Novel chiral ferrocenylpyrazolo[1,5-a][1,4]diazepin-4-one derivatives-Synthesis, characterization and inhibition against lung cancer cells

Article abstract of DOI:10.1016/j.ejmech.2013.02.016

A series of novel 2-ferrocenyl-7-hydroxy-5-phenethyl-5,6,7,8-tetrahydro-4H- pyrazolo[1,5-a][1,4]diazepin-4-one derivatives with optical activity (2) was synthesized in the microwave-assisted condition and characterized by means of IR, ¹H NMR and mass spectroscopy, and furthermore confirmed by X-ray analysis of a representative compound (R)-2a. Preliminary biological evaluation showed that some compounds could suppress the growth of A549, H322 and H1299 lung cancer cells. Among the tested compounds, 2b-d were more effective and might perform their action through cell cycle arrest for A549 cell. Whereas these compounds inhibited growth of H1299 and H322 cells by inducing apoptosis. The anti-tumor activities of these compounds were related to the nature of substituents in benzene moiety. In addition, the results indicated also that compounds 2b-d possessed notable cytotoxicity and selectivity for A549 vs H1299 and H322 lung cancer cells.

Full text of DOI:10.1016/j.ejmech.2013.02.016

European Journal of Medicinal Chemistry 63 (2013) 256e268
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel chiral ferrocenylpyrazolo[1,5-a][1,4]diazepin-4-one derivatives e Synthesis,
characterization and inhibition against lung cancer cells
,
a,
*
Shi-Li Shen a, Jin-Hui Shao b, Ji-Zhuang Luo b, Jin-Ting Liu a, Jun-Ying Miao b , Bao-Xiang Zhao
*
a Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Shanda Nanlu 27, Jinan, Shandong 250100, PR China
b Institute of Developmental Biology, School of Life Science, Shandong University, Shanda Nanlu 27, Jinan, Shandong 250100, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A
series of novel 2-ferrocenyl-7-hydroxy-5-phenethyl-5,6,7,8-tetrahydro-4H-pyrazolo[1,5-a][1,4]dia-
Received 24 October 2012
Received in revised form
19 January 2013
Accepted 13 February 2013
Available online 24 February 2013
zepin-4-one derivatives with optical activity (2) was synthesized in the microwave-assisted condition
and characterized by means of IR, 1H NMR and mass spectroscopy, and furthermore confirmed by X-ray
analysis of a representative compound (R)-2a. Preliminary biological evaluation showed that some
compounds could suppress the growth of A549, H322 and H1299 lung cancer cells. Among the tested
compounds, 2bed were more effective and might perform their action through cell cycle arrest for A549
cell. Whereas these compounds inhibited growth of H1299 and H322 cells by inducing apoptosis. The
anti-tumor activities of these compounds were related to the nature of substituents in benzene moiety.
In addition, the results indicated also that compounds 2bed possessed notable cytotoxicity and selec-
tivity for A549 vs H1299 and H322 lung cancer cells.
Keywords:
Pyrazole
Ferrocene
Chiral diazepinone
Lung cancer cells
Cell cycle arrest
Apoptosis
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
Diazepanone ring is a core structure of the liposidomycins and
caprazamycins which are unique nucleoside-type antibiotics iso-
Cancer is the second major cause of deaths after cardio and
cerebrovascular diseases. Non-small cell lung cancer (NSCLC) ac-
counts for 80% of lung cancers, and is the leading cause of cancer
mortality. Overall, the 5-year survival rate of NSCLC patients is poor
and hardly reaches 15%, due to NSCLC is relatively resistant to
chemotherapy and radiation therapy [1,2]. Furthermore, the
adverse effects of chemotherapeutic compounds often hamper the
quality of life of lung cancer patients. Therefore, the discovery of
effective novel prophylactic, diagnostic, and therapeutic treatments
for NSCLC is urgently needed.
Pyrazole represents an important class of nitrogen-containing
heterocycles and compounds with pyrazole framework display
diverse biological activities such as anti-cancer, antimicrobial,
antiinflammatory, antiangiogenic, antidepressant, antioxidant and
antiinfluenza activities [3e9]. Recently, much attention has been
focused on pyrazoles [10e15]. Pyrazole-containing structures have
also been found among naturally occurring compounds such as the
antibiotic pyrazofurin which has been documented to possess
potent anti-cancer and antimicrobial activities [16].
lated from Streptomyces [17e19]. These natural products have eli-
cited intense interest in the scientific community because of their
antibiotic properties. In addition to their striking antibiotic profiles,
their complex and unprecedented molecular architectures have
appealed to synthetic chemists to investigate these molecules as
synthetic targets of great biological interest [20]. Various analogs of
them have been synthesized and showed extent bioactivity such as
anti-tuberculosis [21].
Metal-based complexes have been playing a prominent role in
the medicinal chemistry and drug development since the discovery
of the anti-tumor properties of square-planar platinum (II) complex
(cisplatin) reported by Rosenberg et al. in 1969 [22]. Though plat-
inum complex continue to play a central role in the treatment of
patients suffering from cancer [23], they suffer from two main
disadvantages: side effects such as nephrotoxicity and ineffective-
ness against platinum-resistant tumors [24]. There is an urgent
need to develop cisplatin analogs and design other new metal-
based complexes. During the past years, numerous promising
new organometallic complexes were reported to exhibit high anti-
cancer activity, such as Au complexes [25,26], Rh complexes [27],
Ru complexes [28,29], Ir complexes [30], Ti complexes [31,32], Os
complexes [33], ferrocene derivatives [34,35] and so on. Among
them, ferrocene derivatives have captured the attention of chemists
* Corresponding authors. Tel.: þ86 531 88366425; fax: þ86 531 88564464.
E-mail addresses: miaojy@sdu.edu.cn (J.-Y. Miao), bxzhao@sdu.edu.cn,
sduzhao@hotmail.com (B.-X. Zhao).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.02.016

