Remarkable anti-breast cancer activity of ferrocene tagged multi-functionalized 1,4-dihydropyrimidines

Article abstract of DOI:10.1016/j.ejmech.2013.04.021

A novel series of ferrocene tagged multi-functionalized 1,4-dihydropyrimidines is synthesized by base catalyzed cyclocondensation between ferrocenyl chalcones and amidines. The structures of synthesized compounds were established on the basis of ¹H NMR, ¹³C NMR, FTIR spectroscopy as well as by mass spectrometry. The compounds were evaluated for in vitro anticancer activity. The most active compounds from the series displayed GI₅₀ value equal to doxorubicin against the strain of human breast cancer cell line MDA-MB-435.

Full text of DOI:10.1016/j.ejmech.2013.04.021

European Journal of Medicinal Chemistry 65 (2013) 232e239
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Remarkable anti-breast cancer activity of ferrocene tagged
multi-functionalized 1,4-dihydropyrimidines
Jagannath Jadhav a, Aarti Juvekar b, Rajanikant Kurane a, Sharanabasappa Khanapure a,
,
Rajashri Salunkhe a, Gajanan Rashinkar a
*
a Department of Chemistry, Shivaji University, Kolhapur 416004, M.S., India
b Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Navi Mumbai 410210, M.S., India
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of ferrocene tagged multi-functionalized 1,4-dihydropyrimidines is synthesized by base
catalyzed cyclocondensation between ferrocenyl chalcones and amidines. The structures of synthesized
compounds were established on the basis of 1H NMR, 13C NMR, FTIR spectroscopy as well as by mass
spectrometry. The compounds were evaluated for in vitro anticancer activity. The most active compounds
from the series displayed GI50 value equal to doxorubicin against the strain of human breast cancer cell
line MDA-MB-435.
Received 27 January 2013
Received in revised form
29 March 2013
Accepted 8 April 2013
Available online 28 April 2013
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
Ferrrocene
1,4-Dihydropyrimidines
Ferrocenyl chalcones
Amidines
Anti breast cancer activity
1. Introduction
more effective than tamoxifen against both estrogen-dependent
and -independent breast cancer. These findings have stimulated
The advent of ferrocene through the serependitious discovery
by Pauson and Kealy in 1951 has sparked off a significant inter-
disciplinary research activity [1]. The unique structure with Feþ2
ion sandwiched between two pentahapto cyclopentadienyl ligands
and three dimensional aromatic system has made ferrocene a
versatile scaffold for covalent functionalization. Owing to its unique
geometry, tunable redox properties and the high reactivity, ferro-
cenyl compounds find wide applications in diverse areas such as
catalysis [2], materials science [3], analytical sensors [4], non linear
optics [5], bio-organometallic and biological chemistry [6], organic
synthesis [7] and in electrochemistry [8]. The relative stability of
ferrocene in biological media and low levels of in vivo toxicity of its
derivatives has spurred considerable interest in the development of
ferrocene based compounds for cancer therapeutics. The inclusion
of ferrocene in anticancer drug design strategies received a decisive
impetus as Jaouen and co-workers [9] reported on ferrocenyl an-
alogs of tamoxifen viz ferrocifen (1, Fig. 1) and hydroxyferrocifen (2,
Fig. 1). In vitro cytotoxicity assays have revealed that ferrocifen is
enormous interest in design and synthesis of new lead structures
based on ferrocenyl moiety in drug discovery programs aimed at
the development of potential anticancer agents [10]. It is believed
that ferrocene tagged organic frameworks can provide new
impetus for building structures with unprecedented attributes to
probe and modulate anticancer activities.
The 1,4-dihydropyrimidine (1,4-DHPM) core is ubiquitous in both
naturally occurring and synthetic biologically active molecules and
many of its derivatives display interesting anti-viral, anti-tumor, anti-
bacterial, and anti-inflammatory activities [11,12]. In addition, they
are also regarded as calcium channel blockers [13], a-adrenergic an-
tagonists as well as HIV gp-120-CD4 inhibitors [14]. Recently, two
prototype members of 1,4-DHPM family viz Bay 41-4109 [methyl-4-
(2-chloro-4-fluorophenyl)-2-(3,5-difluoro-2-pyridinyl)-6-methyl-1,
4-dihdro pyrimidine-5-carboxylate] (3, Fig. 1) and Bay 39-5493
[methyl-4-(2-chloro-4-fluorophenyl)-2-(-2-thiazyl)-6-methyl-1,4-di
hdro-pyrimidine-5-carboxylate] (4, Fig. 1) have displayed prominent
anti-hepatitis B replication activity [15]. Moreover, 1,4-DHPM de-
rivatives such as 5 and 6 (Fig. 1) have been proved as selective ROCK1
inhibitors. ROCK1 is a potential therapeutic target in the treatment of
cardiovascular diseases [16]. The anticancer potential of 1,4-DHPMs
has been explored recently [17]. These compounds owe their anti-
cancer activity to their ability to inhibit human kinesin Eg5 activity.
* Corresponding author. Tel.: þ91 231 260 9169; fax: þ91 231 2692333.
E-mail addresses: gsr_chem@unishivaji.ac.in, gajanan.rashinkar@rediffmail.com
(G. Rashinkar).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.04.021

J. Jadhav et al. / European Journal of Medicinal Chemistry 65 (2013) 232e239
233
R
F
F
Cl
O
Cl
N
O
MeO
N
MeO
N
F
Fe
S
Me
N
H
Me
N
H
O
( )
n
N
N
F
3
4
1
2
R= H Ferrocifen
Bay 41-4109
Bay 39-5493
R= OH Hydroxyferrocifen
F
F
H
N
F
H
O
N
O
N
N
N
H
N
N
H
N
Cl
N
Me
N
H
Me
N
H
5
6
Fig. 1. Pharmacologically important ferrocene and 1,4-DHPM based compounds.
