Synthesis, structure characterization and preliminary biological evaluation of novel 5-alkyl-2-ferrocenyl-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one derivatives

Article abstract of DOI:10.1016/j.jorganchem.2008.01.043

A series of novel 5-alkyl-2-ferrocenyl-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one derivatives were synthesized by the reaction of ethyl 1-(2-bromoethyl)-3-ferrocenyl-1H-pyrazole-5-carboxylate with non-aromatic primary amines in one-pot procedure and characterized by ¹H NMR, ¹³C NMR, IR, HRMS and X-ray diffraction analysis. The effects of all the compounds on A549 cell growth were investigated. The results showed that all compounds had almost inhibitory effects on the growth of A549 cells.

Full text of DOI:10.1016/j.jorganchem.2008.01.043

Available online at www.sciencedirect.com
Journal of Organometallic Chemistry 693 (2008) 1367–1374
www.elsevier.com/locate/jorganchem
Synthesis, structure characterization and preliminary biological
evaluation of novel 5-alkyl-2-ferrocenyl-6,7-
dihydropyrazolo[1,5-a]pyrazin-4(5H)-one derivatives
a
b
a,
a,
c
*
*
Yong-Sheng Xie , Xiao-Hong Pan , Bao-Xiang Zhao , Jin-Ting Liu , Dong-Soo Shin ,
Jin-Hua Zhang a, Liang-Wen Zheng a, Jing Zhao b, Jun-Ying Miao b,*
a Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China
b Institute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100, China
c Department of Chemistry, Changwon National University, Changwon 641-773, Republic of Korea
Received 23 December 2007; received in revised form 30 January 2008; accepted 30 January 2008
Available online 5 February 2008
Abstract
A series of novel 5-alkyl-2-ferrocenyl-6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one derivatives were synthesized by the reaction of
ethyl 1-(2-bromoethyl)-3-ferrocenyl-1H-pyrazole-5-carboxylate with non-aromatic primary amines in one-pot procedure and character-
1
ized by H NMR, 13C NMR, IR, HRMS and X-ray diffraction analysis. The effects of all the compounds on A549 cell growth were
investigated. The results showed that all compounds had almost inhibitory effects on the growth of A549 cells.
Ó 2008 Elsevier B.V. All rights reserved.
Keywords: Ferrocene; Pyrazole-fused pyrazinone; Synthesis; X-ray structure; Bioactivity; A549 cell
1. Introduction
compatible with other treatment [5–7]. In this sense, the
integration of one or more ferrocene units into a heterocy-
clic ring molecule has been recognized as an attractive way
to endow a novel molecule functionally [8–12].
Since the discovery of ferrocene in 1951 [1], its fascinat-
ing sandwich structure has captured the imagination of
chemists, to the point of being nowadays among the most
important structural motifs in organometallic chemistry,
materials science, and, especially, catalysis. Recently, ferro-
cene and its derivatives have been attracted much more
attention in the biological activities [2,3]. Incorporation
of a ferrocene fragment into a molecule of an organic com-
pound often obtained unexpected biological activity, which
is rationalized as being due to their different membrane
permeation properties and anomalous metabolism [4].
Furthermore, the stability and non-toxicity of the ferroce-
nyl moiety is of particular interest rendering such drugs
The pyrazole unit is one of the core structures in a num-
ber of natural products. Many pyrazole derivatives are
known to exhibit a wide range of biological properties such
as anti-hyperglycemic, analgesic, anti-inflammatory, anti-
pyretic, anti-bacterial, antifungal hypoglycemic, sedative-
hypnotic activity, antitumor and anticoagulant activity
[13–15]. The incorporation of heterocyclic rings into
prospective pharmaceutical candidates is a major tactic to
gain activity and safety advantages. Although much work
has been directed toward the design and synthesis of
fused-pyrazole derivatives [16–21], a search of the litera-
tures revealed very few reports concerning pyrazolo-pyraz-
inones [22]. In the previous papers, we synthesized a series
of novel pyrazole derivatives including ethyl 1-(20-hydroxy-
30-aroxypropyl)-3-aryl-1H-pyrazole-5-carboxylate deriva-
tives [23], 6-(aroxymethyl)-2-aryl-6,7-dihydropyrazolo[5,1-
*
Corresponding authors. Tel.: +86 531 88366425; fax: +86 531
88564464.