S.-L. Shen et al. / European Journal of Medicinal Chemistry 63 (2013) 256e268
257
so intensively since the first report of ferrocene in 1951 [36].
Incorporation of a ferrocene fragment into a molecule of an organic
compound often resulted in excellent biological activity due to its
similarities in aromaticity, lipophilicity, and morphology to a
phenyl ring, along with the unusual redox properties [37]. For
example, ferroquine, which is obtained by the replacement of a CH2
group with a ferrocenyl group in the anti-malarial chloroquine, is
active against both chloroquine-sensitive and chloroquine-
resistant Plasmodium falciparum [38], and is about to complete
phase II clinical trials as a treatment for uncomplicated malaria
[39]. In addition, the stability and non-toxicity of the ferrocene
moiety is of particular interest rendering such drugs compatible
with other treatment. A lot of ferrocenyl compounds display anti-
tumor, antioxidant and antimycobacterial activity [40e43].
In an attempt to search for novel anti-tumor compounds, we
focused our attention to the design, synthesis and bioactivity
evaluation of pyrazole derivatives with structure diversity [44e57].
In a continuation of an ongoing program aiming at finding new
structural leads with potential chemotherapeutic activities, it was
rationalized to synthesize some novel pyrazole fused heterocycle
that would produce anti-cancer activity. The design of pyrazolo-
diazepanone scaffolds should be the most suitable plan to reach
large families of compounds with structure diversity because it can
take advantage of multiple functional groups which facilitate the
further introduction of pharmacophoric groups. Furthermore,
scaffolds with cyclic structures can reduce the entropic cost asso-
ciated with the loss of conformational degrees of freedom upon
binding to the target protein. We would like to report the synthesis
of novel 2-ferrocenyl-7-hydroxy-5-phenethyl-5,6,7,8-tetrahydro-
4H-pyrazolo[1,5-a][1,4]diazepin-4-one derivatives with optical ac-
tivity by microwave-assisted reaction, and preliminary biological
evaluation against A549, H322 and H1299 lung cancer cells.
and 2-arylethanamine as outlined in Scheme 1. Starting materials
(R) or (S)-1 were easily obtained from ethyl 3-ferrocenyl-1H-pyr-
azole-5-carboxylate and (R)- or (S)-oxiran-2-ylmethyl 4-
methylbenzenesulfonate according to previous reported method
[14]. The synthesis was performed both under classical convective
and microwave-assisted heating techniques. Firstly, the reaction
was carried out under the condition of reflux in ethanol. Although
desired compounds were obtained, the yields were lower (20e28%)
because byproducts 3 and 4 were also formed as shown in Scheme
2. In order to prepare the target compounds in approving yields, the
reaction was performed in a sealed tube under higher temperature
(140 ꢀC). As expected, the compounds were obtained in higher yield
(62e82%).
Microwave-assisted chemistry has blossomed into a useful
technique for a variety of applications in organic synthesis and
inorganic synthesis. The use of microwave heating can dramatically
cut down reaction time, increase product purity and yields, and
allow precise control of reaction conditions. Thus, we focused our
attention on the microwave-assisted synthesis technique after
obtaining compounds 2 by classical heating method. These re-
actions are performed in a microwave synthesis lab station. In a
typical experiment, ethyl 3-ferrocenyl-1-(oxiran-2-ylmethyl)-1H-
pyrazole-5-carboxylate 1 and amine were mixed in an open vessel
and irradiated keeping the reaction temperature at 70 ꢀC for 10e
15 min without any solvent. After work-up, desired compounds
were obtained. Comparing two methods, microwave-assisted
synthesis technique dramatically cut down reaction time, in-
crease product yields as shown in Table 1. Similarly, we synthesized
two compounds (R)-5 and (S)-5 with phenyl instead of ferrocene
(Scheme 3) in order to compare effects of the compounds with and
without ferrocene against lung cancer cells.
The structures of 2-ferrocenyl-7-hydroxy-5-phenethyl-5,6,7,8-
tetrahydro-4H-pyrazolo[1,5-a][1,4]diazepin-4-one derivatives
2
were determined by IR, 1H NMR and mass spectroscopy.
For example, (R)-2-ferrocenyl-7-hydroxy-5-phenethyl-5,6,7,8-
tetrahydro-4H-pyrazolo[1,5-a][1,4]diazepin-4-one ((R)-2a), ob-
tained in 81% yield as yellow solid, gave a [M þ H]þ-ion peak at m/z
456.1375 in HRMS in accord with the molecular formula
2. Chemistry
2.1. Synthesis
The target compounds, 2-ferrocenyl-7-hydroxy-5-phenethyl-
C
25H26FeN3O2 ([M þ H]þ: 456.1374). In the IR spectra, the band at
5,6,7,8-tetrahydro-4H-pyrazolo[1,5-a][1,4]diazepin-4-one
rivatives, were synthesized by the reaction of (R) or (S)-3-
ferrocenyl-1-(oxiran-2-ylmethyl)-1H-pyrazole-5-carboxylate
de-
3398 cmꢁ1 is typical stretching vibration absorption of hydroxyl
group. The two weak bands appeared at 2923 and 2869 cmꢁ1 may
1
Scheme 1. Synthesis of pyrazolo[1,5-a][1,4]diazepin-4-one derivatives 2.

258
S.-L. Shen et al. / European Journal of Medicinal Chemistry 63 (2013) 256e268
Scheme 2. The formation of byproducts 3 and 4.
be ascribed to the CeH absorptions. The absorption of carbonyl
group was observed at 1607 cmꢁ1, and the CaN absorption was
observed at 1526 cmꢁ1. The torsional or swing vibration absorption
of CpFeCp appeared at 484 cmꢁ1. The 1H NMR spectra indicated
intramolecular C18AeH18B/O1 weak hydrogen bond (Fig. 1,
Table 4).
Two Cp rings of the ferrocene fragment are perfectly planar but
deviate slightly from being parallel with an angle of 1.9(4)ꢀ and are
almost in the eclipsed conformation by the torsion angle CeCg2e
Cg3eC varies between ꢁ5.09 and ꢁ6.20ꢀ, where Cg2 and Cg3 are
the substituted and unsubstituted Cp ring centroids, respectively.
chemical shift of the OH at
(J ¼ 3.9 Hz). One proton signal in pyrazole moiety appeared at
¼ 6.82 ppm. The protons of one methylene in the phenethyl
moiety appeared at
¼ 2.92 ppm as triplet peaks (J ¼ 7.5 Hz), the
other methylene protons in the phenethyl moiety appeared at
d
¼ 5.47 ppm as doublet peaks
d
ꢀ
d
The Fe1eCg2 and Fe1eCg3 distances are 1.644(3) and 1.650(4) A
respectively, and the Cg2eFe1eCg3 angle is 178.75(17)ꢀ. The FeeC
d
¼ 3.58 ppm (dt, J ¼ 13.5 Hz, 7.5 Hz) and
d
¼ 3.86 ppm (dt,
distances in unsubstituted Cp ring are all similar to those found in
ꢀ
J ¼ 13.5 Hz, 7.5 Hz), respectively. Furthermore, the structure was
confirmed by the X-ray diffraction as shown in Fig. 1.
ferrocene itself [59], ranging from 2.020(7) to 2.033(7) A [mean
ꢀ
2.024(1) A], while those in substituted Cp ring are different and
ꢀ
ꢀ
range from 2.027(5) to 2.040(5) A [mean 2.032(1) A] (Table 3), thus
indicating distortion of the ferrocene moiety caused by substitution
2.2. Single-crystal structure
ꢀ
at C5. The longest FeeC5 distance of 2.040(5) A shows displace-
ment of the atom away from the Fe atom.
Single crystals of (R)-2a were all grown via slow evaporation of
ethyl acetate solution. The molecular view of (R)-2a is shown in
Fig. 1. Details of crystal data, data collection and refinement pa-
rameters are compiled in Tables 2 and 3.
The seven-membered diazepanone ring displays a twisted boat
conformation, the atoms of C14, C16, C17 and N3 form the bottom
of the boat (Maximum deviation from the mean N3/C14/C16/C17
ꢀ
plane ¼ 0.155 A), C15 the prow, and N2 and C13 the stern [distances
Fig. 1 shows that (R)-2a contains a ferrocenyl bound to pyrazole
ring which fused with diazepanone ring. All of the bond lengths
and bond angles in pyrazole and diazepanone rings are in the
normal range (Table 3). The dihedral angle between the substituted
cyclopentadienyl (Cp) ring (C1eC5) and the pyrazole ring (N2/N3/
ꢀ
from the N3/C14/C16/C17 mean plane ¼ 0.557, 0.813, 0.912 A,
respectively].
The crystal packing of structure (R)-2a was stabilized by strong
intermolecular hydrogen bonds and weak CeH/
p interactions
(Table 4). In the crystal lattice, the carbonyl groups of the dia-
zepanone interact with the hydroxy groups of the adjacent mole-
cules through linear O2eH2/O1 hydrogen bonds to form an
infinite one-dimensional chain along the (100) direction. It is
interesting to find that a pairs of C17eH17A/Cg3 hydrogen
teractions join the adjacent molecules into centrosymmetric
R22(16) dimers. Moreover, pairs of CeH/ interactions of the
C17eH17A/Cg4 (Cg4 is the centroid of the benzene ring) join the
dimers with one another to result in formation of the three-
dimensional structure (Fig. 2).
ꢀ
C11/C12/C13) is 13.9(3) A that is similar to that found in related
ꢀ
structures [58]. The C5eC11 distance is 1.464(7) A. While the
phenyl ring (C20eC25) and the pyrazole ring in the molecule is
nearly perpendicular with an angle of 87.0(3)ꢀ. The two methylene
groups of the phenethyl moiety in 2a were dually disordered at
298 K (Fig. 1) resulting in conformational synmorphism. The dis-
tribution of conformers A and B in the crystal is found in number
ratio 0.60:0.40. The molecular conformation A is stabilized by
p in-
p
Table 1
The yields of compounds 2 in three conditions.
2.3. Cyclic voltammetry
Entry Products Classical heatinga In sealed tubeb
Microwave-assistedc
The electrochemical characterization of the synthesized com-
pounds was performed by means of cyclic voltammetry (CV). The
compounds (R)- and (S)-2aed were dissolved in CH2Cl2 to give a
solution containing 0.5 mM of the analyte and 0.1 M Bu4N[PF6] as
the supporting electrolyte. The redox properties of ferrocene de-
rivatives are of particular interest as they have been implicated in
the production of reactive oxygen species (ROS) responsible for
their anti-cancer activity [60,61]. Meanwhile CV measurements
provide an additional proof of purity of our biologically investi-
gated compounds. Pertinent data are presented in Table 5. Fig. 3
shows the voltammograms of (R)- and (S)-2aed.
Time (h) Yields (%) Time (h) Yields (%) Time (min) Yields (%)
1
2
3
4
5
6
7
8
(S)-2a
(S)-2b
(S)-2c
(S)-2d
(R)-2a
(R)-2b
(R)-2c
(R)-2d
10
10
10
10
10
10
10
10
25
21
21
20
28
22
20
21
10
8
12
12
10
8
74
80
65
72
78
82
62
71
10
10
15
15
10
10
15
15
81
74
72
75
81
74
74
77
12
12
a
b
c
Alcohol, reflux.
Alcohol, 140 ꢀC.
No solvent, 70 ꢀC.

