Synthesis and?SAR study of?acridine, 2-methylquinoline and?2-phenylquinazoline analogues as?anti-prion agents

Article abstract of DOI:10.1016/j.ejmech.2006.05.002

Transmissible spongiform encephalopathies (TSEs) are thought to arise from aggregation of a protease resistant protein denoted PrP^Sc, which is a misfolded isoform of the normal cellular prion protein PrP^C. Using virtual high-throughp

Full text of DOI:10.1016/j.ejmech.2006.05.002

European Journal of Medicinal Chemistry 41 (2006) 1124–1143
http://france.elsevier.com/direct/ejmech
Original article
Synthesis and SAR study of acridine, 2-methylquinoline
and 2-phenylquinazoline analogues as anti-prion agents
H. Cope, R. Mutter, W. Heal, C. Pascoe, P. Brown, S. Pratt, B. Chen *
Department of Chemistry, University of Sheffield, Dainton Building, Brookhill, Sheffield, S3 7HF, UK
Received 23 January 2006; accepted 4 May 2006
Available online 19 June 2006
Abstract
Transmissible spongiform encephalopathies (TSEs) are thought to arise from aggregation of a protease resistant protein denoted PrPSc, which
is a misfolded isoform of the normal cellular prion protein PrPC. Using virtual high-throughput screening we have selected structures analogous
to acridine, 2-methyquinoline and 2-phenylquinazoline as potential therapeutic candidates for the treatment of TSEs. From the synthesis and
screening of constructed libraries we have shown that an electron-rich aromatic ring attached through an amine linker to the position para to
the ring nitrogen is beneficial to both binding to PrPC and the suppression of PrPSc accumulation for acridine and 2-methylquinoline analogues.
2-Phenylquinazoline analogues appear to utilise a different mode of action by binding at a different location and/or pose. We report IC50s in the
nanomolar range.
© 2006 Elsevier Masson SAS. All rights reserved.
Keywords: Prion protein; Acridine; Quinoline; Quinazoline; TSE; vCJD
1. Introduction
digestion. PrPC is widely distributed throughout mammalian
cells as a glycosylated cell-surface protein of apparent impor-
tance due to its genetic and structural conservation between spe-
cies [5]. The function of PrPC is currently undetermined but
reports suggest it to be involved in copper transport, metabolism
or cell signalling, along with other potential roles [6,7]. Never-
theless, PrP-knockout mice models showed no signs of neurode-
generation or histopathological changes for up to 15 months,
endorsing PrPC as a target for therapeutic intervention [8]. The
conversion of the prion protein from a soluble protease sensitive
form to that of an insoluble protease resistant form occurs con-
comitantly with a conformation change in the tertiary structure,
where regions of α-helicity are converted into β-sheet [9]. The
trigger for this change is little understood but most likely
involves destabilisation of the native isoform. It has been
hypothesised that a ‘protein X’ might act as a molecular chaper-
one and aid the conversion [10].
To date, there are no therapies available for the treatment of
prion diseases and as such all recorded cases have proven fatal.
One possible mode of action for therapeutic candidates is that of
binding to PrPC to increase its stability towards conformational
change [11]. Quinacrine was shown to be a potent inhibitor of
prion disease in cultured neuroblastoma cells during a recent
study, which also showed inhibition brought about by lysomo-
tropic agents and cysteine protease inhibitors [12]. Although
Transmissible spongiform encephalopathies (TSEs) are fatal
disorders of the central nervous system (CNS) that become
symptomatic due to the aggregation of misfolded protein affect-
ing cerebral function. In humans these maladies include Creutz-
feldt–Jakob disease (CJD), Gerstmann–Straussler–Scheinker
disease, and fatal familial insomnia. TSEs in the animal king-
dom include bovine spongiform encephalopathy (BSE), scrapie
and chronic wasting disease among others. According to the
protein-only hypothesis [1] the infectious particle responsible
for progression and transmission of TSEs is a partially protease
resistant protein known as PrPSc or PrP-res [2]. Insoluble PrPSc
is susceptible to accumulation forming fibres, which tend to
aggregate in the CNS tissue and instigate a degeneration of
brain function, leading to progressive dementia or ataxic illness
[3,4]. The normal cellular isoform of the prion protein, denoted
PrPC or PrP-sen, is intrinsically soluble and sensitive to protease
Abbreviations: PrPC, normal cellular prion protein or PrP-sen; PrPSc
disease-causing isoform or PrP-res; SMB, Scrapie-infected mouse brain;
SPR, surface plasmon resonance; vCJD, variant Creutzfeldt–Jakob disease;
VHTS, virtual high-throughput screening.
* Corresponding author. Tel.: +44 114 222 9467; fax: +44 114 222 9346.
E-mail address: b.chen@sheffield.ac.uk (B. Chen).
,
0223-5234/$ - see front matter © 2006 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.05.002