Human Eg5 has been an interesting drug target for the development
of cancer chemotherapeutics as it plays a vital role in mitosis by
establishing the bipolar spindle, the inhibition of which leads to
mitotic arrest and cell death. Because of interesting biological prop-
erties and several degrees of structural diversity, development of
novel methods for synthesis of 1,4-DHPMs as well as new analogs is
an intensively investigated field of utmost importance.
Based on these precedents, we sought to construct hybrid mo-
lecular architectures by combining ferrocene and pyrimidine
pharmocophores. We envisioned that resultant conjugates by vir-
tue of the presence of critical structural features might serve as a
prototype for new drugs that could be used in anticancer research.
In continuation of research work related to applications of ferro-
cene [18], we report herein synthesis and in vitro anti breast cancer
activity of novel ferrocene tagged multi-functionalized 1,4-
dihydropyrimidines.
of bases required for cyclocondensation. A series of experiments
were conducted using various bases in different solvents. The use of
sodium metal in isopropanol was found to be most efficient catalytic
system as it afforded the desired 6-ferrocenyl-4-phenyl-2-pyridin-
2-yl-1-o-tolyl-1,4-dihydropyrimidine in 80% yield within 3 h under
reflux conditions (Table 1, entry 1). The use of inorganic bases such
as NaOH and KOH showed remarkably poor catalytic activity as the
corresponding product was obtained in a scarce yield along with the
formation of undesired tarry material after prolonged reaction time
(Table 1, entries 2e5). On the other hand, the use of Na in ethanol
furnished the anticipated product in moderate yield (Table 1, entry
5). Thus, use of sodium in IPAunder refluxconditions was selected as
optimal reaction conditions for further studies.
After the optimization of reaction conditions, we evaluated the
scope of the protocol by reacting structurally diverse ferrocenyl
chalcones with different amidines (Scheme 2). The results are
summarized in Table 2. In general, the corresponding ferrocene
tagged multi-functionalized 1,4-DHPMs were obtained in good to
excellent yields in all the investigated cases. No significant elec-
tronic effects were observed for the electron donating as well as
electron withdrawing substituents on phenyl ring in ferrocenyl
chalcones. It was interesting to observe that Na/IPA catalytic system
is tolerant to a halogen substitution on the phenyl ring thus
providing a handle for additional synthetic manipulation via cross
coupling chemistry.
2. Results and discussion
The initially planned retrosynthesis of ferrocene tagged multi-
functionalized 1,4-DHPMs is depicted in Scheme 1 and is based on
cyclocondensation between chalcones and amidine. This conver-
gent route was envisaged to be highly significant due to easy
accessibility of large number of structurally diverse ferrocenyl
chalcones which can be easily prepared by reacting acetyl ferrocene
with large variety of commercially available aryl aldehydes under
basic conditions [19]. In addition, we hypothesized that use of
substituted benzamidines that have already displayed promising
biological activities [20] will offer a valuable addendum in biolog-
ical profile.
R"
R'
O
N
N
R"
R'
NH
NH
R
Fe
+
Fe
R
To test the validity of our disconnection approach, a preliminary
survey of reaction conditions was conducted using 1-ferrrocenyl-3-
phenyl-2-propen-1-one (a prototype ferrocenyl chalcone) and N-o-
tolylpicolinamidine (Table 1). Our initial studies focused on scrutiny
Scheme 1. Retrosynthetic analysis of ferrocene tagged multi-functionalized 1,4-
DHPMs.

234
J. Jadhav et al. / European Journal of Medicinal Chemistry 65 (2013) 232e239
Table 1
Optimization of reaction conditions.a
N
O
N
NH
NH
base
N
N
+
Fe
Fe
solvent
Entry
Base
Temp (ꢀC)
Solvent
Time (h)
Yieldb (%)
1
2
3
4
5
6
Na
80
80
60
80
80
80
Isopropanol
Ethanol
Methanol
Ethanol
DMF
3
10
7
10
10
5
80
30
25
35
40
58
NaOH
NaOH
KOH
KOH
Na
Ethanol
a
All products were characterized by IR, 1H and 13C NMR spectroscopy as well as by mass spectrometry.
Isolated yields after chromatography.
b
The structures of all the ferrocene tagged multi-functionalized
All the synthesized ferrocene tagged multifunctionalized 1,4-
DHPMs (3aej) were evaluated for their in vitro cytotoxicity in hu-
man breast cancer cell line MDA-MB-435 by employing the sulfo-
rhodamine B (SRB) assay method [22]. The results are described in
Table 4. It is worthy of note that all the compounds except 3hej
were significantly cytotoxic against MDA-MB-435 compared to the
standard drug tested, i.e. Doxorubicin, with the concentration of
the drug that produced 50% inhibition of cell growth (GI50) ranging
1,4-DHPMs were elucidated on the basis of 1H NMR and 13C NMR
spectroscopy as well as by FTIR and mass spectrometry. The spec-
tral data is in agreement with the proposed structures. All the
synthesized compounds are air and moisture stable for several
weeks without any decomposition and are soluble in normal
organic solvents.
The UVevis absorption spectra of all the synthesized com-
pounds were measured in methanol (Fig. 2) and the optical char-
acteristics are summarized in Table 3. The studies revealed that the
absorption peaks of ferrocene tagged multi-functionalized 1,4-
DHPMs were different from precursor ferrocenyl chalcones as the
from 17.4 to 152.9
turned out to be the most attractive since they demonstrated the
lowest GI50 values of 18 and 17.4 M respectively in the cell line
examined which was almost equal to the standard anticancer agent
mM. With regard to anticancer activity, 3b and 3c
m
former display peaks in the range 270e318 nm due to
p
ep
* tran-
Doxorubicin (18.4 mM).
sitions of the ferrocene chromophore while latter exhibit the same
in the range 487e517 nm [21]. The hypsochromic shift observed
may be attributed to the disturbance in enone system of ferrocenyl
chalcones during the cyclo-condensation with amidines. In addi-
tion, the less intensity absorption peaks in the region 450e550 nm
were also observed. These peaks may be assigned to the metal to
ligand charge transfer (MLCT) transitions from Fe to either anti-
bonding or nonbonding orbitals of Cp rings.