S.-L. Shen et al. / European Journal of Medicinal Chemistry 63 (2013) 256e268
259
Scheme 3. Synthesis of pyrazolo[1,5-a][1,4]diazepin-4-one derivatives 5.
Under the above-described experimental conditions, com-
pounds (R)- and (S)-2aed undergo one-electron oxidations at half-
wave potentials of ca. 450 mV against the ferrocene/ferrocenium
redox couple. As shown in Table 5, almost the same half-wave po-
tentials of compounds (R)- and (S)-2aed indicate that the different
substituent in benzene moiety nearly don’t have any influence on
the redox properties due to the lack of electronic interactions be-
tween the ferrocenyl moiety and benzene moiety. The cyclic vol-
tammetry behavior showed that the redox property of compounds
(R)- and (S)-2aed is similar to other ferrocene derivatives reported
[62]. Moreover, the uncomplicated voltammogram confirmed
electrochemical purity of our synthesized compounds [63].
compounds at the concentration of 40
m
M for 48 h. These com-
pounds are stable in the media used for biological evaluation dur-
ing the time of testing. The results showed that the compounds (S)-
and (R)-2bed displayed great effects on the growth of the cells as
shown in Fig. 4. Although the effects of these compounds are
inferior to cisplatin, it is interesting that the compounds substituted
with eOMe and eF persistently show different behavior than the e
H one. The reason might be in electronic and steric characteristics
of compounds as well as hydrophilicehydrophobic balance of the
molecules. Furthermore, in order to compare the different biolog-
ical properties of the compounds with and without ferrocene, we
performed the anti-cancer activity evaluation of them. We can see
from Fig. 4 that the effects of compounds (S)- and (R)-2b with
ferrocene on the growth of the cells excel that one of the com-
pounds (R)-5 and (S)-5 without ferrocene. Moreover, the starting
materials, compounds (R)-1 and (S)-1, have only weak effects on the
growth of A549 cells.
3. Pharmacology
Screening of synthesized substances and cisplatin used as
reference was carried out using lung cancer A549, H322 and H1299
cell lines. Proliferation percentage was determined by the sulfo-
rhodamine B (SRB) assay. Cells were incubated with substances at
Furthermore, Fig. 5 indicates the cytotoxic effects of the com-
pounds tested at different concentration on the viability of A549,
H322 and H1299 lung cancer cells that were incubated for 48 h. The
viability of 3 cell lines was significantly dose-dependently sup-
20, 40 and 60
mM for 48 h and the cell proliferation/viability
determination using the survival percentage obtained with the cell
treated only with the solvent (DMSO at 0.1%) as reference. The re-
sults are expressed as the average of triplicate assays.
pressed by these compounds. Growth inhibitory properties (IC50
,
Table 2
4. Results and discussion
Summary of crystal data and structure refinement.
Compound
(R)-2a
C25H25FFeN3O2
455.33
Triclinic
P-1
7.6782(7)
10.4352(9)
13.2430(12)
87.263(2)
4.1. Effects of the compounds on the viability of A549 lung cancer
cells
Empirical formula
Formula weight
Crystal system
Space group
In order to evaluate the inhibitory effects of the compounds on
growth of A549 cells we carried out the SRB assay with the
ꢀ
a (A)
ꢀ
b (A)
ꢀ
c (A)
a
b
g
Z
(ꢀ)
(ꢀ)
(ꢀ)
87.660(2)
86.4970(10)
2
Calculated density [Mg/m3]
Crystal size [mm]
1.430
0.08 ꢂ 0.1 ꢂ 0.1
Absorption coefficient [mmꢁ1
F (000)
]
0.741
476
Reflection collected
Data/restraints/parameters
q
3554
2194/54/362
1.54e20.79
Range for data collection [ꢀ]
Ranges of indices h, k, l
ꢁ7 ꢃ h ꢃ 6, ꢁ10 ꢃ k ꢃ 9, ꢁ13 ꢃ l ꢃ 9
Final R indices [I > 2
R indices (all data)
s
(I)]
R1 ¼ 0.0459,
R1 ¼ 0.0554,
1.026
u
u
R2 ¼ 0.1148
R2 ¼ 0.1227
Fig. 1. X-ray crystal structure of compound (R)-2a. The CeH/O intramolecular
hydrogen bond is shown with orange dashed line.(For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)
Goodness of fit on F2
ꢀꢁ3
Dr (max/min) [e A
]
0.748; ꢁ0.351

