N-ferrocenylpyridazinones and new organic analogues: Synthesis, cyclic voltammetry, DFT analysis and in vitro antiproliferative activity associated with ROS-generation

Article abstract of DOI:10.1016/j.tet.2017.09.015

Employing an optimized Pd-catalyzed cross-coupling reaction promoted by CuI, novel N-ferrocenylpyridazinones along with N-phenyl- and N-(2-pyridyl) analogues were synthesized from readily available heterocyclic precursors, iodoferrocene, iodobenzene and 2

Full text of DOI:10.1016/j.tet.2017.09.015

Accepted Manuscript
N-ferrocenylpyridazinones and new organic analogues: Synthesis, cyclic voltammetry,
DFT analysis and in vitro antiproliferative activity associated with ROS-generation
Tamás Jernei, Szilvia Bősze, Rita Szabó, Ferenc Hudecz, Katalin Majrik, Antal
Csámpai
PII:
S0040-4020(17)30937-7
10.1016/j.tet.2017.09.015
TET 28966
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 25 May 2017
Revised Date: 21 August 2017
Accepted Date: 11 September 2017
Please cite this article as: Jernei Tamá, Bősze S, Szabó R, Hudecz F, Majrik K, Csámpai A, N-
ferrocenylpyridazinones and new organic analogues: Synthesis, cyclic voltammetry, DFT analysis and
in vitro antiproliferative activity associated with ROS-generation, Tetrahedron (2017), doi: 10.1016/
j.tet.2017.09.015.
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N-Ferrocenylpyridazinones and new organic
analogues: synthesis, Cyclic Voltammetry,
DFT analysis and in vitro antiproliferative
activity associated with ROS-generation
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Tamás Jernei, Szilvia Bősze, Rita Szabó, Ferenc Hudecz, Katalin Majrik and Antal Csámpai

1
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Tetrahedron
journal homepage: www.elsevier.com
N-Ferrocenylpyridazinones and new organic analogues: synthesis, Cyclic
Voltammetry, DFT analysis and in vitro antiproliferative activity associated with
ROS-generation
a
b
b
b,c
d
a
Tamás Jernei , Szilvia Bősze , Rita Szabó , Ferenc Hudecz , Katalin Majrik and Antal Csámpai ∗
a
Department of Inorganic Chemistry, Eötvös Loránd University (ELTE) Budapest Pázmány P. sétány 1/A, H-1117, Hungary.
MTA-ELTE Research Group of Peptide Chemistry, Budapest Pázmány P. sétány 1/A,H-1117, Hungary.
b
c
Department of Organic Chemistry, Eötvös Loránd University (ELTE) Budapest Pázmány P. sétány 1/A, H-1117, Hungary.
d
Research Centre of Natural Sciences, Hungarian Academy of Sciences, H-1117 Budapest, Magyar Tudósok körútja 2.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Employing an optimized Pd-catalyzed cross-coupling reaction promoted by CuI, novel N-
ferrocenylpyridazinones along with N-phenyl- and N-(2-pyridyl) analogues were synthesized
from readily available heterocyclic precursors, iodoferrocene, iodobenzene and 2-
bromopyridine. With exception of the ferrocenylation of 6-ferrocenylpyridazin-3(2H)-one
yielding both N- and O-substituted products, the studied reactions exclusively afforded N-aryl
lactams. The novel compounds exhibited cytotoxicity towards HEPG2 and HT-29 human
malignant cells under in vitro conditions. The measured IC50 values supplemented with the
results of cyclic voltammetry and DFT calculations suggest that the cytotoxic activity of the N-
and O-ferrocenyl-substituted derivatives and the decreased effect of the N-phenyl analogues
seem to be at least partly associated with the potential to generate reactive oxygen species
Available online
Keywords:
Pyridazine, Lactams, N-arylation, Ferrocene, (ROS). This interpretation, allowing the prediction of characteristic substituent-dependent SAR,
was supported by the results of related studies on the practically inactive N-(2-
pyridyl)pyridazinones assumed to be present in protonated chelate forms with highly a decreased
propensity to undergo ionization.
Cytotoxicity, SAR, Cyclic Voltammetry, HOMO
energy, Ionization energy
2
009 Elsevier Ltd. All rights reserved.
1
. Introduction
plethora of organic ferrocene derivatives were found to exhibit
promising antitumor activities. In most cases their effect is based
5
It is a generally accepted view that chemotherapy is one of the
essential tools for treatment of malignancies. During the last
decades, important advances have been made leading to the
approval of well-known therapeutic agents, such as cisplatin,
carboplatin and oxaliplatin , along with a variety of emblematic
organic compounds which can be represented by eg.
on the generation of radicals through Fenton pathway that causes
6
DNA damage and consequent apoptosis of cells. In this regard, a
variety of N-acyl derivatives of aminoferrocene emerged as an
intriguing class of cytotoxic agents with considerable selectivity
toward cancer cells, capable of playing a highly important role in
1
7
ROS-production in tumorous cells,. It was also demonstrated
2
3
daunomycin, doxorubicin, vinblastine and vincristine. However,
in order to overcome the frequent toxic limitations and to
broaden the scope of treatable malignancies, an intense search
was initiated for alternative therapeutic agents with enhanced
activity and bioavailability. As a result, besides the afore
mentioned classical metal complexes and organic molecules,
organometallics have also emerged as potential anticancer agents.
Among them, ferrocene derivatives with diverse molecular
architectures are of pronounced importance. It is well
documented that the replacement of an aromatic nucleus in
certain organic compounds for a ferrocene unit can lead to
that aminoferrocene-based carbamates can be activated by
intracellular redox processes which finally increase the oxidative
stress in cancer cells leading to their death. On the other hand,
8
although the majority of pyridazinone-based heterocycles of
9
10
biological relevance are known for anti-HIV, antiviral,
11
12
13
antibacterial,
antihypertensive
and anti-inflammatory
activities, a number of them have been identified as anti-cancer
14
15
drug candidates, cyclooxygenase and PFKFB3 disphosphatase
16
inhibitors with simultaneous anti-tumouractivity, showing their
promising potential in fighting cancer. Since the chemistry and
biological applications of ferrocene- and pyridazine derivatives
are the focus of our interest, we have demonstrated that the
4
compounds with enhanced biological activity. Accordingly, a
—
——
∗
Corresponding author. csampai@chem.elte.hu

2
combination of ferrocene and pyridazinone mo
in highly promising hybrids exhibiting marked antiproliferative
A
iet
C
ies
C
m
E
ig
P
h
T
t r
E
es
D
ult MAth
N
e
U
th
S
r
C
ee-
R
di
I
mensional iodoferrocene (2), the observed
P
T
regioselectivity is in good agreement with the well-established
view that Cu-catalyzed C-heteroatom bond-forming reactions are
strongly influenced by the steric hindrance on the aryl halide
17,18
activity against human malignant cell lines.
precedent prompted us to envisage the synthesis and preliminary
in vitro evaluation of small library of N-
All of this
2
2
component.
a
ferrocenylpyridazinones, the cyclic derivatives of N-
acylhydrazinoferrocene, expected to have significant potential in
ROS-generation associated with antiproliferative activity.
2
. Results and Discussion
Since the synthesis and characterization of hydrazinoferrocene, a
reagent with expected enhanced tendency to undergo oxidative
degradation, have not been reported in the literature so far, we
envisaged accessing the target N-ferrocenylpyridazinone
derivatives through the cross-coupling of selected pyridazinone
components with the easily available iodoferrocene (2). In order
to optimize reaction conditions, a series of experiments were
Scheme 1
Since the copper-mediated transformations provided low isolated
yields with small share of the targeted N-
ferrocenylpyridazinone 3, we hoped to enhance the efficiency
and N-selectivity of the model coupling reaction through the
application of Pd-complexes (PPh ) PdCl and (dppf)PdCl as
single metal source or components in mixed metal systems
(Table 2).
The Pd-catalysed reactions carried out in the absence of CuI led
again to the formation of mixtures of 3 and 4 in low isolated
yields with ca. 1:8 ratio which proved to be practically invariant
to the reaction conditions (Table 2: entries 2-4). Changing to a
Sonogashira type protocol (entry 5), a minimal increase was
observed in the yield of the regioisomeric products (38%), with a
doubled ratio of 3 in the isolated mixture. The third series of Pd-
a
19
carried out employing 6-ferrocenylpyridazin-3(2H)-one (1) as
substrate (Scheme 1). First, we attempted to utilize copper-
mediated reactions conducted in different solvents using CuI as
metal source in catalytic or stoichiometric amount (Table 1). It is
of interest that contrary to the general expectations regarding the
3 2 2 2
20
regioselectivity of Ullmann-Goldberg type amide-N-arylations
independently on the conditions, the majority of the reactions
afforded ca. 2:11 mixtures of N- and O-ferrocenyl-substituted
products 3 and 4, respectively, in low isolated yields. When the
reactions were performed in dioxane, 3 and 4 could be
exclusively or partly isolated as DMEDA-coordinated copper
catalyzed reactions, carried out in the presence of
stoichiometric amount of CuI and K CO in DMF:TEA (1:1
a
1
complexes (Table 1: entries 3 and 5), as indicated by H-NMR
2
3
spectroscopy measurements. In this series of experiments, the
coupling reaction conducted in DMF containing potassium
phosphate and a stoichiometric amount of CuI along with
DMEDA seemed to be the best choice to effect ferrocenylation of
V/V), afforded the mixture of regioisomeric products in mediocre
yields (59-65%: entries 7-9) with substantially increased share of
3. It must be pointed out here that DMF seems essential to effect
the desired conversions, as indicated by the fact that no any
coupling reaction took place when toluene or TEA was used as
single solvent (entries 1 and 6).
1
(Table 1: entry 9).
The simultaneous formation of 3 and 4 is closely related to
copper(I)-mediated O-arylation 2 of 2-hydroxypyridinesbwith
1
sterically demanding aryl halides. Considering the steric bulk of
Table 1: Yields of the mixture of 3 and 4 isolated after the reactions conducted under different conditions employing catalytic or
stoichiometric amounts of CuI
reagent or
catalyst
a
isolated yield
Entry
1
ligand
-
solvent
DMF
base
T [°C]
110
b
(%)
1
0% CuI
K
2
CO
5
0
3
2
3
4
5
6
7
8
9
10% CuI
10% CuI
10% CuI
DMEDA
DMEDA
DMEDA
DMEDA
DMEDA
-
DMEDA
DMEDA
DMEDA
TEA
toluene
dioxane
DMF
dioxane
DMF
TEA
DMF
DMF
DMF
K
2
K
2
K
2
K
2
K
2
K
2
K
2
CO
CO
CO
CO
CO
CO
CO
3
3
3
3
3
3
3
100
90
110
90
110
80
90
110
110
80
80
110
110
c
(8 )
7
c
100% CuI
100% CuI
100% CuI
100% CuI
100% CuI
100% CuI
100% CuI
100% CuI
100% CuI
100% CuI
18 (9 )
25
0
15
33
0
5
3
30
16
K
KO Bu
PO
3
4
t
1
1
1
1
1
0
1
2
3
4
DMF
DMF
DMF:TEA 1:1
DMF:DIPA 1:1
K
2
K
2
K
2
K
2
CO
CO
CO
CO
3
3
3
3
DIPA
-
-
a
Performed with 2 eq. of DMEDA or 4 eq. of TEA or 4 eq. of DIPA as ligand.
The product ratio 3:4 was ca. 2:11 as determined by H-NMR spectroscopy.
Isolated mixture of stoichiometric mono-copper complexes of 3 and 4 as indicated by the CH - and CH signals of DMEDA
b
1
c
2
3
presumably coordinated to the Cu(I)-centre.