H. Cope et al. / European Journal of Medicinal Chemistry 41 (2006) 1124–1143
1125
quinacrine inhibits PrPSc accumulation it is considered an unsui-
table drug candidate due to its toxicity [13]. Analogues of acri-
dine, phenothiazine and other tricyclic compounds, including
quinacrine, bind to PrPC via specific amino acid residues [14].
High-throughput screening assays have also highlighted a range
of potent inhibitors from natural products and known drugs that
include analogues of bicyclic- and tricyclic- nitrogen containing
aromatic systems such as quinoline, acridine and phenothiazine;
some of which display IC50s in the nanomolar range [15]. These
prior studies into the activities of phenothiazines, acridines and
quinolines have focused almost exclusively on compounds con-
taining aliphatic side chains [16,17].
Using virtual high-throughput screening (VHTS) a library
of commercially available drug-like molecules were docked
into a hypothetical binding pocket within PrPC, identified
from SYBYL-Molcad surfaces generated from the PrPC NMR
structures available from the Brookhaven protein data bank
(1QM3, conformer 15). Consensus scoring was used to rank
chemical structures according to their predicted binding affinity
using a list of independent scoring functions. These include
LUDI, PLP1&2, PMF, Jain and functions developed for
LigandFit (ligScore1 and LigScore2) [18]. A selection of the
highest ranked compounds was sourced and screened for bind-
ing to huPrPC using a surface plasmon resonance (SPR) bind-
ing assay [19]. Within the hit compounds structures A1–A4
were identified as quinacrine-like substances, each consisting
of nitrogen containing fused aromatic systems (Fig. 1). A
small number of commercially available analogues of these
parent compounds that displayed variation in the location and
nature of side chains were assayed for binding to hu- and
moPrPC. The results of this binding study indicated that aroma-
ticity at the side chain para to the ring nitrogen could be of
benefit. Using a rational design approach, libraries of analo-
gous compounds were synthesised. Structure–activity relation-
ships relating both to binding to PrPC and inhibition of PrPSc
accumulation were investigated. Acridines, 2-methylquinolines
and a further class of potential prion therapeutics based upon 2-
phenylquinazoline were varied by substitution of a side chain
para to the ring nitrogen. The effects of this change on binding
affinity to PrPC and inhibition of PrPSc accumulation in scra-
pie-infected mouse brain (SMB) cell lines are reported here.
phenylquinazoline 3 (Fig. 2). In all three lead compounds the
side chain is attached through an amine, which provided simple
synthetic routes involving substitution at a halogenated carbon.
Former studies into compounds comparable to those reported
here lack detailed SAR studies and focus on aliphatic side
chains extending from the 9 and 4 positions of acridine and
quinoline, respectively [16,17]. To avoid repetition, the acri-
dine and 2-methylquinoline libraries were based solely on aro-
matic side chains. For 2-phenylquinazoline, a larger library was
constructed encompassing both aliphatic and aromatic moi-
eties. Indeed, our results suggest that aromaticity in these posi-
tions is of benefit to binding over aliphatic side chains. Obser-
vations as to the effect that the π-system itself has on binding
and activity was probed by altering the functional groups
attached to the side chain ring.
2.1. Library synthesis
Libraries were synthesised using 9- and 4-chlorinated lead
structure where appropriate. 9-Chloroacridine 4, 4-chloro-2-
methylquinoline 5 and 4-chloro-2-phenylquinazoline 6 were
commercially available compounds from which analogues
were synthesised by reaction of the appropriate amine via
SN2 type reactions.
In most cases, acid catalysis was utilised to activate the
chlorine bearing carbon towards nucleophilic attack by the
amine. Exceptions to this rule were those pairings of starting
materials in which the amine was more basic than the skeletal
nitrogen. For library 1, based on analogues of acridine, synth-
eses were carried out as reported by Sun et al. [20] (Scheme 1).
The reactions were acid catalysed at room temperature and fol-
lowed by TLC until completion. Reaction times for this library
were typically 24 hours, although some were complete within
90 min. An exception to this methodology, within the acridine-
based library, led to the synthesis of product 1l. The amine
used for synthesis of 1l was protonated preferentially to 9-
chloroacridine under the usual acidic conditions resulting in a
failure to produce the desired product. Phenol was utilised as
reported by Castello et al. [21] to form a phenolic intermediate
2. Chemistry
The lead structures selected by analysis of in vitro screening
Fig. 2. Acridine, 2-methylquinoline and 2-phenylquinazoline—core structures
data were analogous to acridine 1, 2-methylquinoline 2, and 2-
for the libraries as selected by analysis of VHTS hit compounds.
Fig. 1. Hit compounds A1–A4. Binding toward huPrPC as determined by SPR binding assay.

