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Synthesis and Antiproliferative Activitiy of Novel Diaryl Ureas Possessing a 4H-Pyrido[12-a]pyrimidin-4-one Group.

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Arch. Pharm. Chem. Life Sci. 2010, 343, 17 – 23
P. Yao et al.
17
Full Paper
Synthesis and Antiproliferative Activitiy of Novel Diaryl Ureas
Possessing a 4H-Pyrido[1,2-a]pyrimidin-4-one Group
Peng Yao, Xin Zhai, Dong Liu, Bao Hui Qi, Hai Liang Tan, Yong Cai Jin, and Ping Gong
Key Lab of New Drugs Design and Discovery of Liaoning Province, School of Pharmaceutical Engineering,
Shenyang Pharmaceutical University, Shenyang, P. R. China
We herein disclose a series of novel diaryl urea derivatives possessing a 4H-pyrido[1,2-a]pyrimidin-4-one group as novel potent anticancer compounds. The structures were confirmed by IR, 1HNMR, and MS. All the compounds were screened for their antiprofilerative activity agaist the
human breast cancer cell line (MDA-MB-231). The pharmacological results indicated that most of
the compounds showed moderate activity. The best of this series is compound 4c (IC50 = 0.7
lmol/L), with a potency 3.6-fold higher than Sorafenib (IC50 = 2.5 lmol/L), which was approved in
2005.
Keywords: Antiprofilerative activity / 4H-Pyrido[1,2-a]pyrimidin-4-one / Synthesis /
Received: June 24, 2009; accepted: September 11, 2009
DOI 10.1002/ardp.200900130
Introduction
Cancer is considered to be one of the major killer diseases
worldwide. It is caused by mutations in critical genes
that alter normal cell functioning. Since kinases are
involved in many critical biological signaling pathways,
which are essential for the cell cycle regulation [1], the
development of selective protein kinase inhibitor is
widely considered a promising approach for antitumor
drug development. Since the discovery of Sorafenib,
which had been approved by FDA for use in patients with
hepatocellular carcinoma and renal cell carcinoma in
2005 [2], derivatives with a diaryl urea framework constituted an interesting class of compounds as multi-target
protein kinase inhibitors [3].
In this context, many derivatives based on the essential
diaryl urea scaffold were synthesized [3 – 5], however,
there are hardly any studies referring to compounds
bearing a 4H-pyrido[1,2-a]pyrimidin-4-one group so far.
After reading many references, we had concluded that
Correspondence: Key Lab of New Drugs Design and Discovery of
Liaoning Province, School of Pharmaceutical Engineering, Shenyang
Pharmaceutical University, Ping Gong, 103 Wenhua Road, Shenhe District, 110016 Shenyang, Liaoning, P. R. China.
E-mail: gongpinggp@126.com
Fax: +86 24 2398-6429
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2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Structures of Sorafenib and target compounds.
the selective introduction of a 4H-pyrido[1,2-a]pyrimidin4-one group and the modification of the aryl substituents
on the 1-position at the diaryl urea framework should be
of significant importance for the antitumor activity.
Thus, in our efforts developing new antitumor agents
with improved solubility and better biological interactions, 20 new diaryl urea derivatives possessing a 4H-pyrido[1,2-a]pyrimidin-4-one group were designed and synthesized (Fig. 1). All the prepared compounds, which are
represented by the general structure of 4a – 4f, 8a – 8g,
13a – 13g in Tables 1 and 2, were identified as potent antitumor agents and screened for their antiprofilerative
activity against the human breast cancer cell line (MDAMB-231; Sorafenib had been tested against MDA-MB-231
by Chunrong Yu et al.).
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P. Yao et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 17 – 23
Table 1. The substituents and IC50 values against the human
breast cancer cell line of compounds 4a – 4f and 8a – 8g.
Compound
R1
R2
IC50 (lmol/L)
MDA-MB-231
4a
4b
4c
4d
4e
4f
8a
8b
8c
8d
8e
8f
8g
Sorafenib
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
2,6-difluoro
2-fluoro
3-fluoro
3-trifluoromethyl
3-chloro-4-fluoro
4-fluoro
4-fluoro
2,6-difluoro
3-chloro-4-fluoro
3,5-dichloro
2-fluoro
3-fluoro
3-trifluoromethyl
9.5
2.1
0.7
1.8
4.2
1.6
5.1
20.0
8.2
14.0
5.9
3.9
4.4
2.5
Reagents and conditions: (a) Polyphosphate, 45 – 1258C, 2 h; (b) K2CO3, DMF, 4nitrophenol, 758C, 6 h; (c) Fe/AcOH, 95% EtOH, reflux, 4 h; (d) substituted arylisocyanate, DMF, 308C, 2 – 3 h.
Scheme 1. Synthesis of the compounds 4a – 4f and 8a – 8g.
Table 2. The substituents and IC50 values against the human
breast cancer cell line of compounds 13a – 13g.
Compound
R
IC50 (lmol/L)
MDA-MB-231
13a
13b
13c
13e
13f
13g
Sorafenib
3-fluoro
2,6-difluoro
2-fluoro
4-fluoro
3-trifluoromethyl
3-chloro-4-fluoro
188.0
102.0
71.0
46.0
37.0
41.0
2.5
Results and discussion
Chemistry
The title compounds 4a – 4f were synthesized via a convenient four-step method outlined in Scheme 1. The commercially available 4-methylpyridin-2-amine in polyphosphate was cyclizated with ethyl 4-chloro-3-oxobutanoate
at 1258C to provide compound 1 as a grey solid [6], which
was then reacted with 4-nitrophenol in the presence of
K2CO3 in DMF to afford 2 [7]. Subsequently, the intermediate 2 underwent reduction with Fe in ethanol to produce
compound 3 [8]. The target compounds 4a – 4f were syn-
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2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Reagents and conditions: (a) Conc. sulfuric acid (98%), EtOH, reflux, 8 h; (b) DMF/
DMA, reflux, 4 – 5 h; (c) Pyridin-2-amine, AcOH, reflux, 6 h; (d) Fe/AcOH, EtOH,
reflux, 8 h; (e) substituted arylisocyanate, DMF, 308C, 2 – 4 h.