E-mail addresses: bxzhao@sdu.edu.cn, sduzhao@hotmail.com (B.-X.
Zhao), jintliu@sdu.edu.cn (J.-T. Liu).
0022-328X/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2008.01.043

1368
Y.-S. Xie et al. / Journal of Organometallic Chemistry 693 (2008) 1367–1374
c][1,4]oxazin-4-one derivatives [24], ethyl 1-arylmethyl-3-
aryl-1H-pyrazole-5-carboxylate derivatives [25], 1-aryl-
methyl-3-aryl-1H-pyrazole-5-carbohydrazide derivatives
[26] and 1-arylmethyl-3-aryl-1H-pyrazole-5-carbohydraz-
ide hydrazone derivatives [27]. The evaluation of biological
activity showed that these compounds can inhibit A549
lung cancer cell growth.
ate and hydrazine hydrate in the presence of acetic acid at
room temperature [23]. Thus, ethyl 3-ferrocenyl-1H-pyra-
zole-5-carboxylate (4) was readily synthesized in 76% yield
by the reaction of ethyl 2,4-dioxo-4-ferrocenylbutanoate
(3), which can be obtained from acetylferrocene (2) and
diethyl oxalate, with hydrazine hydrate in the presence of
acetic acid at room temperature as shown in Scheme 1.
One would expect that the introduction of additional
ferrocenilic fragments into molecules of pyrazoles of this
class will afford products possessing a broader spectrum
of useful biological characteristics. In our ongoing interest
in the preparation of novel pyrazole derivatives, herein, we
would like to report the synthesis, structure characteriza-
tion and preliminary biological evaluation of a series of
novel 5-alkyl-2-ferrocenyl-6,7-dihydropyrazolo[1,5-a]pyra-
zin-4(5H)-one derivatives.
2.2. Synthesis of ethyl 1-(2-bromoethyl)-3-ferrocenyl-1H-
pyrazole-5-carboxylate (6)
It is known that nitrogen atom in 1st position of a pyr-
azole moiety possesses nucleophilic ability to react with
alkyl halide under a suitable condition [25]. The N-alkyl-
ation reaction between ethyl 3-ferrocenyl-1H-pyrazole-5-
carboxylate (4) and excess 1,2-dibromoethane was achieved
in the presence of potassium carbonate as the base in
acetonitrile. After flash chromatography on silica gel, the
ethyl 1-(2-bromoethyl)-3-ferrocenyl-1H-pyrazole-5-carbox-
ylate (6) and the isomer, ethyl 1-(2-bromoethyl)-5- ferroce-
nyl-1H-pyrazole-3-carboxylate (5) were obtained in 57%
and 25% yields, respectively (Scheme 1). It should be easily
understood that owing to annular tautomerism, pyrazoles
can exist in two tautomeric forms such as ethyl 3-ferroce-
nyl-1H-pyrazole-5-carboxylate (4) and ethyl 5-ferrocenyl-
1H-pyrazole-3-carboxylate (40) to lead two isomers of
product. It should be noted that it was difficult to distin-
guish two isomers in general because there is a lack of spe-
cific spectroscopic data in the literature [12]. In present
work, we differentiated successfully two isomers on X-ray
diffraction analysis. The general view of the molecule 6 is
given in Fig. 1 [29]. Thus, we can compare the chemical
2. Results and discussion
The synthetic strategies adopted to obtain the target
compounds are depicted in Scheme 1. The key intermediate
in the present study is the ethyl 1-(2-bromoethyl)-3-ferroce-
nyl-1H-pyrazole-5-carboxylate (6).
2.1. Synthesis of ethyl 3-ferrocenyl-1H-pyrazole-5-
carboxylate (4)
Firstly, acetylferrocene (2) was prepared from ferrocene
(1) and acetic anhydride with the catalytic phosphoric acid
in 80% yield according to the known method [28]. It has
proven to be successful that synthesis of ethyl 3-aryl-1H-
pyrazole-5-carboxylate from ethyl 2,4-dioxo-4-arylbutano-
O
O
(CO2Et)2
O
Ac2O
Fe
Fe
Fe
NaOC2H5/C2H5OH
rt, 24 h
CO2Et
1
2
3
N
NH
BrCH2CH2Br
NH2NH2/H2O
AcOH, rt, 24 h
CO2Et
Fe
K2CO3/ CH3CN
reflux, 4 h
4
Br
N
N
N
N
N
N
Br
CO2Et
RNH2
N
7
R
*
+
Fe
Fe
CH3CN/KI
reflux, 2-8 h
CO2Et
Fe
O
6
8
5
7a-7f
8a-8f
b
and
R =
*
*
*
*
*
MeO
Cl
N
OMe
e
a
c
d
f
Scheme 1.

