CONTENTS
PREFACE .............................................................................................................................................................. 2
SYNTHESIS OF G-QUADRUPLEX LIGANDS AS POTENTIAL TELOMERASE INHIBITORS ........... 3
INTRODUCTION .................................................................................................................................................... 3
THE CHEMISTRY OF PHENANTHROLINE ............................................................................................................. 18
CHEMISTRY ....................................................................................................................................................... 22
HETEROARYLATION .......................................................................................................................................... 33
COUPLING ......................................................................................................................................................... 40
MACROCYCLIC LIGANDS ................................................................................................................................... 43
RATIONAL DESIGN, SYNTHESIS AND BIOLOGICAL EVALUATION OF BIS(PYRIMIDO[5,6,1DE]ACRIDINES) AND BIS(PYRAZOLO[3,4,5-KL]ACRIDINE-5-CARBOXAMIDES) AS POTENTIAL
TOPOISOMERASE INHIBITORS ................................................................................................................... 66
INTRODUCTION TO THE CHEMISTRY .................................................................................................................. 66
CHEMISTRY ....................................................................................................................................................... 69
RESULTS AND DISCUSSION ................................................................................................................................ 72
DNA-Binding Properties .............................................................................................................................. 72
Cytotoxic Activity ......................................................................................................................................... 77
Hollow Fiber Assay for Preliminary in Vivo Testing ................................................................................... 81
SYNTHESIS AND BIOLOGICAL EVALUATION OF INDAZOLO[4,3BC][1,5]NAPHTHYRIDINES(10-AZA-PYRAZOLO[3,4,5-KL]ACRIDINES) AS POTENTIAL
TOPOISOMERASE INHIBITORS ................................................................................................................... 83
INTRODUCTION TO THE CHEMISTRY .................................................................................................................. 83
CHEMISTRY ....................................................................................................................................................... 85
RESULTS AND DISCUSSION ................................................................................................................................ 87
DNA-Binding Properties .............................................................................................................................. 87
In Vitro Cytotoxic Activity ........................................................................................................................... 90
Apoptosis Assays .......................................................................................................................................... 91
DNA Fragmentation Assays ......................................................................................................................... 94
RATIONAL DESIGN, SYNTHESIS AND BIOLOGICAL EVALUATION OF 3H-NAPHTHO[1,2,3DE]QUINOLINE-2,7-DIONES AS POTENTIAL TOPOISOMERASE INHIBITORS .............................. 95
INTRODUCTION TO THE CHEMISTRY .................................................................................................................. 95
CHEMISTRY ....................................................................................................................................................... 97
RESULTS AND DISCUSSION ................................................................................................................................ 98
HT29 human colon adenocarcinoma cell line ............................................................................................. 98
Human ovarian carcinoma cell line panel ................................................................................................. 100
EXPERIMENTAL SECTION.......................................................................................................................... 101
CONCLUSIONS ................................................................................................................................................ 131
REFERENCES .................................................................................................................................................. 133
1
Preface
Herein it is described the goal of the proposed research, which is the synthesis of
model phenanthroline derivatives, capable of stabilizing the G-quadruplex structure and
interfering with the action of telomerase. The human single stranded telomere end can
potentially fold into a number of four-repeat quadruplexes. Stabilization of these
quadruplexes by small molecules will destabilize telomere maintainance in cancer cells and
lead to potential agents capable of destroying cancerous growth. A number of other novel
acridine derivatives, know as topoisomerase inhibitors and relative cytotoxic activity on
different cell lines has also been investigated. This class of compounds is potentially capable
of forming a stable complex with DNA-topoisomerase, which prevents the enzyme from
completing his cycle, therefore causing destabilization, protein disruption or denaturation.
Most of the newly synthesized compounds reveal a high DNA affinity, an excellent cytotoxic
potency and interesting preliminary in vivo activity.
2
Synthesis of G-Quadruplex Ligands as Potential
Telomerase Inhibitors
Introduction
The DNA region at the end of a linear chromosome is called telomere. Its function is
necessary for the chromosome’s replication and stability. Telomeres must act as
chromosomes “stabilizers”, since at their ends mutations may occur. According to species,
telomeres have specialized sequences which can be divided into two subtypes: 1) simple
telomeric sequences at the end of chromosomal DNA molecules, made of tandem repeat units,
unique to each organism; 2) complex sequences, often of a few kilobases long, located at the
proximal regions of the chromosomes’ ends.1
In most organisms, telomeres consist of a short repetitive motif, which can differ
greatly between species and particularly in humans they contain up to several thousand arrays
of the short sequence TTAGGG.2
Telomeres must be fully replicated. Before replication takes place, the DNA double
helix must be unwound by an enzyme known as DNA helicase, producing two forks (Fig. 1).
Since two copies are generated, each one containing one parental strand and one entirely new
polimerized strand, DNA replication is called semiconservative.
As the two antiparallel strands separate by breaking the weak hydrogen bonds that
link the paired bases, the enzyme DNA polymerase reaches the point where the synthesis will
begin. The starting place for replication depends on small primers, which adhere to the end of
each strand and initiate the DNA synthesis.
During the process, DNA polymerase “reads” the sequence of bases on the template
strand in the 3’ to 5’ direction and “synthesizes” the complementary one, proceeding from the
5’ to 3’ direction.
Since the two original DNA strands are complementary and antiparallel, only a new
one can replicate continuously from the 3’ end, while the other so-called lagging strand is
replicated discontinuously.
3
In fact, DNA polymerase III adds a multitude of small segments of nucleotides
named Okazaki fragments, primed by short stretches of RNA primers.3,4
In the next step, another type of enzyme, DNA polymerase I, removes RNA primers,
replacing the ribonucleotides with deoxyribonucleotides by extending the strand from the
adjacent Okazaki fragments.
Later, the enzyme DNA ligase joins the 5’ end of one DNA strand to the 3’ end of
another (or the same) polynucleotide chain. The primers can be replaced everywhere except at
the extreme 5’ end, which makes this new strand slightly shorter than the parental one. This
situation is the molecular basis of the “end replication problem”.5
Figure 1. The DNA replication fork
4
It was predicted that after 20-40 generations of cell doubling, terminal degradation
and fusion of chromosomal DNA would eventually lead to loss of genetic stability and
prevent cells from further duplicating, causing cellular senescence and irreversible cell death.6
This counting mechanism has been variously related to ageing and its counterpart,
cellular immortality, since the telomeres of immortalized cells do not shorten upon division.
In order to overcome this incomplete “end-replication” problem, immortal eukaryotic
cells, including transformed human cells, apparently use telomerase as a key enzyme that
elongates telomeres by RNA-templated addition of tandemly repeated, often G-rich
sequences.7
Telomerase is a high molecular weight complex, which uses an RNA template for
the addition of (GGTTAG)n telomere repeats to the 3’ termini of the chromosomes.
In humans, the RNA subunit is known as hTR (human Telomerase RNA), whereas
the protein subunit is known as hTERT (human Telomerase Reverse Transcriptase).
The hTR contains eleven nucleotides with a unique sequence of bases 5’CUAACCCUAAC-3’ that binds to the end of the telomere. After a first recognition of the
telomere end, hTERT synthesizes new telomeric DNA repeats, GGTTAG and finally the
RNA subunit translocates back to the newly synthesized 3’ end, for a further round of
elongation.1,2,4
Although telomeric sequences vary between species, fundamentally, telomeres and
telomerase work very similarly throughout the eukaryotes. Telomerase is absent for the
normal function of most somatic cells that usually have longer telomeres, whereas it is widely
express in immortal cells.7 In fact, telomeres are stably maintained in length in 80-85% of
human tumour cells, which divide indefinitely by the action of telomerase.8
Therefore, since telomerase is necessary for the immortality of many cancer types, it
is thought to be a potentially highly selective and attractive drug target for several anti-tumour
strategies. Its action is detected in most primary human tumour specimens and tumourderived cell lines, such as those of the prostate, breast, colon, lung and liver.9 Hence,
inactivation of telomerase may play an important role in cancer therapy.
5
Figure 2. Schematic representation of basic principles for processive synthesis of telomeric
DNA by the human telomerase enzyme. a) DNA primer (or 3'-end of chromosome in vivo)
binds to telomerase by base-pairing to the RNA template (shown in blue) and by interaction
with the anchor site (dotted domain). b) In the second step, reverse transcriptase activity of
hTERT extends the DNA until the end of the template is reached. The newly synthesised
DNA is shown in green. The telomerase RNA and telomeric DNA form a DNA–RNA hybrid
that might involve up to 11 base pairs. c) In the third step, the DNA is translocated back to the
beginning of the template and positioned for a subsequent round of elongation (dashed
arrow).
6
In contrast, activation of telomerase, is proposed to be an essential step in cell
immortalization and human ageing. Since human cells can divide only a limited number of
times in culture (about 50), many researchers wondered whether there is a “clock” that
measures cell divisions. The hypothesis that telomere shortening could be the cell division
clock in human cells has been validated by several investigations. The search for ageing and
longevity genes has been a focus for many years, in biomedical research.
One of the most recent theories of ageing was first discovered by scientists at the
Geron Corporation, California, indicating that telomeres shorten every time a cell divides.
This shortening of telomeres is believed to lead to cellular damage, due to the inability of the
cell to duplicate itself correctly. At each round of replication, cells begin to exhibit further
signs of old age, of cellular dysfunction, ageing and death.
Cells from various tissues in the body, such as osteoblasts in the bone, endothelial
cells in the blood vessels, retinal pigment epithelial cells in the eye, fibroblasts in the skin,
and lymphocytes in the blood are mortal, that is to say, they divide 20-100 times (depending
on the tissue and age of the donor) and then cease dividing in a process called cell senescence.
This phenomenon of cell ageing was first described by Leonard Hayflick in 1961,
and therefore the limit of cell proliferation is often called the “Hayflick Limit”.
The telomere hypothesis proposes that as mortal cells divide, terminal DNA or
telomeres are progressively lost with each cell division, causing telomere shortening.
When a critical amount of telomere shortening has occurred, the genetic program of
cell senescence, or cell ageing, is triggered. Among normal cells, only the reproductive cells
do not senesce; the telomere clock does not "tick", telomeres do not shorten, and the cells can
apparently divide indefinitely, a characteristic referred to as immortality. Cellular immortality
does not mean that the cells cannot die; like all cells, they must be carefully nourished to
remain viable.
Instead, immortality refers to the fact that these cells are not limited to a finite
number of doublings. Immortal cells, provided they are properly fed and maintained, can
divide indefinitely.
Senescent cells are not only incapable of dividing, they also exhibit an altered pattern
of gene expression, leading to a number of changes in their structure and function. The effects
of senescence on cell function can damage the surrounding tissues, contributing to age-related
pathologies.
7
For example, senescent skin fibroblasts produce lesser amounts of important skin
matrix elements such as collagen and elastin and elevated levels of enzymes such as
collagenase that break down the skin matrix. These changes contribute to atrophy of the skin
and, ultimately, age-related skin disorders. Similarly, metabolic changes in senescent retinal
pigmented epithelium cells and thrombotic factors in endothelial cells are considered
contributors to the pathologies of age-related macular degeneration (AMD) and
atheriosclerosis.
Skin disorders, AMD, and atherosclerosis are major diseases in the ageing
population. Skin atrophy, for example, affects virtually all ageing individuals, with 40 percent
of the population over 75 years of age seeking treatment for at least one skin disorder. These
disorders range from photoageing and wrinkling to increased wounding and, ultimately, skin
ulceration, which can be life-threatening. In the case of AMD, one third of the population at
age 70 is affected and, in most patients, the disease is currently untreatable.
The delay or prevention of cell senescence through the extension of cell life-span is
expected to have important beneficial effects in diseases to which cell senescence contributes.
In addition to skin disorders, AMD and atherosclerosis, this group is thought to include
osteoporosis, immune dysfunction, arthritis, and neurodegenerative disorders.
The medical and biological advances that will be achieved over the next ten to
twenty years will hopefully allow humans to live long past one hundred.
The improvement in the health of the elderly through the more effective treatment of
age-related diseases is expected to increase the length of healthy life. The use of telomerasebased cell immortalization may offer a variety of practical applications in medical research
and may also provide new therapies or improve treatments such as bone marrow
transplantations and skin grafts.10
There are a number of strategies that have been reported for telomerase inhibition,
which use the catalytic hTERT protein and the hTR template RNA, components of the
enzyme complex, as well as other accessory proteins, including Hsp90, p23 and TEP-1 as
potential molecular targets.
Some research was focused on the regulation of genes for hTR and hTERT. It has
been demonstrated that the mechanism for the elongation of telomeres is dependant on the
sequence of the hTR template. By mutating this template, any subsequent addition of
nucleotides to form the new telomeric repeats will show a corresponding mutation. In this
manner the telomere tips also become mutated, hence destroying the characteristic catalytic
8
binding site of hTERT. Without this catalytic function of hTERT, the cells progressively lose
DNA and undergo senescence. With regards to the inhibition of the RNA subunit (hTR), in
vivo studies have shown antitumour effects.
While the hTERT messenger RNA has been targeted with oligonucleotides, several
studies have demonstrated that if the interaction of Hsp90 and p23 with the catalytic subunit
of telomerase is hindered, the assembly of telomerase is blocked in vitro.
Guanine rich sequences of telomeric DNA tend to associate and interact forming a
G-tetrad, stabilized by Watson-Crick and Hoogsteen hydrogen bonds. Successive layering of
two or more G-tetrads, which are connected by two lateral loops and one central diagonal
loop11 form a box-like arrangement called G-quadruplex (Fig. 3). The space between Gtetrads is well suited to coordinate monovalent cations (K+ and occasionally Na+) which are
required in order to stabilize the structure through their coordination to the guanyl oxygens.12
The connecting loops can also present structural diversity. The loops may be either
parallel or antiparallel. They may connect with guanine tracts either edgewise or diagonally,
on opposite ends or on the same side. The capping bases or base pairs are generally not very
flexible. The bases directly adjacent to the tetrads often stack in planar structures and
complementary base pairs can form from the same or different strands.13
Figure 3. G-Tetrads and G-Quadruplexes.13
9
Figure 4. G-quartet structure as
part of the telomeric repeat unit.
The intercalation of planar aromatic chromophores between adjacent base pairs is a
well-studied mechanism of binding to the DNA double helix, and several thousand
intercalating molecules are known.
On the basis of observations that potassium ions can stabilize these structures, small
or large molecule ligands that mimic their effect, have been designed and found to inhibit
telomerase activity, such as anthraquinones, fluorenones, substituted acridines, cationic
porphyrins,
perylenes,
ethidium
derivatives,
dibenzophenanthrolines,
fluoroquinoanthroxazines, telomestatine (Fig. 5). Many of these compounds have been
described in literature and it was demonstrated that the more potent may produce replicative
senescence and telomere shortening in cell culture.
The common structural feature of all these ligands is an extended polyaromatic
(often heteroaromatic) planar chromophore, generally composed of three or four fused rings,
bearing one or more positive charge(s). A noteworthy exception is NMM, an anionic
porphyrin, which binds selectively to quadruplexes. The first G-quadruplex interactive
compounds were relatively non potent (IC50 in the micromolar range), but recently, more
potent and selective molecules have been designed or discovered.
The natural product telomestatine, composed of one thiazoline and seven oxazole
rings, was reported to be a potent nanomolar telomerase inhibitor, with evidence indicating
that its mechanism of action also involves a G-quadruplex complex.14
10
N
O
R1 R2
N
O
N
NH
O
HN
O
N
N
benzo[b]naphto[2,3-d]furan-6,11-dione
dibenzo[b,j](1,7)phenanthroline
O
R2N
O
N
H
N
H
O
O
Fluorenones (FL)
OH
O
N
N
H
N
OH
HN
N
NR2
H
N
N
NH
N
HN
H
N
H
N
Bisacridine
NMM (anionic porphyrine)
O
H
N
O
N
N
H
N
O
O
BSU-1051 (2,6-diaminoanthraquinone)
O
O
N
N
O
O
NH
NH
PIPER (perylenetetracarboxylix diimide derivative)
O
N
O
N
DODC (carbocyanines)
Figure 5. Chemical structures of some telomerase inhibitors that target telomeres.
11
The best of the porphyrins and anthraquinones show 50% telomerase inhibition
(IC50), using a standard assay at 1-25 M; in vitro studies have shown that the cationic
porphyrin TMPyP4 is able to shorten telomeres, induce a delayed cell crisis (2-4 weeks) and
induce apoptosis.15 On the basis of molecular modelling data, the perylene derivative PIPER,
containing two positive charges at the extreme nitrogens of the molecule, was designed and
later synthesized, exhibiting very good telomerase and DNA polymerase activity.16
In addition, the 9-anilino proflavine derivative was designed to optimize its
interaction with the intramolecular G-quadruplex from the human telomere and minimize its
interaction with duplex DNA. This compound has demonstrated to have a potency of 60-100
nM, corresponding to a low cytotoxicity. Moreover, the fluoroquinophenoxazines known to
be topoisomerase II poisons were found to be able to interact more specifically with Gquadruplex structures.17
An essential requirement for telomerase inhibition and antitumour effect is the low
concentration of inhibitor, which needs to be below acute toxicity levels, otherwise
generalized cytotoxic effect outside tumours will take place. In some cases, it was reported
that antitumor agents that bind to duplex DNA, may develop their cytotoxic activity by
interfering with transcription or with the correct function of DNA topoisomerases or other
enzymes involved in DNA replication.
First-generation G-quadruplex-interactive agents, like some substituted porphyrins,
have demonstrated affinity for duplex DNA rather than complete quadruplex selectivity. In
order to demonstrate that telomerase inhibition occurs via a quadruplex-mediated mechanism,
two main features must be taken into account: 1) G4 selectivity over duplex DNA affinity; 2)
increased potency for telomerase inhibition along with a low level of acute cytotoxic
activity.18
Regardless of a number of studies that have reported spectroscopic investigations of
drug interactions with G-quadruplexes, no definite structural information has been so far
available. Recent modelling studies suggested that four different binding modes of a G-4
interacting molecule can generally be observed: 1) “true” intercalation; 2) end stacking; 3)
“minor” groove binding; 4) binding to the loops.
In some cases it was reported that the intercalation of a chromophore between base
pairs may be disfavoured because of the high energy involved to separate the two quartets and
to eject a monocation. Conversely, other NMR studies suggested that the presence of a
12
positive charge, located above the channel of negative electrostatic potential in the
quadruplex, may enhance overall interaction. However, several experimental data tend to
favour the possibility of an efficient external stacking by π-π interactions onto each terminal
G-plane and the bases that flank quartet structures (Fig. 6). The presence of substituent chains
may form also favourable hydrophobic interactions with the sides of the grooves.
Nonetheless, interactions such as groove binding have been identified, since Gquartets are likely to form 4 different grooves and expose adenine/thymine loops that may be
specifically recognized by ligands. With respect to the loop binding orientation, some studies
still need to be fully demonstrated.2,11,19
In order to detect telomerase activity in human cancer cell, in the area of ligand-DNA
interactions, different strategies have been developed. A biochemical method as telomeric
repeat amplification protocol (TRAP) assay, which provides the effective concentration for
50% inhibition (EC50 values) for ligands from dose-response studies and two biophysical
assays, such FRET (Fluorescence Resonance Energy Transfer) and SPR (Surface Plasmon
Resonance Studies) are now widely used. Moreover, molecular dynamics simulations are
often carried out to examine qualitative structure-activity relationships and possible
interactions with quadruplex DNA.20
Figure
6.
NMR
derived
structure of PIPER-G4 complex.
Color coding: adenine, purple;
guanine, yellow; PIPER, green;
thymine, cyan.16
13
As it was previously mentioned, protein components that function at or near the replication
fork include: DNA polymerases, single-stranded binding proteins, helicases, primase and
DNA ligase. Among these, the enzymes topoisomerases I and II can be also included, which
act during the DNA replication process. At this stage DNA is supercoiled due to the
replication fork. The major role of topoisomerases is to prevent DNA tangling through
transient DNA nicking-closing (Figure 7).21,22,23,24
Figure 7. The topoisomerases function to maintain the
preferred degree of underwinding. The two colors of DNA
correspond to the temporarily broken strand (blue) and the
unbroken strand (orange). The break is the blue strand is
visible in the center of the picture The magenta structure is
the tyrosine phosphorylated to a deoxyribose.25
The two major classes of DNA topoisomerases, types I and II, tend to be considered
as different variants of a group of enzymes which exclusively serve to change DNA
conformation. Each topoisomerase has different catalytic activities for relaxing torsioned
DNA strands chromatin, distinct properties and biological functions. Recent results suggest
that topoisomerase I is a multifunctional protein with at least two different catalytic activities,
whereas topoisomerase II, in addition to its catalytic roles, is a structural protein which serves
14
as a chromatin-docking protein for different molecular partners. The different catalytic
activities for relaxing torsioned DNA strands on chromatin are: the ATP-indipendent breaking
of DNA single strands of chromatine (topoisomerase I) and the ATP-dependent breaking of
DNA double strands (toposomerase II).
Eukaryotic topoisomerase I, prokaryotic topoisomerase I (first discovered by
Champoux and Dulbecco in the early 1970s26) and topoisomerase III (isolated from yeast and
human cells) are all type I enzymes. This monomeric protein catalyzes interconversions
between different topological states of DNA by transiently breaking the phosphodiester
backbone of one strand of DNA, thereby allowing the passage of the intact DNA strand
through the break. This is followed by religation of the nicked DNA strand leading to
reformation of an intact DNA helix. Recent studies show, that topoisomerase I also has a
kinase activity and can phosphorylate certain splicing factors, such as SF2/ASF1.27
Topo-II enzyme catalyzes processes of DNA binding, strand breakage, passage and
reunion acting, however, over the two double strands of DNA. the cleavable complex is
crucial not only for DNA relaxation, but also for other topoisomerase-mediated reactions such
as recombination and recognition of DNA damage. Moreover, it is the target for clinically
used antineoplastic agents, which act by preventing the religation step, thereby resulting in the
accumulation of cleavable complexes and of the enzyme with covalent binding, followed by
respective breakage of single or double strands of DNA.
Recently, a variety of inhibitors of topo-I or topo-II and of both have been found.
Many of them are known to have anti-tumor activities and have been used in cancer
chemiotherapy, although their anti-tumor mechanism are not yet clear. Those already reported
are: camptothecin analog (CPT-11) and topotecan as inhibitors of topo-I; 4’-(9acrydinylamino)methane-sulfon-m-aniside (m-AMSA), adriamycin, daunomycin, etoposide
(VP-16), anthracenediones (mitoxantrone) and teniposide (VM-26) as inhibitors of topo-II;
and saintopin as inhibitor of both. Today, there’s a clear evidence of a close association
between the efficient induction of chromosome-type aberrations (in normal and malignant
cells) by topo-inhibitors, and their ability to stabilize the cleavable complex. 28,29
Cleavable complexes are also formed in the proximity of DNA lesions and in the
presence of the antitumor agent. While formation of cleavable complexes may be necessary
for the initial stages of the DNA damage response, these complexes are also potentially
dangerous to the cell due to their ability to mediate illegitimate recombination, which can lead
to genomic instability and oncogenesis. Thus the levels and stability of these complexes have
15
to be strictly regulated. This is obtained by maintaining the enzyme levels relatively constant,
by limiting the stability of the cleavable complexes through physical interaction with the
oncogene suppressor protein p53 and by degradation of the topoisomerase I by the
proteasome system. Emerging evidence suggest that these regulatory functions are perturbed
in tumor cells, explaining at the same time why topoisomerase I activities so often are
increased in certain human tumors, and why these cells are sensitized to the cytotoxic effects
of camptothecins.30
O
O
OH
OH
NH(CH2)3N(CH3)2
OH
N
OMe O
OH
O
Adriamycin
O
Nitracrine
H3C
H3CO
H
NH2
OH
NHSO2CH3
N
N
CONH(CH2)2N(CH3)2
N
m-AMSA
DACA
Figure 8. Some topoisomerase inhibitors.
A crucial reaction intermediate during relaxation of DNA is the formation of a
covalent DNA-topoisomerase complex (the cleavable complex) where topoisomerase is
covalently linked to a 3’-end of DNA thereby creating a single or double stranded DNA break
(Figure 8). A precise understanding of the mechanism of action of topoisomerases is not only
important because their inibition cancels cell proliferation (human topo I is the major anticancer target)31 but also because the chemical reaction performed by them induces movement
of DNA segments several orders of magnitude larger than the size of the protein. For this
reason, topoisomerases are prime case studies for understanding the triggering of large-scale
16
mechanical motions induced by biomolecular motors, and in particular the effect that external
forces or torques have in tuning such machines at the single-molecule level.32
Figure 9. Mechanism of action of DNA-topoisomerase intercalating agents.
17
The Chemistry of Phenanthroline
The goal of the present work is the synthesis of phenanthroline derivatives, a class of
chelating ligands, whose coordination chemistry and discovery date back to the early
nineteenth century. Throughout the years, these compounds have been described for their
utility as colorimetric indicators for many transition metals or more recently, for their use as
sensitizer for dye sensitized solar-cells.
Lately, studies performed on copper complexes of phenanthroline derivatives, have
demonstrated a strong interest in the use of these compounds as candidates for the
development of photonic devices, including sensors, photovoltaic devices and switches.33
The photochemical and electrochemical properties of copper(I) phenanthroline
compounds have also been used to study their interaction with biological systems, in
particular DNA intercalation and scission.34 There have also been numerous studies of these
types of complexes in relation to their biomimetic behavior.35
Their chemistry has been widely investigated and still continues to be explored. The
versatility is mostly due to the possible formation of a variety of stable complexes with
metals.
Their stability may be deducted from the ability of acting both as π acids and σ bases;
moreover, the possibility of forming π bonds among complete metal orbitals d and their
empty orbitals π‫ ٭‬may enhance these properties.
5
6
4
7
8
3
2
N
1
N
10
1
18
9
1,10-phenanthroline 1 is available from commercial suppliers, but the synthesis of
this ligand from its various precursors is often necessary in the preparation of more complex
structures.
With a general procedure, Skraup reaction or the very similar Doebner-von Miller
reaction, have demonstrated to be two efficient routes to obtain a quinoline core 4 (Scheme
1).36 The first type involves aniline 2 and glycerol 3, in the presence of a strong acid and an
oxidizing agent.
NH2
+
OH
HO
Ph-NO2
HO
2
N
H2SO4
3
4
Scheme 1
The first step is the acid-catalyzed conversion of glycerol into acrolein. Indeed,
variations of the Skraup synthesis use preformed acroleins, instead of glycerol. The reaction
sequence then proceeds through a Michael addition and an intramolecular aromatic
substitution, followed by a final oxidation (Scheme 2).37
19
H
OH
OH
O
H
Michael
NH2
OH2
H
N
H
N
H2
H3O / H2O
OH
O
H
H
Acrolein
N
H
OH
H2O
H OH
PhNO2
N
N
H
N
H
N
H
Scheme 2
In an analogous manner, reaction of 5 with glycerol 3 or acrolein 6 in the presence of
sulphuric or phosphoric acid and arsenic pentoxide produces 1 directly (Scheme 3).
+
N
OH
HO
H
O
or

HO
5
NH2
3
H2SO4, As2O5
6
N
N
1
Scheme 3
An alternative attractive approach for the direct construction of a pyridine ring is the
Friedländer condensation, which does not require the use of troublesome oxidizing agents and
20
provides
higher
yields.
According
to
Scheme
4,
starting
from
8-amino-7-
quinolinecarbaldehyde 7, the second pyridine ring is closed by coupling with an enolizable
ketone, with concomitant loss of water.
O
O
KOH
H
+
R2
NH2
N
R1
EtOH, 
R1
N
N
R2
7
1a
8
Scheme 4
For substances of type 1, carrying substituents at 5 or 5 and 6 positions, a Skraup
condensation synthesis or Friedländer condensation have been used, starting from appropriate
substituted precursors, to give several phenanthroline derivatives.
The synthesis of phenanthroline derivatives has played an important role in the
development of supramolecular and coordination chemistry. The increasing interest in the
preparation of complex structures stems in the possibility of using the 2,9-dichloro-1,10phenanthroline (whose preparation will be later described) as a key and versatile
intermediate.38
The results herewith presented, suggest that indeed the 2,9-dichloro-1,10phenanthroline can be considered the starting point for the design of telomerase inhibitors and
new derivatives. The development of new highly functionalized compounds by appropriate
modifications of the phenanthroline nucleus hopefully will provide further support for the
theory of G4 mediation in the inhibition of telomerase activity.
21
Chemistry
The goal of the proposed research, performed in collaboration with Prof. A. Paul
Krapcho at the University of Vermont, U.S.A., is the synthesis of molecules related to
structure 9, based on in silico data of model system from Stefano Moro, University of Padua.
R
R
N(H2C)2HNH2C
X
R
R
CH2NH(CH2)2N
N
N
N
N
N(H2C)2HNH2C
X = S, O
R
CH2NH(CH2)2N
X
R
R
R
9
The retrosynthesis of 9 is outlined in Scheme 5.
