Synthesis, crystal structure, and biological activity of diethyl-2-[5-(4-fluorophenyl)-7-(trifluoromethyl)pyrazolo[1,5-a] pyrimidine-3-carboxamido]pentanedioate

Article abstract of DOI:10.3184/174751915X14206280982837

The title compound, diethyl-2-[5-(4-fluorophenyl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine-3-carboxamido]pentanedioate (C₂₃H₂₂F₄N₄O₅, Mr = 510.45) was synthesised and structurally characterised by

Full text of DOI:10.3184/174751915X14206280982837

4 RESEARCH PAPER
VOL. 39 JANUARY, 4–6
JOURNAL OF CHEMICAL RESEARCH 2015
Synthesis, crystal structure, and biological activity of
diethyl-2-[5-(4-fluorophenyl)-7-(trifluoromethyl)pyrazolo[1,5-a]
pyrimidine-3-carboxamido]pentanedioate
Liu Jua, Ge Hong-Guangb and Lu Jiu-Fub*
aCollege of Pharmacy, Liaoning University, Shenyang, 110036,Liaoning, P.R. China
bShaanxi University of Technology, Chemical Engineering College, Hanzhong,723001, P.R. China
The title compound, diethyl-2-[5-(4-fluorophenyl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine-3-carboxamido]pentanedioate
1
(C23H22F4N4O5, Mr=510.45) was synthesised and structurally characterised by element analysis, IR, H NMR and single
crystal X-ray diffraction. The crystal belongs to the monoclinic system, space group C121 with a=27.001(3) Å, b=7.161(1) Å,
c=12.0900 Å, α=90.00 °, β=92.379 (7) °, γ=90.00 °. In the crystal structure, the dihedral angles between the benzene ring and
pyrazolopyrimidine are 53.618(162)°. Through C–H…F and C–H…O weak hydrogen bonds among molecules, the whole molecule
is stacked into a three-dimensional structure. In addition, the compound possesses a moderate antituberculosis activity.
Keywords: synthesis, X‑ray diffraction, biological activity
spectra were recorded on Bruker AV‑600 in DMSO‑d6 using TMS
as an internal standard (chemical shifts in ppm). Crystal data was
obtained on a Bruker P4 X‑diffractometer. All solvents and reagents
were obtained from commercial sources and were used without
purification. 4‑Ethoxycarbonyl‑5‑amino‑pyrazole 1 was purchased
from Aldrich. 4,4,4‑Trifluoro‑1‑4‑fluorophenyl)butane‑1,3‑dione was
obtained as reported.9
There is a critical need for the development of new drugs to
treat cancer due to the recent and rapid appearance of numerous
single, multiple, and extensively drug‑resistant forms of
the disease. L‑Glutamic acid plays an important role in the
biosynthesis of purine and pyrimidine bases of DNA and RNA.1–4
It is metabolised to L‑glutamine by L‑glutamine synthetase and
this metabolic process is essential for normal maintenance of
cells. The synthesis of L‑glutamine is hindered in neoplastic
cells due to lower reactivity of L‑glutamine synthetase. Thus
antagonists of this enzyme can interfere with the metabolic role
of L‑glutamine and act as anticancer agents,5and also used as
antifungals and antituberculostatics drugs.6–8
Synthesis of 5-(4-fluorophenyl)-7-(trifluoromethyl)pyrazolo[1,5-a]
pyrimidine-3-carboxylic acid
A solution of ethyl 5‑amino‑1H‑pyrazole‑4‑carboxylate (15.5 g, 0.1 mol)
and 4,4‑trifluoro‑1‑(4‑fluorophenyl)‑butane‑1,3‑dione (23.4 g, 0.1 mol)
in acetic acid (50 mL) was heated at reflux for 6 h. After cooling to
room temperature, the formed precipitate was filtered off, washed
with water, and dried. The resulting ethyl carboxylate was added to
a mixture of NaOH (5.6 g, 0.14 mol) in EtOH/water (1:3) (120 mL),
and the reaction mixture was kept at 65 °C for 5 h. The mixture was
cooled to room temperature and acidified with concentrated HCl
until pH 1 was reached. The formed precipitate was filtered off,
washed with water, and recrystallised from MeCN to give 20.6 g pure
5‑(4‑fluorophenyl)‑7‑(trifluoromethyl)pyrazolo[1,5‑a]pyrimidine‑3‑
carboxylic acid in 63.4% yield m.p. 252–253 °C; 1H NMR (500 MHz):
δ 2.35–2.55 (s, 1H, COOH), 7.25 (d, J=8.2 Hz, 2H, ArH), 8.2 (s, 1H,
ArH), 8.5 (m, 2H, ArH), 8.52 (s, 1H, ArH); 13C NMR (125 MHz): δ
166.23 (COOH), 120.16 (CF3), 163.41, 2×116.12, 2×130.27, 133.73
(4‑fluorophenyl), 104.21, 106.33, 132.10, 145.66, 148.31, 155.14
(pyrazolo[1,5‑a]pyrimidine); LC/MS m/z 326 (M+1).
Therefore, we now report the design and synthesis of a new
glutamic acid compound, diethyl 2‑[5‑(4‑fluorophenyl)‑7‑
(trifluoromethyl)pyrazolo[1,5‑a]pyrimidine‑3‑carboxamido]
pentanedioate, with its synthetic route depicted in Scheme 1.
In order to confirm its structure and investigate its
stereoconfiguration, a single crystal of the title compound
was obtained from ethyl acetate and the molecular structure
1
was determined by element analysis, IR, H NMR and X‑ray
diffraction.
Experimental
Melting points (°C) were measured with Koeffler melting point
apparatus and are uncorrected. TLC was performed on aluminium
sheets precoated with silica gel (Merck, Kieselgel 60 F‑254). 1H NMR
CF3
N
O
O
N
N
NH
NH2
AcOH, reflux
+
CF3
N
EtO
OEt
F
O
F
O
2
3
1
CF3
CF3
N
N
1)NaOH/H2O/EtOH, 65°C
2)
N
N
COOEt
N
N
HCl
OH
N
COOEt
F
O
F
O
H
4
5
Scheme 1 Synthetic route of diethyl-2-[5-(4-fluorophenyl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine-3-carboxamido]pentanedioate.
* Correspondent. E‑mail: jiufulu@163.com