3
Table 2: Yields of the mixture of 3 and 4 isola
and a catalytic or stoichiometric amount of CuI.
A
ted
C
af
C
te
E
r t
P
he
T
re
E
ac
D
tio
M
ns c
A
on
N
du
U
ct
S
ed
C
u
R
nd
I
e
P
r different conditions employing Pd-complexes
T
Entry
reagent or catalyst
solvent
toluene
DMF
base
T [°C]
100
110
110
110
isolated yield (%)
0
1
2
3
4
5% (PPh
5% (PPh
3
)
)
2
PdCl
PdCl
2
K
2
2
2
2
CO
CO
CO
CO
3
3
3
3
a
K
K
K
27
33
34
3
2
2
a
5% (dppf)PdCl
5% (PPh PdCl
0%. CuI,
.5% (PPh ) PdCl
2
DMF
DMF:TEA 1:1
2
a
)
2
3
2
1
b
5
6
7
8
DMF:TEA 1:1
K
2
K
2
K
2
K
2
CO
CO
CO
CO
3
3
3
3
110
80
38
2
3
2
1
00%CuI,
% (dppf)PdCl2
00%CuI,
% (PPh ) PdCl
2
TEA
0
5
1
c
DMF:TEA 1:1
DMF:TEA 1:1
110
110
61
5
3
2
1
00%CuI,
c
65
5
% (dppf)PdCl2
c
5
9
1
00%CuI,
.5% (PPh ) PdCl
2
9
DMF:TEA 1:1
K
2
CO
3
110
(applied for further
reactions)
2
3
2
a
1
The product ratio 3:4 was ca.1:8 as determined by H-NMR spectroscopy.
b
1
The product ratio 3:4 was ca. 1:4 as determined by H-NMR spectroscopy.
c
1
The product ratio 3:4 was ca. 5:9 as determined by H-NMR spectroscopy.
The conditions featuring a satisfactory overall yield (59%) for the
mixture of 3 and 4 and a reasonable loading (2.5%) of the
cheaper Pd-catalyst [(PPh ) PdCl ] (entry 9) was our choice to
3
2
2
accomplish coupling reactions of condensed pyridazinones 5a-p
and 7a,b (Scheme 1). These targets were equipped with such
substituents that might enable diversity-oriented synthesis by a
variety of further functionalizations (5a-h,k-n and 7a,b have
23
been reported, while the procedures affording 5i,j and 5o,p are
described in this contribution). It is worth pointing out that on the
bicyclic substrates the coupling reactions took place on the
sterically more accessible lactam nitrogen allowing the exclusive
isolation of N-ferrocenyl derivatives 6a-p and 8a,b in low-to-
mediocre yields as presented in Scheme 2. In order to investigate
the effect of the free OH-group in the hydroxyethylamino chain
attached to the phthalazinone scaffold in position 4 (a potential
donor site in 5h), a representative substrate was protected by
simple acetylation (5h→5i). This modification increased the
efficiency of the coupling protocol (cf. the isolated yields 16%
and 36% obtained for conversions 5h→6h and 5i→6i,
respectively). On the other hand, in the presence of the intact 4-
(2-hydroxyethyl) chain, the replacement of the 8-nitro group to
phenylthio substituent (5h→5j) obviously makes N-
ferrocenylation more feasible (cf. isolated yield 41% obtained for
conversion 5j→6j). It must also be pointed out that – probably
due to the participation of the basic free amino group in the
cross-coupling
process
affording
highly
unstable
hydrazinoferrocene type intermediate inclined to undergo
decomposition – a complex mixture of components was isolated
when
4-((2-hydroxyethyl)amino)-8-(1-methylhydrazinyl)
phthalazin-1(2H)-one (5n) was used as substrate under the
studied conditions. However, hydrazones 5o and 5p, obtained by
condensations with benzaldehyde and formylferrocene,
underwent N-ferrocenylation affording 5o and 6p in low yields
Scheme 2: N-Ferrocenylation of polyfunctional fused
pyridazinones using reaction conditions presented in entry 9 of
Table 2
(19% and 33%, respectively). The extremely low yield of 8a can
probably be attributed to the chelation-induced deactivation of
the metal-containing catalyst species by the carbonyl-oxygen and
the adjacent pyridine nitrogen in precursor 7a. In general, the
unreacted components were partially recovered from the reaction
mixture.
In order to establish structure-activity relationships regarding the
contribution of the N-aryl group to the cytostatic effect, the
analogues
of
a
representative
selection
of
N-
ferrocenylpyridazinones were also prepared to serve as references
in the biological assays. Thus, under the conditions employed for
the ferrocenylation reactions, 1 and 5a were coupled with
iodobenzene and 2-bromopyridine affording N-phenyl- and N-(2-

4
pyridyl)pyridazinones (3a/6aa and 3b/6ab, respectively: Scheme
experienced effects might be associated with the production of
ACCEPTED MANUSCRIPT
3
), as exclusively isolable products which were not contaminated
reactive oxygen species (ROS) possibly playing a crucial role in
30
by the corresponding aryloxy-substituted pyridazines. Employing
iodobenzene as coupling partner, phthalazinone 5l was converted
into 6la serving as a further reference in the in vitro assays.
However, the reaction conducted under the same conditions in
the presence of 2-bromopyridine afforded a complex mixture of
products of which separation has been unsuccessful so far.
the observed cytotoxicity.
Accordingly, the compounds
evaluated in MTT tests were also subjected to comparative cyclic
voltammetry (CV) affording half-wave potentials E (1) (Table
3). However, in the case of 3, 3a,b, 6g, 6la and Tamoxifen, the
measurements could not be accomplished due to unrevealed
irreversible reactions producing solid hardly removable
undefined coatings on the electrode surface. It is also worth
noting that two consecutive electron transfers characterized by
half-wave potentials E (1) and E (2) (235.0 mV and 404.5 mV,
1/2
1/2
1/2
respectively) could be detected only for 3 with spatially separated
ferrocene redox centers linked to the aromatic pyridazine core.
Nevertheless, taking into account that besides ROS-generation
the cytotoxic activity of the investigated compounds might also
be controlled by diverse factors including e.g. bioavailability and
binding affinity to specific receptors, a satisfactory correlation
can be established between the relevant E (1) potentials and the
1/2
measured IC50 values. For instance, 6l and 6m, displaying the
most pronounced cytotoxic effects unambiguously emerged as
the most efficient reductants in the series of the investigated
model compounds. On the other hand, since only limited
experimental data could be collected, and it is reasonable to
assume that the measured half-wave potentials (particularly
E (2) obtained for 4) are also affected by solvation, diffusion (a
Scheme 3: Synthesis of N-arylated pyridazinones to serve as
reference organic compounds in the in vitro tests.
1/2
non-thermodynamic effect), ionic strength, chemical and physical
interactions with the electrode surface and – under the employed
experimental conditions – an enhanced degree of coordination of
the solvent acetonitrile molecules with highly structure-
1
13
The IR- H- and C-NMR spectroscopic data of the novel
compounds listed in the Experimental part are consistent with
their structure, but the following remarks about the unambiguous
discrimination between the alternative sites of the amide-
arylation reactions are necessary. The characteristic amide-I band
31
dependent modes to the cationic species. To eliminate the
effects of previously listed, not ROS-generation dependent
properties, which have significant effect on the measured
cytotoxic activity or on the measured redox potential, we
undertook complementary DFT studies on the redox properties of
the tested compounds at B3PW91 level of theory using DGTZVP
basis set on the neutral compounds and the corresponding radical
cationic species with doublet electron configuration generated by
-
1
discernible in the range 1620-1640 cm of the IR spectra of
pyridazinones 3, 6a-p, 8a,b, 3a,b, 6aa, 6ab and 6la refers to their
N-arylated structure. Accordingly, the C1‘ resonance was
identified around 100 ppm in the C-NMR spectra registered for
, 6a-p and 8a,b, while in the C-NMR spectrum of
ferrocenyloxypyridazine 4 the same signal was detected at 119.9
ppm. The assignment of the C1‘-signal of pronounced diagnostic
importance was confirmed by comparative GIAO analysis
carried out for the optimized structures of 3 and 4, respectively,
by B3LYP hybrid functional
1
3
1
3
3
removing one electron from the HOMO of the parent molecules.
24
32
The first adiabatic ionization energy [E (1)] values, presumably
i
relevant under biological conditions were obtained as the
difference in the total electron energy values calculated for the
optimized structures of the analyzed compounds and the
appropriate radical cationic species, respectively. Supporting our
view regarding the above-discussed cytotoxic activity, at least
particularly associated with ROS-generation, the measured
E (1) potentials, the calculated HOMO levels and E (1) values
2
5
using the extended 6-
26
3
11++G(2d,p) basis set, a well-established protocol for the
calculation of NMR data (the measured/calculated shifts: 99.6
ppm/107.2 ppm and 119.9 ppm/126.2 ppm for 3 and 4,
respectively). The optimized structures of 3 and 4 were generated
27
by the B3PW91 hybrid functional employing the DGTZVP
2
8
1/2
i
basis set. Note that B3PW91 has been demonstrated to provide
an improvement over B3LYP regarding the bonding
(
Table 3) seem to be in acceptable correlation with the IC data
50
derived
from in vitro assay.
For instance,
N-
29
characterization of metal-containing fragments.
ferrocenylpyridazinones 3 and 6l with higher HOMO levels
We have studied the cytostatic activity in vitro using human
HepG2 hepatoma and HT-29 colorectal adenocarcinoma cell
cultures for a reasonable selection of the novel ferrocene-
containing- and the corresponding organic reference compounds.
The cells were treated at 10 to 10 µM concentration range and
the viability of the cells was determined by MTT-assay. The
activity was characterized by the IC50 values and compared with
Tamoxifen as positive control.
undergo ionization more readily [EHOMO(1)/E (1)= -5.47 eV/6.15
i
eV and = -5.12 eV/5.64 eV, resp.] than their N-phenyl-substituted
counterparts 3a and 6la [EHOMO(1)/E (1)= -5.80 eV/7.21 eV and -
i
5
.37 eV/6.34 eV, resp.]. However, it must be commented that N-
-
4
2
(2-pyridyl)pyridazinone 3b exhibiting no detectable effect in the
test can be characterized by somewhat higher HOMO-level and
lower ionization energy [EHOMO(1)= -5.73 eV and E (1)=7.02 eV]
i
than its N-phenyl-substituted counterpart 3a [EHOMO(1)= -5.80 eV
The data summarized in Table 3 show that each representative
compound with an N- or O-ferrocenyl substituent produces a
characteristic in vitro cytostatic effect, in average comparable to
that exhibited by Tamoxifen, on both cell cultures investigated.
Highlighting the essential contribution of the heteroatom-bonded
ferrocene unit to cytostatic activity, significantly decreased
effects could be detected for N-phenylpyridazinones 3a, 6aa and
and E (1)=7.21 eV]. This finding can be explained by the facile
alternative chelate-forming modes of protonation of 3b probably
i
taking place under biological conditions to afford cationic pair
+
+
3
bH1 and 3bH2 (Scheme 4) of which DFT analysis revealed
their relative stability and highly decreased propensity to undergo
ionization probably associated with ROS-generation [cf.
enhanced HOMO levels and E (A) data in Table 3.]. According to
i
6
la relative to those measured for their N-ferrocenyl-substituted
+
the relative energetics 3bH2 stabilized by six-membered chelate
analogues 3, 6a and 6l. These results related to a preliminary
structure-activity relationship (SAR) suggesting that the
ring is expected to be overpopulated relative to its isomeric pair