260
S.-L. Shen et al. / European Journal of Medicinal Chemistry 63 (2013) 256e268
Table 3
Selected bond lengths [A] and angles [ ] for Compound (R)-2a.
between R and S enantiomers are mostly not so significant, so the
chirality of the synthesized compounds almost doesn’t have any
influence in the inhibition on the tested cancer cells. The results
indicated also that compounds 2bed possessed notable cytotox-
icity and selectivity for A549 vs H1299 and H322 lung cancer cells.
ꢀ
ꢀ
Fe(1)eC(10)
Fe(1)eC(9)
Fe(1)eC(8)
Fe(1)eC(7)
Fe(1)eC(6)
Fe(1)eC(5)
Fe(1)eC(4)
Fe(1)eC(3)
Fe(1)eC(2)
Fe(1)eC(1)
O(2)eC(16)
O(1)eC(14)
C(15)eN(3)eC(14)
C(17)eN(2)eC(13)
N(1)eN(2)eC(17)
N(1)eN(2)eC(13)
N(2)eN(1)eC(11)
N(2)eC(17)eC(16)
O(2)eC(16)eC(17)
O(2)eC(16)eC(15)
C(17)eC(16)eC(15)
N(3)eC(15)
N(3)eC(14)
N(2)eN(1)
N(2)eC(17)
N(2)eC(13)
N(1)eC(11)
C(17)eC(16)
C(16)eC(15)
C(14)eC(13)
2.021(7)
2.020(7)
2.033(7)
2.025(6)
2.025(6)
2.040(5)
2.027(5)
2.030(5)
2.034(5)
2.033(6)
1.365(7)
1.242(6)
121.1(5)
126.9(5)
121.0(5)
111.9(4)
104.7(4)
113.8(6)
109.0(6)
112.6(6)
112.1(6)
1.470(8)
1.339(7)
1.346(5)
1.453(7)
1.360(6)
1.345(6)
1.495(9)
1.518(11)
1.473(7)
1.365(7)
1.387(7)
1.464(7)
117.5(7)
122.2(5)
119.7(5)
118.2(5)
123.8(5)
106.6(4)
105.8(5)
111.0(4)
4.2. Compounds induced apoptosis in H322 and H1299 cells
The mode of cell killing induced by most anti-cancer agents is
apoptotic cell death. To detect whether selected compounds result
in apoptosis of the cells, we performed Hoechst 33258 staining
assay. It is well known that DNA fragmentation, chromatin
condensation, cell shrinkage, and membrane blebbing are the
characteristics of apoptotic cells. The chromatin condensation and
DNA fragmentation in the cells were determined by Hoechst 33258
staining under a fluorescence microscope. It was observed that
compounds (S)- and (R)-2bed at the concentration of 40
induced apoptosis in H322 cells greatly (p < 0.05 vs control, Fig. 6).
The compounds (S)- and (R)-2bed at the concentration of 60 M for
mM for 48 h
m
48 h induced apoptosis in H1299 cells greatly (p < 0.05 vs control,
Fig. 7). However, the compounds tested did not induce apoptosis in
A549 cells (data not shown).
4.3. Flow cytometry analysis of cell cycle distribution
The eukaryotic cell cycle consists of alternating rounds of DNA
replication (S phase) and cell division (M phase) separated by the
gap phases G0eG2. It is known that regulation of proteins to
mediate critical events in the cell cycle may be a useful anti-tumor
strategy [64]. Thus, what we need to know is whether the selected
compounds induce A549 cell cycle arrest. The results of cytometry
analysis showed that tested compounds treatment with 40 mM at
48 h induced G1-phase arrest effectively as shown in Fig. 8 which is
one representative experiment from three independent experi-
C(13)eC(12)
C(12)eC(11)
C(11)eC(5)
N(3)eC(15)eC(16)
O(1)eC(14)eN(3)
O(1)eC(14)eC(13)
N(3)eC(14)eC(13)
N(2)eC(13)eC(14)
N(2)eC(13)eC(12)
C(13)eC(12)eC(11)
N(1)eC(11)eC(12)
ments. After 48 h treatment with 40 mM compounds tested, the G1
population was noticeably enhanced by 21e34% compared with
control. The increase in the G1-phase cell population was accom-
panied by a decrease in the S and G2-phase cell populations. The
strong G1-phase arrest induced by tested compounds is concomi-
tant with the growth inhibitory effect.
The compounds might perform their action through inducing a
strong G1-phase arrest rather than apoptosis for A549 cell, whereas
by inducing apoptosis for H322 and H1299 cells.
m
M) of the compounds and cisplatin at 48 h in A549, H322 and
H1299 cell were shown in Table 6. The results indicated that these
compounds had selective effects on the proliferation of A549, H322
and H1299 cells, though their inhibitory effects are inferior to
cisplatin. The compounds possessed more effective inhibition for
A549 than other cell lines. Among them, (R)-2b with methoxy
group in 3-position of benzene moiety showed the most potent
inhibitory effect on A549 cells growth. However, (S)-2d with fluo-
rine group in 2-position of benzene moiety displayed a most potent
growth inhibition for H322 and H1299 cells. The results showed
that the anti-tumor activities of these compounds were related to
the nature of substituents in benzene moiety. Moreover, (R)-2b
possessed more effective inhibition than (S)-2b, for which their IC50
are 27.5 and 38.9 for A549 cell, meanwhile (S)-2d showed more
effective inhibition than (R)-2d, for which their IC50 are 43.8 and
53.9 for H322 cell, respectively. But, the inhibition differences
4.4. Necrosis detection by LDH activity assay
In order to determine if the growth inhibitory effects were due
to necrosis that is believed to be an unwanted side effect of cancer-
fighting agents, LDH assay was performed on cells treated with
compounds (S)- and (R)-2bed or 0.1% DMSO (as control). As shown
in Fig. 9, there were no significant differences in LDH release be-
tween the cells in the control group and the compounds treatment
groups. The results indicated that the compounds at the test range
of concentration did not cause necrosis in the cells.
5. Conclusion
Table 4
We have described a facile approach to prepare 2-ferrocenyl-
7-hydroxy-5-phenethyl-5,6,7,8-tetrahydro-4H-pyrazolo[1,5-a]
[1,4]diazepin-4-one derivatives with optical activity by the reac-
tion of substituted phenylethylamine and ethyl 3-ferrocenyl-1-
(oxiran-2-ylmethyl)-1H-pyrazole-5-carboxylate under the gen-
eral heating condition and the microwave-assisted condition.
Microwave-assisted reaction in solvent-free is fast 50 fold than
general heating method in sealed tube. A representative single-
Hydrogen-bond geometry for (R)-2a.
XeH/A/pa
XeH H/A/
p
X/A/
p
XeH/A/
p
Symmetry codes
O2eH2A/O1
0.82 2.13
0.96 2.28
2.860(6)
2.704(10)
148
106
157(5)
121
1 þ x, y, z
C18AeH18B/O1
C17eH17A/Cg3
C19AeH19A/Cg4
2.70(6) 3.551(8)
2.68 3.274(10)
ꢁx, 1 ꢁ y, ꢁz
1 ꢁ x, ꢁy, 1 ꢁ z
a
Cg3 and Cg4 are the centroids of the cyclopentadienyl ring and the benzene ring,
respectively.

S.-L. Shen et al. / European Journal of Medicinal Chemistry 63 (2013) 256e268
261
Fig. 2. Packing diagram of (R)-2a. Hydrogen bonds and CeH/p interactions are shown with orange, red and black dashed lines.(For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
crystal structural characterization of the compound (R)-2a by X-
ray is valuable for further investigation. Although (R)-2b dis-
played more effective inhibition than (S)-2b for A549 cells, and
(S)-2d was more effect than (R)-2d for H322 cells, the inhibition
differences between R and S enantiomers are mostly not so sig-
nificant. So for the compounds synthesized, chirality almost
doesn’t have any influence in the inhibition on the tested cancer
cells. The anti-tumor activities of these compounds were related
to the nature of substituents in benzene moiety and the com-
pounds substituted with eOMe or eF were more effective than
the eH one. The compounds 2bed possessed notable cytotoxicity
and selectivity for A549 vs H1299 and H322 lung cancer cells due
to the compounds could inhibit growth of A549, H322 and H1299
cells in different mechanism. Compounds (S)- and (R)-2ae
d inhibited growth of A549 cells by inducing a strong G1-phase
arrest. Whereas these compounds inhibited growth of H1299
and H322 cells by inducing apoptosis.
electrode cell equipped with a glassy carbon (GC) electrode
(A ¼ 0.2 cm2) as the working electrode, a platinum sheet with a
surface of 4 cm2 served as the counter electrode, and a saturated
calomel electrode (SCE) as the reference electrode. The CH2Cl2 for
electrochemical use was distilled from CaH2 and deoxygenated by
saturation with N2.
6.2. General procedure for the synthesis of 5-arylethyl-2-ferrocenyl-
7-hydroxy-5,6,7,8-tetrahydro-4H-pyrazolo[1,5-a][1,4]diazepin-4-
one 2 [(S)-2 and (R)-2] by classical heating technique
The mixture of ethyl 3-ferrocenyl-1-(oxiran-2-ylmethyl)-1H-
pyrazole-5-carboxylate 1 (3 mmol) and the corresponding 2-
arylethanamine (3.6 mmol) in absolute alcohol (50 mL) was stir-
red and heated at reflux for 10 h under N2. Then the reaction
mixture was cooled to room temperature and concentrated under
reduced pressure. The residue was purified by column chroma-
tography on silica gel with the eluent system of ethyl acetate/pe-
troleum ether (v/v ¼ 1:1) to afford the product 2 in 20e28% yield.
6. Experimental
6.1. General
Analytical TLC was performed on silica gel 60 F254 plates
(Merck KGaA). Melting points were determined on an XD-4 dig-
ital micromelting point apparatus. 1H NMR spectra were recorded
on a Bruker Avance 300 (300 MHz) spectrometer, using DMSO as
solvent and tetramethylsilane (TMS) as internal standard. IR
spectra were recorded with an IR spectrophotometer Avtar 370
FT-IR (Thermo Nicolet). Electrospray ionization mass spectrom-
etry (ESI-MS) spectra were recorded on a LTQ Orbitrap Hybrid
mass spectrograph. All microwave-assisted reactions were carried
out in a START SYNTH Microwave Synthesis Lab station. Cyclic
voltammetry measurements were performed on
a CHI760C
electrochemical workstation (Shanghai China) using a three-
Table 5
Electrochemical data for compounds (R)- and (S)-2aed obtained at a scan rate of
0.2 V/s at RT in CH2Cl2/Bu4NPF6 (0.1 M) as the supporting electrolyte.
Compound (S)-2a (S)-2b (S)-2c (S)-2d (R)-2a (R)-2b (R)-2c (R)-2d
0
E0 [V]a
0.451 0.453 0.452 0.459 0.456 0.455 0.456 0.454
Fig. 3. Cyclic voltammograms of (S)- and (R)-2a-d in CH2Cl2/NBu4PF6 (0.1 M) at a scan
rate of 0.2 V/s at RT.
0
a
E0 ¼ (Epa þ Epc)/2.

