Design, synthesis and biological evaluation of benzo[e][1,2,4]triazin-7(1H) -one and [1,2,4]-triazino[5,6,1-jk]carbazol-6-one derivatives as dual inhibitors of beta-amyloid aggregation and acetyl/butyryl cholinesterase

Article abstract of DOI:10.1016/j.ejmech.2012.10.003

Alzheimer's disease (AD) onset and progression are associated with the dysregulation of multiple and complex physiological processes and a successful therapeutic approach should therefore address more than one target. Two new chemical entities, the easily

Full text of DOI:10.1016/j.ejmech.2012.10.003

European Journal of Medicinal Chemistry 58 (2012) 84e97
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of benzo[e][1,2,4]triazin-7(1H)-one
and [1,2,4]-triazino[5,6,1-jk]carbazol-6-one derivatives as dual inhibitors of
beta-amyloid aggregation and acetyl/butyryl cholinesterase
Marco Catto a, Andrey A. Berezin b, Daniele Lo Re b, Georgia Loizou b, Marina Demetriades b,
,
Angelo De Stradis c, Francesco Campagna a, Panayiotis A. Koutentis b, Angelo Carotti a
*
a Dipartimento di Farmacia, Università degli Studi di Bari “Aldo Moro”, Via Orabona 4, 70125 Bari, Italy
b Department of Chemistry, University of Cyprus, PO Box 20537, 1678 Nicosia, Cyprus
c Istituto di Virologia Vegetale del CNR, Università degli Studi di Bari “Aldo Moro”, Via Amendola 165/A, 70126 Bari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Alzheimer’s disease (AD) onset and progression are associated with the dysregulation of multiple and
complex physiological processes and a successful therapeutic approach should therefore address more
than one target. Two new chemical entities, the easily accessible heterocyclic scaffolds 1,3-diphenylbenzo
[e][1,2,4]triazin-7(1H)-one (benzotriazinone I) and 2-phenyl-6H-[1,2,4]triazino[5,6,1-jk]carbazol-6-one
Received 23 July 2012
Received in revised form
27 September 2012
Accepted 3 October 2012
Available online 11 October 2012
(triazafluoranthenone II), were explored for their multitarget-directed inhibition of beta-amyloid (Ab)
fibrillization and acetyl- (AChE) and/or butyryl- (BChE) cholinesterase, three valuable targets for AD
therapy. Introduction of appropriate amine substituents at positions 6 and 5 on scaffold I and II,
respectively, allowed the preparation of a series of compounds that were tested as Ab1e40 aggregation
Keywords:
Benzotriazinones
Triazafluoranthenones
Alzheimer’s disease
and cholinesterase inhibitors. Potent inhibitors of A
benzotriazinone 7 exhibited an outstanding IC50 equal to 0.37
b
self-aggregation were discovered and among them
M. Compounds bearing a basic amine
m
linked to the heterocyclic scaffold through a linear alkyl chain of varying length also afforded good ChE
b-Amyloid aggregation inhibitors
Cholinesterase inhibitors
Multitarget-directed ligands
inhibitors. In particular, benzotriazinone 24 and triazafluoranthenone 38 were endowed with an inter-
esting multiple activity, the former displaying IC50 values of 1.4, 1.5 and 1.9
AChE and BChE inhibition, respectively, and the latter showing IC50 values of 1.4 and an outstanding
0.025 M in the A aggregation and BChE inhibition, respectively. Benzotriazinone 24 and triaza-
fluoranthenone 29, selected owing to their suitable aqueous solubility and A aggregation inhibition,
mM on Ab aggregation and
m
b
b
were submitted to a time course kinetic assay followed with thioflavin T (ThT) spectrofluorimetry,
circular dichroism (CD) and transmission electron microscopy (TEM). Experimental data indicated that
24 acted at a low concentration ratio (10
mM 24 vs. 50 mM Ab), stabilizing the unstructured Ab peptide
and inhibiting fibrillogenesis, and that 29 also acted as fibrillization inhibitor, but likely enhancing and
stabilizing the
b
-sheet arrangement of A
b
to yield protofibrillar species as detected by TEM.
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
Alzheimer’s disease (AD) is by far the most common form of
senile dementia, affecting almost ten million people only in Europe
[2]. Along with many other NDs, AD is a typical multifactorial
disease and its insurgence and progression may be caused by the
dysregulation of multiple pathophysiological processes [3], and by
environmental [4] and genetic factors [5]. The hallmark of AD is the
neuronal degeneration in selected areas of the brain, caused either
Neurodegenerative diseases (NDs) are, along with cancer and
cardiovascular diseases, the most important cause of illness and
death in developed countries. The high social and clinical costs of
healthcare and (familiar) assistance for ND suffering patients [1] are
exasperated by the lack of disease-modifying therapies. Despite the
wide efforts of both academic and industrial researchers, there are
still only a few pharmacological treatments for NDs.
by overproduction and accumulation of beta amyloid peptide (Ab)
in extracellular aggregates (neuritic plaques), and intracellular
formation of neurofibrillary tangles, constituted mainly by hyper-
phosphorylated tau protein, a microtubule-associated protein.
Since the cholinergic transmission is heavily impaired by the loss of
* Corresponding author. Tel.: þ39 0805442782; fax: þ39 0805442230.
E-mail addresses: angelo.carotti@uniba.it, carotti_angelo@hotmail.com(A. Carotti).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.10.003

M. Catto et al. / European Journal of Medicinal Chemistry 58 (2012) 84e97
85
cholinergic neurons in basal forebrain, current pharmacological
therapies for AD rely chiefly on restoration of acetylcholine levels
with acetylcholinesterase inhibitors (AChEIs) [6].
palladium catalyzed cyclizations at C8 gave the triazafluoranthenone
II [22], which is structurally related to biologically active canthinone
alkaloids [24,25], II was also chosen as the second planar and more
rigid scaffold for comparison.
A
b
peptide, normally present as a soluble species in extra-
cellular fluids, is mainly constituted by b1e40 and b1e42
(composed of 40 and 42 amino acids, respectively), originated
from the enzymatic cleavage of amyloid precursor protein (APP)
A
A
Benzotriazinones 3e28 (Table 1) and triazafluoranthenones 31e38
(Table 2) bearing mostly differently shaped amino substituents, were
therefore prepared with the aim to i) discover potent Ab aggregation
operated by secretases
a
and
b
. A
b
polymerizes through
b
-sheet
inhibitors exhibiting an additional strong AChE and/or BChE inhibition
to target AD, along the strategy of the multitargeted-ligands approach
[26,27]; ii) delineate the structureeactivity relationships (SARs) for
further molecular optimization; and iii) improve physicochemical
properties relevant for molecular bioactivity, including water solu-
bility for easier and more reliable biochemical and pharmacological
assays.
ordered structures, in a multistep process involving the forma-
tion of several intermediate species, including soluble, low
molecular weight oligomers and protofibrillar aggregates [7].
Several studies attribute the neurotoxicity and the progressive
neurodegeneration of AD to these soluble intermediates rather
than to mature amyloid fibrils or neuritic plaques [8]. Agents able
to prevent or reverse fibrillization or, even better, the early
All compounds were assessed for their A
b antiaggregating
oligomerization of A
treatment of AD [9].
b
may then have therapeutic potential in the
activity through the thioflavin T (ThT) spectrofluorimetric assay
[28] and for their inhibitory activity toward AChE and BChE by the
classical spectrophotometric test of Ellman [29].
Suitably selected compounds were subjected to a time course
kinetic study by means of ThT fluorescence, CD signal at 215 nm
and TEM micrographs to gain insights on the mechanism of inhi-
bition and possibly on the targeted oligomeric species.
Protein misfolding and aggregation are common hallmarks in
many NDs [10]. As such, the inhibition of protein aggregation and
related toxic events are under intense investigation, but to date, no
valuable therapeutic agents have been discovered and all clinical
trials with compounds acting on different targets have been
unsuccessful [11].
Many chemical agents, either naturally occurring or produced
by synthesis, and often containing planar (hetero)aromatic resi-
2. Chemistry
dues, can inhibit A
b
fibrillogenesis [12,13]. Antiamyloidogenic
Readily available 1,3-diphenylbenzo[e][1,2,4]triazin-7(1H)-one
1 [30] and 2-phenyl-6H-[1,2,4]triazino[5,6,1-jk]carbazol-6-one 29
[22] were reacted with a series of nucleophiles to give the corre-
sponding 6-substituted benzotriazinones 2e23, 25, 27 and 28 and
5-substituted triazafluoranthenones 30e37, according to reported
procedures [21,22] (Scheme 1).
activity has been reported by some of us for glycine-based oligo-
peptides [14], indoles [15], isatins [16], tricyclic heteroaromatics
[17] and anthraquinone drugs [18]. Recently, we also reported on
a dynamic model of inhibition of A
that specific , hydrophobic and electrostatic/polar interactions,
including hydrogen bonds, involving the backbone and some side
chains of A , led to the A assembly. Disruption and/or prevention
b aggregation [19], suggesting
pep
Efforts to prepare the 6-({6-[benzyl(methyl)amino]alkyl}(methyl)
amino)-1,3-diphenylbenzo-[e][1,2,4]triazin-7(1H)-ones 24 (alkyl chain
n ¼ 5) or 26 (alkyl chain n ¼ 8) by direct nucleophilic addition of N1-
b
b
of these interactions by small molecules could trigger the inhibition
of fibril formation.
benzyl-N1,N6-dimethylalkane-1,6-diamines to benzotriazinone
1
Keeping in mind this array of interactions, as well as the ones
taking place in the inhibition of cholinesterases, largely studied by
some of us [20], benzo[e][1,2,4]triazin-7(1H)-ones I and [1,2,4]-
triazino[5,6,1-jk]carbazol-6-ones II (Chart 1) were selected as
were unsuccessful owing to difficulties encountered in the preparation
of the required diamine. Furthermore, although 6-[8-(N-benzyl-N-
methylamino)octylamino]-1,3-diphenylbenzo[e][1,2,4]triazin-7(1H)-
one 25 could be prepared directly from benzotriazinone 1 and the
readily accessible N-methyl-N-benzyloctane-1,8-diamine [31], the
subsequent N-methylation with either MeI or Me2SO4 suffered
from overmethylation leading to the formation of quaternary salts.
Therefore, the desired 6-({6-[benzyl(methyl)amino]alkyl}(methyl)
amino)-1,3-diphenylbenzo[e][1,2,4]triazin-7(1H)-ones 24 and 26
were prepared via a stepwise synthesis that involved preparing
the 6-(methylamino)-1,3-diphenylbenzo[e][1,2,4]triazin-7(1H)-one
40, its bromoalkyl derivatives 6-[(6-bromoalkyl)(methyl)amino]-
1,3-diphenylbenzo[e][1,2,4]triazin-7(1H)-ones 42 and 43 (alkyl
chain n ¼ 5 and 8, respectively) and their subsequent benzylme-
thylamination to give the desired products 24 and 26 (Scheme 2).
For the preparation of 5-{N-[8-(N-benzyl-N-methylamino)octyl]-
N-methylamino}-2-phenyl-6H-[1,2,4]triazino[5,6,1-jk]carbazol-6-
one (38) an additional difficulty was faced since the desired starting
material 5-(methylamino)triazafluoranthenone could not be
prepared directly because the treatment of triazafluoranthenone 29
with aqueous methylamine gave only very complex reaction
mixtures. As the consequence, a strategy similar to that described
above was adopted. Treatment of readily available 8-iodo-1,3-
diphenylbenzo[e][1,2,4]triazin-7(1H)-one 39 [22], with aqueous
methylamine gave 8-iodo-6-(methylamino)-1,3-diphenylbenzo[e]
[1,2,4]triazin-7(1H)-one 41 in 70% yield and subsequent reaction
with 1,8-dibromooctane gave 6-[(8-bromooctyl)(methyl)amino]-8-
iodo-1,3-diphenylbenzo[e][1,2,4]triazin-7(1H)-one 44 in 61% yield
(Scheme 2). At this point attempted benzylmethylamination led
mainly to protodeiodination. To avoid this undesirable side reaction
attractive heterocycles to investigate Ab fibrillization inhibition,
since they share a quinone/quinonimine moiety and an extended
planar azaheterocyclic system, that are important molecular
determinants for establishing
polar interactions with the aggregating A
a recent study we demonstrated that interactions of a quinone
moiety with the A peptide backbone can efficiently hamper the A
-sheet strands, thus blocking the A aggregation
p
ꢀp
, hydrophobic and electrostatic/
b
peptide. Indeed, in
b
b
self-assembly to
process [19].
b
b
Scaffold I is amenable for molecular decoration because it
undergoes facile regioselective nucleophilic addition and electro-
philic substitution, at the C6 and C8 positions, respectively [21e23],
thus allowing the attainment of
a large molecular diversity.
Furthermore, as oxidative and non-oxidative silver-mediated
8
9
7
10
Ph
N 1
8
5
O
R
N 2
7
O
R
N
N 1
6
6
3
N
Ph
5
4
2
N
Ph
4
3
I
II
Chart 1. General structures of the examined benzotriazinones
fluoranthenones II.
I and triaza-