OHC
X
CHO
H3C
X
CH3
N
N
N
N
N
N
N
N
9
OHC
X
CHO
H3C
10
H3C
X
CH3
11
X
Cl
NH
CH3
CH3
N
N
N
N
+
NH
X
H3C
12
Cl
CH3
CH3
13
Scheme 5
22
14
Initially, in order to evaluate a procedure for the introduction of a chloro substitutent
at position 2 in the 1,10-phenanthroline moiety, 1,10-phenantholine (1) (Scheme 6) was
treated according to a literature procedure39 with urea-hydrogen peroxide (UHP) in formic
acid to afford the corresponding N-oxide (15).Treatment of this oxide with POCl3 in the
presence of triethylamine40 led predominantly to 2-chloro-1,10-phenanthroline (16). One also
notes in the 1H NMR spectra the presence of an additional product in this reaction which
might be 4-chloro-1,10-phenanthroline (17).
Cl
N
O
N
ii
N
+
N
i
Cl
N
15
N
17
16
N
N
O
iii
I
N
1
iv
N
CH3
N
Cl
v
N
CH3
N
18
19
N
16
Reagents: (i) UHP/HCOOH; (ii) POCl3, Et3N, iii) CH3I, CH3CN, ;
(iv) K3(FeCN)6, NaOH/H2O; (v) POCl3/PCl5
Scheme 6
The alternative route to get solely compound 16 is a three step procedure, which
involves one troublesome oxidation with ferricyanide. Johnson41 obviated the problem of
producing toxic waste, by replacing this oxidizing agent with activated MnO2, which was
found to be an effective and mild reagent for a number of oxygen atom transfer reactions. The
only inconvenience was represented by the concurrent oxidation of the iodide and the
necessity of washing the resulting I2 with thiosulfate.
23
In the current study, compound 1 was converted into the methiodide salt 18 with
methyl iodide in acetonitrile, at reflux for 1.25 hrs. Product 18, upon oxidation with alkaline
ferricyanide, yielded compound 19 quantitatively, which was then transformed into the
desired 2-chloro-1,10-phenanthroline 16 (68% yield) by refluxing with a mixture of
phosphorous oxychloride and phosphorous pentachloride.
With the success of the introduction of a Cl substituent at C-2, the attention was
turned to the chemistry of 4,8-dimethyl-1,10-phenanthroline (14) (Scheme 7). Treatment of
this compound with urea-hydrogen peroxide (UHP) in formic acid led to 4,7-dimethyl-1,10phenanthroline N-oxide (20) as a pink solid (74%). Treatment of this N-oxide with POCl3 in
the presence of triethylamine led to a crude purple solid 21 (52%). Chromatography of a small
portion on silica gel led to a white solid whose 1H NMR was consistent with this structure.
Attempts to oxidize the more basic nitrogen of 21 to afford 22 using the UHP-formic
acid reagent combination was unsuccessful. In this case the starting material 21 was the only
identifiable product via 1H NMR analysis along with a small amount of the desired N-oxide
22. The oxidation of 21 to 22 was then performed using acetic acid-hydrogen peroxide.42 One
notes in this case a singlet in the proton NMR at δ 6.79 which might cast some doubt on this
structural formulation.
H3C
H3C
H3C
N
N
i
O
N
H3C
N
ii
H3C
Cl
N
iii
iv
O
N
N
H3C
H3C
14
Cl
H3C
21
20
13
N
22
Reagents: (i) UHP/HCOOH; ii) POCl3, Et3N, ;
(iii) CH3COOH/H2O2, (iv) POCl3/Et3N
Scheme 7
Two attempts using POCl3 for the preparation of the dichloro analogue 13 from 22
were not successful. However, in the third attempt (use of POCl3 in the absence of Et3N) a
low yield of product was obtained which exhibited 3 singlets in its 1H NMR spectrum, which
24
is consistent with structure 13. Other impurities were also present and the low yield precluded
further study.
Because of difficulties encountered on the preparation of 13, the attention was turned
to the preparation of the mono-substituted analogue 23 shown in a retrosynthetic fashion in
Scheme 8.
O
O
(H3C)2N(H2C)2HNH2C
Cl
N
N
NH
N
N
N
NH
N
O
O
24
23
CH3
Cl
25
Scheme 8
The retrosynthesis of 25 from commercially available 26 is illustrated in Scheme 9.
Cl
N
N
N
CH3
N
CH3
Cl
25
26
Scheme 9
Following a literature procedure for the preparation of bis-salts from 1,10phenanthroline, treatment of 26 with 1,3-dibromopropane led to 27.38 Oxidation of 27 with
potassium ferricyanide under basic conditions led to the bis-amide 28 in about 50% yield. The
25
conversion of 28 to 25 has been accomplished by treatment with POCl3 and PCl5 in
reasonable yields (Scheme 10). In an analogous manner, compound 25a was derived from its
precursor 1.
O
N
X
N
i
X
N
N
ii
·2 Br
N
X
Cl
N
iii
N
X
N
O
X = CH3: 26
X = H:
28
28a
27
27a
1
Cl
25
25a
Reagents: (i) Br(CH2)3Br, ; (ii) K3Fe(CN)6, NaOH/H2O; (iii) POCl3/PCl5, 
Scheme 10
The synthesis of 24 was attempted according to the procedures outlined in Scheme
11, while compound 25a was derived from its precursor 1, following the same route outlined
in Scheme 10, for the preparation of the 2,9-dichloro-5 methyl-1,10-phenanthroline (25).
A first investigation to assess the possible formation of compound 24 was
undertaken, starting from the synthesis of the analogue 30. The same strategies were then
applied for the preparation of the bis-amide 24. Following a literature procedure,43 compound
16 was treated with a solution of sodium methoxide, followed by reflux for 19 hours to afford
product 29a (79%).
The intermediate 29a was readily converted to the desired 30, pursuing two
approaches. By treatment of 29a with HI [57%] and subsequent basification with NaOH,44 a
purple solid was obtained, whose 1H NMR was consistent with structure 30. One also noted a
broad singlet at δ 11.19, relative to the -NH group, but the dark color precluded further study.
A second attempt of demethylating compound 29a was done using HBr [48%],
which afforded a yellow solid (Scheme 11). Also in this case 1H NMR showed that the only
identifiable product was 30. Presumably the protonated phenanthroline nucleus is activating
the facile cleavage of the O-C bond in the use of HBr.
26
Compound 24 was derived in an analogous manner. By applying the same
procedures described above, involving a solution of NaOCH3, from the precursor 25a,
compound 29b was obtained quantitatively and it was efficiently demethylated with HBr
[48%].
O
NH
30
OCH3
Cl
N
i
N
from 29a
from 29b
N
N
N
ii
ii
X
16 (X = H)
25a(X = Cl)
O
X
NH
29a (X = H)
29b (X = OCH3)
NH
24
O
Reagents: (i) CH3ONa, ; (ii) HI 57% or HBr 48%, 
Scheme 11
Several papers appeared dealing with the proton-shift tautomerism, which can be
deducted also from structure 30. For most simple phenols this equilibrium lies to the side of
the OH group, where the aromatic character is maintained. Therefore, for phenol itself, there’s
no evidence of the keto form.45 However, the keto form becomes important: (1) where certain
groups, such as a second OH group or an N=O group are present;46 (2) in systems of fused
aromatic rings;47 and (3) in heterocyclic systems. In many heterocyclic in the liquid phase or
in solution, the keto form is more stable, although in the vapour phase the positions of many
of these equilibria are reversed.48
For example, in the equilibrium between 4-pyridone I and 4-hydroxypyridine II
(Figure 10)49, I is the only detectable form in ethanolic solution, while II predominates in the
vapor phase.48 In other heterocycles, the hydroxy form prevails.
27
2-Hydroxypyridone III and pyridone-2-thiol V are in equilibrium with their
tautomers, IV and VI, respectively. In both cases, the most stable form is the hydroxyl
tautomer, III and V.
O
OH
N
H
N
I
II
N
OH
N
H
III
N
O
IV
SH
N
H
V
S
VI
Figure 10
With regards to compound 30, 1H NMR evidence is in accordance with the
observation provided by Zhang50, upon which the proton is bonded to the nitrogen atom,
instead of the oxygen atom. In other words, since there is no peak that can be attributed to the
hydroxy tautomer, it’s possible to conclude that the keto form, as it is the only detectable one,
exists largely in this structure rather than its tautomer, and it may therefore predominate
(Figure 11).
28
N
N
HN
N
OH
O
30
30a
Figure 11
At the same time, experiments pertaining the synthesis of sulphur analogues, similar
in structure to compounds 30 and 24 were carried out. Since their chemistry appeared to be
particularly attractive, as evidenced by several works published over the last thirty years, the
second main goal of this project became the formulation of a strategy for the preparation of
30, whose retrosynthetic pathway is shown in Scheme 12:
S
S
(H3C)2N(H2C)2HNH2C
Cl
N
N
NH
N
N
N
NH
N
S
S
32
31
CH3
Cl
25
Scheme 12
As a prelude to investigations of sulphur derivatives, initially three different routes
for the preparation of monothione 33 were patterned (Scheme 13). Subsequently, the same
procedures for the obtainment of the dithione 32 were also attempted.
Several studies reported the DMSO oxidation as a standard procedure for the
conversion of aromatic thiols into disulfides. However, the application of this method resulted
in failure, when Hunziker51 attempted the conversion of the bisthione 32 from the monothione
33. This obstacle was later overcome by the introduction of KHS as an efficient reagent, using
elevated pressure and temperature.
29
Herewith, alternative and successful synthetic methods are described.
S
Cl
HN
N
v
from 25a
HN
S
from 16
N
S
N
X
32
33
16 (X = H)
25a (X = Cl)
iii
S
from 25a
ii
S
HN
NH
i
S
HN
iv
S
NH
NH
+
HN
N
S
N
Cl
32
NH
Cl
34
34
S
32
Reagents: (i) (NH2)2CS, EtOH, , 34% or Na2S·xH2O or Na2S·9H2O, DMF, ;
(ii) (NH2)2CS (5 eq.), EtOH, ; (iii) (NH2)2CS (20 eq.), EtOH, , 30%;
(iv) Na2S·xH2O, DMF, r.t., 67% or, 58%;
(v) Na2S·xH2O, DMF, , 67%.
Scheme 13
The initial procedure was carried out by reacting 16 with an excess of thiourea, using
ethanol as a solvent and refluxing the mixture for 2 hours, to give an isothiuronium salt (16a).
This intermediate was readily cleaved to the thiol (16b) by subsequent thermal treatment with
alkali (Scheme 14):
30
Cl
N
Cl
S
NH2
S
H2N C NH2
N
S
NH
1) OH
NH2
2) pH=7
N
N
N
16
33
16a
Scheme 14
Unfortunately, after extractions with CHCl3, the displacement of the chloride in 16
gave a poor yield (34%).
By analogy with the tautomeric forms of 30 and 30a, the envisaged conclusions
could be applied to the sulphur derivatives too, as reported in Figure 12. In fact, 1H NMR
analysis showed a broad peak at δ 12.35, as expected for structure 33.
S
SH
NH
N
N
N
33
33a
Figure 12
The low conversion prompted an investigation towards an alternative procedure for
the preparation of the desired compound, which was obtained in almost quantitative yield,
using sodium hydrosulfide hydrate in DMF, with heating, followed by acidification with
glacial acetic acid, which led to a yellow solid. Similarly, compound 33 was afforded with
reasonable yields (56%) by treatment of 16 with sodium sulfide nonahydrate in DMF with
heating. The work-up was the same as the one described for the previous procedure. These
routes were later extended to the preparation of the bisthione.
31
Preliminary experiments showed that compound 25a could be converted into
necessary intermediate 34 to get 32 exclusively.
Partial transformation of 25a into 32 was accomplished using an excess of thiourea
in EtOH at reflux for about 5.5 hours. After evaporation of EtOH, followed by basification
with NaOH and extraction with CHCl3, a mixture of chlorothione 34 and dithione 32 was
obtained and the two compounds were partially isolated by gravity column chromatography.
The best results were achieved when 2,9-dichloro-1,10-phenanthroline 25a was
treated with thiourea in ethyl alcohol to afford the corresponding 9-chloro-1H-1,10phenanthroline-2-thione (34). Treatment of this chlorothione with sodium hydrosulfide
hydrate in DMF, at r.t., led to the desired 1,10-phenanthroline-2,9(1H,10H)-dithione 32
(67%). The low solubility in chloroform, prompted a proton NMR analysis which could only
be recorded in DMSO-d6, showing three pairs of equivalent aromatic protons and a broad
singlet, relative to the two thioamide hydrogens, as expected for compound 32.
However, it could not be excluded a possible interaction with the solvent (DMSOd6), known to be a hydrogen bond acceptor, that could favour the tautomeric form 32 rather
than the thione form at one side only, 32a (Figure 13).51
A further investigation was directed toward the obtainment of 32, starting from
precursor 25a. The successful reaction of 25a with about 8 molar equivalents of hydrate
sodium hydrosulfide in DMF, with heating, afforded 32 in good yields (67%).
S
N
H
N
SH
32a
Figure 13
32
Heteroarylation
The addition of alkyl and aryl groups to the 2- and 9- positions of 1,10phenanthroline 1, proceeds most commonly by using organolithium reagents.
Pallenberg et al.52 investigated the 2,9-dialkyl-1,0-penanthrolines and their copper(I)
complexes. These derivatives can be easily obtained via the nucleophilic aromatic substitution
of 1,10-phenanthroline 1 with the corresponding alkyllithium reagent, following a
rearomatization by oxidation with an excess of manganese dioxide.
Nonetheless, since 2,9-substituted 1,10-phenanthrolines have been and continue to be
a primary area of interest in contemporary transition metal coordination chemistry, it’s worth
mentioning one of the numerous applications that these derivatives can bring about, in areas
like electroresponsive reversible membranes and controlled transport materials. To this extent,
Bernhard and co-workers studied novel polymeric copper (I) systems containing 2,9dimethyl-1,10-phenanthroline moieties, linked by either p-xylene or an n-decane bridge.
These reactions were afforded through a 2-fold alkylation reaction of ,’-dibromo-p-xylene
and 1,10-diiododecane respectively, with 2,9-dimethyl-1,10-phenanthroline.
It was also reported that the addition of aryl groups to the 3- and 8- positions of 1
may proceed starting from the easily accessible 3,8-dibromo-1,10-phenanthroline, using a
phenylboronic ester, under Suzuki cross coupling conditions.53
Alkylation of sulphur phenanthroline derivatives at 2- and 9- positions can likewise
take place. The evidence is provided by further studies conducted in the present work.
The onset approach to this chemistry was conducted on compound 33 and then
proceeded to the bisthione.
One initial experiment was designed to directly introduce a methyl group on
compound 33. A first attempt of alkylating the 1,10-phenanthroline-2(1H)-thione was made
using CH3I at room temperature for 2 hrs, which led to the hydroiodide salt 35 (Scheme 15).
This salt, on treatment with aqueous potassium carbonate and extraction with CH2Cl2, led to
the free base 35a, which was the only identifiable product, by 1H NMR evidence. In fact,
absorption at 2.88 ppm (s, 3 H) indicated the presence of the protons belonging to the SCH3
group.
33
S
S
ii
N
S
N
iv
N
·HBr
N
35a
N
iii
i
S
35
N
S
36
NH
Br
N
vi
36a
N
33
N
iii
v
S
I
S
N
·HI
N
Br
S
N
N
·HI
Br
N
39
38
Reagents: (i) CH3I, r.t.; (ii) K2CO3, CH2Cl2; (iii) Br(CH2)3Br, ;
(iv) K2CO3, CH2Cl2; (v) Br(CH2)2Br, ; (vi) I(CH2)6I, r.t.
Scheme 15
34
37
Br
With the success of these experiments, further studies were conducted, introducing
1,3-dibromopropane and 1,2-dibromoethane for S-alkylation purpose. Two first attempts of
forming a ring using 1,3-dibromopropane to afford compound 36 were unsuccessful, as
starting material 33 was the predominant product. In the first case THF was introduced as a
solvent and the mixture was heated for about 3 hours. 1H NMR analysis showed that the
reaction did not go to completion, since starting material seemed to be still present, although 3
small peaks between δ 4 and 2 ppm may have revealed the beginning of a possible formation
of the chain RS-CH2-CH2-CH2-Br. In the second pathway, in a similar manner, CH3CN was
used but also in this case the reaction failed. Evidence of the formation of compound 36 was
proved in the third attempt, when 1,3-dibromopropane was used in the absence of solvent.
Unfortunately, spectral data confirmed structure 36 and ruled out cyclized product 37.
Treatment of salt 36 with aqueous potassium carbonate and extraction with CH2Cl2 led to the
free base 36a.
A successful and interesting experiment which afforded the expected cyclized
product 38 was obtained reacting 33 with 1,2-dibromoethane in excess, with heating. While
using CH3CN as a solvent under reflux partially afforded compound 38 along with some
starting material, also for this reaction it was noted that the best result was obtained when no
solvent was involved. Evidence of this possible structural formulation was proved by 1H
NMR analysis, showing absorptions at δ 6.40 and 4.01 ppm, which appeared to be consistent
with chemical shifts for the methylene protons adjacent to the quaternary nitrogen and the
sulfur, respectively.
Based upon these results, it was reasonable to expect that reaction with
phenanthroline-2-thione 33 and 1,6-iodohexane at room temperature (6 hours), would give the
desired compound 39 (Scheme 17). In fact, proton NMR analysis gave evidence of this
formulation, showing 3 multiplet structures at δ 3.60, 3.27 and 1.79 that could be assigned to
the methylene protons, although the presence of other peaks interfered with a correct
interpretation.
In order to obtain sulfur bis alkylation, an interesting pathway was followed, starting
from 2,9-dichloro-1,10-phenanthroline phenanthroline 25a which was taken to the 2,9-bismethylsulfanyl-1,10-phenanthroline 40a (Scheme 16). By treatment of 25a with sodium
thiomethoxide (1:5 molar ratios) in DMSO at r.t., the two haloelements were displaced and
upon addition of water, compound 40a was obtained in good yield. Also in this case the
35
proton NMR showed a singlet at 2.9 ppm, which indicated the presence of 6 protons relative
to the 2 methyl groups.
Cl
N
i
25a
N
S R
Cl
from i: R = -CH3
N
N
S
40a
from ii: R = -CH2CH3 40b
S R
NH
ii
32
NH
S
Reagents: (i)CH3SNa, DMSO, r.t., 20 hrs; (ii) CH3CH2I, 
Scheme 16
Another way of obtaining bis alkylation was possible beginning with the bisthioamide 32, which was treated with ethyl iodide with heating. Work-up with aq. K2CO3 and
extraction with dichloromethane led to a product 40b with an identical 1HNMR as the
hydroiodide salt. This suggested that the original product was not the HI salt.
With a variety of substituted sulphur phenanthrolines in hand and successful and
easy routes to obtain them, the study was later focused on the preparation of heteroaryl
sulfides, derived 1,10-phenanthroline 2-thiol, 33 and its analogue 32. Before this goal was
achieved, it was thought that a better understanding of sulphur reactivity was necessary,
starting from trial experiments in which less complex structures were involved.
For this purpose, the chemistry of commercially available 2-mercapto-pyridine and
2-chloro-pyridine was investigated and particularly their coupling.
Two different experimental conditions were examined (Scheme 17). The first
successful experiment was done by treatment of 41 with a solution of sodium ethoxide. After
removal of EtOH the resulting thiolate salt 42 was made to react with the 2-chloropyridine in
36
DMA with heating followed by acidification. The proton NMR indicated that there was
evidence of the desired product 43.
A second attempt was made reacting 41 and 2-chloropyridine with potassium
carbonate in DMF with heating. Also in this case, after treatment with water it was noted that
43 was the identifiable product. The reaction gave positive results with the introduction of
Hunig’s base, when the 2-mercaptopyridine 41 was made to react with the 2-Cl-pyridine (1:1
molar ratio) in DMF under reflux.
41
i
S
N
H
iii, iv
ii
NaS
N
H
N
S
N
43
42
Reagents: (i) NaOEt; (ii) 2-Cl-py, DMA, ; (iii) 2-Cl-py;
(iv) K2CO3 or Hunig's base, DMF, 
Scheme 17
A similar approach was then applied to the phenanthroline nucleus, with the aim of
forming symmetrical thioethers. Methods for the preparation of aryl thioethers have generally
suffered from limited applicability, due to the involvement of activated reactants, severe
reaction conditions, complicated procedures or a combination of all these requirements.54
Aryl sulfides (thioethers) may generally be prepared by nucleophilic substitution of
aryl halide by thiolate anion (salts of thiols). The R’ group (Scheme 18) may be alkyl or aryl
and organolithium bases can be used to deprotonate the thiol.
37
RX +
R'S
RSR'
Scheme 18
Campbell54 reported difficulties in the preparation of aryl ethers, involving Ullmanlike conditions. In fact, when aryl halide and potassium aryl thiolate were heated in excess
thiol at temperatures above 200°C in the presence of copper salts, no reaction was observed.
Presumably drastic conditions were not sufficient for an increase of the solubility of
potassium thiolate. Therefore it was found that this reaction was promoted by high boiling
point solvents, in particular, best results were achieved when DMF or DMA were employed.
Following this general procedure, some examples of diaryl sulfides were undertaken
and are reported in Scheme 19.
Two reactions gave positive results when 2-chloro-1,10-phenanthroline 16 was
treated with the 2-mercaptopyridine (1:1 molar ratio) for the first attempt and with 2mercaptopyrimidine for a second one, refluxing with DMF and using potassium carbonate.
After removal of the solvent by distillation, followed by treatment with water, both products,
44a and 44b, were easily purified by column chromatography. In particular, for compound
44a, recrystallization from acetonitrile afforded nice crystals.
Cl
N
S
N
i, ii
N
N
X
N
16
from i
44a
(X = C)
from ii
44b
(X = N)
Reagents: (i) 2-mercaptopyridine, DMF, K2CO3,  ;
(ii) 2-mercaptopyrimidine, DMF, K2CO3, .
Scheme 19
38
When the same approach was followed for the introduction of pyrimidine or the
pyridine moiety on both sides of the phenanthroline dithiol nucleus, unexpectedly the
reactions outlined in Scheme 20 did not take place:
Cl
S
i, ii
N
N
Cl
N
N
X
N
X
S
25a
N
from i
45a
(X = C)
from ii
45b
(X = N)
Reagents: (i) 2-mercaptopyridine, DMF, K2CO3,  ;
(ii) 2-mercaptopyrimidine, DMF, K2CO3, 
Scheme 20
39
Coupling
Several approaches exist for coupling 1,10-phenanthroline 1 with itself. Fused-ring
oligophenanthrolines exhibit strong electronic coupling (Figure 14). Tetrapyridophenazine A,
tetraazatetrapyridpentacene B and bispyridoquinoxalinepyrene C are planar bitopic ligands
prepared by condensation analogous to dipyridophenazine D. The latter is a common
intercalating ligand, whose ruthenium(II) complex shows “light switch”-type luminescence
enhancement upon intercalation with double helical DNA. These derivatives often serve as
bridging ligands in more complex structures. Eliatin E, a ligand of biological origin, has been
shown to induce dimerization of its (bis) heteroleptic ruthenium(II) complexes. The tritopic
ligand hexaazatriphenylene F, allows the formation of polynuclear complexes in three
directions.55
A
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
B
N
N
N
N
N
N
N
N
C
N
N
N
N
N
N
N
N
N
N
N
N
N
E
N
F
Figure 14
40
D
Hunziker51 reported the preparation of the symmetrical compound related to structure
46 (Scheme 21). His procedure involved the coupling of substrates 33 and 16, promoted by
KOH and DMA with heating. This product was fully characterized both by spectroscopic
methods and X-ray structure analysis. A variation to this synthetic methodology which was
equally successful, is hereby demonstrated:
Cl
S
S
i
N
NH
+
N
33
N
N
N
N
N
46
16
Reagents: (i) K2CO3, DMF,  or Hunig's base, DMA, 
Scheme 21
In order to obtain compound 46, two attempts were made, choosing different
strategies.
Treatment of 33 with 16 in the presence of potassium carbonate and N,Ndimethylformamide, led to a yellow product, which was purified by column chromatography.
1
H NMR of the pure solid showed that 46 is the only identifiable product. Besides, a complete
characterization by MS, elemental analysis,
13
C and proton NMR is in agreement with the
predicted structure. Wang et al.56 proposed that the same reaction would also proceed
smoothly from 16 and with H2S in good yields.
With the success of this reaction, a second endeavour was made, reacting 33 and 15
with N,N-diisopropyl-ethylamine with heating. 1H NMR of the crude product may be
consistent with structure 46, although other impurities were also present.
41
As part of the goal was achieved, it was thought that some of the intermediates so far
described, could be considered as attractive candidates for their further synthetic elaboration.
Thus, the focus was narrowed to the construction of the most interesting objective of the
present work, that is, the so called “sulphur macrocycle”.
42
Macrocyclic ligands
Many studies have been conducted on the synthesis of macrocyclic ligands, which
have been prepared by reactions with 2,9-disubstituted phenanthrolines and other suitable
species. Cyclic bis-phenanthrolines have been prepared through amino-, aza- and thiol bridges
at the 2,9-positions.
These ligands possess a coordination environment similar to porphyrins and form
stable complexes with several metals. Ethylene-bridged bis(phenanthrolines) produce helicate
dimetallic complexes.55 Yao and co-workers57 were able to tie together 2,9-dimethyl-1,10phenanthroline ligands, according to the procedures outlined by Lehn 58 and then could
attempt to prepare copper(I), copper(II) complexes for photophysical properties enhancement
(G). Complexes with Cu(I) take advantage of the stabilizing effect of the 2,9-substituents
which effectively prohibit the planarization of the complex which would occur upon oxidation
to the Cu(II) species. Among the species so far investigated, complexes of branchedmacrocyclic ligands have demonstrated to absorb incident light very efficiently and shield, to
a great extent, the metal ion from interaction with solvent molecules, which would cause
nonradiative decay of the luminescent level.59
2+
N
N
N
Cu
N
N
Cu
N
N
N
G
Figure 15
Bridged bis-phenanthroline macrocyclic compounds are of great interest, owing their
potential applications in the catalytic reduction of CO2 and in the possible activity towards
43
DNA. With regards to DNA binding, the great deal of interest derives from the different
ability of each metal complex to interact with DNA, to induce its cleavage with a particular
conformation and sequence.60
Preparation of the aza-bridged bis-phenanthroline macrocycle was first reported by
Ogawa et al.61 For azacyclophane, bridged by carbon H and nitrogen I, tautomerization to the
fully conjugated forms Ha and Ia respectively, could be predicted.
H
H
N
N
N
N
H
H
H
N
N
N.
H.
H
..
H
N
.
N
N
N
N
N
N
N.
..
.
N
Ha
H.
H
.
..
..
N
H
N
I
Ia
N
.
N
Figure 16
The aza-macrocycle was obtained either by thermolysis of 2,9-diamino,1-10phenanthroline (at 280-230°C) or by condensation of the latter with the dichloro analogue,
using potassium carbonate as a deprotonating agent and nitrobenzene as a solvent, followed
by heating. Further studies were also conducted for coordination chemistry in order to form
nickel, cobalt and copper complexes. Their properties were later investigated and it was
postulated the planar or nearly planar structure which appeared to endow them with a unique
chemical character. However, because of the low solubility of these complexes in water,
studies with DNA were impossible.60 Wang and co-workers62 later developed an alternative
one-pot synthesis of the aza-bridged 1,10-phenanthroline, starting from 2,9-dichloro-1,10phenanthroline, which at 250°C, under a stream of ammonia gas, afforded the expected azamacrocycle as a bis hydrochloride salt. The free base was obtained as a precipitate by treating
the aqueous solution with alkali.63
44
In this case, molecular modelling studies were in disagreement with the
aforementioned non planar structure, indicated by Ogawa.
In addition, X-ray crystallographic analysis revealed that the two central hydrogen
atoms, were localized on two of the opposite central nitrogen atoms (Ia), similar to those of
free base porphyrin. A later study by Ogawa et al.,64 reported that as well as for porphyrin,
aza-bridged macrocycle has a delocalized structure with an ionic character, that is, it contains
two cationic hydrogens. Therefore, macrocycle hydrochloride Ib, is supposed to have the
electronic charge of six nitrogen atoms, equalized by hydrochlorination (Figure 17).
H
N
.
N
H
H.....
N
N
.
...
.
N....
...
.
2Cl
.
N
H
Ib
Figure 17
In the present work, three different strategies are presented, which describe
interesting routes for the obtainment of a macrocycle, consisting of two 1,10-phenanthroline
molecules, bridged by sulphur.
Some initial experiments were unsuccessful, when a condensation of bis thione 32
with the dichloro 25a was attempted (Scheme 22), using the same procedure outlined for the
preparation of the mono-sulfur bridged macrocycle (Scheme 21) or simply with the
employment of Hunig’s base with heating.
45
S
Cl
S
HN
N
i
N
N
N
N
+
HN
N
S
Cl
S
32
25a
47
Reagents: (i) K2CO3, DMF,  or Hunig's base, DMA, or Hunig's base, 
Scheme 22
Starting from the chlorothione 34 (Scheme 23), different procedures were used,
which provided primising results. With the introduction of sodium hydroxide as a base (1:10
molar ratio) with DMF and heating the mixture, the solid obtained 47a was subjected to
proton NMR analysis. The 2 doublets and 1 singlet cast no doubts concerning the macrocyclic
structure. The only concern could be represented by the four nitrogen atoms in the centre of
the molecule, which may form a bond to the sodium ion. Such molecule has a structure that
recalls a multidentate (tetradentate) ligand, as it contains a chelating agent, represented by
four nitrogen atoms bonded to a sodium ion. Four of these bonds my tie it irreversibly to the
entire molecule.