Scheme 2. Synthesis of the compounds 13a – 13g.
thesized from 3 with a variety of substituted arylisocyanates [9]. The new compounds 8a – 8g were prepared successfully according to the same method as described for
4a – 4f when the initial material was replaced with pyridin-2-amine (Scheme 1).
Compounds 13a – 13g were synthesized as shown in
Scheme 2. 2-(4-Nitrophenyl)acetic acid was refluxed in
ethanol with 0.15 equivalents of 98% sulfuric acid to
afford compound 9 in good yield [10], which was then
treated with DMF/DMA to give 10 [11]. The cyclization of
10 with pyridine-2-amine was carried out in acetic acid to
obtain the intermediate 11 [12, 13]. The intermediate 11
was subsequently reduced with powdered iron in ethanol
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Arch. Pharm. Chem. Life Sci. 2010, 343, 17 – 23
Reagents and conditions: (a) Bis(trichloromethyl)carbonate(BTC), ethyl acetate,
reflux, 12 h.
Scheme 3. Synthesis of the compounds 14a – 14g.
to give compound 12 [8], which was reacted with substituted arylisocyanate to afford the target compounds
13a – 13g [9]. In addition, the intermediates 14a – 14g
were prepared from substituted anilines with bis(trichloromethyl)carbonate (BTC) in ethyl acetate or 1,4dioxane at 808C [14] (Scheme 3).
Antiproliferative activity and discussion
The antiproliferative activity of the synthesized compounds 4a – 4f, 8a – 8g, and 13a – 13g against MDA-MB-231
(human breast cancer cell lines) were determined by MTT
assay. Sorafenib was used as positive control, and the
results expressed as IC50 values are summarized in Table
1 and Table 2.
As listed in Tables 1 and 2, most of the compounds
showed moderate activity, while compounds 4b, 4c, 4d,
and 4f, having IC50 values ranging from 0.7 to 2.1 lmol/L
with the R2 moieties 2-fluoro, 3-fluoro, 3-trifluomethyl,
and 4-fluoro, respectively, exhibited an activity more
potent than that of Sorafenib (IC50 = 2.5 lmol/L).
All the compounds in Table 1 were more potent than
those in Table 2, which suggests that the oxymethylene
group on the 4-position of the benzene ring was essential
for the activity. In our opinion, the oxygen atom is able
to act as a receptor of hydrogen bonds; hydrogen bonds
are formed with some amino acid residues of a variety of
enzymes.
On the other hand, compounds 4a – 4f and 8a – 8g were
chosen as examples to study the effect of different substituents on the antitumor activity. The activity of 4a – 4g
bearing a methyl group on the 8-position of the 4H-pyrido[1,2-a]pyrimidin-4-one moiety was decreased nearly 1
to 7-times compared to 8a – 8f substituted with a hydrogen atom (4b vs. 8e, 4c vs. 8f, and 4d vs. 8g). One can conclude that a methyl group is favorable in this region.
The substitution position of groups on the 1-anilino
moiety also exerts an important effect on the pharmacological activity. Generally, substituents on the 3position were optimal (4c, IC50 = 0.7 lmol/L and 8f, IC50
= 3.9 lmol/L), followed by the 4-position (4f, IC50 = 1.6
lmol/L and 8a, IC50 = 5.1 lmol/L), while the introduction of substituents both on the 2- and 6-positions will
cause a dramatically decreased activity (4a, IC50 = 9.5
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2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
4H-Pyrido[1,2-a]pyrimidin-4-one
19
lmol/L and 8b, IC50 = 20 lmol/L). In addition, the bulkiness of the substituent can also affect the activity, a
flouro atom was superior to a trifluoromethyl group
(4c vs. 4d and 8f vs. 8g).
In conclusion, a series of diaryl urea derivatives possessing a 4H-pyrido[1,2-a]pyrimidin-4-one group were
designed and synthesized in order to find potent antitumor compounds. All the compounds were screened for
their antiproliferative activity against the human breast
cancer cell line MDA-MB-231. The pharmacological
results indicated that most of the compounds showed
moderate activity, and the best one of them was compound 4c (IC50 = 0.7 lmol/L), with potency a 3.6-fold
higher potency than Sorafenib (IC50 = 2.5 lmol/L). The preliminary structure-activity relationship (SAR) showed
that the oxymethylene group on the 4-position of benzene ring was essential for the activity, and a methyl
group was favorable on the 8-position of 4H-pyrido[1,2-a]
pyrimidin-4-one. Furthermore, the position of the substitutent and its size also contributed important effects to
the activity. The SAR results of this study will provide useful information for the design of novel diaryl urea antitumor derivatives bearing fused heterocyclic groups.
Experimental
Chemistry
All melting points were obtained on a Bchi Melting Point B-540
apparatus (Bchi Labortechnik, Flawil, Switzerland) and are
uncorrected. Mass spectra (MS) were taken in ESI mode on Agilent 1100 LC-MS (Agilent, Palo Alto, CA, USA). Proton (1H) nuclear
magnetic resonance spectroscopy was performed using Bruker
ARX-300, 300 MHz spectrometers (Bruker Bioscience, Billerica,
MA, USA) with TMS as an internal standard. IR spectra (KBr disks)
were recorded with a Bruker IFS 55 instrument (Bruker). Elemental analysis was determined on a Carlo-Erba 1106 Elemental
analysis instrument (Carlo Erba, Milan, Italy). The oxygen analysis was carried out using the Unterzaucher principle by sample
pyrolysis in a helium stream at 16008C. Unless otherwise noted,
all solvents and reagents were commercially available and used
without further purification.
