1 and 2. The data in 5-Chloromethyl-3-cyano-7-hydroxypyrazolo[1,5-
Table 1 indicated that the di-substituted anilino group at a]pyrimidine 4
A mixture of 3 (10.8 g, 0.1 mol), ethyl 4-chloroacetoacetate
(20 mL, 0.15 mol), and glacial acetic acid (100 mL) was refluxed
for 4 h. After cooling to room temperature, the pale white solid
was filtered and washed with ethanol, dried with suction, and
recrystallized from ethyl acetate to obtain the solid product
position 7 of pyrazolo[1,5-a]pyrimidine core structure
enhanced the anti-tumor activity. In the best case,
potency was almost improved six-fold over the corre-
sponding 3-chloroaniline substituted compound (8 vs.
11). Introduction of an ethyl group to the amino alkoxy
moiety of C-5 decreased the anti-tumor activity, indi-
cating non-tolerance for longer chains in this region.
With the discovery of the di-substituted anilino group
at position 7 as an advance in the anti-tumor activity,
we next synthesized a second set of analogues, 15–21,
by varying the substituent at the 7-position and exam-
ined the effect of extension from the basic nitrogen of
the amino alkoxy moiety. From the data in Table 2,
compound 19 was the most potent one against two can-
cer cell lines. The pyrazolo[1,5-a]pyrimidine derivative
17 possessed potent anti-tumor effect against Bel-7402,
and their IC50 were three times less than that of the
positive control. It could be concluded that incorpora-
tion of the bis(trifluoromethyl)aniline functionality
enhanced the anticancer activity. Comparing the activ-
ity of compounds 15–21 with various cyclic basic
groups at the 29-position of the alkoxy moiety, a 4-
methylpiperzinyl group was detrimental to the activ-
ity, although compound 16 was active against the can-
cer cell lines Bel-7402.
(19.7 g, 95%) [7, 8]. MS [MH+] (m/z): 209.6; IR (KBr) cm– 1
:
3450.1(tOH), 2230.3 (tC=N), 1639.7, 1590.6 (tC=C, tC=N); 1H-NMR
(DMSO-d6) d: 4.71 (s, 2H, CH2), 6.77 (s, 1H, C6-H), 8.85 (s, 1H, C2-H),
11.83 (s, 0.78H, OH); 13C-NMR (DMSO-d6) d: 55.8, 75.2, 99.5, 112.7,
145.3, 149.6, 155.2, 174.1; Anal. calcd. for C8H5ClN4O: C 46.07, H
2.42, N 26.86; Found: C 46.16, H 2.34, N 26.55.
5-Chloromethyl-3-cyano-7-chloropyrazolo[1,5-a]-
pyrimidine 5
Phosphorus oxychloride (55 mL, 0.59 mol) was added dropwise
into a solution of 3 (82.0 g, 0.39 mol) in pyridine (35 mL,
0.43 mol). The mixture was heated to 858C slowly, and then stir-
red at 1208C for 1 h. After it was cooled to 608C, chloroform
(500 mL) was added and stirred for 1 h. Cold water (300 mL) was
added to the solution. The insoluble materials was filtered off,
the organic layer of the filtrate was washed with cold water to
pH 7, concentrated, and recrystallized from ethyl acetate/cyclo-
hexane to give 4 (70 g, 78%) [7, 8]. MS [MH+] (m/z): 228.0; IR (KBr)
1
cm– 1: 2232.3 (tC=N), 1612.7, 1541.5, 1499.8 (tC=C, tC=N); H-NMR
(DMSO-d6) d: 4.72 (s, 2H, CH2), 6.83 (s, 1H, C6-H), 8.86 (s, 1H, C2-H);
13C-NMR (DMSO-d6) d: 53.8, 78.2, 103.5, 116.7, 151.3, 159.6, 165.2,
169.1; Anal. calcd. for C8H4Cl2N4: C 42.32, H 1.78, Cl 31.23, N
24.68; Found: C 42.23, H 1.67, Cl 31.31, N 24.55.
General procedure for the synthesis of 5-Chloro-
methyl-3-cyano-7-subsituted-pyrazolo[1,5-a]-
pyrimidine 6
Experimental
A mixture of 5 (8.0 g, 35 mmol), R1NH2 (45 mmol), and isopropa-
nol (50 mL) was heated at 608C for 2 h. After cooling to room
temperature, a massive solid precipitated. Recrystallization
from methol gave 6 as slight yellow powder (80–86%).