86
M. Catto et al. / European Journal of Medicinal Chemistry 58 (2012) 84e97
Table 1 (continued)
Table 1
Inhibition data of Ab1e40 aggregation, AChE and BChE of benzotriazinones 1e28.
Entry
R
A
b
aggregation Cholinesterase
inhibitiona inhibitionb
AChE
(49)
BChE
O
R
N
N
N
21
(42)
(6)
S
N
CH3
22
23
24
25
1.0
2.1
1.4
21
(56)
(53)
1.5
(22)
(13)
1.9
Entry
R
Ab aggregation Cholinesterase
inhibitiona
inhibitionb
N
AChE
BChE
CH2CH3
1
2
3
H
CH3O
(C2H5)2N
(39)
(60)
17
(47)
(47)
(55)
(27)
(15)
(19)
N
CH3
N
CH3
4
(45)
(46)
(18)
H3C
CH3
N
N
N
(45)
(28)
N
H
CH3
CH3
CH3
N
N
N
5
6
(68)
7.1
(54)
(39)
(15)
(7)
26
65
(50)
0.31
N
CH3
N
27
28
6.7
(41)
(47)
(10)
(14)
CH3
N
7
0.37
(0)
(22)
(68)
N
H3C
N
N
8
9
0.73
(68)
(55)
(48)
(25)
(11)
Data are means of three independent experiments; SEM < 10%.
a
IC50
IC50
(
(
m
m
M) or inhibition % at 100
M) or inhibition % at 10
m
M concentration (in parentheses).
b
m
M concentration (in parentheses).
H3C
H3C
N
the non oxidative Pd and AgF mediated cyclization [21], to give 5-[N-
(8-bromooctyl)-N-methylamino]-2-phenyl-6H-[1,2,4]triazino[5,6,1-
jk]carbazol-6-one 45 in moderate yield (39%), was carried out. The
triazafluoranthenone 45 readily underwent benzylmethylamination
to give the desired product 38 in 90% yield (Scheme 3).
10
11
1.2
(46)
(55)
(18)
(12)
CH3
N
(55)
N
H3C
O
N
N
12
13
(48)
(62)
(42)
(42)
(0)
(0)
3. Biological assays
In vitro inhibition of Ab1e40 aggregation was assessed following
a previously reported ThT fluorescence-based method involving
the use of hexafluoroisopropanol (HFIP) as aggregation enhancer
S
N
14
15
1.5
(35)
2.2
(10)
(15)
[14e16]. Samples of A
b were coincubated with test molecules at
100 M in phosphate buffered saline (PBS) containing 2% HFIP and
m
up to 10% v/v of DMSO as the cosolvents, and antiaggregating
N
activities were measured after 2 h of incubation at 25 ꢁC. For the
N
(53)
most active compounds (ꢂ80% A
b aggregation inhibition) IC50
values were determined under the same assay conditions as
already described [14e16]; data are reported in Tables 1 and 2. In
order to confirm the antiaggregating activity of these new classes of
compounds, inhibitors 7, 24, 29 and 38 were also tested for their
activity on Ab1e42 aggregation (Fig. 4).
N
16
17
(61)
2.8
(56)
(46)
(18)
(47)
N
N
Inhibitory activities on AChE (from electric eel) and BChE (from
equine serum) were determined by the spectrophotometric
method of Ellman et al. [29] and are reported in Tables 1 and 2 as
18
19
20
4.0
(55)
(57)
(57)
(37)
(9)
N
CH3
IC50
(
mM) for the most active compounds, or as percentage of
inhibition at 10
mM for compounds with low activity.
(13)
(51)
N
H
4. Transmission electron microscopy (TEM)
(11)
TEM analysis was performed at 39,000-fold magnification for
N
CH3
samples of coincubated Ab1e40 (50
mM) with compounds 24

M. Catto et al. / European Journal of Medicinal Chemistry 58 (2012) 84e97
87
Table 2
Inhibition data of Ab1e40 aggregation, AChE and BChE of triazafluoranthenones 29e38.
5. CD spectroscopy
CD spectra were recorded in the spectral range 195e250 nm,
following the conformational random coil to
revealed by the increase of the negative peak of CD signal at
215 nm. Reference A , and coincubated samples of A /compound
/compound 29 were prepared as above described for TEM
b-sheet transition as
O
R
N
N
N
b
b
24 and A
b
studies.
6. Results and discussion
Entry
R
A
b
aggregation
Cholinesterase
inhibitionb
inhibitiona
Inhibition data listed in Tables 1 and 2 were analyzed to derive
structureeactivity relationships and eventual insights in potential
mechanism of action toward the different targets.
Some interesting considerations can be made by comparing
biochemical data in Tables 1 and 2 for the two sets of products
bearing the same substituent. With some notable exceptions, tri-
azafluoranthenones (Table 2) were more active than the corre-
sponding benzotriazinones (Table 1). This is evident for lead
AChE
BChE
29
30
31
H
CH3O
(C2H5)2N
6.1
(33)
1.4
(46)
(52)
(46)
(59)
(11)
(54)
H3C
N
CH3
N
CH3
32
33
34
7.6
2.0
5.5
(49)
(44)
(27)
4.0
N
(52)
(53)
compounds (compare 29, IC50 ¼ 6.1
m
M with 1, 39% inhibition at
M), 32 (7.6 M) vs. 4 (45%), 33
M) vs. 26 (65 M). In contrast, other
100
(2.0
m
M), 31 (1.4
m
M) vs. 3 (17
m
m
mM) vs. 5 (68%), 38 (1.4
m
m
H3C
N
couples of equally substituted inhibitors showed an inverted order
of activity (compare 34 vs. 8, 36 vs. 20 and 37 vs. 25) or were nearly
equipotent (30 vs. 2, 35 vs. 14). Although restricted to fewer
derivatives than benzotriazinones, triazafluoranthenones could be
considered as a very active class of compounds showing, with only
N
35
3.9
(48)
(11)
two exceptions (comps. 30 and 37) an Ab antiaggregating activity in
the low micromolar range.
N
CH3
36
37
4.1
(53)
(46)
(26)
(25)
Within the set of benzotriazinones, compounds 7 and 8 emerged
ashighlypotent inhibitors ofA fibrillization, withIC50 values of0.37
and 0.73 M, respectively, significantly better than the average IC50
values reported so far in the literature either for inhibitors of A self-
aggregation (direct inhibitors) or for inhibitors of A aggregation
derived from the blocking of the peripheral binding site of AChE that
promotes/accelerates A aggregation itself (indirect inhibitors) [32].
CH
N
b
(60)
N
H
m
b
CH
N
b
38
1.4
(55)
0.025
N
CH
b
Data are means of three independent experiments; SEM < 10%.
Withintheclass ofbenzotriazinones, piperidinederivatives 6,10 and
14 showed interesting activities with IC50 values of 7.1, 1.2 and
a
IC50
IC50
(
(
m
m
M) or inhibition % at 100
M) or inhibition % at 10
m
M concentration (in parentheses).
M concentration (in parentheses).
b
m
1.5
mM, respectively. Although benzotriazinones accounted for
a high degree of substituent diversity, only some limited SAR could
be derived. While small (1e5) and aromatic (19e21) substituents
characterized low active compounds, the unsubstituted piperidine
derivative 6 displayed an activity higher than the corresponding
(10 mM) and 29 (50 mM), compared with a free incubation sample of
A
b
. Incubations were run using ethanol as the co-solvent (10% v/v)
in PBS at the temperature of 37 ꢁC, in order to achieve the complete
A
b
aggregation within 10 days.
pyrrolidine derivative 5 (IC50 ¼ 7.1
mM vs. 68% inhibition at 100 mM).
Very surprisingly, while the 2- and 3-methylpiperidine derivatives 7
and 8 resulted the most active inhibitors, the 4-methylregioisomer 9
exhibited a much lower inhibitory potency (68% inhibition), in
contrast also to the 4-phenyl analog 14 that had high activity (IC50
Ph
N
Ph
N
O
O
N
N
NuH
1.5 mM). Isosteric substitution in the piperidine ring was also detri-
N
Ph
Nu
N
Ph
mental as can be seen by comparing the poor activity of 1-
morpholinyl and 1-thiomorpholinyl derivatives 12 and 13 (48 and
62% inhibition, respectively) with the piperidinyl one 6. Piperazine
derivatives showed a mixed and somehow unpredictable behavior:
while comp. 11 retained the same low activity of the piperidine
analog 9, 4-phenylpiperazine derivative 15, unlike its close 4-
phenylpiperidine analog 14, held the same low potency (53% inhi-
bition), similar to its 4-N-benzyl homologue 16 (61%).
1
2-23, 25, 27, 28
The introduction of larger cyclic (comp. 17) or bicyclic amines
(comps. 27 and 28) had the same controversial effect. While
1-azepinyl and 1-quinolinyl derivatives 17 and 27 were good
O
N
N
O
N
N
N
N
NuH
Ph
Nu
Ph
inhibitors of A
b
fibrillization (IC50 ¼ 2.8
mM and 6.7 mM, respec-
tively), 2-isoquinolinyl analog 28 again suffered a strong drop of
29
30-37
activity (68% inhibition). In this case, when comparing the activity
of 28 with open-chain analogs 22 (IC50 ¼ 1.0
mM) and 23 (2.1 mM),
a detrimental effect due to a molecular rigidification could be
Scheme 1. Reagents and conditions: suitable amine or alcohol (NuH, excess), anhy-
drous THF, reflux.

88
M. Catto et al. / European Journal of Medicinal Chemistry 58 (2012) 84e97
X
Ph
N
X
Ph
N
MeNH2,
THF, rt
O
O
N
N
N
Ph
HN
N
Ph
Me
1
40
(X = H)
(X = H), 84%
39 (X = I)
41 (X = I), 70%
X
Ph
N
X
Ph
N
Br-(CH2)n-Br,
NaH, DMF, rt
BnNHMe,
toluene, refl.
O
O
N
N
Bn
Br
nN
N
Ph
N
nN
N
Ph
Me
Me Me
42
24
(X = H, n = 5), 54%
(X = H, n = 5), 30%
43 (X = H, n = 8), 73%
44 (X = I, n = 8), 61%
26 (X = H, n = 8), 32%
Scheme 2. Synthesis of compounds 24 and 26.
claimed. Finally, it is worth noting that N-alkyl-N-benzyl deriva-
tives 22 and 23 were significantly more active than the corre-
sponding anilino derivative 20, and that the substitution of phenyl
ring of 20 with a cyclohexyl in comp. 18 led to a potent inhibitor
activities, with the notable exception of comp. 26 and its fused
analog 38, which exhibited a very potent and selective inhibition of
BChE (IC50 ¼ 0.31 and 0.025
mM, respectively). The inhibitory
potency of 38 was outstanding compared with the average litera-
ture data on selective BChE inhibitors [33]. Very surprisingly, its N-
(IC50 ¼ 4.0
Compounds 22e26, 37 and 38 were designed and prepared
with the aim to fully address, besides A aggregation process, the
inhibition of AChE and/or BChE. They all bore the N-methyl-N-
benzylamino moiety as typical structural determinant for
mM) of Ab aggregation.
demethylated analog 37 lacked both anti-A
b and cholinesterase
b
activity. A similar behavior was observed for the benzotriazinones
25 and 26: The N-Me derivative 26 displayed a BChE inhibition
much stronger than the corresponding NH analog 25 and this may
suggest some critical and selective interactions of the 6-NMe group
of 26 and 38 into the BChE binding site.
The attractiveness of some of the most active benzotriazinones
and triazafluoranthenones as potential hit molecules for an in vivo
screening on animal models of AD [34] and for the evaluation of
additional biopharmacological activities may be limited by their
poor bioavailability and aqueous solubility.
Taking into account that the ThT test does not provide any
evidence about the actual mechanism of inhibition, being only
indicative of the presence of fibrillar aggregates, and that false
positives might emerge [35], a battery of parallel ThT/CD/TEM
experiments was designed and performed to assess the effects of
a
cholinesterase inhibition, due to a likely binding at the catalytic
site, either connected directly to the benzotriazinone scaffold (22
and 23) or spaced by linear alkyl chains of different length (24e26,
37 and 38). Compounds 22 and 23 and the pentamethylene-
bridged compound 24, all showed a very potent activity against
A
b
aggregation (IC50 values in the range 1e2
compound 24 was very active also as AChE and BChE dual inhibitor
showing IC50 values of 1.5 and 1.9 M, respectively. The very well
mM). Interestingly,
m
balanced and strong inhibitory activities of 24 make this compound
a promising candidate for further in vitro and in vivo studies in AD
animal models.
The elongation of the bridging linker to an octamethylene
resulted in the lowering of both antiaggregating and cholinesterase
selected inhibitors over the temporal evolution of
Ab1ꢀ40
I
Ph
N
Pd(Ph3P)2Cl2 (5mol%)
AgF (1.5 equiv), DMF
O
O
N
N
N
N
Br 8 N
Ph
Br 8 N
Ph
N
Me
Me
45, 39%
44
BnNHMe,
toluene, refl.
O
N
N
Bn
N
8 N
N
Ph
Me Me
38, 90%
Scheme 3. Synthesis of compound 38.