S
S
N
N
...
.. ...
.
.
.. ....
...
.
NH
i
Na
N
N
S
Cl
47a
34
Reagents: (i) NaOH, DMF, 
Scheme 23
46
· Cl
Hirai60 demonstrated, by a full characterization, that the product obtained through
this synthetic method is indeed a chloride of sodium complex. Subsequent reaction with
copper acetate salt was also explored, providing a high water soluble copper(II) complex
which is supposed to have a planar structure, that may favour the interaction with DNA.
The problem of having Na ion irreversibly immobilized in the molecule, was later
circumvented by simply heating the 9-chloro-1H-1,10-phenanthroline-2-thione (34) up to
about 320°C. Whether the product was quenched into water and sodium hydroxide was
added, or no treatment was followed, the proton NMR was in agreement with the assigned
structure 47 (Scheme 24).
S
NH
S
N
i
N
· HCl
N
N
Cl
N
S
34
47
Reagents: (i)  (320°C) or Ph2(O), .
Scheme 24
It was then decided to introduce the high boiling point diphenylether, to promote this
type of reaction (Scheme 24). Compound 47 which precipitated from a solution obtained
when the original mixture reached 170°C, was simply washed with ethyl ether and its proton
NMR this time showed still two doublets and one singlet, but their chemical shifts were
different from the ones relative to product 47a, Scheme 23.
Hirai et al.60 also demonstrated by proton NMR analysis, MS and elemental analysis,
that it was possible to obtain the same compound 47, metal free, from 34, using a combination
of DBU and DMA with heating.
It’s somewhat interesting the difference in chemical shifts provided by Wang, when
he claims to obtain the same sulphur bridge macrocycle, starting from the dichloro derivative
25a, heated up to 170° and under an atmosphere of H2S. The lack of agreement derives from
47
the proton NMR, performed in deuterated trifluoro acetic acid, which in the present study
clearly shows that this type of solvent protonates the compound, causing a reduction of the
deshileding ring current of the aromatic ring, by comparison with the spectrum of the same
compound in DMSO-d6. This effect produces a change in terms of chemical shifts, which are
moved downfield, but still showing that the two doublets and one singlet remain in the same
order (d, s, d), in accordance with Ogawa’s result.60
Besides, the proposed compound was compared to symmetrical model system 40a
(Scheme 16), whose 1H NMR in CF3COOD produces chemical shifts from DMSO of +0.40
for the doublet at low field, +0.32, relative to the singlet and +0.35 for the doublet at high
field. It’s worth pointing out that also in this case the d, s, d pattern stays in the same order.
On the contrary, Wang’s published work reports a different chemical shift pattern, (s,
d, d) which make his conclusions doubtable.
Further extensive characterization of the product, obtained with the convenient route
herewith presented, rationalized the observed structure. IR analysis revealed traces of water
(peaks at 3333 cm-1 and at 1659 cm-1), while titration proved the presence of chloride, which
confirmed the structure of macrocycle 47 as an HCl salt. All these data validate elemental
analysis results. Moreover, Professor C. Pettinari’s research group performed chelation
chemistry on the obtained sulphur macrocycle, which is in support of the hypothesised
structure. In fact, preliminary studies for a complexation of the potential ligand 47 with
equimolar amount of CoCl2 in ethanol, at room temperature, afforded product 48a. In a
similar manner a complex with Zn(II), 48b was obtained from another metal bromide, ZnBr2
(Scheme 25). Additional experiments with other metals such as titanium and copper are
currently under investigation.
48
S
S
N
N
N
i, ii
... ..
. ..
....
N
X
.
...
N
N
N
N
S
II
S
47
from i
48a
X = Co
from ii
48b
X = Zn
Reagents: (i) CoCl2, EtOH, r.t.
(ii) ZnBr2, EtOH, r.t.
Scheme 25
Whilst Hunziker51 asserts the possible condensation of 25a with 32 in the presence of
DMA and Na2S, providing 47 in small yields, along with polymeric and other side products,
as follows a third convenient synthetic route to pure 47 is displayed (Scheme 26), using
diphenylether with heating at 170°C for 3 hours.
S
Cl
S
HN
N
i
+
X
Cl
(X = H)
X
HN
N
25a
N
N
N
N
S
S
32
(X = CH3) 25
(X = H)
47
(X = CH3)
47b
Reagent: (i) Ph2(O), 
Scheme 26
Since this chemistry gave positive results, the research was branced into a similar
direction, finalized to the introduction of substituents on macrocycle 47.
49
In order to avoid any complication represented by initial transformation of derivative
25 into a possible mixture of regioisomers 47c,d, which would have been difficult to isolate,
as patterned in retrosynthetic fashion (Scheme 27), methyl derivative 25 was treated with
compound 32, aiming to obtain 47b exclusively.
S
N
S
N
+
H3C
N
CH3
N
H3C
S
N
N
N
N
CH3
S
47c
47d
S
Cl
Cl
NH
N
N
+
H3C
N
HN
Cl
34a
CH3
H3C
N
Cl
S
34b
25
Scheme 27
Using the same reaction conditions outlined for condensation of 25a with 32, from
derivative 25, compound 47b was not identified as a pure sample. MS analysis reported a
number of other unidentified products, along with the expected compound, possibly as an
hydrochloride salt, by analogy with the unsubstituted 47, while proton NMR spectrum was
difficult to interpret.
In order to explore the chemistry of methyl derivative 25, it was decided to shift
emphasis in the anti-telomerase inhibition, but still keeping the sulphur chemistry in first
place, by proposing a new compound which could potentially arouse interest in terms of
biological activity. By analogy with the compound shown in Figure 15, which demonstrated
to be inactive with duplex DNA, while inhibits telomerase starting from a concentration of 5
50
M, it was designed a new compound 48 and later also 49 could be discerned, with
similar structural features, as described in Figure 16.
N
H
N
N(CH3)2
·HCl
N
H
N
N(CH3)2
L
Figure 18. Compound synthesised by
Prof. A. P. Krapcho’s research.group.
S
N(CH3)2
N
·HCl
(H3C)2N
N
H
N
S
N(CH3)2
48
H
N
N(CH3)2
N
(H3C)2N
H
N
·HCl
N
N
H
N(CH3)2
49
Figure 19. The proposed two new compounds.
The retrosynthesis of 48 and 49 is illustrated in Scheme 28.
51
S(CH2)2N(CH3)2
(H3C)2N(H2C)2HN
N
N
· HCl
· HCl
(H3C)2N(H2C)2HNH2C
N
N
S(CH2)2N(CH3)2
CH2NH(CH2)2N(CH3)2
(H3C)2N(H2C)2HN
49
48
Cl
Cl
(H3C)2N(H2C)2HNH2C
N
N
N
N
Cl
Cl
51
50
Cl
H3C
Cl
N
N
N
N
Cl
CHO
Cl
25
52
Scheme 28
52
CH=N(CH2)2N(CH3)2
The synthetic strategy was based on the oxidation of methyl group in 25 into
aldehyde 52, followed by nucleophilic attack of N,N-dimethylethylenediamine, which would
afford the imine (Schiff base) derivative 51, to be reduced to amine 50 through sodium boro
hydride. Compound 48 would result from the employment of sodium hydride in DMF to
remove HCl from key intermediate 50 and to form sulphur ion for the displacement of the
chloride. Modification of this final step by the introduction of N,N-dimethylethylene diamine
for the displacement of chlorides in 50, would lead to the analogue 49.
In order to assess the potential use of 25, initial investigations were performed on the
commercially available 5-methyl-1,10-phenanthroline 26.
Since oxidations of methyl at 2-, 4- 7- and 8- of the phenanthroline nucleus are know
in the literature,65,66 likewise it was supposed that methyl group at 5- position could easily
undergo oxidation to aldehyde.
A preliminary experiment was performed following a literature procedure, 67 using
SeO2 in dioxane with water (4%) under 3 hours reflux. After hot filtration through celite, it
was expected to observe crystals precipitating from the filtrate. The progress of this type of
reaction is generally followed by the appearance of a black or reddish selenium that coats the
walls of the flask.
In fact, while 4-methyl-1,10-phenanthroline on heating quickly deposited red Se(0),
even on long heating, 5-methyl-1,10-phenanthroline did not appear to react. Therefore the
initial assumption analysis proved that the position of the methyl clearly controls the rate of
oxidation and based on the initial result, it was supposed that by analogy, also 2,9-dichloro-5methyl-1,10-phenanthroline 25 would not react.
These conclusions turned out to be inaccurate, when a second attempt was carried
out in a manner similar to that reported by Baudoin et al.68, that is in hot naphthalene with
controlled amounts of selenium dioxide.
Such conditions were considered an improvement in the oxidation of aromatic
methyls as it was reported that the reaction would not proceed in the usual solvent mixture
dioxane/water. In accordance with what it was described, this approach afforded compound
53, which was also purified by column chromatography over alumina to provide reasonable
yields.
53
Another convenient method was also envisaged for the preparation of 53. The use of
SeO2 was reconsidered, under more drastic conditions, heating the mixture with 26 for two
hours at reflux and using o-dichlorobenzene as a solvent.69
The product of this reaction afforded a proton NMR spectrum comparable to the one
obtained in the previous experiment and showing the absorption expected for a typical
carboxaldehyde, a sharp singlet at δ 10.41.
N
H3C
N
i, ii
H
N
N
O
26
53
Reagents: (i) SeO2, naphtalene, ;
(ii) SeO2, o-DCB, .
Scheme 29
Based on these preliminary experiments, it was hoped that at least one of the
proposed routes could be applied also to derivative 25 with the intention of achieving product
52. The three proposed pathways were investigated and unexpectedly gave disappointing
results (Scheme 30).
54
Cl
i, ii, iii
N
H3C
Cl
N
H
N
N
O
Cl
Cl
52
25
Reagents: (i) SeO2, dioxane/H2O (96:4), ;
(ii) SeO2, naphtalene, ;
(iii) SeO2, o-DCB, .
Scheme 30
The first procedure involved the use of SeO2 in combination with dioxane/water,
under reflux and for the second one, SeO2 was used with naphthalene, still under reflux.
Evidence of this failure was proved by 1H NMR analysis, which in both cases showed a broad
peak at ~δ 10.5. For the peculiar shape, this peak looked more consistent with an -NH group
rather than with an aldehydic function. Therefore it was assumed the potential formation of a
complex with selenium, chelated by the two nitrogens of starting material.
In addition, the presence of the a peak at ~2.7 ppm proves that no oxidation took
place. Unlike the singlet relative to the -CH3 group belonging to 25, for both pathways, this
peak appeared as a multiplet structure, which may cast some doubts concerning the prospect
of recovering starting material. This formulation can be also proved by the presence of a
multiplet structure at δ 6.7 which is not consistent with structure 25. Similarly, no evidence of
product 52 was observed when SeO2 was used in combination with ortho-DCB at reflux.
Hence, conclusions seemed to favour the hypothesis that the presence of the chloro at
position 2- and 9- in the phenanthroline nucleus may control the rate of oxidation and may
also deactivate the aldehyde formation, at least when methyl group is in 5- position.
As a consequence to these inconclusive results, studies were geared towards different
strategic routes. It was envisioned that products 25 and 25a could still be considered as a key
compounds for the preparation of new derivatives, bypassing the synthesis of troublesome
aldehyde (Scheme 31).
55
58
57
59
Cl
Cl
Cl
N
N
N
O
HOOC
NC
N
Cl
Cl
Cl
49
N
N
(n = 0, from 57)
H
N
25a (X = H, from 49)
Cl
N(CH3)2
N
H
N
(H3C)2N
N
·HCl
(n)
N
N
N
H
49a
N(CH3)2
X
Cl
25 (X = CH3, from 49a)
(n = 1, from 54)
Cl
Cl
N
HOOC
Cl
N
NC
N
N
Br
N
Cl
N
Cl
54
55
Scheme 31
56
Cl
56
The retrosynthetic analysis of Scheme 31, describes two different strategies,
involving key derivatives 25 and 25a.
As a first hypothesis, target molecule 49a would be obtained from precursors 50 and
51, described earlier in Scheme 28. A straightforward reduction of carboxylic acid 54 would
afford the resulting imine 51, or alternatively previous reduction of 54 to aldehyde, followed
by addition of appropriate Schiff base, would lead to 51. Compound 56 could be deducted
from 25 by simple bromination, which could later be transformed to intermediate 55 via
cyano-de-halogenation (The Rosenmund-von Braun Reaction). Nitrile hydrolysis of 55 would
give carboxylic acid 54.
Another suggestion would be the acquisition of 49b from 57, by similarity with
compound 54. Product 57 would be afforded by hydrolysis of 58, obtained through ring
opening of 59 via SN2 mechanism. Phase transfer catalysis of 26 would give intermediate 59.
A trial experiment was commenced on derivative 25a for the conversion of the
methyl group into the bromomethyl analogue 56.
It’s widely known that methyl benzene and other substrates with methyl groups on
aromatic rings can be chlorinated or brominated. The bromine atom is much more selective
than the chlorine, as it can substitute tertiary and benzylic positions selectively. Fluorine has
also been used,70 but seldom, because it’s too reactive and hard to control.71 Besides, it often
breaks carbon chains down into smaller units, a side reaction that sometimes becomes
troublesome in chlorinations too. For this type of reaction, known as Wohl-Ziegler
bromination, by far, the most common reagent is the N-bromo amide NBS. To a much lesser
extent, chlorination can be carried out with NCS or tert-butyl hypochlorite.72 With any
reagent, a non polar solvent and an initiator is needed, which is usually a benzoyl peroxide or
UV light. Following a literature procedure,73 25 was treated with NBS, dibenzoyl peroxide
with light (Scheme 32). After work-up and purification by column chromatography, product
56 was obtained in 56% yield. 1H NMR gave evidence of the formation of the desired
compound, but further characterization was made difficult by the presence of unreacted
starting material, which was difficult to isolate by chromatography, since Rf values of 25 and
56 were similar.
57
Cl
N
H3C
Cl
N
i
Br
N
N
Cl
Cl
56
25
Reagents: (i) NBS, (C6H5CO)2O2, C6H6, h.
Scheme 32
Despite negative developments towards oxidation of methyl group to aldehyde, for a
simple investigation and to provide insight in the chemistry of phenanthroline, initially benzyl
halide 56 was taken to aldehyde following two pathways. This nitroparaffin salt is easy to
prepare and it is generally an efficient reagent that gives carbon-alkylation, starting from
certain benzyl halides (p-nitrobenzyl chloride, o-nitrobenzyl-chloride and 2,4-dinitrobenzylchloride) or oxygen alkylation from p-cyano-benzyl chloride or benzyl chloride. Data in the
literature on the reaction between nitroparaffin salts and halides other than benzyl halides,
indicate predominantly oxygen-alkilation.
This method is also a general procedure for the preparation of para-substituted
banzaldehyde, with the lone exception of p-nitrobenzaldehyde. This synthesis, in general is
comparable to the Sommelet reaction, where hexamethyltetramine is used, followed by water.
However although the former gives slightly higher yields, there are also some advantages
when sodium salt of 2-nitroporpane is involved. First, it was reported that substituted
benzaldehydes can be produced by halogenation, halomethylation, hydrohalogenation of the
appropriate compound, followed by treatment with sodium 2-propanenitronate. Second, this is
a convenient synthetic method to use when substances are sensitive to oxidative conditions.74
As a first experiment, starting material 56 was treated with sodium 2propanenitronate in ethanol, according to a literature procedure (Scheme 33).75
After stirring the mixture at room temperature for 48 hours, the precipitated sodium
bromide salt was filtered, while the filtrate was treated with water and extracted with ether.
The ethereal solution was washed with alkali to remove acetoxime and excess of
nitropropane, dried over magnesium sulphate, filtered and distilled off. Attempts to purify the
residue by column chromatography over alumina, were unsuccessful, as 1H NMR indicated
58
the presence of precursor 25, which was still interfering in the present reaction, although one
singlet at  10.3 may have been consistent with the aldehyde peak.
Cl
i,ii
N
Br
Cl
N
+
H
N
+
NaBr
N
O
Cl
NOH
Cl
56
52
Cl
N
iii
55
NC
N
Cl
Reagents: (i) [(CH3)2CNO2]Na, r.t., 48h;
(ii) [(CH3)2CNO2]Na, DMSO, r.t., 3h;
(iii) NaCN, EtOH, .
Scheme 33
In a similar fashion, a second pathway involved the same conditions outlined above,
but this time the procedure necessitated DMSO and 3 h stirring at room temperature. 76 It was
devised that for this type of reaction, additional solvent (DMSO or DMF) had to be employed.
In addition, it was envisioned that the anion of 2-nitropropane is the active species and that
ethanol probably prevents undesirable side reactions.
Unfortunately also under these conditions the reaction did not occur, although a
small peak at  10.3 may have revealed the beginning of a possible aldehyde formation.
Neither the conversion of 56 into cyanomethyl derivative 55 gave significant results.
One attempt was made introducing NaCN instead of KCN77 as a reagent and EtOH as a
solvent.
59
Three hours reflux followed by work up with water and dichloromethane afforded an
unidentified product, by 1H NMR analysis, which revealed a number of peaks that could not
be assigned to structure 55.
Since pursuing the straightforward methyl oxidation or bromination did not appear to
be much productive, the research was focused on the preparation of reactive precursor 1,10phenanthroline-5,6-epoxide and it was anticipated that the chemistry leading to this
derivative, could be applied also for the obtainment of analogue 59 from Scheme 31.
A versatile and preparative route was followed according to a literature procedure, 78
which presented a slight variation of the method by Krishnan et al.79.
It was reported that oxidation of 1 with commercial bleach (hypochlorite), in the
presence of a phase transfer catalyst,79,80 would give the 1-5,6-epoxide, 60, which is quite
versatile in the preparation of a variety of 5-substituted phenanthrolines, including cyano-,
hydroxyl-, dimethylamino-, aza-18-crown-6- and methoxy-(1).
Phase transfer catalysis is generally a convenient method for nucleophilic
substitutions. For this type of reactions the substrate is usually insoluble in water and other
polar solvents, while the nucleophile is often an anion, which is soluble in water but not in the
substrate or other organic solvents. Therefore, when the two reactants are brought together,
their concentrations in the same phase are too low for convenient reaction rates. One way to
overcome this problem is to use a solvent that is able to dissolve both species. One way could
be the use of dipolar aprotic solvent, another one could be the phase transfer catalysis. In the
former method, a catalyst has the function of carrying the nucleophile from the aqueous to the
organic phase. There are two principal types of phase transfer catalyst, whose effects are the
same: quaternary ammonium salts or phosphonium salts and crown ethers or other
cryptands.81
For the synthesis shown in Scheme 34, tetrabutylammonium hydrogen sulphate and
1 dissolved in CHCl3, was added to a stirred mixture of bleach and water, carefully adjusted
to pH 8.5 with conc. HCl. Despite literature reports a reaction period that may vary from 15
minutes up to 24 hours, in the present experiment it was proved that starting material had not
completely reacted.
60
Observations based on 1H NMR analysis led to the conclusion that according to peak
areas, the expected compound 60 and starting material were present approximately in 42%
and 58% ratio respectively. When the same procedure was also adapted to the dichloro
derivative 25a, it was noted that an extended time period (48 hrs) was not changing the course
of reaction. Even in this case, starting material was the only recovered product.
N
i
N
O
N
+
N
N
1
60
58%)
N
1
42%)
Reagents: (i) TBAHSO4, bleach, pH = 8.5, 18°C.
Scheme 34
At this state of the investigations, the proposed preparative routes to analogues 49a,b
were questionable. Thus, the attention was directed towards exploring the possible
functionalization at position 5- of dichloro derivative 25a.
The literature reports direct nitration of 1,10-phenanthroline or its monohydrate,82,83
describing also the concurrent formation of side product 1-5,6-dione, whose colorless iron(II)
complex was of some interest at the time of its discovery. The most common combination
reagent is a mixture of concentrated nitric and sulphuric acid. By analogy with the synthesis
of 5-nitro-neocuproine, described by Gallagher and co-workers73, it was assumed that also
derivative 25a could undergo nitration at 5- position. In fact, the expected novel compound 61
was obtained, as outlined in Scheme 35:
61
Cl
N
Cl
N
i
O2N
N
N
Cl
Cl
25a
61
Reagents: HNO3/H2SO4, 
Scheme 35
Treatment of 25a in a mixture of HNO3/H2SO4 refluxed for one hour, followed by
work up with 2M NaOH, led to a solid, subsequently purified by column chromatography,
which afforded the corresponding 2,9-dichloro-5-nitro-1,10-phenanthroline (61, 43%). It
might also be noted that the low yield could be ascribed to the possible formation of
unidentified secondary oxidation products.
Consequently, this successful outcome was subjected to further evaluation, which
provided a set of other interesting molecules. It was estimated that product 61 could be a key
compound in the formation of a new functionalized macrocycle. In addition, an alternative
route to the acquisition of 61 was designed (Scheme 36).
As outlined in the following retrosynthetic pathway, it was hypothesized that
derivative 61 could be afforded from its precursor 64, the commercially available 5-nitro1,10-phenanthroline, according to the same route reported in the aforementioned Scheme 10.
In a similar fashion, also the acetylated 61a was supposed to be achieved from 66.
The former, could be deducted from the acetylation of 65, accessible by reduction of nitro
derivative 64.
After the obtainment of 61 and 61a, it was observed that inevitably, a condensation
with 32 would necessitate to be explored.
62
Cl
O
N
N
N
N
2Br
X
N
X
N
Cl
X
for a:
62 (X = NO2)
62a
63 (X = NO2)
63a
NHCOCH3
66
S
Cl
N
N
N
S
67 (X = NO2)
67a
N
X
S
+
N
HN
HN
Cl
S
61 (X = NO2)
61a
Scheme 36
63
32
64
N
N
X = -NHCOCH3
N
X
N
O
61 (X = NO2)
61a
N
O2N
N
N
NH2
65
Initially, compound 64 was subjected to preliminary experiments (Scheme 37).
Its first transformation into the bis-quaternary salt 63, using 1,3-dibromopropane
resulted in failure as presumably the nitro group was deactivating the salt formation. Instead,
it was performed in 63% yield after an extended time period of reflux and with the addition of
acetonitrile as a solvent. Reduction of 64 was accomplished using 10% Pd/C and hydrazine,84
leading to product 65 in 69% yield. Two procedures were followed, in order to acetylate
compound 65. A first experiment was carried out adapting a literature method by Yoshida et
al.,85 using acetic anhydride in dry benzene at room temperature, which afforded 66 in 42%.
The yield was significantly improved (85%) when benzene was replaced by pyridine. 86
A final investigation on compound 65 was directed to the modification of 5-amino
derivative with a different tether. According to Chen and co-workers87 under standard
conditions, compound 66a was accomplished using iodoacetic acid and carbodiimide
coupling reagent DCC.
N
O2N
N
i
N
·2Br
O2N
N
64
63
ii
N
H2N
N
iii, iv
N
X
65
from iii: 66
from iv: 66a
Reagents: (i) Br(CH2)3Br, CH3CN, ;
(ii) 10% Pd/C, NH2-NH2, EtOH, ;
(iii) Ac2O, C6H5N or C6H6, EtOH, ;
(iv) ICH2COOH, DCC, AcOEt, CH3CN, r.t.
Scheme 37
64
N
X = -NHCOCH3
X = -NHCOCH2I
The procedures described for products 66 and 66a, will be later undertaken on the
dichloro derivative too.
At the same time the effective condensation of 63 with 32 was experimented, using
diphenilether with heating (Scheme 38). From this reaction a brownish/yellow solid was
afforded, which was subjected to 1H, 13C NMR, elemental analysis and MS.
At present all the collected data are insufficient to assess the possible formation of
67. It was envisioned that purification will be later attempted in order to be able to extrapolate
significant conclusions.
Cl
S
S
N
HN
DPE, 
N
N
N
N
+
O2N
N
Cl
61
O2N
HN
?
S
32
S
67
Scheme 38
65
Rational Design, Synthesis and Biological
Evaluation of Bis(pyrimido[5,6,1-de]acridines) and
Bis(pyrazolo[3,4,5-kl]acridine-5-carboxamides) as
Potential Topoisomerase Inhibitors
Introduction to the Chemistry
Over the past years, interest in symmetric bifunctional intercalators has been growing
and a number of derivatives employing different chromophores have been studied
exstensively.88 The noticeable results in the field may be exemplified by LU 79553 (68,
Figure 20) and WMC-26 (69, Figure 20), both showing high effectiveness against tumor
xenografts in vivo.88f,g
O
H
N
N
O
O
H
68
N
N
N
O
H
N
N
O
N
O
N
69
N
H
N
Figure 20. Structures of LU 79553 (68) and WMC-26 (69).
66
Herein is described the synthesis and biological properties of two novel interesting
classes of antitumor agents, belonging to this type of derivatives: the bis(acridine-4carboxamides) 70 and 71 (Figure 18).89 Among them, 70a [X = H, Y = (CH2)3N(Me)(CH2)3],
70d [X = NO2, Y = (CH2)3N(Me)(CH2)3], 71a [X = H, Y = (CH2)3N(Me)(CH2)3], and 71b [X
= OMe, Y = (CH2)3N(Me)(CH2)3] emerged as lead derivatives.89 Compounds 70a,d and 71a,b
have fulfilled the purpose of enhancing the outstanding biological response shown by the
corresponding bis-functionalized acridone-4-carboxamide monomers.90 Prompted by the
above results, the synthesis of two new classes of potential bis intercalators was designed: the
bis(pyrimidoacridines) 72 and the bis(pyrazoloacridinecarboxamides) 73, which can be
regarded as cyclized derivatives of 70 and 71, respectively (Figure 18). On the other hand, the
chromophore moiety of 72 and 73 being constituted by the pyrimido[5,6,1-de]acridine-1,3,7trione and by the pyrazolo[3,4,5-kl]acridine, respectively (Figure 18), were expected to show
an increase of the distinguished antitumor properties relatively to the corresponding
monomers 7491 and 75.92 Finally, it was extensively investigated the relevance of the linker Y
for the biological activity of these compounds.
67
O
HN
Y
NH
O
X
N
H
HN
70 O
O
O
N
H
NH
N
N
X
NH
HN
X
X
71
N
H
N
H
N
N
O
HN
Y
NH
O
NH
O
X
O
72
N
O
N
N
O
N
HN
Y
O
N
N
X
N
N
N
X
N
X
73
O
N
H
N
H
N
O
N
O
NH
N
N
N
N
HN
Y
O
N
HN
X
O
O
O
74a (X = H)
74b (X=9-OMe)
74c (X=9,10-OMe)
74d (X=9-OH)
74e (X=9-NO2)
N
75a (X = H)
75b (X=9-OMe)
75c (X=9-OH)
X
N
H
O
N
N
H
N
Figure 21. Rational design of 72 and 73 which can be regarded either as cyclized derivatives
of the bis(acridine-4-carboxamides) 70 and 71 or as bis derivatives of the corresponding
monomers 74 and 75.
68
Chemistry
Schemes 40 and 41 show the synthetic pathways leading to target derivatives 72 and
73.
According
to
Scheme
40,
reaction
of
the
proper
6-chloro-2-[2-
(dimethylamino)ethyl]pyrimido[5,6,1-de]acridine-1,3,7-trione (74)91a,b with the appropriate
,diamine in ethoxyethanol, in the presence of triethylamine at 80°C, afforded the target
bis(pyrimidoacridines) 72aj. All the diamines were commercially available, except the
N1,N2-bis(2-aminoethyl)-N1,N2-dimethyl-1,2-ethanediamine, necessary for 72g, which was
prepared according to the literature.88e Cleavage of the methoxy derivatives 72h,i with
aqueous HBr gave the hydroxy derivatives 72k,l, respectively. Target compound 72m could
not be prepared in the same way, due to the difficulty for the achievement of 9-nitro
derivative of 74.91b However, direct cyclization of 71b, performed in CHCl3 with COCl2 and
triethylamine at room temperature, afforded 72m.
As outlined in Scheme 41, the bis(pyrazolo[3,4,5-kl]acridinecarboxamides) 73ad
were prepared by reaction of the appropriate bis(acridine-4-carboxamides) 76ad89 with [(2hydrazino)ethyl]dimethylamine in 2-ethoxyethanol at 120°C. The hydroxy derivatives 73e,f
were obtained by refluxing the corresponding methoxy derivatives 73c,d in aqueous HBr.
All the target compounds 72 and 73 were examined as water-soluble hydrochloride
salts, afforded with the usual methods, according to their DNA-binding properties and their
antineoplastic activity.
69
O
Cl
O
X
HN
Y
NH
O
O
X
X
N
N
O
O
HN
N
N
74f (X = H)
74g (X=9-OMe)
74h (X=9,10-OMe)
O
NH
O
OH
O
N
N
(from 72h,i)
O
O
N
N
NH
N
N
O
O
N
N
N
O
O
HN
N
N
NH
O
O2N
NO2
NO2
iii
O
N
71b
O
N
N
H
NH
O
N
N
O
72m
O
N
N
N
a
O
72k,l
72a-j
O2N
N
H
HN
O
ii
N
N
Y
HO
i
O
HN
Reagents: (i) H2N-Y-NH2, N(Et)3; (ii) HBr 48%; (iii) COCl2, N(Et)3. Linker Y: (CH2)3N(Me)(CH2)3 for 72a,h,j
(CH2)2N(Me)(CH2)2 for 72b,i,l; (CH2)3 for 72c; (CH2)6 for 72d; (CH2)8 for 72e; (CH2)12 for 72f;
(CH2)2N(Me)(CH2)2N(Me)(CH2)2 for 72g. Substituents X: H for 72a-g; 9,9'-OCH3 for 72h,i;
9,9',10,10'-OCH3 for 72j.