JOURNAL OF CHEMICAL RESEARCH 2015 5
Fig. 1 The structure of C23H22F4N O5 with all non-H atom-labelling scheme
Fig. 2 A packing diagram of C23H22F4N4O5.
and ellipsoids drawn at the 30% p4robability level.
temperature for 0.5 h, a solution of L‑glutamic acid diethyl ester
hydrochloride (1.77 g, 7.38 mmol) in 30 mL of dried DMF was added.
After the mixture was stirred for 12 h at room temperature, it was
diluted with water (200 mL), extracted by ethyl acetate three times
(100 mL×3), and dried over anhydrous sodium sulfate filtered, and
concentrated under reduced pressure to give crude product, which was
purified by column chromatography to give 2.1 g of the title compound
Synthesis of diethyl-2-[5-(4-fluorophenyl)-7-(trifluoromethyl)pyrazolo
[1,5-a]pyrimidine-3-carboxamido]pentanedioate
Under a nitrogen atmosphere, a mixture of 5‑(4‑fluorophenyl)‑7‑
(trifluoromethyl)pyrazolo[1,5‑a]pyrimidine‑3‑carboxylic acid (2.0 g,
6.15 mmol), EDCI (1.42 g, 7.38 mmol), HOBt (0.83 g, 6.15 mmol),
DMAP (0.75 g, 6.15 mmol) in 30 mL dried DMF was stirred at room
1
as yellow–green powder, yield: 66.90%. m.p. 154–157 °C; H NMR
Table 1 Crystal data for diethyl-2-[5-(4-fluorophenyl)-7-(trifluoromethyl)
pyrazolo[1,5-a]pyrimidine-3-carboxamido]pentanedioate
(600 MHz, DMSO‑d6): δ 8.73 (s,1H, ArH), 8.54 (m, 2H, ArH), 8.51
(s,1H, –NH), 8.37 (s, 1H,ArH), 7.44 (t, J=9.0 Hz, 2H, PhH), 4.71 (m,
1H, CH), 4.20 (q, J=7.2 Hz, 2H, CH2), 3.98 (m,2H, CH2), 2.46 (m, 2H,
CH2), 2.23 (m, 1H, CH2), 2.09 (m, 1H, CH2), 1.22 (t, J=7.2 Hz, 3H,
CH3–H), 1.08 (t, J=7.2 Hz, 3H, CH3–H). IR (KBr) ν: 3325, 3114, 3049,
2996, 2952, 1736, 1655, 1634, 1600, 1568, 1550, 1517, 1485, 1473, 1408,
1383, 1326, 1290, 1258, 1167, 1076, 1033, 845, 764, 632. Anal. calcd for
C23H22F4N4O5: C, 54.12; H, 4.34; N, 10.98; found: C, 54.10; H, 4.35; N,
11.03%.
Crystal size
Formula
fw
0.14 mm×0.06 mm×0.12 mm
C23H22F4N4O5
510.45
T/K
293(2)
Crystal system
Space group
Monoclinic
C121
a/Å
b/Å
c/Å
α/°
27.001(3)
7.161(1)
12.0900
90.00
Crystal data structure determination
A
colourless crystal (C23H22F4N4O5) with dimensions of
0.14 mm×0.06 mm×0.12 mm was selected for data collection
which was performed on a Bruker P4 diffractometer equipped
β/°
γ/°
V/Å3
92.379 (7)
90.00
2335.7 (5)
4
1.452
1056
with
by using an
a
graphite‑monochromatic CuKα radiation (λ=1.54187 Å)
scan mode at 298(2) K. total of 10466
ω
A
reflections were collected in the range of 3.3<θ<72.0° (index
ranges: –33<h<32, –7<k<8, –14<l<14) and 3735 were independent
(Rint =0.064), of which 3466 observed reflections with I>2σ (I) were
used in the structure determination and refinements. The structure
was solved by direct methods with SHELXS‑97 program10 and
expanded by the Fourier technique. The non‑hydrogen atoms were
refined anisotropically. The hydrogen atoms bound to carbon were
determined with theoretical calculations and those attached to nitrogen
and oxygen were determined with successive difference Fourier
syntheses. The structure was refined by full‑matrix Least‑squares
techniques on F2 with SHELXL‑9710. The final refinement gave the
Z
Dc/g.cm–3
F(000)
GOF on F2
Reflection/unique
R1,wR2 [I>2 (I)]
R1,wR2 (all data)
1.140
13125/3735
0.0531, 0.1072
0.0552, 0.1089
R=∑(||Fo |–|Fc ||)/∑|Fo |
2
wR=[∑w(Fo –|Fc2)2 /∑w(Fo)2]1/2
2
final R=0.0552 and wR=0.1072 (w=1/ [σ2 (Fo2)+(0.0696P) ] where
Table 2 Hydrogen bond lengths (Å) and bond angles (°)
P=(Fo2 +2Fc2)/3). S=1.14, (Δ /σ) max=0.001, (Δ ρ) max=0.49 and (Δ
ρ) min=–0.55 e Å–3. All calculations were performed using the Crystal
Structure crystallographic software package except for the refinement.
Crystallographic data and experimental details of structural analyses
for compound are summarised in Table 1. The hydrogen bond data of
title compound is listed in Table 2. CCDC 882212.
D–H…A
d(D–H)
d(H…A)
d(D…A)
∠DHA
C(22)–H(22B)…F(4)
C(10)–H(10)O(1)
C(6)–H(6)…O(3)
N(4)–H(1)…N(1)
0.9900
0.9500
0.9500
0.83(3)
2.489
2.503
2.644
2.25(3)
3.261
3.410
3.459
134.53
159.69
144.12
2.942(3) 140(2)