262
S.-L. Shen et al. / European Journal of Medicinal Chemistry 63 (2013) 256e268
Table 6
Growth inhibitory properties for the compounds at 48 h in A549, H322 and H1299
lung cancer cells.
Compounds
IC50
(
mM) ꢅ SD
A549
H322
H1299
(S)-2b
(S)-2c
(S)-2d
(R)-2b
(R)-2c
(R)-2d
(S)-1
(R)-1
(S)-5
(S)-5
Cisplatin
38.9 ꢅ 4.05
35.4 ꢅ 2.50
33.2 ꢅ 6.68
27.5 ꢅ 7.29
35.6 ꢅ 9.37
34.6 ꢅ 4.33
96.9 ꢅ 0.04
135.7 ꢅ 0.03
118.9 ꢅ 0.02
100.5 ꢅ 0.03
14.79 ꢅ 0.45
60.8 ꢅ 3.22
53.6 ꢅ 12.1
43.8 ꢅ 15.6
61.5 ꢅ 9.53
57.7 ꢅ 6.91
53.9 ꢅ 10.0
e
70.6 ꢅ 5.59
62.7 ꢅ 3.73
52.3 ꢅ 11.9
65.3 ꢅ 1.43
62.7 ꢅ 4.36
56.4 ꢅ 4.16
e
e
e
e
e
Fig. 4. Effects of the compounds on A549 cell viability. A549 cells were treated with
compounds (S)- and (R)-2aed, (S)- and (R)-1, (S)- and (R)-5, cisplatin or 0.1% DMSO
(control) for 48 h at the concentration of 40 mM.
e
e
21.15 ꢅ 0.69
45.42 ꢅ 1.67
Data are shown as the mean values of three independent experiments with the
corresponding standard deviation (SD), “e” not determined.
6.3. General procedure for the synthesis of 5-arylethyl-2-ferrocenyl-
7-hydroxy-5,6,7,8-tetrahydro-4H-pyrazolo[1,5-a][1,4]diazepin-4-
one 2 [(S)-2 and (R)-2] in sealed tube
stirred in a sealed tube and heated in an oil bath at 140 ꢀC for 8e
12 h. Then the reaction mixture was cooled to room temperature
and concentrated under reduced pressure. The residue was purified
by column chromatography on silica gel with the eluent system of
ethyl acetate/petroleum ether (v/v ¼ 1:1) to afford the product 2 in
62e82% yield.
The solution of compound 1 (0.5 mmol) and the corresponding
2-arylethanamine (0.6 mmol) in absolute alcohol (10 mL) was
6.4. General procedure for the synthesis of 5-arylethyl-2-ferrocenyl-
7-hydroxy-5,6,7,8-tetrahydro-4H-pyrazolo[1,5-a][1,4]diazepin-4-
one 2 [(S)-2 and (R)-2] by microwave-assisted technique
To a glass vessel, compound 1 (0.5 mmol) and the corresponding
2-arylethanamine (0.6 mmol) was added and then irradiated in the
microwave cavity, keeping the reaction temperature at 70 ꢀC for
10e15 min. Then the reaction mixture was cooled to room tem-
perature and purified by column chromatography on silica gel with
the eluent system of ethyl acetate/petroleum ether (v/v ¼ 1:1) to
afford the product 2 in 72e81% yield.
6.4.1. (S)-2-ferrocenyl-7-hydroxy-5-phenethyl-5,6,7,8-tetrahydro-
4H-pyrazolo[1,5-a][1,4]diazepin-4-one [(S)-2a]
Yellow solid, mp 218e220 ꢀC, ½a D30
¼ þ20:2 (c ¼ 0.4, CHCl3). IR
ꢄ
(KBr), n
/cmꢁ1: 3425 (OeH), 2941, 2869 (CeH), 1609 (CaO), 1526
(CaN), 1454, 1269, 1177, 810, 756, 506, 485 (CpFeCp). 1H NMR:
d
2.92 (t, J ¼ 7.5 Hz, 2H, CH2), 3.06 (dd, J ¼ 14.8 Hz, 7.0 Hz, 1H, CH2),
3.38 (dd, J ¼ 14.8 Hz, 4.6 Hz, 1H, CH2), 3.58 (dt, J ¼ 13.5 Hz, 7.5 Hz,
1H, CH2), 3.87 (dt, J ¼ 13.5 Hz, 7.5 Hz, 1H, CH2), 4.04 (s, 5H, C5H5),
4.07 (dd, J ¼ 12.0 Hz, 3.8 Hz, 1H, CH2), 4.20e4.30 (m, 2H, CH2, CH),
4.27 (t, J ¼ 1.8 Hz, 2H, C5H4), 4.70 (t, J ¼ 1.8 Hz, 2H, C5H4), 5.47 (d,
J ¼ 3.9 Hz, 1H, OH), 6.82 (s, 1H, 4-H), 7.20e7.35 (m, 5H, ArH).
HRMS calcd for
456.1363.
C25H26FeN3O2 [M
þ
H]þ: 456.1374, found:
6.4.2. (S)-2-ferrocenyl-7-hydroxy-5-(3-methoxyphenethyl)-5,6,7,8-
tetrahydro-4H-pyrazolo[1,5-a][ꢀ1,4]diazepin-4-one [(S)-2b]
a 30
Yellow solid, mp 155e158 C, ½ ꢄD ¼ þ21:9 (c ¼ 0.4, CHCl3). IR
(KBr), n
/cmꢁ1: 3440 (OeH), 2923, 2832 (CeH), 1643 (CaO), 1522
(CaN), 1455, 1481, 1258, 998, 786, 507, 482 (CpFeCp). 1H NMR:
2.89 (t, J ¼ 7.5 Hz, 2H, CH2), 3.05 (dd, J ¼ 15.0 Hz, 6.9 Hz, 1H, CH2),
d
3.38 (dd, J ¼ 15.0 Hz, 4.8 Hz, 1H, CH2), 3.57 (dt, J ¼ 13.5 Hz, 7.5 Hz,
1H, CH2), 3.75 (s, 3H, CH3), 3.86 (dt, J ¼ 13.5 Hz, 7.5 Hz, 1H, CH2),
4.04 (s, 5H, C5H5), 4.02e4.10 (m, 1H, CH2), 4.20e4.33 (m, 2H, CH2,
CH), 4.28 (t, J ¼ 1.8 Hz, 2H, C5H4), 4.70 (t, J ¼ 1.8 Hz, 2H, C5H4), 5.48
(d, J ¼ 3.9 Hz,1H, OH), 6.78e6.87 (m, 3H, ArH), 6.82 (s,1H, 4-H), 7.23
(t, J ¼ 8.1 Hz, 1H, ArH). HRMS calcd for C26H28FeN3O3 [M þ H]þ:
486.1480, found: 486.1485.
Fig. 5. Effects of the compounds on A549, H322 and H1299 cell viability. Cells were
treated with compounds (S)- and (R)-2bed and cisplatin (reference) or 0.1% DMSO
(control) for 48 h at the concentration of 20, 40 or 60 mM respectively. A: A549; B:
H322; C: H1299. Cell viability was analyzed by SRB assay. Data are mean ꢅ SD from
three independent experiments (*, p < 0.05 and **, p < 0.01 vs control).