Scheme 40a
70
O
N
O
Cl
Cl
O
N
X
X
N
H
i
N
H
O
N Y N
H
H
O
N
N
N
N
X
X
N
H
N Y N
H
H
a
O
71
N
N
N
OH
HO
73a-d
Reagents: (i) H2N-NH-(CH2)2N(CH3)2; (ii) HBr 48%. Linker Y:
(CH2)3N(Me)(CH2)3 for 73a,c,e; (CH2)2N(Me)(CH2)2 for 73b,d,f.
Substituents X: H for 73a,b; 9,9'-OMe for 73c,d.
Scheme 41a
N
ii
(from c,d)
N
H
O
76a-d
N
N
N
N
H
N
H
O
N Y N
H
H
O
73e,f
Results and Discussion
DNA-Binding Properties
As shown in Table 1, competitive displacement (C50) fluorometric assays93 with
DNA-bound ethidium was used (a) to determine “apparent” equilibrium constants (Kapp) for
drug binding, as the C50 value is approximately in inverse proportion to the binding
constant,94 and (b) to establish possible base- or sequence-preferential binding.95 In the
present study, fluorescence displacement assays were performed at pH 7 to enable comparison
in biological conditions.
The Kapp values of the new derivatives 72am and 73af, of related bis 70 and 71,
and of corresponding monomers 74 and 75, with CT-DNA, AT, and GC are reported in Table
1. The results indicate that target compounds possess excellent DNA affinity, generally higher
than ethidium, but lower than mitoxantrone (Mx). Some observations can be made about the
CT-DNA Kapp values. (a) With regards to the linker Y: in the unsubstituted sub series 72aj,
we can observe the effect of the linker on binding; many Kapp values are in the range 23 
107, indicating that the nature of the linker is not decisive for binding; anyway, the weakest
ligand is 72f (Y = (CH2)12), with a Kapp value of 0.36  107, indicating that the length and
flexibility of the linker are detrimental for DNA binding; the best results are obtained with
72a,g (Y = (CH2)3N(CH3)(CH2)3 and Y = (CH2)2N(CH3)(CH2)2, respectively), with
corresponding Kapp values of 7.1  107 and 15  107, suggesting that basic nitrogen atoms,
protonated at pH 7, may increment the binding potency; considering the homologue pairs (Y
= (CH2)3N(CH3)(CH2)3 and Y = (CH2)2N(CH3)(CH2)2, respectively), 72a is 3.4 times more
efficient than 72b, and the same trend is confirmed for 72h,i, 72k,l, and 73c,d; for the pairs
73a,b and 73e,f, the behavior is opposite. (b) Regarding the substituents X: in series 72 the
rank in binding potency, for derivatives with Y = (CH2)3N(CH3)(CH2)3, is 9,9'NO2 >
9,9'OH > 9,9'H >9,9',10,10'OMe > 9,9'OMe, and, similarly, for derivatives with Y =
(CH2)2N(CH3)(CH2)2, it is 9,9'OH > 9,9'H > 9,9'OMe; in series 73 the behavior is
different; for derivatives with Y = (CH2)3N(CH3)(CH2)3, the rank is 9,9'H > 9,9'OMe >
72
9,9'OH, whereas, for derivatives with Y = (CH2)2N(CH3)(CH2)2, the rank is 9,9'H >
9,9'OH > 9,9'OMe. (c) With regards to the chromophore: comparing the analogue pairs
72a,73a, 72b,73b, 72i,73d, and 72l,73f, the pyrazoloacridine chromophore seems more
efficient than the pyrimidoacridone chromophore; only in the analogue pair 72k,73e we have
an opposite trend. (d) Concerning a comparison with monomers: the bis derivatives 72 and
73, with Y = (CH2)3N(CH3)(CH2)3, result always more efficient than corresponding
monomers 74 and 75, with the only exception of 73c and 75b. (e) Comparing related
bis(acridine-4-carboxamides): the new bis derivatives 72 and 73 result always less efficient
than related bis(acridine-4-carboxamides) 70 and 71, with the only exception of 73a and 71a.
Generally, the binding behavior of target compounds with synthetic polynucleotides
reflects what it was observed for CT-DNA. However, in some cases, a clear, remarkable,
preference for binding to AT rich duplexes is to be noted in both series 72 and 73. On the
contrary, compounds related to bis derivatives 72 have previously shown a strong GC
preference (monomers 74) or a borderline AT preference (dimers 70), whereas compounds 73
show a binding site preference very similar to that of related bis derivatives 71 and monomers
75.
73
Table 1. Melting Points,a Yields, Formula,b DNA Binding,c and Cytotoxic Activity against Human Colon Adenocarcinoma (HT29) of Target
Compounds 72am and 73af, of Related Bis(acridine-4-carboxamides) 70 and 71, of Corresponding Monomers 74 and 75, and of
Mitoxantrone (Mx).
Kappd  10-7 M-1
IC50 (nM) f
AT
CTDNA
GC
binding site
preference e
126
7.1
5.0
AT (25)
<0.1
70ah
9.3
10
7.5
none
0.43
74ai
0.68
1.7
1.2
GC (0.57)
22
compd
72a
mp, °C
yield, %
formula
143-144 (259-260 d)
59
C45H49N9O6
HT29
72b
183-184 (275-277 d)
60
C43H45N9O6
4.0
2.1
0.94
AT (4.3)
110
72c
222-224 (273-274)
50
C41H40N8O6
2.5
2.8
0.28
AT (8.9)
390
72d
275-277 (239-241)
66
C44H46N8O6
8.2
3.3
0.27
AT (30)
43
72e
242-244 (185-187)
64
C46H50N8O6
6.9
3.6
0.17
AT (41)
360
72f
187-189 (254-256)
81
C50H58N8O6
0.49
0.36
0.041
AT (12)
3500
72g
182-184 (> 350)
29
C46H52N10O6
31
15
0.37
AT (84)
390
72h
183-185 (258-259 d)
44
C47H53N9O8
7.1
2.1
3.4
AT (2.1)
<0.1
70bh
26
6.2
14
AT (1.9)
39
74bi
0.73
1.3
3.3
GC (0.22)
67
72i
200-202 (253-254 d)
48
C45H49N9O8
0.90
0.34
0.88
none
720
72j
74ci
190-192 (240-242 d)
30
C49H57N9O10
11
6.2
2.4
AT (4.6)
250
0.40
1.5
3.2
GC (0.12)
370
72k
263-264 (265-267 d)
95
C45H49N9O8
13
7.8
AT
(1.7)
<0.1
70ch
263-264 (265-267 d)
95
C45H49N9O8
13
7.8
AT (1.7)
<0.1
74
8.5
8.5
74di
72l
0.79
3.5
3.2
GC (0.25)
22
279-280 (270-272 d)
97
C43H45N9O8
3.9
3.7
0.43
AT (9.1)
390
148-149 (262-263)
59
C45H47N11O10
26
14
11
AT (2.4)
<0.1
70dh
19
14
23
none
<0.1
74ei
0.31
0.79
2.6
GC (0.12)
310
17
10
8.2
AT (2.1)
3.0
71ah
8.2
6.3
0.79
AT (10)
57
75aj
2.5
4.9
0.73
AT (3.4)
210
72m
(> 350)g
73a
74
C43H51N11O2 · 3HCl · 3H2O
73b
(230-231)g
34
C41H47N11O2 · 3HCl · 2H2O
20
23
5.2
AT (4.3)
360
73c
(204-205)g
67
C45H55N11O4 · 3HCl · 3H2O
9.9
6.3
1.8
AT (5.5)
3.0
71bh
13
20
5.2
AT (2.5)
2.0
75bj
4.1
17.5
2.6
AT (1.6)
120
73d
(245-247)g
60
C43H51N11O4 · 3HCl · 2H2O
5.0
1.7
1.8
AT (2.8)
150
73e
239-240 (260-261)
40
C43H51N11O4 · 3HCl · 3H2O
25
4.3
19
AT (1.3)
780
71ch
13
4.4
8.3
AT (1.6)
800
75cj
9.7
3.4
1.9
AT (5.1)
600
8.5
4.9
3.0
AT (2.8)
> 10000
73f
282-283 (255-257)
39
C41H47N11O4 · 3HCl · 2H2O
Mx
34
a
10
In parentheses hydrochlorides melting points, d = decomposition. bAnalyses for C, H, and N. cCT-DNA, AT, and GC refer to calf
thymus DNA, [poly(dAdT)]2, and [poly(dGdC)]2, respectively. dKapp = 1.26/C50  107 in which 1.26 is the concentration (M) of ethidium
in ethidium-DNA
75
Table 2. Cytotoxic Screening against Six Human Cancer Cell Lines of Selected Compounds 72 and 73 after 1h and 144 h of Drug Exposure.a
H460M
1h
144h
MKN45
1h
144h
PC3
1h
144h
HCT116
1h
144h
LoVo
1h
144h
LoVo/Dxb
1h
144h
72a
< 10-3
< 10-3
0.011
< 10-3
4.1
1.6
0.72
0.10
5.2
0.31
11 (2.1)
3.1 (10)
72h
0.066
< 10-3
0.057
0.0028
4.7
1.2
0.85
0.66
18
0.95
76 (4.2)
7.6 (8.0)
72k
< 10-3
< 10-3
< 10-3
< 10-3
2.0
0.011
0.060
< 10-3
98
3.9
810 (8.3)
59 (15)
72m
0.93
0.19
1.3
0.29
11
4.5
2.9
1.1
42
7.6
380 (9.0)
67 (8.8)
73a
7.2
4.4
6.8
0.60
12
6.0
12
19
150
1.1
3600 (24)
400 (360)
73c
26
4.3
51
13
> 100
39
120
43
53
5.1
3100 (58)
290 (57)
Mx
0.85
0.75
6.8
12
75
7.0
110
5.8
12
3.3
810
22
9100 (11)
2300 (100)
Dx
a
Activity expressed as IC50 (nM). bIn parentheses RI = resistance index is the IC50 ratio of LoVo/Dx on LoVo.
76
Cytotoxic Activity
The target compounds 72am and 73af, the related bis 70 and 71, the
corresponding monomers 74 and 75, and the reference drug mitoxantrone (Mx) were
examined for antiproliferative activity against the human colon adenocarcinoma cell line
(HT29). The results shown in Table 1 indicate that compounds 72a,h,k,m and 73a,c possess
very potent antiproliferative activity, with IC50 values in the low/sub nM range, being
remarkably more potent than Mx itself.
The following remarks can be made:
(i) Regarding the linker: (a) In the unsubstituted (X = H) sub series 72ag, with
seven different linkers, 72a (Y = (CH2)3N(CH3)(CH2)3 appears to be the most potent
derivative, with IC50 value < 0.1 nM (at least 3 order of magnitude lowest in the sub series),
but also 72d (Y = (CH2)6) is very active (IC50 43 nM); on the opposite site is 72f (Y =
(CH2)12) with the highest IC50 (3.5 M); the other derivatives possess similar activity (IC50
range 0.11-0.39 M). It seems that compounds 72a and 72d (Y = (CH2)3N(CH3)(CH2)3 and Y
= (CH2)12, respectively ensure an optimal linker length, but also the presence of a basic
nitrogen atom, compound 72a, is important in terms of cytotoxicity. (b) Comparison of the
homologous pairs (72a,b, 72h,i, 72k,l, 73a,b, 73c,d, and 73e,f) clearly indicates that the best
results are always obtained with Y = (CH2)3N(CH3)(CH2)3 for both series 72 and 73,
according to what is observed for related bis derivatives 70 and 71.89 The difference in
potency is 3 order of magnitude, at least, in series 72 and 2 order of magnitude in series 73 for
derivatives with Y = (CH2)3N(CH3)(CH2)3.
(ii) Regarding the substituents X: (a) In the series 73 the cytotoxicity rank order of
derivatives with the same linker is 73c,d (X = 9,9'OMe)  73a,b (X = H) >> 73e,f (X =
9,9'OH). (b) The nature of substituents in 9,9' positions in the series 72 does not influence
the activity significantly. In the sub series with Y = (CH2)3N(CH3)(CH2)3, all derivatives
72a,h,k,m, possess very potent cytotoxicity (IC50 < 0.1 nM); in the subseries with Y =
(CH2)2N(CH3)(CH2)2, derivatives 72b,i,l, possess IC50 in the range 0.110.72 M, with a
weak influence showing the following rank order 72b (X = H) > 72l (X = OH) > 72i (X =
OMe). (c) The only derivative with substituents in 9,9',10,10' positions, 72j (Y =
(CH2)3N(CH3)(CH2)3, X = 9,9',10,10'OMe), possesses IC50 = 0.25 M, indicating that this
kind of substitution is not detrimental for binding, but greatly diminishes cytotoxic potency.
77
(iii) Regarding the chromophore: Comparing IC50 values of the pairs with Y =
(CH2)3N(CH3)(CH2)3 and the same X, 72a,73a, 72h,73c, and 72k,73e, it can be underlined
that series 72 (pyrimido[5,6,1-de]acridine chromophore) is much more cytotoxic than series 6
(pyrazolo[3,4,5-kl]acridine chromophore), especially considering the pair 72k,73e. The results
are in agreement with what it is observed with the corresponding monomers 74and 75.
(iv) Finally, it should be noted that the target derivatives with Y =
(CH2)3N(CH3)(CH2)3, 72a, 72h, 72j, 72k, 72m, 73a, 73c, and 73e, are all more potent
cytotoxic agents than the corresponding monomers 74 and 75; they are more potent than or
equally potent to related bis derivatives 70 and 71.
Generally, there is not a great correlation between IC50 and Kapp values. However,
some considerations can be made: (a) In the homologous pairs of series 72, the linker Y =
(CH2)2N(CH3)(CH2)2 always corresponds to an inferior cytotoxicity and DNA affinity with
respect to the linker Y = (CH2)3N(CH3)(CH2)3. (b) Also in the homologous pairs of series 73,
the shortest linker, Y = (CH2)2N(CH3)(CH2)2, always corresponds to an inferior cytotoxicity
with respect to the longest linker, Y = (CH2)3N(CH3)(CH2)3, but for the DNA affinity the
trend is different. (c) Compounds 72f,I, the weakest DNA ligands, along with 72g, 73b, the
most potent DNA ligands are scarcely cytotoxic; it can be deduced that not only DNA binding
is determinant for cytotoxic activity; other factors, e.g. cellular uptake, may influence
cytotoxicity.
Compounds 72a,h,k,m, and 73a,c, the most potent in the series, were selected for a
cytotoxic screening against six human cancer cell lines (large cell lung carcinoma H460M,
gastric cancer MKN45, prostatic carcinoma PC3, colon adenocarcinoma HCT116, LoVo,
sensitive, and LoVo/Dx, doxorubicin-resistant). The IC50 (nM) values after 1 and 144 h drug
exposure are reported in Table 2. Reference drugs are Mx and doxorubicin (Dx). From the
results the following considerations can be described: (i) The target derivatives are extremely
potent cytoxic agents, especially compounds 72, which often presents IC50 values inferior to
the minimum drug concentration tested (10-3 nM). (ii) As previously noted with the HT29 cell
line, compounds 72 are more potent than compounds 73, but here we can also discriminate
among the potency of selected derivatives 72, according to the nature of substituents in 9,9'
positions. The rank order in potency seems to be 72k (X = OH) > 72a (X = H) > 72h (X =
OMe) > 72m (X = NO2). (iii) Finally, it can be pointed out that both derivatives 72 and 73 are
cross resistant with Dx, but the grade of cross resistance of compounds 72 appears to be
inferior to that of derivatives 73.
78
Compounds 72a,h,k,m, and 73a,c, were also selected by the National Cancer
Institute (NCI) for a screening on a panel of 60 human tumor cell lines. This screen is
designed to discover spectrum of activity and, eventually, drugs selectivity. The data from this
assay can be presented in several different formats.96 Since it is not practical to report all
experimental data available, we choose to describe in Table 3, in one of the possible formats,
the antiproliferative activity of selected compounds against one cell line of each NCI sub
panel and the mean of the activity on these nine cell lines. Therefore, for each compound, the
percentage growth of the cell lines exposed for 48h at three different increasing drug
concentrations (10-8, 10-6, and 10-5 M, respectively) has been reported. Positive values
represent the percentage growth of each cell line with respect to the nontreated control (100%
growth) and give an idea of the cytostatic action. Negative values represent the percentage of
cell deaths with respect to the initial number and give an idea of the derivative’s cell killing
capacity. The data at 10 nM show that all compounds possess a strong cytostatic action, about
75% average of cell growth inhibition, except 73c, which however has an average of 49%;
compounds 72 demonstrate very similar cytostatic potency and are shown to be more
cytostatic than compounds 73. At 10-6 M not only is 100% of cellular growth inhibition
achieved but also a significant reduction of the initial cell number often occurs (cell killing):
the activity rank order is 72m > 72a > 72k > 72h > 73a = 73c. At 10-5 M a very marked cell
killing capacity can be noted: the activity rank order is 72a >> 72h > 72m >> 72k  73a 
73c. It is worth noting the potency and the broad spectrum of activity of all selected
compounds.
79
Table 3. Percent Growth of Some NCI Cell Lines Exposed for 48 h at Three Increasing Concentrations (10-8, 10-6, and 10-5 M) of Selected
Compounds.a
cell line
72a
10-8 10-6
10-5
72h
72k
10-8 10-6 10-5 10-8 10-6
10-5
72m
72a
73c
10-8 10-6 10-5 10-8 10-6 10-5 10-8 10-6 10-5
leukemia: MOLT-4
9
-26
-71
6
-33
-62
-4
-37
-60
-2
-31
-50
28
7
-27
19
-28
-28
lung-NSC: NCI-H460
13
-22
-95
14
-20
-87
11
-23
-60
10
-36
-60
12
-6
-53
23
3
-58
colon: SW-620
26
-25
-89
30
-2
-49
36
10
-8
42
5
-16
37
14
-45
44
3
-39
CNS: SNB-19
32
7
-53
38
14
-48
34
-4
-17
37
-25
-66
40
-7
-31
47
0
-37
melanoma: LOX IMVI
25
-43
-82
26
-34
-91
20
-50
-77
16
-56
-69
22
5
-57
46
3
-80
ovarian: OVCAR-4
15
-53
-77
0
-37
-72
42
-37
-36
31
-54
-65
41
-17
-40
71
-5
-54
renal: 786-0
34
4
-100
37
10
-79
36
1
-52
33
-4
-63
30
5
-48
53
11
-33
prostate: DU-145
37
-13
-98
40
3
-66
32
-6
-42
35
-13
-83
25
-23
-40
70
-8
-21
breast: MCF7
31
0
-96
39
-3
-63
16
-1
-32
21
-2
-55
18
2
-35
66
7
-18
mean of the cell lines
25
-19
-85
26
-11
-69
25
-16
-43
25
-24
-59
28
-2
-42
49
-2
-41
a
The negative values indicate the percent of cells killed.
80
Hollow Fiber Assay for Preliminary in Vivo Testing.96
On the basis of these results, derivative 73a was selected for preliminary in vivo
testing. In this assay human tumor cells are cultivated in hollow fibers, which are implanted in
mice. Some mice are treated with tested compound at various concentrations, whereas the
control mice receive only the compound diluent. The fiber cultures are collected on the day
following the last day of treatment, and the effect of antitumor activity of the tested
compounds is calculated on the basis of the net increase in the tumor cell mass. Thus, the
cytostatic and cytocidal capacities of the test compound can be assessed. The compound was
tested against 12 human cancer cell lines. This represents a total of 4 experiments since each
experiment contains three cell lines. The data are reported as %T/C for each of the two
compound doses against each of the cell lines with separate values calculated for the
intraperitoneal and subcutaneous samples.
A compound is selected for further in vivo testing in standard subcutaneous
xenograft models on the basis of several hollow fiber assay criteria. These include: (a) a %
T/C of 50 or less in 10 of the 48 possible test combinations (12 cell lines  2 sites  2
compound doses); (b) activity at a distance (intraperitoneal drug/subcutaneous culture) in a
minimum of 4 of the 24 possible combinations; and/or (c) a net cell kill of one or more cell
lines in either implant site. To simplify evaluation, a points system has been adopted which
allows rapid viewing of the activity of a given compound. For this, a value of 2 is assigned for
each compound dose which results in a 50% or greater reduction in viable cell mass. The
intraperitoneal and subcutaneous samples are scored separately so that criteria (a) and (b) can
be evaluated. A compound with a combined ip+sc score  20, a sc score  8 or a net cell kill
of one or more cell lines is referred for xenograft testing.
In Table 4 ip score, sc score, ip + sc score, and net cell kill for compound 73a are
presented. It can be seen that 73a largely exceed the limits of the combined IP+SC score  20
and of the SC score  8 indicating an interesting preliminary in vivo activity worthy of in vivo
testing in standard subcutaneous xenograft models.
81
Table 4. Results of the in Vivo Hollow
Fiber Assay for 73a.a
ip score
40
sc score
10
total score
50
cell kill
N
Ip = intraperitoneal implants, sc =
subcutaneous implants, total score = ip+sc
score, N = no cell kill.
a
82
Synthesis and Biological Evaluation of Indazolo[4,3bc][1,5]naphthyridines(10-Aza-pyrazolo[3,4,5kl]acridines) as Potential Topoisomerase Inhibitors
Introduction to the Chemistry
Within the class of DNA-intercalating anticancer drugs, structurally characterized
by the presence of a planar or semi-planar chromophore portion, possibly capable of
intercalation into DNA, the acridine and the 9-acridone chromophores play an important
role.97,98 Often, the acridine/acridone ring system is fused with a five or six-membered
heterocyclic ring to yield interesting polycyclic derivatives.97,98 Among them, a noticeable
example is represented by the 5-nitropyrazolo[3,4,5-kl]acridines (78, Figure 22).99 The 2[3-(dimethylamino)propyl]-9-methoxy-5-nitro-2,6-dihydropyrazolo[3,4,5-kl]acridine
(PZA; 77a: R = CH2N(CH3)2, X = OMe) combines the DNA binding properties of acridine
with the reducible nitro group, responsible for hypoxic cell selectivity.
Recent studies suggest that PZA might be a dual inhibitor of DNA topoisomerase
I and DNA topoisomerase II that exerts its effects by diminishing the formation of
topoisomerase-DNA
adducts.
Moreover,
PZA-mediated
DNA
fragmentatiom,
accompanied by induction of very high molecular weight DNA fragments (0.5 to 1 Mb)
was detected by pulsed-field gel electrophoresis in human breast cancer cells100, and bone
marrow mononuclear cells from metastatic colorectal cancer patients.101 Consistent with
these mechanisms of action, PZA exhibits broad spectrum antitumor activity in preclinical
models in vivo. In addition, this agent displays several unique properties including solid
tumor selectivity, activity against hypoxic cells, and cytotoxicity in noncycling cells. PZA
also retains full activity against cells that are resistant to other agents on the basis of
overexpression of P-glycoprotein or the multidrug resistance-associated protein (MRP) and
with loss of p53 function.102 Moreover, recently, the combination of PZA with
conventional anticancer agents such as doxorubicin, etoposide, and topotecan increased the
cytotoxicity against drug-resistant tumor cells.103
83
PZA has been studied in phase I trials in adults and children, and is currently
undergoing broad phase II trials.104 These phase II studies of PZA in patients with previously
treated colorectal cancer, pancreatic cancer, transitional cancer of the bladder, germ cell and
renal cancer have shown no objective responses, whereas myelosuppression was prominent.
In contrast, evidence of clinical activity has been seen in platinum-sensitive ovarian cancer
and hormone refractory prostate cancer.
In the former case, 1 out of 17 patients with androgen-independent prostate cancer
shows a 96% decrease in the prostatic specific antigen level (PSA) accompanied by an
improvement of bone scan.105
1
N
N
10
X
2
1
R
2
N
10
O
9
R
N
N
9
6N
H
6N
5
H
NO2
5
NO2
78e-f
77
Figure 22. Structures and ring numbering of parent 5nitropyrazolo[3,4,5-kl]acridines 77 and of target 5nitroindazolo[4,3-bc][1,5]naphthyridines
(5-nitro-10-
aza-pyrazolo[3,4,5-kl]acridines) 78ef.
In the continuous search of new classes of anticancer agents, it was decided to
further investigate a novel chromophore represented by an aza-acridine skeleton fused with a
five-membered
heterocyclic
ring
to
form
the
new
heterocycle
indazolo[4,3-
bc][1,5]naphthyridine. As shown in Figure 19, the target 2-[-(alkylamino)alkyl]-9-methoxy5-nitro-2,6-dihydroindazolo[4,3-bc][1,5]naphthyridines (2-[-(alkylamino)alkyl]-9-methoxy5-nitro-2,6-dihydro-10-aza-pyrazolo[3,4,5-kl]acridines)
(78af)
represent
the
10-aza
analogues of the parent 5-nitropyrazolo[3,4,5-kl]acridines 77. Prompted by the above
rationale, compounds 78af were synthesized in order to investigate some biologically
relevant aspects such as (i) DNA-binding properties, (ii) cytotoxic activity, (iii) apoptotic cell
death, and (iv) DNA fragmentation.
84
Chemistry
Scheme 42 shows the synthetic pathway leading to target derivatives 78af. Thus,
the new 6-chloro-2-[(6-methoxy-3-pyridyl)amino]-3-nitrobenzoic acid (81) was obtained by
heating at 75 °C the 2,6-dichloro-3-nitrobenzoic acid (80) in 6-methoxy-3-pyridinamine (79).
Compound 81 was heated at 100°C in H2SO4, the resulting mixture was poured into crushed
ice to yield the 9-chloro-2-methoxy-6-nitro-5,10-dihydrobenzo[b][1,5]naphthyridin-10-one
(82) as an orange
precipitate. Condensation of
82
with the appropriate (-
aminoalkyl)hydrazine106 in tetrahydrofurane-methanol (1:1) at room temperature afforded the
desired
2-[-(alkylamino)alkyl]-9-methoxy-5-nitro-2,6-dihydroindazolo[4,3-
bc][1,5]naphthyridines, or 2-[-(alkylamino)alkyl]-9-methoxy-5-nitro-2,6-dihydro-10-azapyrazolo[3,4,5-kl]acridines, (78af) as orange precipitates.
Cl
O
HOOC
N
NH2
i
O
N
O Cl
HO
NO2
O
ii
N
H
Cl
O
Cl
N
82
N
H
NO2
NO2
iii
79
80
81
Reagents: (i) ; (ii) H2SO4; (iii) NH2NHCH2CH2R.
N(CH3)2
78b
CH2N(CH3)2
78c
N(C2H5)2
78d
1-pyrrolidinyl
78e
Piperidino
78f
Morpholino
O
78a-f
Scheme 42
85
N
N
N
H
R
78a
N
NO2
R
In order to assess the DNA-binding properties and the antineoplastic activity of these
agents, the free base forms of the target derivatives 77 were converted into their water-soluble
hydrochlorides by the usual methods.
86
Results and Discussion
DNA-Binding Properties
'Apparent' binding constant (Kapp) values were determined, using a competitive
fluorometric ethidium displacement method that has been used extensively for other DNAbinding ligands, particularly intercalants.107,108,93a In this assay, the relative Kapp affinity for
calf thymus DNA (CTDNA) is defined by Kapp (drug) = 1.26/C50  Kapp (ethidium), were
1.26 is the concentration (μmol) of ethidium in ethidium-DNA complex, C50 represents the
concentration (μmol) of added compound required to reduce the fluorescence of the ethidiumDNA complex by 50%, and the Kapp (ethidium) binding constant is taken as 107 M-1.107,125 In
the present study, fluorescence displacement assays were performed at pH 7 to enable
comparison in biological conditions.
Under these relations, the Kapp values can be regarded as indicative of the strength
and extent of binding to this 'pseudo-random' DNA sequence, but not of the mode of
interaction (e.g. intercalation and/or groove binding mechanism). However, Table 5 shows
that all compounds 77 have a significant binding with DNA, a little more efficient than that of
parent compound PZA. The Kapp values are in the range1.41.6  107, indicating that the
target derivatives are more DNA-affinic than ethidium itself. The only exception is
constituted by the morpholino derivative 78f, which is the weakest ligand in the series, as we
expected from previous results.90,109 We suggested that the poor effect of 78f is due to the
protonation status of the pendant side chain, since the morpholine residue provides the least
basic amine residue within this series and would be only ~10–20% protonated at pH 7.90,109
An equivalent competitive fluorometric assay method was similarly used to assess
the binding of derivatives 78 to the synthetic polynucleotides [poly(dAdT)]2 (AT) and
[poly(dGdC)]2 (GC) in order to examine the possible binding site preferences and to
compare 77 to the parent compound PZA. The Kapp values determined for the compounds
with each duplex are collected in Table 5, together with the binding side preferences and, in
parentheses, the GC/AT or AT/GC affinity ratio. The possible binding site selectivity is
considered to be significant only for an GC/AT or AT/GC affinity ratio differing by
more than 30% from the sequence-neutral unity value (i.e. >1.3). It can be observed that, also
87
in these cases, the Kapp values of compounds 78 are indicative of a strong binding with these
synthetic DNA. For derivatives 78a,b,c,f, the site-dependent behavior is significantly (78f) or
highly GC preferential, differently from derivatives 78d,e and parent compound PZA, which
show very markedly AT binding site preference.