1126
H. Cope et al. / European Journal of Medicinal Chemistry 41 (2006) 1124–1143
terion permitting the amine group to exist in its protonated
+
form RNH3 and thus prohibiting reaction with the electro-
phile. By basifying the reaction mixture the freed electron
pair of the amine could react. Once the reaction was considered
complete it was neutralised with acetic acid before being
reduced to dryness and the crude product recrystallised from
ethyl acetate to yield 3w in a 90% yield (Table 3).
Scheme 1. Synthesis of an acridine-based library. (a) NMP, HCl (cat.), 25 °C.
(b) Phenol 120 °C.
of heightened reactivity relative to 9-chloroacridine. The
increased reactivity of this intermediate allowed 6-methoxypyr-
imidin-4-ylamine 7l to attack the appropriate carbon centre.
The yields obtained during synthesis of the acridine-based
library fell between 17% and 89% (Table 1).
3. Biological screening
3.1. Binding affinities toward huPrPC and moPrPC
Binding affinities toward huPrPC and moPrPC were deter-
mined using a BIAcore 3000, which utilises surface plasmon
resonance (SPR) technology. The BIAcore system operates by
measuring the real time change in the angle of reflected light
upon association of ligand and displays the results as a senso-
gram. The size of this change is directly proportional to the
mass of the immobilised ligand and so the binding affinity
was measured as percentage of the maximum response
expected for a 1:1 binding ratio with units % RUmax. The
detection limits of SPR means that the detection of large mole-
cular weight entities is more reliable, such as protein–protein
interactions. Detection of small molecule–protein interactions
is therefore highly dependant on the level of protein immobili-
sation. However, a lack of homogeneity in protein immobilisa-
tion has led to poor reproducibility between runs. For this rea-
son compounds were initially analysed by triplicate injection.
All binding compounds were then screened in one run (single
injection only) to allow direct comparison of quantitative bind-
ing data. The proteins under discussion were bound to the car-
boxymethylated dextran surface of a sensor chip and specific
concentrations of ligands (typically 40 μM) were passed over
the chip in phosphate buffer (6.5% DMSO, 150 mM NaCl,
3 mM EDTA, 0.005% surfactant P20). The binding ligands
then dissociate in the continued stream of phosphate buffer.
The synthesis of library 2, based on 2-methylquinoline, used
methodology laid in place by Baig and Stevens (Scheme 2)
[22]. The amines utilised in the construction of the library
were reacted with 4-chloro-2-methylquinoline in acetone at
reflux with an acid catalyst to afford the products as their
hydrochloride salts. Reaction times were typically 18 hours
but with monitoring by TLC some were deemed complete
after as little as 90 min. Yields ranged from 36% to 99%
(Table 2). The exception to this methodology was that
employed for product 2y by which 4-chloro-2-methylquinoline
was reacted with 6-methoxypyrimidin-4-ylamine 7l in phenol,
allowing synthesis via a phenolic intermediate in a similar
manner to compound 1l. Following characterisation by NMR
and ESI-MS the product obtained was shown not to be the
desired 4-amino-2-methylquinoline but the phenolic intermedi-
ate. The reaction afforded a 55% yield of 8 (Table 2).
The 2-phenylquinazoline-based library was larger than the
previous two. Those products derived from aromatic amines
were synthesised almost exclusively under acid catalysed con-
ditions in acetone (Scheme 3). Reaction times were typically
around 20 hours, although 3b only required 5 hours reaction
time to reach completion. Again, amine 6-methoxypyrimidin-
4-ylamine 7l would not react under these conditions so another
route was employed. Syntheses involving aliphatic amines did
not favour acid catalysed conditions as the increased basicity of
the aliphatic amines meant that they were protonated more
readily than 6, thus preventing activation of the carbon-chlor-
ine bond to nucleophilic attack. An adaptation of a procedure
reported by Ananthan et al. [23] in which the reaction takes
place in dimethylformamide (DMF) with an excess of amine
was employed to avoid using acid catalysis. The reactions
were carried out in dioxane for easier solvent removal with a
1:5 ratio of 4-chloro-2-phenylquinazoline to amine. The excess
of amine drives the reaction to completion by consuming the
HCl released during the reaction and forming its hydrochloride
salt. Reaction times ranged from 10 min to 94 hours and yields
were typically above 80%. This methodology was applied to
the coupling reaction of 6-methoxypyrimidin-4-ylamine 7l
that had been problematic, the reaction reaching completion
after 70 hours. Product 3l was isolated form the mixed crude
products by flash column chromatography in a disappointing
3% yield. For the synthesis of 3w, procedure C was modified
to include the basification of the reaction mixture using sodium
carbonate. At pH 7 ε-amino-n-caproic acid 7w exists as a zwit-
3.2. SMB cell line screening
The compounds were assessed for activity and toxicity in
SMB cell lines. The dosing of cells was carried out in duplicate
with one experiment utilised for toxicity studies and the
remaining cells assessed for activity. The viability of cells
and the concentration of PrPSc per experiment were measured
as a percentage of untreated controls. Pentosan polysulphate
(PPS) at 100 ng ml–1 acted as a positive control. Compounds
are deemed toxic if the viability of cells falls below 70% of the
untreated control. Compounds were deemed active if PrPSc
levels fall below 70% of the untreated control.
4. Results and discussion
The results of BIAcore screening and SMB cell line assays
were evaluated in relation to a compound’s structure to inves-
tigate the significance these changes might have to binding
and/or activity (Tables 1–3).
To allow direct comparison of the backbone structures 1–3,
10 amines were used in the construction of each of the three

H. Cope et al. / European Journal of Medicinal Chemistry 41 (2006) 1124–1143
1127
Table 1
Synthesis and binding data for an acridine based library
a
Amine
Product
Method
A
Yield (%)
88
Binding (% RUmax)
huPrPC
60
moPrPC
7a
7b
1a
16
1b
1c
1d
1e
A
A
A
A
59
17
31
37
84
23
93
19
42
7c
7d
7e
151
62
82
7f
1f
A
A
42
46
71
53
7g
1g
159
130
7h
1h
A
89
69
30
7i
7j
1i
1j
A
A
79
85
59
81
14
43
7k
1k
A
18
19
0
7l
1l
B
A
A
14
31
83
20
86
62
0
7m
7n
1m
1n
107
29
7o
7x
1o
1x
A
A
73
61
336
32
326
1
a
Synthetic method. Refer to experimental section.