M. Catto et al. / European Journal of Medicinal Chemistry 58 (2012) 84e97
89
fibrillization. Besides ThT fluorescence, circular dichroism (CD)
spectroscopy, which accounts for conformational changes of the
monomeric random coil Ab1e40 to b-sheet ordered amyloidogenic
TEM micrographs (Fig. 3) highlighted the fibrillization of Ab1e40
control peptide that gave rise after 7 days to an extensive formation
of thin and long fibrils (Fig. 3B). While the effects of compound 24
were consistent with ThT and CD measures, leading only to isolated
fibrillar aggregates (Fig. 3C), samples co-incubated with compound
29 showed an extended fibrillar weft (Fig. 3D). Since ThT results
intermediates, was used [36]. The characterization of quantity,
shape and dimension of aggregates formed was finally investigated
with transmission electron microscopy (TEM). Since coincubation
had to be made in aqueous buffer (PBS) and only a limited aliquot
(10%) of the ethanol as cosolvent can be allowed, the selection of
molecules to be tested was made after an accurate preliminary
measure of their solubility in the assay medium, by means of UV
spectroscopy (data not shown). From this solubility screening
benzotriazinone 24 and triazafluoranthenone 29 emerged as the
clearly stated for both coincubation samples A
a very similar and significant decrease of fluorescence compared
with reference A (Fig. 1), it must be deduced that filaments in
b/24 and Ab/29
b
Fig. 3D could be protofibrillar species that are clearly detected by
CD, although being transparent to ThT fluorescence measures.
Resolution of TEM micrographs, however, did not allow a precise
evaluation of diameter and shape of such aggregates, which appear
most promising compounds in terms of aqueous solubility and A
b
antiaggregating activity; they were therefore chosen and tested at
their allowed maximum solubility in the incubation assay medium
as quite similar to fibrils formed in free Ab aggregation. Once again,
the TEM results for triazafluoranthenone 29 are controversial and
(10 and 50
mM, respectively).
need further biophysical evaluation to assess the mechanism of
Stock solutions of 24 and 29 were prepared in ethanol, avoiding
Ab1e40 aggregation inhibition and the actual intermediate species
the use of DMSO which is non-transparent for CD measures. Free
formed/targeted. Unfortunately, the poor water solubility of our
compounds precluded the use of capillary electrophoresis (CE) to
clarify such an issue. CE indeed has been an efficient separation and
detection technique largely used by us to identify in a non-
aggregation samples, containing Ab1e40 (50 mM) alone or with test
inhibitors, were incubated at 37 ꢁC without agitation. Aliquots were
removed at various time points and analyzed by ThT fluorescence
and CD spectroscopy (Figs. 1 and 2). The fibrillization evolved with
ambiguous manner the oligomeric species of Ab targeted by
a
typical sigmoidal increase of fluorescence, accounting for
inhibitors of different classes of compounds [18].
a nucleation-dependent process, with an initial lag phase of three
days and an exponential growth going to completion between days
3 and 6. Both 24 and 29 acted as strong inhibitors of the fibrilli-
zation process that lacked the exponential growth phase and
reached only 30e40% of fluorescence of the control peptide (full
lines in Fig. 1). Evolution of the CD spectra showed for the free
incubation control sample the same sigmoidal shape with attain-
Amyloid peptide Ab1e42 represents the predominant form in AD
brain, respect to Ab1e40, and it is also more toxic and prone to
aggregation. It is also well known that Ab1e42 quickly aggregates in
common in vitro tests, leading to fibril formation in shorter time
than Ab1e40. Although Ab1e40 is more handable and commercially
cheaper, and for these reasons is often preferred for in vitro tests,
the measure of antiaggregating effect even on Ab1e42 should give
a more complete information on the inhibitory behavior of any new
class of substances. For these reasons we did also perform a suitable
inhibition test of Ab1e42 aggregation for few selected compounds,
by measuring the ThT fluorescence of a coincubation sample of
ment of plateau at day 6. The random coil to b-sheet transition was
measured as the absolute value of the typical negative peak of
ellipticity at 215 nm (dotted lines in Fig. 1). CD data of the coincu-
bated samples were contrasting and highlighted an inhibitory
behavior quite different for the two examined inhibitors. While 24
100 mM inhibitor with 30 m
M Ab1e42 in PBS/10% DMSO at 37 ꢁC [37].
acted on
b
-sheet transition in a parallel way to fibrillization process,
-sheet content to
Along with the most active inhibitor 7, we tested compounds 24
and 29 already selected for time course kinetic experiments, and
delaying the exponential phase and lowering the
b
40% of control peptide, the co-incubated sample of 29 showed
finally 38 as the most interesting dual Ab/BChE inhibitor. As refer-
a dramatic increment between days 3 and 4, reaching and stabi-
ence compound to validate the reliability of our results, we used
quercetin, a well established inhibitor of amyloid aggregation [14].
Very satisfactorily, experimental results (Fig. 4) were in full
agreement with those determined for Ab1e40. In particular, ThT
fluorescence of coincubation samples was always quite lower than
self aggregating peptide used as control (Fig. 4) at both 8 h and 24 h
lizing a
reference A
hypothesizing for compound 29 a strong stabilization of its
complex with -sheet arranged peptide, hampering the further
b
-sheet configuration even at a greater extent than
b
alone (Fig. 2). This feature can be rationalized
b
aggregation to mature fibrils, while compound 24 appeared instead
to act as stabilizer of random coil arranged, unstructured peptide.
time points, thus confirming the Ab antiggregating activity of these
two classes of substances.
7. Conclusions
120
100
80
60
40
20
0
Benzotriazinones 1e28 and triazafluoranthenones 29e38 were
designed and prepared to address two major therapeutic targets
involved in AD, beta-amyloid self-aggregation and cholinesterase
activity. The presence of a quinonimine motif in both planar aza-
heterocyclic scaffolds suggested a possible activity either as
b-sheet
disrupting or -sheet formation preventing agents, while their easy
b
synthetic decoration allowed the introduction of different substit-
uents, in particular protonatable amines able to interact with the
catalytic site of cholinesterases. The underlying goal was to provide
new chemical entities able to act as dual active agents, within the
so-called multitarget approach to handle different pathological
features of AD and possibly other multifactorial NDs.
0
1
2
3
4
5
6
7
8
9
10
11
days
From the ThT-based fluorimetric test, a number of compounds
were identified as potent inhibitors of Ab1e40 aggregation, with IC50
Fig. 1. Time-course aggregation kinetics of Ab1e40 control (diamonds), A
b/24 (squares)
and A /29 (circles). Results are expressed as percentage of the final value of ThT
b
values spanning from 0.37 to 65
mM. 6-(2-Methylpiperidin-1-yl)-
fluorescence (full lines) and CD ellipticity at 215 nm (dotted lines), both attained at the
plateau of the fibrillization reaction of control peptide.
benzotriazinone 7 emerged as the most potent inhibitor (IC50

90
M. Catto et al. / European Journal of Medicinal Chemistry 58 (2012) 84e97
1100
00
A
B
C
-1100
--2200
-30
1100195
205
215
225
235
245
255
00
-1100
--2200
-30
1100195
205
215
225
235
245
255
00
--1100
--2200
--3300
nm
195
205
215
225
235
245
255
Fig. 2. Circular dichroism spectra of aggregating Ab1e40 alone (A) and coincubated with inhibitors 24 (B) and 29 (C) in PBS/10% ethanol at 37 ꢁC. Dotted lines: CD spectrum at time
point zero; full lines: CD spectra after 7 days.
0.37
m
M), while several other compounds from both series exhibited
The high BChE inhibitory activity of triazafluoranthenone 38
also emerged as an outstanding result, even considering its very
good antiaggregating activity, although the exact role of BChE and
its selective inhibition in AD is still debated [33].
The poor aqueous solubility and the potential low bioavailability
would be important drawbacks of the two classes of compounds
examined herein, only partially overcome by their high potencies.
In this perspective, the evaluation of derivatives bearing more
hydrophilic substituents will be a mandatory evolution of the study
reported herein along with an assessment (and optimization) of
ADMET properties. Eventually, pharmacological data from animal
models of AD are needed to confirm the validity of the multi-
targeted ligands approach in AD.
low to submicromolar IC50 values. With some notable exceptions,
triazafluoranthenones were more active than the corresponding
benzotriazinones bearing the same substituents, suggesting a favor-
able role of a more extended planarity and rigidity in hydrophobic
interactions with aggregation-prone Ab peptide sequence.
Anticholinergic activity was measured on both AChE and BChE
through the classical Ellman’s test. Two closely related compounds
26 and 38, bearing the heterocyclic scaffolds tethered to an N(Me)
Bn moiety through an octamethylene linker, showed a very potent
and selective inhibition of BChE. In particular triazafluoranthenone
38 had an impressive IC50 (25 nM) and a high BChE over AChE
selectivity. Only a few other compounds, all bearing a basic moiety
as substituent, gave good and somewhat selective inhibition of
AChE (15), BChE (32) or acted as dual inhibitors (24).
8. Experimental
To gain insights on the inhibitory mechanism of Ab aggregation,
benzotriazinone 24 and triazafluoranthenone 29 were selected for
a time course kinetic study by means of ThT fluorescence, CD signal
at 215 nm and TEM micrographs. Experimental data suggested that
8.1. General methods and materials
All commercial chemicals and reagents were purchased from
SigmaeAldrich and used without further purification unless
otherwise specified. DCM was freshly distilled from CaH2 under
argon. Reactions were protected from atmospheric moisture by
CaCl2 drying tubes. Anhydrous Na2SO4 was used for drying organic
extracts, and all volatiles were removed under reduced pressure. All
reaction mixtures and column eluents were monitored by TLC
using commercial glass backed thin layer chromatography (TLC)
plates (Merck Kieselgel 60 F254). The plates were observed under
UV light at 254 and 365 nm. The technique of dry flash chroma-
tography was used throughout for all non-TLC scale chromato-
graphic separations using Merck Silica Gel 60 (less than 0.063 mm).
Melting and decomposition points were determined using either
a PolyTherm-A, Wagner & Munz, Koefler-Hotstage Microscope
apparatus or a TA Instruments DSC Q1000 with samples hermeti-
cally sealed in aluminum pans under an argon atmosphere, using
24 might act as
b-sheet disrupter while 29 might enhance and
stabilize the -sheet arrangement of A
b
b
. In both cases the ultimate
effect was the inhibition of fibrillization, as detected by ThT and
confirmed, at least for 24, by TEM.
The antiaggregating behavior of these two classes of compounds
was finally confirmed also on Ab1e42 peptide, by means of a coin-
cubation test that allowed to assess that compounds 7, 24, 29 and
38 also acted as strong inhibitors of Ab1e42 fibrillogenesis.
Since prefibrillar oligomers of Ab, and not mature fibrils, are
responsible for neuronal damage in AD [8], it could be claimed that
compound 24 and some of its close analogs may deserve further
investigations, aimed at discovering the nature and toxicity of the
oligomeric intermediates targeted by 24. The good inhibitory
activity of 24 on both cholinesterases represents another qualifying
feature of this compound.

M. Catto et al. / European Journal of Medicinal Chemistry 58 (2012) 84e97
91
Fig. 3. Effects of inhibitors 24 and 29 on Ab1e40 aggregation. Ab1e40 (50
m
M) was incubated under fibrillization conditions alone or in the presence of 10
m
M 24 or 50
mM 29. Aliquots
of each reaction were assayed by TEM (39 000-fold magnification): for control reaction at time point zero (A) and after 7 days (B); for coincubated A
b/24 (C) and Ab/29 (D) after 7
days.
heating rates of 5 ꢁC/min. Solvents used for recrystallization are
indicated after the melting point. UV spectra were obtained using
a PerkineElmer Lambda-25 UV/vis spectrophotometer and inflec-
tions are identified by the abbreviation ‘inf’. IR spectra were
recorded on a Shimadzu FTIR-NIR Prestige-21 spectrometer with
a Pike Miracle Ge ATR accessory and strong, medium and weak
peaks are represented by s, m and w, respectively. 1H and 13C NMR
spectra were recorded on a Bruker Avance 300 machine (at 300 and
75 MHz, respectively) and on a Bruker Avance 500 machine (at 500
and 125 MHz, respectively). Deuterated solvents were used for
homonuclear lock and the signals are referenced to the deuterated
solvent peaks. Low resolution (EI) mass spectra were recorded on
a Shimadzu Q2010 GC/MS with direct inlet probe.
Benzotriazinones 1 [30], 2 [22], 3 [22], 5 [22], 6 [21], 11 [21], 15
[21],18e21 [21], 28 [21], 39 [22], triazafluoranthenones 29e31 [22],
33 [22] and N-methyl-N-benzyloctane-1,8-diamine [31] were
prepared according to the literature methods.
20
15
control
7
8.2. Preparation of 6-substituted benzotriazinones 4, 7e10, 12e14,
16, 17, 22, 23, 25, 27
10
24
29
38
A stirred mixture of 1,3-diphenylbenzo[e][1,2,4]triazin-7(1H)-
one (1) (100 mg, 0.334 mmol) and alkylamine (7.5 mmol) in THF
(1 mL) was heated at ca. 80 ꢁC for 1 h. The reaction mixture was
allowed to cool to rt, diluted with DCM (20 mL) and washed with
10% HCl (2 ꢃ 10 mL). The organic layer was separated, dried
(Na2SO4), filtered, adsorbed onto silica and chromatographed to
give the desired product.
5
quercetin
0
5
10
15
20
25
-5
time (h)
8.2.1. 6-{N-[3-(Dimethylamino)propyl]-N-methylamino}-1,3-
diphenylbenzo[e][1,2,4]-triazin-7(1H)-one (4)
Fig. 4. ThT fluorescence assay for the inhibition of Ab1e42 aggregation of compounds 7,
24, 29 and 38. Points are ThT fluorescence values (arbitrary units) registered at various
Orange needles (98%). Mr: 233e234ꢁS (from EtOH), Rf (DCM/
time points for 30
mM peptide alone (control) and coincubated with 100 mM test
MeOH, 1:5) 0.39; lmax(DCM)/nm 278 (log
3 , 3.22), 298 (3.29), 318
compounds. Quercetin was used as reference compound [14,37]. Data are means of
three independent experiments ꢄ SEM.
inf (3.17), 342 inf (2.80), 358 inf (2.72), 431 inf (3.10), 449 (3.20);