S.-L. Shen et al. / European Journal of Medicinal Chemistry 63 (2013) 256e268
263
Fig. 6. Compounds induced H322 cell apoptosis significantly. A: Fluorescent micrographs of Hoechst 33258 staining (200ꢂ). The cells were treated with DMSO 0.1% (v/v) as a vehicle
control and were treated with the compounds (S)- and (R)-2bed at concentrations of 40
(n ¼ 3. *, p < 0.05 vs control).
m
M respectively; B: Ratio of apoptotic cells vs control. Results are presented as mean ꢅ SD;
Fig. 7. Compounds induced H1299 cell apoptosis significantly. A: Fluorescent micrographs of Hoechst 33258 staining (200ꢂ). The cells were treated with DMSO 0.1% (v/v) as a
vehicle control and were treated with the compounds (S)- and (R)-2bed at concentrations of 60
mean ꢅ SD; (n ¼ 3. *, p < 0.05 vs control; **, p < 0.01 vs control).
mM respectively. B: Ratio of apoptotic cells vs control. Results are presented as

Fig. 8. Effect of compounds (S)- and (R)-2bed on cell cycle distribution of A549 lung cancer cells. Cells were exposed to the compounds at 40
mM and incubated for 48 h, and then were stained with PI. Values are expressed as
percentage of the cell population in the G1, S, and G2 phase of cell cycle. A: Control; B: (S)-2b; C: (S)-2c; D: (S)-2d; E: (R)-2b; F: (R)-2c; G: (R)-2d.

S.-L. Shen et al. / European Journal of Medicinal Chemistry 63 (2013) 256e268
265
Fig. 9. Effects of the compounds on LDH release in A549, H322 and H1299 cells. The cells were treated with compounds (S)- and (R)-2bed at concentrations of 40
untreated (control) for 48 h. LDH assay results are presented as mean ꢅ SD; n ¼ 3.
mM or left
6.4.3. (S)-2-ferrocenyl-5-(4-fluorophenethyl)-7-hydroxy-5,6,7,8-
tetrahydro-4H-pyrazolo[1,5-a][1,4]diazepin-4-one [(S)-2c]
128.4(2C), 126.2, 105.8, 78.0, 69.4, 69.2(5C), 68.2(2C), 66.2, 66.1,
55.3, 52.7, 49.2, 33.7. HRMS calcd for C25H26FeN3O2 [M þ H]þ:
Yellow solid, mp 162e165 ꢀC, ½a D30
ꢄ
¼ þ15:6 (c ¼ 0.4, CHCl3). IR
456.1374, found: 456.1375.
(KBr), n
/cmꢁ1: 3342 (OeH), 3098, 2936 (CeH), 1613 (CaO), 1529
(CaN), 1507, 1364, 1218, 815, 503, 486 (CpFeCp). 1H NMR:
d
2.91 (t,
6.4.6. (R)-2-ferrocenyl-7-hydroxy-5-(3-methoxyphenethyl)-
J ¼ 7.5 Hz, 2H, CH2), 3.07 (dd, J ¼ 15.0 Hz, 4.2 Hz, 1H, CH2), 3.39 (dd,
J ¼ 15.0 Hz, 5.1 Hz, 1H, CH2), 3.57 (dt, J ¼ 13.2 Hz, 7.5 Hz, 1H, CH2),
3.83 (dt, J ¼ 13.2 Hz, 7.5 Hz, 1H, CH2), 4.04 (s, 5H, C5H5), 4.07 (dd,
J ¼ 14.1 Hz, 3.9 Hz, 1H, CH2), 4.17e4.20 (m, 1H, CH), 4.27 (t,
J ¼ 1.8 Hz, 2H, C5H4), 4.29 (dd, J ¼ 14.1 Hz, 5.4 Hz, 1H, CH2), 4.69 (t,
J ¼ 1.8 Hz, 2H, C5H4), 6.81 (s, 1H, 4-H), 5.49 (d, J ¼ 3.9 Hz, 1H, OH),
7.14 (t, J ¼ 8.9 Hz, 2H, ArH), 7.33 (t, J ¼ 7.1 Hz, 2H, ArH). HRMS calcd
for C25H25FFeN3O2 [M þ H]þ: 474.1280, found: 474.1286.
5,6,7,8-tetrahydro-4H-pyrazolo[1,5-a][1,4]diazepin-4-one [(R)-2b]
Yellow solid, mp 158e160 ꢀC, ½a D30
¼ ꢁ22:3 (c ¼ 0.4, CHCl3). IR
ꢄ
(KBr), n
/cmꢁ1: 3420 (OeH), 3097, 2927, 2869 (CeH), 1644 (CaO),
1526 (CaN),1457,1258,1161,1029, 787, 508, 483 (CpFeCp). 1H NMR:
2.89 (t, J ¼ 7.5 Hz, 2H, CH2), 3.05 (dd, J ¼ 15.0 Hz, 6.9 Hz, 1H, CH2),
d
3.38 (dd, J ¼ 15.0 Hz, 4.8 Hz, 1H, CH2), 3.57 (dt, J ¼ 13.5 Hz, 7.5 Hz,
1H, CH2), 3.75 (s, 3H, CH3), 3.86 (dt, J ¼ 13.5 Hz, 7.5 Hz, 1H, CH2),
4.04 (s, 5H, C5H5), 4.02e4.10 (m, 1H, CH2), 4.20e4.33 (m, 2H, CH2,
CH), 4.28 (t, J ¼ 1.8 Hz, 2H, C5H4), 4.70 (t, J ¼ 1.8 Hz, 2H, C5H4), 5.47