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H. Cope et al. / European Journal of Medicinal Chemistry 41 (2006) 1124–1143
Scheme 3. Synthesis of a 2-phenylquinazoline-based library. (a) Acetone, HCl
(cat.), reflux. (b) Dioxane, reflux.
Scheme 2. Synthesis of a 2-methylquinoline-based library. (a) Acetone, HCl
(cat.), reflux. (b) NMP, HCl (cat.), 25 °C. (c) Phenol, 120 °C.
Table 2
Synthesis and binding data for a 2-methylquinoline based library
a
Amine
Product
Method
C
Yield (%)
85
Binding (% RUmax)
huPrPC
37
moPrPC
10
7a
7b
2a
2b
2c
2d
2e
C
C
C
C
99
68
36
77
46
54
51
46
14
25
22
17
7c
7d
7e
7f
2f
C
C
74
95
42
33
15
11
7h
2h
7i
7j
2i
2j
C
C
68
82
35
41
12
15
7k
2k
A
37
23
7
7m
7n
2m
2n
C
C
61
46
78
36
29
12
8
B
55
24
1
Structure
a
Synthetic method. Refer to experimental section.

H. Cope et al. / European Journal of Medicinal Chemistry 41 (2006) 1124–1143
1129
Table 3
Synthesis and binding data for a 2-phenylquinazoline based library
a
Amine
Product
Method
C
Yield (%)
80
Binding (% RUmax)
huPrPC
58
moPrPC
37
7a
7b
3a
3b
3c
3d
3e
C
C
C
C
92
78
99
86
48
56
69
71
56
52
50
47
7c
7d
7e
b
b
7f
3f
C
C
96
96
–
–
7g
3g
55
62
36
45
7h
3h
C
99
7i
7j
3i
3j
C
C
89
89
67
18
51
4
7k
7l
3k
3l
C
D
98
3
35
18
b
b
–
–
7m
7n
3m
3n
C
C
70
80
47
52
b
b
–
–
7o
7p
3o
3p
C
D
17
88
50
40
26
16
7q
3q
D
84
40
13
(continued)

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H. Cope et al. / European Journal of Medicinal Chemistry 41 (2006) 1124–1143
Table 3 (continued)
a
Amine
Product
Method
D
Yield (%)
86
Binding (% RUmax)
huPrPC
21
moPrPC
0
7r
7s
3r
3s
D
84
33
2
7t
3t
D
D
90
83
32
21
0
0
7u
3u
7v
3v
D
D
61
90
45
23
17
2
7w
3w
7x
3x
C
87
57
43
a
Synthetic method. Refer to experimental section.
Immeasurable result. Interaction with flow cell 1.
b
libraries. For analysis, the BIAcore data for the 30 compounds
is ranked in descending order of binding scores for the 2-phe-
nylquinazoline library (Fig. 3). The libraries of acridine and 2-
methylquinoline analogues show similar trends as R is varied.
In the majority of cases, a variation that is beneficial to binding
in the acridines is also beneficial to the 2-methylquinolines. An
example might be the difference in binding affinity observed
toward huPrPC between derivatives of amine 7m and 7k,
resulting in a decrease in binding. Conversely, changing the
amine substituent from 7k to 7j increases binding in both
cases. No such pattern is observed within the 2-phenylquinazo-
line as the same amine substituents would be ranked
7m > 7k > 7j in terms of their effect on binding of the 2-phe-
nyquinazolines. Although this is an isolated example, the trend
is displayed more fully for binding to both hu- and moPrPC
and suggests that altering the amine substituents has a different
effect on the acridines and 2-methylquinolines than the effect
the same alterations have on the 2-phenylquinazolines (Fig. 3).
Indeed, the effect of altering the amine substituents in the 2-
phenylquinazoline library is not very marked at all, suggesting
that this class of compounds might bind to the protein in a
different location and/or pose. Following this observation it
becomes clear that the libraries must be discussed separately.
4.1. Acridines
Our library of acridine-based analogues displays persistently
better binding affinities toward hu- and moPrPC compared with
the other libraries discussed here. The majority of binding
scores were found to be within the range of 50–100% RUmax
(Table 1). In exception to this trend compounds 1k and 1l dis-
played binding of approximately 20% RUmax towards huPrPC
Fig. 3. BIAcore screening results in % RUmax for compounds 1, 2 and 3 derived
from common amines. A: Binding affinities toward moPrPC. B: Binding
affinities toward huPrPC. In each case scores are ranked in decreasing order of
affinity for the 2-phenylquinazolines. Acridines (■) 2-methylquinolines (●) 2-
phenylquinazolines (▲).