2-(Chloromethyl)-8-methyl-4H-pyrido[1,2-a]pyrimidin-4one 1
A mixture of 4-methylpyridin-2-amine (38 g, 0.35 mol) and ethyl
4-chloro-3-oxobutanoate (55 mL) in PPA (250 g) was stirred at
1258C for 2 h and then cooled to room temperature, poured into
water, and extracted with CHCl3. The extract was washed with
water and brine, dried (MgSO4), filtered and concentrated to provide pale solid (48 g, 64.7%). M.p.: 110 – 1128C; MS (ESI) m/z: 210.1
[M + H+].
8-Methyl-2-((4-nitrophenoxy)methyl)-4H-pyrido[1,2-a]
pyrimidin-4-one 2
A mixture of 1 (21 g, 0.1 mol), 4-nitrophenol (15.3 g, 0.11 mol)
and K2CO3 (41.4 g, 0.3 mol) in DMF was heated at 758C for 5 h. It
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P. Yao et al.
was then poured into water, filtered and washed with water,
and dried to give the title compound without further purification (25.1 g, 80%). M.p.: 156 – 1598C; MS (ESI) m/z: 312.1 [M + H+].
2-((4-Aminophenoxy)methyl)-8-methyl-4H-pyrido[1,2-a]
pyrimidin-4-one 3
A mixture of 3 (21.8 g, 0.07 mol), Fe (23.5 g, 0.42 mol), and
CH3COOH (1 mL) in 95% ethanol (300 mL) was refluxed for 4 h. The
mixture was filtered immediately and washed three times with
hot ethanol. The combined filtrates were evaporated in vacuo to
yield a light yellow solid, which was then crystallized from ethanol and water. Compound 3 was afforded as a light yellow solid
(15.9 g, 81%). M.p.: 172 – 1758C. MS (ESI) m/z: 282.1 [M + H+].
General procedure for preparation of compounds 4a – 4f
Compound 3 (0.5 g, 1.78 mmol) was dissolved in N,N-dimethylformamide (10 mL) at 308C. Then, substituted isocyanate (2.67
mmol) was added and the mixture was stirred for 3 h, poured
into water, filtered, and washed with water. The crude product
was dissolved in refluxed ethanol and chloroform, then, methane sulfonic acid (approx. 2 mmol) was added. The mixture was
refluxed for 0.5 h, filtered, and dried to afford yellow solid 4a –
4f, 8a – 8g, and 13a – 13g.
1-(2,6-Difluorophenyl)-3-(4-((8-methyl-4-oxo-4Hpyrido[1,2-a]pyrimidin-2-yl)methoxy)phenyl)urea 4a
Arch. Pharm. Chem. Life Sci. 2010, 343, 17 – 23
1-(4-((8-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)
methoxy)phenyl)-3-(3-(trifluoromethyl)phenyl)urea 4d
Prepared from 3 and 14f, 73% yield. M.p.: A3308C; IR (KBr) [cm – 1]:
3332.3, 3089.7, 2660.2, 1737.0, 1656.5, 1555.3, 1512.6, 1443.7,
1319.9, 1248.8, 1184.9, 1043.1; 1H-NMR (DMSO-d6) d [ppm]: 9.21
(s, 1H), 8.94 – 8.97 (d, J = 7.2 Hz, 1H), 8.90 (s, 1H), 8.02 (s, 1H), 7.65
(s, 1H), 7.57 – 7.59 (d, J = 8.4 Hz, 1H), 7.48 – 7.51 (d, J = 7.8 Hz, 1H),
7.41 – 7.44 (d, J = 8.7 Hz, 3H), 7.26 – 7.29 (d, J = 7.5 Hz, 1H), 7.00 –
7.03 (d, J = 8.7 Hz, 2H), 6.53 (s, 1H), 5.13 (s, 2H), 2.55 (s, 3H); MS m/
z: 469.1 [M + H+]. Anal. calcd. for C24H19F3N4O3: C, 61.54; H, 4.09; N,
11.96; O, 10.25. Found: C, 61.48; H, 4.07; N, 12.01; O, 10.19.
1-(3-Chloro-4-fluorophenyl)-3-(4-((8-methyl-4-oxo-4Hpyrido[1,2-a]pyrimidin-2-yl)methoxy)phenyl)urea 4e
Prepared from 3 and 14g, 74% yield. M.p.: A3308C; IR (KBr) [cm – 1]:
3279.3, 3084.8, 1736.9, 1652.6, 1603.4, 1555.0, 1500.4, 1384.4,
1313.4, 1207.5, 1168.6, 1047.4; 1H-NMR (DMSO-d6) d [ppm]: 9.02
(s, 1H), 8.95 – 8.97 (d, J = 7.2 Hz, 1H), 8.83 (s, 1H), 7.78 – 7.80 (d, J =
7.2 Hz, 1H), 7.65 (s, 1H), 7.39 – 7.45 (t, J1 = 9.6 Hz, J2 = 9 Hz, 3H),
7.30 – 7.33 (t, J1 = 6.6 Hz, J2 = 1.2 Hz, 2H), 7.00 – 7.03 (d, J = 9 Hz,
2H), 6.53 (s, 1H), 5.13 (s, 2H), 2.56 (s, 3H); MS m/z: 454.1 [M + H+].