A tentative mechanism for the formation of ferrocene tagged
multi-functionalized 1,4-DHPMs is depicted in Scheme 3. The
reaction is likely to proceed via sodium isopropoxide induced 1,4-
nuclepohilic addition of amidine on ferrocenyl chalcone. An
intramolecular displacement leads to nucleophilic addition
furnishing cyclized intermediate. Under experimental reaction
conditions, the cyclized intermediate loses water forming the
final product.
3. Conclusion
In summary, we have developed a reliable and generally
applicable approach for the synthesis of a novel series of ferrocene
tagged multi-functionalized 1,4-DHPMs from easily accessible
ferrocenyl chalcones and amidines. The structures of all the
compounds were established on the basis of analytical and spec-
tral data. The synthesized compounds were evaluated for their
in vitro anticancer activities against the strain of human breast
cancer cell line MDA-MB-435. The compounds 3b and 3c with a
GI50 values 18 and 17.4 mM respectively were found to be the most
active. We believe that reported protocol offers scope for exten-
sion to variety of other substrates to form products that might
serve as an interesting alternative in endemic area of anticancer
research.
O
N
R'
NH
NH
R
N
+
Na Metal
N
N
Fe
IPA, 80-90 O
C
Fe
R
R'
1
2
3
Scheme 2. Na/IPA catalyzed cyclocondensation of ferrocenyl chalcones with amidines.

J. Jadhav et al. / European Journal of Medicinal Chemistry 65 (2013) 232e239
235
Table 2
Synthesis of ferrocene tagged multi-functionalized 1,4-DHPMs.a
Entry
Ferrocenyl chalcone (1)
Amidine (2)
Product (3)
Yieldb (%)
N
N
O
N
NH
NH
a
80
N
Fe
Fe
N
N
O
N
N
NH
NH
b
88
Fe
Fe
NO2
NO2
N
O
N
Cl
N
NH
NH
N
c
85
Fe
Fe
NO2
Cl
NO2
N
O
O
O
N
NH
NH
N
N
N
N
d
e
f
82
Fe
Fe
Fe
Cl
Fe
Cl
N
N
N
NH
NH
72
Fe
Cl
Cl
Fe
NH
NH
N
N
70
Fe
N
Fe
(continued on next page)

236
J. Jadhav et al. / European Journal of Medicinal Chemistry 65 (2013) 232e239
Table 2 (continued )
Entry
Ferrocenyl chalcone (1)
Amidine (2)
Product (3)
Yieldb (%)
O
Fe
NH
NH
N
N
N
N
g
75
Fe
Fe
Fe
Fe
O
N
NH
NH
N
N
h
65
68
76
Fe
Fe
N
Fe
Cl
Cl
Fe
O
N
NH
N
N
i
NH
Fe
Fe
N
Fe
Cl
Cl
N
O
N
N
NH
NH
N
j
Fe
Fe
a
All products were characterized by IR, 1H and 13C NMR spectroscopy as well as by mass spectrometry.
Isolated yields after chromatography.
b
4. Experimental part
synthesized following the literature procedure. All other chemical
were obtained from local chemical; suppliers and were used
without further purification.
4.1. General
1H NMR and 13C NMR spectra were recorded on a Brucker AC
4.2. General procedure for the synthesis of ferrocene tagged multi-
(300 MHz for 1H NMR and 75 MHz for 13C NMR) spectrometer using
CDCl3 as solvent and tetramethylsilane (TMS) as an internal stan-
dard. Infrared spectra were recorded on a PerkineElmer FTIR
spectrometer. The samples were examined as KBr discsw5% w/w.
Mass spectra were recorded on a Perkin Elmer Flexar SQ 300 LCMS.
Elemental analyses were performed on EURO EA3000 vectro
model. Melting points were determined on MEL-TEMP capillary
melting point apparatus and are uncorrected. Precoated aluminum
sheets (silicagel 60 F254, Merck Germany) were used for thin-layer
chromatography (TLC) and spots were visualized under UV light. All
the ferrocenyl chalcones [19] and benzamidines [20] were
functionalized 1,4-dihydropyrimidines (3aej)
A mixture of sodium metal (69 mg, 3 mmol) in isopropanol
(25 ml) was refluxed for 30 min. Amidine (1 mmol) and ferrocenyl
chalcone (1 mmol) were sequentially charged into reaction flask at
intervals of 30 min. The reaction mixture was heated under reflux
conditions for 3e5 h. After completion of the reaction as monitored
by thin layer chromatography (TLC), the reaction mixture was
poured into ice-water and extracted with ethyl acetate (3 ꢁ 25 ml).
The combined organic layers were washed with brine, dried over
anhydrous Na2SO4 and concentrated under reduced pressure to

J. Jadhav et al. / European Journal of Medicinal Chemistry 65 (2013) 232e239
237
3.5
3
O
3a
3b
R
2.5
2
3c
Fe
3d
3e
3f
1.5
1
N
R'
3g
3h
0.5
0
N
NH
O
N
.
.
Na
3i
3j
+
IPA
N
R
H
NH
..
+
220
320
420
Wavelength
Fig. 2. UVevis absorption spectra of 1,4 DHPMs in methanol.
520
620
NaOPr
-0.5
Fe
R'
give crude residue which was purified by column chromatography
over silica gel using hexane/ethyl acetate (4:1 v/v) to afford the
pure compound.