6 JOURNAL OF CHEMICAL RESEARCH 2015
Table 3 In vitro anticancer activity testa of diethyl-2-[5-(4-fluorophenyl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine-3-carboxamido]pentanedioate on
DU-145, PC-3 and COLO-205 cell lines
% Inhibition at 80 µg mL–1 in
Compound
DU-145
PC-3
COLO-205
Dethyl-2-[5-(4-fluorophenyl)-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine-3-carboxamido]pentanedioate
Adriamycin
84.5
86.5
70
98.2
42.5
97.4
aTest MTT colourimetric assay in DU-145, PC-3(prostate) and COLO-205(colon) human cancer cell lines.
In vitro anticancer activity test of the title compound on DU-145, PC-3
Electronic Supplementary Information
and COLO-205 cell lines
Figures S1 and S2 have been deposited in the ESI available
through: stl.publisher.ingentaconnect.com/content/stl/jcr/supp‑data
The title compound exhibited significant inhibitory activity in PC‑3
cell line, DU‑145 cell line and COLO‑205 cell line. The title compound
was slightly less potent than adriamycin in DU‑145 but obviously less
potent than adriamycin in PC‑3, COLO‑205. The percentage inhibition
determined is reported in Table 3.
The authors thank the National Natural Science Foundation
of China for financial support (project no. 21373132).
Received 16 October 2014; accepted 24 November 2014
Paper 1402964 doi: 10.3184/174751915X14206280982837
Published online: 9 January 2015
Results and discussion
The element analysis, IR (Fig. S1 in the electronic
supplementary information, ESI), H NMR (Fig. S2, ESI) and
1
References
X‑ray diffraction data for the product are in good agreement
with the structure of the title compound. A perspective view
of the crystal structure and packing diagram are shown in
Figs 1 and 2, the F(1)–C(11), O(4)–C(21) and O(5)–C(21)
bonds are 1.354(2), 1.203(3) and 1.339(3) Å respectively. The
bond length of O(1)–C(14) is 1.234(2) Å and belongs to the
typical C=O double bond. The dihedral angle between the
benzene ring and pyrazolopyrimidine is 53.618(162)°. On
the other hand, there is weak intramolecular hydrogen bond
N(4)–H(1)…N(1). Meanwhile, intermolecular hydrogen bond
C(22)–H(22B)…F(4)ˎN(4)–H(4)…Cl(2) and C(10)–H(10)…O(1)
are also present as shown in Table 2. These interaction forces
give the molecules a three‑dimensional structure. In addition,
preliminary bioassay indicated that the title compound
possessed some anti‑tuberculosis activity, and the activity data
are still waiting for further analysis.
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