Anal. calcd. for C23H18ClFN4O3: C, 61.00; H, 4.01; N, 12.37; O,
10.60. Found: C, 60.95; H, 4.07; N, 12.21; O, 10.57.
1-(4-Fluorophenyl)-3-(4-((8-methyl-4-oxo-4H-pyrido[1,2-a]
pyrimidin-2-yl)methoxy)phenyl)urea 4f
Prepared from 3 and 14b, 81% yield. M.p.: A3308C; IR (KBr) [cm – 1]:
3272.6, 1707.6, 1646.6, 1605.3, 1559.2, 1508.7, 1479.6, 1332.3,
1240.7, 1172.0, 1071.9, 1002.6; 1H-NMR (DMSO-d6) d [ppm]: 8.87
(s,1H),8.79 (s, 1H), 8.05 (s, 1H), 7.53 (s, 1H), 7.37 (s, 2H), 7.27 (s, 2H),
7.13 (s, 2H), 6.99 (s, 2H), 6.39 (s, 1H), 5.04 (s, 2H), 2.49 (s, 3H); MS
m/z: 437.1 [M + H+]. Anal. calcd. for C23H18F2N4O3: C, 63.30; H, 4.16;
N, 12.84; O, 11.00. Found: C, 63.24; H, 4.19; N, 12.91; O, 11.04.
Prepared from 3 and 14e, 77% yield. M.p.: A3308C; IR (KBr) [cm – 1]:
3282.9, 1735.8, 1641.6, 1560.1, 1507.2, 1336.6, 1213.9, 1166.7,
1039.2; 1H-NMR (DMSO-d6) d [ppm]: 8.96 – 8.98 (d, J = 7.2 Hz, 1H),
8.80 (s, 1H), 8.70 (s, 1H), 7.67 (s, 1H), 7.39 – 7.46 (m, 5H), 7.07 – 7.13
(t, J = 9 Hz, 2H), 6.99 – 7.02 (d, J = 9 Hz, 2H), 6.54 (s, 1H), 5.13 (s,
2H), 2.58 (s, 3H); MS m/z: 419.0 [M + H+]. Anal. calcd. for
C23H19FN4O3: C, 66.02; H, 4.58; N, 13.39; O, 11.47. Found: C, 65.98;
H, 4.60; N, 13.41; O, 11.44.
1-(2-Fluorophenyl)-3-(4-((8-methyl-4-oxo-4H-pyrido[1,2-a]
pyrimidin-2-yl)methoxy) phenyl)urea 4b
2-(Chloromethyl)-4H-pyrido[1,2-a]pyrimidin-4-one 5
Prepared from 3 and 14c, 80% yield. M.p.: A3308C; IR (KBr) [cm – 1]:
3277.0, 3062.4, 1736.1, 1661.4, 1601.4, 1545.7, 1510.1, 1454.6,
1335.5, 1218.5, 1185.8, 1164.3, 1034.2; 1H-NMR (DMSO-d6) d
[ppm]: 9.03 (s, 1H), 8.97 – 8.99 (d, J = 7.2 Hz, 1H), 8.54 (s, 1H), 8.08 –
8.13 (t, J = 8.1 Hz, 1H), 7.70 (s, 1H), 7.48 – 7.50 (d, J = 7.2 Hz, 1H),
7.4 – 7.43 (d, J = 8.7 Hz, 2H), 7.18 – 7.25 (dd, J1 = 11.4 Hz, J2 = 8.4 Hz,
1H), 7.09 – 7.14 (t, J = 7.5 Hz, 1 H), 6.97 – 7.04 (m, J1 = 8.7 Hz, J2 = 6.3
Hz, 3H), 6.57 (s, 1H), 5.16 (s, 2H), 2.57 (s, 3H); MS m/z: 419.1 [M +
H+]. Anal. calcd. for C23H19FN4O3: C, 66.02; H, 4.58; N, 13.39; O,
11.47. Found: C, 66.00; H, 4.55; N, 13.41; O, 11.44.
2-((4-Nitrophenoxy)methyl)-4H-pyrido[1,2-a]pyrimidin-4one 6
1-(3-Fluorophenyl)-3-(4-((8-methyl-4-oxo-4H-pyrido[1,2-a]
pyrimidin-2-yl)methoxy)phenyl)urea 4c
Prepared from 3 and 14a, 78% yield. M.p.: A3308C; IR (KBr) [cm – 1]:
3271.7, 3086.8, 1734.3, 1661.3, 1607.7, 1553.8, 1513.2, 1439.9,
1384.0, 1335.0, 1218.3, 1160.8, 1089.8, 1036.8; 1H-NMR (DMSOd6) d [ppm]: 9.03 (s, 1H), 8.96 – 8.98 (d, J = 7.2 Hz, 1H), 8.80 (s, 1H),
7.67 (s, 1H), 7.44 – 7.51 (m, 2H), 7.40 – 7.43 (d, J = 9 Hz, 2H), 7.24 –
7.32 (dd, J1 = 8.1 Hz, J2 = 15 Hz, 1H), 7.11 – 7.13 (d, J = 8.1 Hz, 1H),
7.0 – 7.03 (d, J = 9.3 Hz, 2H), 6.72 – 6.77 (m, 1H), 6.55 (s, 1H), 5.14 (s,
2H), 2.57 (s, 3H); MS m/z: 419.1 [M + H+]. Anal. calcd. for
C23H19FN4O3: C, 66.02; H, 4.58; N, 13.39; O, 11.47. Found: C, 65.98;
H, 4.57; N, 13.41; O, 11.51.
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2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Prepared in a similar procedure as described for 1, prepared
from pyridin-2-amine and ethyl 4-chloro-3-oxobutanoate, 67%
yield. M.p.: 98 – 1018C; MS (ESI) m/z: 196.1 [M + H+].
Prepared in a similar procedure as described for 2, prepared
from 5 and 4-nitrophenol, 81% yield. M.p.: 148 – 1518C; MS (ESI)
m/z: 298.1 [M + H+].
2-((4-Aminophenoxy)methyl)-4H-pyrido[1,2-a]pyrimidin4-one 7
Prepared in a similar procedure as described for 3, prepared
from 6, 80% yield. M.p.: 205 – 2088C; MS (ESI) m/z: 268.1 [M + H+].
General procedure for preparation of compounds 8a – 8g
Prepared in a similar procedure as described for 4a – 4f.