92
M. Catto et al. / European Journal of Medicinal Chemistry 58 (2012) 84e97
nmax/cmꢀ1 1587 m, 1535 s, 1510 m, 1481 m, 1450 m, 1393 m, 1360 m,
1315 m,1298 m, 814 m, 775 m; dH (300 MHz, SDCl3) 8.30e8.27 (2O,
m, Ar H), 7.62e7.54 (5O, m, Ar H), 7.48e7.45 (3O, m, Ar H), 6.75 (1O,
s, Ar H), 6.00 (1O, s, Ar H), 4.09 (2O, t, J 6.9, SH2), 3.21 (3O, s, SH3),
3.20e3.12 (2O, m, SH2), 2.82 (6H, s, 2SH3), 2.45e2.32 (2H, m, SH2);
dC (75 MHz, SDCl3) 1 ꢃ Ar CH peak missing,176.5 (s),154.6 (s),152.7
(s), 152.4 (s), 141.6 (s), 135.2 (s), 135.0 (s), 130.1 (d), 129.8 (d), 128.6
(d), 126.9 (d), 125.9 (d), 103.2 (d), 97.2 (d), 55.7 (t), 50.6 (t), 43.1 (q),
40.4 (q), 24.5 (t); m/z (EI) 413 (Mþ, 4%), 355 (2), 342 (36), 327 (15),
313 (15), 285 (2), 180 (3), 168 (2), 84 (5), 77 (11), 58 (100). Anal calcd
for C25H27N5O (413.51): C 72.61, H 6.58, N 16.94. Found: C 72.56, H
6.49, N 17.03.
31.0 (d), 21.6 (q); m/z (EI) 396 (Mþ, 100%), 353 (7), 341 (5), 339 (7),
327 (9), 314 (12), 299 (15), 271 (8), 198 (5), 168 (7), 118 (4), 77 (28),
51 (7). Anal calcd for C25H24N4O (396.48): C 75.73, H 6.10, N 14.13.
Found: C 75.85, H 6.09, N 14.13.
8.2.5. 6-(3,5-Dimethylpiperidin-1-yl)-1,3-diphenylbenzo[e][1,2,4]
triazin-7(1H)-one (10)
Orange prisms (60%). Mr: 172e175 ꢁC (from cyclohexane), Rf
(THF/hexane, 1:1) 0.54; lmax(DCM)/nm 281 (log 3.52), 304 (3.58),
3
322 inf (3.46), 346 inf (3.08), 439 inf (3.44), 460 (3.52); nmax/cmꢀ1
1593 m, 1535 s, 1503 m, 1493 m, 1450 m, 1315 m, 1285 m, 1238 m,
1217 m, 1171 m, 1099 m, 905 m, 849 m, 824 m, 779 m; dH (300 MHz,
acetone-d6) 8.35e8.22 (2O, m, Ar H), 7.79e7.59 (5O, m, Ar H), 7.55e
7.40 (3O, m, Ar H), 6.84 (1O, s, Ar H), 5.75 (1O, s, Ar H), 4.73 (2O, d, J
11.1, SH2), 2.58 (2O, t, J 12.1, SH2), 1.95e1.72 (3O, m, SH, SH2),
1.05e0.83 (7O, m, SH, SH2); dC (75 MHz, acetone-d6) 1 ꢃ Ar CH
peak missing, 177.3 (s), 156.0 (s), 153.9 (s), 151.7 (s), 143.0 (s), 136.6
(s), 135.5 (s), 130.7 (d), 130.5 (d), 129.3 (d), 127.3 (d), 127.0 (d), 104.9
(d), 97.6 (d), 56.7 (t), 43.1 (t), 32.5 (d), 19.3 (q); m/z (EI) 410 (Mþ,
100%), 395 (12), 355 (22), 339 (5), 327 (14), 314 (23), 299 (11), 271
(5), 205 (6), 168 (6), 77 (21), 55 (6). Anal calcd for C26H26N4O
(410.51): C 76.07, H 6.38, N 13.65. Found: C 76.22, H 6.23, N 13.53.
8.2.2. 6-(2-Methylpiperidin-1-yl)-1,3-diphenylbenzo[e][1,2,4]
triazin-7(1H)-one (7)
Orange needles (49%). Mr: 158e159 ꢁS (EtOH aq.), Rf (Et2O)
0.56; lmax(DSN)/nm 281 inf (log 3.63), 302 (3.84), 349 inf (3.18),
3
365 inf (3.01), 442 (3.51), 458 (3.55); nmax/cmꢀ1 1584 m, 1530 s,
1493 m, 1449 m, 1315 m, 1306 m, 1258 m, 1180 m, 1140 m, 1126 m,
891 m, 835 m, 820 m, 781 m, 773 m; dH (300 MHz, SDCl3) 8.29e
8.24 (2O, m, Ar H), 7.59e7.53 (5O, m, Ar H), 7.49e7.42 (3O, m, Ar
H), 6.85 (1O, s, Ar H), 6.05 (1O, s, Ar H), 4.97 (1O, br s, SHN), 4.44
(1O, br d, J 11.7, SHN), 3.29e3.19 (1O, m, CHMe), 1.97e1.44 (6H, m,
SH2), 1.37e1.32 (3O, d, J 6.6, SH3); dC (75 MHz, SDCl3) 177.5 (s),
156.5 (s), 152.9 (s), 151.8 (s), 141.8 (s), 135.6 (s), 134.7 (s), 129.8 (d),
129.7 (d), 129.6 (d), 128.5 (d), 126.8 (d), 125.9 (d), 104.8 (d), 97.6 (d),
51.5 (d), 43.45 (t), 30.8 (t), 25.9 (t), 18.7 (t), 15.6 (q); m/z (EI) 396
(Mþ, 100%), 381 (10), 353 (8), 339 (14), 327 (10), 314 (27), 299 (11),
271 (6), 198 (9), 168 (7), 144 (5), 118 (6), 104 (8), 91 (11), 77 (46), 63
(5), 55 (10), 51 (10). Anal calcd for C25H24N4O (396.48): C 75.73, H
6.10, N 14.13. Found: C 75.78, H 6.12, N 14.04.
8.2.6. 6-Morpholino-1,3-diphenylbenzo[e][1,2,4]triazin-7(1H)-
one (12)
Orange needles (93%). Mr: 204e206ꢁS (from EtOH/
cyclohexane), Rf (Et2O) 0.24; lmax(DCM)/nm 279 (log
3
3.40), 300
(3.41), 319 inf (3.36), 339 inf (3.10), 360 inf (2.93), 451 (3.32);
nmax/cmꢀ1 1597 m, 1589 m, 1547 s, 1510 m, 1491 m, 1456 m,
1315 m, 1285 m, 1240 m, 1200 m, 1121 s, 1097 m, 934 m, 901 s,
837 m, 820 m, 768 m, 718 m; dH (300 MHz, SDCl3) 8.29e8.23
(2O, m, Ar H), 7.65e7.50 (5O, m, Ar H), 7.50e7.41 (3O, m, Ar H),
6.84 (1O, s, Ar H), 6.10 (1H, s, Ar H), 3.87 (8O, br s, SH2); dC (75 MHz,
SDCl3) 176.7 (s), 155.7 (s), 152.9 (s), 151.7 (s), 141.4 (s), 134.9 (s),
134.6 (s), 130.0 (d), 129.8 (d), 129.75 (d), 128.5 (d), 126.7 (d), 125.8
(d), 105.6 (d), 97.6 (d), 66.6 (t), 49.0 (t); m/z (EI) 384 (Mþ, 100%), 367
(5), 353 (6), 341 (87), 327 (52), 313 (16), 299 (17), 271 (9), 192 (7),
180 (8), 168 (12), 140 (7), 118 (10), 103 (12), 91 (8), 77 (53), 63 (9), 51
(14). Anal calcd for C23H20N4O2 (384.43): C 71.86, H 5.24, N 14.57.
Found: C 71.92, H 5.22, N 14.75.
8.2.3. 6-(3-Methylpiperidin-1-yl)-1,3-diphenylbenzo[e][1,2,4]
triazin-7(1H)-one (8)
Orange needles (86%). Mp: 170e171ꢁS (from cyclohexane), Rf
(Et2O) 0.53; lmax(DSN)/nm 280 inf (log
3
3.35), 303 (3.41), 321
inf (3.29), 347 inf (2.90), 361 inf (2.78), 459 (3.33); nmax/cmꢀ1
1591 m, 1535 s, 1495 m, 1449 m, 1315 m, 1294 m, 1285 m,
1236 m, 1204 m, 968 m, 901 m, 845 m, 824 m, 779 m, 745 m; dH
(300 MHz, SDCl3) 8.28e8.25 (2O, m, Ar H), 7.58e7.52 (5O, m, Ar
H), 7.49e7.42 (3O, m, Ar H), 6.85 (1O, s, Ar H), 6.06 (1O, s, Ar H), 4.63
(1O, d, J 11.7, SH2), 4.48 (1O, d, J 12.3, SH2), 3.09 (1O, ddd, J 12.3,
12.3, 3.2, SH2), 2.82 (1O, dd, J 12.9, 10.8, SH2), 1.91e1.65 (4O, m,
SH2), 1.31e1.19 (1O, m, SH), 0.98e0.95 (3O, d, J 6.3, SH3); dC
(75 MHz, SDCl3) 177.2 (s), 155.7 (s), 152.7 (s), 151.8 (s), 141.7 (s),
135.4 (s), 134.7 (s), 129.8 (d), 129.7 (d), 129.6 (d), 128.5 (d), 126.75
(d), 125.9 (d), 104.5 (d), 97.5 (d), 56.8 (t), 50.0 (t), 33.2 (t), 31.7 (d),
25.6 (t), 19.1 (q); m/z (EI) 396 (Mþ, 100%), 381 (8), 353 (6), 341 (14),
314 (19), 299 (13), 271 (8), 198 (6), 168 (9), 116 (5), 104 (6), 84 (5), 77
(37), 55 (8), 51 (7). Anal calcd for C25H24N4O (396.48): C 75.73, H
6.10, N 14.13. Found: C 75.83, H 6.24, N 14.28.
8.2.7. 6-Thiomorpholino-1,3-diphenylbenzo[e][1,2,4]triazin-7(1H)-
one (13)
Purple needles (51%). Mr: 246e247ꢁS (from EtOH/
cyclohexane), Rf (Et2O) 0.56; lmax(DCM)/nm 281 (log
3
3.64), 292
(3.64), 320 inf (3.46), 347 inf (3.14), 362 inf (2.98), 431 inf (3.36),
433 (3.45); nmax/cmꢀ1 1584 m, 1530 s, 1504 m, 1493 m, 1450 m,
1315 m, 1294 m, 1219 m, 1182 m, 961 m, 878 m, 826 m, 777 m;
dH (300 MHz, SDCl3) 8.29e8.26 (2O, m, Ar H), 7.60e7.55 (5O, m,
Ar H), 7.48e7.45 (3O, m, Ar H), 6.90 (1O, s, Ar H), 6.12 (1H, s, Ar H),
4.25e4.22 (4O, m, SH2), 2.86e2.82 (4O, m, SH2); dC (75 MHz,
SDCl3) 176.7 (s), 155.2 (s), 152.8 (s), 151.9 (s), 141.5 (s), 135.0 (s),
134.7 (s), 130.1 (d), 129.9 (d), 129.8 (d), 128.6 (d), 126.8 (d), 125.8 (d),
105.6 (d), 97.8 (d), 52.0 (t), 27.5 (t); m/z (EI) 400 (Mþ, 100%), 383 (6),
367 (56), 341 (42), 327 (33), 314 (10), 299 (16), 271 (6), 200 (5), 186
(6), 168 (9), 144 (6), 116 (7), 104 (9), 91 (6), 77 (61), 51 (13). Anal
calcd for C23H20N4OS (400.50): C 68.98, H 5.03, N 13.99. Found: C
69.02, H 5.17, N 14.09.
8.2.4. 6-(4-Methylpiperidin-1-yl)-1,3-diphenylbenzo[e][1,2,4]
triazin-7(1H)-one (9)
Orange needles (99%). Mr: 170e171 ꢁS (FtOH aq.), Rf (Et2O)
0.51; lmax(DSN)/nm 280 inf (log 3.34), 303 (3.40), 322 inf (3.28),
3
347 inf (2.89), 362 inf (2.74), 458 (3.33); nmax/cmꢀ1 1585 m, 1530 s,
1485 m, 1447 m, 1315 m, 1233 m, 1200 m, 1076 m, 829 m, 820 m,
775 m, 746 m; dH (300 MHz, SDCl3) 8.27e8.24 (2O, m, Ar H), 7.57e
7.49 (5O, m, Ar H), 7.44e7.42 (3O, m, Ar H), 6.85 (1O, s, Ar H), 6.05
(1O, s, Ar H), 4.63 (2O, d, J 12.3, SH2), 3.09 (2O, t, J 12.3, SH2), 1.99
(1O, br s, SH), 1.80e1.70 (2O, m, SH2), 1.45e1.33 (2O, m, SH2), 0.97
(3O, d, J 6.3, SH2); dC (75 MHz, SDCl3) 177.2 (s), 155.8 (s), 152.7 (s),
151.7 (s), 141.7 (s), 135.4 (s), 134.7 (s), 129.8 (d), 129.6 (d), 129.5 (d),
128.4 (d), 126.7 (d), 125.8 (d), 104.7 (d), 97.5 (d), 49.7 (t), 34.3 (t),
8.2.8. 6-(4-Phenylpiperidin-1-yl)-1,3-diphenylbenzo[e][1,2,4]
triazin-7(1H)-one (14)
Orange needles (93%). Mp: 200e201ꢁS (from cyclohexane), Rf
(Et2O) 0.51; lmax(DCM)/nm 281 (log
3
3.47), 302 (3.53), 321 inf
(3.42), 345 inf (3.08), 361 inf (2.95), 436 inf (3.61) 451 (3.46);
nmax/cmꢀ1 1585 m, 1535 s, 1504 m, 1493 m, 1450 m, 1416 m,