(d, J ¼ 3.9 Hz,1H, OH), 6.78e6.87 (m, 3H, ArH), 6.82 (s,1H, 4-H), 7.23
(t, J ¼ 8.1 Hz, 1H, ArH). HRMS calcd for C26H28FeN3O3 [M þ H]þ:
486.1480, found: 486.1468.
6.4.4. (S)-5-(2-fluorophenethyl)-7-hydroxy-2-ferrocenyl-5,6,7,8-
tetrahydro-4H-pyrazolo[1,5-a][1,4]diazepin-4-one [(S)-2d]
Yellow solid, mp 177e178 ꢀC, ½a D30
¼ þ15:0 (c ¼ 0.4, CHCl3). IR
ꢄ
(KBr), n
/cmꢁ1: 3402 (OeH), 3085, 2949, 2866 (CeH), 1621 (CaO),
1532 (CaN), 1461, 1420, 1104, 758, 510, 486 (CpFeCp). 1H NMR:
2.96 (t, J ¼ 7.5 Hz, 2H, CH2), 3.08 (dd, J ¼ 15.0 Hz, 6.9 Hz, 1H, CH2),
6.4.7. (R)-2-ferrocenyl-5-(4-fluorophenethyl)-7-hydroxy-5,6,7,8-
tetrahydro-4H-pyrazolo[1,5-a][1,4]diazepin-4-one [(R)-2c]
d
3.37 (dd, J ¼ 15.0 Hz, 5.1 Hz, 1H, CH2), 3.61 (dt, J ¼ 13.2 Hz, 7.5 Hz,
1H, CH2), 3.82 (dt, J ¼ 13.2 Hz, 7.5 Hz, 1H, CH2), 4.04 (s, 5H, C5H5),
4.08 (dd, J ¼ 14.1 Hz, 4.2 Hz,1H, CH2), 4.20e4.27 (m,1H, CH), 4.28 (t,
J ¼ 1.8 Hz, 2H, C5H4), 4.30 (dd, J ¼ 14.1 Hz, 5.4 Hz, 1H, CH2), 4.70 (t,
J ¼ 1.8 Hz, 2H, C5H4), 5.51 (d, J ¼ 4.2 Hz, 1H, OH), 6.81 (s, 1H, 4-H),
7.15(d, J ¼ 7.2 Hz, 1H, ArH), 7.19 (d, J ¼ 8.1 Hz, 1H, ArH), 7.29 (t,
J ¼ 7.2 Hz, 1H, ArH), 7.38 (t, J ¼ 7.5 Hz, 1H, ArH). HRMS calcd for
Yellow solid, mp 172e175 ꢀC, ½a D30
¼ ꢁ14:2 (c ¼ 0.4, CHCl3). IR
ꢄ
(KBr), n
/cmꢁ1: 3348 (OeH), 3098, 2936 (CeH), 1613 (CaO), 1530
(CaN), 1408, 1365, 1217, 817, 503, 486 (CpFeCp). 1H NMR:
d 2.91 (t,
J ¼ 7.5 Hz, 2H, CH2), 3.07 (dd, J ¼ 15.0 Hz, 4.2 Hz, 1H, CH2), 3.39 (dd,
J ¼ 15.0 Hz, 5.1 Hz, 1H, CH2), 3.57 (dt, J ¼ 13.2 Hz, 7.5 Hz, 1H, CH2),
3.83 (dt, J ¼ 13.2 Hz, 7.5 Hz, 1H, CH2), 4.04 (s, 5H, C5H5), 4.07 (dd,
J ¼ 13.8 Hz, 3.9 Hz, 1H, CH2), 4.17e4.20 (m, 1H, CH), 4.27 (t,
J ¼ 1.8 Hz, 2H, C5H4), 4.29 (dd, J ¼ 14.1 Hz, 5.4 Hz, 1H, CH2), 4.69 (t,
J ¼ 1.8 Hz, 2H, C5H4), 6.81 (s, 1H, 4-H), 5.48 (d, J ¼ 3.9 Hz, 1H, OH),
7.14 (t, J ¼ 8.9 Hz, 2H, ArH), 7.33 (t, J ¼ 7.1 Hz, 2H, ArH). HRMS calcd
for C25H25FFeN3O2 [M þ H]þ: 474.1280, found: 474.1263.
C
25H25FFeN3O2 [M þ H]þ: 474.1280, found: 474.1283.
6.4.5. (R)-2-ferrocenyl-7-hydroxy-5-phenethyl-5,6,7,8-tetrahydro-
4H-pyrazolo[1,5-a][1,4]diazepin-4-one [(R)-2a]
Yellow solid, mp 219e220 ꢀC, ½a D30
¼ ꢁ20:4 (c ¼ 0.4, CHCl3). IR
ꢄ
(KBr),
n
/cmꢁ1: 3398 (OeH), 2923, 2869 (CeH), 1607 (CaO), 1526
6.4.8. (R)-2-ferrocenyl-5-(2-fluorophenethyl)-7-hydroxy-5,6,7,8-
tetrahydro-4H-pyrazolo[1,5-a][1,4]diazepin-4-one [(R)-2d]
(CaN), 1454, 1266, 1177, 810, 756, 506, 484 (CpFeCp). 1H NMR:
d
2.92 (t, J ¼ 7.5 Hz, 2H, CH2), 3.06 (dd, J ¼ 15.0 Hz, 7.0 Hz, 1H, CH2),
Yellow solid, mp 180e183 ꢀC, ½a D30
¼ ꢁ15:6 (c ¼ 0.4, CHCl3). IR
ꢄ
3.38 (dd, J ¼ 14.8 Hz, 4.8 Hz, 1H, CH2), 3.58 (dt, J ¼ 13.5 Hz, 7.5 Hz,
1H, CH2), 3.86 (dt, J ¼ 13.5 Hz, 7.5 Hz, 1H, CH2), 4.04 (s, 5H, C5H5),
4.07 (dd, J ¼ 12.0 Hz, 3.6 Hz, 1H, CH2), 4.19e4.30 (m, 2H, CH2, CH),
4.27 (t, J ¼ 1.8 Hz, 2H, C5H4), 4.70 (t, J ¼ 1.8 Hz, 2H, C5H4), 5.47 (d,
J ¼ 3.9 Hz, 1H, OH), 6.82 (s, 1H, 4-H), 7.20e7.35 (m, 5H, ArH); 13C
(KBr), n
/cmꢁ1: 3401 (OeH), 3086, 2944, 2870 (CeH), 1613 (CaO),
1529 (CaN), 1456, 1232, 1102, 757, 508, 486 (CpFeCp). 1H NMR:
d
2.96 (t, J ¼ 7.5 Hz, 2H, CH2), 3.08 (dd, J ¼ 15.0 Hz, 6.6 Hz, 1H, CH2),
3.37 (dd, J ¼ 15.0 Hz, 4.8 Hz, 1H, CH2), 3.61 (dt, J ¼ 13.5 Hz, 7.5 Hz,
1H, CH2), 3.82 (dt, J ¼ 13.5 Hz, 7.5 Hz, 1H, CH2), 4.04 (s, 5H, C5H5),
4.08 (dd, J ¼ 14.1 Hz, 3.9 Hz,1H, CH2), 4.20e4.27 (m, 1H, CH), 4.28 (t,
NMR (DMSO, 75 MHz):
d 161.6, 148.5, 138.9, 138.5, 128.7(2C),