Y.-S. Xie et al. / Journal of Organometallic Chemistry 693 (2008) 1367–1374
1369
N
N
N
NEt3, PhCH2NH2
N
Br
CO2Et
8c
Fc
+
Fc
CH3CN
reflux, 12 h
CO2Et
9
6
Scheme 2.
Table 1, we can find that the more the steric hindrance of
amine was, the lower the yield of compound 8. For exam-
ple, in the case of 8a and 8b, because the steric hindrance of
isopropylamine is larger than n-butylamine, the yields of 8a
(23%) is less than 8b (76%). Furthermore, as shown in
Scheme 3, in the case of (R)-1-phenylethanamine, (R)-ethyl
3-ferrocenyl-1-(2-(1-phenylethylamino)ethyl)-1H-pyrazole-
5-carboxylate (10) was only obtained, which could not
undergo cyclization reaction in the same conditions to give
desired compound.
Fig. 1. The molecular structure of compound 6, with displacement
ellipsoids drawn at the 50% probability level and H atoms omitted.
shift of two isomers and the absolute value of chemical
shift difference between two isomers. It should be found
that C17–H and two protons in substituted Cp of Fc moiety
peaks in 6 are relatively downfield and appear around 4.92
and 4.69 ppm while corresponding protons in 5 are rela-
tively upfield and resonate around 4.64 and 4.51 ppm. In
contrast, five protons in the unsubstituted Cp (C1–C5) of
Fc moiety peak in 6 is relatively upfield and appear around
4.08 ppm, while corresponding protons in 5 is relatively
downfield and resonate around 4.20 ppm. Moreover, in
the isomer 6, the absolute value of chemical shift difference
between protons in C17 and C1–C5 is mostly larger than
that between respective protons in C17 and C1–C5 in isomer
5, i.e. jDd(H–H) isomer 6j > jDd(H–H) isomer 5j (4.92 ꢀ
4.08 = 0.84 > 4.64 ꢀ 4.40 = 0.24).
2.4. Crystal structure
The spatial structure of compound 8c was determined
by using X-ray diffraction analysis. The single crystals were
grown from acetone at room temperature. The molecular
view of 8c is shown in Fig. 2, whereas Table 3 lists its
selected bond lengths and angles.
Fig. 2 shows that 8c contains a ferrocenyl bound to pyr-
azole ring which fused with pyrazinone. The molecular
structure is dominated by the arrangement of the rings of
the pyrazole, pyrazinone, two cyclopentadienyl of the ferr-
ocenyl moiety and the phenyl. Steric hindrance of the pro-
tons in the C14 a-position of the Cp-ring and the proton at
C12 and N3 of the pyrazole could be decreased by a per-
pendicular arrangement of the Cp and the pyrazole ring.
In addition, an optimal electronic overlap of the p-system
demands a coplanar arrangement. The dihedral angle
between the substituted Cp ring (C14–C18) and the pyra-
zole ring (N2/N3/C10/C12/C13) of 16.08(11)° is less than
that of the complex of ferrocenyl substituted pyrazolyl pyr-
idine (20.5°) [30], but larger than that of 6 (8.20°). The
2.3. Synthesis of 5-alkyl-2-ferrocenyl-6,7-dihydro-
pyrazolo[1,5-a]pyrazin-4(5H)-one (8)
Ethyl 1-(2-bromoethyl)-3-ferrocenyl-1H-pyrazole-5-car-
boxylate (6) reacted with excess non-aromatic primary
amines 7 in acetonitrile to afford pyrazolo-pyrazinones
derivatives 8 in moderate yields (Scheme 1). For example,
8b was obtained in 76% yield by the reaction of 6 with
excess n-butylamine 7b (10 equiv.) that was indispensable
as the substrate and base. To reduce the amount of reagent
amine, we tried to use cheaper base, such as triethylamine.