5
+
3
bH1 with a five-membered chelate residue. It must be noted
modelled by B3PW91/DGTZVP method (Table 3). However,
ACCEPTED MANUSCRIPT
here that contrary to N-ferrocenylphthalazinone 6a with a
the second adiabatic ionization energy values [E (2)] obtained as
i
relatively high EHOMO(1) level (-5.46 eV) and somewhat
the relative energetics of the optimized structures of dications and
the appropriate radical cations (Table 3) do not suggest an
outstanding propensity of 4 to undergo oxidation. This apparent
increased first adiabatic ionization energy (E (1)=6.42 eV)
i
+
enabling to exert remarkable activity against both cell lines
investigated, neither N-phenyl-substituted analogue 6aa nor N-
contradiction might be attributed to an enhanced degree of
coordination of the solvent acetonitrile molecules to the cationic-
(2-pyridyl)phthalazinone 6ab with substantially lowered
31
EHOMO(1) levels (-6.34 eV and -6.54 eV, respectively) and
and particularly to dicationic species which interaction was not
taken into account for theoretical studies as a highly demanding
uncertain explicit solvent model. Nevertheless, we attempted to
support this concept by a highly preliminar theoretical study on
acetonitrile-promoted stepwise ionization of 6a, a characteristic
representative of the studied compounds with reasonable
structural complexity (Scheme 5). The modified ionization
energies E (1)* and E (2)* unambiguosly referring to solvent-
increased E (1) (-7.98 eV and -7.74 eV, respectively) values
i
proved to be practically inactive in the in vitro tests. Moreover,
6
ab is also expected to readily bind proton in alternative chelated+
fashions to afford expectably highly deactivated cations 6abH1
and 6abH2 [cf. lowered EHOMO(1) and enhanced E (1) values in
+
i
Table 3] with a dominance of the latter one as suggested by their
calculated relative energetics (Scheme 4).
i
i
promoted SET processes were obtained as the appropriate
differences of the energetics of the optimized structures of non-
+
coordinated 6a and cationic mono-acetonitrile complexes 6a .L
2+
and 6a .L featuring slightly and substantially distorted
metallocene structure with negligible and strong Fe-N
interactions (interatomic distances: 4.547 Å and 1.944 Å,
respectively). Selected bonding interactions inside the modeled
acetonitrile complexes are visualized by representative delocalized
MO’s with energy levels referring to a much stronger coordination of
the ligand in dication 6a .L than in radical cation 6a .L (Scheme 5).
The method used (B3PW91/DGTZVP) could not disclose any
interaction between 6a and MeCN.
Scheme 4: Relative energy values calculated for isomeric pairs of
protonated N-(2-pyridyl)pyridazinones, the species assumed to be
present under biological conditions, with high ionization energy
associated with significantly decreased tendency to generate
ROS.
2+
+
In order to obtain additional information about the structural
characteristics that might provide plausible interpretation for
redox properties of the prepared compounds and, in particular,
for the unique propensity of ferrocenyloxypyridazin 4 to undergo
sequentional SET processes under the CV conditions employed,
a reasonable selection of diamagnetic dications were also
Table 3. In vitro cytostatic effects on two human malignant cell lines expressed as IC values, measured for the selected compounds
5
0
a
b
c
d
and Tamoxifen (serving as reference) along with experimental [E (1) and E (2) ] and theoretical parameters [E (1) / EHOMO(2)
and E (1) / E (2) ] characterizing their redox properties of potential relevance in ROS-generation, one of the possible mechanisms
1/2
1/2
HOMO
e
f g
i
i
responsible for the observed antiproliferative effects.
IC ± SD [µM]
50
Compound
E (1)
E (2)
[mV]
EHOMO(1)
[eV]
EHOMO(2)
[eV]
-8.16
-9.20
-9.38
-
E (1)
E (2)
i
f,g
1/2
1/2
i
HepG2
HT-29
a
b
c,g
d,g
e,g
[
mV]
[eV]
6.15
7.21
7.02
9.89
9.86
6.10
6.91
7.74
7.97
11.90
11.53
6.03
6.05
5.64
6.34
5.84
6.09
8.46
6.51
[eV]
10.79
10.72
11.00
-
3
19.6 ± 2.4
43.8 ± 2.0
>100
11.0 ± 3.2
41.7 ± 3.5
>100
-
-
-
-
-
-
-
-
-
-5.47
-5.80
-5.73
-8.61
-8.47
-5.43
-5.46
-6.34
-6.54
-10.47
-10.54
-5.35
-5.40
-5.12
-5.39
-5.16
-5.45
-8.10
-5.55
3
3
a
b
+
h
h
[
[
3bH ]
3bH ]
-
-
-
-
1
+
-
-
-
2
4
19.6 ± 0.5
16.4 ± 0.5
>100
>100
-
18.6 ± 7.9
20.7 ± 7.0
>100
>100
-
235.0
239.5
-
265.5
-
-
256.5
-
168.0
-
172.5
207.0
-
-
404.5
-7.84
-8.22
-10.76
-10.94
-
10.74
11.16
12.16
12.34
-
6
a
-
-
-
-
-
-
-
-
-
-
-
-
-
6
6
aa
ab
+
h
[
[
6abH ]
1
+
h
6abH ]
-
-
-
-
2
6
6
b
g
16.5 ± 4.3
19.9 ± 10.0
5.7 ± 0.1
79.8 ± 18.9
11.3 ± 4.0
24.1 ± 23.2
-
25.2 ± 5.0
54.8 ± 11.1
9.8 ± 1.2
27.1 ± 8.0
15.5 ± 5.4
35.9 ± 7.5
-
-8.93
-9.53
-8.09
-8.56
-8.33
-9.15
-
10.74
11.44
10.03
10.01
10.18
11.05
-
6
l
6
6
la
m
8
b
+
h
[
8bH ]
Tamoxifen
20.0 ± 6.5
15.6 ± 5.7
-8.39
9.77
a
b
c
d
e
f
First half-wave potential obtained in CV-measurements.
Second half-wave potential obtained in CV-measurements.
HOMO level of the parent compounds and the presented protonated forms.
HOMO level of the corresponding radical cations.
Adiabatic ionization energy of the parent compounds and the presented protonated forms
Adiabatic ionization energy of the corresponding radical cations.
Resulted from B3PW91/DGTZVP calculations.
g
h
Assumed to be at least partially present under the conditions of in vitro assays.

6
ACCEPTED MANUSCRIPT
Scheme 5. DFT-modelled stepwise oxidations of 6a taking place in the absence and in the presence of 1 equivalent of
acetonitrile as represented by the optimized structures of the parent molecule and the resulting non-coordinated and
coordinated cationic intermediates supplemented with representative bonding MO’s identified for cationic complexes. The
calculated ionization energies refer to the promoter role of the solvent stabilizing the resulting cations.
than the HOMO’s of other studied radical cations with N-
ferrocenyl/aryl s keleton [note as characteristic examples:
+
+
E
(2)=-8.16 eV for 3 , -8.09 eV for 6l and -8.33 eV for
HOMO
+
6
m ].
As indicated by the delocalization of the HOMO’s of the
selection of the modelled products (Figure 1), compound 4
undergoes an O-ferrocenyl-centered primary SET, while
substantially extended parts of the whole molecular skeletons are
involved in the first ionization of N-ferrocenyl/arylpyridazinones.
+
The delocalization of the HOMO of radical cation 4 indicates
that the second electron transfer detectable in CV experiments
carried out with 4 is centered on the C-ferrocenyl group.
Moreover, in this radical cation the HOMO-1 and HOMO-2, with
energy levels higher to those identified for the isomeric radical
+
cation 3 , are also concentrated on the same metallocene
fragment assumably also increasing the chance of the second
ionization.
In this context the redox chemistry of reference Tamoxifen must
also be commented on. Recent CV studies conducted under non-
aqueous conditions disclosed that its irreversible primary
oxidation generates a very short-lived amine radical cation which
readily abstracts a H-atom from the solvent or an adventitious
3
3
reactant finally affording the corresponding ammonium ion.
This finding is in accordance with the mainly amine-centered
HOMO we identified for the radical cation generated from
Tamoxifen (Figure 1). On the other hand, the reletively high-
energy HOMO of Tamoxifen is delocalized over the whole
phenylstyrene skeleton with slight extension toward the basic
side-chain.
Besides the obvious electronic- and chelate-forming properties of
the N/O-ferrocenyl/aryl group, the relative tendency of the tested
fused pyridazinones type 5 to undergo ionization is controlled by
the electronic effects of the substituents bonded in positions 4
and 8 which can significantly contribute to the stabilization of the
radical cationic species, as clearly indicated by the markedly
increased cytotoxic activity of 6l relative to that of 6a. The
HOMO-raising activation of 6l to behave as a potential reductant
can be ascribed to the donor-type C-substituents significantly
Figure 1. Optimized structures and relevant high-energy
occupied MO’s of selected pyridazine-based products and
Tamoxifen along with their appropriate radical cations.
Besides relative ionization energies HOMO levels of the radical
cations can also be considered as the measure of their propensity
to undergo ionization affording dications. Thus, in accordance
+
with the experimental findings, radical cation 4 features a
+
HOMO lying substantially higher in energy [EHOMO(2)=-7.84 eV]
contributing to the stabilization of ground-state 6l , as

7
represented by HOMO, HOMO-1 and HOMO-2 with
pronounced delocalization involving the side chains. This view
gains support from oxidation-induced shortening of the C4-N and
C8-N bond lengths discernible in the optimized structures of this
determined with a Boethius microstage. Merck Kieselgel (230-
ACCEPTED MANUSCRIPT
400 mesh, 60 Å) was used for flash column chromatography. The
IR spectra were run by the ATR (Attenuated Total Reflectance)
method on a Bruker IFS-55 FT-spectrometer controlled by Opus
3.0 software. The exact mass measurements were performed
using a Q-TOF Premier mass spectrometer (Waters Corporation,
34 Maple St, Milford, MA, USA) in positive electrospray mode.
redox pair (C4-N/C8-N: 1.381 Å/1.358 Å and 1.361 Å/1.350 Å in
+
6
l and 6l , respectively) pointing to intensified lone-pair
delocalization from the directly attached nitrogen atoms towards
+
1
13
the heterocyclic skeleton in 6l .
The H and C NMR spectra were recorded in DMSO-d or
6
The important contribution of the amine-based side-chains to the
ROS-induced cytotoxic effect is reflected in the comparison of
CDCl solution in 5 mm tubes at RT, on a Bruker DRX-500
3
1
13
spectrometer at 500 ( H) and 125 ( C) MHz, with the deuterium
signal of the solvent as the lock and TMS as internal standard
IC -, E
(1)- and E (1) values found for inactive 6aa and
5
0
HOMO
i
1
13
active 6la, respectively (Table 3). It is also worth mentioning the
IC -, E (1)-, E (1)- and E (1) values obtained for 6l and 6m
( H, C). The 2D-COSY, HSQC and HMBC spectra were
obtained by using the standard Bruker pulse programs.
Optimized structures are available from the authors.
50
1/2
HOMO
i
with an almost negligible structural difference in the terminal
region of their C8-substituents. The lowered cytotoxic activity of
6
m relative to that of 6l, assumed at least partly associated with
Preparation of 4-((2-acetoxyethyl)amino)-8-nitrophthalazin-
the slightly decreased level of ROS-generation, might be the
consequence of the decreased basicity of the hydroxyl group in
1(2H)-one (5i) from 5h
6
m relative to that of the piperidinyl group in 6l which is
Phthalazinone 5h (2.50 g, 10mmol) was dissolved in glacial
acetic acid (20 mL) and the solution was refluxed for 2 h. The red
colored reaction mixture was concentrated in vacuum to half of
its volume then diluted with water. The precipitated solid was
filtered off and recrystallized from water (ca. 130-150 mL).
Yellow solid; Yield: 2.12 g (73%); mp.: 231.1-232.6 °C (from
involved in a five-membered chelate ring of a bifurcated H-bond
system (with the carbonyl-oxygen as the second donor site),
capable of strengthening the abovementioned lone pair
delocalization from the directly attached nitrogen towards the
electron-deficient heterocyclic skeleton in 6l . This view gains
support from the spectacularly different extensions and energies
of the two highest-level occupied orbitals (HOMO and HOMO-
+
-
1
H O); IR (ATR, cm ): 3385, 3352, 1748, 1730, 1658, 1589,
2
1529, 1455, 1431, 1372, 1339, 1309, 1248, 1233, 1219, 1179,
+
+
1
) visualized for 6l and 6m , respectively, consistent with the
1154, 1139, 1032, 981, 951, 851, 822, 746, 712, 663, 605, 574,
1
small but definite difference in the C8-N bond lengths found in
their optimized structures (1.349 Å and 1.353 Å in 6l and 6m ,
respectively). Accordingly, the same C4-N atomic distance
387, 370; H-NMR (DMSO-d ): 11.95 (1H, s, H2), 8.31 (1H, dd,
6
+
+
J = 6.5 Hz, 2.9 Hz, H5), 8.08 (1H, d, J = 7.7 Hz, H7, overlapped
by H6), 8.07 (1H, t, J = 7.6 Hz, H6, overlapped by H7), 6.98 (1H,
t, J = 5.4 Hz, CH NH), 4.21 (2H, t, J = 5.8 Hz, CH O), 3.48 (2H,
(1.361 Å) is discernible in both of these cations carrying a 2-
2
2
13
hydroxyethylamino group in position 4.
q, J = 5.7 Hz, CH NH), 1.99 (3H, s, COCH ); C-NMR (DMSO-
2
3
d ): 171.0 (COCH ), 154.6 (C1), 149.2 (C8), 144.3 (C4), 134.5
6
3
3
. Conclusions
(
(
C6), 126.7 (C4a), 126.2 (C5), 125.7 (C7), 119.0 (C8), 62.3
CH O), 40.6 (CH NH). HRMS exact mass calcd. for
2
2
Starting from easily available pyridazinone-based precursors and
iodoferrocene (2), Cu-mediated cross-coupling protocol
+
C H N O [MH] , requires m/z 293.0886, found m/z 293.0879.
12
13
4
5
a
employing Pd-catalysis provided 3-ferrocenyl-6-
Preparation
of
4-((2-hydroxyethyl)amino)-8-
ferrocenyloxypyridazine and a series of novel N-ferrocenyl-
substituted lactams, the first representatives of novel classes of
functionalized organometallics with promising antitumor actions
at micromolar concentrations on HepG2 and HT-29 tumor cell
cultures. The use of a diverse range of pyridazinones and related
lactams is expected to result in a library of related ferrocene-
containing cytostatic heterocycles of which activity seems to be –
at least partly – associated with ROS-generation. This view was
supported by the well-correlated results of comparative biological
tests, cyclic voltammetry (CV) experiments and DFT studies
carried out on selected organometallic derivatives and on their N-
phenyl- and N-(2-pyridyl)-substituted analogues. Besides the
feasibility of further coupling reactions including conjugation to
biomolecules enabled by the functional groups attached to the
heterocyclic scaffolds, the established structure-activity
relationships, disclosing the importance of highly substituent-
dependent fine-tunable redox properties, might be taken into
(
phenylthio)phthalazin-1(2H)-one (5j) from 5h
Phthalazinone 5h (2.50 g, 10 mmol), thiophenol (1.32 g, 12
mmol) and K CO (2.21 g, 1.6 mmol) were dissolved in DMF (15
mL) and the solution was refluxed for 5 h. The yellow reaction
mixture was diluted with water and the precipitated solid was
filtered off and recrystallized from EtOH (ca. 80-100 mL).
White solid; Yield: 1.92 g (61%) ; mp.: 307.4-310.7 °C (from
2
3
-
1
EtOH); IR (ATR, cm ): 3330, 2997, 2863, 1625, 1578, 1534,
1
9
4
509, 1460, 1438, 1333, 1320, 1229, 1175, 1136, 1068, 1022,
98, 915, 810, 779, 757, 705, 693, 666, 606, 540, 476, 428, 415,
1
01, 362; H-NMR (DMSO-d ): 11.61 (1H, s, H2), 7.73 (1H, d, J
6
=
8.0 Hz, H5), 7.58-7.51 (6H, m, H6 and H2”-H6”), 6.76 (1H, d,
J = 8.0 Hz, H7), 6.45 (1H, t, J = 5.3 Hz, CH NH), 4.69 (1H, t, J =
5
J = 5.8 Hz, CH NH); C-NMR (DMSO-d ): 159.8 (C1), 145.2
2
.5 Hz, CH OH), 3.59 (2H, q, J = 5.8 Hz, CH OH), 3.29 (2H, q,
2 2
13
2
6
(C4), 144.0 (C8), 136.4 (C3” and C5”), 132.8 (C6), 132.4 (C1”),
account for
a rational fragment-based design of related
1
1
30.8 (C2” and C6”), 130.2 (C4”), 127.4 (C7), 127.0 (C4a),
23.5 (C8a), 59.1 (CH OH), 44.6 (CH NH). HRMS exact mass
organometallic- and purely organic drug candidates.
2
2
+
4
. Experimental section
calcd. for C16H N O S [MH] , requires m/z 314.0963, found m/z
3
16 3 2
14.0970.
All chemicals (expect iodoferrocene (2), which was prepared
34
according to the method of Fish and Rosenblum ) were obtained
from commercially available sources (Aldrich, Fluka) and used
without further purification, expect solvents. Methanol, dioxane
and triethylamine (TEA) were distilled from sodium, N,N-
dimethylformamide was distilled from 4A Molecular sieves
under reduced pressure. Melting points (uncorrected) were