266
S.-L. Shen et al. / European Journal of Medicinal Chemistry 63 (2013) 256e268
J ¼ 1.8 Hz, 2H, C5H4), 4.30 (dd, J ¼ 14.1 Hz, 5.4 Hz, 1H, CH2), 4.70 (t,
J ¼ 1.8 Hz, 2H, C5H4), 5.51 (d, J ¼ 4.2 Hz, 1H, OH), 6.81 (s, 1H, 4-H),
7.15(d, J ¼ 7.2 Hz, 1H, ArH), 7.19 (d, J ¼ 8.1 Hz, 1H, ArH), 7.29 (t,
J ¼ 7.2 Hz, 1H, ArH), 7.38 (t, J ¼ 7.5 Hz, 1H, ArH). HRMS calcd for
approximate dimensions of 0.08 mm ꢂ 0.10 mm ꢂ 0.08 mm for 2a
were mounted on a Bruker Smart Apex II CCD equipped with a
ꢀ
graphite monochromated MoK
a
radiation (
l
¼ 0.71073 A) by using
4 and
u
scan modes and the data were collected at 298(2) K. The
C
25H25FFeN3O2 [M þ H]þ: 474.1280, found: 474.1246.
structure of the crystal was solved by direct methods and refined
by full-matrix least-squares techniques implemented in the
SHELXTL-97 crystallographic software [65]. The non-hydrogen
atoms were refined anisotropically. The hydrogen atoms bound
to carbon were located by geometrical calculations, with their
position and thermal parameters being fixed during the structure
refinement. Molecular graphics were designed by using XP and
DIAMOND 3.2.
6.4.9. Diethyl 1,10-((2R,20R)-(phenethylazanediyl)bis(2-
hydroxypropane-3,1-diyl))bis(3-ferrocenyl-1H-pyrazole-5-
carboxylate) [(R)-3a]
Red oil. IR (KBr),
n
/cmꢁ1: 3415 (OeH), 3088, 2939 (CeH), 1719
(CaO), 1457, 1258, 1085, 816, 764, 699, 486 (CpFeCp). 1H NMR:
d
1.32 (t, J ¼ 7.2 Hz, 6H, CH3), 2.48e2.56 (m, 4H, CH2), 2.58e2.71 (m,
4H, CH2), 3.99e4.05 (m, 2H, CH), 4.01 (s, 10H, C5H5), 4.27 (t,
J ¼ 1.8 Hz, 4H, C5H4), 4.29 (q, J ¼ 7.2 Hz, 4H, CH2), 4.48e4.60 (m, 4H,
CH2), 4.74 (t, J ¼ 1.8 Hz, 4H, C5H4), 4.81 (d, J ¼ 4.8 Hz, 2H, OH), 6.97
(s, 2H, 4-H), 7.20 (m, 5H, ArH). HRMS calcd for C46H52Fe2N5O6
[M þ H]þ: 882.2616, found: 882.2600.
6.6. Cell culture
A549, H322 and H1299 lung cancer cells were cultured in RPMI
(Roswell Park Memorial Institute) 1640 medium at 37 ꢀC with 5%
CO2, and 95% air, supplemented with 10% (v/v) bovine calf serum
and 80 U/mL gentamicin. The cells were seeded onto 96-well plates
or other appropriate dishes containing the medium at the cell
density of 6250/cm2.
6.4.10. (R)-ethyl 3-ferrocenyl-1-(2-hydroxy-3-(phenethylamino)
propyl)-1H-pyrazole-5-carboxylate [(R)-4a]
Red oil. IR (KBr),
1719 (CaO), 1564 (CaN), 1457, 1384, 1260, 1085, 1006, 817, 699, 486
(CpFeCp). 1H NMR:
n
/cmꢁ1: 3420 (OeH), 3088, 2936, 2833 (CeH),
d
1.33 (t, J ¼ 7.2 Hz, 3H, CH3), 2.65e2.72 (m, 2H,
6.7. Cell viability assay
CH2), 2.75e2.81 (m, 2H, CH2), 2.86e2.94 (m, 2H, CH2), 4.00e4.08
(m, 1H, CH), 4.04 (s, 5H, C5H5), 4.29 (t, J ¼ 1.8 Hz, 2H, C5H4), 4.32
(q, J ¼ 7.2 Hz, 2H, CH2), 4.47 (dd, J ¼ 13.5, 5.7 Hz, 1H, CH2), 4.57 (dd,
J ¼ 13.5, 6.6 Hz, 1H, CH2), 4.74 (t, J ¼ 1.8 Hz, 2H, C5H4), 7.00 (s, 1H, 4-
H), 7.17e7.31 (m, 5H, ArH). HRMS calcd for C27H32FeN3O3, [M þ H]þ:
502.1793, found: 502.1786.
Cells were seeded in 96-well plates. After 48 h, cells were
treated with 0.1% DMSO (as control) or the compounds at indicated
concentrations for specified time durations. The compounds were
dissolved in DMSO as stock solution and applied to cells such that
the final concentration of DMSO in the culture medium was below
0.1% (v/v). DMSO at a concentration of 0.1% (v/v) did not affect the
viability of the cells. Cell viability was evaluated by sulforhodamine
B (SRB) assay. Briefly, the medium was poured off and the cells were
6.4.11. (R)-7-hydroxy-5-(3-methoxyphenethyl)-2-phenyl-5,6,7,8-
tetrahydropyrazolo[1,5-a][1,4]diazepin-4-one [(R)-5]
White solid, yield 66%, mp 149e150 ꢀC, ½a D30
¼ ꢁ18:8 (c ¼ 0.4,
ꢄ
fixed by adding 100
incubating for 1 h at 4 ꢀC. The supernatant was discarded and then
the plates were washed 5 times with deionized water. 50 L of 0.4%
mL of cold 10% trichloroacetic acid (TCA) and
CHCl3). IR (KBr), n
/cmꢁ1: 3422 (OeH), 2922 (CeH), 1665 (CaO),
1438, 1252, 1050, 772, 689. 1H NMR:
d
2.90 (t, J ¼ 7.5 Hz, 2H, CH2),
m
3.08 (dd, J ¼ 15.0 Hz, 6.9 Hz, 1H, CH2), 3.41 (dd, J ¼ 15.0 Hz, 5.1 Hz,
1H, CH2), 3.59 (dt, J ¼ 13.2 Hz, 7.5 Hz, 1H, CH2), 3.75 (s, 3H, OCH3),
3.87 (dt, J ¼ 13.2 Hz, 7.5 Hz, 1H, CH2), 4.16 (dd, J ¼ 13.8 Hz, 3.0 Hz,
1H, CH2), 4.20e4.29 (m, 1H, CH), 4.34 (dd, J ¼ 13.8 Hz, 4.8 Hz, 1H,
CH2), 5.53 (d, J ¼ 3.9 Hz, 1H, OH), 6.80 (d, J ¼ 9.0 Hz, 1H, ArH), 6.86
(d, J ¼ 6.9 Hz, 1H, ArH), 6.87 (s, 1H, 4-H), 7.17 (s, 1H, ArH), 7.23 (t,
J ¼ 7.8 Hz,1H, ArH), 7.31 (t, J ¼ 7.2 Hz,1H, ArH), 7.41 (t, J ¼ 7.5 Hz, 2H,
ArH), 7.84 (d, J ¼ 7.8 Hz, 2H, ArH). HRMS calcd for C22H24N3O3
[M þ H]þ: 378.1818, found: 378.1806.
(W/V) SRB solution in 1% acetic acid was added to each well and
shaken for 5 min on a titer plate shaker. The plates were washed 5
times with 1% acetic acid and subsequently 100 mL of 10 mM un-
buffered Tris base (pH 10.5) was added to dissolve the bound dye.
After being mixed for 5 min on a microtiter plate shaker, optical
densities were read at the wavelength of 510 nm using a Spec-
traMAX 190 microplate spectrophotometer (GMI Co., USA).
6.8. Hoechst 33258 staining to detect apoptosis
6.4.12. (S)-7-hydroxy-5-(3-methoxyphenethyl)-2-phenyl-5,6,7,8-
For A549, the living cells were stained with 10 mg/mL of Hoechst
tetrahydropyrazolo[1,5-a][1,4]diazepin-4-one [(S)-5]
33258 in the medium for 10 min at 37 ꢀC. For H322 and H1299, cell
White solid, yield 64%, mp 147e148 ꢀC, ½a D30
¼ þ18:1 (c ¼ 0.4,
ꢄ
CHCl3). IR (KBr), n
/cmꢁ1: 3422 (OeH), 2923 (CeH), 1665 (CaO),
fixation was done with 4% formaldehyde in phosphate buffered
saline (PBS) for 10 min before staining with 2 mg/mL of Hoechst
1438, 1152, 1050, 772, 690. 1H NMR:
d
2.90 (t, J ¼ 7.5 Hz, 2H, CH2),
33258 at 37 ꢀC for 30 min. Subsequently, the cells were gently
washed once with PBS, and were then observed under a fluores-
cence microscope (Nikon). The condensed DNA of apoptotic cells
was identified by intense local staining in the nucleus, in contrast to
diffused staining of DNA in normal cells. A minimum of 500 cells
was counted, and each experiment was performed in triplicate.
3.08 (dd, J ¼ 15.0 Hz, 7.2 Hz, 1H, CH2), 3.41 (dd, J ¼ 15.0 Hz, 5.1 Hz,
1H, CH2), 3.59 (dt, J ¼ 13.2 Hz, 7.5 Hz, 1H, CH2), 3.75 (s, 3H, OCH3),
3.87 (dt, J ¼ 13.2 Hz, 7.5 Hz, 1H, CH2), 4.16 (dd, J ¼ 13.8 Hz, 3.3 Hz,
1H, CH2), 4.20e4.29 (m, 1H, CH), 4.34 (dd, J ¼ 13.8 Hz, 4.8 Hz, 1H,
CH2), 5.53 (d, J ¼ 3.9 Hz, 1H, OH), 6.80 (d, J ¼ 9.0 Hz, 1H, ArH), 6.86
(d, J ¼ 7.2 Hz, 1H, ArH), 6.87 (s, 1H, 4-H), 7.17 (s, 1H, ArH), 7.23 (t,
J ¼ 7.8 Hz,1H, ArH), 7.31 (t, J ¼ 7.2 Hz,1H, ArH), 7.41 (t, J ¼ 7.5 Hz, 2H,
ArH), 7.84 (d, J ¼ 7.5 Hz, 2H, ArH). HRMS calcd for C22H24N3O3
[M þ H]þ: 378.1818, found: 378.1807.
6.9. Flow cytometry analysis of cell cycle distribution
Cells were harvested and then fixed with 70% ethanol, stained
6.5. X-ray crystallography
with 50 mg/mL propidium iodide (PI) containing 10 mg/mL RNase A
at 4 ꢀC for 1 h. The stained cells were analyzed using a FACS
(Fluorescence Activated Cell Sorter) Calibur flow cytometer (BD
Bioscience, USA). Cell cycle distribution was analyzed by ModiFit
software (BD Bioscience, USA).
Suitable single crystals of 2a for X-ray structural analysis were
obtained by slow evaporation of a solution of the solid in ethyl
acetate at room temperature for 10 days. The crystals with

S.-L. Shen et al. / European Journal of Medicinal Chemistry 63 (2013) 256e268
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activity
relationships
of
novel
1-arylmethyl-3-aryl-1H-pyrazole-5-