M. Catto et al. / European Journal of Medicinal Chemistry 58 (2012) 84e97
93
1315 m, 1290 m, 1220 m, 1192 m, 1155 m, 1003 m, 910 m, 869 m,
8.2.12. 6-(N-Benzyl-N-ethylamino)-1,3-diphenylbenzo[e][1,2,4]
triazin-7(1H)-one (23)
Orange prisms (31%). Mr: 180e182 ꢁC (from cyclohexane), Rf
(Et2O) 0.62; lmax(DCM)/nm 299 (log 3.66), 320 inf (3.51), 345
841 m, 818 m, 770 m, 750 m; dH (300 MHz, SDCl3) 8.30e8.27
(2O, m, Ar H), 7.61e7.57 (5O, m, Ar H), 7.47e7.45 (3H, m, Ar H),
7.35e7.30 (3H, m, Ar H), 7.25e7.23 (2O, m, Ar H), 6.95 (1O, s, Ar H),
6.10 (1O, s, Ar H), 4.84 (2O, br d, J 12.6, SH2), 3.21 (2O, ddd, J 12.3,
12.3, 3.3, SH2), 2.93e2.86 (1O, hept, J 5.6, SH), 2.01e1.43 (4O, m,
SH2); dC (75 MHz, SDCl3) 2 ꢃ Ar CH peaks missing, 177.3 (s), 156.0
(s), 153.0 (s), 151.8 (s), 145.1 (s), 141.7 (s), 135.4 (s), 134.8 (s), 129.9
(d),129.8 (d),129.7 (d),128.6 (d),126.8 (d),126.5 (d),125.9 (d),105.2
(d), 97.6 (d), 50.0 (t), 42.9 (d), 33.5 (t); m/z (EI) 458 (Nþ, 100%), 389
(14), 354 (11), 339 (12), 327 (8), 313 (20), 301 (13), 299 (11), 271
(10), 177 (14), 168 (9), 163 (10), 155 (19), 140 (5), 128 (6), 115 (10),
104 (11), 91 (13), 77 (43), 63 (6). Anal calcd for C30H26N4O (458.55):
C 78.58, H 5.72, N 12.22. Found: C 78.42, H 5.48, N 12.08.
3
(3.17), 360 inf (3.08), 431 inf (3.49), 452 (3.58); nmax/cmꢀ1 1587 m,
1531 s, 1493 m, 1449 m, 1354 m, 1315 s, 1285 m, 1238 m, 970 m,
874 m, 831 m, 779 m; dH (300 MHz, acetone-d6) 8.28e8.21 (2H, m,
Ar H), 7.78e7.60 (5H, m, Ar H), 7.50e7.23 (8H, m, Ar H), 6.70 (1H, s,
Ar H), 5.79 (1H, s, Ar H), 5.23 (2H, s, CH2), 3.86 (2H, dd, J 13.5, 6.5,
CH2CH3), 1.34 (3H, t, J 7.0, CH2CH3); dC (75 MHz, CDCl3) 176.8 (s),
153.5 (s), 152.7 (s), 152.0 (s), 141.7 (s), 137.1 (s), 135.5 (s), 134.6 (s),
129.74 (d), 129.69 (d), 129.6 (d), 128.6 (d), 128.4 (d), 127.2 (d), 127.1
(d), 126.8 (d), 125.9 (d), 102.5 (d), 97.5 (d), 54.7 (t), 47.2 (t), 12.5 (q);
m/z (EI) 432 (Mþ, 23%), 403 (34), 341 (100), 180 (8), 104 (5), 91 (12),
77 (22), 65 (5). Anal calcd for C28H24N4O (432.52): C 77.75, H 5.59, N
12.95. Found: C 77.95, H 5.40, N 12.92.
8.2.9. 6-(4-Benzylpiperazin-1-yl)-1,3-diphenylbenzo[e][1,2,4]
triazin-7(1H)-one (16)
Orange needles (16%). Mr: 210e212 ꢁC (from cyclohexane), Rf
8.2.13. 6-[8-(N-Benzyl-N-methylamino)octylamino]-1,3-
diphenylbenzo[e][1,2,4]triazin-7(1H)-one (25)
(Et2O) 0.18; lmax(DCM)/nm 281 (log
3
3.51), 301 (3.56), 320 inf
(3.47), 345 inf (3.16), 432 inf (3.39), 454 (3.47); nmax/cmꢀ1 1537 s,
1315 m, 1296 m, 1231 m, 1204 m, 897 m; dH (300 MHz, acetone-d6)
8.33e8.22 (2H, m, Ar H), 7.79e7.61 (5H, m, Ar H), 7.53e7.38 (8H, m,
Ar H), 6.85 (1H, s, Ar H), 5.76 (1H, s, Ar H), 3.94 (4H, t, J 4.2, 2CH2),
3.56 (2H, s, CH2Ph), 2.61 (4H, t, J 4.8, 2CH2); dC (75 MHz, CDCl3) 177.0
(s),155.8 (s),152.9 (s),151.6 (s),141.6 (s),137.5 (s),135.2 (s),134.6 (s),
129.8 (d),129.7 (d),129.6 (d), 129.1 (d), 128.5 (d),128.2 (d),127.2 (d),
126.7 (d), 125.8 (d), 105.3 (d), 97.5 (d), 62.8 (CH2Ph), 52.8 (t), 48.7
(CH2); m/z (EI) 473 (Mþ, 21%), 382 (5), 340 (18), 327 (9), 314 (16),
146 (100), 117 (7), 91 (94), 77 (15). Anal calcd for C30H27N5O
(473.57): C 76.09, H 5.75, N 14.79. Found: C 76.03, H 5.62, N 14.69.
Orange needles (59%). Mr: 124e126 ꢁC (from DCM/n-pentane),
Rf (THF/hexane, 1:1) 0.22; lmax(DCM)/nm 290 (log
3
3.52), 300
(3.52), 314 inf (3.47), 333 (3.12), 414 inf (3.35), 432 (3.44), 488 inf
(2.54); nmax/cmꢀ1 1562 s, 1549 s, 1516 m, 1489 s, 1476 s, 1450 m,
1310 m, 1294 m, 773 m; dH (300 MHz, CDCl3) 8.35e8.26 (2H, m, Ar
H), 7.65e7.52 (5H, m, Ar H), 7.51e7.41 (3H, m, Ar H), 7.36e7.19 (5H,
m, Ar H), 6.89 (1H, br t, J 5.0, NH), 6.63 (1H, s, Ar H), 6.12 (1H, s, Ar H),
3.48 (2H, s, CH2Ph), 3.43e3.36 (2H, m, CH2), 2.36 (2H, t, J 7.5, CH2),
2.18 (3H, s, CH3), 1.82e1.68 (2H, m, CH2), 1.58e1.24 (10H, m, 5CH2);
dC (75 MHz, CDCl3) 1 ꢃ CH2 peak missing, 172.9 (s), 152.4 (s), 152.3
(s), 152.0 (s), 141.8 (s), 139.2 (s), 136.0 (s), 135.4 (s), 130.0 (d), 129.80
(d), 129.76 (d), 129.0 (d), 128.5 (d), 128.1 (d), 126.9 (d), 126.8 (d),
126.0 (d), 95.5 (d), 94.0 (d), 62.3 (t), 57.4 (t), 42.8 (t), 42.2 (q), 29.4
(t), 29.2 (t), 28.5 (t), 27.3 (t), 27.0 (t); m/z (EI) 545 (Mþ, 46%), 454
(19), 424 (7), 327 (7), 315 (5), 134 (21), 91 (100), 77 (9). Anal calcd
for C35H39N5O (545.72): C 77.03, H 7.20, N 12.83. Found: C 76.91, H
7.07, N 12.77.
8.2.10. 6-(Azepan-1-yl)-1,3-diphenylbenzo[e][1,2,4]triazin-7(1H)-
one (17)
Orange needles (71%). Mr: 191e192ꢁS (aq. EtOH), Rf (Et2O) 0.42;
lmax(DSN)/nm 281 inf (log 3.31), 301 (3.43), 324 inf (3.21), 349 inf
3
(2.80), 364 inf (2.71), 456 (3.36); nmax/cmꢀ1 1584 m, 1531 s, 1504 m,
1485 m, 1454 m, 1356 m, 1317 m, 1298 m, 1265 m, 1248 m, 978 m,
905 m, 885 m, 829 m, 820 m, 785 m, 772 m, 745 m; dH (300 MHz,
SDCl3) 8.28e8.25 (2O, m, Ar H), 7.58e7.49 (5H, m, Ar H), 7.487.38
(3O, m, Ar H), 6.72 (1O, s, Ar H), 6.01 (1O, s, Ar H), 4.01 (4O, br s,
SH2N), 1.96e1.88 (4O, br m, SH2), 1.65e1.59 (4O, br m, SH2); dC
(75 MHz, SDCl3) 1 Ar CH & 1 CH2 peaks missing, 177.0 (s), 153.8 (s),
152.5 (s), 152.0 (s), 141.8 (s), 135.7 (s), 134.7 (s), 129.7 (d), 129.5 (d),
128.4 (d),126.8 (d),125.9 (d),101.4 (d), 97.4 (d), 53.3 (t), 26.6 (t); m/z
(EI) 396 (Nþ, 100%), 379 (5), 353 (21), 339 (11), 325 (6), 314 (18),
299 (13), 271 (5), 168 (7), 144 (5), 116 (5), 104 (8), 77 (48), 55 (9), 51
(9). Anal calcd for C25H24N4O (396.48): C 75.73, H 6.10, N 14.13.
Found: C 75.90, H 6.13, N 14.06.
8.2.14. 6-[3,4-Dihydroquinolin-1(2H)-yl]-1,3-diphenylbenzo[e]
[1,2,4]triazin-7(1H)-one (27)
Brown needles (59%). Mp: 213e215ꢁS (from EtOH/
cyclohexane), Rf (Et2O) 0.42; lmax(DCM)/nm 281 (log
3
3.43), 301
(3.55), 325 inf (3.38), 330 inf (3.32), 368 inf (2.74), 451 (3.65);
nmax/cmꢀ1 1585 m, 1531 s, 1508 m, 1489 m, 1456 m, 1362 m,
1315 m, 1285 m, 1248 m, 1192 m, 1171 m, 1163 m, 984 m, 870 m,
853 m, 827 m, 781 m, 767 m; dH (300 MHz, SDCl3) 8.27e8.25
(2O, m, Ar H), 7.66e7.52 (5O, m, Ar H), 7.45e.43 (3O, m, Ar H),
7.34 (1O, s, Ar H), 7.21e7.05 (4O, m, Ar H), 6.28 (1O, s, Ar H), 4.10
(2O, dd, J 5.8, 5.8, SH2), 2.88 (2O, t, J 6.8, SH2), 2.05e1.96 (2O, m,
CH2); dC (75 MHz, CDCl3) 176.9 (s),155.7 (s), 152.9 (s), 152.0 (s), 141.6
(s), 141.3 (s), 135.1 (s), 134.9 (s), 130.6 (s), 130.1 (d) 130.0 (d), 129.9
(d),129.6 (d),128.6 (d),126.8 (d),126.3 (d),125.9 (d),124.4 (d),123.3
(d), 112.8 (d), 97.9 (d), 49.1 (t), 26.9 (t), 26.5 (t), 23.6 (t); m/z (EI) 430
(Mþ, 100%), 415 (12), 326 (5), 300 (14), 222 (7), 201 (18), 194 (6), 168
(6), 117 (7), 89 (6), 77 (34), 51 (8). Anal calcd for C28H22N4O
(530.50): C 78.12, H 5.15, N 13.01. Found: C 77.91, H 5.42, N 12.91.
8.2.11. 6-(N-Benzyl-N-methylamino)-1,3-diphenylbenzo[e][1,2,4]
triazin-7(1H)-one (22)
Red prisms (38%). Mr: 180e183 ꢁC (from 2-PrOH), Rf (Et2O) 0.58;
lmax(DCM)/nm 277 inf (log
3
3.51), 298 (3.60), 319 inf (3.48), 344
(3.15), 359 inf (3.05), 431 inf (3.45), 450 (3.52); nmax/cmꢀ1 1584 m,
1530 s, 1503 m, 1493 m, 1449 m, 1391 m, 1314 m, 1292 m, 1194 m,
920 m, 818 m, 781 m; dH (300 MHz, CDCl3) 8.32e8.22 (2H, m, Ar H),
7.65e7.51 (5H, m, Ar H), 7.49e7.40 (3H, m, Ar H), 7.39e7.23 (5H, m,
Ar H), 6.76 (1H, s, Ar H), 6.09 (1H, s, Ar H), 5.33 (2H, s, CH2), 3.22 (3H,
s, CH3); dC (75 MHz, CDCl3) 176.8 (s), 154.5 (s), 152.7 (s), 151.8 (s),
141.6 (s), 137.4 (s), 135.4 (s), 134.7 (s), 129.8 (d), 129.7 (d), 129.6 (d),
128.6 (d), 128.4 (d), 127.4 (d), 127.2 (d), 126.7 (d), 125.8 (d), 103.1 (d),
97.4 (d), 56.9 (t), 40.6 (q); m/z (EI) 418 (Mþ, 41%), 403 (23), 327
(100), 209 (10), 180 (10), 91 (16), 77 (30), 65 (6), 51 (6). Anal calcd
for C27H22N4O (418.49): C 77.49, H 5.30, N 13.39. Found: C 77.35, H
5.26, N 13.24.
8.3. Preparation of 5-substituted triazafluoranthenones 32, 34e37
To a stirred solution of 2-phenyl-6H-[1,2,4]triazino[5,6,1-jk]
carbazol-6-one (29) (110 mg, 0.37 mmol) in THF (1 mL) at rt,
alkylamine (22.2 mmol) was added. The mixture was stirred at rt
for 12 h, then diluted with water (2 mL) and the precipitate that
formed filtered, and washed sequentially with water, Et2O and n-
pentane. The residue was crystallized to give the product.