N
N
4.2.1. 6-Ferrocenyl-4-phenyl-1-pyridin-2-yl-2-o-tolyl-1,4-dihydro-
pyrimidine (3a)
R'
Fe
R'
Fe
Reddish brown solid; m.p: 144e146 ꢀC; Anal. calc. for
N
N
HO
-H2O
N
R
N
R
C
32H27FeN3: C 75.44, H 5.34; N 8.24%. Found: C 75.56, H 5.77, N
8.93%; 1H NMR (300 MHz, CDCl3):
d
8.6 (d, 2H, J ¼ 4 Hz, Py-H), 7.91e
7.84 (m, 4H, AreH), 7.79 (m, 3H, AreH), 7.67 (m, 2H, AreH), 7.28 (m,
3H, AreH), 7.13 (d,1H, J ¼ 6.3 Hz, AreH), 4.93 (s, 2H, ferrocene), 4.60
H
(s, 2H, ferrocene), 4.23 (s, 5H, ferrocene), 2.24 (s, 3H, methyl-H); 13
C
NMR (75 MHz, CDCl3):
133.7, 129.9, 129.4, 129.2, 127.9, 125.7, 123.4, 121.0, 119.1(AreC),
d 159.0, 145.9, 139.4, 138.0, 135.9, 134.7,
Scheme 3. Proposed mechanism of Na/IPA catalyzed cyclocondensation of ferrocenyl
chalcones with benzamidines.
80.45, 72.9, 70.14, 69.7 (ferrocene-C), 15.3 (methyl-C); IR (KBr, thin
film):
y
¼ 3052 (AreH), 2951 (CeH), 1532 (C]N), 1456 (C]C), 1244
4.2.3. 4-(3-Nitro-phenyl)-6-ferrocenyl-1-pyridin-2-yl-2-(2-
chlorophenyl)-1,4-dihydropyrimidine (3c)
(CeN); MS (EI): m/z ¼ 509.
Reddish brown solid; m.p: 130e132 ꢀC; Anal. calc. for
4.2.2. 4-(3-Nitro-phenyl)-6-ferrocenyl-1-pyridin-2-yl-2-o-tolyl-
1,4-dihydro-pyrimidine (3b)
C
31H23FeN4O2Cl: C 64.77, H 4.02; N 9.74%. Found: C 64.83, H 4.09; N
9.84%; 1H NMR (300 MHz, CDCl3):
d
8.33 (s, 1H, PyeH), 8.26e8.23
Reddish brown solid; m.p: 85e87 ꢀC; Anal. calc. for
(m, 1H, AreH), 7.96e7.92 (m, 1H, AreH), 7.89e7.83 (d, 2H, AreH),
7.78e7.70 (m, 2H, AreH), 7.68–7.58 (m, 2H, AreH), 7.41e7.38 (m,
3H, AreH), 7.26e7.23 (m, 1H, AreH), 6.76e6.74 (d, 1H, pyrimidine-
H), 4.95 (s, 2H, ferrocene), 4.66 (s, 2H, ferrocene), 4.23 (d, 5H,
C
32H26FeN4O2: C 69.32, H 4.72; N 10.10%. Found: 69.35, H 4.75; N
10.13%; 1H NMR (300 MHz, CDCl3):
d
8.51 (s, 1H, Py-H), 8.34 (s, 1H,
AreH), 8.26 (d, 1H, J ¼ 7.8 Hz, AreH), 7.92 (d, 1H, J ¼ 6.9 Hz, AreH),
7.83 (d, 1H, AreH), 7.64e7.58 (t, 2H, J ¼ 7.5 Hz, AreH), 7.4 (s, 1H,
AreH), 7.2e7.13 (m, 5H), 6.97e6.95 (d, 1H, pyrimidine-H), 4.94 (s,
2H, ferrocene), 4.65 (s, 2H, ferrocene), 4.24 (s, 5H, ferrocene), 2.2
ferrocene); 13C NMR (75 MHz, CDCl3):
d
161.1, 158.0, 137.8, 134.2,
130.9, 129.8, 129.4, 129.2, 127.6, 125.3, 123.4, 121.9, 118.6 (AreC),
80.5, 72.9, 70.1, 69.7 (ferrocene-C); IR (KBr, thin film):
y
¼ 3078 (Are
(s, 3H, methyl-H); 13C NMR (75 MHz, CDCl3):
d 162.1, 161.2145.8,
137.8, 137.5, 135.5, 134.2, 130.8, 129.9, 129.3, 127.7, 125.9, 125.6,
124.16, 121.9, 121.6, 118.1 (AreC), 84.2, 80.3, 73.1, 70.1, 69.8
Table 4
In vitro cytotoxicities of ferrocene tagged multi-functionalized 1,4-DHPMs against
human breast cancer cell line MDA-MB-435.a
(ferrocene-C), 19.7 (methyl-C); IR (KBr, thin film):
H), 2959 (CeH), 1587 (N]O), 1487 (C]C), 1243 (CeN); MS (EI): m/
z ¼ 554.
y
¼ 3054 (Are
TGIc
138.8
85.3
61.6
143.0
99.2
125.4
98.5
>125.5
>125.5
>152.9
<18.4
GI50
b
d
Compound
LC50
3a
3b
3c
3d
3e
3f
3g
3h
>157.1
>144.4
116.5
63.2
<18.0
<17.4
52.3
40.6
16.85
30.4
>125.5
>125.5
>152.9
<18.4
Table 3
>147.3
>147.3
>129.6
>129.6
>125.5
>125.5
>152.9
73.2
The maximum wavelength of absorption spectra of ferrocene tagged multi-
functionalized 1,4-DHPMs in methanol.
Compound
lmax for
p
ep
* transition (nm)
lmax for MLCT (nm)
3a
3b
3c
3d
3e
3f
3g
3h
3i
290
270
280
276
288
308
310
310
312
318
506
515
515
510
516
506
512
514
510
506
3i
3j
Doxorubicin
a
Concentrations in mM.
b
Concentration of drug resulting in a 50% reduction in the measured protein at
the end of the drug treatment as compared to that at the beginning) calculated from
[(Ti ꢂ Tz)/Tz] ꢁ 100 ¼ ꢂ50.
c
Drug concentration resulting in total growth inhibition (TGI) will calculated
from Ti ¼ Tz.