The result showed that the elimination reaction proceeded
and afforded a by-product, which was assigned to be
ethyl 3-ferrocenyl-1-vinyl-1H-pyrazole-5-carboxylate (9)
˚
C13–C14 distance 1.464(3) A is same as that in 6 [29],
but slightly shorter than that of substituted phenyl pyrazole
derivatives (1.472(3) and 1.4785(19) A) [31,32]. In the ferr-
˚
ocenyl moiety, the cyclopentadienyl (Cp) rings are perfectly
planar but deviate slightly from being parallel, the angle
between the plane normals being 1.66(13)°, and twisted
from the eclipsed conformation by 13.41–14.00°. The angle
Cg2–Fe1-Cg3 (where Cg is the centre of the plane defined
by five C-atoms) is 178.16(5)°. The distances Cg2 –Fe
and Cg3–Fe are 1.6420(13) A and 1.6452(14) A. The C–C
bond lengths and C–C–C angles in the cyclopentadienyl
rings show electronic overlap of the p-system-induced vari-
ations. The mean C–C distance involving the substituted
C(14) atoms is the longest in the rings at 1.434(3) A; adja-
cent C–C bonds are 1.424(3) and 1.416(3) A, with the final
C(16)–C(17) length being 1.422(3) A. C–C–C angle at
substituted atom averages 107.15(16)°, slightly reduced
from the ideal 108°, with the other angles averaging
1
1
by H NMR. The H NMR spectra indicated the presence
of a double bond and the chemical shift of the double bond
protons appeared at d = 7.43 (J = 15.3 and 9.7 Hz) in the
form double doublet peaks, 6.01 (J = 15.3 Hz) and 5.04
(J = 9.7 Hz) ppm in the form of doublet peaks respectively,
the coupling constants are consistent with trans and cis
vinylic protons, respectively. The competition reaction
resulted in the decrease of desired compound 8 (Scheme
2). On the other hand, the yield of compound 8 was
depended on the structure of reagent amine 7. From
˚
˚
˚
˚
˚

1370
Y.-S. Xie et al. / Journal of Organometallic Chemistry 693 (2008) 1367–1374
Table 1
Physical properties, HRMS and IR spectrum data for compounds 8
Compound
Yield (%)
M.p. (°C)
Formula
19H21FeN3O
C20H23FeN3O
23H21FeN3O
C27H29FeN3O
C26H27FeN3O3
HRMS: found
MS: require
IR (KBr) m (cmꢀ1
)
8a
8b
8c
8d
8e
8f
23
76
65
42
65
34
220–222
129–130
177–178
214–215
180–181
230–232
C
[M+H+]: 364.1113
[M+H+]: 378.1264
[M+Na+]: 434.0934
[M+H+]: 468.1735
[M+H+]: 486.1478
[M+H+]: 447.0672
364.1107
378.1263
434.0926
468.1733
486.1475
447.0670
1650
1650
1662
1649
1655
1648
C
C
22H1ClFeN3O
Table 2
1H NMR and 13C NMR data for compound 8
H
N
NH2
N
N
1H NMR (300 MHz, d, CDCl3) and 13C NMR (100 MHz,
d, CDCl3)
N
N
Compound
Fc
Br
CO2Et
Fc
KI, CH3CN
reflux, 4 h
CO2Et
8a
1H NMR: 6.85 (s, 1H, PyzH), 5.08–4.95 (m, 1H, CH3CH),
4.73 (s, 2H, C5H4), 4.34 (br s, 4H, C5H4 and NCH2CH2N),
4.11 (s, 5H, C5H5), 3.67 (s, 2H, NCH2CH2N), 1.23 (d,
J = 6.6 Hz, 6H, 2CH3); 13C NMR: 157.2, 151.5, 135.4,
104.8, 77.2, 69.5, 68.7, 66.5, 46.1, 43.4, 38.8, 19.7
1H NMR: 6.85 (s, 1H, PyzH), 4.72 (s, 2H, C5H4), 4.39–
4.32 (m, 4H, C5H4 and NCH2CH2N), 4.12 (s, 5H, C5H5),
3.76 (t, J = 6.3 Hz, 2H, NCH2CH2N), 3.57 (t, J = 7.2 Hz,
2H, NCH2CH2CH2CH3), 1.70–1.57 (m, 2H,
6
10
Scheme 3.