94
M. Catto et al. / European Journal of Medicinal Chemistry 58 (2012) 84e97
8.3.1. 5-{N-[3-(Dimethylamino)propyl]-N-methylamino}-2-phenyl-
6H-[1,2,4]triazino[5,6,1-jk] carbazol-6-one (32)
1481 m, 1452 m, 1391 m, 1314 m, 1261 m, 1229 m, 1190 m, 1109 m,
945 m, 901 m, 789 m, 752 m; dH (300 MHz, CDCl3) 8.53e8.47 (2H, m,
Ar H), 8.30 (2H, dd, J 9.0, 7.9, Ar H), 7.62e7.47 (5H, m, Ar H), 7.42e7.27
(5H, m, Ar H), 6.52 (1H, s, Ar H), 5.28 (2H, s, CH2), 3.27 (3H, s, CH3); dC
(75 MHz, CDCl3) 1 ꢃ Ar CH peak missing,174.3 (s), 158.6 (s),156.8 (s),
152.3 (s), 137.1 (s), 136.1 (s), 134.9 (s), 130.3 (d), 128.7 (d), 128.6 (d),
127.8 (d), 127.5 (d), 127.3 (d), 126.5 (s), 125.2 (s), 124.6 (d), 122.3 (d),
113.0 (d), 104.3 (s), 101.0 (d), 58.0 (t), 41.5 (q); m/z (EI) 416 (Mþ, 50%),
401 (10), 325 (100), 256 (6), 208 (8), 151 (5), 140 (6), 125 (5), 118 (6),
105 (7), 91 (12), 77 (10). Anal calcd for C27H20N4O (416.47): C 77.87, H
4.84, N 13.45. Found: C 77.77, H 4.68, N 13.37.
Orange needles (58%). Mp: 82e86ꢁS (from DCM/MeOH), Rf
(DCM/MeOH, 1:5) 0.12; lmax(DCM)/nm 222 (log
3 , 3.96), 275
(3.39), 306 (3.27), 413 inf (2.51), 437 (3.12), 460 (3.18); nmax/cmꢀ1
2953 m, 2922 m, 2853 m, 1641 m, 1620 m, 1612 m, 1595 m,
1562 m, 1537 s, 1514 s, 1493 s, 1479 m, 1468 m, 1450 s, 1391 m,
1366 m, 1315 s, 1296 s, 1261 m, 1192 m, 1173 m, 1105 m, 1069 m,
1049 m, 1028 m, 924 m, 822 m, 781 m; dH (300 MHz, SDCl3)
8.54e8.49 (2O m, Ar H), 8.27 (2H, dd, J 8.0, 8.0, Ar H), 7.62e7.44
(5O, m, Ar H), 6.42 (1O, s, Ar H), 4.02 (2O, t, J 7.4, SH2), 3.35 (3O,
s, CH3), 2.50 (2O, t, J 7.1, SH2), 2.34 (6O, s, 2SH3), 2.07 (2O, pent,
J 7.3, SH2); dC (75 MHz, SDCl3) 174.2 (s), 157.9 (s), 156.7 (s), 152.0 (s),
136.2 (s), 134.8 (s), 130.2 (d), 128.5 (d), 127.7 (d), 127.4 (d), 126.4 (s),
125.2 (s), 124.3 (d), 122.1 (d), 112.9 (d), 104.1 (s), 99.4 (d), 56.7 (t),
53.5 (t), 45.4 (q), 42.2 (q), 26.5 (t); m/z (EI) 411 (Mþ, 2%), 340 (19),
325 (7), 311 (8), 84 (6), 58 (100). Anal calcd for C25H25N5O (411.50):
C 72.97, H 6.12, N 17.02. Found: C 72.90, H 6.19, N 16.92.
8.3.5. 5-({8-[Benzyl(methyl)amino]octyl}amino)-2-phenyl-6H-
[1,2,4]triazino[5,6,1-jk]-carbazol-6-one (37)
Orange prisms (51%). Mr: 148e149 ꢁC (from DCM/n-pentane), Rf
(THF/hexane, 1:1) 0.31; lmax(DCM)/nm 231 (log
(3.57), 267 (3.62), 276 (3.65), 304 (3.54), 312 inf (3,53), 353 (2.85),
368 (2.84), 394 inf (3.0), 417 (3.40), 440 (3,58), 466 inf (2.62); nmax
3
3.59), 256 inf
/
cmꢀ1 1638 m, 1555 s, 1526 s, 1493 m, 1472 s, 1454 m, 1319 m,
1306 m; dH (300 MHz, CDCl3) 8.52 (2H, d, J 7.8, Ar H), 8.28 (2H, d,
J 7.8, Ar H), 7.65e7.47 (5H, m, Ar H), 7.36e7.20 (5H, m, Ar H), 7.09
(1H, t, J 4.8, NH), 6.31 (1H, s, Ar H), 3.49 (2H, s, CH2Ph), 3.46e3.36
(2H, m, CH2), 2.42e2.31 (2H, m, NHCH2), 2.18 (3H, s, CH3), 1.87e
1.73 (2H, m, NCH2), 1.59e1.27 (10H, m, 5CH2); dC (75 MHz, CDCl3)
1 ꢃ Cq peak missing, 170.5 (s), 157.1 (s), 155.0 (s), 152.7 (s), 136.1 (s),
135.6 (s), 130.4 (d), 129.1 (d), 128.7 (d), 128.2 (d), 128.1 (d), 127.9 (d),
126.9 (d), 126.34 (s), 126.31 (s), 124.8 (d), 122.4 (d), 113.3 (d), 102.2
(s), 92.6 (d), 62.3 (t), 57.4 (t), 43.5 (t), 42.2 (q), 29.4 (t), 29.2 (t), 28.4
(t), 27.31 (t), 27.28 (t), 27.1 (t); m/z (EI) 543 (Mþ, 38%), 452 (25), 134
(48), 91 (100). Anal calcd for C35H37N5O (543.70): C 77.32, H 6.86, N
12.88. Found: C 77.26, H 6.97, N 12.76.
8.3.2. 5-(3-Methylpiperidin-1-yl)-2-phenyl-6H-[1,2,4]triazino
[5,6,1-jk]carbazol-6-one (34)
Red prisms (45%). Mr: 151e154 ꢁC (from cyclohexane), Rf (Et2O)
0.73; lmax(DCM)/nm 235 (log 3.62), 270 inf (3.65), 280 (3.66), 306
3
(3.52), 363 (2.83), 380 (2.83), 413 inf (3.07), 441 (3.42), 466 (3.54),
527 inf (2.70); nmax/cmꢀ1 1641 m, 1620 s, 1524 s, 1514 s, 1479 m,
1452 m, 1391 m, 1369 m, 1317 m, 1258 m, 1234 s, 1138 m, 1126 m,
943 m, 853 m, 797 m; dH (500 MHz, CDCl3) 8.49 (2H, d, J 7.4, Ar H),
8.30 (1H, d, J 8.0, Ar H), 8.27 (1H, d, J 8.0, Ar H), 7.61e7.47 (5H, m, Ar
H), 6.61 (1H, s, Ar H), 4.53 (1H, br d, J 11.2, CH2), 4.43 (1H, br d, J 12.0,
CH2), 3.16 (1H, t, J 13.6, CH2), 2.87 (1H, t, J 11.7, CH2), 2.01e1.77 (4H,
m, Ar H), 1.34e1.23 (1H, m, CH), 1.01 (3H, d, J 6.3, CH3); dC (75 MHz,
CDCl3) 1 ꢃ Ar CH peak missing, 174.6 (s), 160.2 (s), 156.8 (s), 152.5
(s), 136.2 (s), 134.9 (s), 130.3 (d), 128.6 (d), 127.8 (d), 126.6 (s), 125.3
(s), 124.5 (d), 122.3 (d), 113.1 (d), 104.5 (s), 102.9 (d), 58.0 (t), 51.2 (t),
33.1 (t), 31.7 (d), 25.7 (t) 19.2 (q); m/z (EI) 394 (Mþ, 100%), 351 (10),
339 (14), 325 (10), 312 (18), 297 (20), 285 (5), 269 (6), 197 (8), 139
(5), 98 (13), 84 (9). Anal calcd for C25H22N4O (394.47): C 76.12, H
5.62, N 14.20. Found: C 76.28, H 5.71, N 14.18.
8.4. Preparation of N-[(N-benzyl-N-methylamino)alkyl]-N-
methylamino substituted benzotriazinones 24 and 26 and
triazafluoranthenone 38
8.4.1. General procedure for the preparation of 6-
methylaminobenzotriazinones 40 and 41
To
a stirred solution of 1,3-diphenylbenzo[e][1,2,4]triazin-
8.3.3. 2-Phenyl-5-(4-phenylpiperidin-1-yl)-6H-[1,2,4]triazino
[5,6,1-jk]carbazol-6-one (35)
7(1H)-one (1) or 8-iodo-1,3-diphenylbenzo[e][1,2,4]triazin-7(1H)-
one (39) [22] (0.27 mmol) in THF (2 mL), methylamine (0.4 mL, 40%
aq. soln) was added. The mixture was stirred at rt for 30 min. The
mixture was diluted with water (2 mL) and the precipitate was
collected by filtration, washed sequentially with water, Et2O and
n-pentane. Crystallization of the residue gave the product.
Red prisms (79%). Mr: 171e173 ꢁC (from cyclohexane), Rf (Et2O)
0.73; lmax(DCM)/nm 234 (log
3
3.63), 260 inf (3.61), 273 (3.65), 280
(3.66), 305 (3.53), 362 (2.79), 383 (2.84), 413 inf (3.11), 440 (3.43),
464 (3.54); nmax/cmꢀ1 1641 m, 1624 s, 1524 s, 1514 s, 1481 m, 1452 m,
1443 m, 1433 m, 1393 m, 1312 m, 1258 m, 1209 s, 1179 m, 1099 m,
1009 m, 945 m, 827 m, 795 m; dH (500 MHz, CDCl3) 8.51 (2H, d, J 8.0,
Ar H), 8.31 (1H, d, J 8.0, Ar H), 8.28 (1H, d, J 8.5, Ar H), 7.62e7.49 (5H,
m, Ar H), 7.39e7.31 (2H, m, Ar H), 7.29e7.22 (3H, m, Ar H), 6.69 (1H, s,
Ar H), 4.77e4.67 (2H, m, CH2), 3.32e3.23 (2H, m, CH2), 2.96e2.87
(1H, m, CH), 2.11e2.00 (4H, m, CH2); dC (125 MHz, CDCl3) 1 ꢃ Ar
CH peak missing, 174.5 (s), 160.3 (s), 156.7 (s), 152.4 (s), 145.0 (s),
136.0 (s), 134.8 (s), 130.4 (d), 128.6 (d), 127.8 (d), 127.7 (d), 126.8 (d),
126.6 (s), 126.5 (d), 125.1 (s), 124.6 (d), 122.2 (d), 113.1 (d), 104.5 (s),
103.6 (d), 51.1 (t), 42.6 (d), 33.5 (t); m/z (EI) 456 (Mþ, 100%), 387 (7),
352 (12), 337 (12), 325 (8), 311 (37), 297 (13), 269 (5), 176 (17), 162
(13), 115 (7), 77 (5), 56 (5). Anal calcd for C30H24N4O (456.54): C
78.92, H 5.30, N 12.27. Found: C 79.06, H 5.48, N 12.26.
8.4.1.1. 6-(Methylamino)-1,3-diphenylbenzo[e][1,2,4]triazin-7(1H)-
one (40). Orange needles (84%). Mr: 307e311 ꢁC (from CHCl3), Rf
(THF/hexane, 1:1) 0.24; lmax(DCM)/nm 247 inf (log
(3.46), 288 (3.49), 300 (3.49), 313 inf (3.46), 332 inf (3.14), 350 inf
(3.04), 409 inf (3.31), 430 (3.40), 496 inf (2.50), 535 inf (2.22); nmax
3 3.20), 276 inf
/
cmꢀ1 1547 s, 1491 s, 1477 s, 1449 m, 1416 m, 1396 m, 1383 m, 1314 s,
1298 m, 1279 m, 988 m, 858 m, 812 m, 781 m, 752w; dH (300 MHz,
CDCl3) 8.35e8.26 (2H, m, Ar H), 7.65e7.53 (5H, m, Ar H), 7.52e7.42
(3H, m, Ar H), 6.89 (1H, br s, NH), 6.62 (1H, s, Ar H), 6.11 (1H, s, Ar H),
3.13 (3H, d, J 5.5, CH3); dC (75 MHz, CDCl3) 173.0 (s), 153.0 (s), 152.5
(s), 152.4 (s), 141.8 (s), 136.1 (s), 135.3 (s), 130.1 (d), 129.9 (d), 129.8
(d), 128.6 (d), 127.0 (d), 126.0 (d), 95.5 (d), 94.0 (d), 29.4 (q); m/z (EI)
328 (Mþ,100%), 300 (18),168 (5), 164 (5),120 (9), 104 (5), 77 (37), 51
(14). Anal calcd for C20H16N4O (328.37): C 73.15, H 4.91, N 17.06.
Found: C 73.08, H 4.80, N 16.90.
8.3.4. 5-(N-Benzyl-N-methylamino)-2-phenyl-6H-[1,2,4]triazino
[5,6,1-jk]carbazol-6-one (36)
Red prisms (21%), Mr: 153e155 ꢁC (from cyclohexane), Rf (Et2O)
0.69; lmax(DCM)/nm 233 (log
3
3.65), 260 inf (3.66), 271 (3.70), 279
8.4.1.2. 8-Iodo-6-(methylamino)-1,3-diphenylbenzo[e][1,2,4]triazin-
(3.69), 306 (3.54), 363 (2.82), 379 (2.84), 409 inf (3.08), 435 (3.44),
7(1H)-one (41). Red needles (70%). Mr: 294e296 ꢁC (from CHCl3),
459 (3.58), 525 inf (2.52); nmax/cmꢀ1 1645 m, 1622 m, 1614 m, 1514 s,
Rf (Et2O) 0.38; lmax(DCM)/nm 256 (log
3
3.43), 300 (3.56), 349 inf