3j
d
Growth inhibition of 50% (GI50) calculated from [(Ti ꢂ Tz)/(C ꢂ Tz)] ꢁ 100 ¼ 50.

238
J. Jadhav et al. / European Journal of Medicinal Chemistry 65 (2013) 232e239
H), 2953 (CeH), 1597 (N]O), 1456 (C]C), 1248 (CeN); MS (EI): m/
z ¼ 574.
6.53%; 1H NMR (300 MHz, CDCl3):
d 8.36 (s, 1H, py-H), 7.98 (s, 1H,
AreH), 7.84 (s, 1H, AreH), 7.74e7.72 (d, 2H, J ¼ 6.6 Hz, AreH), 7.48e
7.42 (m, 3H, AreH), 7.0 (s, 1H, AreH), 6.77e6.75 (d, 1H, J ¼ 6.3 Hz,
pyrimidine-H), 4.8 (s, 2H, ferrocene), 4.6e4.5 (d, 4H, ferrocene), 4.4
(s, 2H, ferrocene), 4.2e4.18 (d, 10H, ferrocene); 13C NMR (75 MHz,
4.2.4. 4-(4-Chloro-phenyl)-6-ferrocenyl-2-pyridin-2-yl-1-o-tolyl-
1,4-dihydro-pyrimidine (3d)
Reddish brown solid; m.p: 124e126 ꢀC; Anal. calc. for
CDCl3):
120.2, 118.9 (AreC), 80.7, 79.5, 72.5, 71.0, 70.1,69.7, 69.6, 68.7
(ferrocene-C); IR (KBr, thin film):
d 145.9, 142.1, 138, 134.6, 131.3, 129.9, 127.7, 125.6, 121.3,
C
32H26FeN3Cl: C 70.66, H 4.81; N 7.74%. Found: C 70.71, H 4.87; N
7.84%; 1H NMR (300 MHz, CDCl3):
d
8.60e8.59 (d, 2H, Py-H), 7.88e
y
¼ 3076 (AreH), 2965 (CeH),
7.83 (t, 1H, AreH), 7.77e7.66 (d, 1H, J ¼ 6.8 Hz, AreH), 7.59e7.57 (m,
2H, AreH), 7.41e7.39 (m, 3H, AreH), 7.26e7.24 (m, 2H, AreH), 7.11 (s,
1H, AreH), 7.06e7.04 (d, 1H, AreH), 6.96e6.94 (d, 1H, J ¼ 6.4 Hz,
pyrimidine-H), 4.91 (s, 2H, ferrocene), 4.61 (s, 2H, ferrocene), 4.22 (s,
5H, ferrocene), 2.2 (s, 3H, methyl-H); 13C NMR (75 MHz, CDCl3):
1537 (C]N), 1432 (C]C), 1268 (CeN); MS (EI): m/z ¼ 637.
4.2.9. 2-(3-Chloro-phenyl)-4,6-diferrocenyl-1-pyridin-2-yl-1,4-
dihydro-pyrimidine (3i)
Reddish brown solid; m.p: 170e172 ꢀC; Anal. calc. for
d
161.5, 145.9, 139.3, 137.6, 135.9, 135.6, 133.7, 130.8, 129.4, 129.3, 129.1,
127.7,125.8,123.4,121.4,118.2 (AreC), 80.5, 72.7, 70.0, 69.7(ferrocene-
C), 19.6 (methyl-C); IR (KBr, thin film):
C
35H28Fe2N3Cl: C 65.91, H 4.42; N 6.58%. Found: C 65.93, H 4.46; N
6.62%; 1H NMR (300 MHz, CDCl3):
d
8.36 (s, 1H, py-H), 7.98 (s, 1H,
y
¼ 3089 (AreH), 2934 (CeH),
AreH), 7.83 (s, 1H, AreH), 7.74e7.71 (d, 2H, J ¼ 6.6 Hz, AreH), 7.44
(m, 3H,AreH), 7.0 (s, 1H, AreH), 6.76e6.74 (d, 1H, J ¼ 6.3 Hz,
pyrimidine-H), 4.8 (s, 2H, ferrocene), 4.6e4.5 (d, 4H, ferrocene), 4.4
(s, 2H, ferrocene), 4.2e4.19 (d, 10H, ferrocene); 13C NMR (75 MHz,
CDCl3):
121.5,12.3, 118.85, (AreC), 80.7, 79.5, 72.5, 71.0, 70.1, 69.7, 69.6, 68.8
(ferrocene-C); IR (KBr, thin film):
1566 (C]N), 1455 (C]C), 1292 (CeN); MS (EI): m/z ¼ 543.
4.2.5. 4-(4-Chloro-phenyl)-6-ferrocenyl-1-pyridin-2-yl-2-o-tolyl-
1,4-dihydro-pyrimidine (3e)
d 145.9, 142.1, 137.9, 134.7, 131.2, 129.9, 127.7, 125.5,
Reddish brown solid; m.p: 134e136 ꢀC; Anal. calc. for
32H26FeN3Cl: C 70.66, H 4.81; N 7.74%. Found: C 70.69, H 4.81; N
C
y
¼ 3052 (AreH), 2960 (CeH),
7.80%; 1H NMR (300 MHz, CDCl3):
d
8.38 (s, 1H, Py-H), 7.75e7.70 (m,
1553 (C]N), 1490 (C]C), 1242 (CeN); MS (EI): m/z ¼ 637.