1-(4-Fluorophenyl)-3-(4-((4-oxo-4H-pyrido[1,2-a]
pyrimidin-2-yl)methoxy)phenyl)urea 8a
Prepared from 7 and 14e, 78% yield. M.p.: A3308C; IR (KBr) [cm – 1]:
3317.5, 3077.9, 1725.6, 1658.0, 1611.0, 1563.3, 1503.7, 1446.4,
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Arch. Pharm. Chem. Life Sci. 2010, 343, 17 – 23
4H-Pyrido[1,2-a]pyrimidin-4-one
1339.1, 1313.7, 1232.1, 1205.4, 1162.3, 1038.0; 1H-NMR (DMSO-d6)
d [ppm]: 9.03 – 9.05 (d, J = 6.9 Hz, 1H), 8.81 (s, 1H), 8.71 (s, 1H),
8.17 – 8.22 (t, J = 7.5 Hz, 1H), 7.82 – 7.85 (d, J = 8.7 Hz, 1H), 7.50 –
7.55 (t, J = 6.9 Hz, 1H), 7.44 – 7.48 (dd, J1 = 9 Hz, J2 = 5.1 Hz, 2H),
7.39 – 7.42 (d, J = 8.7 Hz, 2H), 7.06 – 7.12 (t, J = 9 Hz, 2H), 6.99 – 7.02
(d, J = 8.7 Hz, 2H), 6.57 (s, 1H), 5.13 (s, 2H); MS m/z: 404.1 [M + H+].
Anal. calcd. for C22H17FN4O3: C, 65.34; H, 4.24; N, 13.85; O, 11.87.
Found: C, 65.38; H, 4.20; N, 13.91; O, 11.84.
1-(2,6-Difluorophenyl)-3-(4-((4-oxo-4H-pyrido[1,2-a]
pyrimidin-2-yl)methoxy)phenyl)urea 8b
Prepared from 7 and 14b, 85% yield. M.p.: A3308C; IR (KBr) [cm – 1]:
3283.2, 1712.8, 1698.2, 1645.0, 1619.7, 1604.1, 1560.9, 1510.1,
1467.0, 1383.4, 1241.6, 1074.3, 1003.1; 1H-NMR (DMSO-d6) d
[ppm]: 8.94 – 8.97 (d, J = 6.9 Hz,1H), 8.79 (s, 1H), 8.04 (s, 1H) 7.97 –
8.02 (t, J1 = 8.4 Hz, J2 = 7.2 Hz, 1H) 7.68 – 7.71 (d, J = 9.0 Hz, 1H),
7.35 – 7.38 (t, J1 = 3 Hz, J2 = 5.7 Hz, 3H), 7.26 – 7.32 (t, J = 7.8 Hz, 1H),
7.11 – 7.16 (t, J = 8.1 Hz, 2H) , 6.97 – 7.0 (d, J = 9.0 Hz, 2H) , 6.45 (s,
1H), 5.01 (s, 2H). MS m/z: 415.1 [M + H+]. Anal. calcd. for
C22H16F2N4O3: C, 62.56; H, 3.82; N, 13.26; O, 11.36. Found: C,
62.58; H, 3.80; N, 13.21; O, 11.40.
1-(3-Chloro-4-fluorophenyl)-3-(4-((4-oxo-4H-pyrido[1,2-a]
pyrimidin-2-yl)methoxy)phenyl)urea 8c
Prepared from 7 and 14g, 74% yield. M.p.: A3308C; IR (KBr) [cm – 1]:
3363.2, 1747.1, 1661.4, 1601.7, 1552.8, 1498.2, 1384.3, 1297.9,
1206.4, 1042.5; 1H-NMR (DMSO-d6) d [ppm]: 9.03 – 9.05 (d, J = 3.9
Hz, 2H), 8.85 (s, 1H), 8.17 – 8.23 (m, 1H), 7.78 – 7.86 (m, 2H), 7.51 –
7.55 (t, J = 6.9 Hz, 1H), 7.39 – 7.42 (d, J = 9 Hz, 2H), 7.30 – 7.33 (d, J =
7.8 Hz, 2H), 7.0 – 7.03 (d, J = 9 Hz, 2H), 6.57 (s, 1H), 5.14 (s, 2H); MS
m/z: 440.0 [M + H+]. Anal. calcd. for C22H16ClFN4O3: C, 60.21; H,
3.67; N, 12.77; O, 10.94. Found: C, 60.18; H, 3.70; N, 12.81; O,
10.91.
–1
Prepared from 7 and 14d, 82% yield. M.p.: A3308C; IR (KBr) [cm ]:
3368.0, 3278.6, 3084.7, 1739.5, 1663.8, 1589.6, 1545.7, 1511.0,
1451.1, 1242.5, 1197.3, 1074.1, 1047.9; 1H-NMR (DMSO-d6) d
[ppm]: 9.23 (s, 1H), 9.02 – 9.04 (d, J = 6.6 Hz, 1H), 8.97 (s, 1H), 8.14 –
8.17 (t, J1 = 7.8 Hz, J2 = 7.2 Hz, 1H), 7.80 – 7.83 (d, J = 8.7 Hz, 1H),
7.50 – 7.53 (d, J = 9.3 Hz, 3H), 7.39 – 7.42 (d, J = 8.7 Hz, 2H), 7.12 (s,
1H), 7.0 – 7.03 (d, J = 8.7 Hz, 2H), 6.56 (s, 1H), 5.13 (s, 2H); MS m/z:
456.1 [M + H+]. Anal. calcd. for C22H16Cl2N4O3: C, 58.04; H, 3.54; N,
12.31; O, 10.54. Found: C, 58.08; H, 3.50; N, 12.33; O, 10.51.