8
Preparation of imines 5o and 5p from 4-((2-
hydroxyethyl)amino)-8-(1-methylhydrazinyl)phthalazin-1(2H)-
mixtures as eluent) and subsequent crystallization from MeOH,
i t
ACCEPTED MANUSCRIPT
EtOH, PrOH, BuOH, MeOH-water or EtOH-water.
one (5n)
2
,6-Diferrocenylpyridazin-3(2H)-one (3)
Benzaldehyde (0.531 g, 5 mmol) or formylferrocene (1.070 g, 5
mmol) in MeOH (1 mL) was added to 5n (1.250 g, 5 mmol)
suspended in MeOH (5 mL) and the resulting suspension was
stirred at 65 °C for 12 h. The reaction mixture was cooled down
to room temperature, and the solid precipitate formed was filtered
off washed with water and dried over P O in vacuum.
Red solid, Yield: 72.1 mg (16%); mp.: 197.3-199.2 °C (from
EtOH); IR (ATR, cm ): 3095, 1654, 1597, 1551, 1488, 1411,
1394, 1368, 1304, 1270, 1230, 1120, 1105, 1078, 1058, 1026,
-
1
1000, 932, 890, 843, 817, 676, 579, 545, 503, 484, 443, 391, 381;
1
H-NMR (DMSO-d ): 7.74 (1H, d, J = 9.6 Hz, H5), 6.94 (1H, d,
6
J = 9.6 Hz, H4), 5.16 (2H, s, H2’ and H5’), 4.97 (2H, t, J = 1.8
Hz, H2’’ and H5’’), 4.52 (2H, t, J = 1.8 Hz, H3’’ and H4’’), 4.26
2
5
5
5
(E)-8-(2-Benzylidene-1-methylhydrazinyl)-4-((2-
(2H, s, H3’ and H4’), 4.21 (5H, s, 2-η -C H ), 4.18 (5H, s, 6-η -
5 5
13
hydroxyethyl)amino)phthalazin-1(2H)-one (5o)
C H ); C-NMR (DMSO-d ): 158.8 (C3), 146.3 (C6), 130.9
5 5 6
Light yellow solid; Yield: 1.459 g (86%); mp.: 274.2-276.9 °C
(C5), 129.7 (C4), 99.6 (C1’), 80.4 (C1’’), 70.9 (C3’’ and C4’’),
70.0 (2-η -C H and 6-η -C H ), 67.3 (C2’’ and C5’’), 66.3 (C3’
5 5 5 5
-
1
5 5
(from MeOH); IR (ATR, cm ): 3266, 1628, 1589, 1562, 1533,
1
1
7
477, 1445, 1418, 1393, 1377, 1309, 1291, 1252, 1200, 1162,
130, 1100, 1089, 1067, 995, 922, 882, 873, 853, 821, 811, 790,
69, 757, 700, 691, 674, 653, 615, 581, 552, 520, 460; H-NMR
and C4’), 64.4 (C2’ and C5’). HRMS exact mass calcd. for
56 +
C H N O Fe [MH] , requires m/z 465.0353, found m/z
24
21
2
2
1
465.0345.
(
DMSO-d ): 11.31 (1H, s, CONH), 7.78 (1H, t, J = 7.8 Hz, H6),
6
7
.76 (1H, d, J = 7.5 Hz, H5), 7.72 (1H, dd, J = 7.0 Hz, 2.0 Hz,
H7), 7.68 (1H, s, CHPh), 7.67 (2H, d, J = 7.7 Hz, H2’ and H6’),
.38 (2H, t, J = 7.6 Hz, H3’ and H5’), 7.27 (1H, t, J = 7.3 Hz,
H4’), 6.33 (1H, t, J = 5.4 Hz, NH), 4.66 (1H, t, J = 5.7 Hz, OH),
.64 (2H, q, J = 6.0 Hz, CH OH), 3.33 (2H, q, J = 5.8 Hz,
3-Ferrocenyl-6-ferrocenyloxypyridazine (4)
Dark red solid, Yield: 201.9 mg (43%); mp.: 185.4-186.7 °C
-
1
7
(from EtOH); IR (ATR, cm ): 1660, 1592, 1522, 1457, 1392,
1302, 1282, 1236, 1219, 1173, 1130, 1104, 1096, 1070, 1026,
3
998, 916, 882, 849, 820, 801, 687, 625, 503, 483, 454, 440, 398;
2
1
CH NH, overlapped by HDO signal of the solvent), 3.28 (3H, s,
CH3); C-NMR (DMSO-d ): 157.0 (C1), 149.6 (C8), 145.2 (C4),
1
1
H-NMR (DMSO-d ): 7.92 (1H, d, J = 9.2 Hz, H4), 7.29 (1H, d,
2
6
13
J = 9.2 Hz, H5), 5.03 (2H, t, J = 1.8 Hz, H2’ and H5’), 4.56 (2H,
s, H2’’ and H5’’), 4.49 (2H, t, J = 1.8 Hz, H3’’ and H4’’), 4.27
6
37.0 (C1’), 134.2 (CHPh), 132.9 (C6), 129.0 (C3’ and C5’),
28.0 (C4’), 127.8 (C4a), 126.2 (C7 and C2’ and C6’), 119.2
5
5
(5H, s, 6-η -C H ), 4.07 (7H, s, H3’’ and H4’’ and 3-η -C H );
5
5
5
5
13
(C5), 118.2 (C8a), 59.8 (CH OH), 44.7 (CH NH), 42.2 (CH ).
C-NMR (DMSO-d ): 165.0 (C6), 158.3 (C3), 128.8 (C4), 119.9
6
5
2
2
3
+
HRMS exact mass calcd. for C H N O [MH] , requires m/z
2
(C1’), 117.7 (C5), 80.9 (C1’’), 70.8 (C3’ and C4’), 70.0 (3-η -
18
20
5
2
5
93.0886, found m/z 293.0892.
C H ), 69.7 (6-η -C H ), 67.7 (C2’ and C5’), 63.6 (C3’’ and
5
5
5
5
C4’’), 61.7 (C2’’ and C5’’). HRMS exact mass calcd. for
5
6
+
(E)-8-(2-Ferrocenylidene-1-methylhydrazinyl)-4-((2-
C H N O Fe [MH] , requires m/z 465.0353, found m/z
24 21 2 2
hydroxyethyl)amino)phthalazin-1(2H)-one (5p)
465.0348.
Reddish orange solid; Yield: 1.93 g (87%); mp.: 235.2-238.6
-
1
(
degr.) °C (from MeOH); IR (ATR, cm ): 3210, 1618, 1582,
2-Ferrocenylphthalazin-1(2H)-one (6a)
1
1
6
1
540, 1466, 1387, 1293, 1249, 1215, 1177, 1154, 1124, 1104,
Reddish orange solid; Yield: 73.0 mg (22%); mp.: 133.6-135.8
-
1
050, 1039, 990, 939, 875, 850, 822, 801, 765, 703, 682, 644,
°C (from EtOH); IR (ATR, cm ): 1656, 1593, 1483, 1458, 1449,
1399, 1360, 1344, 1324, 1309, 1275, 1240, 1227, 1157, 1134,
1117, 1102, 1080, 1034, 1024, 1014, 996, 966, 929, 907, 989,
1
21, 569, 520, 493, 482, 450, 440, 419; H-NMR (DMSO-d ):
6
1.29 (1H, s, CONH), 7.77 (1H, t, J = 7.9 Hz, H6), 7.70 (1H, d, J
1
=
6
(
7.9 Hz, H5), 7.67 (1H, d, J = 7.9 Hz, H7), 7.49 (1H, s, FcCH),
.34 (1H, t, J = 5.2 Hz, NH), 4.70 (1H, t, J = 5.4 Hz, OH), 4.62
2H, br s, H2’ and H5’), 4.33 (2H, br s, H3’ and H4’), 4.19 (5H,
979, 844, 834, 815, 805, 763, 726, 686, 598, 537, 503; H-NMR
(DMSO-d ): 8.57 (1H, br s, H4), 8.33 (1H, d, J = 7.9 Hz, H8),
6
7.97 (1H, d, J = 7.9 Hz, H5, overlapped by H5), 7.96 (1H, td, J =
7.9 Hz, 1.3 Hz, H6, overlapped by H6), 7.90 (1H, td, J = 7.9 Hz,
1.8 Hz, H7), 5.13 (2H, t, J = 2.0 Hz, H2’ and H5’), 4.24 (2H, t, J
5
s, η -C H ), 3.63 (2H, q, J = 5.3 Hz, CH OH), 3.33 (2H, q, J =
5
5
5
2
13
.3 Hz, CH NH), 3.18 (3H, s, CH ); C-NMR (DMSO-d ): 157.0
2
3
6
5
13
(
(
(
(
C1), 150.0 (C8), 145.2 (C4), 133.0 (C6), 127.8 (C4a), 125.3
C7), 118.5 (C8a), 117.3 (C5), 134.7 (FcCH), 83.0 (C1’), 69.4
η -C H ), 69.3 (C3’ and C4’), 66.9 (C2’ and C5’), 59.8
CH OH), 44.6 (CH NH), 42.4 (CH ). HRMS exact mass calcd.
= 2.0 Hz, H3’ and H4’), 4.16 (5H, s, η -C H ); C-NMR
5 5
(DMSO-d ): 158.4 (C1), 139.2 (C4), 134.1 (C6), 133.0 (C7),
6
5
129.1 (C4a), 128.2 (C8a), 127.6 (C5), 126.6 (C8), 100.3 (C1’),
5
5
5
69.8 (η -C H ), 66.0 (C3’ and C4’), 64.0 (C2’ and C5’). HRMS
2
2
3
5
5
56
+
56
+
for C H N O Fe [MH] , requires m/z 446.1279, found m/z
exact mass calcd. for C H N O Fe [MH] , requires m/z
22
24
5
2
18 15 2
4
46.1282.
331.0534, found m/z 331.0540.
General method for the coupling reactions of pyrydazin-based
lactams with aryl halides iodoferrocene (2), iodobenzene and 2-
bromopyridine
4-(Diethylamino)-2-ferrocenylphthalazin-1(2H)-one (6b)
Orange solid; Yield: 137.3 mg (34%); mp.: 131.5-132.5 °C (from
-
1
EtOH); IR (ATR, cm ): 2973, 1656, 1584, 1551, 1484, 1464,
448, 1407, 1397, 1382, 1370, 1331, 1316, 1290, 1234, 1204,
1
The precursor lactam (1 mmol) was dissolved in DMF-TEA 1:1
1171, 1140, 1117, 1105, 1085, 1074, 1062, 1021, 999, 967, 888,
(
2 mL) ,then K CO (0.410 g, 3 mmol), CuI (190 mg, 1 mmol),
856, 827, 809, 780, 745, 719, 695, 678, 600, 555, 502, 492, 461,
2
3
1
PdCl (PPh ) (17.5 mg, 0.025 mmol) and aryl halide (1.1 mmol)
429, 401; H-NMR (DMSO-d ): 8.33 (1H, br d, J = 7.8 Hz, H8),
2
3 2
6
were added to the solution. The reaction mixture was stirred
under argon, at 110 °C for overnight. The reaction mixture was
filtered and brine (30 mL) was added to the solution. The
precipitated solid was filtered off then washed with brine and
water. The crude product was purified by column
chromatography (on silica, using DCM or DCM-MeOH 100-5:1
7.98-7.92 (2H, m, H5 and H6), 7.87 (1H, ddd, J = 7.8 Hz, 6.5 Hz,
1.9 Hz, H7), 5.14 (2H, t, J = 1.9 Hz, H2’ and H5’), 4.21 (2H, t, J
5
= 1.9 Hz, H3’ and H4’), 4.14 (5H, s, η -C H ), 3.32 (4H, q, J =
5
5
1
3
7.0 Hz, NCH ), 1.19 (6H, t, J = 7.0 Hz, CH ); C-NMR (DMSO-
2
3
d ): 157.4 (C1), 147.9 (C4), 133.6 (C6), 132.2 (C7), 129.2 (C8a),
6
5
127.4 (C8), 126.8 (C4a), 125.4 (C5), 100.3 (C1’), 69.6 (η -C H ),
5
5
6
5.7 (C3’ and C4’), 63.6 (C2’ and C5’), 45.6 (NCH ), 12.6
2