H. Cope et al. / European Journal of Medicinal Chemistry 41 (2006) 1124–1143
1131
and did not bind to moPrPC. These compounds contain elec-
tron poor substituents which obviously disfavour the binding
of the acridines. In contrast, compounds 1i and 1j, which
bear electron-withdrawing groups, bind reasonably well to
both hu- and moPrPC. This may be due to the meta position
of the electron withdrawing substituents disfavouring forma-
tion of a δ-positive charge at the carbon adjacent to the NH
linker. Although 1k houses a meta-methoxy group this see-
mingly does not overcome the intrinsic electron-poor nature
of the pyrimidine ring and also contributes to a δ-positive
charge at the aforementioned carbon centre.
Compound 1x is comparable in structure to 1a, differing
only by the substitution of a methyl group at the nitrogen linker
in place of hydrogen. The effect of this change demonstrates
the importance of a hydrogen atom at this position since its
removal reduces binding dramatically. Additionally, during
analysis of the VHTS and in vitro assay hits prior to analogue
synthesis, structures containing aliphatic R groups were found
not to bind to hu- and moPrPC. With this in mind, we suggest
that an aromatic moiety is essential for binding in this class of
compounds and the inclusion of an NH linker is significantly
beneficial. Compounds 1b to 1o are variations on this rela-
tively simple structure. Compounds in which the π-system of
the phenyl ring is made electron deficient with respect to 1a
have comparatively diminished binding affinities. Our data
suggests that including groups on the phenyl ring that enrich
the electron density of the π-system increase the binding
beyond that of 1a.
pounds 3a–3o bear an aromatic side chain and as such display
the highest binding (Table 3). Conversely, those compounds
with aliphatic substituents (3r–3x) showed markedly dimin-
ished binding. This pattern is well illustrated by comparison
of 3a with 3r, where binding is reduced from 58% to 21%
RUmax with only the substitution of R is phenyl for R is cyclo-
hexanyl. The absence of an NH linker brought about no sig-
nificant change in binding. Comparing 3a with 3x, which have
an NH and NMe linker, respectively, illustrates this observa-
tion, as the binding remains the same. Of the 21 analogues
within this library only five compounds show binding higher
than that of 3a including 3l, which binds non-specifically.
These compounds contain R groups with electron withdrawing
substituents, electron pushing substituents or even both. 3d, 3e,
3h and 3i therefore show no correlation and as such no influ-
ential characteristics could be determined.
Analysis of the results from the BIAcore screening and the
subsequent SAR study has demonstrated that altering the sub-
stituents of the aromatic amine substituent does little to
enhance the binding with respect to phenylamine. Together
with the observation that the NH linker is not essential in the
library of 2-phenylquinazoline series, these analyses support
the hypothesis that their binding region and/or pose are differ-
ent. Whether this effect is due to the additional nitrogen atom
in the structure of 6 or the non-fused phenyl group is yet to be
shown.
Further to the compounds discussed here a small group of 4-
aminopyridine analogues were also synthesised and screened
but displayed no binding towards hu- or moPrPC and no anti-
prion activity in cell lines (data not shown). This observation
suggests that at least two fused aromatic rings are necessary for
biological activity as shown by the increased activity of 2-
methylquinoline analogues.
4.1.1. Methylquinolines
The library of 2-methylquinoline analogues did not perform
as well as the acridines in the BIAcore screening with the
majority of results within the range of 30–60% RUmax
(Table 2). Previous studies surrounding quinoline derivatives
as therapeutic candidates for TSEs have focused on aliphatic
side chains extending from the position para to the ring nitro-
gen (in the work by Murakami-Kubo et al. SPR screening was
carried out at 100 μM) [17]. In a similar manner to the acri-
dines cluster, the data suggests that aromaticity at this position
is of significant benefit to binding. The removal of the NH
linker was also shown to be detrimental to binding as seen by
comparing 2a with 2y, a phenolic derivative (Table 2), whereas
the 2-methylquinoline analogues show little sensitivity to the
inclusion of electron withdrawing substituents attached to the
phenyl ring. Compounds 2h, 2i, 2k, and 2n also contain elec-
tron withdrawing groups, but in comparison to the effect on the
acridines, are only marginally detrimental to binding. The
inclusion of electron pushing functional groups on the phenyl
ring was beneficial to binding and the most dramatic change is
seen with compound 2m, in which the amine-derived substitu-
ent is bi-aromatic. However, 2m was shown, in later kinetics
studies, to bind non-specifically to huPrPC.
4.2. Inhibition of PrPSc formation
Each of the drug candidates was tested for activity in SMB
cell lines by quantifying their ability to suppress PrPSc forma-
tion relative to an untreated control. For each experiment the
level of total cell protein was determined along with the viabi-
lity of the cells relative to an untreated control. If a compound
was seen to affect the viability of the cells they were classed as
cytotoxic and rescreened at lower concentrations. This was to
ensure that a reduction in PrPSc levels could only be attributed
to the activity of the compounds and not a reduction in cell
number. The parents of the acridine and 2-methyquinoline
libraries displayed activity at 1 μM whereas the 2-phenylquina-
zoline parent A1 was inactive at 10 μM. The lowest concentra-
tion at which any parent displayed cytotoxicity was at 10 μM.
IC50s of parents A2–A4 were within the range of 1–5 μM with-
out exception (data not shown).
Following the screening of the parents, the libraries of com-
pounds were initially screened at 8 μM for toxicity and activ-
ity, a concentration below that at which cytotoxicity was first
observed for the parents. Compounds that showed no activity
or toxicity were rescreened at the same concentration for con-
firmation. The cytotoxicity and activity of those compounds
4.1.2. Phenylquinazolines
As mentioned, initial analyses suggested that the 2-phenyl-
quinazoline analogues might occupy a different binding region
and/or pose to the acridines and 2-methylquinolines. Com-