1-(2-Fluorophenyl)-3-(4-((4-oxo-4H-pyrido[1,2-a]
pyrimidin-2-yl)methoxy)phenyl)urea 8e
Prepared from 7 and 14c, 79% yield. M.p.: A3308C; IR (KBr) [cm – 1]:
3340.7, 1738.3, 1663.9, 1620.5, 1601.6, 1536.6, 1511.7, 1454.5,
1320.9, 1214.7, 1171.3, 1079.7, 1039.9; 1H-NMR (DMSO-d6) d
[ppm]: 8.96 – 9.01 (d, J = 6.3 Hz, 2H), 8.51 (s, 1H), 8.10 – 8.17 (d, J =
7.5 Hz, 2H), 7.79 – 7.81 (d, J = 8.7 Hz, 1H), 7.47 – 7.51 (t, J = 6.9 Hz,
1H), 7.38 – 7.41 (d, J = 8.7 Hz, 2H), 7.18 – 7.25 (t, J1 = 8.7 Hz, J2 = 7.8
Hz, 1H), 7.09 – 7.14 (t, J1 = 7.8 Hz, J2 = 7.2 Hz, 1H), 6.97 – 7.03 (t, J =
8.4 Hz, 3H), 6.54 (s, 1H), 5.12 (s, 2H); MS m/z: 405.1 [M + H+]. Anal.
calcd. for C22H17FN4O3: C, 65.34; H, 4.24; N, 13.85; O, 11.87.
Found: C, 65.38; H, 4.20; N, 13.87; O, 11.84.
2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1-(3-Fluorophenyl)-3-(4-((4-oxo-4H-pyrido[1,2-a]
pyrimidin-2-yl)methoxy)phenyl)urea 8f
Prepared from 7 and 14a, 76% yield. M.p.: A3308C; IR (KBr) [cm – 1]:
3274.0, 3100.5, 1736.7, 1661.4, 1554.0, 1513.4, 1440.8, 1336.2,
1219.2, 1160.0, 1039.6; 1H-NMR (DMSO-d6) d [ppm]: 9.02 – 9.04 (d,
J = 8.7 Hz, 2H), 8.80 (s, 1H), 8.14 – 8.19 (t, J = 7.5 Hz, 1H), 7.81 – 7.83
(d, J = 8.7 Hz, 1H), 7.47 – 7.53 (m, 2H), 7.39 – 7.42 (d, J = 9 Hz, 2H),
7.23 – 7.31 (dd, J1 = 15 Hz, J2 = 7.8 Hz, 1H), 7.10 – 7.13 (d, J = 8.1 Hz,
1H), 7.00 – 7.03 (d, J = 9 Hz, 2H), 6.72 – 6.77 (m, 1H), 6.56 (s, 1H),
5.13 (s, 2H); MS m/z: 405.1 [M + H+]. Anal. calcd. for C22H17FN4O3: C,
65.34; H, 4.24; N, 13.85; O, 11.87. Found: C, 65.36; H, 4.21; N,
13.88; O, 11.84.
1-(4-((4-Oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)methoxy)
phenyl)-3-(3-(trifluoromethyl)phenyl)urea 8g
Prepared from 7 and 14f, 72% yield. M.p.: A3308C; IR (KBr) [cm – 1]:
3308.9, 3100.5, 1749.8, 1675.9, 1566.9, 1513.6, 1447.0, 1383.8,
1337.9, 1223.1, 1163.8, 1111.0, 1046.2; 1H-NMR (DMSO-d6) d
[ppm]: 9.06 (s, 1H), 8.98 – 9.0 (d, J = 6.9 Hz, 1H), 8.73 (s, 1H), 8.04 –
8.09 (t, J = 7.5 Hz, 1H), 8.01 (s, 1H), 7.73 – 7.76 (d, J = 8.7 Hz, 1H),
7.55 – 7.58 (d, J = 8.1 Hz, 1H), 7.46 – 7.52 (t, J = 7.5 Hz, 1H), 7.38 –
7.41 (d, J = 8.7 Hz, 2H), 7.27 – 7.29 (d, J = 7.2 Hz, 1H), 7.0 – 7.03 (d,
J = 8.7 Hz, 2H), 6.50 (s, 1H), 5.10 (s, 2H); MS: 455.0 [M + H+]. Anal.
calcd. for C23H17F3N4O3: C, 60.79; H, 3.77; N, 12.33; O, 10.56.
Found: C, 60.78; H, 3.79; N, 12.37; O, 10.54.
Ethyl 2-(4-nitrophenyl)acetate 9
To a solution of 2-(4-nitrophenyl)acetic acid (50 g, 0.276 mol) and
ethanol (250 mL), conc. H2SO4 (12 mL, 0.22 mol) was added dropwise in 30 min. The mixture was refluxed for 8 h, after that, it
was cooled to room temperature. Ethanol was evaporated in
vacuo, then poured into water, filtered, washed with water, and
dried to afford a white solid (53.4 g, 92.5%). M.p.: 64 – 668C; MS
m/z: 210.0 [M + H+].
Ethyl 3-(dimethylamino)-2-(4-nitrophenyl)acrylate 10
1-(3,5-Dichlorophenyl)-3-(4-((4-oxo-4H-pyrido[1,2-a]
pyrimidin-2-yl)methoxy)phenyl)urea 8d
i
21
Compound 9 (50 g, 0.239 mol) was refluxed in DMF/DMA (48 mL)
and toluene (350 mL) for 4 – 5 h, the solvent was evaporated in
vacuo. The residue was extracted twice with 125 mL CH2Cl2. The
combined extracts were washed with brine and water, dried
(MgSO4), filtered, and evaporated in vacuum, followed by column chromatographic purification to afford the desired compound 10 (43.6 g, 69.0%). M.p.: 139 – 1428C; MS (ESI) m/z: 265.1 [M
+ H+].