Chemistry
Melting points were determined with capillary tube method,
and the thermometer was uncorrected. Mass spectra were
obtained with a Finnigan LCQ HPLC-MS instrument (Thermo
Electron Corporation, Bremen, Germany). IR spectra were col-
lected on a Bruker IFS 55 instrument (Bruker, Rheinstetten, Ger-
many). 1H-NMR and 13C-NMR spectra were run on a Bruker ARX-
300 instrument, TMS as the internal standard. Elemental anal-
ysis was performed by a Carlo-Erba 1106 Elemental analysis
instrument (Carlo Erba, Milan, Italy). Compound 7 was prepared
according to the literature [9].
General procedure for the synthesis of 8–21
NaH (0.3 g, 6.2 mmol, 60%) was added to the solution of 7
(6.2 mmol) in dried DMF (20 mL). The mixture was stirred for
30 min. Then 6 (2.84 mmol) was added and the resulting mix-
ture was stirred for 2 h at 408C. It was then poured into water
and recrystallized to afford 8–21.
3-Amino-4-cyano-pyrazole 3
A mixture of malononitrile (16.5 g, 0.25 mol), triethyl orthofor-
mate (50 mL, 0.25 mol), and acetic anhydride (51 mL, 0.54 mol)
was heated to reflux. After 2 h., the mixture was cooled to 208C,
then hydrazine hydrate (19.4 g, 0.31 mol, 80%) was added drop-
wise at 20–258C. The resulting mixture was stirred at 25–308C
for 18 h, and neutralized with aqueous sodium hydroxide. Fil-
tration and washing with water gave 19.2 g (71%) of 3 as white
powder, m.p.171–1728C, (lit. 166–1698C [6]). MS [MH+] (m/z):
109.1; IR (KBr) cm– 1: 3308.5 (tNH2), 2240.3 (tC=N), 1645.0 1569.6
1523.3 (tC=C, tC=N); Anal. calcd. for C4H4N4: C 44.44, H 3.73, N
51.83; Found: C 44.45, H 3.73, N 51.84.
7-(3,4-Dichlorophenylamino)-3-cyano-5-((2-(dimethyl-
amino)ethoxy)methyl)pyrazolo[1,5-a]pyrimidine 8
Yield: 85%; m. p. 187–1888C; MS [MH+] (m/z): 406.2; IR (KBr) cm– 1
:
3440.5 (tNH), 2928.1 (tCH3), 2229.6 (tC=N), 1612.7, 1576.9, 1542.4
(tC=C, tC=N). 1H-NMR (DMSO-d6) d: 2.81 (s, 6H, N(CH3)2), 3.31 (t, J = 5.7
Hz, 2H, NCH2CH2O), 3.84 (t, J = 5.7 Hz, 2H, NCH2CH2O), 4.63 (s, 2H,
CH2), 6.64 (s, 1H, C6-H), 7.51 (m, J = 8.7 Hz, 1H, ph-H), 7.55–7.59
(m, 2H, ph-2H), 8.81 (s, 1H, C2-H); Anal. calcd. for C18H18Cl2N6O: C
53.34, H 4.48, N 20.74; Found: C 53.21, H 4.55, N 20.59.
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7-(3,4-Dichlorophenylamino)-3-cyano-5-((2-
7-(3-Chlorophenylamino)-3-cyano-5-((2-(4-
(diethylamino)ethoxy)methyl)pyrazolo[1,5-a]pyrimidine 9
methylpiperazin-1-yl)ethoxy)methyl)pyrazolo[1,5-
Yield: 81%; m.p. 181-1838C; MS [MH+] (m/z): 434.0; IR (KBr) cm– 1
:
a]pyrimidine 14
Yield: 79%; m. p. 180–1818C; MS [M+] (m/z): 426.0; IR (KBr) cm-1:
3444.4 (tNH), 2931.7 (tCH2), 2222.4 (tC=N), 1623.4, 1585.3, 1559.4,
3443.5 (tNH), 2963.1 (tCH2), 2230.9 (tC=N), 1590.0, 1556.5, 1473.4
(tC=C, tC=N). 1H-NMR (DMSO-d6) d: 0.82 (t, J = 7.0 Hz, 6H, 26CH2CH3),
2.40 (q, J = 7.0 Hz, 4H, 2 x CH2CH3), 2.55 (t, J = 6.0 Hz, 2H,
NCH2CH2O), 3.48 (t, J = 6.0 Hz, 2H, NCH2CH2O), 4.31(s, 2H, CH2),
6.06 (s, 1H, C6-H), 7.55–7.58 (m, 3H, ph-3H), 8.26 (s, 1H, C2-H);
Anal. calcd. for C20H22Cl2N6O: C 55.43, H 5.12, N 19.39; Found: C
55.29, H 5.18, N 19.27.