All together, binding data indicate that the aza substitution of 5-nitropyrazolo[3,4,5kl]acridines 77, leading to derivatives 78, does not affect the strength, the extent, and the site
preference of binding, while the nature of pendent side chain deeply influences the binding
properties of the new compounds 78.
88
Table 5. DNA Binding Properties and Cytotoxic Activity (PC-3) of Target Compounds 78af in
comparison with parent compound PZA.a
Kapp  10-7 M-1
a
Cytotoxic activity
AT
CTDNA
GC
binding site
preference
78a
0.36
1.6
1.7
GC (4.7)
600
11.7
63.1
78b
0.22
1.6
4.5
GC (20)
891
14.1
108
78c
0.16
1.4
4.0
GC (25)
562
10.7
58.9
78d
2.4
1.4
0.37
AT (6.5)
380
10.0
53.7
78e
3.0
1.4
0.63
AT (4.8)
646
3.40
126
78f
0.12
0.19
0.24
GC (2.0)
N.A.b
N.A.b
N.A.b
PZA
1.7
1.3
0.36
AT (4.7)
355
11.2
73.6
Compd
GI50 (nM)
TGI (μM) LC50 (μM)
For the meaning of Kapp, binding site preference, GI50, TGI, and LC50see text. b N.A. = not active.
89
In Vitro Cytotoxic Activity
In vitro cytotoxic potencies of target 5-nitro-10-aza-pyrazolo[3,4,5-kl]acridines
(78af) in comparison with parent 5-nitro-pyrazolo[3,4,5-kl]acridine (77a, PZA) against
human hormone-refractory prostate adenocarcinoma cell line (PC-3) are reported in Table 5.
Growth Inhibition 50, GI50 (nM), and Total Growth Inhibition, TGI (μM), represent the drug
concentration required to inhibit cell growth by 50% and by 100%, respectively, giving an
idea of the cytostatic action of the drugs. The Lethal Concentration 50, LC50 (μM), represents
the drug concentration required to kill 50% of the initial cell number, giving an idea of the
cytotoxic action of the drugs. Each quoted value is the mean of triplicate experiments.
The results indicate that: (a) As shown by GI50 values, all compounds 78 possess a
good antiproliferative activity in the high nanomolar range, similar, but a little lower than
parent PZA; the only exception is represented by 78f, which does not inhibit the cell growth
of 50% even at highest concentration (10-4 M) tested. (b) The TGI values show that the new
derivatives completely inhibit the cell growth at micromolar concentration even lower,
especially 78e, than PZA. (c) The LC50 values indicate that compounds 78 posses cytotoxicity
similar to that of PZA, 78c,d being the most potent compounds in the series. The data
obtained for a limited number of derivatives do not allow us to formulate structureactivity
relationships but some considerations about pendent side chain can be made: (i) The optimal
distance between the two nitrogen atoms is of two methylene units, as indicated by the
difference in all the three parameters, GI50, TGI, and LC50, in the pair 78a,b. (ii) Bulky
substituents at the terminal nitrogen atoms do not decrease the cytotoxicity, as can be seen
from the GI50, TGI, and LC50 values of the pair 78a,c. (iii) When the distal nitrogen atom is
part of an heterocycle, compounds 78df, the best results are obtained with a pyrrolidine, but
a piperidino yielded the best TGI value in the series; instead, a morpholino resulted in a
complete loss of activity.
There is no quantitative correlation between DNA-binding and in vitro activity, but
the lack of activity of 78f, which exhibit weakest Kapp values, confirms that an efficient
binding is a necessary, also if not sufficient, condition for cytotoxic activity. The binding site
preference does not seem to play an important role on cytotoxicity.
90
Apoptosis Assays
Recent evidence suggested that the characteristics of tumor cell death may be the
most important feature for successful chemotherapy. Apoptosis is defined by morphological
and biochemical changes resulting in cell loss and has been found to be relevant to a wide
spectrum of biological processes, including neoplasia and cancer chemotherapy.
Concentration on the mechanisms of action of chemotherapeutic agents has allowed to
establish that most of these agents exert their biological effects by triggering apoptotic cell
death. The ability of these agents to induce apoptosis in tumor cells has now become a
rationale for therapeutic approaches and suggests the possibility that apoptosis may be
enhanced in tumors for therapeutic benefit. Although available data demonstrate the induction
of apoptosis in tumor cells, the information is limited on the correlation of apoptotic cell death
to the success of chemotherapy and resistance. Recent findings have supported and confirmed
the presence of chemotherapy-induced DNA fragmentation in tumor cells following treatment
with PZA in vivo, however the mechanisms responsible to apoptotic and/or necrotic cell death
are so far poorly addressed.110
Thus, the most cytotoxic target derivatives 78c,d, the in vitro inactive 78f, and
reference compound PZA were selected, in order to investigate whether in vitro treatment
with these drugs was able to induce necrotic and/or apoptotic cell death. To this end, the
percentage of apoptotic cells in PC-3 treated with the target compounds 78c,d,f and reference
compound PZA, was evaluated, by Annexin V staining and Propidium iodide (PI)/Annexin V,
biparametric flow cytometric analysis and agarose gel electhrophoresis.
A characteristic feature of necrotic cell death is the loss of plasma membrane
integrity, whereas early during apoptosis phosphatidylserine translocates from the inner to the
outer plasma membrane layer.111 Thus, it was evaluated whether the PZA treatment of PC-3
cells induced externalization of PS residues from the inner to the outer leaflet of the plasma
membrane. To this end, annexin V binding on PC-3 cells by flow cytometric analysis was
investigated. As shown in Figure 23, treatment with target compound 78d induce an early (2
h) and extensive traslocation of PS. PS exposure on target compound 78d-treated PC-3 cells
increased in a time-dependent manner until 6 h and the declined at 8 h. Similar, but less
intensively (2.5-fold less) PS exposure was observed on target compound 78c-treated PC-3
91
cells. The target compound 78f and reference compound PZA did not affected PS expression
on PC-3 cells.
Annexin V-FITC (MFI)
600
500
400
PZA
2c
300
2d
200
2f
100
0
0
2
4
6
8
10
time (h)
Figure 23. The expression of Annexin V on PC-3 cells treated at
different times (2, 4, 6 and 8h) with target compounds 78c,d,f
and
reference
compound
PZA,
was
evaluated
by
immunofluorescence and FACS analysis. Data expressed as
mean fluorescence intensity (MFI) are representative of one out
of three separate experiments. (For 2c: 78c; 2d: 78d; 2f: 78f).
Data obtained by Annexin V staining were further streinghed by biparametric flow
cytometric analysis. Thus, 6 h after target compounds 78d and 78c treatment about 36% and
15% of PC-3 cells, respectively were Annexin V+ PI, whereas negligible PS expression was
observed on PC-3 cells treated with the target compound 78f and reference compound PZA
(Table 6); moreover, about 25% and 20% of the PC-3 cells treated with the target compound
78f and reference compound PZA, were PI+ Annexin V respectively.
92
Table 6. Treatment of PC-3 cells for 6 h with target compounds 78c,d,f and parent
compound PZA. The target compounds 78c,d induce apoptotic cell death.
Compounds
PI+ Annexin V
PI Annexin V
PI Annexin V+
78c
25.1  1.9a
71.6  3.2
2.3  0.2
1.1  0.1
78d
20.1  1.5
63.0  2.1
15.0  0.8
1.8  0.3
78f
3.8 + 0.3
56.2 + 2.5
36.2 + 2.1
3.8 + 0.4
PZA
8.2 + 0.3
90.6 + 2.8
0.1 + 0.0
1.1 + 0.1
a
PI+ Annexin V+
Percentage of positive cells. Results are representative of one out of three separate
experiments.
93
DNA Fragmentation Assays
DNA fragmentation during apoptosis leads to extensive loss of DNA content
resulting in a characteristic internucleosomal DNA ladder.112 In accordance with
cytofluorimetric analysis, agarose gel electrophoresis evidenced necrosis or apoptosis of PC-3
cells depending on the different compounds used (Figure 24). Target compound 78d at 50 μM
concentration induced apoptosis as evidenced by a characteristic ladder pattern of DNA
fragments, whereas as previously described101 very high molecular weight (about 0.8 Mb)
DNA fragments were observed in PC-3 cells treated with the reference compound PZA. In
addition, PC3 cells treated with the target compound 78c showed both necrotic/apoptotic cell
death as evidenced by 20% and 15% of PC-3 cells being PI+ Annexin V and PI Annexin V+.
Neither apoptosis nor necrosis was observed in PC-3 cells treated with the compound 78f.
Overall, these results indicate that the cytotoxic activity of target compound 78d on
androgen-independent PC3 prostate cancer cells detected by SRB assay, is the result of its
ability to induce oligonucleosomal DNA fragmentation and apoptotic cell death.
MW 77f PZA 77c 77d
ff
Figure 24. Analysis of DNA fragmentation
from
PC-3
cells
treated
with
target
compounds 78c,d,f and reference compound
PZA at 6 h after treatment, was performed by
agarose gel (1.7%) electrophoresis. MW =
molecular
weight.
Data
shown
are
representative of three separate experiments.
94
Rational Design, Synthesis and Biological Evaluation
of 3H-Naphtho[1,2,3-de]quinoline-2,7-diones as
Potential Topoisomerase Inhibitors
Introduction to the Chemistry
The antitumor drugs that intercalate DNA have demonstrated a growing interest in
the field of anticancer derivatives. Generally, they are characterized by a planar chromophore,
often made up of three or four condensed rings, which can intercalate into base pairs, and at
least, they may have attached one flexible basic side chain, which can improve the DNA
binding and/or can interact with enzymes such as topoisomerases. Some examples of
derivatives undergoing clinical trials endowed with only one side chain are acridine-4carboxamide DACA,113 mitonafide,114 azonafide115 (83), imidazoacridones.116 Among
intercalating anticancer agents with two basic chains, it is worth mentioning mitoxantrone, 117
anthrapyrazoles,118 aza-anthracendiones,131 aza-anthrapyrazoles,119 clinically used or currently
in clinical trials and also a number of interesting acridine derivatives synthesized by our
research group: the bis functionalized acridone-4-carboxamides,90 the bis functionalized
acridine-4-carboxamides,120
the
pyrazolo[3,4,5-kl]acridine-5-carboxamides,92a
the
pyrazolo[3,4,5-mn]pyrimido[5,6,1-de]acridines,121 and, in particular, the pyrimido[5,6,1de]acridines91a,b,122,123 (84).
In the continuous search for new classes of antitumor agents, it was decided to
investigate the 6-[(-aminoalkyl)amino]-3H-naphtho[1,2,3-de]quinoline-2,7-diones (85) as
analogues related to the azonafide 83 and especially, to the pyrimido[5,6,1-de]acridines 84,
whose chromophores present similar ring systems, as shown in Figure 25. Prompted by the
above rationale, compounds 85aj were synthesized and their relative cytotoxic activity on
different cell lines was also studied.
95
8
7
4
11
O
7
H
O
8
6
4
11
R
N
7
6
4
11
N
O
1
O
N
R
N
X
8
12 3
N
H
O
6
2
N
1
3
O
R1
83
Ph
3
2 NH
O
84
85
Naphtho[1,2,3-de]quinoline-2,7-diones
Figure 25. Structures and ring numbering of parent compounds azonafide
(83) and pyrimido[5,6,1-de]acridines (84) in comparison with the target
naphto[1,2,3-de]quinoline derivatives (85).
96
Chemistry
Scheme 43 shows the synthetic pathway leading to target derivatives 85aj.
Compounds 85ai were obtained by nucleophilic substitution of the commercially available
6-Bromo-4-methyl-1-phenyl-3H-naphtho[1,2,3-de]quinoline-2,7-dione with the appropriate
(-aminoalkyl)amine in different experimental conditions. The preparation of 85j was
achieved by nucleophilic substitution of 6-Bromo-4-methyl-1-phenyl-3H-naphtho[1,2,3de]quinoline-2,7-dione with tert-butyl N-(2-aminoethyl)carbamate and subsequently Boc
(Boc = tert-butoxycarbonyl) deprotection in dioxane and hydrochloric acid.
O
Br
O
R
N
R1
(a)
NH
Ph
NH
Ph
O
O
85a-j
Reagents (a): NHR(CH2)2R1 for 85a-i; NH2(CH2)2NHC(O)OC(CH3)3, then H+ for 85j.
Substituents: R = Me for 54a; R = H for 54b-j. R1 = N(CH3)2 for a and b, CH2N(CH3)2 for c,
N(C2H5)2 for d, CH2N(C2H5)2 for e, CH2NH(CH2)2OH for f, piperidino for g, 1-pyrrolidinyl for h,
morpholino for i, NH2 for j.
Scheme 43
The free base forms of target derivatives were converted, by usual methods, into their
more soluble hydrochlorides, to examine the cytotoxic activity of these agents.
97
Results and Discussion
HT29 human colon adenocarcinoma cell line
In vitro cytotoxic potencies of target 6-[(-aminoalkyl)amino]-3H-naphtho[1,2,3de]quinoline-2,7-diones (85) in comparison with reference drug doxorubicin (Dx) against
human colon adenocarcinoma cell line (HT29) are reported in Table 7. Parent 6-[2(dimethylamino)ethyl]amino-2,3-dihydro-1H,7H-pyrimido[5,6,1-de]acridine-1,3,7-trione
(84a, Figure 22: R = N(CH3)2, X = R1 = H) was also included in Table 7 to allow a direct
comparison between compounds 85 and the unique derivative 84 possessing only a basic side
chain in position 6.91b IC50 represents the drug concentration (M) required to inhibit cell
growth by 50%.
The results indicate that: (a) 85b,c,e emerge as the most potent among the new
derivatives with IC50 values of  0.3 micromolar and with cytotoxicity higher than related
84a; (b) many compounds 85 possess a moderate antiproliferative activity in the micromolar
range; (c) none of the new derivatives posses potency similar to that of reference drug
doxorubicin (Dx, IC50 = 26 nanomolar), but only 85g,i are devoid of cytotoxic activity.
The data obtained allow us to formulate some considerations regarding the side
chains: (i) the side chain of 85a has been selected to check whether the intramolecular
hydrogen bond between the carbonyl in position 7 and the hydrogen of amine in position 6 for
compounds 85 is important as in the case of derivatives 8491b and of analogous acridine
derivatives.90 In agreement to what it previously observed,90,91b the difference in activity
between the pair 85a85b, with 85b being 12 times more cytotoxic than its the N-methyl
derivative 85a, clearly indicates the importance of the cited intramolecular hydrogen bond
also for these compounds. (ii) In contrast with what it was noted earlier with derivatives 84,91b
especially for bulky substituents, the optimal distance between the two nitrogen atoms seems
to be of three methylene units, as indicated by the difference in potency between the pair
85d85e whose substituents at distal nitrogen atom are two ethyl groups. (iii) A side chain
similar to that of the clinically useful drug mitoxantrone, compound 85f, leads to a fair
activity, but not excellent as for the parent compounds 84.91b (iv) In the sub series 85g85i, in
which the distal nitrogen atom is part of a cycle, the best result are obtained with 85h (R1 = 1-
98
pyrrolidinyl, Scheme 42) endowed of a good activity, while 85g,i (R1 = piperidino or
morpholino, respectively) are not active. The good activity of 86h is not surprising in
comparison with derivatives bearing similar side chain;92a,121 also expected was the inactivity
of 86i, for which the distal nitrogen atom is not sufficiently basic to be protonated at
physiological conditions;90,120 on the contrary, it was surprising the inactivity of 85g bearing a
side chain that was found to be efficient in many other derivatives.90121 (v) Finally,
compound 85j, bearing a side chain similar to that of BBR 2778,126 an aza-anthracendione in
phase II of clinical trial,124 possesses border line cytotoxic activity, showing that this kind of
side chain is not optimal for 84.
Table 7. Cytotoxic activity of 85a-j in comparison with parent pyrimido[5,6,1-de]acridine
84a and with reference drugs doxorubicin (Dx) and cisplatin (Cs) versus human colon
adenocarcinoma cell line a (HT29) and versus ovarian carcinoma cell line panel b (A2780 and
A2780/Cs cisplatin resistant, CH1 and CH1/Cs cisplatin resistant, and SKOV3).
IC50 (M) c
HT29
A2780
A2780/Cs d
CH1
CH1/Cs d
SKOV-3
85a
3.3
9.0
11 (1.2)
12
12 (1.0)
15
85b
0.27
1.6
3.0 (1.9)
2.3
9.8 (4.3)
2.7
85c
0.32
2.4
3.7 (1.5)
5.4
12 (2.2)
3.5
85d
3.3
2.1
>25 (>12)
>25
>25 (n.c.)
9.0
85e
0.27
1.2
2.5 (2.1)
2.4
2.3 (1.0)
2.4
85f
1.4
0.7
8.6 (12)
3.2
8.0 (2.5)
7.4
85g
>10
>25
>25 (n.c.)
>25
>25 (n.c.)
>25
85h
0.97
1.6
3.2 (2.0)
5.6
18 (3.2)
6.0
85i
>10
>25
>25 (n.c.)
>25
>25 (n.c.)
>25
85j
3.2
5.6
12 (2.2)
11
9.0 (0.8)
12
84a e
0.90
2.1
2.0 (0.95)
1.4
2.45 (1.7)
3.85
Dx
0.026
0.89
3.4 (3.8)
0.15
2.4 (16)
3.4
Comp.
Cs
99
Human ovarian carcinoma cell line panel
In vitro cytotoxic potencies of the new 6-[(-aminoalkyl)amino]-3H-naphtho[1,2,3de]quinoline-2,7-diones (85), in comparison with parent compound 84a and reference drug
Cisplatin (Cs), against five human ovarian carcinoma cell lines, A2780 (sensitive), A2780/Cs
(cisplatin resistant), CH1 (sensitive), CH1/Cs (cisplatin resistant), and SKOV-3, are shown
also in Table 7. In the resistant cell line columns, besides the IC50 values, in parentheses are
reported the resistance index (RI = IC50 ratio of resistant line on sensitive one) values.
In general, results are parallel to what it was found for HT29. On sensitive cell lines
the best compounds seem to be 85b,e, which possess activity comparable to that of reference
drug Cs; also 85c,f,h are endowed of fair activity on some cell lines. In addition, some
derivatives 85, especially, 85a,e,j are scarcely or not cross resistant with Cs on resistant cell
lines.
100
Experimental Section
Materials and Methods. All chemicals were commercial grade, purchased from Sigma
Aldrich and GFS Chemicals and were used as received. TLC analyses were performed on
silica gel plates with fluorescent indicator SIL G/UV254 (Merck) and over alumina plates
Stratcrom ALF254 (Carlo Erba). Column chromatography purifications were run over silica
gel (Kieselgel 40, 0.040-0.063 mm; Kieselgel 60, 0063-0.200 mm - Merck) and over
aluminium oxide (0.05-0.15 mm - Fluka). 1H and 13C NMR spectra were recorded operating
at room temperature, on a Bruker ARX 500 MHz at the University of Vermont, VT, USA. At
the University of Camerino they were recorded on a Varian 200 MHz Gemini and Varian 400
MHz Mercury. H and C chemical shifts () are reported in parts per million (ppm) from
SiMe4. Peak multiplicities are abbreviated as follows: broad singlet, brs; singlet, s; doublet, d;
triplet, t; multiplet, m. Elemental analyses (C, H, S and N) were performed with a Fison
Instrument 1108 CHNS-O elemental analyzer. IR spectra were recorded with a Perkin-Elmer
system 2000 Fourier transform IR instrument. The negative and positive ESIMS spectra were
obtained with a Hewlett Packard series 1100 MSI detector spectrometer using reagent-grade
methanol as a mobile phase. Melting points are uncorrected and were taken on a Mel-Temp II
and Fisher-Johns apparatus (use of microscope slides for high temps) at the University of
Vermont, VT, USA and on a BÜCHI B-50 scientific instrument at the University of
Camerino.
1,10-phenanthroline-1-oxide (15). To a water-cooled solution of urea-hydrogen peroxide
(2.0 g, 200 mmol) in formic acid (6 g, 5 mL, 130 mmol) the 1,10-phenanthroline anhydrous
(1) (0.5 g, 3 mmol) was added in small portions over a period of 3 minutes. The reaction
mixture was stirred for 3.5 hours at room temperature. The solution was poured into water (20
mL), made alkaline with solid Na2CO3 (pH = 9.0) and more water was added as an emulsion
occurred. The aqueous layer was extracted using CH2Cl2 (2 x 30 mL). The combined organic
extracts were dried over Na2SO4, filtered and evaporated to dryness under reduced pressure to
afford the product as an orange solid (0.26 g, 44%) as a hydrate.(lit.,125 m.p.: 180-181°C).
101
1
H NMR (DMSO-d6): δ 9.12 (brs, 1 H), 8.68 (brs, 1 H), 8.50 (brs, 1 H), 8.01 (brs, 3H), 7.80
(brs 1 H), 7.67 (brs, 1H).
2-chloro-1,10-phenanthroline (16). To a stirred solution of 1, 10-phenanthroline-1-oxide
(15) (0.24 g, 1.2 mmol) and triethylamine (0.15 g, 1.4 mmol, 0.2 mL) in dichloromethane (1
mL), a solution of phosphorous oxychloride (0.2 g, 1.4 mmol, 0.13 mL) in dichloromethane
(1 ml) was added drop-wise at 10° C and it was kept under a nitrogen atmosphere. The
reaction mixture was stirred for 30 minutes at room temperature, refluxed for 1 hour and it
was held overnight at room temperature. The brown mixture was poured into water (2 mL)
and basified with 2M NaOH. The aqueous layer was then extracted with dichloromethane (2 x
60 mL). The combined dichloromethane layers were dried over sodium sulfate, filtered, taken
to dryness under vacuum to afford a brown solid (0.17 g, yield 65%). m.p.: 190-193°C. The
extrapolated 1H NMR for this product could be compared with an authentic sample.
1
H NMR (CDCl3): δ 9.22 (t, J = 3.97 Hz, 1 H), 8.25 (d, J = 8.6 Hz, 1 H), 8.19 (d, J = 8.34 Hz,
1 H), 7.80 (m, 2 H), 7.64 (m, 2H).
1-methyl-1,10-phenanthrolin-1-ium iodide (18). The 1,10-phenanthroline (1) (10.0 g, 55.5
mmol) was added to round bottom flask followed by dry acetonitrile (50 ml). Methyl iodide
(23.63 g, 166.5 mmol) was added to the stirring solution, which was then heated to reflux for
1.25 hr. The solution was cooled to room temperature and the yellow precipitate was collected
by filtration (16.78 g, 93%): m.p.: 205 – 210°C.(lit.,126 m.p.: 210-213oC).
1
H NMR (DMSO-d6):  9.57 (d, J = 5.74 Hz, 1H), 9.40 (d, J = 7.96 Hz, 1H), 9.32 (m, 1H),
8.80 (m, 1H), 8.44 (m, 3H), 8.07 (m, 1H), 5.29 (s, 3H).
1-methyl-1,10-phenanthrolin-2(1H)-one (19). The methiodide (18) (5 g, 0.016 mol) was
added to water (about 180 mL, and still not all dissolved) and placed in an addition funnel
attached to a 2-necked flask equipped with a second addition funnel. Sodium hydroxide [18 g
in water (90 mL)] was placed in the other addition funnel. The potassium ferricyanide (13 g in
90 mL of water) was placed in the flask and the flask cooled in an ice bath. The NaOH
solution and the salt were then added to the ferricyanide solution dropwise over a period of
0.5 h. The mixture was stirred for an additional hour and the yellow solid was collected by
filtration. The product was washed with ice water and allowed to air dry (3.2 g). The solid
was dissolved in dichloromethane (40 mL), filtered to remove a trace of insoluble and the
102
solvent evaporated to afford the desired product as a pale yellow product (3.1 g). (lit., 41 m.p.:
122-124°C; lit.,126 m.p.: 123-124°C).
1
H NMR (CDCl3): δ 8.95 (dd, J = 4.0/1,8 Hz, 1H), 8.17 (dd, J = 8.2 Hz/1.8 Hz, 1H), 7.77 (d, J
= 9.3 Hz, 1H), 7.55 (m, 2H), 7.49 (m, 1H), 6.90 (dd, J = 9.3 Hz, 1H), 4.50 (s, 3H).
2-chloro-1,10-phenanthroline (16). 1-methyl-1,10-phenanthrolin-2(1H)-one (19) (5.00 g,
23.8 mmol) and PCl5 (6.19 g, 29.8 mmol) were added to an oven-dried round bottom flask.
The POCl3 (40 ml) was then added and the flask was fitted with an oven-dried reflux
condenser. The suspension was refluxed for 8 hrs during which time the suspension turned
very pale yellow. The POCl3 was removed under reduced pressure without heating. Ice chips
were then added to the residual solid and “fizzing” ceased. The mixture was then basified
with aqueous ammonium hydroxide (pH 10). The pale tan precipitate was collected by
filtration. The solid was extracted with chloroform, filtered to removes some insoluble
material and the chloroform was concentrated to yield a tan solid (3.1 g). The aqueous filtrate
was extracted twice with chloroform, dried over sodium sulfate and concentrated to yield 0.42
g of a tan solid for a combined yield of 68%. (lit.,126,127 m.p.: 129-130°C).
1
H NMR (CDCl3): δ 9.22 (m, 1 H), 8.24 (m, 1 H), 8.18 (d, J = 8.55 Hz, 1 H), 7.78 (m, 2 H),
7.63 (m, 2 H).
4,7-dimethyl-1,10-phenanthroline-1-oxide (20). To a water-cooled solution of ureahydrogen peroxide (3.0 g, 33 mmol) in formic acid (13.42 g, 11 mL, 300 mmol), the 4,7–
dimethylphenanthroline anhydrous (14) (1 g, 48 mmol) was added in small portions in 3
minutes. The reaction mixture was stirred for 3 hours at room temperature. The solution was
poured into water (20 mL), made alkaline (pH = 8.0) with solid Na2CO3 and washed with
more water as emulsion occurred. Dichloromethane (50 mL) was then added and the resulting
mixture was stirred overnight. The aqueous layer was decanted and treated several times with
CH2Cl2 (5  70 mL). The combined organic extracts, separated by decantation, were dried
over Na2SO4, filtered and evaporated to dryness under reduced pressure to afford the product
as a pink solid (0.79 g, 74% recovery). m.p.: 165-168ºC.
1
H NMR (CDCl3): δ 9.15 (d, J = 4.4 Hz, 1H), 8.65 (d, J = 6.4 Hz, 1H), 8.10 (d, J = 9.3 Hz,
1H), 7.90 (d, J = 9.3 Hz, 1H), 7.49 (d, J = 4.39 Hz, 1H), 7.29 (d, J = 6.4 Hz, 1H) 2.80 (s, 3H),
2.73 (s, 3H).
103
2-chloro-4,7-dimethyl-1,10-phenanthroline (21). To a stirred solution of 4,7-dimethyl-1,10phenanthroline-1-oxide (20) (0.5 g, 22 mmol) and triethylamine (0.26 g, 26 mmol) in
dichloromethane (1.6 mL), a solution of phosphorous oxychloride (0.4 g, 0.24 mL. 26 mmol)
in dichloromethane (1 mL) was added dropwise at 10°C and it was kept under a nitrogen
atmosphere. The reaction mixture was stirred for 30 minutes at room temperature, refluxed for
1 hour and it stirred overnight at room temperature. The mixture was poured into water (2
mL) and basified with 2M NaOH. The aqueous layer was then extracted with
dichloromethane (2  60 mL). The combined dichloromethane layers were dried over sodium
sulfate, filtered, concentrated to dryness under vacuum to give a purple solid (0.34 g, 52%).
The crude product (0.1 g) was purified by gravity column chromatography on silica gel using
CHCl3 as eluent followed by CHCl3/MeOH 99:1 to afford the product as a white solid (0.046
g, 46% recovery). m.p.: 190-193ºC.
1
H NMR (CDCl3): δ 9.06 (d, J = 4.3 Hz, 1H), 8.03 (d, J = 9.3 Hz, 1H), 7.96 (d, J = 9.2 Hz,
1H), 7.45 (m, 2H), 2.79 (s, 3H), 2.77 (s, 3H).
2,9-dichloro-4,7-dimethyl-1, 10-phenanthroline (13). To a stirred solution of 2-chloro-4, 8dimethyl-1,10-phenanthroline 10-oxide (22) (0.08 g, 0.31 mmol) in CH2Cl2 (2 mL), a solution
of phosphorous oxychloride (0.054 g, 0.033 mL, 0.36 mmol) in dichloromethane (1 mL) was
added drop-wise at 10º C and it was kept under a nitrogen atmosphere. The reaction mixture
was stirred for 30 minutes at room temperature, refluxed for 1 hour. The mixture was then
poured into water (2 mL) and extracted with dichloromethane (5 mL). The organic layer was
dried over anhydrous Na2SO4, filtered, concentrated to dryness under vacuum to afford a
small amount of a tan solid. Chromatography over silica gel using as eluent chloroform
followed by chloroform : methanol (9:1) led to a small amount of solid whose 1H NMR was
somewhat better than the crude material.