9
5
6
+
(
CH ). HRMS exact mass calcd. for C H N O Fe [MH] ,
1463, 1443, 1412, 1398, 1366, 1340, 1329, 1309, 1287, 1260,
3
22
24
ACCEPTED MANUSCRIPT
3
requires m/z 402.1269, found m/z 402.1260.
1195, 1173, 1154, 1134, 1118, 1105, 1089, 1035, 1026, 1016,
82, 929, 858, 815, 795, 768, 720, 680, 643, 629, 605, 505, 496,
481, 457, 441, 422, 401; H-NMR (DMSO-d ): 8.32 (1H, d, J =
9
1
2
-Ferrocenyl-4-(pyrrolidin-1-yl)phthalazin-1(2H)-one (6c)
6
Orange solid; Yield: 107.6 mg (27%); mp: 140.2-142.5 °C (from
EtOH); IR (ATR, cm ): 2960, 2930, 2861, 1641, 1609, 1575,
7.9 Hz, H8), 8.14 (1H, d, J = 5.1 Hz, H5), 7.92 (1H, t, J = 7.6 Hz,
H6), 7.85 (1H, t, J = 7.6 Hz, H7), 7.03 (1H, t, J = 4.9 Hz, NH),
-
1
1
1
7
4
1
526, 1492, 1466, 1438, 1407, 1363, 1330, 1241, 1231, 1161,
148, 1104, 1076, 1030, 1019, 997, 949, 928, 912, 893, 856, 817,
5.19 (2H, br s, H2’ and H5’), 4.20 (2H, t, J = 6.3 Hz, CH O),
2
5
4.17 (2H, br s, H3’ and H4’), 4.13 (5H, s, η -C H ), 3.49 (2H, q,
5
5
88, 778, 756, 718, 686, 655, 643, 628, 615, 595, 535, 498, 485,
J = 6.0 Hz, CH NH), 2.10 (2H, p, J = 6.3 Hz, CH CH CH ), 2.04
2 2 2 2
13
1
59, 446, 408; H-NMR (DMSO-d ): 8.34 (1H, dd, J = 7.8 Hz,
(3H, s, CH3); C-NMR (DMSO-d ): 171.0 (CH COO), 156.4
6 3
6
.5 Hz, H8), 8.15 (1H, d, J = 8.0 Hz, H5), 7.91 (1H, td, J = 7.6
(C1), 144.6 (C4), 133.2 (C7), 132.0 (C6), 128.6 (C8a), 127.4
5
Hz, 1.5 Hz, H6), 7.85 (1H, td, J = 7.5 Hz, 1.2 Hz, H7), 5.13 (2H,
t, J = 2.0 Hz, H2’ and H5’), 4.18 (2H, t, J = 2.0 Hz, H3’ and
H4’), 4.14 (5H, s, η -C H ), 3.61 (4H, t, J = 6.5 Hz, H2’’ and
(C8), 124.4 (C4a), 123.4 (C5), 100.7 (C1’), 69.4 (η -C H ), 65.4
5
5
(C3’ and C4’), 63.5 (C2’ and C5’), 62.8 (CH O), 38.7 (CH NH),
2
2
5
27.9 (CH CH CH ), 21.3 (CH ). HRMS exact mass calcd. for
2 2 2 3
56 +
5
5
13
H5’’), 1.96 (4H, tt, J = 6.5 Hz, 2.0 Hz, H3’’and H4’’); C-NMR
DMSO-d ): 157.0 (C1), 147.2 (C4), 133.2 (C6), 132.0 (C7),
C H N O Fe [MH] , requires m/z 446.1167, found m/z
23 24 3 3
(
1
446.1174.
6
5
29.2 (C8a), 127.7 (C8), 126.1 (C4a), 125.9, (C5), 69.7 (η -
C H ), 65.5 (C3’ and C4’), 63.6 (C2’ and C5’), 51.0 (C2’’ and
2-Ferrocenyl-4-(2-hydroxyethoxy)phthalazin-1(2H)-one (6g)
5
5
C5’’), 25.5 (C3’’ and C4’’). HRMS exact mass calcd. for
Orange solid; Yield: 103.1 mg (26%); mp.: 152.6-153.5 °C (from
EtOH); IR (ATR, cm ): 3428, 3087, 2944, 1636, 1619, 1587,
56
+
-1
C H N O Fe [MH] , requires m/z 400.1112, found m/z
22
24
3
4
00.1107.
1495, 1454, 1410, 1384, 1348, 1316, 1240, 1187, 1102, 1079,
1
027, 1017, 1000, 967, 930, 899, 858, 825, 809, 794, 778, 728,
1
4
-(2,6-Dimethylmorpholino)-2-ferrocenylphthalazin-1(2H)-one
690, 644, 574, 485, 457, 442; H-NMR (DMSO-d ): 8.31 (1H, d,
6
(
6d)
J = 7.7 Hz, H8), 8.09 (1H, d, J = 7.6 Hz, H5), 7.97 (1H, td, J =
7.5 Hz, 1.5 Hz, H6), 7.93 (1H, td, J = 7.5 Hz, 1.5 Hz, H7), 5.14
(2H, t, J = 2.0 Hz, H2’ and H5’), 5.03 (1H, t, J = 5.7 Hz, OH),
Yellowish orange solid; Yield: 100.0 mg (23%); mp.: 160.6-
-
1
1
1
1
1
6
62.8 °C (from EtOH); IR (ATR, cm ): 2977, 2853, 1656, 1587,
547, 1487, 1468, 1452, 1422, 1398, 1379, 1363, 1329, 1317,
269, 1234, 1123, 1187, 1174, 1144, 1104, 1084, 1072, 1054,
038, 1024, 1000, 968, 930, 895, 860, 817, 790, 778, 767, 724,
4.46 (2H, t, J = 4.9 Hz, COCH ), 4.21 (2H, t, J = 2.0 Hz, H3’ and
2
5
H4’), 4.18 (5H, s, η -C H ), 3.90 (2H, q, J = 5.2 Hz, CH OH);
C-NMR (DMSO-d ): 157. (C1), 149.5 (C4), 134.0 (C6), 133.0
(C7), 127.2 (C8), 129.3 (C8a), 124.1 (C5), 123.9 (C4a), 69.8 (η -
5
5
2
13
6
1
5
98, 678, 658, 648, 599, 503, 489, 462, 428, 406; H-NMR
(
7
CDCl ): 8.45 (1H, br s, H8), 7.88 (1H, br d, J = 4.1 Hz, H5),
C H ), 69.2 (COCH ), 65.0 (C3’ and C4’), 63.8 (C2’ and C5’),
3
5
5
2
5
6
.80 (1H, br s, H6), 7.76 (1H, br s, H7), 5.46 (2H, s, H2’ and
60.0 (CH OH). HRMS exact mass calcd. for C H N O Fe
2 20 19 2 3
+
5
H5’), 4.45 (2H, s, H3’ and H4’), 4.41 (5H, s, η -C H ), 4.04 (2H,
[MH] , requires m/z 391.0745, found m/z 391.0752.
5
5
br s, CHCH O), 3.44 (2H, br d, J = 8.2 Hz, H3’’a and H5’’a),
3
2
.79 (2H, br t, H3’’b and H5’’b), 1.31 (6H, s, CH , overlapped by
2-Ferrocenyl-4-((2-hydroxyethyl)amino)-8-nitrophthalazin-
1(2H)-one (6h)
3
13
HDO signal of the solvent); C-NMR (CDCl ): 157.8 (C1),
3
1
1
6
1
48.3 (C4), 132.5 (C6), 131.4 (C7), 129.6 (C4a), 127.8 (C8),
Dark red solid; Yield: 70.4 mg (16%); mp.: 199.9-201.4 °C (from
EtOH); IR (ATR, cm ): 3574, 3336, 1643, 1582, 1538, 1521,
5
-1
25.6 (C8a), 124.5 (C5), 71.7 (η -C H ), 71.6 (C2’’ and C6’’),
5
5
6.9 (C3’ and C4’), 64.3 (C2’ and C5’), 57.0 (C3’’ and C5’’),
1455, 1399, 1376, 1343, 1331, 1299, 1238, 1168, 1122, 1106,
1076, 1045, 1032, 1002, 951, 876, 864, 849, 824, 812, 803, 779,
56
+
9.2 (CH ). HRMS exact mass calcd. for C H N O Fe [MH] ,
3
24 26
3
2
1
requires m/z 444.1374, found m/z 444.1379.
761, 725, 705, 676, 642, 540, 496, 486, 456, 434, 387, 375; H-
NMR (DMSO-d ): 8.39 (1H, d, J = 7.4 Hz, H5), 8.12 (1H, d, H7,
6
2
-Ferrocenyl-4-(phenethylamino)phthalazin-1(2H)-one (6e)
overlapped by H6), 8.09 (1H, t, H6, overlapped by H7), 7.14
Orange solid; Yield: 67.4 mg (15%); mp: 170.9-173.3 °C (from
EtOH); IR (ATR, cm ): 3345, 1632, 1579, 1537, 1497, 1478,
(1H, br t, NH), 5.06 (2H, s, H2’ and H5’), 4.78 (1H, t, J = 5.3 Hz,
-
1
5
OH), 4.18 (2H, s, H3’ and H4’), 4.15 (5H, s, η -C H ), 3.76 (2H,
5
5
1
3
1
1
6
7
452, 1428, 1381, 1328, 1313, 1229, 1185, 1167, 1151, 1119,
q, J = 5.6 Hz, CH OH), 3.49 (2H, q, J = 5.5 Hz, CH NH); C-
2
2
108, 1048, 1026, 1003, 925, 801, 771, 747, 719, 701, 687, 646,
NMR (DMSO-d ): 152.6 (C1), 149.4 (C8), 143.9 (C4), 134.3
6
1
13, 586, 474, 455, 406; H-NMR (DMSO-d ): 8.33 (1H, d, J =
(C6), 131.4 (C4a), 126.3 (C7), 126.0 (C5), 118.9 (C8a), 99.9
6
5
.5 Hz, H5), 8.14 (1H, d, J = 8.1 Hz, H8), 7.92 (1H, t, J = 7.3 Hz,
(C1’), 69.7 (η -C H ), 65.7 (C3’ and C4’ and CH OH), 63.8 (C2’
5
5
2
H6), 7.85 (1H, t, J = 7.5 Hz, H7), 7.39-7.33 (4H, m, H2’’ and
H3’’ and H5’’ and H6’’), 7.24 (1H, tt, J = 7.3 Hz, 2.0 Hz, H4’’),
and C5’), 44.8 (CH NH); HRMS exact mass calcd. for
2
56 +
C H N O Fe [MH] , requires m/z 435.0756, found m/z
20
19
4
4
7
.11 (1H, t, J = 5.0 Hz, NH), 5.20 (2H, br s, H2’ and H5’), 4.19
435.0765.
5
(2H, br s, H3’ and H4’) 4.13 (5H, s, η -C H ), 3.64 (2H, td, J =
5
5
13
7
.4 Hz, 5.0 Hz, CH NH), 3.11 (2H, t, J = 7.4 Hz, CH Ph); C-
4-((2-Acetoxyethyl)amino)-2-ferrocenyl-8-nitrophthalazin-1(2H)-
one (6i)
2
2
NMR (DMSO-d ): 156.5 (C1), 144.6 (C4), 140.6 (C4a), 133.3
6
(
1
1
C6), 132.1 (C7), 129.1 (C2’’ and C6’’), 128.9 (C3’’ and C5’’),
Dark red solid; Yield: 172.6 mg (36%); mp.: ~220 (degr.) °C
-
1
28.6 (C8a), 127.3 (C5), 126.5 (C4’), 124.4 (C1’’), 123.4 (C8),
(from EtOH); IR (ATR, cm ): 3418, 1723, 1641, 1588, 1534,
1463, 1454, 1444, 1410, 1380, 1363, 1333, 1308, 1230, 1174,
1156, 1139, 1105, 1027, 1000, 958, 931, 914, 886, 851, 812, 801,
5
00.7 (C1’), 69.6 (η -C H ), 65.5 (C3’ and C4’), 63.5 (C2’ and
5
5
C5’), 43.8 (CH NH), 34.7 (CH Ph). HRMS exact mass calcd. for
C H N O Fe [MH] , requires m/z 450.1269, found m/z
2
2
56
+
1
775, 761, 725, 699, 676, 643, 610, 570, 494, 459, 441, 405; H-
26
24
3
4
50.1277.
NMR (DMSO-d ): 8.36 (1H, d, J = 7.5 Hz, H5), 8.14 (1H, br d, J
6
=
8.4 Hz, H7), 8.10 (1H, t, J = 8.0 Hz, H6), 7.34 (1H, t, J = 5.4
4
-((2-Acetoxypropyl)amino)-2-ferrocenyl-phthalazin-1(2H)-one
Hz, NH), 5.08 (2H, s, H2’ and H5’), 4.18 (2H, s, H3’ and H4’),
5
(6f)
4.16 (5H, s, η -C H ), 4.40 (2H, t, J = 5.7 Hz, CH OH), 3.67 (2H,
5
5
2
13
Yellow solid; Yield: 71.6 mg (16%); mp.: 133.9-135.2 °C (from
EtOH); IR (ATR, cm ): 3420, 1715, 1651, 1590, 1544, 1483,
q, J = 5.5 Hz, CH NH), 2.04 (3H, s, CH ); C-NMR (DMSO-d6):
2
3
-
1
171.0 (CH COO), 154.7 (C1), 149.4 (C8), 143.8 (C4), 134.4
3