1
1473.0 (tC=C, tC=N). H-NMR (DMSO-d6) d: 2.51 (s, 3H, CH3), 2.71 (b,
4H, piperazinyl-3,5-1CH2), 2.82 (b, 4H, piperazinyl-2,6-2CH2), 3.14
(t, J = 5.4 Hz, 2H, NCH2CH2O), 3.57 (t, J = 5.4 Hz, 2H, NCH2CH2O),
4.57 (s, 2H, CH2), 6.59 (s, 1H, C6-H), 7.41–7.58 (m, 4H, ph-4H), 8.80
(s, 1H, C2-H); Anal. calcd. for C21H24ClN7O: C 59.22, H 5.68, N
23.02; Found: C 59.37, H 5.74 N 23.26.
7-(3,4-Dichlorophenylamino)-3-cyano-5-((2-(4-
methylpiperazin-1-yl)ethoxy)methyl)pyrazolo[1,5-
a]pyrimidine 10
7-(4-Bromo-3-cholrophenylanimo)-3-cyano-5-((2-
(pyrrolidin-1-yl)ethoxy)methyl)pyrazolo[1,5-a]pyrimidine
Yield: 78%; m. p. 170–1738C; MS [MH+] (m/z): 461.0; IR (KBr) cm– 1
:
15
Yield: 77%; m. p. 175–1768C; MS [M+] (m/z): 459.0; IR (KBr) cm– 1
:
3443.0 (tNH), 2923.7 (tCH2), 2228.8 (tC=N), 1586.5, 1557.2, 1472.8
(tC=C, tC=N). 1H-NMR (DMSO-d6) d: 2.51 (s, 3H, CH3), 2.73 (b, 4H,
piperazinyl-3,5-2CH2), 2.87 (brs, 4H, piperaziny-2,6-2CH2), 3.04 (t,
J = 5.7 Hz, 2H, NCH2CH2O), 3.67 (t, J = 5.7 Hz, 2H, NCH2CH2O), 4.57
(s, 2H, CH2), 6.59 (s, 1H, C6-H), 7.51 (d, J = 6.6 Hz, 1H, ph-H), 7.77–
7.80 (m, 2H, ph-2H), 8.79 (s, 1H, C2-H); Anal. calcd. for
C21H23Cl2N7O: C 54.79, H 5.04, N 21.30; Found: C 54.87, H 4.98, N
21.46.
3445.4 (tNH), 2923.7 (tCH2), 2221.5 (tC=N), 1624.0, 1575.0, 1483.3
(tC=C, tC=N). 1H-NMR (DMSO-d6) d: 3.23 (b, 4H, pyrrolinyl-3,5-2CH2),
3.34 (b, 4H, pyrrolinyl-2,6-2CH2), 3.39 (t, J = 5.7 Hz, 2H,
NCH2CH2O), 3.79 (t, J = 5.7 Hz, 2H, NCH2CH2O), 4.63 (s, 2H, CH2),
6.67 (s, 1H, C6-H), 7.34 (d, J = 8.7 Hz, 1H, ph-H), 7.56 (d, J = 8.4 Hz,
1H, ph-H), 7.83 (t, J = 8.7 Hz, 1H, ph-H), 8.79 (s, 1H, C2-H); Anal.
calcd. for C20H20BrFN6O: C 52.30, H 4.39, N 18.30; Found: C 52.19,
H 4.26 N 18.29.