4H, AreH), 7.58e7.56 (m, 2H, AreH), 7.41 (s, 2H, AreH), 7.28e7.24
(m, 2H, AreH), 7.10e7.05 (m, 2H, AreH), 6.97 (d, 1H, J ¼ 6.4 Hz,
pyrimidine-H), 4.90 (s, 2H, ferrocene), 4.59 (s, 2H, ferrocene), 4.21
(s, 5H, ferrocene), 2.5 (s, 3H, methyl-H); 13C NMR (75 MHz, CDCl3):
4.2.10. 6-Ferrocenyl-1-pyridin-2-yl-2-o-tolyl-4-p-tolyl-1,4-
dihydro-pyrimidin (3j)
Reddish brown solid; m.p: 154e156 ꢀC; Anal. calc. for
d
147.7, 139.4, 136.8, 135.8, 133.7, 130.9, 129.4, 129.4, 129.1, 126.9,
125.1,123.4,121.0,114. (AreC), 77.4, 77.0, 76.6, 70.0, 69.7 (ferrocene-
C), 19.7 (methyl-C); IR (KBr, thin film):
C
33H29FeN3: C 75.71, H 5.58; N 8.02%. Found: C 75.70, H 5.52; N
8.00%; 1H NMR (300 MHz, CDCl3):
d
8.53 (d, 2H, J ¼ 4.2 Hz, py-H),
y
¼ 3076 (AreH), 2945 (Ce
8.51e8.49 (d, 1H, J ¼ 7.5 Hz, AreH), 7.85e7.75 (m, 2H, AreH), 7.56
(d, 2H, J ¼ 7.8 Hz, AreH), 7.42e7.38 (t, 1H, AreH), 7.28e7.17 (m, 3H,
AreH), 7.11e7.09 (d, 1H, J ¼ 7.8 Hz, AreH), 7.02e6.97 (t, 1H, AreH),
6.92e6.90 (d, 1H, J ¼ 6.6 Hz, pyrimidine-H), 4.91 (s, 2H, ferrocene),
4.56 (s, 2H, ferrocene), 4.21 (s, 5H, ferrocene), 2.41 (s, 3H, methyl-H),
H), 1555 (C]N), 1436 (C]C), 1262 (CeN); MS (EI): m/z ¼ 543.
4.2.6. 4,6-Diferrocenyl-1-pyridin-2-yl-2-o-tolyl-1,4-dihydro-
pyrimidine (3f)
Reddish brown solid; m.p: 146 ꢀC; Anal. calc. for C36H31Fe2N3: C
2.20 (s, 3H, methyl-H); 13C NMR (75 MHz, CDCl3):
d 151.4, 147.8,
70.03, H 5.06; N 6.80%. Found: C 70.10, H 5.00; N 6.89%; 1H NMR
140.9, 140.3, 136.6, 132.5, 130.8, 129.6, 128.3, 126.8, 125.0, 123.2,
(300 MHz, CDCl3):
d
8.3 (d, 1H, J ¼ 3.4 Hz, py-H), 7.72e7.70 (d, 2H,
121.9, 121.7, 120.7 (AreC), 80.7, 72.5, 70.0,69.7, 69.7(ferrocene-C),
J ¼ 6.5 Hz, AreH), 7.47 (s,1H, AreH), 7.30e7.25 (m, 4H, AreH), 6.98 (t,
1H, AreH), 6.77e6.75 (d, 1H, J ¼ 6.4 Hz, pyrimidine-H), 4.87 (s, 2H,
ferrocene), 4.59e4.55 (d, 4H, ferrocene), 4.46 (s, 2H, ferrocene), 4.2e
4.18 (d, 10H, ferrocene), 2.5 (s, 3H, methyl-H); 13C NMR (75 MHz,
21.5, 17.7 (methyl-C); IR (KBr, thin film):
H), 1542 (C]N), 1451 (C]C), 1249 (CeN); MS (EI): m/z ¼ 523.
y
¼ 3057 (AreH), 2949 (Ce
4.3. Procedure of the SRB-assay
CDCl3):
120.2, 120.0, 119.3 (AreC), 80.8, 79.5, 72.5, 71.0, 70.1,69.7, 69.6, 68.7
(ferrocene-C), 19.8 (methyl-C); IR (KBr, thin film):
2976 (CeH),1535 (C]N),1425 (C]C),1237 (CeN); MS(EI): m/z ¼ 617.
d 145.9, 142.1, 138.2, 135.9, 134.7, 130.9, 130.2, 128.0, 125.9,
Tumor cells (human breast cancer cell line MDA-MB-435) were
grown in tissue culture flasks in growth medium (RPMI-1640 with
2 mM glutamine, pH 7.4, 10% fetal calf serum, 100 mg/mL strepto-
y
¼ 3056 (AreH),
mycin, and 100 units/mL penicillin) at 37 ꢀC under the atmosphere
of 5% CO2 and 95% relative humidity employing a CO2 incubator.
The cells at subconfluent stage were harvested from the flask by
treatment with trypsin (0.05% trypsin in PBS containing 0.02%
EDTA) and placed in growth medium. The cells with more than
97% viability (trypan blue exclusion) were used for cytotoxicity
4.2.7. 4,6-Diferrocenyl-2-pyridin-2-yl-1-o-tolyl-1,4-dihydro-
pyrimidine (3g)
Reddish brown solid; m.p: 146 ꢀC; Anal. calc. for C36H31Fe2N3: C
70.03, H 5.06; N 6.80%. Found: C 70.08, H 5.09; N 6.77%; 1H NMR
(300 MHz, CDCl3):
d
8.6 (d,1H, J ¼ 4.2 Hz, py-H), 8.5 (d,1H, J ¼ 7.2 Hz,
AreH), 7.8 (t, 1H, AreH), 7.7 (d, 1H, AreH), 7.43e7.40 (t, 1H, AreH),
7.26e7.21 (m, 2H, AreH), 7.0 (t, 1H, AreH), 6.9 (d, 1H, AreH), 6.77e
6.75 (d, 1H, J ¼ 6.4 Hz, pyrimidine-H), 4.8 (s, 2H, ferrocene), 4.6 (d,
2H, ferrocene), 4.5 (s, 2H, ferrocene), 4.47 (s, 2H, ferrocene), 4.2e4.18
(d, 10H, ferrocene), 2.2 (s, 3H, methyl-H); 13C NMR (75 MHz, CDCl3):
studies. An aliquot of 100 mL of cells were transferred to a well of
96-well tissue culture plate. The cells were allowed to grow for one
day at 37 ꢀC in a CO2 incubator as mentioned above. The test
materials at different concentrations were then added to the wells
and cells were further allowed to grow for another 48 h. Suitable
blanks and positive controls were also included. Each test was
performed in triplicate. The cell growth was stopped by gently
d
147.9, 142.0, 136.9, 130.8, 126.9, 125.3, 121.9, 120.3, (AreC), 80.8,
79.5, 72.4, 70.9, 70.0,69.7, 69.6, 68.7 (ferrocene-C), 17.6 (methyl-C);
IR (KBr, thin film):
y
¼ 3074 (AreH), 2985 (CeH), 1542 (C]N), 1446
layering of 50 mL of 50% trichloroacetic acid. The plates were
(C]C), 1259 (CeN); MS (EI): m/z ¼ 617.