3-(4-Nitrophenyl)-4H-pyrido[1,2-a]pyrimidin-4-one 11
A solution of 10 (25 g, 94.7 mmol) and pyridin-2-amine (27.8 g,
284 mmol) in glacial acetic acid (150 mL) was refluxed for 6 h,
then cooled to 08C and filtered to give a light yellow solid which
was used without further purification (17.2 g, 68.0%). M.p.: 164 –
1678C; MS (ESI) m/z: 268.1 [M + H+].
3-(4-Aminophenyl)-4H-pyrido[1,2-a]pyrimidin-4-one 12
Prepared in a similar procedure as described for 3, prepared from
11, 58.5% yield. M.p.: 226 – 2298C; MS (ESI) m/z: 238.1 [M + H+].
General procedure for preparation of compounds 13a –
13g
Prepared in a similar procedure as described for 4a – 4f.
www.archpharm.com
22
P. Yao et al.
1-(3-Fluorophenyl)-3-(4-(4-oxo-4H-pyrido[1,2-a]
pyrimidin-3-yl)phenyl)urea 13a
Prepared from 12 and 14a, 73% yield. M.p.: A3308C; IR (KBr)
[cm–1]: 3273.7, 3089.7, 1725.0, 1601.0, 1534.7, 1490.5, 1384.1,
1324.3, 1220.8, 1182.0, 1043.4; 1H-NMR (DMSO-d6) d [ppm]: 9.18 –
9.26 (m, 3H), 8.75 (s, 1H), 8.30 – 8.35 (t, J = 7.5 Hz, 1H), 7.93 – 7.96
(d, J = 8.7 Hz, 1H), 7.76 – 7.79 (d, J = 8.7 Hz, 2H), 7.66 – 7.71 (t, J = 6.9
Hz, 1H), 7.60 – 7.63 (d, J = 9 Hz, 2H), 7.50 – 7.54 (t, J1 = 12 Hz, J2 = 1.8
Hz, 1H), 7.27 – 7.35 (m, 1H), 7.16 – 7.18 (d, J = 8.1 Hz, 1H), 6.75 –
6.82 (m, 1H); MS m/z: 375.1 [M + H+]. Anal. calcd. for C21H15FN4O2:
C, 67.37; H, 4.04; N, 14.97; O, 8.55. Found: C, 67.38; H, 4.08; N,
14.92; O, 8.54.
1-(2,6-Difluorophenyl)-3-(4-(4-oxo-4H-pyrido[1,2-a]
pyrimidin-3-yl)phenyl)urea 13b
Prepared from 12 and 14b, 68% yield. M.p.: A3308C; IR (KBr)
[cm–1]: 3261.1, 1725.0, 1649.7, 1599.1, 1535.0, 1466.6, 1384.4,
1296.6, 1191.8, 1042.0; 1H-NMR (DMSO-d6) d [ppm]: 9.28 (s, 1H),
9.23 – 9.26 (d, J = 6.9 Hz, 1H), 8.74 (s, 1H), 8.35 (brs, 2H), 7.94 – 7.97
(d, J = 9 Hz, 1H), 7.75 – 7.78 (d, J = 8.7 Hz, 2H), 7.67 – 7.72 (t, J = 6.9
Hz, 1H), 7.58 – 7.61 (d, J = 8.7 Hz, 2H), 7.27 – 7.37 (m, 1H), 7.12 –
7.20 (m, 2H); MS m/z: 394.1 [M + H+]. Anal. calcd. for C21H14F2N4O2:
C, 64.28; H, 3.60; N, 14.28; O, 8.16. Found: C, 64.26; H, 3.61; N,
14.24; O, 8.14.
1-(2-Fluorophenyl)-3-(4-(4-oxo-4H-pyrido[1,2-a]
pyrimidin-3-yl)phenyl)urea 13c
Prepared from 12 and 14c, 76% yield. M.p.: >3308C; IR (KBr)
[cm–1]: 3272.2, 1729.9, 1651.5, 1596.4, 1536.4, 1384.1, 1302.7,
1255.5, 1190.3, 1039.5; 1H-NMR (DMSO-d6) d [ppm]: 9.30 (s, 1H),
9.21 – 9.23 (d, J = 6.9 Hz, 1H), 8.73 (s, 1H), 8.65 – 8.66 (d, J = 2.1 Hz,
1H), 8.23 – 8.28 (t, J1 = 7.2 Hz, J2 = 8.4 Hz, 1H), 8.13 – 8.18 (dd, J1 =
8.1 Hz, J2 = 6.9 Hz, 1H), 7.89 – 7.92 (d, J = 8.7 Hz, 1H), 7.78 – 7.87 (d,
J = 8.7 Hz, 2H), 7.63 – 7.66 (d, J = 6.9 Hz, 1H), 7.57 – 7.60 (d, J = 8.7
Hz, 2H), 7.22 – 7.28 (m, 1H), 7.13 – 7.18 (t, J = 7.5 Hz, 1H), 6.99 –
7.05 (m, 1H); MS m/z: 375.0 [M + H+]. Anal. calcd. for C21H15FN4O2:
C, 67.37; H, 4.04; N, 14.97; O, 8.55. Found: C, 67.34; H, 4.01; N,
14.99; O, 8.60.
1-(3,5-Dichlorophenyl)-3-(4-(4-oxo-4H-pyrido[1,2-a]
pyrimidin-3-yl)phenyl)urea 13d
Prepared from 12 and 14d, 71% yield. M.p.: >3308C; IR (KBr)
[cm–1]: 3330.5, 3082.9, 1722.7, 1646.5, 1586.5, 1531.5, 1448.6,
1383.8, 1297.4, 1194.1; 1H-NMR (DMSO-d6) d [ppm]: 9.36 (s, 1H),
9.30 (s, 1H), 9.23 – 9.26 (d, J = 7.2 Hz, 1H), 8.75 (s, 1H), 8.29 – 8.34
(m, 1H), 7.91 – 7.94 (d, J = 9 Hz, 1H), 7.77 – 7.80 (d, J = 8.7 Hz, 2H),
7.65 – 7.70 (t, J = 6.9 Hz, 1H), 7.59 – 7.62 (d, J = 8.7 Hz, 2H), 7.57 –
7.58(d, J = 1.5 Hz, 2H), 7.17 (s, 1H); MS m/z: 426.0 [M + H+]. Anal.
calcd. for C21H14Cl2N4O2: C, 59.31; H, 3.32; N, 13.17; O, 7.52.