7-(3-Chlorophenylamino)-3-cyano-5-((2-
(dimethylamino)ethoxy)methyl)pyrazolo[1,5-a]pyrimidine
7-(4-Bromo-3-cholrophenylanimo)-3-cyano-5-((2-(4-
methylpiperazin-1-yl)ethoxy)methyl)pyrazolo[1,5-
a]pyrimidine 16
11
Yield: 84%; m. p. 180–1818C; MS [MH+] (m/z): 371.1; IR (KBr) cm– 1
:
Yield: 75%; m. p. 194–1968C; MS [M+] (m/z): 488.0; IR (KBr) cm– 1
:
3426.9 (tNH), 2925.1 (tCH2), 2228.2 (tC=N), 1622.9 1566.9 1537.0
1478.5 (tC=C, tC=N). 1H-NMR (DMSO-d6) d: 2.79 (s, 6H, 26CH3), 3.29
(t, J = 5.4 Hz, 2H, NCH2CH2O), 3.82 (t, J = 5.4 Hz, 2H, NCH2CH2O),
4.64 (s, 2H, CH2), 6.59 (s, 1H, C6-H), 7.40–7.58 (m, 4H, ph-4H), 8.81
(s, 1H, C2-H); Anal. calcd. for C18H19ClN6O2: C 58.30, H 5.16, N
22.66; Found: C 58.43, H 5.29, N 22.76.
3440.4 (tNH), 2924.0 (tCH2), 2227.0 (tC=N), 1615.2, 1566.9, 1483.1
(tC=C, tC=N). 1H-NMR (DMSO-d6) d: 2.67 (s, 3H, CH3), 2.75 (b, 4H,
piperazinyl-3,5-2CH2), 2.69 (b, 4H, piperazinyl-2,6-2CH2), 3.24 (t, J
= 5.4 Hz, 2H, NCH2CH2O), 3.83 (t, J = 5.4 Hz, 2H, NCH2CH2O), 4.58
(s, 2H, CH2), 6.65 (s, 1H, C6-H), 7.33 (d, J = 8.7 Hz, 1H, ph-H), 7.57 (d,
J = 8.4 Hz, 1H, ph-H), 7.85 (t, J = 8.7 Hz, 1H, ph-H), 8.79 (s, 1H, C2-
H); Anal. calcd. for C21H23BrFN7O: C 51.65, H 4.75, N 20.08; Found:
C 51.49, H 4.74, N 20.18.
7-(3-Chlorophenylamino)-3-cyano-5-((2-
(diethylamino)ethoxy)methyl)pyrazolo[1,5-a]pyrimidine
7-(Benzo[d][1,3]dioxol-5-yl-amino)-3-cyano-5-((2-
pyrrolidin-1-yl)ethoxy)methyl)pyrazolo[1,5-a]pyrimidine
12
Yield: 81%; m. p. 176–1778C; MS [M+] (m/z): 399.0; IR (KBr) cm– 1
:
3443.5 (tNH), 2926.7 (tCH2), 2226.3 (tC=N), 1622.8, 1588.5, 1476.8
(tC=C, tC=N).1H-NMR (DMSO-d6) d: 1.14 (t, J = 7.2 Hz, 6H, 26CH2CH3),
3.14 (q, J = 7.2 Hz, 4H, 26CH2CH3), 3.31 (t, J = 5.7 Hz, 2H,
NCH2CH2O), 3.83 (t, J = 5.7 Hz, 2H, NCH2CH2O), 4.64 (s, 2H, CH2),
6.54 (s, 1H, C6-H), 7.41–7.57 (m, 4H, ph-4H), 8.81 (s, 1H, C2-H);
Anal. calcd. for C20H23ClN6O2: C 60.22, H 5.81, N 21.07; Found: C
60.18, H 5.72 N 21.16.
17
Yield: 84%; m. p. 201–2028C; MS [MH+] (m/z): 407.1; IR (KBr) cm– 1
:
3442.8 (tNH), 2961.1 (tCH3), 2227.0 (tC=N), 1623.0, 1584.7, 1485.7
(tC=C, tC=N). 1H-NMR (DMSO-d6) d: 3.23 (b, 4H, pyrrolinyl-3,5-2CH2),
3.34 (b, 4H, pyrrolinyl-2,6-2CH2), 3.39 (t, J = 5.7 Hz, 2H,
NCH2CH2O), 3.79 (t, J = 5.7 Hz, 2H, NCH2CH2O), 4.63 (s, 2H, CH2),
6.12 (s, 2H, OCH2O), 6.36 (s, 1H, C6-H), 6.92 (d, J = 8.4 Hz, 1H, ph-H),
7.04–7.07 (m, 2H, ph-2H), 8.74 (s, 1H, C2-H); Anal. calcd. for
C21H22N6O3: C 62.06, H 5.46, N 20.68; Found: C 62.15, H 5.33, N
20.52.