incubated at 4 ꢀC for an hour to fix the cells attached to the bottom
of the wells. Liquids of all the wells were gently pipette out and
discarded. The plates were washed five times with doubly distilled
water to remove TCA, growth medium, etc and were air-dried.
4.2.8. 2-(2-Chloro-phenyl)-4,6-diferrocenyl-1-pyridin-2-yl-1,4-
dihydro-pyrimidine (3h)
Dark brown solid; m.p: 160e162 ꢀC; Anal. calc. for
100
mL of SRB solution (0.4% in 1% acetic acid) was added to
C
35H28Fe2N3Cl: C 65.91, H 4.42; N 6.58%. Found: C 65.89, H 4.38; N
each well and the plates were incubated at ambient temperature

J. Jadhav et al. / European Journal of Medicinal Chemistry 65 (2013) 232e239
239
(c) A. Hottin, F. Dubar, A. Steenackers, P. Delannoy, C. Biot, J.B. Behr, Org.
Biomol. Chem. 10 (2012) 5592e5597;
(d) G. Gasser, I. Ott, N. Metzler-Nolte, J. Med. Chem. 54 (2011) 3e25;
for half an hour. The unbound SRB was quickly removed by
washing the wells five times with 1% acetic acid. Plates were air
dried, tris-buffer (100 mL of 0.01 M, pH 10.4) was added to all the
wells and plates were gently stirred for 5 min on a mechanical
stirrer. The optical density was measured on ELISA reader at
540 nm. The cell growth at absence of any test material was
considered 100% and in turn growth inhibition was calculated. GI50
values were determined by regression analysis.
_
ꢀ
(e) K. Kowalski, A. Koceva-Chyla, A. Pienia˛zek, J. Bernasinska, J. Skiba,
ꢀ
A.J. Rybarczyk-Pirek, Z. Józwiak, J. Organomet. Chem. 700 (2012) 58e68;
(f) K.N. Tiwari, J.P. Monserrat, A. Hequet, C. Ganem-Elbaz, T. Cresteil, G. Jaouen,
A. Vessières, E.A. Hillard, C. Jolivalt, Dalton Trans. 41 (2012) 6451e6457;
(g) A.C. De Oliveira, E.A. Hillard, P. Pigeon, D.D. Rocha, F.A.R. Rodrigues,
R.C. Montenegro, L.V. Costa-Lotufo, M.O.F. Goulart, G. Jaouen, Eur. J. Med.
Chem. 46 (2011) 3778e3787;
(h) B. Long, S. Liang, D. Xin, Y. Yang, J. Xiang, Eur. J. Med. Chem. 44 (2009)
2572e2576;
(i) J.C. Swarts, D.M. Swarts, D.M. Maree, E.W. Neuse, C. La Madeleine, J.E. Van
Lier, Anticancer Res. 21 (2001) 2033e2037.
Acknowledgments
[11] (a) C.O. Kappe, Tetrahedron 49 (1993) 6937e6963;
Authors are thankful to ACTREC, TATA Memorial Centre for
Anticancer Screening and IISC, Bangalore for spectral analysis.
(b) C.O. Kappe, Eur. J. Med. Chem. 35 (2000) 1043e1052.
[12] H. Cho, M. Ueda, K. Shima, A. Mizuno, M. Hayashimatsu, Y. Ohnaka,
Y. Takeuchi, M. Hamaguchi, K. Aisaka, J. Med. Chem. 32 (1989) 2399e2406.
[13] D. Nagarathnam, S.W. Miao, B. Lagu, G. Chiu, J. Fang, T.G.M. Dhar, J. Zhang,
S. Tyagarajan, M.R. Marzabadi, F.Q. Zhang, W.C. Wong, W.Y. Sun, D. Tian,
J.M. Wetzel, C. Forray, R.S.L. Chnag, T.P. Broten, R.W. Ransom, T.W. Schorn,
T.B. Chen, S. O’Malley, P. Kling, K. Schneck, R. Benedesky, C.M. Harrell,
K.P. Vyas, C. Gluchowski, J. Med. Chem. 42 (1999) 4764e4777.
[14] A.D. Patil, N.V. Kumar, W.C. Kokke, M.F. Bean, A.J. Freyer, C. De Brosse, S. Mai,
A. Truneh, D.J. Faulkner, B. Carte, A.L. Breen, R.P. Hertzberg, R.K. Johnson,
J.W. Westley, B.C.M. Ports, J. Org. Chem. 60 (1995) 1182e1188.
[15] K. Deres, C.H. Schröder, A. Paessens, S. Goldmann, H.J. Hacker, O. Weber,
T. Krämer, U. Niewöhner, U. Pleiss, J. Stoltefuss, E. Graef, D. Koletzki,
R.N.A. Masantschek, A. Reimann, R. Jaeger, R. Gross, B. Beckermann,
K.H. Schlemmer, D. Haebich, H. Rübsamen-Waigmann, Science 299 (2003)
893e896.
References
[1] T.J. Kealy, P.L. Pauson, Nature 168 (1951) 1039e1040.