1
H NMR (CDCl3): δ 8.01 (s, 1H), 7.49 (s, 1H), 1.25 (s, 3 H).
12-methyl-6,7-dihydro-5H-[1,4]diazepino[4,3,2,1-lmn]-1,10-phenanthroline-4,8-diium
bromide (27). The 1,3-dibromopropane (29.83 g, 15 mL, 140 mmol) was added to 5-methyl1,10-phenanthroline (26) (2 g, 10 mmol) and the mixture was refluxed for 2 hours. The
solution was cooled to room temperature. The precipitate was filtered and washed thoroughly
with ethyl ether to afford a yellow solid (3.84 g, 97%). A small amount was crystallized from
9:1 ethanol/water. m.p.: 268-273oC (dec., black) (lit.128,38, m.p.: 286°C, dec.).
104
1
H NMR (DMSO-d6): δ 9.93 (d, J = 5.5 Hz, 1 H), 9.84 (d, J = 5.6 Hz, 1 H), 9.60 (d, J = 8.5
Hz, 1 H), 9.45 (d, J = 8.3 Hz, 1 H), 8.70 (m, 1 H), 8.63 (m, 1 H), 8.55 (s, 1 H), 5.17 (m, 4 H),
3.26 (m, 2 H), 2.97 (s, 3 H).
C NMR (DMSO-d6/H2O): δ 150.62, 150.51, 145.73, 143.66, 138.03, 134.11, 133.64,
13
133.37, 132.55, 128.73, 127.47, 127.08, 60.025, 59.82, 31.39, 18.85.
3,6,7,9-tetrahydro-5H-[1,4]diazepino-[1,2,3,4-lmn][1,10]-5-methyl-1,10-phenanthroline3,9-dione (28). To an ice-cooled solution of potassium hexacyanoferrate (III) (22 g, 67 mmol)
and water (30 mL), a solution of bis quaternary salt (27) (3.0 g, 7.5 mmol) in H2O (30 mL)
and a solution of NaOH (10.29 g, 257 mmol) in H2O (30 mL) were alternatively added in
small portions. After the addition was complete, the reaction mixture was stirred for 30’ and
then neutralized by a drop-wise addition of concentrated hydrochloric acid while in ice bath.
The dark red suspension was then extracted with CH2Cl2 (3 x 50 mL). The combined extracts
were dried over anhydrous sodium sulfate and concentrated to dryness under vacuum to yield
1.2 g of brown solid (59%). The product was treated with CH3CN and it was filtered off to
afford a yellow solid (0.75 g, 37%). m.p.: 311-315ºC.
1
H NMR (CDCl3): δ 7.91 (d, J = 9.7 Hz, 1H), 7.64 (d, J = 9.4 Hz, 1 H), 7.18 (s, 1 H), 6.80 (m,
2H), 4.29 (m, 4H), 2.57 (s, 3H), 2.45 (m, 2H).
C NMR (DMSO-d6): δ 161.3, 138.9, 135.9, 129.5, 123.1, 122.6, 121.9, 121.8, 122.2, 44.1,
13
44.5, 25.4, 17.9.
2,9-dichloro–5-methyl-1,10-phenanthroline (25). To an ice-cooled mixture of 3,6,7,9tetrahydro-5H-[1,4]diazepino-[1,2,3,4-lmn][1,10]-5-methyl-1,10-phenanthroline-3,9-dione
(28) (0.75 g, 2.8 mmol) and phosphorous pentachloride (1.32 g, 16.3 mmol), phosphoryl
chloride (5.69 g, 8.25 mL, 39 mmol) was added drop-wise and the system was flushed with
nitrogen. The solution was left at room temperature for 5 minutes and then was refluxed for
two hours. The reaction mixture was stirred overnight at room temperature, refluxed for 6
more hours and again left to stir overnight at room temperature. The POCl3 was then removed
through vacuum distillation. The dark suspension was poured into water (15 mL) and basified
(pH = 9.0) with NH4OH 2M while in ice bath. The brown solid was collected by suction
filtration to yield 0.75 g (76%). A portion of the crude product was subjected to gravity
column chromatography on silica gel eluting with CH2Cl2 to afford a pale pink solid (21%
recovery). m.p. 181-184ºC.
105
1
H NMR (CDCl3): δ 8.34 (d, J = 8.60 Hz, 1H), 8.11 (d, J = 8.34 Hz, 1 H), 7.68 (d, J = 8.54
Hz, 1 H), 7.61 (s, 1H), 7.60 (d, J = 8.35 Hz, 1H), 2.76 (s, 3 H).
C NMR (CDCl3): δ 152.1, 145.1, 138.9, 138.2, 135.6, 127.9, 126.4, 125.7, 125.0, 124.7,
13
19.4.
An. Calcd. for C, 59.34; H, 3.06; Cl, 26.95; N, 10.65. Found: C: 59.12, H: 3.11, N: 10.67.
6,7-dihydro-5H-[1,4]diazepino[4,3,2,1-lmn]-1,10-phenanthroline-4,8-diium
bromide
(27a). 1,10-phenanthroline (1) (20 g, 101 mmol) was added to a round bottom flask, followed
by 1,3-dibromopropane (125 mL) and was heated to reflux for 2 hrs. The solution was cooled
to room temperature and the yellow precipitate was collected by suction filtration.
Recrystallization from 95:5 ethanol:water gave 36.3 g as yellow needles (94%). m.p.: 256° C
(dec.).
1
H NMR (DMSO-d6): δ 9.87 (d, J = 5.5 Hz, 2 H), 9.56 (d, J = 8.4 Hz, 2 H), 8.70 (s, 2 H), 8.68
(m, 2 H), 5.13 (t, J = 6.7 Hz, 4 H), 3.23 (m, 2 H).
6,7-dihydro-5H-[1,4]diazepino[4,3,2,1-lmn]-1,10-phenanthroline-3,9(81H,11aH)-dione
(27a). To an ice cooled solution of potassium hexacyanoferrate (III) (12.4 g, 38 mmol), and
water (10 mL), a solution of bis quaternary salt (27 a) (1.0 g, 7.5 mmol) in H2O (30 mL) and a
solution of NaOH (3.3 g, 2.6 mmol) in H2O (10 mL) were alternatively added in small
portions. After the addition was complete, the reaction mixture was stirred for 30’ and then
basified by a drop-wise addition of concentrated hydrochloric acid while in ice bath. The dark
mixture was then extracted with CH2Cl2 (3 x 30 mL). The combined extracts were dried over
sodium sulfate and concentrated to dryness under vacuum to yield 0.25 g of brown solid
(39%). The crude product was subjected to gravity column chromatography on silica gel,
using CH2Cl2:MeOH/ 95:5 as an eluent, to afford a
yellow solid
(0.13 g,
20%).Recrystallization from MeOH/CHCl3 gave some pale yellow solid. m.p.>320°C (lit.38,
m.p.: >320°C)
1
H NMR (DMSO-d6): δ 7.97 (d, J = 9.5 Hz, 2 H), 7.55 (s, 2 H), 6.72 (d, J = 9.4 Hz, 2 H), 4.18
(t, J= 5.9 Hz, 4 H), 2.22 (q, J = 6.5 Hz, 2 H).
C NMR (DMSO-d6): δ 161.7, 139.2, 131.7, 122.6, 122.4, 122.3, 44.8, 25.3.
13
2,9-dichloro-1,10-phenanthroline (25a). The dione (28a) (3.00 g, 11.9 mmol) and PCl5
(5.20 g, 25.0 mmol) were added to an oven dried round bottom flask and fit with a reflux
106
condenser. The system was flushed with nitrogen and POCl3 (35 mL) was quickly added. The
mixture was refluxed for 8 hours. The POCl3 was then evaporated and ice chips were added to
the brown oil residue. The solution was placed in an ice bath and basified with concentrated
ammonium hydroxide. The brown precipitate was removed by suction filtration and then
extracted with chloroform. The chloroform was removed and the residue was subjected to
column chromatography with 99:1, CH2Cl2:MeOH, to yield 2.14 g of light brown solid
(76%). m.p.: 250° C (lit.129, m.p.: 249-250°C).
1
H NMR (CDCl3): δ 8.19 (d, J = 8.18, 2 H), 7.81 (s, 2 H), 7.63 (d, J = 8.41 Hz, 2 H).
C NMR (CDCl3): δ 152.1, 145.1, 138.9, 127.9, 126.4, 125.1.
13
An. Calcd. for C, 57.86; H, 2.43; Cl, 28.47; N, 11.25. Found: C: 57.61, H: 2.44, N: 11.27
2-methoxy-1,10-phenanthroline (29a). To a solution of MeOH (17 mL) and Na (0.9 g, 39
mmol), the 2-chloro-1,10-phenanthroline (16) (0.5 g, 2.3 mmol) was added in one portion.
The yellow mixture was refluxed for 19 hours and it was then taken to dryness on a rotary
evaporator. The yellow oil was quenched into H2O (8 mL) and the flask was placed in an ice
bath under a stirring. The product that precipitated was collected by filtration to yield a brown
solid (0.38g, 79%). The product could be recrystallized from aqueous methanol to afford a
white solid. m.p.: 69-73 °C (lit.,130 m.p.: 74-76°C; lit.,44 m.p.: 86-89°C).
1
H NMR (DMSO-d6): δ 9.11 (dd, J = 1.8/4.2 Hz, 1 H), 8.46 (dd, J = 1.7/8.0 Hz, 1 H), 8.39 (d,
J = 8.7 Hz, 1 H), 7.95 (d, J = 8.7 Hz, 1 H), 7.86 (d, J = 8.7 Hz, 1 H), 7.74 (m, 1 H), 7.24 (d, J
= 8.6 Hz, 1 H) 4.16 (s, 3H).
C NMR (DMSO-d6): δ 162.3, 149.5, 144.5, 143.7, 139.4, 135.9, 128.7, 126.1, 124.5, 123.8,
13
122.8, 113.2, 53.4.
1H-1,10-phenanthroline-2-one (30). A yellow solution of 2 methoxy -1,10-phenanthroline
(29a) (0.20 g, 0.95 mmol) and hydrobromic acid [48%] (2 mL, 2.98 g, 36.82 mmol) was
heated at 90° C for 18 hours. The yellow solid was collected by filtration, washed with its
filtrate and left to dry. It was then quenched into ethyl alcohol (5 mL), and left to stir for ½
hour. The solid was collected by filtration to yield a yellow product (0.10 g, 57%). m.p.: 310320°C.
1
H NMR (DMSO-d6): δ 9.00 (dd, J = 1.5/4.1 Hz, 1 H), 8.50 (dd, J = 1.5/8.2 Hz, 1 H), 8.13 (d,
J = 9.4 Hz, 1 H), 7.83 (d, J = 8.6 Hz, 1 H), 7.74 (m, 2 H), 6.76 (d, J = 9.4 Hz, 1 H)
107
C NMR (DMSO-d6): δ 161.3, 148.9, 140.6, 137.6, 135.6, 135.1, 128.3, 125.9, 123.5, 122.6,
13
120.9, 117.9.
2,9-dimethoxy-1,10-phenanthroline (29b). To a solution of NaOCH3, prepared by reacting
Na metal (0.26 g, 11 mmol) with MeOH (7 mL), the 2,9-dichloro-1,10-phenanthroline (25a)
(0.2 g, 0.80 mmol) was added in one portion and the mixture was refluxed for 19 hours. The
yellow solution was taken to dryness under vacuum and then CHCl3 (8 mL) was added. The
insoluble solid material was removed by filtration, washed with little CHCl3 and the filtrate
was concentrated on a rotary evaporator to give a pale yellow product (0.17 g, 88%). A
portion of the crude product was crystallized from pentane to afford white needles. m.p.: 8385°C (lit.,43 m.p.: 110°C).
1
H NMR (CDCl3): δ 8.09 (d, J = 8.7 Hz, 2 H), 7.62 (s, 2 H), 7.09 (d, J = 8.7 Hz, 2 H), 4.29 (6
H).
1
H NMR (DMSO-d6): δ 8.33 (d, 8.7 Hz, 2 H), 7.78 (s, 2 H), 7.17 (d, J = 8.6 Hz, 2 H), 4.15 (s,
6 H).
C NMR (DMSO-d6): δ 161.7, 142.4, 139.3, 124.9, 123.3, 113.0, 52.8.
13
C NMR (CDCl3): δ 162.7, 143.1, 138.9, 125.2, 123.3, 113.4, 53.3.
13
1,10-dihydro-1,10-phenanthroline-2,9-dione (24). A yellow solution of 2,9-dimethoxy1,10-phenanthroline (29b) (0.22 g, 0.91 mmol) and hydrobromic acid [48%] (2 mL, 2.98 g,
36.82 mmol) was heated at 90°C for 18 hours. The solid was collected by filtration and
washed with ethyl alcohol to afford a pale brown solid (0.10 g, 53 %). m.p.>320°C.
1
H NMR (DMSO-d6): δ 11.73 (brs, 2 H), 7.98 (d, J = 9.5 Hz, 2 H), 7.46 (s, 2 H), 6.64 (d, J =
9.3 Hz, 2 H).
1,10-phenanthroline-2(1H)-thione (33) - Procedure 1. A solution of 2-chloro-1,10phenanthroline (16) (0.1 g, 0.46 mmol) and sodium hydrosulfide (hydrate) (0.1 g, 1.70 mmol)
in N, N-dimethylformamide (25.0 mmol, 2 mL) was heated at 120º C for 3 hours. It was then
left to stir at room temperature overnight. The DMF was removed through vacuum distillation
and after cooling, the yellow residue was treated with diluted glacial acetic acid (5 mL), then
filtered to afford a yellow solid (0.090 g, yield 93%). m.p.: 214-217º C (red dec.). (lit.,51 m.p.:
221-222°C).
1
H NMR (CDCl3): δ 12.35 (s, 1 H), 8.95 (m, 1 H), 8.22 (m, 1 H), 7.65 (m, 5 H).
108
C NMR (CDCl3): δ 180.99, 149.75, 136.05, 135.09, 135.94, 134.40, 134.12, 128.40, 124.85,
13
123.84, 123.13, 121.14.
1,10-phenanthroline-2(1H)-thione (33) - Procedure 2. To a yellow solution of 2-chloro1,10-phenanthroline (16) (0.10 g, 0.46 mmol) and N,N-dimethylformamide (12.8 mmol, 1
mL), [98%] sodium sulfide nonahydrate (0.22, 0.92 mmol) was added in one portion and the
green mixture was left to stir at 60° C for 4 hours. It was then quenched into water (2 mL) and
treated with a dropwise addition of 1 M glacial acetic acid until solid precipitated. The yellow
mixture was left to stir for 1 hour at room temperature and the solid was collected by filtration
to yield a yellow product (0.085 g, 90%). The crude product was purified by gravity column
chromatography over silica gel using CH2Cl2:MeOH (99:1) as the eluents. The fractions were
allowed to evaporate to afford beautiful yellow needles, 0.055 g, 56%. m.p. 218-221oC.
1
H NMR (DMSO-d6): δ 12.25 (brs), 9.07 (dd, J = 1.5/4.3 Hz, 1 H), 8.55 (dd, J = 1.4/8.2 Hz, 1
H), 8.07 (d, J = 9.1 Hz, 1 H), 7.90 (m, 2 H), 7.83 (m, 1 H), 7.55 (d, J = 9.0 Hz, 1H).
9-chloro-1,10-phenanthroline-2(1H)-thione
(34).
A
mixture
of
2,9-dichloro-1,10-
penanthroline (25a) (0.20 g, 0.80 mmol), thiourea (0.12 g, 1.60 mmol) and ethyl alcohol (9
mL) was refluxed for 3 hours and it was left to stir at room temperature overnight. The ethyl
alcohol was evaporated on a rotary evaporator and the residue was treated with CH2Cl2 (20
mL). The yellow mixture was left to stir for 40’, after which the solid was collected by
filtration. The filtrate was evaporated on a rotary evaporator to yield a yellow product (0.057
g, 30%). The solid that was insoluble in CH2Cl2 was left to stir for 1 hour with warm 0.2 M
NaOH (8 mL) and it was then extracted with CH2Cl2. The organic phase was decanted and
evaporated under reduced pressure, but very little material was obtained. m.p. > 300°C.
1
H NMR (CDCl3): δ 12.04 (brs, 1H), 8.16 (d, J = 8.5 Hz, 1 H), 7.65 (m, 4 H), 7.56 (d, J = 8.5
Hz, 1 H).
C NMR (DMSO-d6): δ 186.1, 155.5, 145.9, 140.6, 140.5, 139.8, 139.4, 132.7, 131.3, 130.5,
13
128.5, 127.4.
Anal. Calcd for C13H8ClNS·1/2H2O: C: 56.36; H: 3.15; N: 10.95; S: 12.54. Found: C: 55.98;
H: 3.20; N: 11.36; S: 11.20.
1,10-phenanthroline-2,9(1H,10H)-dithione (32). The 2,9-dichloro-1,10-phenanthroline
(25a) (0.109 g, 0.44 mmol) and the NaSH·xH2O (0.200 g, 3.40 mmol) were placed in a small
109
flask. The DMF (2 mL, 1.88 g, 25.75 mmol) was added to form a bright green mixture, and
the mixture was heated in an oil bath (130-135oC) for 3.5 h. After standing overnight, a
yellow precipitate formed in a dark red-orange liquid. The DMF was removed under reduced
pressure on the water aspirator to leave a yellow-orange solid. Water (2 mL) was added to the
flask, then acetic acid (2 mL, 2 M) was added to form a bright yellow solid. The product was
collected by filtration, then heated in chloroform. The insoluble material was removed by hot
filtration, and the filtrate was rotary evaporated to yield an orange solid (0.071 g, 67%). m.p.>
300°C (lit.,51 m.p.: 325°C, dec.).
1
H NMR (DMSO-d6):  9.7 (brs, 2H), 7.91 (d, J = 9.05 Hz, 2H), 7.67 (s, 2H), 7.41 (d, J = 9.05
Hz, 2H).
C NMR (DMSO-d6):  182.4, 135.1, 133.5, 126.8, 123.6, 123.5.
13
2-(methylthio)-1,10-phenanthroline (35a). A mixture of 1,10-phenanthroline-2(1H)-thione
(33) (0.01 g, 0.046 mmol) and iodomethane (3 mL, 48 mmol) was left to stir at room
temperature for two hours, under a nitrogen atmosphere. The yellow solid that was formed
(35), was collected by filtration and it was treated with H2O (4 mL), then with K2CO3 (pH =
9.0). The aqueous layer was extracted with CH2Cl2 (3 x 5 mL) and the combined organic
extracts were dried over Na2SO4, filtered, to yield very little fine solid.
1
H NMR (DMSO-d6): δ 9.11 (m, 1H), 8.46 (m, 1 H), 8.31 (d, J = 8.4 Hz, 1 H), 7.92 (m, 2 H),
7.75 (m, 1 H), 7.65 (d, J = 8.4 Hz, 1 H), 2.79 (s, 3 H).
10,11-dihydro[1,3]thiazolo[3,2-a]-1,10-phenanthrolin-12-ium bromide (38). A yellow
suspension of 1,10-phenanthroline-2(1H)-thione (33) (0.10 g, 0.46 mmol) and 1,2dibromoethane (20 mL, 40.3 g, 230 mmol) was heated at 120° C for for 7 minutes on which
an orange solution developed. The mixture was heated for 5 hours and an aliquot analyzed by
1
H NMR still indicated starting material. The mixture was heated for an additional 2.5 hours
and allowed to stand at room temperature overnight. The solid was collected by filtration to
afford 0.13 g (93%) of a yellow product. m.p.> 300ºC. The 1H NMR still indicated traces of
starting thione.
1
H NMR (DMSO-d6): δ 9.26 (m, 1H), 9.03 (d, J = 8.8 Hz, 1 H), 8.45 (d, J = 8.6 Hz, 1 H), 8.30
(m, 2 H), 8.01 (m, 1 H), 6.40 (t, J = 8.4 Hz, 1 H), 4.02 (t, J = 8.4 Hz, 2 H), 4.02 (t, J = 8.4 Hz,
2 H).
110
2,9-bis(methylthio)-1,10-phenanthroline (40a). The 2,9-dichloro-1,10-phenanthroline (25a)
(0.046 g, 0.18 mmol) was dissolved in DMF (0.5 mL). The sodium thiomethoxide (0.63 g, 0.9
mmol) was added on which a suspension formed. A slight exothermicity could be noted. The
mixture was stirred for 20 hours at room temperature and a solid filled the flask. Upon
addition of water the white solid which formed was collected by filtration, washed with water
and dried to yield the product (0.43 g, 88%). m.p.: 180-184°C.
1
H NMR (CDCl3): δ 7.95 (d, J = 8.4 Hz, 2H), 7.62 (s, 2H), 7.44 (d, J = 8.4 Hz, 2H), 2.91 (s,
6H).
1
H NMR (DMSO-d6): δ 8.30 (d, J = 8.5 Hz, 2 H), 7.86 (s, 2 H), 7.65 (d, J = 8.5 Hz, 2 H), 2.83
(s, 6 H).
1
H NMR (CF3COOD): δ 8.7 (dd, J = 1.5/8.8 Hz, 2 H), 8.18 (s, 2 H), 8.00 (dd, J = 1.8/8.8 Hz,
2 H), 3.02 (s, 6 H)
C NMR (CDCl3): δ 160.0, 144.9, 135.3, 126.1, 124.7, 122.1, 121.8.
13
2,9-bis(ethylthio)-1,10-phenanthroline (40b). The ethyl iodide (2 mL, 3.9 g, 14 mmol) and
1,10-dihydro-1,10-phenanthroline-2,9-dithione (32) (0.020 mg, 0.081 mmol) were placed in a
flask and stirred at room temperature. The suspension after 1.5 hours turned from yellow to
orange. The mixture was heated at 60oC for 5 hours on which the mixture turned brown. The
product was collected by filtration to yield 0.025 g (97%) of a brown solid.
1
H NMR (DMSO-d6) δ 8.24 (d, J = 8.5 Hz, 2H), 7.83 (s, 2H), 7.56 (d, J = 8.46 Hz, 2H), 3.49 (
q, J = 7.3 Hz, 4H), 1.49 (t, J = 7.3 Hz, 6H).
2-(pyridin-2-ylthio)-1,10-phenanthroline (44a). To a brown suspension of 2-chloro-1,10phenanthroline (16) (0.30 g, 1.40 mmol) and 2-mercapto-pyrdine (0.15 g, 1.4 mmol) in N,Ndimethylformamide (3 mL), potassium carbonate (0.19 g, 1.4 mmol) was added. The reaction
mixture was refluxed for 3.5 hours and it was left to stand overnight. Refluxing was continued
for further 3.5 hours and then the N,N–dimethylformamide was removed through water
aspirator with heating at 60º C. The brown residue was quenched into water (3 mL) and the
aqueous layer was extracted with CHCl3 (2 x 5 mL). The combined organic extracts were
evaporated to dryness under reduced pressure to afford a brown oil. After treatment with ethyl
ether, a brown solid precipitated (0.25 g, 62%). A portion (0.15 g) was purified by gravity
column chromatography over silica gel using CH2Cl2 as eluent followed by CH2Cl2:MeOH
111
96:4 and then CH2Cl2:MeOH 9:1 to afford 0.11 g (73% recovery) as an orange solid. This
material was recrystallized from acetonitrile to yield orange crystals. m.p.: 110-112°C.
1
H NMR (DMSO-d6): δ 9.10 (m, 1 H), 8.58 (d, J = 4.8 Hz, 1 H), 8.50 (m, 1 H), 8.43 (d, J =
8.4 Hz, 1 H), 8.00 (m, 2 H), 7.86 (m, 2 H), 7.78 (m, 1 H), 7.72 (d, J = 8.43, 1 H), 7.37 (m, 1
H).
C NMR (DMSO-d6): δ 157.3, 156.0, 150.5, 140.4, 145.8, 144.9, 138.1, 137.6, 136.5, 129.1,
13
127.0, 126.9, 126.5, 126.2, 124.7, 123.8, 122.8.
MS (ESI) m/z 601 [(2M+Na)+]
2-(pyrimidin-2-ylsulfanyl)-1,10-henanthroline (44b). To a yellow solution of 2-chloro1,10-phenanthroline (16) (0.10 g, 0.46 mmol) in N,N-dimethylformamide (1 mL), potassium
carbonate (0.06 g, 0.46 mmol) and 2-mercaptopyrimidine (0.05 g, 0.46 mmol) were added and
the yellow mixture was refluxed for 3.5 hours. The N,N-dimethylformamide was removed
through vacuum distillation with heating at 60°C, then the brown residue was quenched into
water (3 mL). The mixture was left to stir for one hour and the product was collected by
filtration to yield a pale brown solid (0.10 g, 77%). The crude product was purified by flash
column chromatography on silica gel, using first CH2Cl2 as an eluent, then CH2Cl2:MeOH
99:1 and after CH2Cl2:MeOH 98:2 to yield a pale yellow solid (0.064 g, 49%). m.p.: 179183°C.
1
H NMR (DMSO-d6): δ 9.11 (m, 1 H), 8.68 (d, J = J = 4.8, 2 H), 8.52 (m, 2 H), 8.17 (d, J =
8.8 Hz, 1 H), 8.03 (m, 2 H), 7.78 (m, 1 H), 7.34, (t, J = 4.8, 1 H)
C NMR (DMSO-d6): δ 158.3, 153.8, 150.1, 145.5, 144.8, 137.2, 136.3, 128.7, 128.2, 127.5,
13
127.4, 126.2, 124.6, 123.5, 118.6.
MS (ESI) m/z 603 [(2M+Na)+].
di(1,10-phenanthrolin-2-yl)sulfane (46) – Procedure 1. To a dark yellow suspension of 1H1,10-phenanthroline-2-thione (33) (0.10 g, 0.46 mmol) and N,N-dimethylformamide (2 mL),
first potassium carbonate (0.06 g, 0.06 mmol) was added, followed by 2-chloro-1,10phenanthroline (16)(0.10 g, 0.46 mmol). The reaction mixture was refluxed for 5 hours and
then it was left to stand overnight. The DMF was removed through water aspirator with
heating at 70ºC. The brown residue was quenched into water and it was collected by filtration
to yield a dark yellow solid (0.13 g, 76%). The crude product was purified by gravity column
112
chromatography on silica gel, using CH2Cl2 and then with CH2Cl2:MeOH 99:1 to yield pale
yellow solid (0.051 g, 39% recovery). m.p.: 265-270°C.
1
H NMR (CDCl3): δ 9.23 (dd, J = 1.7/4.3 Hz, 1 H), 8.25 (m, 1 H), 8.15 (d, J = 8.5 Hz, 1 H),
8.02 (d, J = 8.5 Hz, 1 H), 7.78 (m, 2 H), 7.64 (m, 1 H).
1
H NMR (DMSO-d6): δ 9.12 (dd, J = 1.7/4.1 Hz, 1 H), 8.52 (m, 2 H), 8.17 (d, J = 8.4 Hz, 1
H), 8.03 (brs, 2 H), 7.80 (m, 1 H).
C NMR (DMSO-d6): δ 156.8, 150.1, 145.5, 144.6, 137.5, 136.2, 128.8, 126.9, 126.7, 126.2,
13
124.6, 123.5.
Anal. Calcd for C24H14N4S · 1/2H2O: C: 72.16; H: 3.78; N: 14.02; S: 8.03. Found: C: 72.40;
H: 3.74; N: 14.03; S: 6.88.
di(1,10-phenanthrolin-2-yl)sulfane (46) – Procedure 2. To 1H-1,10-phenanthroline-2thione (32) (0.05 g, 0.23 mmol) first N,N-diisopropyl-ethylamine (0.78 g, 1 mL, 6.0 mmol)
was added, then 2-chloro-1,10-phenanthroline (16) (0.05 g, 0.23 mmol). The yellow mixture
was refluxed for 3 hours and it was then cooled at room temperature. The Hünig’s base was
removed through water aspirator with heating at 55°C and the dark residue was quenched into
water (3 mL). The aqueous layer was extracted with CHCl3 (2 x 5 mL) and the combined
extracts were dried over anhydrous sodium sulfate, filtered and evaporated on a rotary
evaporator to yield a dark brown solid (0.054 g, 60%). The crude product was purified by
flash column chromatography over silica gel, using CH2Cl2 and then with CH2Cl2:MeOH,
98:2 and afterwards CH2Cl2:MeOH, 96:4 to afford the product as a light brown solid (0.034 g,
38% recovery).
1
H NMR (DMSO-d6): δ 9.11 (m, 1 H), 8.52 (m, 2 H), 8.16 (d, J = 8.4 Hz, 1 H), 8.02 (brs, 2
H), 7.80 (m, 1 H).
bis(1,10-phenanthroline)[2,1,10,9-bcdef:2’,1’,10’,9’ijklm][1,8]dithia[3,6,10,13]tetraazacyclotetradecine sodium chloride (47a). A yellow
mixture of 9-Chloro-1H-1,10-phenanthroline-2-thione (34) (0.05 g, 0.20 mmol) and
pulverized sodium hydroxide (0.008g, 0.020 mmol) in DMF (2 mL) was heated at 80° C, after
which the reaction mixture became a complete solution. The temperature was kept at 120°C
for 2.5 hours. The orange solution was left to stand overnight at room temperature. Some fine
yellow solid was collected by filtration and washed with ethyl ether, but 1H NMR showed
peaks that were not well resolved. Upon addition of water to the filtrate, the yellow solid
113
which formed was collected by filtration and it was washed thoroughly with ethyl ether and
dried to yield the product (0.013 g, 15%). m.p.> 270°C (lit.,60 m.p.: 372°C).