7-(3-Chlorophenylamino)-3-cyano-5-((2-(pyrrolidin-1-
yl)ethoxy)methyl)pyrazolo[1,5-a]pyrimidine 13
Yield: 85%; m. p. 190–1918C; MS [M+] (m/z): 397.1; IR (KBr) cm– 1
:
7-(Benzo[d]-[1,3]dioxol-5-yl-amino)-3-cyano-5-((2-(4-
methylpiperazin-1-yl)ethoxy)methyl)pyrazolo[1,5-
a]pyrimidine 18
3441.7 (tNH), 2930.6 (tCH2), 2226.7 (tC=N), 1623.8, 1586.4, 1535.7,
1477.2 (tC=C, tC=N). 1H-NMR (DMSO-d6) d: 1.89 (b, 4H, pyrrolidinyl-
3,4-2CH2), 3.23 (b, 4H, pyrrolidinyl-2,5-2CH2), 3.34 (t, J = 5.7 Hz,
2H, NCH2CH2O), 3.79 (t, J = 5.7 Hz, 2H, NCH2CH2), 4.63 (s, 2H, CH2),
6.57 (s, 1H, C6-H), 7.41–7.58 (m, 4H, ph-4H), 8.80 (s, 1H, C2-H);
Anal. calcd. for C20H21ClN6O: C 60.53, H 5.33, N 21.18; Found: C
60.67, H 5.44 N 21.29.
Yield: 82%; m. p. 191–1928C; MS [MH+] (m/z): 436.0; IR (KBr) cm– 1
:
3443.5 (tNH), 2962.1 (tCH3), 2227.1 (tC=N), 1629.5, 1585.8, 1538.4,
1486.5 (tC=C, tC=N). 1H-NMR (DMSO-d6) d: 2.71 (s, 3H, CH3), 3.13 (b,
4H, piperazinyl-3,5-2CH2), 3.36 (b, 4H, piperazinyl-2,6-2CH2), 3.29
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Arch. Pharm. Chem. Life Sci. 2006, 339, 593–597
Novel Pyrazolo[1,5-a]pyrimidines
597
(t, J = 5.7 Hz, 2H, NCH2CH2O), 3.76 (t, J = 5.7 Hz, 2H, NCH2CH2O),
4.30 (s, 2H, CH2), 6.1 1(s, 2H, OCH2O), 6.57 (s, 1H, C6-H), 6.93 (d, J =
7.5 Hz, 1H, ph-H), 7.02–-7.05 (m, 2H, ph-2H), 8.82 (s, 1H, C2-H);
Anal. calcd. for C22H25N7O3: C 60.68, H 5.79, N 22.51; Found: C
60.80, H 5.84, N 22.52.
Pharmacology
The anticancer activities of compounds 8–21 were evaluated
with Bel-7402 (Human Liver Cancer Cell Lines) and HT-1080
(Human Fibro Sarcoma Cell Lines) by the MTT method in vitro
[10], with cisplatin as the positive control. The cells were seeded
in 96-well plate at the concentration of 4000 cells per well in
100 mL RPM I 1640 medium. After cultured for 12 h at 378C and
5% CO2, cells were incubated with various concentrations of
samples for 24 h. MTT was added at a terminal concentration of
5 lg/mL and incubated with cells for 4 h. The formazan crystals
were dissolved in 100 lL DMSO each well, and the optical den-
sity was measured at 492 nm (for absorbance of MTT formazan)
and 630 nm (for the reference wavelength). The IC50 was calcu-
lated by Bliss method.
7-(3,5-Bis(trifluoromethyl)phenylamino)-3-cyano-5-((2-
(piperidin-1-yl)ethoxy)methyl)pyrazolo[1,5-a]pyrimidine
19
Yield: 81%; m. p. 170–1738C; MS [MH+] (m/z): 513.0; IR (t, KBr,
cm– 1): 3409.6 (tNH), 2924.1 (tCH2), 2214.4 (tC=N), 1581.7, 1543.9,
1466.4 (tC=C, tC=N). 1H-NMR d: 1.39 (b, 6H, piperidinyl-3,4,5-3CH2),
2.33 (b, 4H, piperidinyl-2,6-2CH2), 2.73 (t, J = 5.4 Hz, 2H,
NCH2CH2O), 3.62 (t, J = 5.4 Hz, 2H, NCH2CH2O), 4.53 (s, 2H, CH2),
6.56 (s, 1H, C6-H), 7.93 (1H, ph-H), 8.06 (2H, ph-2H), 8.67 (s, 1H, C2-
H); Anal. calcd. for C22H22F6N6O: C 53.91, H 4.33, N 16.40; Found:
C 53.77, H 4.32, N 16.29.