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H. Cope et al. / European Journal of Medicinal Chemistry 41 (2006) 1124–1143
showing a reduction in cell viability at 8 μM were reassessed at
lower concentrations. Those compounds that showed activity
without toxicity at 8 μM were either rescreened at an appropri-
ate concentration range to determine approximate IC50s or IC50
ranges (Table 4), or confirmed active by rescreening at either
the same concentration (where standard deviations are shown)
Table 4
SMB cell line screening data for compounds that displayed activity. Cell viability is > 70% of the untreated control at the active concentrations presented
a
b
c
Entry
1b
Structure
Conc. (μM)
8 (10)
PrPSc level (%)
24 (63)
Toxic conc. (μM)
15
IC50 range (μM)
8.0–10.0
1d
1e
1
1
64
48
9
5
8
2
> 1.0
~ 1.0
1g
1h
1
40 11
8
1
≤ 1.0
0.25
57
9
~ 0.25
1m
1n
2.5 (5)
2.5 (5)
62 (18)
32 (23)
10
15
2.5–5.0
1.0–2.5
2b
2d
2h
2.5
64 13
53 10
51 (48)
> 10
2.5–5.0
~ 10.0
~ 7.5
10
15
7.5 (8)
15
(continued)

H. Cope et al. / European Journal of Medicinal Chemistry 41 (2006) 1124–1143
1133
Table 4 (continued)
a
b
c
Entry
2m
Structure
Conc. (μM)
0.5
PrPSc level (%)
52
Toxic conc. (μM)
5
IC50 range (μM)
~ 0.5
3j
2.5
57
65
8
2
> 15
~ 2.5
3e
5
8
5.0–8.0
a
Level of PrPSc relative to untreated control present at the stated concentration.
Concentration at which cells are < 70% viable relative to controls.
b
c
IC50, approximate concentration giving 50% inhibition of PrPSc formation.
or at another concentration (shown in brackets). Compounds
without activity were omitted from the Table for brevity and
thus any compound not included was considered inactive.
An initial observation was that compound 1h, a tri-aromatic
ring structure displayed increased activity above compound 2h,
containing a bi-aromatic ring system. Further to the three
libraries discussed a small group of 4-aminopyridine analo-
gues, including a structure derived from amine 7h, were
screened and displayed no binding or activity (data not
shown). These observations suggest that while reducing the
aromatic system from 3 to 2 fused rings might be detrimental
to activity, reducing the system to one ring removes all anti-
prion activity.
In the case of all three libraries the NH linker is seemingly
essential, compounds 1x, 3x and 8 displaying no activity. The
2-methylquinoline and acridine libraries contain the majority of
the active compounds discussed herein and share many simila-
rities. Each of the amine derived side chains that provoke activ-
ity in the 2-methylquinoline library do so within the acridine
library. In general, side groups with oxygen containing, elec-
tron pushing substituents promote the activity of these classes
of compounds. Whether these groups act as hydrogen bond
acceptors or if the effect of the substituents is on the electronic
nature of the aromatic ring is yet to be seen. As discussed pre-
viously, these functional groups also contribute to the binding
of the acridines and 2-methylquinolines and so data suggests
there to be a relationship between binding and activity. Con-
trary to this observation, acridine analogue 1n contains an elec-
tron withdrawing substituent on the aromatic side group and
yet displays activity. The cyano functionality of 1n appears at
the meta position relative to the NH linker and so the activity
might occur because, although electrons are withdrawn from
the aromatic ring, the δ-positive charge is directed away from
the carbon adjacent to the linker avoiding electron deficiency
where electron density was shown to be important to binding.
Alternatively, the cyano group might be metabolised by the
cell.
The phenylquinazoline library clearly shows no such corre-
lation of activity with the acridine or 2-methylquinoline
libraries. Just two compounds, 3j and 3e, show activity below
cytotoxic concentrations (Table 4). Compound 3j houses a
strongly electron withdrawing group unseen in any other active
compounds save for compound 1n discussed previously.
Again, the ketone group might be metabolised by the cell
before reaching the site of action, but nevertheless, data
strongly suggest that factors which control the activity of the
2-phenylquinazoline library might differ from those that con-
trol the activity of the acridine and 2-methylquinoline libraries.
It appears that electron-donating groups do not have any con-
trol over activity in this class and this observation strengthens
the suggestion that these compounds utilise a different mode of
action against prion protein accumulation. Compounds within
the 2-phenylquinazoline library are more cytotoxic than the
other libraries containing the only compounds showing toxicity
at 1 μM, 3d and 3w (data not shown). Worth noting is the
toxicity of 3d which includes a functional group that promotes
activity in libraries 1 and 2.
The majority of the compounds discussed herein are toxic at
concentrations of 15 μM or below. Bearing in mind that many
of the compounds are also active within the range of 1–15 μM
it becomes clear that the activity threshold for many com-
pounds is extremely close to that for cytotoxicity. For instance,
compound 1e displays activity at 1 μM but when the concen-
tration is increased to 2 μM the cell viability begins to be
affected. An example of an IC50 curve is shown for compound
2b (Fig. 4). The toxicity of this compound is above 10 μM and
so is a clear example of a compound whose activity increases
in a concentration-dependant manner. Similar curves for 1h,
1m and 1n were constructed and although their IC50s were
judged to be below 5 μM, they displayed an increase in PrPSc
levels when nearing their cytotoxic concentrations. It is possi-