10
+
56
(
C6), 126.2 (C5), 126.1 (C7), 125.8 (C4a), 119.0 (C8a), 99.9
(C4’’); HRMS exact mass calcd. for C H N O Fe [MH] ,
27 34 5 2
requires m/z 516.2062, found m/z 516.2056.
ACCEPTED MANUSCRIPT
5
(C1’), 69.6 (η -C H ), 65.8 (C3’ and C4’), 63.8 (C2’ and C5’),
5
5
6
2.0 (CH O), 41.0 (CH NH), 21.4 (CH ). HRMS exact mass
2 2 3
56 +
calcd. for C H N O Fe [MH] , requires m/z 477.0861, found
2-Ferrocenyl-4,8-bis((2-hydroxyethyl)amino)phthalazin-1(2H)-
22
21
4
5
m/z 477.0856.
one (6m)
Yellow solid; Yield: 31.2 mg (7%); mp.: 222.3-224.1 (degr.) °C
-
1
2
(
-Ferrocenyl-4-((2-hydroxyethyl)amino)-8-
phenylthio)phthalazin-1(2H)-one (6j)
Orange solid; Yield: 203.1 mg (41%); mp.: 165.3-167.9 °C (from
(from EtOH); IR (ATR, cm ): 3374, 2841, 1634, 1588, 1570,
1528, 1470, 1450, 1404, 1377, 1350, 1323, 1299, 1248, 1234,
1214, 1187, 1165, 1134, 1105, 1092, 1063, 1024, 1000, 980, 893,
-
1
EtOH); IR (ATR, cm ): 3297, 1625, 1595, 1570, 1534, 1458,
874, 837, 812, 802, 762, 701, 686, 672, 633, 574, 539, 498, 491,
462, 448, 418; H-NMR (DMSO-d ): 9.48 (1H, t, J = 4.4 Hz, 8-
NH), 7.57 (1H, t, J = 7.8 Hz, H6), 7.09 (1H, d, J = 7.8 Hz, H5),
6.86 (1H, d, J = 8.3 Hz, H7), 6.56 (1H, t, J = 4.4 Hz, 4-NH), 5.08
(2H, br s, H2’ and H5’), 4.88 (1H, t, J = 4.9 Hz, 8-OH), 4.74 (1H,
1
1
9
4
438, 1307, 1237, 1194, 1166, 1135, 1104, 1070, 1053, 1024,
6
98, 944, 910, 837, 818, 805, 778, 751, 698, 690, 647, 567, 534,
1
93, 459, 433, 411; H-NMR (DMSO-d ): 7.80 (1H, d, J = 5.6
6
Hz, H5), 7.65-7.51 (6H, m, H6 and H2’’– H6’’), 6.84 (1H, d, J =
.4 Hz, H7), 6.79 (1H, br t, NH), 5.13 (2H, s, H2’ and H5’), 4.74
5
6
t, J = 5.3 Hz, 4-OH), 4.13 (5H, s, η -C H ), 3.74 (2H, q, J = 5.8
5
5
5
(1H, br t, OH), 4.17 (7H, s, H3’ and H4’ and η -C H ), 3.75 (2H,
Hz, 4-CH OH), 3.66 (2H, q, J = 5.2 Hz, 8-CH OH), 3.45 (2H, q,
2 2
13
5
5
13
br s, CH OH), 3.48 (2H, br s, CH NH); C-NMR (DMSO-d6):
J = 5.5 Hz, 4-CH NH), 3.27 (2H, q, J = 5.0 Hz, 8-CH NH); C-
2 2
2
2
1
56.8 (C1), 144.6 (C4 and C8), 136.4 (C3’’ and C5’’), 132.7
NMR (DMSO-d ): 159.6 (C1), 151.1 (C8), 145.1 (C4), 134.7
6
(
(
(
(
C6), 132.6 (C1’’), 130.9 (C2’’ and C6’’), 130.3 (C4’’), 128.1
(C6), 125.9 (C4a), 111.7 (C7), 110.3 (C8a), 107.6 (C5), 100.6
5
C7), 126.0 (C4a), 123.5 (C8a), 119.0 (C5), 100.5 (C1’), 69.5
(C1’), 69.4 (η -C H ), 65.2 (C3’ and C4’), 63.7 (C2’ and C5’),
5
5
5
η -C H ), 65.4 (C3’ and C4’), 63.7 (C2’ and C5’), 59.4
59.7 (8-CH OH), 59.6 (4-CH OH), 45.3 (4-CH NH), 44.7 (4-
2 2 2
56 +
5
5
CH OH), 44.8 (CH NH); HRMS exact mass calcd. for
CH NH); HRMS exact mass calcd. for C H N O Fe [MH] ,
2 22 25 4 3
2
2
56
+
C H N O S Fe [MH] , requires m/z 498.0939, found m/z
requires m/z 449.1276, found m/z 449.1270.
26
24
3
2
4
98.0946.
(E)-8-(2-Benzylidene-1-methylhydrazinyl)-2-ferrocenyl-4-((2-
2
-Ferrocenyl-4-((2-hydroxyethyl)amino)-8-(pyrrolidin-1-
hydroxyethyl)amino) phthalazin-1(2H)-one (6o)
yl)phthalazin-1(2H)-one (6k)
Yellowish orange solid; Yield: 104.4 mg (23%); mp.: 198.1-
Reddish orange solid; Yield: 96.6 mg (19%); mp.: 185.9-187.5
-
1
°C (from EtOH); IR (ATR, cm ): 3311, 1629, 1593, 1569, 1543,
1468, 1448, 1431, 1382, 1360, 1318, 1228, 1192, 1178, 1155,
1125, 1105, 1087, 1059, 1028, 992, 936, 920, 888, 811, 804, 790,
-
1
1
1
1
8
4
7
99.5 °C (from EtOH); IR (ATR, cm ): 3320, 2871, 2819, 1618,
528, 1470, 1459, 1436, 1392, 1376, 1356, 1338, 1319, 1298,
290, 1247, 1224, 1194, 1161, 1147, 1119, 1105, 1027, 997, 867,
1
768, 752, 703, 692, 669, 642, 620, 586, 542, 498, 475, 451; H-
48, 814, 797, 787, 757, 711, 702, 665, 634, 566, 532, 494, 841,
NMR (DMSO-d ): 7.83-7.82 (2H, m, H5 and H7), 7.78 (1H, q, J
6
1
49, 420; H-NMR (DMSO-d ): 7.57 (1H, t, J = 8.0 Hz, H6),
= 4.6 Hz, H6), 7.74 (1H, s, CHPh), 7.71 (2H, d, J = 7.4 Hz, H2’’
and H6’’), 7.40 (2H, t, J = 7.5 Hz, H3’’ and H5’’), 7.29 (1H, t, J
= 7.3 Hz, H4’’), 6.75 (1H, t, J = 5.4 Hz, NH), 5.12 (2H, s, J = 2.0
Hz, H2’ and H5’), 4.80 (1H, br s, OH), 4.15 (2H, t, H3’ and H4’,
6
.26 (1H, d, J = 7.7 Hz, H5), 7.07 (1H, d, J = 8.5 Hz, H7), 6.44
(1H, br t, J = 5.5 Hz, NH), 5.09 (2H, s, H2’ and H5’), 4.73 (1H,
5
br t, J = 5.6 Hz, OH), 4.11 (7H, s, H3’ and H4’ and η -C H ),
5
5
5
5
3
.75 (2H, br q, J = 6.1 Hz, CH OH), 3.45 (2H, br q, J = 5.9 Hz,
overlapped byη -C H signal), 4.14 (5H, s, η -C H , overlapped
2
5 5 5 5
CH NH), 3.34 (4H, s, H2’’ and H5’’), 1.90 (4H, br s, H3’’ and
by H3’ and H4’), 3.78 (2H, t, J = 7.8 Hz, CH OH), 3.47 (2H, q, J
2
2
13
H4’’); C-NMR (DMSO-d ): 156.1 (C1), 149.5 (C8), 144.9
= 6.0 Hz, CH NH), 3.31 (3H, s, CH , overlapped by solvent
6
2
3
13
(
C4), 132.6 (C6), 127.2 (C4a), 115.6 (C7), 113.9 (C8a), 109.9
signal); C-NMR (DMSO-d ): 155.0 (C1), 149.8 (C8), 144.5
6
5
(C5), 101.5 (C1’), 69.2 (η -C H ), 65.0 (C3’ and C4’), 63.1 (C2’
(C4), 137.0 (C1’’), 134.4 (PhCH), 133.0 (C6), 129.1 (C3’’ and
C5’’), 128.1 (C4’’), 126.8 (C5), 126.7 (C4a), 126.3 (C2’’ and
5
5
and C5’), 59.7 (CH OH), 52.0 (C2’’ and C5’’), 44.8 (CH NH),
2
2
56
+
5
2
5.9 (C3’’ and C4’’); HRMS exactmassC H N O Fe [MH] ,
C6’’), 118.9 (C8a), 118.2 (C7), 101.2 (C1’), 69.4 (η -C H ), 65.3
24
27
4
2
5
5
requires m/z 459.1483, found m/z 459.1489.
(C3’ and C4’), 63.7 (C2’ and C5’), 59.6 (CH OH), 44.8
2
(
CH NH), 41.9 (CH ); HRMS exact mass calcd for
2 3
56 +
2
-Ferrocenyl-4-((2-hydroxyethyl)amino)-8-((2-(piperidin-1-
C H N O Fe [MH] , requires m/z 522.1592, found m/z
28 28 5 2
yl)ethyl)amino)phthalazin-1(2H)-one (6l)
522.1599.
Yellow solid; Yield: 100.1 mg (19%); mp.: 180.5-182.0 °C (from
-
1
EtOH); IR (ATR, cm ): 3361, 2924, 2853, 1640, 1595, 1570,
(E)-8-(2-Ferrocenylidene-1-methylhydrazinyl)-2-ferrocenyl-4-
((2-hydroxyethyl)amino) phthalazin-1(2H)-one (6p)
1
1
8
537, 1467, 1404, 1363, 1324, 1297, 1269, 1255, 1220, 1209,
189, 1160, 1129, 1117, 1105, 1092, 1072, 1036, 1020, 1000,
60, 837, 812, 802; H-NMR (DMSO-d ): 9.50 (1H, t, J = 4.6
Orange solid; Yield: 204.5 mg (33%); mp.: 189.7-192.8 °C (from
1
-1
EtOH); IR (ATR, cm ): 3358, 2918, 1626, 1567, 1550, 1463,
6
Hz, 8-NH), 7.59 (1H, t, J = 7.9 Hz, H6), 7.09 (1H, d, J = 7.7 Hz,
H5), 6.86 (1H, d, J = 8.7 Hz, H7), 6.55 (1H, t, J = 5.4 Hz, 4-NH),
1434, 1413, 1383, 1363, 1321, 1250, 1224, 1209, 1175, 1153,
1121, 1106, 1090, 1068, 1022, 994, 935, 880, 810, 793, 772, 700,
1
5
.09 (2H, t, J = 2.0 Hz, H2’ and H5’), 4.75 (1H, t, J = 5.0 Hz,
669, 581, 497, 483, 457, 420; H-NMR (DMSO-d ): 7.81-7.68
6
5
OH), 4.14 (7H, m, H3’ and H4’ and η -C H ), 3.74 (2H, q, J =
(3H, m, H5 and H6 and H7), 7.52 (1H, s, CHFc), 6.68 (1H, br t,
NH), 5.11 (2H, s, H2’ and H5’), 4.75 (1H, br t, OH), 4.62 (2H, s,
5
5
5
.5 Hz, CH OH), 3.46 (2H, q, J = 5.7 Hz, CH 4-NH), 3.30 (2H,
2
2
5
q, J = 5.5 Hz, CH 8-NH), 2.58 (2H, t, J = 6.4 Hz, CH Pip), 2.43
H2’’ and H5’’), 4.32 (2H, s, H3’’ and H4’’), 4.19 (5H, s, 8-η -
2
2
5
(
4H, br s, H2’’ and H6’’), 1.55 (4H, p, J = 5.6 Hz, H3’’ and
C H ), 4.13 (7H, s, H3’ and H4’ and 2-η -C H ), 3.77 (2H, br q, J
5
5
5
5
13
H5’’), 1.42 (2H, q, J = 5.6 Hz, H4’’); C-NMR (DMSO-d ):
= 5.1 Hz, CH OH), 3.47 (2H, br q, J = 4.6 Hz, CH NH), 3.21
6
2
2
13
1
1
6
59.5 (C1), 150.9 (C8), 145.1 (C4), 134.6 (C6), 125.9 (C4a),
(3H, s, CH3); C-NMR (DMSO-d ): 155.2 (C1), 150.2 (C8),
6
5
11.8 (C7), 110.4 (C8a), 107.6 (C5), 100.7 (C1’), 69.4 (η -C H ),
144.6 (C4), 134.7 (FcCH) 133.0 (C6), 126.7 (C4a), 125.9 (C5),
5
5
5
5.2 (C3’ and C4’), 63.7 (C2’ and C5’), 59.6 (CH OH), 57.1
118.2 (C8a), 117.2 (C7), 101.2 (C1’), 83.1 (C1’’), 69.5 (1-η -
2
5
(
CH Pip), 54.4 (C2’’ and C6’’), 44.7 (CH 4-NH), 40.3 (CH 8-
C H ), 69.4 (8-η -C H ) 69.3 (C3’’ and C4’’), 67.0 (C2’’ and
2
2
2
5
5
5
5
NH, overlapped by solvent signal), 26.1 (C3’’ and C5’’), 24.6
C5’’), 65.2 (C3’ and C4’), 63.7 (C2’ and C5’), 59.6 (CH OH),
2
4
4.8 (CH NH), 42.1 (CH ); HRMS exact mass calcd.for
2 3