1
H NMR (DMSO-d6): δ 8.26 (d, J = 8.4 Hz, 2 H), 8.03 (s, 2 H), 7.29 (d, J = 8.4 Hz, 2 H).
1
H NMR (CF3COOD): δ 9.16 (d, J = 9.1 Hz, 2 H), 8.59 (s, 2 H), 8.34 (d, J = 13.9 Hz, 2 H).
bis(1,10-phenanthroline)[2,1,10,9-bcdef:2’,1’,10’,9’ijklm][1,8]dithia[3,6,10,13]tetraazacyclotetradecine (47) – Procedure 1. 9-Chloro-1H1,10-phenanthroline-2-thione (34) (0.06 g, 0.24 mmol) was placed in a small flask and under
a nitrogen atmosphere it was slowly heated using an oil bath, until the temperature of 120°C
was reached. At this point the color changed from yellow to orange. Within 20’ the
temperature reached 300°C and then the thermomether was removed from the oil bath. The
system was heated for further 10’. By this time the temperature may have reached
approximately 320°C. The brown solid was allowed to cool and it was then crystallized from
MeOH to afford a yellow product (0.04 g, 40%). m.p. > 270° C.
1
H NMR (DMSO-d6): δ 8.53 (d, J = 8.8 Hz, 2 H), 7.95 (s, 2 H), 7.88 (d, J = 8.4 Hz, 2 H).
C NMR (DMSO-d6): δ 155.4, 139.3, 139.0, 127.7, 126.9, 124.1.
13
MS (ESI) m/z 421 [M+H]+.
bis(1,10-phenanthroline)[2,1,10,9-bcdef:2’,1’,10’,9’ijklm][1,8]dithia[3,6,10,13]tetraazacyclotetradecine (47) – Procedure 2. A mixture of 9chloro-1H-1,10-phenanthroline-2(1H)-thione (34) (0.06 g, 0.24 mmol) in diphenylether 99%
(3 mL, 3.2 g, 18.9 mmol) was heated until it reached the temperature of 160°C, during which
the mixture turned into a complete orange solution. The temperature was taken up to 170° C
and it was kept for 3 hours. Some solid started to precipitate within the first 10 minutes.
The mixture was left to sit at room temperature for 4 hours. The solid was then collected by
filtration, washed with ether and dried using an oil pump to afford a yellow product (0.05 g,
50%). The crude product was crystallized from MeOH. m.p. > 270° C.
1
H NMR (DMSO-d6): δ 8.57 (d, J = 8.8 Hz, 2 H), 7.99 (s, 2 H), 7.92 (d, J = 8.8 Hz, 2H).
1
H NMR (DMSO-d6 + CF3COOD): δ 8.25 (d, J = 8.8 Hz, 2 H), 7.68 (s, 2 H), 7.57 (d, J = 9.2
Hz, 2 H).
1
H NMR (CF3COOD): δ 8.92 (d, J = 9.2 Hz, 2H), 8.35 (s, 2 H), 8.17 (d, J = 8.8 Hz, 2 H).
C NMR (DMSO-d6): δ 155.3, 139.0, 138.8, 127.6, 126.9, 123.9.
13
MS (ESI) m/z 421 [M+H]+.
114
Anal. Calcd. for C24H12N4S2·2H2O·HCl: C, 58.47; H, 3.48; N, 11.36; S, 13.01, Cl 7.19.
Found: C: 58.49, H: 3.14, N: 11.47, S: 13.31.
Titration for chloride analysis revealed 5.77% chloride.
bis(1,10-phenanthroline)[2,1,10,9-bcdef:2’,1’,10’,9’ijklm][1,8]dithia[3,6,10,13]tetraazacyclotetradecine
cobalt
(2+)
chloride
(48a).
Macrocycle (47) (0.015 g, 0.036 mmol) was treated with CoCl2 (0.005 g, 0.036 mmol) in
ethanol (2 mL) and the red mixture was stirred at room temperature; after 5’ it turned into a
complete solution, while within 15’ some solid deposited, which was collected by filtration
and washed with EtOH after 24 hours, to afford 0.008 g of product (yield 40%).
Anal. Calcd. for C24H12N4S2CoCl2·H2O: C, 50.72; H, 2.48; N, 9.86; S, 11.28. Found: C:
50.93, H: 2.51, N: 10.19, S: 11.00.
IR: a water molecule is confirmed by one absorption at 3200 cm-1, relative to (OH). The
spectrum shows both breathing bands of the heterocyclic ring (1574 cm-1) and a distinct
absorption at 248 cm-1, which can be ascribed to Co-Cl bands.
bis(1,10-phenanthroline)[2,1,10,9-bcdef:2’,1’,10’,9’ijklm][1,8]dithia[3,6,10,13]tetraazacyclotetradecine zinc(2+) bromide (48b). Macrocycle
(47) (0.015 g, 0.036 mmol) was treated with ZnBr2 (0.008 g, 0.036 mmol) in ethanol (2 mL)
and the mixture became instantly cloudy. After 24 hrs stirring at room temperature, a solid
deposited, which was collected by filtration and washed with EtOH, to afford 0.01 g of
product (yield 45%).
Anal. Calcd. for C24H12N4S2ZnBr2·1/2EtOH: C, 44.90; H, 2.26; N, 8.38; S, 9.59. Found: C:
45.59, H: 2.40, N: 9.43, S: 10.25.
IR: the presence of one molecule of EtOH is confirmed by one absorption at 3200 cm-1,
relative to (OH) and one bending at 1660 cm-1. The spectrum reveals also breathing bands of
the heterocyclic ring (1609, 1595s, 1574, 1555 cm-1). Moreover, an intense peak at 248 cm-1
may be ascribed to ZnBr2 bands, between 300 and 280 cm-1.
bis(1,10-phenanthroline)[2,1,10,9-bcdef:2’,1’,10’,9’ijklm][1,8]dithia[3,6,10,13]tetraazacyclotetradecine (47) – Procedure 3. A mixture of
1,10-phenanthroline-2,9(1H,10H)-dithione (32) (0.05 g, 0.20 mmol) and 2,9-dichloro-1,10phenanthroline (25a) (0.051, 0.20 mmol) in diphenylether 99% (2 mL, 2.1 g, 12.3 mmol) was
115
heated until the temperature of 120°C was reached. The suspension became a complete
orange solution and at 140°C some dark yellow solid started to precipitate. The temperature
was taken up to 170°C and it was kept for 3 hours. The orange mixture was left to sit a room
temperature for 5 hours. The precipitate was collected by filtration, washed thoroughly with
ethyl ether and dried on an oil pump to yield 0.11 g of crude product (theor.yield: 0.08 g). It
was then crystallized from MeOH to afford a nice yellow solid (0.05 g, 62%). m.p. > 270° C.
1
H NMR (DMSO-d6): δ 8.48 (d, J = 8.8 Hz, 2 H), 7.90 (s, 2 H), 7.82 (d, J = 8.8 Hz, 2 H).
1,10-phenanthroline-5-carbaldehyde (53) - Procedure 1. A mixture of 5-methyl-1,10phenanthroline (26) (0.04 g, 0.20 mmol) and SeO2 (0.038 g, 0.35 mmol) in naphthalene (0.6
g, 4.68 mmol) was gently heated under a nitrogen atmosphere. When the temperature of
110°C was reached, the yellow solution slowly turned orange and some dark solid started to
precipitate. The mixture was then refluxed for 2.5 h. The dark grey residue was suspended in
CH2Cl2/MeOH (9:1) and the red suspension was filtered through celite. The yellow filtrate
was then evaporated to dryness on a rotary evaporator and the resulting yellow residue was
washed with hexane, to remove naphthalene to yield 0.03 g (71%) of a light pink solid. The
crude solid was purified by gravity column chromatography over neutral Al2O3 eluting with
CH2Cl2: EtOH, 99:1, to yield a tan solid which turned brown in 3 days (0.015 g, 50%
recovery). m.p.: 206-209°C (black dec.).
1
H NMR (CDCl3): δ 10.41 (s, 1 H), 9.74 (dd, J = 1.6/8.4 Hz, 1 H), 9.34 (dd, J = 2.0/4.0 Hz, 1
H), 9.26 (m, 1 H), 8.43 (dd, J = 1.6/8.4 Hz, 1 H), 8.37 (s, 1 H), 7.76 (m, 2 H).
1,10-phenanthroline-5-carbaldehyde (53)– Procedure 2. A suspension of 5-methyl-1,10phenanthroline (26) (0.04 g, 0.2 mmol) and SeO2 (0.07 g, 0.6 mmol) in 1,2-dichlorobenzene
99% (9.3 mL, 12.14 g, 82 mmol) was heated at reflux under a nitrogen atmosphere. At 30°C
all the solid went into solution and at 180°C some dark solid slowly started to precipitate.
After two hours, the orange mixture was cooled to room temperature and a dark orange solid
was filtered off through a pad of celite, which promptly turned red. The filtrate was acidified
with little HCl 1N. The aqueous phase was washed with CH2Cl2 and then neutralized by
saturated aqueous sodium bicarbonate. Dichloromethane was added (7 mL) and extractions
were repeated 3 times. The combined organic layers were dried over magnesium sulphate,
filtered and evaporated on a rotary evaporator. The remained o-dichlorobenzene was taken off
by distillation at 140°C to afford a light grey solid (0.03 g, 75%).
116
5-(bromomethyl)-2,9-dichloro-1,10-phenanthroline (56). An orange mixture of 2,9dichloro-5-methyl-1,10-phenanthroline (25) (0.03 g, 0.14 mmol), benzoyl peroxide (0.004 g,
0.016 mmol) and N-bromosuccinimmide (0.027 g, 0.15 mmol) in dry benzene (2 mL) was
illuminated under a nitrogen atmosphere, with a 300 W incandescent light bulb placed about 5
in. from the flask. When the temperature reached 50°C the mixture turned into a complete
yellow solution. The temperature within the flask was kept at 60°C for 2 hours, after which
the reaction mixture was cooled on ice for 1 hour. The light orange solid that precipitated
(TLC indicated the presence of the succinimide) was collected by filtration and it was washed
with benzene. The filtrate was evaporated under vacuum on a rotary evaporator to yield an
orange oil (0.08 g, theor. yield 0.048 g). The crude product was purified by gravity column
chromatography on silica gel (cyclohexane: dichloromethane: ethylacetate, 6: 2: 2) to afford a
white solid (0.027 g, 56 %). The purified product, dissolved in the eluent mixture outlined
above, on standing overnight in an open dish in hood, appeared as white needles. m.p. : 205.5209.2°C.
1
H NMR (CDCl3): δ 8.55 (d, J = 8.8 Hz, 1 H), 8.19 (d, J = 8.4 Hz, 1 H), 7.90 (s, 1 H), 7.72
(m, 2H), 4.94 (s, 2 H).
2,9-dichloro-5-nitro-1,10-phenanthroline (61).
2,9-dichloro-1,10-phenanthroline (25a)
(0.75 g, 3.0 mmol) in a mixture of concentrated H2SO4 (5.42 mL) and fuming HNO3 (3.3 mL)
was heated to reflux for 1 hour under a nitrogen atmosphere and left to cool to room
temperature. The orange solution was quenched into ice water (10 mL), then it was basified
with a solution of 2 M NaOH until pH = 8-9 was reached. The solid that precipitated, was
filtered over a gooch funnel, washed with H2O and left to dry overnight to yield 0.65 g of a
red solid. Crude product was purified by column chromatography over silica gel, using CHCl 3
as an eluent first and later a mixture CHCl3:MeOH, 99:1, to afford 0.30 g (43%) of a fine pale
yellow solid. m.p.: 241-243°C.
1
H NMR (CDCl3): δ 9.04 (d, J = 9.2 Hz, 1 H), 8.74 (s, 1 H), 8.34 (d, J = 8.8 Hz, 1 H), 7.82
(m, 2 H).
C NMR (DMSO-d6): δ 154.2, 151.9, 145.9, 144.6, 143.8, 136.6, 126.6, 126.3, 125.7, 125.3,
13
120.5.
Anal. Calcd. for C12H5Cl2N3O2·CH3OH: C, 47.88; H, 2.78; Cl, 21.74; N, 12.88; O, 14.72.
Found: C: 47.68, H: 2.41, N: 12.08.
117
MS (ESI) m/z 609 [(2M+Na)+].
12-nitro-6,7-dihydro-5H-[1,4]diazepino[4,3,2,1-lmn][1,10]phenanthroline-4,8-diium (63).
The 5-nitro-1,10-phenanthroline (64) (0.1 g, 0.44 mmol) and acetonitrile (20 mL) were placed
in a flask, then 1,3-dibromopropane (39.79 g, 20 mL, 20 mmol) was added in one portion to
the mixture. The mixture was heated in an oil bath and at the temperature of 40°C it turned
into a complete yellow solution. The reaction mixture was refluxed for four days, on which a
greenish solid precipitated. The product was collected by filtration and washed with Et2O
first, then with CHCl3 to form a yellow-green solid (0.12 g, 63 %); m.p.: 270-275°C (black
dec.)
1,10-phenanthrolin-5-amine (65). 5-Nitro-1,10-phenanthroline (64) (0.2g, 0.89 mmol) and
10% Pd/C (0.056 g, 0.52 mmol) were placed in a tri neck round bottom flask, under a
nitrogen atmosphere. First, absolute ethanol (15.8 mL) was added via syringe while stirring at
r.t., then hydrazine monohydrate, 98% (0,22 mL, 0.23 g, 4.45 mmol) was added dropwise.
The yellow mixture was refluxed for four hours and it was then left to cool at room
temperature. Pd/C was filtered over celite and washed with EtOH, while the filtrate was
concentrated under reduced pressure on a rotary evaporator. The brown-yellow residue was
treated with ether and collected by filtration, to yield 0.18 g (69%) of product. m.p.: 250253°C (lit.84, m.p.: 250°C).
1
HNMR (DMSO-d6): δ 9.04 (dd, J=1.8/4.4 Hz, 1 H), 8.67 (m, 2 H), 8.04 (m, 1 H), 7.73 (m, 1
H), 7.50 (m, 1 H), 6.16 (s, 2 H).
N-(1,10-phenanthrolin-5-yl)acetamide (66) – Procedure 1. To a suspension of 1,10phenanthroline-5-amine (65) (0.04 g, 0.20 mmol) in dry benzene (2 mL), acetic anhydride
(0.021 g, 0.26 mL, 0.21 mmol) was added in one portion at r.t., under a nitrogen atmosphere,
to form a yellow solid in a red liquid. After two hours stirring, the mixture turned into a light
orange color. Reaction progress was monitored by TLC over alumina plate (CHCl3: MeOH,
98:2, Rf = 0.21), showing the disappearance of starting material (Rf = 0.47) after 6 hours. The
mixture was dried under reduced pressure on a rotary evaporator to yield a pink solid (0.02 g,
42%); a sample was crystallized from acetonitrile to give pale pink crystals; m.p.: 236-240°C.
1
H NMR (DMSO-d6): δ 10.15 (brs, 1 H), 9.12 (m, 1 H), 9.02 (m, 1 H), 8.63 (dd, J = 1.4/8.4
Hz, 1 H), 8.44 (m, 1 H), 8.17 (s, 1 H), 7.78 (m, 2 H), 2.24 (s, 3 H).
118
N-(1,10-phenanthrolin-5-yl)acetamide (66) – Procedure 2. To a suspension of 5-amino1,10-phenanthroline (65) (0.04 g, 0.20 mmol) in pyridine(0.1 mL), acetic anhydride (0.021 g,
1 mL, 0.21 mmol) was added in one portion at r.t., under a nitrogen atmosphere, to form a
yellow solid in a red liquid. After one hour stirring, the mixture became pale orange in color.
Reaction progress was monitored by TLC over alumina plate (CHCl3: MeOH, 98:2, Rf =
0.21), showing the disappearance of starting material (Rf = 0.47) after 4 hours. The mixture
was dried under reduced pressure on a rotary evaporator and the obtained reddish oil was
triturated with ethyl ether. The residue was then filtered to give a pink-brown solid (0.04 g,
85%). A sample was crystallized from acetonitrile to yield pale pink crystals; m.p.: 236240°C.
1
H NMR (DMSO-d6): δ 10.15 (brs, 1 H), 9.12 (m, 1 H), 9.02 (m, 1 H), 8.63 (dd, J = 1.4/8.4
Hz, 1 H), 8.44 (m, 1 H), 8.17 (s, 1 H), 7.78 (m, 2 H), 2.24 (s, 3 H).
C NMR (DMSO-d6): δ 169.8, 150.2, 149.6, 146.2, 144.1, 136.1, 132.2, 132.0, 128.4, 124.9,
13
123.9, 123.2, 120.2, 23.9.
Anal. Calcd for C14H11N3O·CH3CN: C, 69.05; H, 5.07; N, 20.13; O, 5.75. Found: C: 69.69;
H: 4.84; N: 17.66.
2-iodo-N-(1,10-phenanthrolin-5-yl)acetamide (68). A mixture of iodoacetic acid (0.4 g,
2.17 mmol) and dicyclohexylcarbodiimide (0.2 g, 0.98 mmol) in ethylacetate (5 mL), was
stirred at room temperature for 3 hours under a nitrogen atmosphere. The resulting urea was
collected by filtration, washing with little ethylacetate, while the filtrate was concentrated on a
rotary evaporator and then dried on an oil pump. To the orange residue (0.36 g), previously
dissolved in acetonitrile (2.5 mL), 1,10-phenanthrolin-5-amine (65) (0.05 g, 0.26 mmol) in
acetonitrile (3.3 mL) was added in one portion and the reaction mixture was stirred at room
temperature for 17 hours under a nitrogen atmosphere, after which some orange/reddish solid
coated the flask. The reaction progress was monitored by TLC over alumina plate (CHCl 3:
MeOH, 98:2), showing the disappearance of starting material and the concurrent formation of
a new spot (Rf = 0.58).
The mixture was then left to sit for 15’ and the supernatant was collected using a pasteur
pipet, while the residue was stirred with cold 5% NaHCO3 (5 mL) at r.t. for 10’. The solid
was collected by filtration, rinsed with little water and dried in a desiccator under vacuum for
one day, to yield a purple product (0.05 g, 53%). m.p.: 206-230° C.
119
1
HNMR (DMSO-d6): δ 10.55 (s, 1 H), 9.10 (m, 2 H,), 8.58 (m, 2 H), 8.18 (s, 1 H), 7.83 (m, 2
H), 4.05 (s, 2 H).
1,9-bis{2-[2-(dimethylamino)ethyl]-1,3,7-trioxo-2,3-dihydro-1H,7H-pyrimido[5,6,1de]acridin-6-yl}-5-methyl-1,5,9-triazanonane (72a). Example of general procedure for
the preparation of 72aj. The 6-chloro-2-[2-(dimethylamino)ethyl]-2,3-dihydro-1H,7Hpyrimido[5,6,1-de]acridine-1,3,7-trione91a
(74f,
0.3
g,
0.81
mmol),
5-methyl-1,5,9-
triazanonane (0.07 mL, 0.405 mmol), and triethylamine (0.5 mL) were stirred in 2ethoxyethanol (10 mL) at 80°C for 2 h. The resulting mixture was extracted with CHCl3 (2 
30 mL) and an excess of 1 M aqueous Na2CO3 (30 mL). The organic layer was worked up to
give a residue which was chromatographed on a silica gel column eluted with CHCl3/MeOH
(4:1 v/v) to give pure 72a.
1
H NMR (CDCl3):  1.902.07 (m, 4H, 2  CH2), 2.32 (s, 3H, CH3), 2.40 (s, 12H, 4  CH3),
2.60 (t, 4H, 2  CH2), 2.72 (t, 4H, 2  CH2), 3.25-3.39 (m, 4H, 2  CH2), 4.28 (t, 4H, 2 
CH2), 6.43 (d, 2H, ar), 7.28 (t, 2H, ar), 7.53 (t, 2H, ar), 7.97 (d, 2H, ar), 8.21 (d, 2H, ar), 8.63
(d, 2H, ar), 10.75 (t, 2H, 2  NH, ex).
Derivative 72bj were prepared in a similar manner from the appropriate 6-chloro-2-[2(dimethylamino)ethyl]-2,3-dihydro-1H,7H-pyrimido[5,6,1-de]acridine-1,3,7-trione91a,b
and
the suitable H2NYNH2.
1,9-bis{2-[2-(dimethylamino)ethyl]-9-hydroxy-1,3,7-trioxo-2,3-dihydro-1H,7Hpyrimido[5,6,1-de]acridin-6-yl}-5-methyl-1,5,9-triazanonane (72k). Example of general
procedure for the preparation of 72k,l and 73e,f. Compound 72h (0.24 g, 0.28 mmol) was
suspended in aqueous HBr 48% (3 mL) and refluxed for 1 h. The mixture was cooled at room
temperature, extracted with CHCl3 (4  20 mL) and an excess of 1 M aqueous Na2CO3 (20
mL). The organic layer was worked up to give a residue, which was flash-chromatographed
on a silica gel column eluted first with CHCl3/MeOH (1:1 v/v) and then with CHCl3/MeOH
(1:1 v/v) and 32% aqueous NH3 (15 mL for 1 L of eluent) to give pure 72h.
1
H NMR (DMSO-d6):  1.79-1.98 (m, 4H, 2  CH2), 2.32 (s, 3H, CH3), 2.36 (s, 12H, 4 
CH3), 2.56-2.73 (m, 8H, 4  CH2), 3.22-3.52 (m, 4H, 2  CH2), 4.05-4.18 (m, 4H, 2  CH2),
6.50 (d, 2H, ar), 7.06 (d, 2H, ar), 7.43 (s, 2H, ar), 7.78 (d, 2H, ar), 8.48 (d, 2H, ar), 10.01 (b t,
2H, 2  OH, ex), 10.67 (t, 2H, 2  NH, ex).
120
Derivatives 72l and 73e,f were prepared in a similar manner from 72i and 73c,d, respectively.
1,9-bis{2-[2-(dimethylamino)ethyl]-9-nitro-1,3,7-trioxo-2,3-dihydro-1H,7Hpyrimido[5,6,1-de]acridin-6-yl}-5-methyl-1,5,9-triazanonane (72m). To a mixture of 71b89
(0.23 g, 0.81 mmol) and triethylamine (0.75 mL) in CHCl3 (10mL) COCl2 (20% in toluene,
0.7 mL, 1.4 mmol) in CHCl3 (10mL) was added dropwise with stirring at 0 °C. The stirring
was protracted for 20 min at room temperature. The mixture was extracted with CHCl3 (2 
20 mL) and an excess of 1 M aqueous Na2CO3 (30 mL). The organic layer was worked up to
give a residue, which was chromatographed on a silica gel column eluted with CHCl3/MeOH
(1:1 v/v) to give pure 72m.
1
H NMR (CDCl3):  1.89-2.10 (m, 4H, 2  CH2), 2.36 (s, 15H, 5  CH3), 2.59-2.73 (m, 8H, 4
 CH2), 3.32-3.48 (m, 4H, 2  CH2), 4.26 (t, 4H, 2  CH2), 6.50 (d, 2H, ar), 7.99 (d, 2H, ar),
8.28 (d, 2H, ar), 8.79 (d, 2H, ar), 8.96 (s, 2H, ar), 10.66 (t, 2H, 2  NH, ex).
1,9-bis{2-[2-(dimethylamino)ethyl]-2,6-dihydropyrazolo[3,4,5-kl]acridine-5-carbonyl}-5methyl-1,5,9-triazanonane · 3HCl (73a). Example of general procedure for the
preparation of 73ad. The hydrochloride salt of 77a89 (0.25 g, 0.36 mmol) and [(2hydrazino)ethyl]dimethylamine (0.37 g, 3.6 mmol) were stirred in 2-ethoxyethanol (10 mL) at
120 °C for 1 h. The resulting mixture was extracted with CHCl3 (2  30 mL) and an excess of
1 M aqueous Na2CO3 (30 mL). The organic layer was worked up to give a residue which was
flash-chromatographed on a silica gel column eluted with CHCl3/MeOH (1:1 v/v) and 32%
aqueous NH3 (10 mL for 1 L of eluent) to give pure 73a, which was directly transformed into
a hydrochloride salt and, likewise characterized.
1
H NMR (DMSO-d6):  1.90-2.04 (m, 4H, 2  CH2), 2.73 (s, 3H, CH3), 2.81 (s, 12H, 4 
CH3), 2.97-3.27 (m, 4H, 2  CH2), 3.29-3.41 (m, 4H, 2  CH2), 3.50-3.63 (m, 4H, 2  CH2),
4.72 (t, 4H, 2  CH2), 6.82 (d, 2H, ar), 7.10 (t, 2H, ar), 7.32 (t, 2H, ar), 7.50 (d, 2H, ar), 7.727.87 (m, 4H, ar) 8.50-8.60 (m, 2H, 2  CONH, ex), 10.55-10.83 (m, 5H, 2  6H + 3  N+H,
ex).
Derivative 73bd were prepared in a similar manner from the appropriate bis(acridine-4carboxamide) 7789 and [(2-hydrazino)ethyl]dimethylamine.
121
Biophysical Evaluation. 1. Fluorescence Binding Studies. The fluorometric assays have
been described previously.93 The C50 values for ethidium displacement from CT-DNA and
from synthetic [poly(dA-dT)]2 (AT) and [poly(dG-dC)]2 (GC) oligonucleotides were
determined using aqueous buffer (10 mM Na2HPO4, 10 mM NaH2PO4, 1 mM EDTA, pH 7.0)
containing 1.26 M ethidium bromide and 1 M CT-DNA, AT, and GC, respectively.93,94
All measurements were made in 10-mm quartz cuvettes at 20 °C using a Perkin-Elmer LS5
instrument (excitation at 546 nm; emission at 595 nm) following serial addition of aliquots of
a stock drug solution (~5 mM in DMSO). The C50 values are defined as the drug
concentrations which reduce the fluorescence of the DNA-bound ethidium by 50%, and are
calculated as the mean from three determinations.
2. In Vitro Cytotoxicity. Cell Culturing. Human colon adenocarcinoma HT29 and HCT116
cell lines. The cells were cultured in D-MEM medium supplemented with 10% FBS, 2 mM
glutamine, 100 units/mL penicillin, and 100 mg/mL streptomycin in a humidified atmosphere
with 5% CO2 at 37°C. Human large cell lung carcinoma H460M, gastric cancer MKN45, and
prostate carcinoma PC-3 cell lines. The cells were seeded into 75 cm2 culture flasks in RPMI
1640 medium with supplemented with L-glutamine, 10% fetal calf serum, and penicillinstreptomycin (100 units/mg). Culturing details of human colon adenocarcinoma carcinoma
LoVo sensitive and LoVo/Dx resistant cell lines have been previously described.131
Cell Growth Inhibition Assays. For the cytotoxicity evaluation of the compounds the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay was
used.132 Briefly, cells at appropriate densities, depending on the growth characteristics of
particular cell lines, were seeded in 96 microwell plates and preincubated for 24 h. After this
time, the cells were exposed to drugs and reincubated for 144 h (drug exposure time) at 37 °C
in an atmosphere of 5% CO2. The IC50 values were determined as previously described.133 All
assays were performed in duplicate.
NCI Assays.96 The NCI uses the sulforhodamine B assay for assessing the cytotoxicity of test
agents in their panel of 60 cell lines.134 Briefly, cell lines were inoculated into a series of 96well microtiter plates, with varied seeding densities depending on the growth characteristics
of particular cell lines. Following a 24-h drug-free incubation, test agents were added
routinely at five 10-fold dilutions with a maximum concentration of 10-4 M. After 2 days of
122
drug exposure, the change in protein stain optical density allowed the inhibition of cell growth
to be analyzed.
3. In Vivo Hollow Fiber Assay.96 Human tumor cells were cultivated in polyvinylidene
fluoride (PVDF) hollow fibers, and samples of the cells were implanted into two physiologic
compartments of each mouse. The mice were treated by the intraperitoneal route with the
analogues over time, the fibers were harvested the day following the last treatment, and the
degree of cell kill compared to controls was measured by optical density. Each compound was
tested at two different doses against a minimum of 12 human cancer cell lines.
6-chloro-2-[(6-methoxy-3-pyridyl)amino]-3-nitrobenzoic acid (81). The 2,6-dichloro-3nitrobenzoic acid (80, 0.5 g, 2.12 mmol) was added in portions to 6-methoxy-3-pyridinamine
(79, 1.05 g, 8.48 mmol) within 10 min. The resulting mixture was heated at 75°C under
stirring for 24 h. After cooling, 1 N aqueous Na2CO3 (5 mL) was poured in the mixture and
the precipitated solid was filtered off. The solution, acidified with 2 M aqueous HCl, was
extracted with CHCl3 (3  30 mL). The organic layer was worked up to give 81 as a solid
residue (0.55 g, 80%) enough pure. TLC over silica gel using CHCl3/MeOH (4:1) as an eluent
mixture, was used for next step. A small sample was purified by column chromatography over
silica gel, first dissolving the sample in methanol (2 mL) and then eluting with CHCl 3/MeOH
(30:1), to afford pure 81; m.p. 104-105°C.