8b
NCH2CH2CH2CH3), 1.47–1.33 (m, 2H,
NCH2CH2CH2CH3), 0.97 (t, J = 7.2 Hz, 3H, CH3); 13C
NMR: 157.8, 151.6, 135.4, 104.9, 77.2, 69.7, 68.9, 66.7,
46.2, 45.9, 45.5, 29.3, 20.1, 13.8
8c
8d
1H NMR: 7.37–7.27 (m, 5H, PhH), 6.93 (s, 1H, PyzH),
4.78 (s, 2H, PhCH2), 4.70 (s, 2H, C5H4), 4.40–4.27 (m, 4H,
C5H4 and NCH2CH2N), 4.10 (s, 5H, C5H5), 3.67 (t,
J = 6.3 Hz, 2H, NCH2CH2N); 13C NMR: 158.0, 151.7,
136.3, 135.0, 128.9, 128.2, 127.9, 105.1, 77.2, 69.5, 68.7,
66.5, 49.3, 45.8, 44.6
1H NMR: 7.38 (d, J = 8.0 Hz, 2H, PhH), 7.27 (d,
J = 8.0 Hz, 2H, PhH), 6.92 (s, 1H, PyzH), 4.75–4.70 (m,
4H, C5H4 and PhCH2), 4.32 (s, 4H, C5H4 and
NCH2CH2N), 4.10 (s, 5H, C5H5), 3.68 (s, 2H,
NCH2CH2N), 1.32 (s, CH3, 9H); 13C NMR: 158.0, 151.7,
150.9, 135.1, 133.2, 128.0, 125.8, 105.1, 77.2, 69.6, 68.7,
66.6, 49.1, 45.9, 44.6, 34.5, 31.3
1H NMR: 6.86 (s, 1H, PyzH), 6.84–6.77 (m, 3H, PhH),
4.70 (s, 2H, C5H4), 4.32 (s, 2H, C5H4), 4.20 (t, J = 6.6 Hz,
2H, NCH2CH2N), 4.10 (s, 5H, C5H5), 3.87 (s, 6H, OCH3),
3.79 (t, J = 7.2 Hz, 2H, PhCH2CH2), 3.55 (t, J = 6.6 Hz,
2H, NCH2CH2N), 2.93 (t, J = 7.2 Hz, PhCH2); 13C NMR:
157.8, 151.6, 149.0, 147.7, 135.2, 131.1, 120.8, 111.8, 111.2,
104.8, 77.2, 69.5, 68.7, 66.5, 55.9, 55.8, 48.9, 46.6, 45.8,
33.8
1H NMR: 8.38 (s, 1H, PyH), 7.72 (dd, J = 8.1 and 1.8 Hz,
1H, PyH), 7.35 (d, J = 8.1 Hz, 1H, PyH), 6.90 (s, 1H,
PyzH), 4.75 (s, 2H, PhCH2), 4.73 (s, 2H, C5H4), 4.35 (s,
4H, C5H4 and NCH2CH2N), 4.11 (s, 5H, C5H5), 3.71 (br s,
2H, NCH2CH2N); 13C NMR: 158.1, 152.0, 151.4, 149.3,
139.0, 134.6, 131.1, 124.7, 105.4, 77.2, 69.5, 68.8, 66.6,
46.5, 45.8, 45.1
Fig. 2. The molecular structure of compound 8c, with displacement
ellipsoids drawn at the 50% probability level and H atoms omitted.
108.21°. Fe–C distances is longest for the substituted C(14),
˚
mean 2.0484(19) A. All these bond and angle variations are
very similar to those observed in 6. In the pyrazole-fused
pyrazinone ring, N1 and C11 are coplanar with pyrazole
ring, forming p-system. The bond length of C10–C11 and
8e
˚
C11–N1 are 1.471(3) and 1.357(3) A, respectively (see
Table 4).
2.5. Effects of the compounds on the viability of A549 lung
cancer cells
8f
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-
um bromide (MTT) cell proliferation assay is widely
accepted as a reliable way to measure the cell proliferation
rate, and conversely when metabolic events lead to apopto-
sis or necrosis. The data obtained by MTT assay showed
that compounds 8a–8f had inhibitory effects on the growth
of A549 cells in dosage- and time-dependent manners as
indicated by the results in Fig. 3. As typically shown in
Fig. 3, exposure of cells to 8c–8f at 20 lM for 24 h resulted
in cell viability decrease from 100% to 64–51% (p < 0.01).