1
1
56
+
C H N O Fe2 [MH] , requires m/z 630.1255, found m/z
(C5), 130.6 (C4), 124.7 (C5’), 122.3 (C3’), 80.0 (C1’’), 70.7
32
32
5
2
ACCEPTED MANUSCRIPT
5
6
30.1247.
(C3’’ and C4’’), 69.9 (η -C H ), 67.3 (C2’’ and C5’’); HRMS
5 5
56 +
exact mass calcd. for C H N O Fe [MH] , requires m/z
19
16
3
7
8
-Ferrocenyl-5-((2-hydroxyethyl)amino)pyrido[2,3-d]pyridazin-
358.0643, found m/z 358.0651.
(7H)-one (8a)
1
Orange solid; Yield: 13.3 mg (3%); H-NMR (DMSO-d ): 9.06
2-Phenylphthalazin-1(2H)-one (6aa)
6
(
(
1H, br d, J = 4.3 Hz, H2), 8.84 (1H, br d, J = 8.3 Hz, H4), 7.92
1H, dd, J = 8.3 Hz, 4.3 Hz, H3), 7.33 (1H, br t, NH), 5.17 (2H, t,
White solid, Yield: 46.2 mg (21%); mp.: 107.6-109.0 °C (from
-
1
EtOH); IR (ATR, cm ): 3360, 2917, 2848, 1654, 1584, 1488,
1452, 1363, 1326, 1309, 1291, 1241, 1200, 1151, 1135, 1121,
1075, 1059, 1017, 971, 903, 871, 863, 844, 820, 793, 764, 734,
J = 1.9 Hz, H2’ and H5’), 4.98 (1H, br t, J = 6.0 Hz, OH), 4.19
5
(2H, t, J = 1.9 Hz, H3’ and H4’), 4.16 (5H, s, η -C H ), 3.77 (2H,
5 5
q, J = 5.6 Hz, CH OH, overlapped), 3.48 (2H, q, J = 5.6 Hz,
706, 686, 617, 598, 583, 537, 483, 466, 431, 421, 405, 391, 380;
2
13
1
CH NH); C-NMR (DMSO-d ): 154.6 (C8), 154.1 (C2), 144.4
H-NMR (DMSO-d ): 8.59 (1H, s, H4), 8.33 (1H, d, J = 8.0 Hz,
2
6
6
(
C5), 143.6 (C4a), 132.8 (C4), 127.5 (C3), 121.7 (C8a), 101.0
H8), 8.03 (1H, dd, J = 8.4 Hz, 1.4 Hz, H5, overlapped by H6),
8.00 (H6, td, J = 8.3 Hz, 1.2 Hz, 1H, overlapped by H5), 7.92
(1H, ddd, J = 7.9 Hz, 6.8 Hz, 1.8 Hz, H7), 7.62 (2H, dt, J = 7.4
Hz, 1.3 Hz, H2’ and H6’), 7.53 (2H, tt, J = 8.2 Hz, 1.3 Hz, H3’
5
(C1’), 69.7 (η -C H ), 65.6 (C3’ and C4’), 64.0 (C2’ and C5’),
5
5
6
0.0 (CH OH), 45.0 (CH NH); HRMS exact mass calcd. for
2 2
56 +
C H N O Fe [MH] , requires m/z 391.0857, found m/z
3
19
19
4
2
1
3
91.0864.
and H5’), 7.43 (1H, tt, J = 7.4 Hz, 1.7 Hz, H4’); C-NMR
DMSO-d ): 158.8 (C1), 142.3 (C1’), 139.2 (C4), 134.4 (C6),
(
6
2
1
-Ferrocenyl-4-((2-hydroxyethyl)amino)pyrido[3,4-d]pyridazin-
(2H)-one (8b)
132.8 (C7), 129.7 (C4a), 129.0 (C3’ and C5’), 128.2 (C8a), 128.0
(C4’), 127.5 (C5), 126.7 (C8), 126.5 (C2’ and C6’); HRMS exact
+
Red solid; Yield: 83.0 mg (21%); mp.: 209.2-210.3 °C (from
EtOH); IR (ATR, cm ): 3311, 1633, 1608, 1569, 1528, 1461,
mass calcd for C H N O [MH] , requires m/z 223.0871, found
14
11
2
-
1
m/z 223.0867.
1
1
6
9
410, 1394, 1346, 1321, 1300, 1241, 1219, 1175, 1146, 1117,
107, 1081, 1065, 1046, 1025, 999, 929, 901, 847, 804, 793, 741,
2-(Pyridin-2-yl)phthalazin-1(2H)-one (6ab)
1
93, 685, 669, 624, 563, 487, 464, 379; H-NMR (DMSO-d ):
Beige solid, Yield: 245.5 mg (37%); mp.: 120.2-123.7 °C (from
6
-
1
.53 (1H, br s, H5), 9.00 (1H, br s, H7), 8.13 (1H, d, J = 4.6 Hz,
EtOH); IR (ATR, cm ): 3040, 1666, 1587, 1482, 1465, 1450,
1442, 1365, 1333, 1295, 1241, 1163, 1151, 1074, 1043, 992, 915,
H8), 7.24 (1H, t, J = 5.4 Hz, NH), 5.16 (2H, s, H2’ and H5’),
.80 (1H, t, J = 5.4 Hz, OH), 4.20 (2H, s, H3’ and H4’), 4.16
4
876, 802, 787, 761, 739, 711, 682, 623, 598, 589, 541, 486, 469,
5
1
(5H, s, η -C H ), 3.79 (2H, q, J = 5.4 Hz, CH OH), 3.51 (2H, q, J
412, 404; H-NMR (CDCl ): 8.71 (1H, br s, H6’), 8.52 (1H, d, J
5
5
2
3
13
=
5.4 Hz, CH NH); C-NMR (DMSO-d ): 155.1 (C1), 151.7
= 7.9 Hz, H8), 8.37 (1H, d, J = 6.7 Hz, H5), 7.90 (1H, td, J = 7.1
Hz, 1.6 Hz, H5’, overlapped by H6), 7.88 (1H, td, J = 7.1 Hz, 1.1
Hz, H6, overlapped by H5’), 7.83 (1H, td, J = 7.4 Hz, 1.1 Hz,
H7), 7.80 (1H, d, J = 7.1 Hz, H3’, overlapped by H5), 7.78 (1H,
d, J = 6.7 Hz, H5, overlapped by H3’), 7.37 (1H, dd, J = 7.4 Hz,
2
6
(C7), 147.1 (C5), 143.9 (C4), 134.0 (C8a), 119.5 (C8), 119.0
5
(C4a), 100.8 (C1’), 69.6 (η -C H ), 65.7 (C3’ and C4’), 63.7 (C2’
5
5
and C5’), 59.3 (CH OH), 44.6 (CH NH); HRMS exact mass
calcd.for C H N O Fe [MH] , requires m/z 391.0857, found
m/z 391.0850.
2
2
5
6
+
19
19
4
2
13
1.8 Hz, H4’); C-NMR (CDCl ): 159.3 (C1), 153.3 (C2’), 149.3
3
(C6’), 139.0 (C4), 137.8 (C5’), 133.8 (C6), 132.1 (C7), 129.6
6
-Ferrocenyl-2-phenylpyridazin-3(2H)-one (3a)
(C4a), 128.6 (C8a), 127.3 (C8), 126.2 (C5), 123.2 (C4’), 121.3
+
Orange solid, Yield: 184.6 mg (52%); mp.: 156.0-157.9 °C (from
EtOH); IR (ATR, cm ): 1664, 1601, 1489, 1454, 1388, 1303,
(C3’); HRMS exact mass calcd for C H N O [MH] , requires
13
10
3
-
1
m/z 224.0824, found m/z 224.0818.
1
8
4
9
280, 1262, 1207, 1170, 1124, 1106, 1092, 1022, 1007, 912, 897,
39, 817, 764, 727, 689, 655, 642, 623, 610, 581, 564, 513, 491,
4-((2-Hydroxyethyl)amino)-2-phenyl-8-((2-(piperidin-1-
yl)ethyl)amino)phthalazin-1(2H)-one (6la)
1
79, 443, 429, 391, 377; H-NMR (DMSO-d ): 7.81 (1H, d, J =
6
.6 Hz, H5), 7.60 (2H, d, J = 7.8 Hz, H2’ and H6’), 7.51 (2H, t, J
7.8 Hz, H3’ and H5’), 7.42 (1H, t, J = 7.4 Hz, H4’), 7.05 (1H,
Bright yellow solid; Yield: 95.1 mg (23%); mp.: 77.4-80.7 °C
-
1
=
(from EtOH); IR (ATR, cm ):3277, 2929, 2851, 1634, 1601,
1565, 1524, 1487, 1455. 1408, 1292, 1236, 1203, 1155, 1114.
d, J = 9.6 Hz, H4), 4.83 (2H, t, J = 1.9 Hz, H2’’ and H5’’), 4.46
5
13
(2H, t, J = 1.9 Hz, H3’’ and H4’’), 4.18 (5H, s, η -C H ); C-
1087, 1049, 962, 909, 857, 805, 755, 693, 665, 617, 518, 480,
5
5
1
NMR (DMSO-d ): 159.1 (C1), 146.8 (C4), 142.1 (C1’), 132.4
460, 423, 410, 389, 375; H-NMR (DMSO-d ): 9.32 (1H, t, J =
6
6
(
C5), 130.6 (C4), 129.0 (C3’ and C5’), 128.2 (C4’), 126.0 (C1’
5.0 Hz, 8-NH), 7.63 (2H, d, J = 7.9 Hz, H2’ and H6’, overlapped
by H6), 7.62 (1H, t, J = 8.1 Hz, H6, overlapped by H2’ and H6’),
7.46 (2H, t, J = 7.9 Hz, H3’ and H5’), 7.32 (1H, tt, J = 7.4 Hz,
1.3 Hz, H4’), 7.13 (1H, d, J = 7.8 Hz, H5), 6.88 (1H, d, J = 8.4
Hz, H7), 6.48 (1H, t, J = 5.4 Hz, 4-NH), 4.66 (1H, t, J = 5.6 Hz,
5
and C6’), 80.9 (C1’’), 70.7 (C3’’ and C4’’), 69.9 (η -C H ), 67.2
5
5
56
(
[
C2’’ and C5’’); HRMS exact mass calcd. for C H N O Fe
20 17 2
+
MH] , requires m/z 357.0690, found m/z 357.0682.
6
-Ferrocenyl-2-(pyridin-2-yl)pyridazin-3(2H)-one (3b)
OH), 3.63 (2H, q, J = 5.5 Hz, CH OH), 3.33 (2H, q, J = 5.8 Hz,
2
Red solid, Yield: 228.8 mg (64%); mp.: 156.0-157.9 °C (from
EtOH); IR (ATR, cm ): 1662, 1597, 1584, 1570, 1508, 1492,
CH 4-NH), 3.26 (2H, q, J = 5.0 Hz, CH 8-NH), 2.54 (2H, t, J =
2
2
-
1
6.4 Hz, CH Pip), 2.39 (4H, br s, H2’’ and H6’’), 1.49 (4H, p ,J =
2
13
1
1
8
5
462, 1431, 1407, 1387, 1310, 1297, 1285, 1251, 1214, 1178,
149, 1131, 1106, 1097, 1056, 1038, 1026, 1013, 999, 992, 967,
97, 856, 848, 838, 828, 819, 793, 751, 739, 650, 629, 610, 590,
5.5 Hz, H3’’ and H5’’), 1.38 (2H, q ,J = 5.9 Hz, H4’’); C-NMR
(DMSO-d ): 159.9 (C1), 151.2 (C8), 145.6 (C4), 143.1 (C1’),
6
135.0 (C6), 128.7 (C3’ and C5’), 126.7 (C4’), 126.6 (C4a), 126.2
(C2’ and C6’), 111.8 (C7), 110.4 (C8a), 107.7 (C5), 59.7
(CH OH), 57.3 (CH Pip), 54.5 (C2’’ and C6’’), 44.5 (CH 4-NH),
1
82, 571, 523, 498, 484, 449, 434, 414, 389, 379; H-NMR
(
DMSO-d ): 8.62 (1H, ddd, J = 4.8 Hz, 1.9 Hz, 0.9 Hz, H6’),
6
2
2
2
8
.02 (1H, td, J = 7.7 Hz, 1.9 Hz, H4’) 7.86 (1H, d, J = 9.7 Hz,
H5), 7.61 (1H, dt, J = 8.0 Hz, 1.0 Hz, H3’), 7.53 (1H, ddd, J =
.8 Hz, 1.9 Hz, 0.9 Hz, H5’), 7.08 (1H, d, J = 9.7 Hz, H4), 4.79
2H, t, J = 1.9 Hz, H2’’ and H5’’), 4.46 (2H, t, J = 1.9 Hz, H3’’
40.3 (CH 8-NH, overlapped by solvent signal), 28.0 (C3’’ and
2
C5’’), 24.5 (C4’’); HRMS exact mass calcd for C H N O
2
3
30
5
2
+
4
(
[MH] , requires m/z 408.2399, found m/z 408.2391.
5
13
and H4’’), 4.19 (5H, s, η -C H ); C-NMR (DMSO-d ): 159.2
5
5
6
(C1), 153.9 (C2’), 149.5 (C6’), 146.5 (C4), 139.1 (C4’), 133.2