1
H NMR (DMSO-d6): δ 3.82 (s, 3H, CH3), 6.71 (d, 1H, ar), 7.20 (d, 1H, ar), 7.38 (d, 1H, ar),
7.85 (s, 1H, ar), 8.10 (d, 1H, ar), 8.82 (s, 1H, COOH ex), 13.54 (brs, 1H, NH, ex).
9-chloro-2-methoxy-6-nitro-5,10-dihydrobenzo[b][1,5]naphthyridin-10-one (82). To the
6-chloro-2-[(6-methoxy-3-pyridyl)amino]-3-nitrobenzoic acid (81, 100 mg, 0.31 mmol)
H2SO4 (0.5 mL) was added and the mixture was heated at 100°C for 30 min. It was then
poured into crushed ice to yield an orange precipitate, which was filtered, washed with
portions of water and methanol, and dried at room temperature. The resulting solid 82 (80 mg,
84 %) was enough pure. TLC over silica gel, eluted with CHCl3/MeOH (99:1), was used for
the next step. A small sample was purified by column chromatography over silica gel,
dissolving the crude product in hot methanol (1 mL) and then eluting with CHCl3 to afford
pure 82; m.p. 191-193°C.
123
1
H NMR (DMSO-d6): δ 3.97 (s, 3H, CH3), 7.32 (d, 1H, ar), 7.42 (d, 1H, ar), 8.488.58 (m,
2H, ar), 11.62 (s, 1H, NH, ex).
2-[2-(dimethylamino)ethyl]-9-methoxy-5-nitro-2,6-dihydroindazolo[4,3bc][1,5]naphthyridine (2a). Example of general procedure for the preparation of 78af.
N-(2-Hydrazinoethyl)-N,N-dimethylamine101 (0.16 g, 1.33 mmol) was added to a suspension
of compound 82 (0.2 g, 0.65 mmol) in tetrahydrofuran-methanol 1:1 (7 mL). The mixture was
stirred at room temperature for 3 h, the obtained orange precipitate was filtered and washed
with tetrahydrofuran-methanol 1:1. The dried crude solid was suspended in CHCl3 and flash
chromatographed over a silica gel eluting with CHCl3/MeOH (19:1) to yield pure 78a (78 %):
m.p. 257-258°C; hydrochloride m.p. > 300 °C.
1
H NMR (DMSO-d6): δ 2.20 (s, 6H, 2  NCH3), 2.76 (t, 2H, CH2-2'), 3.97 (s, 3H, OCH3),
4.52 (t, 2H, CH2-1'), 6.92 (d, 1H, ar), 7.03 (d, 1H, ar), 7.92 (d, 1H, ar), 8.48 (d, 1H, ar), 11.38
(s, 1H, NH, ex).
Anal. Calcd. for C17H18N6O3: C, 57.62; H, 5.12; N, 23.72. Found: C, 57.23; H, 5.09; N, 23.93.
Derivatives 78bf were prepared in a similar manner.
2-[3-(dimethylamino)propyl]-9-methoxy-5-nitro-2,6-dihydroindazolo[4,3bc][1,5]naphthyridine (78b). (58 %); m.p.: 257-258 °C; hydrochloride 268-270 °C dec.; 1H
NMR (DMSO-d6): δ 1.952.13 (m, 2H, CH2-2'), 2.142.33 (m, 8H, 2  NCH3 + CH2-3'), 3.95
(s, 3H, OCH3), 4.45 (t, 2H, CH2-1'), 6.88 (d, 1H, ar), 7.03 (d, 1H, ar), 7.91 (d, 1H, ar), 8.46 (d,
1H, ar), 11.38 (s, 1H, NH, ex). Anal. calcd for C 18H20N6O3: C, 58.69; H, 5.47; N, 22.81. Found:
C, 58.83; H, 5.29; N, 22.93.
2-[2-(diethylamino)ethyl]-9-methoxy-5-nitro-2,6-dihydroindazolo[4,3bc][1,5]naphthyridine (78c). (58 %); m.p. 249-250°C; hydrochloride m.p. > 300 °C; 1H
NMR (DMSO-d6): δ 0.85 (t, 6H, 2  NCH3), 2.422.52 (m, 4H, 2  diethyl CH2), 2.90 (t,
2H, CH2-2'), 3.98 (s, 3H, OCH3), 4.49 (t, 2H, CH2-1'), 6.90 (d, 1H, ar), 7.06 (d, 1H, ar), 7.95
(d, 1H, ar), 8.48 (d, 1H, ar), 11.40 (s, 1H, NH, ex). Anal. calcd for C19H22N6O3: C, 59.67; H,
5.80; N, 21.98. Found: C, 59.88; H, 5.59; N, 22.13.
9-methoxy-5-nitro-2-[2-(1-pyrrolidinyl)ethyl]-2,6-dihydroindazolo[4,3bc][1,5]naphthyridine (78d). (77 %); m.p.: 249-250°C dec; hydrochloride m.p.: 248-250 °C;
124
1
H NMR (DMSO-d6): δ 2.582.70 (m, 4H, 2  CH2-2"), 2.522.62 (m, 4H, 2  CH2-3"),
3.00 (t, 2H, CH2-2'), 3.98 (s, 3H, OCH3), 4.58 (t, 2H, CH2-1'), 6.93 (d, 1H, ar), 7.04 (d, 1H,
ar), 7.91 (d, 1H, ar), 8.46 (d, 1H, ar), 11.41 (s, 1H, NH, ex). Anal. calcd for C19H20N6O3: C,
59.99; H, 5.30; N, 22.09. Found: C, 59.76; H, 5.48; N, 22.27.
9-methoxy-5-nitro-2-(2-piperidinoethyl)-2,6-dihydroindazolo[4,3-bc][1,5]naphthyridine
(78e). (77 %); m.p.: 290-291°C; hydrochloride m.p. > 300°C; 1H NMR (DMSO-d6): δ
1.201.55 (m, 6H, 2  CH2-2" + CH2-3"), 2.312.54 (m, 4H, 2  CH2-1"), 2.632.90 (m,
2H, CH2-2'), 3.98 (s, 3H, OCH3), 4.424.63 (m, 2H, CH2-1'), 6.88 (d, 1H, ar), 7.02 (d, 1H, ar),
7.90 (d, 1H, ar), 8.46 (d, 1H, ar), 11.40 (s, 1H, NH, ex). Anal. calcd for C20H22N6O3: C, 60.90;
H, 5.62; N, 21.31. Found: C, 60.67; H, 5.89; N, 21.05.
9-methoxy-2-(2-morpholinoethyl)-5-nitro-2,6-dihydroindazolo[4,3-bc][1,5]naphthyridine
(78f). (38 %); m.p.: 242-243 °C; hydrochloride m.p.: 265-267°C dec; 1H NMR (DMSO-d6): δ
2.43 (t, 4H, 2  OCH2), 2.81 (t, 4H, 2  NCH2), 3.45 (t, 2H, CH2-2'), 3.98 (s, 3H, OCH3),
4.57 (t, 2H, CH2-1'), 6.95 (d, 1H, ar), 7.05 (d, 1H, ar), 7.96 (d, 1H, ar), 8.50 (d, 1H, ar), 11.42
(s, 1H, NH, ex). Anal. calcd for C19H20N6O4: C, 57.57; H, 5.09; N, 21.20. Found: C, 57.35; H,
4.80; N, 21.37.
Fluorescence binding studies. The fluorometric assays have been described earlier.93a The
C50 values for ethidium displacement from CTDNA, AT, and GC were determined using
aqueous buffer (10 mM Na2HPO4, 10 mM NaH2PO4, 1 mM EDTA, pH 7.0) containing 1.26
μM ethidium bromide and 1 μM CTDNA, AT, and GC, respectively.93a,94a,94b
All measurements were made in 10-mm quartz cuvettes at 20 °C using a Perkin-Elmer LS5
instrument (excitation at 546 nm; emission at 595 nm) following serial addition of aliquots of
a stock drug solution (~5 mM in DMSO). The C50 values are defined as the drug
concentrations which reduce the fluorescence of the DNA-bound ethidium by 50%, and are
calculated as mean values from three determinations.
In vitro cytotoxicity assay. Human androgen-independent prostate adenocarcinoma cell line
(PC-3) was used for cytotoxicity testing in vitro using the SRB (Sulforhodamine B) assay. 135
Cells were maintained as stocks in DMEM (Gibco) supplemented with 10% fetal bovine
serum (Gibco), 2mM L-glutamine (Gibco). Cell cultures were passaged twice weekly using
125
trypsin-EDTA to detach the cells from their culture flasks. The rapidly growing cells were
harvested, counted, and incubated under the appropriate concentrations (7x105 cells/well) in
96-well microtiter plates. After incubation for 24 h, target and reference compounds dissolved
in culture medium were applied to the culture wells in quadruplicate and incubated for 48 h at
37°C in a 5% CO2 atmospere and 95% relative humidity. At the same time a plate is tested to
value the cell population before the drug addition (Tz). Culture fixed with cold trichloroacetic
acid (TCA) (J.T. Baker B.V., Deventer, Holland), were stained by 0,4% Sulforhodamine B,
(SRB) (Sigma-Aldrich, Milan, Italy) dissolved in 1% acetic acid. Bound stain is subsequently
solubilized with 10 mM Trizma (Sigma-Aldrich, Milan, Italy), and the absorbance read on the
microplate reader Dynatech Model MR 700 at a wavelength of 520 nm. The cytotoxic activity
was evaluated by measuring the drug concentration resulting in a 50% reduction in the net
protein increase (as measured by SRB staining) in control cells during the drug incubation
(GI50), the drug concentration resulting in total growth inhibition (TGI) and the drug
concentration resulting in a 50% reduction in the measured protein at the end of the drug
treatment as compared to that at the beginning (LC50). The percentage of growth inhibition
was calculated as:[(Ti-Tz)/(C-Tz)] x 100 for concentration, where Ti>=Tz; [(Ti-Tz)/Tz] x 100
for concentration, where Ti<Tz, where Tz = absorbance time zero, C = absorbance in
presence of vehicle and Ti = absorbance in presence of drug at different concentration.GI50,
TGI and LC50 were obtained by interpolation in a graph % of growth versus Log(M). Each
quoted value represents the mean of quadruplicates value.
Apoptotic assays. Apoptosis of PC-3 cells treated with about 1  LD50 of target compounds
and reference was evaluated by annexin V binding111 and biparametric PI/Annexin V
cytofluorimetric analysis as well as agarose gel electrophoresis.112 To detect early stages of
apoptosis, the expression of Annexin-V, a Ca2+-dependent phospholipid-binding protein with
high affinity for phosphatidylserine was employed. Moreover, simultaneous staining of cells
with FITC-Annexin-V and with PI, allows the discrimination of intact cells (Annexin V PI),
early apoptotic (Annexin V+ PI) and late apoptotic or necrotic cells (Annexin V+ PI+).23
Apoptotic cells become Annexin V+ after nuclear condensation has started, but before the
cells becomes permeable to PI. Briefly, 2  106 PC-3 cells treated with 1  LC50 of target
compounds 78c,d and 78f (50 μM) and parent compound PZA (70 μM) for 6 h, were
resuspended in 0.2 ml of binding buffer (10 mM Hepes/NaOH, pH 7.4, 150 mM NaCl, 5 mM
KCl, 1 mM MgCl2, 1.8 mM CaCl2) in the presence of 5 μl of FITC-Annexin V (Bender
126
MedSystem, Vienna, Austria), were incubated for 10 min at room temperature in the dark.
Cells were washed, resuspended in 0.2 ml of binding buffer containing 10 μl of PI (20 μg/ml
in PBS) (Molecular Probes, Eugene, OR) and then analyzed as above mentioned.
The percentage of positive cells determined over 10.000 events was analyzed on a FACScan
cytofluorimeter (Becton Dickinson, San Jose’, CA). Fluorescence intensity is expressed in
arbitrary units on a logarithmic scale.
DNA fragmentation assay. Time-course of target compounds 78d,c,f-induced nucleosomal
DNA fragmentation was performed by agarose gel electrophoresis. Briefly, 5  105/ml were
cultured at 37°C, 5 % CO2, treated with 50 μM of target compounds 78c,d,f and 70 μM of
reference compound PZA in different times (2, 4, 6 and 8h). After treatment cells were
washed and DNA was extracted using the Genomix Cells and Tissues Mini Preparations kit
(Talent, Trieste, Italy). The DNA samples were electrophoresed on a 1.7% agarose gel and
stained with ethidium bromide and acquired by a ChemiDoc (BioRad, Milano, Italy).
6-{[(2-dimethylamino)ethyl](methyl)amino}-4-methyl-1-phenyl-3H-naphtho[1,2,3de]quinoline-2,7-dione (3a). Example of general procedure for the preparation of 85fi.
The commercially available 6-Bromo-4-methyl-1-phenyl-3H-naphtho[1,2,3-de]quinoline-2,7dione (300 mg, 0.72 mmol) was suspended in 2-ethoxyethanol (20 ml), added of N1,N1,N2trimethyl-1,2-ethanediamine (0.92 ml, 7.2 mmol), and refluxed for 4 h under stirring.
Reaction progress was monitored by TLC over silica gel using CHCl3/MeOH (30:1 v/v) as an
eluent mixture. The solvent was evaporated and the residue was refluxed in methanol. After
cooling, a solid was obtained, which was washed with methanol and ether, then dried at room
temperature to yield pure 85a (89 %): m.p.: 220-221°C; hydrochloride 113-116°C.
1
H NMR (CDCl3):  2.37 (s, 6H, 2  CH3), 2.54 (s, 3H, 4-CH3), 2.68-2.83 (m, 2H, CH2), 2.90
(s, 3H, 6-NCH3), 3.58 (t, 2H, CH2), 7.07-7.13 (m, 2H, ar), 7.27 (s, 1H, ar), 7.35-7.45 (m, 5H,
ar), 8.16 (d, 1H, ar), 10.78 (brs, 1H, NH ex).
Anal. Calcd. for C28H27N3O2: C, 76.86; H, 6.22; N, 9.60. Found: C, 76.94; H, 6.01; N, 9.94.
Derivatives 85fi were prepared in a similar manner.
6-({2-[(2-hydroxyethyl)amino]ethyl}amino)-4-methyl-1-phenyl-3H-naphtho[1,2,3de]quinoline-2,7-dione (85f). (82 %); m.p.: 159-161°C; hydrochloride 201-203°C; 1H NMR
(DMSO-d6):  2.57-2.77 (m, 5H, CH2  CH3), 2.92 (t, 2H, CH2), 3.43-3.60 (m, 4H, 2 
127
CH2), 4.54 (s, 1H, OH ex), 7.12-7.20 (m, 2H, ar), 7.29-7.58 (m, 9H, 7ar + 2  NH ex), 8.38
(d, 1H, ar), 10.87 (s, 1H, 6-NH ex). Anal. Calcd. for C27H25N3O3: C, 73.78; H, 5.73; N, 9.56.
Found: C, 73.89; H, 5.51; N, 9.42.
4-methyl-1-phenyl-6-[(2-piperidinoethyl)amino]-2,7-dihydro-3H-naphtho[1,2,3de]quinoline-2,7-dione (85g). (51 %); m.p.: 259-260°C; hydrochloride > 300°C;
hydrochloride 1H NMR (DMSO-d6):  1.30-1.92 (m, 6H, 3  CH2), 2.70 (s, 3H, CH3), 2.883.10 (m, 2H, CH2), 3.24-3.41 (m, 2H, CH2), 3.47-3.60 (m, 2H, CH2), 4.00 (t, 2H, CH2), 6.757.70 (m, 10H, 9ar + NH ex), 8.36 (d, 1H, ar), 10.58-10.94 (m, 2H, 2  NH ex). Anal. Calcd.
for C30H29N3O2: C, 77.73; H, 6.31; N, 9.06. Found: C, 77.98; H, 6.53; N, 8.84.
4-methyl-1-phenyl-6-[(2-pyrrolidin-1-yl-ethyl)amino]-2,7-dihydro-3H-naphtho[1,2,3de]quinoline-2,7-dione (85h). (77 %); m.p.: 168-170°C; hydrochloride > 300°C; 1H NMR
(CDCl3):  1.80-1.95 (m, 4H, 2  CH2), 2.57 (s, 3H, CH3), 2.64-2.80 (m, 4H, 2  CH2), 2.93
(t, 2H, CH2), 3.59-3.70 (m, 2H, CH2), 7.07-7.17 (m, 2H, ar), 7.23-7.28 (m, 1H, ar), 7.37-7.50
(m, 6H, ar), 8.42 (d, 1H, ar), 10.08 (brs, 1H, NH ex), 10.68 (t, 1H, 6-NH ex). Anal. Calcd. for
C29H27N3O2: C, 77.48; H, 6.05; N, 9.35. Found: C, 76.29; H, 6.33; N, 9.04.
4-methyl-6-[(2-morpholinoethyl)amino]-1-phenyl-2,7-dihydro-3H-naphtho[1,2,3de]quinoline-2,7-dione (85i). (69 %); m.p.: 264-266°C; hydrochloride 186-189°C;
hydrochloride 1H NMR (DMSO-d6):  2.70 (s, 3H, CH3), 3.13-3.28 (m, 2H, CH2), 3.37-3.47
(m, 2H, CH2), 3.49-3.60 (m, 2H, CH2), 3.78-3.92 (m, 2H, CH2), 3.96-4.09 (m, 4H, 2  CH2),
5.05 (brs, 1H, NH ex), 7.13-7.25 (m, 2H, ar), 7.30-7.40 (m, 2H, ar), 7.42-7.61 (m, 5H, ar),
8.38 (d, 1H, ar), 10.79 (brs, 1H, NH ex), 11.58 (brs, 1H, NH ex). Anal. Calcd. for
C29H27N3O3: C, 74.82; H, 5.85; N, 9.03. Found: C, 74.98; H, 5.62; N, 9.25.
6-{[(2-Dimethylamino)ethyl]amino}-4-methyl-1-phenyl-3H-naphtho[1,2,3-de]quinoline2,7-dione (85b). Example of general procedure for the preparation of 85be. The
commercially
available
6-Bromo-4-methyl-1-phenyl-3H-naphtho[1,2,3-de]quinoline-2,7-
dione (200 mg, 0.48 mmol) was suspended in N1,N1-dimethyl-1,2-ethanediamine (4 ml) and
refluxed for 2 h under stirring. Reaction progress was monitored by TLC over silica gel using
CHCl3/MeOH (9:1 v/v) as an eluent mixture. After cooling, the obtained solid was washed
128
several times with methanol and ether, then it was dried at room temperature to yield pure 85b
(69 %): m.p.: 163-165°C; hydrochloride 263-265°C.
hydrochloride 1H NMR (DMSO-d6):  2.70 (s, 3H, CH3), 2.88 (d, 6H, 2  CH3), 3.30-3.46
(m, 2H, CH2), 3.88-4.06 (m, 2H, CH2), 7.10-7.27 (m, 2H, ar), 7.29-7.41 (m, 2H, ar), 7.437.60 (m, 6H, 5ar + NH ex), 8.37 (d, 1H, ar), 10.70 (brs, 1H, NH ex), 10.90 (brs, 1H, NH ex).
Anal. Calcd. for C27H25N3O2: C, 76.57; H, 5.95; N, 9.92. Found: C, 76.24; H, 6.12; N, 10.14.
Derivatives 85be were prepared in a similar manner.
6-{[(3-dimethylamino)propyl]amino}-4-methyl-1-phenyl-3H-naphtho[1,2,3-de]quinoline2,7-dione (85c). (78 %); m.p. 231-233°C; hydrochloride 170-171°C; 1H NMR (CDCl3): 
1.90-2.10 (m, 2H, CH2), 2.31 (s, 6H, 2  CH3), 2.50 (t, 2H, CH2), 2.57 (s, 3H, CH3), 3.423.58 (m, 2H, CH2), 7.03-7.19 (m, 2H, ar), 7.24-7.32 (m, 2H, ar), 7.36-7.52 (m, 6H, 5ar + NH
ex), 8.45 (d, 1H, ar), 10.67 (t, 1H, NH ex). Anal. Calcd. for C28H27N3O2: C, 76.86; H, 6.22; N,
9.60. Found: C, 76.73; H, 6.31; N, 9.31.
6-{[(2-diethylamino)ethyl]amino}-4-methyl-1-phenyl-3H-naphtho[1,2,3-de]quinoline-2,7dione (85d). (52 %); m.p.: 151-153°C; hydrochloride 269-270°C; 1H NMR (CDCl3):  1.18
(t, 6H, 2  CH3), 2.58 (s, 3H, CH3), 2.67-2.86 (m, 4H, 2  CH2), 2.93 (t, 2H, CH2), 3.523.70 (m, 2H, CH2), 7.07-7.19 (m, 2H, ar), 7.23-7.31 (m, 2H, ar), 7.38-7.52 (m, 6H, 5ar + NH
ex), 8.47 (d, 1H, ar), 10.63 (t, 1H, NH ex). Anal. Calcd. for C29H29N3O2: C, 77.13; H, 6.47; N,
9.31. Found: C, 76.98; H, 6.53; N, 9.64.
6-{[(3-diethylamino)propyl]amino}-4-methyl-1-phenyl-3H-naphtho[1,2,3-de]quinoline2,7-dione (85e). (87 %); m.p.: 193-195°C; hydrochloride 210-211°C; 1H NMR (CDCl3): 
1.10 (t, 6H, 2  CH3), 1.93-2.07 (m, 2H, CH2), 2.57 (s, 3H, CH3), 2.60-2.85 (m, 6H, 3 
CH2), 3.42-3.56 (m, 2H, CH2), 7.01 (s, 1H, ar), 7.06-7.12 (m, 1H, ar), 7.22-7.30 (m, 2H, ar),
7.38-7.48 (m, 6H, 5ar + NH ex), 8.43 (d, 1H, ar), 10.63 (t, 1H, NH ex). Anal. Calcd. for
C30H31N3O2: C, 77.39; H, 6.71; N, 9.03. Found: C, 77.09; H, 6.54; N, 9.24.
6-{[(2-Amino)ethyl]amino}-4-methyl-1-phenyl-3H-naphtho[1,2,3-de]quinoline-2,7-dione
(85j).
The
commercially
available
6-Bromo-4-methyl-1-phenyl-3H-naphtho[1,2,3-
de]quinoline-2,7-dione (300 mg, 0.72 mmol) was suspended in 2-ethoxyethanol (20 ml),
129
added of tert-butyl N-(2-aminoethyl)carbamate (860 mg, 5.4 mmol), and refluxed for 24 h
under stirring. Reaction progress was monitored by TLC over silica gel using CHCl 3/MeOH
(30:1 v/v) as an eluent mixture. After cooling to room temperature, a solid precipitated, which
was washed subsequently with methanol and ether. The solid (85j N-Boc protected) was
suspended in dioxane (20 ml) and 37 % HCl and stirred at room temperature for 7 h. The
volatile was evaporated and the residue was refluxed in ethanol. After cooling, a solid was
obtained which was washed with ethanol and ether, and dried at room temperature to yield
pure 85j (35 %): m.p.: 259-261°C; hydrochloride 264-267°C;
hydrochloride 1H NMR (DMSO-d6):  2.64 (s, 3H, 4-CH3), 3.03-3.20 (m, 2H, CH2), 3.653.87 (m, 2H, CH2), 7.09-7.30 (m, 2H, ar), 7.30-7.59 (m, 7H, ar), 7.83-8.03 (m, 3H, NH3+),
8.30-8.42 (m, 1H, ar), 10.50 (brs, 1H, 3-H ex), 11.50 (brs, 1H, 6-NH ex).
Anal. Calcd. for C25H21N3O2: C, 75.93; H, 5.35; N, 10.63. Found: C, 76.09; H, 5.53; N, 10.78.
In vitro cytotoxicity. HT29 human colon adenocarcinoma. Details regarding HT29 human
colon adenocarcinoma cell line assay have been previously described.133 Drug solutions at
appropriate concentrations were added to a culture containing HT29 cells at 2.5  104 cells/ml
of medium and the drug exposure was protracted for 144 h. All assays were performed in
triplicate, as previously described.133
In vitro cytotoxicity. Human Ovarian Carcinoma Experimental Protocol. Established
details and biological properties of human ovarian carcinoma cell lines (A2780, A2780cisR,
CH1, CH1cisR, and SKOV-3) have been previously described.136 The sulforhodamine B
(SRB) experimental protocol used has been described previously.136,93a Cells were plated
(100–5000 cells) in 96-well microtiter plates and left overnight to adhere prior to drug
treatment. Aqueous drug solutions at pH 7.0 were then added to the cells at various
concentrations following dilution of a stock DMSO solution. After 96 h continuous drug
exposure at 37 °C, growth inhibition was assessed using SRB protein staining. IC50 values, as
mean of two independent assays, (drug dose required for 50% growth inhibition compared to
drug-free controls) were determined by comparing treated and untreated cells.
130
Conclusions
All of the published research dealing with 1,10-phenanthrolines that was herein
reported, show the great versatility of this ligand along with a strong interest as a DNAligand. The applications of metal binding components in different aspects of coordination
chemistry and areas such as supramolecular and bioinorganic chemistry are of interest.
Much research still needs to be done in order to synthesize the target molecule,
which was the initial goal of the current research. Many efforts were directed to the
production of a set of new phenanthroline derivatives, with modifications at the 2- ,9- and 5positions. The preparation of the sulphur macrocycle has been accomplished by treatment of
the thione with dichlorophenanthroline and applied to the 5-methyl analogue and in a
preliminary run to the 5-nitrophen. These latter derivatives would be utilized for side chain
extensions to form the (CH2)2N(CH3)2 side arm and evaluated as a potential G-quadruplex
stabilizer.
The preparation of some mono-substituted sulfur linked heterocycles at position 2- of
1,10-phenanthroline has been performed. The structures of the thiones, dithione, amides and
diamides have been clearly established by NMR spectroscopy. The alkylations of the thione
and the dithiones have also been accomplished.
A number of other substituted derivatives may still be prepared and it cannot be
excluded that some of these may be relevant from a biological standpoint. It is also
fundamental to point out that experiments that appear easy on paper, are often difficult to
perform in the laboratory. Many factors that may limit the rate of reactions need to be taken
into account. It was observed that while the introduction of a nitro group at 5- position of the
2,9-dichloro-1,10-phenanthroline proceeds efficiently and allows modifications into different
functional groups, the presence of the methyl in the same position is resistant to oxidation.
A facile synthetic method for the obtainment of substituted 5-nitro-sulfur macrocycle
is not yet available as it is still under current investigations. Work is currently underway to
prepare macrocycles bearing functional groups. It is hoped that some of these points will be
clarified in the near future and will provide new compounds of the 1,10-phenanthroline series
with sulphur macrocyclic structures and various side arms appended at position 5-.
131
With regards to the two new series of bis acridine derivatives, from the study herewith
presented, some considerations can be made as follows: (i) the expectation to enhance the
remarkable antitumor properties of monomers 74 and 75 was fulfilled with corresponding bis
derivatives 72 and 73. In fact, 72a,h,k,m, and 73a,c exhibit enhanced cytotoxic activity and
higher DNA-affinity than corresponding monomers. (ii) The bis derivatives 72 and 73 are also
more potent cytotoxic agents than related bis 70 and 71. (iii) It was established that the best
linker for this kind of bis derivatives is provided by Y = (CH2)3N(CH3)(CH2)3. (iv) The
compounds 72a,k and 73a, being endowed of a remarkable DNA affinity, a broad spectrum of
activity, an excellent cytotoxic potency, and a interesting preliminary in vivo activity (73a),
represent good candidates for preclinical studies.
Likewise, studies conducted on a new class of aza-acridine derivatives, related to structure 78,
show remarkable antitumor properties. The 10-aza substitution of 5-nitropyrazolo[3,4,5kl]acridines 77 yielded derivatives 78 endowed with enhanced DNA affinity and similar in
vitro cytotoxic activity compared to reference compound PZA. Besides it has exhibited the
potential of inducing oligonucleosomal DNA fragmentation and apoptotic cell death, absent
in PZA. In particular the 9-methoxy-5-nitro-2-[2-(tetrahydro-1H-1-pyrrolyl)ethyl]-2,6dihydroindazolo[4,3-bc][1,5]naphthyridine (78d), which possesses the most relevant
biological characteristics in the series, can be regarded as a new lead in the field of potential
anticancer derivatives. Activation of programmed cell death in cancer cells offers novel and
potentially useful approaches to the improvement of patient responses in conventional
chemotherapy. Thus, the ability of this compound to early induce oligonucleosomal DNA
fragmentation and apoptotic cell death of the hormone-refractory PC-3 prostate cancer cells
may be particulary relevant to overcome drug resistance or sensitize tumor cells to the effects
of other antineoplastic agents.
Finally, with respect to the naphto[1,2,3-de]quinoline derivatives related to structure 85 from
observations based on the presented results, a similar activity profile to that of mono
functionalized 84a can be discerned. Perhaps, the introduction of a second basic side chain in
position 3 may increase biological properties as it was found for reference compounds
84.91a,91b However, the 6-[3-(diethylamino)propyl]-3H-naphtho[1,2,3-de]quinoline-2,7-diones
(85e), which possesses good cytotoxicity and low or none cross resistance with Cs on
resistant cell lines, can be regarded as a new lead in the development of anticancer
derivatives.
132
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