1134
H. Cope et al. / European Journal of Medicinal Chemistry 41 (2006) 1124–1143
SAR studies discussed we also conclude that the 2-phenylqui-
nazoline analogues may bind to huPrPC in a different location
and/or pose to the acridines and 2-methylquinolines. It follows
that these compounds may therefore involve a different mode
of action against PrPSc accumulation. This postulation was
strengthened following activity studies in SMB cell lines,
which demonstrated that factors influencing the activity of the
acridines and 2-methylquinolines do not bring about the same
beneficial impact to the 2-phenylquinazoline library. The
alteration of aromatic side groups attached to structures 1, 2
and 3 has brought about significant improvements to the parent
structure activities. Furthermore, we have discussed the possi-
bility that at least two fused aromatic rings are necessary for
binding to PrPC and also anti-prion activity. Reducing the core
structure from a bicyclic system to a pyridine ring is detrimen-
tal to the biological properties, whereas a tricyclic system, as
found in acridine, may be of benefit.
We have formed a solid argument for the substitution of
aromatic side groups to increase the binding affinity and
drug-like potency of these types of compounds. We have
seen that the NH linker itself is likely to be a significant func-
tional group at the position para to the ring nitrogen. Further
investigation into the hypothesis that having an electron-rich
aromatic group attached to the NH linker at this position
would be most beneficial and may lead to the development of
increasingly potent potential TSE therapies.
Fig. 4. Dose–response curve for compound 2b. Compound affects cell viability
at concentrations > 10 μM.
ble that this increase in PrPSc is due to effects on cell viability,
in that as the cell encounters marginally toxic concentrations of
these compounds the levels of PrPC increases as part of a cel-
lular stress response. As a result of this behaviour, it is possible
that some compounds may be active at concentrations very
close to those at which cell viability is affected, and therefore
some compounds may have been deemed inactive in the initial
screen when they may in fact have some underlying activity at
lower concentrations. This might also explain the anomalous
result for the seemingly reduced activity of 1b at 10 μM
(Table 4). As the compound has displayed activity at two con-
centrations its action against PrPSc accumulation cannot be dis-
puted.
6. Experimental
The active compounds reported by Korth et al. [16] and
Murakami-Kubo et al. [17] were tested in neuroblastoma
cells. However, there is evidence that it is harder to inhibit
PrPSc formation in SMB cells than in neuroblastoma lines. Pen-
Melting points were measured using a Bibby-Sterilin
SMP10 melting point apparatus. Accurate mass and nominal
mass measurements were measured using a Waters-Micromass
LCT electrospray mass spectrometer. Infra red spectra were
recorded using neat compounds on a Perkin-Elmer Spectrum
RX1 FT-IR system equipped with a DuraSamplIR IITM dia-
mond ATR solid sample unit, measuring from 4000 to
400 cm–1.
Thin layer chromatography (TLC) was performed using
glass backed silica gel 60 plates (0.25 mm layer), and the
ascending technique was used with a variety of solvents.
Visualisation was achieved using irradiation at either 254 or
365 nm. Flash column chromatography was carried out using
Fluorochem silica gel 60 Å. Dry solvents, when required, were
obtained from an in house Grubbs solvent purification system.
1H NMR and 13C NMR spectra were recorded (unless
otherwise specified) in deuterio-chloroform in 5 mm tubes on
a Bruker AC-250 machine at 250 MHz and 62.5 MHz, respec-
tively. Chemical shifts are quoted in ppm downfield with
respect to trimethylsilane (δ = 0.00 ppm). Coupling constants
(J) are quoted in Hertz to the nearest 0.1 Hz. Compounds
screened were > 95% pure by NMR.
tosan polysulphate was reported to have an IC50 of 1.5 × 10–7
g
ml–1 in SMB cells [24], whilst Priola and Caughey reported an
IC50 of 1 × 10–9 g ml–1 in ScMNB cells [25]. An EC50 of
300 nM has been determined for quinacrine in ScN2a cells
[16], whilst in SMB cells we have found the IC50 to be two
orders of magnitude higher [26]. If this trend is general, it can
be assumed that the IC50 values of the active compounds
reported here could be two orders of magnitude lower in neu-
roblastoma cells. When applied to compounds such as 1h and
2m (Table 4) this gives hypothetical IC50s of less than 10 nM,
which are quite comparable with the IC50s of QCQH (7.5 nM),
PCQH (4 nM) and 2,2′-biquinoline (3 nM) reported in ScNB
cells [17].
5. Conclusion
Within this study we have synthesised three libraries based
upon lead structures determined by VHTS. The work has
focused mainly on aromatic side chains extending from the
carbon para to the ring nitrogen of acridine and 2-methylquino-
line and a comparable location in a previously unreported
potential prion therapeutic 2-phenylquinazoline. We have
shown that binding to PrPC is affected by electron inducing
substituents attached to this aromatic moiety for the acridine
and 2-methylquinoline libraries to their benefit. From the
6.1. General procedures
A. Aryl chloride 4, 5 or 6 (0.5 mmol) and aryl amine 7
(1.0 mmol) were dissolved in 1-methyl-2-pyrrolidinone
(NMP) containing one drop of concentrated hydrochloric acid