1
2
Cytotoxicity Assay
The adherent HepG2 human hepatoma cells and HT-29 human
5. Acknowledgements
ACCEPTED MANUSCRIPT
35
36
This work was financially supported by the Hungarian Scientific
colorectal adenocarcinoma cells were cultured in RPMI-1640
medium supplemented with 10% FCS (fetal calf serum, Sigma
Ltd.), 2 mM l-glutamine, and 160 ꢀg/mL gentamicin. Cell
cultures were maintained at 37 °C in a humidified atmosphere
with 5% CO2.
Research Fund (OTKA K83874 and K104385).
6
. References
1
(a) Lokich, J.; Anderson, N.; Annals of Oncology, 1998, 9,
3-21; (b) Apps, M. G.; Choi, E. H. Y.; Wheate, N. J.;
The cells were grown to confluency and were distributed into 96-
1
3
well plate with initial cell number of 5.0x10 per well. After 24 h
Endocrine-related Cancer, 2015, 22, 219
o
incubation at 37 C, the cells were treated with the compounds in
2
3
Laginha, K. M.; Verwoert, S.; Charrois, G. J. R.; Allen, T.
M.; Clin. Cancer Res., 2005, 11, 6944
(a) Ferguson, P. J.; Phillips, J. R.; Seiner, M.; Cass, C. E.;
Cancer Res., 1984, 44, 3307; (b) Gerzon, K.; in Anticancer
Agents Based on Natural Product Models, ed. Cassady, J.
M. and Dourous, J. D.; Academic Press, Inc., New York,
2
00 ꢀL final volume containing 1.0 v/v% DMSO. The cells were
-4 2
incubated with the compounds at 10 -10 ꢀM concentration
range for overnight. Control cells were treated with serum free
medium (RPMI-1640) only or with DMSO (c=1.0 v/v %) at 37
o
C for overnight. After incubation the cells were washed twice
with serum free (RPMI-1640) medium. To determine the in vitro
cytostatic effect, the cells were cultured for a further 72 h in 10%
serum containing medium. MTT-solution (45 ꢀL, 2 mg/mL, final
concentration: 0.37 ꢀg/mL) was added to each well. The
1
980, 271.
4
5
Biot, C.; Glorian, G.; Maciejewski, L. A.; Brocard, J. S.; J.
Med. Chem., 1997, 40, 3715.
Representative examples: (a) Dombrowki, K. E.; Baldwin,
W.; Sheats, J. E.; J. Organomet. Chem., 1986, 302, 281;
b) Köpf-Maier, P.; Köpf-Maier, H.; Chem. Rev., 1987, 87,
137; (c) Neuse, E. W.; Meirim, M. G.; Blam, N. F.;
Organometallics, 1988, 7, 2562; (d) Top, S.; Vessières, A.;
Cabestaing, C.; Laios, I.; Leclerq, G.; Provot, C.; Jaouen,
G.; J. Organomet. Chem., 2001, 600, 637; (e) Jaouen, G.;
Top, S.; Vessières, A.; Alberto, R.; J. Organomet. Chem.,
2000, 600, 23; (f) Ma, H.; Hou, Y.; Bai, Y.; Lu, J.; Yang,
B.; J. Organomet. Chem., 2001, 7427, 637; (g) Delhaes, L.;
Abessolo, H.; Biot, C.; Berry, L.; Delcourt, P.;
Maciejewski, L.; Brocard, J.; Camus, D.; Dive, D.;
Parasitol. Res., 2001, 87, 239; (h) Weber, B.; Serafin, A.;
Michie, J.; Van Rensburg, C.; Swarts, J. C.; Bohm, L.;
Anticancer Res., 2004, 24, 763; (i) Joksovic, M. D.;
Markovic, V.; Juranic, Z. D.; Stanojkovic, T.; Jovanovic,
L. S.; Damljanovic, I. S.; Szécsényi, K. M.; Todorovic, N.;
Trifunovic, S.; Vukicevic, R. D.; J. Organomet. Chem.,
3
7
38
respiratory chain and other electron transport systems reduce
MTT and thereby form non-water-soluble violet formazane
39
crystals within the cell. The amount of these crystals can be
determined spectrophotometrically and serves as an estimate for
the number of mitochondria and hence the number of living cells
(
1
40
in the well. After 4 h of incubation the cells were centrifuged
for 5 min (900 g) and the supernatant was removed. The obtained
formazane crystals were dissolved in DMSO (100 ꢀL) and
optical density (OD) of the samples was measured at λ=540 and
6
20 nm, respectively, using ELISA Reader (iEMS Reader,
Labsystems, Finland). OD620 values was substracted from OD540
values. The percent of cytostasis was calculated by using the
following equation:
Cytostatic effect (%) = [1 – (ODtreated/ODcontrol)] x100
Values ODtreated and ODcontrol correspond to the optical densities
of the treated and the control cells, respectively. In each case two
independent experiments were carried out with 4 parallel
measurements. The 50% inhibitory concentration (IC ) values
50
were determined from the dose-response curves. The curves were
defined using Microcal TM Origin1 (version 7.5) software:
cytostasis was plotted as a function of concentration, fitted to a
sigmoidal curve, and based on this curve, the half maximal
inhibitory concentration (IC ) value was determined. IC
2
009, 694, 3935; (j) Corry, A. J.; O’Donovan, N.; Mooney,
50
50
Á.; O’Sullivan, D.; Rai, D. K.; Kenny, P. T. M.; J.
Organomet. Chem., 2009, 694, 880; (k) Ornelas, C.; New J.
Chem., 2011, 35, 1973; (l) Braga, S. S.; Silva, A. M. S.;
Organometallics, 2013, 32, 5626.
represents the concentration of a compound that is required for
0% inhibition in vitro and expressed in micromolar units.
5
Cyclic voltammetry assay
The samples were investigated by CV in a conventional three-
electrode electrochemical glass cell with a Biologic SP150
potentiostat and the EC-LAB software package. The three-
6
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2
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electrolyte
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[0.1
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the solution was then bubbled with Ar for 15 min. The CV
measurements were performed in the potential range -0.15–0.75
–1
V (vs. RHE) at a scan rate of 10 mVꢁs at room temperature.
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