When the exposure continued on to 48 h, compared with
the control group, the cell viability reduced more signifi-
cantly from 100% to 50–28% (p < 0.01). Further, exposure
of cells to 8c–8f at 40 lM for 24 and 48 h, the cell viability
reduced more significantly from 100% to 43–33% and
34–17% (p < 0.01), respectively. To compare with known
anticancer drug, we carried out the assay of the effects of
5-FU on the growth of A549 cells in the same conditions.
From the IC50 of compounds 8a–8f and 5-Fu we can find

Y.-S. Xie et al. / Journal of Organometallic Chemistry 693 (2008) 1367–1374
1371
Table 3
Selected bond lengths (A) and angles (°) of compound 8c
3. Summary
˚
C(8)–N(1)
C(8)–C(9)
C(9)–N(2)
1.469(2)
1.510(3)
1.449(2)
1.352(2)
1.377(3)
1.471(3)
1.225(2)
1.357(3)
1.404(3)
1.341(2)
1.464(3)
C(14)–C(18)
C(14)–C(15)
C(14)–Fe(1)
C(15)–C(16)
C(15)–Fe(1)
C(16)–C(17)
C(16)–Fe(1)
C(17)–C(18)
C(17)–Fe(1)
C(18)–Fe(1)
N(2)–N(3)
1.430(3)
1.434(3)
2.0484(19)
1.424(3)
2.0423(19)
1.422(3)
2.039(2)
1.416(3)
2.038(2)
2.038(2)
1.342(2)
1.6452(14)
In summary, we have described the facile synthesis of
novel 5-alkyl-2-ferrocenyl-6,7-dihydropyrazolo[1,5-a]pyra-
zin-4(5H)-one derivatives from ethyl 1-(2-bromoethyl)-3-
ferrocenyl-1H-pyrazole-5-carboxylate and non-aromatic
primary amines in one-pot procedure. The yield of the
reaction was depended on the structure of reagent amine
and base. These novel compounds were characterized by
1H NMR, 13C NMR, IR, HRMS and X-ray diffraction
analysis. The results from effects of the compounds on
the viability of A549 lung cancer cells showed that the com-
pounds should be potential cancer inhibitor.
C(10)–N(2)
C(10)–C(12)
C(10)–C(11)
C(11)–O(1)
C(11)–N(1)
C(12)–C(13)
C(13)–N(3)
C(13)–C(14)
Cg(2)–Fe(1)
1.6420(13) Cg(3)–Fe(1)
C(11)–N(1)–C(8)
N(1)–C(8)–C(9)
N(2)–C(9)–C(8)
C(10)–N(2)–C(9)
N(2)–C(10)–C(11)
N(1)–C(11)–C(10)
N(2)–C(10)–C(12)
121.56(16)
112.25(16)
107.75(15)
122.57(16)
121.15(16)
115.39(16)
106.59(16)
N(3)–C(13)–C(12)
C(13)–N(3)–N(2)
N(3)–N(2)–C(10)
C(18)–C(14)–C(15) 107.15(16)
C(16)–C(15)–C(14) 108.25(16)
C(17)–C(16)–C(15) 107.77(17)
C(18)–C(17)–C(16) 108.42(17)
C(17)–C(18)–C(14) 108.39(17)
110.83(16)
105.01(14)
112.48(15)
4. Experimental
All solvents were pre-dried and distilled prior to use. All
reactions were carried out under nitrogen and monitored
1
by TLC on silica gel 60 F254 plates (Merck KGaA). H
C(10)–C(12)–C(13) 105.08(16)
Cg(2)–Fe(1)–Cg(3)
178.16(5)
NMR spectra were recorded on a Bruker Avance 300
(300 MHz) spectrometer, using CDCl3 as solvents and
tetramethylsilane (TMS) as internal standard. Melting
points were determined on an XD-4 digital micro melting
point apparatus. IR spectra were recorded with an IR
spectrophotometer Avtar 370 FT-IR (Termo Nicolet).