M. Catto et al. / European Journal of Medicinal Chemistry 58 (2012) 84e97
95
(3.29), 419 inf (3.37), 437 (3.44), 514 inf (2.57); nmax/cmꢀ1 1574 s,
1510 m, 1460 s, 1447 s, 1416 s, 1308 m, 901 m, 781 m; dH (500 MHz,
CDCl3) 8.34e8.28 (2H, m, Ar H), 7.59e7.43 (8H, m, Ar H), 6.89 (1H,
br s, NH), 6.58 (1H, s, Ar H), 3.14 (3H, d, J 5.5, CH3); dC (125 MHz,
CDCl3) 171.5 (s),154.1 (s), 151.7 (s),149.7 (s),142.8 (s),137.3 (s),134.6
(s), 130.3 (d), 129.3 (d), 129.1 (d), 128.6 (d), 127.4 (d), 127.0 (d), 95.5
(d), 68.3 (s), 29.6 (q); m/z (EI) 454 (Mþ, 47%), 327 (100), 312 (7), 299
(7), 284 (17), 181 (7), 168 (5), 155 (10), 149 (14), 140 (5), 127 (7), 116
(10), 91 (5), 77 (30), 51 (9). Anal calcd for C20H15IN4O (454.26): C
52.88, H 3.33, N 12.33. Found: C 53.02, H 3.24, N 12.28.
8.4.2.3. 6-[N-(8-Bromooctyl)-N-methylamino]-8-iodo-1,3-diphenyl-
benzo[e][1,2,4]triazin-7(1H)-one (44). Orange prisms (61%). Mr:
97e99 ꢁC (from cyclohexane/n-pentane), Rf (Et2O) 0.71; lmax(DCM)/
nm 247 (log 3.50), 276 (3.47), 310 (3.65), 363 inf (3.24), 461 (3.59);
3
nmax/cmꢀ1 1562 s, 1530 m, 1487 s, 1474 s, 1454 m, 1443 m, 1404 m,
1364 m, 1312 m, 1290 s, 818 m, 770 m; dH (500 MHz, CDCl3) 8.30e
8.25 (2H, m, Ar H), 7.56e7.51 (4H, m, Ar H), 7.50e7.42 (4H, m, Ar
H), 6.63 (1H, s, Ar H), 3.84 (2H, br s, CH2), 3.41 (2H, t, J 6.8, CH2), 3.34
(3H, br s, CH3), 1.86 (2H, pent, J 6.9, CH2), 1.78 (2H, br t, J 6.8, CH2),
1.48e1.40 (2H, m, CH2),1.40e1.27 (6H, m, CH2); dC (125 MHz, CDCl3)
173.0 (s), 153.7 (s), 151.6 (s), 151.2 (s), 142.7 (s), 137.3 (s), 134.8 (s),
130.0 (d),129.0 (d),128.9 (d),128.5 (d),127.0 (d),126.9 (d),100.8 (d),
73.9 (s), 55.1 (t), 41.3 (q), 33.9 (t), 32.7 (t), 29.2 (t), 28.6 (t), 28.0 (t),
26.8 (t), 26.7 (t); m/z (EI) 646 (Mþ þ 2, 99%), 644 (Mþ,100), 631 (14),
629 (15), 518 (12), 481 (10), 467 (34), 453 (24), 439 (9), 339 (13), 327
(29), 313 (15), 298 (31), 284 (7), 234 (15), 208 (7), 180 (21), 170 (16),
156 (7),140 (11),128 (8),116 (8),104 (9), 77 (44), 69 (7). Anal calcd for
8.4.2. General procedure for the preparation of 6-(N-bromoalkyl-N-
methylamino) substituted benzotriazinones 42e44
To a stirred solution of either 6-(methylamino)-1,3-diphenylbenzo
[e][1,2,4]triazin-7(1H)-one (40) or 8-iodo-6-(methylamino)-1,3-
diphenylbenzo[e][1,2,4]triazin-7(1H)-one (41) (0.31 mmol) in dry
DMF (2 mL) at rt, NaH 60% dispersion in mineral oil, (15 mg,
0.36 mmol) was added. The mixture was stirred at rt for 30 min and
then dibromoalkane (0.91 mmol) was added and the mixture was
stirred for a further 12 h. The reaction mixture was then diluted with
DCM (20 mL) and washed with water (2 ꢃ 20 mL). The organic layer
was separated, dried (Na2SO4), filtered and the volatiles removed. The
residue was then dissolved in DCM (5 mL), adsorbed onto silica and
chromatographed (hexane) to remove excess of dibromoalkane, and
then with t-BuOMe to give the product.
C28H30BrIN4O (645.37): C 52.11, H 4.69, N 8.68. Found: C 51.98, H
4.53, N 8.75.
8.4.3. Preparation of 5-[N-(8-bromooctyl)-N-methylamino]-2-
phenyl-6H-[1,2,4]triazino[5,6,1-jk]carbazol-6-one (45)
To a stirred solution of 6-[N-(5-bromooctyl)-N-methylamino]-8-
iodo-1,3-diphenylbenzo[e] [1,2,4]triazin-7(1H)-one (44) (0.17 mmol)
in DMF (1 mL) at rt, were added Pd(Ph3P)2Cl2 (11 mg, 0.016 mmol,
10 mol %) and AgF (32 mg, 0.25 mmol), and the mixture was heated at
ca. 100 ꢁC for 1 h. The reaction mixture was allowed to cool to rt and
then diluted with Et2O (5 mL) and washed with H2O (5 mL). The
organiclayerwasseparated,dried(Na2SO4), filtered andevaporated in
vacuo. Chromatography (DCM)of the residuegavethe titlecompound
45 as red prisms (39%). Mr: 72e73 ꢁC (from cyclohexane/n-pentane),
8.4.2.1. 6-[N-(5-Bromopentyl)-N-methylamino]-1,3-diphenylbenzo
[e][1,2,4]triazin-7(1H)-one (42). Red needles (54%). Mr: 137e
139 ꢁC (DCM/n-pentane), Rf (Et2O) 0.51; lmax(DCM)/nm 280 inf
(log 3.44), 300 (3.62), 320 inf (3.47), 345 (3.09), 361 inf (3.0), 431
3
inf (3.45), 453 (3.55); nmax/cmꢀ1 1587 m, 1535 s, 1499 m, 1450 m,
1314 m, 1298 m, 1256 m, 827 m, 772 m; dH (300 MHz, CDCl3)
8.32e8.23 (2H, m, Ar H), 7.64e7.50 (5H, m, Ar H), 7.50e7.40 (3H,
m, Ar H), 6.67 (1H, s, Ar H), 6.02 (1H, s, Ar H), 4.00 (2H, br t, J 6.3,
CH2), 3.43 (2H, t, J 6.6, CH2), 3.30 (3H, s, CH3), 1.92 (2H, pent, J 6.8,
CH2), 1.77 (2H, pent, J 7.5, CH2), 1.50 (2H, pent, J 7.4, CH2); dC
(75 MHz, CDCl3) 176.8 (s), 154.2 (s), 152.5 (s), 151.9 (s), 141.7 (s),
135.5 (s), 134.7 (s), 129.8 (d), 129.7 (d), 129.5 (d), 128.5 (d), 126.8
(d), 125.9 (d), 102.0 (d), 97.3 (d), 54.2 (t), 41.3 (q), 33.6 (t), 32.2 (t),
27.7 (t), 25.3 (t); m/z (EI) 478 (Mþ þ 2, 70%), 476 (Mþ, 78), 463 (32),
461 (33), 397 (10), 368 (18), 355 (28), 341 (81), 327 (90), 313 (40),
299 (27), 285 (8), 271 (14), 195 (6), 180 (25), 170 (26), 140 (11), 132
(16), 116 (12), 94 (10), 90 (10), 77 (100), 69 (7), 64 (9), 55 (11), 51
(16). Anal calcd for C25H25BrN4O (477.40): C 62.90, H 5.28, N 11.74.
Found: C 63.04, H 5.19, N 11.90.
Rf (Et2O) 0.71; lmax(DCM)/nm 233 (log
3
3.57), 275 (3.69), 305 inf
(3.53), 363 (2.61), 412 inf (3.12), 437 (3.40), 460 (3.48); nmax/cmꢀ1
1645 m, 1628 m, 1535 s, 1514 s, 1479 m, 1450 m, 1391 m, 1310 m,
1261 m, 787 m, 752 m; dH (300 MHz, CDCl3) 8.46 (2H, dd, J 2.1, 7.5, Ar
H), 8.25 (1H, d, J 7.7, Ar H), 8.20 (1H, d, J 8.1, Ar H), 7.58e7.41 (5H, m, Ar
H), 6.32 (1H, s, Ar H), 3.90 (2H, br t, J 7.0, CH2), 3.41 (2H, t, J 6.8, CH2),
3.32 (3H, s, CH3), 1.92e1.73 (4H, m, 2CH2),1.52e1.29 (8H, m, 4CH2); dC
(75 MHz, CDCl3) 174.4 (s), 158.0 (s), 156.9 (s), 152.1 (s), 136.3 (s), 134.9
(s),130.2 (d),128.5 (d),127.8 (d),127.7 (d),126.5 (s),125.4 (s),124.4 (d),
122.2 (d),113.0 (d),104.2 (s), 99.4 (d), 55.5 (t), 42.3 (q), 34.0 (t), 32.7 (t),
29.2 (t), 28.7 (t), 28.3 (t), 28.1 (t), 26.8 (t); m/z (EI) 518 (Mþ þ 2, 86%),
518 (Mþ, 87), 503 (15), 501 (15), 353 (11), 339 (100), 325 (44), 311 (27),
297 (22), 282 (7), 269 (7), 256 (6), 220 (6),183 (7),169 (55),151 (6),140
(14),125 (6),115 (6), 77 (8), 67 (7), 55 (26). Anal calcd for C28H29BrN4O
(517.46): C 64.99, H 5.65, N 10.83. Found: C 64.84, H 5.56, N 10.74.
8.4.2.2. 6-[N-(8-Bromooctyl)-N-methylamino]-1,3-diphenylbenzo[e]
[1,2,4]triazin-7(1H)-one (43). Red prisms (73%). Mr: 87e88 ꢁC
(from DCM/n-pentane), Rf (Et2O) 0.62; lmax(DCM)/nm 278 inf (log
8.4.4. General procedure for the preparation of N-[5-(N-benzyl-N-
methylamino)alkyl]-N-methylamino substituted benzotriazinones
24 and 26 and triazafluoranthenone 38
3 3.57), 300 (3.67), 320 inf (3.52), 345 (3.12), 362 inf (3.03), 431 inf
(3.49), 453 (3.59); nmax/cmꢀ1 1595 m, 1537 s, 1315 m, 1296 m; dH
(300 MHz, CDCl3) 8.27 (2H, dd, J 7.3, 2.3, Ar H), 7.63e7.51 (5H, m, Ar
H), 7.49e7.41 (3H, m, Ar H), 6.67 (1H, s, Ar H), 6.02 (1H, s, Ar H), 3.96
(2H, br pent, J 4.9, CH2), 3.40 (2H, t, J 6.8, CH2), 3.32 (3H, br s, CH3),
1.85 (2H, pent, J 6.8, CH2), 1.73 (2H, br pent, J 6.8, CH2), 1.49e1.25
(8H, m, CH2); dC (75 MHz, CDCl3) 176.9 (s), 154.2 (s), 152.5 (s),
152.0 (s), 141.7 (s), 135.6 (s), 134.8 (s), 129.73 (d), 129.66 (d), 129.5
(d), 128.5 (d), 126.8 (d), 125.9 (d), 101.9 (d), 97.3 (d), 54.6 (t), 41.3 (q),
33.9 (t), 32.7 (t), 29.1 (t), 28.6 (t), 28.3 (t), 28.0 (t), 26.7 (t); m/z (EI)
520 (Mþ þ 2, 100%), 518 (Mþ, 98), 505 (27), 503 (32), 491 (7), 438
(10), 423 (14), 355 (29), 341 (88), 327 (78), 313 (37), 299 (14), 285
(7), 271 (7), 184 (10), 180 (18), 171 (23), 168 (9), 144 (7), 132 (8), 116
(7), 104 (9), 91 (7), 77 (46), 67 (6), 55 (14). Anal calcd for
To a stirred solution of 6-[N-(bromoalkyl)-N-methylamino]-1,3-
diphenylbenzo[e][1,2,4]triazin-7(1H)-ones 42 or 43, or 5-[N-(8-
bromooctyl)-N-methylamino]-2-phenyl-6H-[1,2,4]triazino[5,6,1-
jk]-carbazol-6-one (45) (0.11 mmol) in toluene (5 mL) at rt, N-
methylbenzylamine (25 mg, 0.21 mmol) was added. The mixture
was heated at ca. 110 ꢁC for 12 h and allowed to cool to rt, diluted
with DCM (15 mL), washed with water (2 ꢃ 20 mL), dried (Na2SO4),
filtered and the volatiles evaporated in vacuo. The residue was then
dissolved in DCM (5 mL), adsorbed onto silica and chromato-
graphed (DCM) to remove traces of amine, and then further elution
with THF gave the product.
8.4.4.1. 6-{N-[5-(N-Benzyl-N-methylamino)pentyl]-N-methylamino}-
1,3-diphenylbenzo[e] [1,2,4]triazin-7(1H)-one (24). Orange needles
(30%). Mr: 88e90 ꢁC (from 2-PrOH/n-pentane), Rf (THF/hexane, 1:1)
C
28H31BrN4O (519.48): C 64.74, H 6.01, N 10.79. Found: C 64.56, H
5.87, N 10.64.

96
M. Catto et al. / European Journal of Medicinal Chemistry 58 (2012) 84e97
0.16; lmax(DCM)/nm 279 inf (log
3
3.47), 300 (3.57), 318 inf (3.44), 346
inhibition, compounds were first tested at 100
were prepared in phosphate buffered saline (PBS; 0.01 M, NaCl
0.1 M, pH 7.4), with 30 M A peptide concentration, and contained
2% HFIP and 10% DMSO. Blank samples were prepared for each
concentration, devoid of peptide, and their fluorescence value
subtracted from the corresponding fluorescence values of coincu-
bation samples. As the control, a sample of peptide was incubated
in the same PBS/2% HFIP/10% DMSO buffer, without inhibitor.
Incubations were run in triplicate at 25 ꢁC for 2 h. Fluorimetric
mM; test samples
(3.01), 361 (2.94), 431 inf (3.39), 453 (3.50); nmax/cmꢀ1 1595 m, 1537 s,
1485 m, 1450 m, 1315 m, 1298 m, 822 m, 779 m; dH (300 MHz, CDCl3)
8.32e8.24 (2H, m, Ar H), 7.64e7.50 (5H, m, Ar H), 7.49e7.41 (3H, m, Ar H),
7.34e7.19 (5H, m, Ar H), 6.66 (1H, s, Ar H), 6.01 (1H, s, Ar H), 3.95 (2H, br s,
CH2), 3.46 (2H, s, CH2), 3.31 (3H, br s, CH3), 2.37 (2H, t, J7.3, CH2), 2.18 (3H,
s, CH3),1.73 (2H, pent, J7.5, CH2),1.55 (2H, pent, J7.5, CH2),1.35 (2H, pent, J
7.3, CH2); dC (75 MHz, CDCl3) 1 ꢃ Ar CH peak missing,176.9 (s),154.2 (s),
152.5 (s), 151.9 (s), 141.7 (s), 139.2 (s), 135.6 (s), 134.7 (s), 129.7 (d), 129.6
(d), 129.5 (d), 128.9 (d), 128.4 (d), 128.1 (d), 126.8 (d), 125.9 (d), 101.9 (d),
97.3 (d), 62.3 (t), 57.2 (t), 54.5 (t), 42.2 (q), 41.3 (q), 28.2 (t), 27.1 (t), 24.6 (t);
m/z (EI) 517 (Mþ, 84%), 396 (14), 381 (8), 341 (11), 329 (43), 313 (7), 300
(5), 292 (5),188 (10),180 (8),160 (10),134 (16),120 (7), 98 (84), 91 (100),
77 (21), 70 (10), 65 (7). Anal calcd for C33H35N5O (517.66): C 76.57, H 6.81,
N 13.53. Found: C 76.44, H 7.00, N 13.54.
m
b
measures were performed in a 700
LS55 spectrofluorimeter, using FLWinlab program. 470
solution were mixed with 30 L of sample, and the resulting fluo-
mL cuvette with a PerkineElmer
mL of ThT
m
rescence measured with parameters set as follows: excitation at
440 nm (slit 5 nm); emission at 485 nm (slit 10 nm); integration
time 2 s. Biological activity was determined as percent of inhibitory
activity Vi for each concentration, according to the formula:
8.4.4.2. 6-{N-[8-(N-Benzyl-N-methylamino)octyl]-N-methylamino}-
1,3-diphenylbenzo[e] [1,2,4]triazin-7(1H)-one (26). Orange needles
(32%). Mr: 97e99 ꢁC (from 2-PrOH), Rf (THF/hexane, 1:1) 0.20;
Vi ¼ 100 ꢀ ½ðFi ꢀ FbÞ=F0ꢅ ꢃ 100
where Fi is the fluorescence value of the sample, Fb its blank value,
and F0 the fluorescence value of Ab control (already subtracted of its
blank). For the most active inhibitors, IC50 values were determined
by testing in duplicate 5e7 concentrations, ranging from 200 to
lmax(DCM)/nm 276 inf (log
3
3.46), 300 (3.57), 346 (3.04), 363 inf
(2.94), 433 inf (3.40), 454 (3.47); nmax/cmꢀ1 1589 m, 1537 s, 1450 m,
1315 m, 1296 m, 1285 m, 820 m; dH (500 MHz, CDCl3) 8.27 (2H, d, J
6.8, Ar H), 7.62e7.50 (5H, m, Ar H), 7.48e7.40 (3H, m, Ar H), 7.33e
7.20 (5H, m, Ar H), 6.66 (1H, s, Ar H), 6.02 (1H, s, Ar H), 3.99e3.88
(2H, m, CH2), 3.46 (2H, s, CH2Ph), 3.33 (3H, br s, CH3), 2.34 (2H, t,
J 7.3, CH2), 2.17 (3H, s, CH3), 1.76e1.67 (2H, m, CH2), 1.53e1.46 (2H,
m, CH2), 1.38e1.34 (8H, m, 4CH2); dC (125 MHz, CDCl3) 1 ꢃ Ar CH
and 1 ꢃ CH2 missing, 176.9 (s), 154.3 (s), 152.5 (s), 152.0 (s), 141.8 (s),
139.3 (s), 135.6 (s), 134.8 (s), 129.73 (d), 129.67 (d), 129.5 (d), 129.0
(d), 128.5 (d), 128.1 (d), 126.8 (d), 125.9 (d), 101.9 (d), 97.3 (d), 62.3
(t), 57.5 (t), 54.7 (t), 42.2 (q), 41.3 (q), 29.5 (t), 29.4 (t), 27.4 (t), 27.3
(t), 26.8 (t); m/z (EI) 559 (Mþ, 73%), 468 (10), 341 (8), 329 (12), 250
(7), 238 (5),180 (6),169 (6),154 (5),147 (6),140 (9),134 (17),120 (5),
108 (6), 91 (92), 77 (15), 65 (6), 57 (5). Anal calcd for C36H41N5O
(559.74): C 77.25, H 7.38, N 12.51. Found: C 77.35, H 7.28, N 12.52.
0.01
mM. Statistics from three independent experiments were
calculated within GraphPad PrismÒ v. 5 software.
Inhibition test of Ab1e42 was performed according to a previous
method [37] with modifications. Ab1e42 (30
PBS/10% DMSO at 37 ꢁC alone as control and with selected
compounds (100 M); spectrofluorimetric lectures with ThT were
mM) was incubated in
m
performed as above described for Ab1e40 at 8 h and 24 h time
points. Fluorescence values are reported as arbitrary units in the
graph of Fig. 4, obtained as the mean ꢄ SEM from three indepen-
dent experiments and calculated within GraphPad Prism software.
8.6. Inhibition of cholinesterases
The in vitro inhibition assays of AChE from electric eel (463 U/
mg) and BChE from equine serum (13 U/mg) were run in phosphate
buffer 0.1 M, pH 8.0. Acetyl- and butyrylthiocholine iodides were
used, respectively, as substrates, and 5,50-dithiobis(2-nitrobenzoic
acid) (DTNB) was used as the chromophoric reagent [29]. Inhibition
assays were carried out on an Agilent 8453E UVevisible spectro-
photometer equipped with a cell changer. Solutions of tested
compounds were prepared starting from 10 mM stock solutions in
DMSO, which were diluted with aqueous assay medium to a final
content of organic solvent always lower than 1%. AChE inhibitory
8.4.4.3. 5-{N-[8-(N-Benzyl-N-methylamino)octyl]-N-methylamino}-
2-phenyl-6H-[1,2,4]-triazino[5,6,1-jk]carbazol-6-one (38). Red prisms
(90%). Mr: 56e57 ꢁC (from hexane), Rf (THF/hexane, 1:1) 0.31;
lmax(DCM)/nm 234 (log 3.57), 274 (3.71), 302 inf (3.53), 369 (2.78),
3
409 inf (3.02), 437 (3.38), 460 (3.45); nmax/cmꢀ1 1645 s, 1626 m,
1537 s, 1514 s, 1479 m, 1452 m, 1389 m, 1310 m, 1265 s, 783 m,
754 m; dH (300 MHz, CDCl3) 8.54e8.44 (2H, m, Ar H), 8.32e8.22 (2H,
m, Ar H), 7.61e7.45 (5H, m, Ar H), 7.37e7.19 (5H, m, Ar H), 6.39 (1H, s,
Ar H), 3.93 (2H, br t, J 7.4, CH2), 3.48 (2H, s, CH2), 3.37 (3H, s, CH3),
2.36 (2H, t, J 7.2, CH2), 2.18 (3H, s, CH3), 1.98e1.76 (4H, m, 2CH2),
1.42e1.28 (8H, m, 4CH2); dC (75 MHz, CDCl3) 174.5 (s), 158.1 (s), 157.0
(s), 152.2 (s), 138.9 (s), 136.3 (s), 135.0 (s), 130.2 (d), 129.1 (d), 128.6
(d), 128.2 (d), 127.8 (d), 127.7 (d), 126.9 (d), 126.6 (s), 125.5 (s), 124.4
(d), 122.3 (d), 113.1 (d), 104.3 (s), 99.4 (d), 62.2 (t), 57.4 (t), 55.7 (t),
42.3 (q), 42.1 (q), 29.7 (t), 29.4 (t), 28.3 (t), 27.4 (t), 27.2 (t), 26.9 (t);
m/z (EI) 557 (Mþ, 56%), 466 (9), 339 (21), 327 (20), 298 (5), 160 (14),
154 (16), 147 (5), 140 (9), 134 (47), 120 (7), 91 (100). Anal calcd for
activity was determined in a reaction mixture containing 100
a solution of AChE (0.9 U/mL in 0.1 M phosphate buffer, pH 8.0),
100 L of a 3.3 mM solution of DTNB in 0.1 M phosphate buffer (pH
mL of
m
7.0) containing 6 mM NaHCO3, 100 mL of a solution of the inhibitor
(six to seven concentrations ranging from 1 ꢃ $10ꢀ8 to 1 ꢃ $10ꢀ4 M),
and 600 m
L of work buffer. After incubation for 20 min at 25 ꢁC,
acetylthiocholine iodide (100 mL of 5 mM aqueous solution) was
added as the substrate, and AChE-catalyzed hydrolysis was fol-
lowed by measuring the increase of absorbance at 412 nm for
5.0 min at 25 ꢁC. The concentration of compound which deter-
mined 50% inhibition of the AChE activity (IC50) was calculated by
nonlinear regression of the response/log(concentration) curve,
using GraphPad Prism, version 5. BChE inhibitory activity was
assessed similarly using butyrylthiocholine iodide as the substrate.
C
36H39N5O (557.73): C 77.53, H 7.05, N 12.56. Found: C 77.65, H 7.17,
N 12.49.
8.5. Thioflavin T fluorescence spectroscopy analysis
A known spectrofluorimetric method [14e16], based on fluo-
rescence emission of ThT, was followed. To obtain batches of Ab1e40
and Ab1e42 free from preaggregates, commercial peptides (purity
>95%; EzBiolab, Carmel, USA) were dissolved in HFIP, lyophilized
8.7. TEM studies
and stored at 20 ꢁC. The solution of ThT (25
m
M) used for fluori-
metric measures was prepared in phosphate buffer 0.025 M, pH 6.0,
filtered through 0.45
m nylon filters and stored at 4 ꢁC. For Ab1e40
Samples for TEM analysis were prepared in PBS with 10%
ethanol as co-solvent and incubated for up to 10 days at 37 ꢁC. Final
m