References
[1] A. Arora, E. M. Scholar, J. Pharmacol. Exp. Ther. 2005, 315,
7-(3,5-Bis(trifluoromethyl)phenylamino)-3-cyano-5-((2-
971–979.
morpholinoethyl)methyl)pyrazolo[1,5-a]pyrimidine 20
Yield: 75%; m. p. 166–1678C; MS [MH+] (m/z): 515.0; IR (t, KBr,
cm– 1): 3442.3. (tNH), 2925.1 (tCH2), 2225.4 (tC=N), 1663.1, 1594.4,
1541.4, 1494.1 (tC=C, tC=N).1H-NMR d: 2.17 (brs, 4H, piperidinyl-2,6-
2CH2), 2.74 (brs, 4H, piperidinyl-3,5-2CH2), 3.34 (t, J = 5.4 Hz, 2H,
NCH2CH2O), 3.53 (t, J = 5.4 Hz, 2H, NCH2CH2O), 4.28 (s, 2H, CH2),
5.99 (s, 1H, C6-H), 7.52 (1H, ph-H), 7.98 (2H, ph-2H), 8.19 (s, 1H, C2-
H); Anal. calcd. for C22H20F6N6O2: C 51.37, H 3.92, N 16.34; Found:
C 51.23, H 3.81, N 16.29.
[2] L. Meijer, E. Ravmond, Acc. Chem. Res. 2003, 36, 417–425.
[3] M. E. Fraley, W. F. Hoffman, R. S. Rubino, R. W. Hungate,
Bioorg. Med. Chem. Lett. 2002, 12, 2767–2770.
[4] M. E. Fraley, R. S. Rubino, W. F. Hoffman, S. R. Ham-
baugh, Bioorg. Med. Chem. Lett. 2002, 12, 3537–3541.
[5] Z. C. Wu, M. E. Fraley, M. T. Bilodeau, E. S. Tasber, Bioorg.
Med. Chem. Lett. 2004, 14, 909–912.
[6] O. S. Wolfbeis, Monatsh. Chem. 1981, 112, 875–877.
[7] N. Thomas, R. K. Robins, M. R. Thomas, J. Med. Chem.
1977, 20, 296–299.
7-(3,5-Bis(trifluoromethyl)phenylamino)-3-cyano-5-((2-(4-
methylpiperazin-1-yl)ethoxy)methyl)pyrazolo[1,5-
a]pyrimidine 21
[8] R. H. Springer, M. B. Scholten, D. E. O’Brien, N. Thomas, J.
Med. Chem. 1982, 25, 235–242.
Yield: 83%; m. p. 176–1778C; MS [MH+] (m/z): 528.0; IR (t, KBr,
cm– 1): 3440.1 (tNH), 2924.5 (tCH2), 2224.1 (tC=N), 1597.6, 1543.1,
1460.6 (tC=C, tC=N). 1H-NMR d: 2.09 (s, 3H, CH3), 2.17 (brs, 4H, piper-
idinyl-3,5-2CH2), 2.20 (brs, 4H, piperidinyl-3,5-2CH2), 2.42 (t, J =
5.7 Hz, 2H, NCH2CH2O), 3.53 (t, J = 5.7 Hz, 2H, NCH2CH2O), 4.35 (s,
2H, CH2), 6.17 (s, 1H, C6-H), 7.61 (1H, ph-H), 7.69 (2H, ph-2H), 8.33
(s, 1H, C2-H); Anal. calcd. for C23H23F6N7O2: C 52.37, H 4.40, N
18.59; Found: C 52.22, H 4.28 N 18.72.
[9] W. W. Hartmam, Org. Synth. 1943, Coll. Vol. 2, 183–183.
[10] Method of Pharmacology (Ed.: S. Y. Xu), Public Health
Publishing House, Beijing, 2002, pp. 1784–1786.
i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com