MS spectra were recorded on a LTQ Orbitrap Hybrid mass
spectrograph. X-ray diffraction data were recorded on a
Bruker Smart Apex2CCD diffractometer. RPMI 1640
was obtained from Gibco BRL Co. (Grand Island, USA)
and bovine calf serum was from Beijing DingGuo Biotech-
nology Co. (China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) was purchased from
Amresco.
Table 4
Crystal data and structure refinement for 8c
Empirical formula
Formula weight
Temperature (K)
C23H22FeN3O
412.29
293(2)
0.71069
Triclinic
˚
Wavelength (A)
Crystal system
Space group
Unit cell dimensions
ꢀ
P1
˚
a (A)
8.461(5)
10.473(5)
11.103(5)
100.356(5)
108.089(5)
93.568(5)
912.6(8)
2
˚
b (A)
˚
c (A)
a (°)
b (°)
c (°)
4.1. Synthesis of ethyl 3-ferrocenyl-1H-pyrazole-5-
carboxylate (4) from (2)
3
˚
Volume (A )
Z
Calculated density (Mg/m3)
Absorption coefficient (mmꢀ1
F(000)
1.500
0.846
430
)
The solution of acetylferrocene (1.14 g, 5.0 mmol) in
absolute alcohol (2.5 ml) was added to a stirred solution
of sodium ethoxide (408 mg, 6 mmol) and diethyl oxalate
(877 mg, 6.0 mmol) in absolute alcohol (5.0 mL) (N2 atmo-
sphere). Stirring was continued for 24 h at room tempera-
ture, then glacial acetic acid (420 mg, 7 mmol) was added,
and stirring was continued for an additional 30 min. 80%
hydrazine hydrate (473 mg, 7 mmol) was added. Stirring
was continued for 24 h at room temperature, then the mix-
ture was combined with H2O (30 mL) and the aqueous
phase was extracted with ethyl acetate (3 ꢁ 25 mL). The
combined organic extracts were dried over anhydrate
magnesium sulfate, and evaporated to give a yellow solid.
Recrystallization from alcohol gave the product 4 (1.23 g,
76%) as yellow crystal. M.p. 196–198 °C. 1H NMR
(300 MHz, CDCl3): d 10.47 (br s, 1H, NH), 6.84 (s, 1H,
pyrazole moiety), 4.65 (s, 2H, C5H4), 4.42 (q, J = 7.2 Hz,
2H, OCH2CH3), 4.33 (s, 2H, C5H4), 4.09 (s, 5H, C5H5),
1.43 (t, J = 7.2 Hz, 3H, OCH2CH3). IR (KBr) m: 1719
Crystal size
h range for data collection (°)
Limiting indices
0.27 ꢁ 0.16 ꢁ 0.10 mm
1.97–27.49
ꢀ10 6 h P 10, ꢀ13 6 k P 13,
ꢀ14 6 l P 14
Reflections collected/unique
Completeness to h = 27.49°
Absorption correction
Maximum and minimum
transmission
13384/4168 [Rint = 0.0220]
99.7%
Semi-empirical from equivalents
0.9232 and 0.8044
Refinement method
Full-matrix least-squares on F2
4168/0/253
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2r(I)]
R indices (all data)
1.055
R1 = 0.0309, wR2 = 0.0820
R1 = 0.0378, wR2 = 0.0863
Largest diffraction peak and hole 0.309 and ꢀ0.596
3
˚
(e A )
that compounds 8b–8f have more effects on the growth of
A549 cells then 5-Fu at 24 h. Furthermore, 8d–8f have
more effects then 5-Fu at 48 h (Table 5).
(C@O) cmꢀ1
.

1372
Y.-S. Xie et al. / Journal of Organometallic Chemistry 693 (2008) 1367–1374
Fig. 3. Effects of the compounds 8a–8f on the viability of A549 lung cancer cells. Control (0), the viability of the cells cultured in the medium without any
compounds. Other bars show the viability of the cells treated with the compound 4a–4f at the concentrations of 2.5–40 lM for 24 h and 48 h, respectively
(n = 3).