M. Catto et al. / European Journal of Medicinal Chemistry 58 (2012) 84e97
97
[3] H.W. Querfurth, F.M. LaFerla, N. Engl. J. Med. 362 (2010) 329e344.
[4] P.V. Moulton, W. Yang, J. Environ. Public Health (2012) 472751.
[5] L. Bertram, R.E. Tanzi, Prog. Mol. Biol. Transl. Sci. 107 (2012) 79e100.
concentration of Ab1e40 was 50
were tested at their maximum of solubility (10 and 50
respectively). A control sample of self-aggregating A was prepared
in the same buffer/cosolvent conditions. For each sample, a little
drop (20 L) of incubated sample solution was applied to carbon
m
M, while compounds 24 and 29
m
M,
b
[6] A. Martorana, Z. Esposito, G. Koch, CNS Neurosci. Ther. 16 (2010) 235e245.
[7] J.C. Rochet, P.T. Lansbury, Curr. Opin. Struct. Biol. 10 (2000) 60e68.
[8] D.M. Walsh, I. Klyubin, J.V. Fadeeva, M.J. Rowan, D.J. Selkoe, Biochem. Soc.
Trans. 30 (2002) 552e557.
[9] K. Ono, M. Yamada, J. Neurochem. 117 (2011) 19e28.
[10] F. Chiti, C.M. Dobson, Annu. Rev. Biochem. 75 (2006) 333e366.
[11] M.N. Sabbagh, Am. J. Geriatr. Pharmacother. 7 (2009) 167e185.
[12] M. Bartolini, V. Andrisano, ChemBioChem 11 (2010) 1018e1035.
[13] A.S. DeToma, S. Salamekh, A. Ramamoorthy, M.H. Lim, Chem. Soc. Rev. 41
(2012) 608e621.
m
coated copper/rhodium grid (400 mesh; TAAB Laboratories
Equipment Ltd, Aldermaston, Berks, GB). The coated grid was
floated for 2 min on the sample drop and rinsed with 200
double distilled water. Negative staining was performed with
200 L of 2% w/v uranyl acetate solution (TAAB Laboratories
mL of
m
[14] S. Cellamare, A. Stefanachi, D.A. Stolfa, T. Basile, M. Catto, F. Campagna, E. Sotelo,
P. Acquafredda, A. Carotti, Bioorg. Med. Chem. 16 (2008) 4810e4822.
[15] M. Catto, R. Aliano, A. Carotti, S. Cellamare, F. Palluotto, R. Purgatorio, A. De
Stradis, F. Campagna, Eur. J. Med. Chem. 45 (2010) 1359e1366.
[16] F. Campagna, M. Catto, R. Purgatorio, C.D. Altomare, A. Carotti, A. De Stradis,
G. Palazzo, Eur. J. Med. Chem. 46 (2011) 275e284.
Equipment Ltd). After draining off the excess of staining solution by
means of a filter paper, the specimen was transferred for exami-
nation in a Philips Morgagni 282D transmission electron micro-
scope, operating at 60 kV. Electron micrographs of negatively
stained samples were photographed on Kodak electron microscope
film 4489 (Kodak Company, New York, USA).
[17] Proceedings of XXIV National Congress of Italian Chemical Society, Lecce (I),
2011 Sept. 11e16, FAR-PL-04, 340-341.
[18] (a) R. Colombo, E.De Lorenzi, A. Carotti, M. Catto, M. Racchi, C. Lanni, L. Verga,
G.Caccialanza, Patent MI2008A366 (2008);
(b) R. Colombo, A. Carotti, M. Catto, M. Racchi, C. Lanni, L. Verga,
G. Caccialanza, E. De Lorenzi, Electrophoresis 30 (2009) 1418e1429.
[19] M. Convertino, R. Pellarin, M. Catto, A. Carotti, A. Caflisch, Protein Sci. 18
(2009) 792e800.
[20] (a) A. Carotti, M. De Candia, M. Catto, T.N. Borisova, A.V. Varlamov, E. Méndez-
Álvarez, R. Soto-Otero, L.G. Voskressensky, C. Altomare, Bioorg. Med. Chem. 14
(2006) 7205e7212;
8.8. CD spectroscopic analysis
CD spectra were recorded in the spectral range 195e250 nm, by
using 0.1 cm path length quartz cells (280
Hellma GmbH & Co KG, Milan, I) with a CD Jasco J-810 single beam
spectropolarimeter. Control and /inhibitor coincubation
mL internal volume, from
A
b
Ab
samples were the same as in TEM studies. CD spectra were recorded
at room temperature at 0.1 nm intervals with 4 nm bandwidth and
100 nm/min scan speed. The baseline was recorded with the buffer/
ethanol blank or, for co-incubation experiments, with buffer/
ethanol solution of inhibitor blank, and subtracted from corre-
sponding spectra. To follow conformational changes and for inhi-
bition studies, the absolute value of CD signal at 215 nm was plotted
vs. time of incubation (Fig. 1).
(b) F. Leonetti, M. Catto, O. Nicolotti, L. Pisani, A. Cappa, A. Stefanachi,
A. Carotti, Bioorg. Med. Chem. 16 (2008) 7450e7456;
(c) L. Pisani, M. Catto, I. Giangreco, F. Leonetti, O. Nicolotti, A. Stefanachi,
S. Cellamare, A. Carotti, Chem. Med. Chem. 5 (2010) 1616e1630;
(d) B. Tasso, M. Catto, O. Nicolotti, F. Novelli, M. Tonelli, I. Giangreco, L. Pisani,
A. Sparatore, V. Boido, A. Carotti, F. Sparatore, Eur. J. Med. Chem. 46 (2011)
2170e2184;
(e) A. Conejo-García, L. Pisani, M. del Carmen Núñez, M. Catto, O. Nicolotti,
F. Leonetti, J. Campos, M. Gallo, A. Espinosa, A. Carotti, J. Med. Chem. 54 (2011)
2627e2645.
[21] P.A. Koutentis, H. Krassos, D. Lo Re, Org. Biomol. Chem. 9 (2011) 5228e5237.
[22] P.A. Koutentis, G. Loizou, D. Lo Re, J. Org. Chem. 76 (2011) 5793e5802.
[23] T.A. Ioannou, P.A. Koutentis, H. Krassos, G. Loizou, D. Lo Re, Org. Biomol. Chem.
10 (2012) 1339e1348.
Acknowledgments
[24] H.A. Ioannidou, A. Martin, A. Gollner, P.A. Koutentis, J. Org. Chem. 76 (2011)
5113e5122.
[25] A. Gollner, P.A. Koutentis, Org. Lett. 12 (2010) 1352e1355.
[26] L. Pisani, M. Catto, F. Leonetti, O. Nicolotti, A. Stefanachi, F. Campagna,
A. Carotti, Curr. Med. Chem. 18 (2011) 4568e4587.
[27] A. Rampa, F. Belluti, S. Gobbi, A. Bisi, Curr. Top. Med. Chem. 11 (2011) 2716e2730.
[28] H. LeVine 3rd, Protein Sci. 2 (1993) 404e410.
[29] G.L. Ellman, K.D. Courtney, V. Andres, R.M. Featherstone, Biochem. Pharmacol.
7 (1961) 88e95.
[30] P.A. Koutentis, D. Lo Re, Synthesis (2010) 2075e2079.
[31] N. Dodic, B. Dumaitre, A. Daugan, P. Pianetti, J. Med. Chem. 38 (1995) 2418e2426.
[32] T. Mohamed, P.P. Rao, Curr. Med. Chem. 18 (2011) 4299e4320.
[33] D. Karlsson, A. Fallarero, G. Brunhofer, C. Mayer, O. Prakash, C.G. Mohan,
P. Vuorela, T. Erker, Eur. J. Pharm. Sci. 47 (2012) 190e205.
[34] M. Kitazawa, R. Medeiros, F.M. Laferla, Curr. Pharm. Des. 18 (2012) 1131e1147.
[35] F. Meng, P. Marek, K.J. Potter, C.B. Verchere, D.P. Raleigh, Biochemistry 47
(2008) 6016e6024.
The authors wish to thank the Cyprus Research Promotion
Foundation [Grants: NEAYPODOMH/NEKYP/0308/02 and YGEIA/
BIOS/0308(BIE)/13], the University of Cyprus (Medium Sized
Grant), and the following organizations in Cyprus for generous
donations of chemicals and glassware: the State General Labora-
tory, the Agricultural Research Institute, the Ministry of Agriculture,
Medochemie Ltd and Biotronics Ltd. Furthermore, we thank the
A.G. Leventis Foundation for helping to establish the NMR facility in
the University of Cyprus.
This work was also supported by a grant from MIUR (Rome,
Italy; PRIN project 20085HR5JK_005).
References
[36] K.H. Lim, H.H. Collver, Y.T.H. Le, P. Nagchowdhuri, J.M. Kenney, Biochem.
Biophys. Res. Commun. 353 (2007) 443e449.
[37] T. Akaishi, T. Morimoto, M. Shibao, S. Watanabe, K. Sakai-Kato,
N. Utsunomiya-Tate, K. Abe, Neurosci. Lett. 444 (2008) 280e285.
[1] R. Luengo-Fernandez, J. Leal, A.M. Gray, J. Alzheimer’s Dis. 27 (2011) 187e196.
[2] A. Wimo, L. Jönsson, A. Gustavsson, D. McDaid, K. Ersek, J. Georges, L. Gulácsi,
K.Karpati,P.Kenigsberg, H.Valtonen, Int.J.Geriatr. Psychiatry26(2011)825e832.