[2] (a) A. Togni, T. Hayashi, Ferrocenes: Homogeneous Catalysis, Organic Syn-
thesis, Material Science, VCH, Weinheim, 1995;
(b) R. Atkinson, V. Gibson, N. Long, Chem. Soc. Rev. 33 (2004) 313e328;
(c) D.L. Xin, H.X. Long, Chiral Ferrocenes in Asymmetric Catalysis: Synthesis
and Applications, Wiley-VCH Verlag, GmbH & Co. KGaA, Weinheim, 2010;
(d) H. Werner, Angew. Chem. Int. Ed. 51 (2012) 6052e6058 (and references
cited therein).
[3] N. Metzler-Nolte, M. Salmain, in: P. Stepnicka (Ed.), Ferrocenes: Ligands,
Materials and Biomolecules, Wiley, New York, 2008, pp. 499e639.
[4] (a) C. Ornelas, J.R. Aranzaes, E. Cloutet, S. Alves, D. Astruc, Angew. Chem. Int.
Ed. 46 (2007) 872e877;
(b) R. Djeda, A. Rapakousiou, L. Liang, N. Guidolin, J. Ruiz, D. Astruc, Angew.
Chem. Int. Ed. 49 (2010) 8152e8156.
[5] I.R. Whittall, A.M. McDonagh, M.G. Humphrey, M. Samoc, Adv. Organomet.
Chem. 42 (1998) 291e362.
[6] (a) D.R. van Staveren, N. Metzler-Nolte, Chem. Rev. 104 (2004) 5931e5985;
(b) M.F.R. Fouda, M.M. Abd-EIzaher, R.A. Abdelsamaia, A.A. Labib, Appl.
Organomet. Chem. 21 (2007) 613e625.
[7] (a) V. Ganesh, V.S. Sudhir, T. Kundu, S. Chandrasekaran, Chem. Asian J. 6
(2011) 2670e2694;
[16] (a) C.A. Sehon, G.Z. Wang, A.Q. Viet, K.B. Goodman, S.E. Dowdell, P.A. Elkins,
S.F. Semus, C. Evans, L.J. Jolivette, R.B. Kirkpatrick, E. Dul, S.S. Khandekar, T. Yi,
L.L. Wright, G.K. Smith, D.J. Behm, R. Bentley, C.P. Doe, E. Hu, D. Lee, J. Med.
Chem. 51 (2008) 6631e6634;
(b) Q. Sun, F. Suzenet, G. Guillaumet, Tetrahedron Lett. 53 (2012) 2694e2698;
(c) V.B. Jadhav, H.V. Holla, S.U. Tekale, R.P. Pawar, Der Chem. Sin. 3 (2012)
1213e1228.
[17] (a) T.U. Mayer, T.M. Kapoor, S.J. Haggarty, R.W. King, S.L. Schreiber,
T.J. Mitchison, Science 286 (1999) 971e974;
(b) S.J. Haggarty, T.U. Mayer, D.T. Miyamoto, R. Fathi, R.W. King, T.J. Mitchison,
S.L. Schreiber, Chem. Biol. 7 (2000) 275e286.
[18] (a) G. Rashinkar, R. Salunkhe, J. Mol. Catal. A: Chem. 316 (2010) 146e152;
(b) G. Rashinkar, S. Kamble, A. Kumbhar, R. Salunkhe, Catal. Commun. 12
(2011) 1442e1447;
(c) R. Kurane, V. Gaikwad, J. Jadhav, R. Salunkhe, G. Rashinkar, Tetrahedron
Lett. 53 (2012) 6361e6366.
[19] X. Wu, P. Wilairat, M.L. Go, Bioorg. Med. Chem. Lett. 12 (2002) 2299e2302.
[20] (a) T.J. Muller, J. Conradie, E. Erasmus, Polyhedron 33 (2012) 257e266;
(b) H. Parveen, F. Hayat, A. Salahuddin, A. Azam, Eur. J. Med. Chem. 45 (2010)
3497e3503;
(c) S. Arya, N. Kumar, P. Roy, S.M. Sondhi, Eur. J. Med. Chem. 59 (2013) 7e14.
[21] (a) J. Zienkiewicz, V.G. Young, P. Kaszynski, J. Org. Chem. 69 (2004) 2551;
(b) A.A. Aly, A.M. Nour-El-Din, ARKIVOC i (2008) 153e194.
[22] P. Skehn, R. Storeng, A. Scudiero, J. Monks, D. McMohan, D. Vistica,
T.W. Jonathan, H. Bokesch, S. Kenney, M.R. Boyd, J. Natl. Cancer Inst. 82 (1990)
1107e1112.
(b) N.H. Evans, P.D. Beer, Org. Biomol. Chem. 9 (2011) 92e100;
(c) R. Gómez Arrayás, J. Adrio, J.C. Carretero, Angew. Chem. Int. Ed. 45 (2006)
7674e7715;
(d) H.B. Kraatz, J. Inorg, Organomet. Polym. Mater. 15 (2005) 83e106;
(e) J.C. Thomas, Chem. Rev. 103 (2003) 310;
(f) L.-X. Dai, T. Tu, S.-L. You, W.-P. Deng, X.-L. Hou, Acc. Chem. Res. 36 (2003)
659e667;
(g) C.J. Richards, A.J. Locke, Tetrahedron Asymmetry 9 (1998) 2377e2407;
(h) A. Togni, Angew. Chem. Int. Ed. 35 (1996) 1475e1477.
[8] D. Astruc, Electron Transfer and Radical Processes in Transition-metal
Chemistry, VCH, New York, 1995.
[9] A. Nguyen, A. Vessiéres, E.A. Hillard, S. Top, P. Pigeon, G. Jaouen, Chimia 61
(2007) 716e724.
[10] (a) C. Ornelas, New J. Chem. 35 (2011) 1973e1985;
(b) D. Plazuk, B. Rychlik, A. B1auz, S. Domaga1a, J. Organomet. Chem. 715
(2012) 102e112;