Table 5
mol) under N2. After refluxed for 4 h, the reaction mixture
Growth inhibitory properties (IC50) for the compounds 8a–8f and 5-FU at
was cooled to room temperature, filtered and evaporated,
24 and 48 h
the residue was purified by chromatography on silica gel
Compound
8a
8b
8c
8d
8e
8f
5-FU
with the solvent system of ethyl acetate/petroleum ether
(v/v = 1:2). The product 6 was obtained as yellow crystal
in 57% yield. M.p. 98–99 °C. 1H NMR (300 MHz, CDCl3):
d 6.85 (s, 1H, pyrazole moiety), 4.92 (t, J = 6.9 Hz, 2H,
NCH2CH2Br), 4.69 (s, 2H, C5H4), 4.38 (q, J = 7.2 Hz,
2H, OCH2CH3), 4.30 (s, 2H, C5H4), 4.08 (s, 5H, C5H5),
3.74 (t, J = 6.9 Hz, 2H, NCH2CH2Br), 1.43 (t,
J = 7.2 Hz, 2H, OCH2CH3). IR (KBr) m: 1717 (C@O)
cmꢀ1. The by-product 5 was obtained as a yellow crystal
in 25% yield. M.p. 131–132 °C. 1H NMR (300 MHz,
CDCl3): d 6.84 (s, 1H, pyrazole moiety), 4.64 (t,
J = 6.9 Hz, 2H, NCH2CH2Br), 4.51 (s, 2H, C5H4), 4.43
24 h (lM)
48 h (lM)
51
44
30
23
22
10
31
12
22
10
45
13
4.2. Synthesis of ethyl 1-(2-bromoethyl)-3-ferrocenyl-1H-
pyrazole-5-carboxylate (6) and ethyl 1-(2-bromoethyl)-5-
ferrocenyl-1H-pyrazole-3-carboxylate (5)
To a stirred solution of 4 (3.24 g, 0.01 mol) and
1,2-dibromoethane (18.79 g, 0.1 mol) in dry acetonitrile
(40 mL) was added potassium carbonate (1.67 g, 0.012

Y.-S. Xie et al. / Journal of Organometallic Chemistry 693 (2008) 1367–1374
1373
(q, J = 7.2 Hz, 2H, OCH2CH3), 4.40 (s, 2H, C5H4), 4.20 (s,
5H, C5H4), 3.73 (t, J = 6.9 Hz, 2H, NCH2CH2Br), 1.42 (t,
J = 7.2 Hz, 3H, OCH2CH3). IR (KBr) m: 1711 (C@O)
cedure described by Price and McMillan [33]. The light
absorptions were measured at 570 nm using SpectraMAX
190 microplate spectrophotometer (GMI Co., USA).
cmꢀ1
.
4.7. Statistical analyses
4.3. General procedure for syntheses of pyrazolo-pyrazinones
derivatives (8)
Data were expressed as means SE, accompanied by
the number of experiments performed independently, and
analyzed by t-test. Differences at p < 0.05 were considered
statistically significant.
To a stirred solution of compound 6 (431 mg, 1.0 mmol)
and amine 7 (10 mmol) in dry acetonitrile (20 mL) was
added catalytic KI (33 mg, 0.2 mmol) under N2. After
refluxed for 2–8 h, the reaction mixture was cooled to room
temperature, filtered and evaporated. The residue was puri-
fied by chromatography on silica gel with the solvent sys-
tem of ethyl acetate/petroleum ether (v/v = 1:1) to afford
desired derivatives 8 in moderate yields. The physical prop-
erties, Mass analysis data, IR spectrum data and 1H NMR
spectra of compounds 8 were listed in Tables 1 and 2,
respectively.
Acknowledgement
This study was supported by the Foundation of the
Ministry of Education (104112) and the Natural Science
Foundation of Shandong Province (Y2005B12).
Appendix A. Supplementary material
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.jorganchem.
2008.01.043.
4.4. Synthesis of (R)-ethyl 3-ferrocenyl-1-(2-(1-phenyl-
ethylamino)ethyl)-1H-pyrazole-5-carboxylate (10)
To a stirred solution of compound 6 (431 mg, 1.0 mmol)
and (R)-1-phenylethanamine (1.21 g, 10 mmol) in dry ace-
tonitrile (20 mL) was added catalytic KI (33 mg, 0.2 mmol)
under N2. After refluxed for 4 h, the reaction mixture was
cooled to room temperature, filtered and evaporated. The
residue was purified by chromatography on silica gel with
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