Found: C, 59.28; H, 3.31; N, 13.21; O, 7.54.
1-(4-Fluorophenyl)-3-(4-(4-oxo-4H-pyrido[1,2-a]
pyrimidin-3-yl)phenyl)urea 13e
Prepared from 12 and 14e, 70% yield. M.p.: A3308C; IR (KBr)
[cm–1]: 3326.3, 3084.4, 1714.8, 1647.5, 1603.4, 1531.3, 1505.4,
1333.8, 1300.0, 1208.1, 1157.6, 1044.3; 1H-NMR (DMSO-d6) d
[ppm]: 9.23 – 9.26 (d, J = 6.9 Hz, 1H), 9.03 (s, 1H), 8.93 (s, 1H), 8.74
(s, 1H), 8.28 – 8.34 (m, 1H), 7.92 – 7.94 (d, J = 8.7 Hz, 1H), 7.75 – 7.78
(d, J = 8.7 Hz, 2H), 7.66 – 7.70 (t, J = 6.9 Hz, 1H), 7.58 – 7.61 (d, J = 9
i
2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Arch. Pharm. Chem. Life Sci. 2010, 343, 17 – 23
Hz, 2H), 7.47 – 7.52 (m, 2H), 7.10 – 7.16 (t, J = 8.7 Hz, 2H); MS m/z:
375.1 [M + H+]. Anal. calcd. for C21H15FN4O2: C, 67.37; H, 4.04; N,
14.97; O, 8.55. Found: C, 67.35; H, 4.02; N, 14.95; O, 8.58.
1-(4-(4-Oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)phenyl)-3(3-(trifluoromethyl)phenyl)urea 13f
Prepared from 12 and 14f, 65% yield. M.p.: A3308C; IR (KBr)
[cm–1]: 3317.8, 3091.7, 1725.8, 1662.1, 1595.9, 1536.8, 1447.1,
1384.1, 1319.5, 1181.5,1119.2, 1047.2; 1H-NMR (DMSO-d6) d
[ppm]: 9.17 – 9.19 (d, J = 6 Hz, 2H), 9.05 (s, 1H), 8.70 (s, 1H), 8.11 –
8.17 (t, J1 = 8.4 Hz, J2 = 7.2 Hz, 1H), 8.05 (s, 1H), 7.85 (s, 2H), 7.79 –
7.82 (d, J = 9 Hz, 2H), 7.58 – 7.61 (d, J = 8.4 Hz, 2H), 7.50 – 7.55 (t, J1
= 7.5 Hz, J2 = 8.7 Hz, 2H), 7.31 – 7.33 (d, J = 7.2 Hz, 1H); MS m/z:
425.1 [M + H+]. Anal. calcd. for C22H15F3N4O2: C, 62.26; H, 3.56; N,
13.20; O, 7.54. Found: C, 62.25; H, 3.52; N, 13.25; O, 7.57.
1-(3-Chloro-4-fluorophenyl)-3-(4-(4-oxo-4H-pyrido[1,2-a]
pyrimidin-3-yl)phenyl) urea 13g
Prepared from 12 and 14g, 81% yield. M.p.: A3308C; IR (KBr)
[cm–1]: 3426.9, 1715.2, 1647.6, 1597.6, 1535.3, 1497.9, 1384.4,
1297.4, 1194.3, 1042.8; 1H-NMR (DMSO-d6) d [ppm]: 9.19 – 9.21 (d, J
= 6.6 Hz, 1H), 9.08 (s, 2H), 8.70 (s, 1H), 8.17 – 8.19 (d, J = 7.2 Hz, 1H),
7.82 – 7.88 (t, J1 = 9.9 Hz, J2 = 8.4 Hz, 2H), 7.77 – 7.80 (d, J = 8.4 Hz,
2H), 7.57 – 7.60 (d, J = 8.7 Hz, 3H), 7.33 – 7.36 (t, J = 6.6 Hz, 2H); MS
m/z: 409.1 [M + H+]. Anal. calcd. for C21H14ClFN4O2: C, 61.70; H,
3.45; N, 13.70; O, 7.83. Found: C, 61.68; H, 3.48; N, 13.69; O, 7.86.
Evaluation of the biological activity
The antiproliferative activities of compounds 4a – 4f, 8a – 8g, and
13a – 13g were evaluated with MDA-MB-231 (a human breast cancer cell line) by the MTT method in vitro, with Sorafenib as positive control. The cancer cells (of MDA-MB-231) were cultured in
minimum essential medium (MEM) supplemented with 10%
fetal bovine serum (FBS).
Approximately 46103 cells, suspended in MEM medium, were
plated onto each well of a 96-well plate and incubated in 5% CO2
at 378C for 24 h. The test compounds were added to the culture
medium at the indicated final concentrations and the cell cultures were continued for 72 h. Fresh MTT was added to each well
at a final concentration of 5 lg/mL and incubated with cells at
378C for 4 h. The formazan crystals were dissolved in 100 lL
DMSO per each well, and the absorbency at 492 nm (for the
absorbance of MTT formazan) and 630 nm (for the reference
wavelength) was measured with the ELISA reader. All of the compounds were tested twice in each of the cell lines. The results
expressed as IC50 (inhibitory concentration of 50%) were the averages of two determinations and were calculated by using the
Bacus Laboratories Incorporated Slide Scanner (Bliss) software.
The authors have declared no conflict of interest.
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4H-Pyrido[1,2-a]pyrimidin-4-one
23
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