H. Cope et al. / European Journal of Medicinal Chemistry 41 (2006) 1124–1143
1135
and stirred at room temperature. The reaction was monitored
by TLC and when complete the mixture was poured into
ethyl acetate (100 ml). The resulting precipitate was collected
by suction filtration. The solids were dissolved in hot methanol
and poured into ethyl acetate (100 ml) to re-precipitate. The
solids formed were collected by suction filtration and dried
under vacuum to yield the product as its hydrochloride salt.
B. To aryl chloride 4 or 5 (0.3 mmol) in phenol (0.6 g) was
added 4-amino-6-methoxypyrimidine 7l (0.3 mmol). After stir-
ring at 120 °C for 2 hours the mixture was poured into acetone
(100 ml). No precipitation was observed and the acetone phase
was separated between aq 1 M HCl solution (100 ml) and ethyl
acetate (100 ml). The pH of the aqueous layer was adjusted to
8 by addition of solid NaHCO3 and extracted with DCM
(2 × 50 ml). The combined DCM layers were dried over
MgSO4 and concentrated under reduced pressure to yield the
product as a solid.
145.9, 154.6; ESI-MS m/z: 299 ([M + H]+); found 299.1552
(C21H19N2 [M + H]+, requires 299.1548).
6.1.1.3. Acridin-9-yl-(3-methylsulphanylphenyl)-amine
(1c).
Conditions were as employed for 1a. 3-(Methylmercapto)-ani-
line c (146.1 mg, 1.05 mmol), 4 (213 mg, 1.0 mmol), reaction
time 4.5 h, gave product 1c. (59 mg, 17%). m.p., 270 °C (dec);
IR (Solid): nmax cm–1, 3460, 2830, 1634, 1570, 1552, 1515,
1
1461, 1437, 1377, 1343, 1276, 1237, 1161, 1092, 1033; H
NMR (250 MHz, d6-DMSO): δ 2.42 (3H, s, SCH3), 7.16
(1H, d, J = 7.3, Ar-H), 7.25 (1H, d, J = 7.9, Ar-H), 7.41 (4H,
m, Ar-H), 7.97 (2H, t, J = 7.6, Ar-H), 8.14 (2H, d, J = 8.2, Ar-
H), 8.27 (2H, d, J = 8.5, Ar-H); 13C NMR (62.5 MHz, d6-
DMSO): δ 14.5, 113.9, 119.2, 120.4, 121.1, 123.8, 124.2
125.8, 130.1, 135.2, 140.1, 140.3, 141.8, 155.0; ESI-MS m/z:
317 ([M + H]+); found 317.1112 (C20H17N2S [M + H]+,
requires 317.1112).
C. Aryl chloride 5 or 6 (0.5 mmol) and aryl amine 7
(1.0 mmol) were refluxed in acetone (11 ml), containing five
drops of concentrated hydrochloric acid and followed to com-
pletion by TLC. The solids formed were collected by suction
filtration and washed with acetone to yield the product as its
hydrochloride salt.
D. To aryl chloride 6 (0.5 mmol) in dioxane (3 ml) was
added amine 7 (2.50 mmol). The reaction mixture was heated
at reflux to completion as determined by TLC. The reaction
mixture was taken up in ethyl acetate (50 ml) and washed
with water (3 × 50 ml). The organic phase was dried over
MgSO4 and then reduced under vacuum to yield the solid pro-
duct.
6.1.1.4. Acridin-9-yl-(3-methoxyphenyl)-amine (1d). Condi-
tions were as employed for 1a. m-Anisidine d (181.9 mg,
1.48 mmol), 4 (213.7 mg, 1.00 mmol), reaction time
24 hours gave product 1d (103.3 mg, 31%) as a solid. m.p.,
170 °C; IR (Solid): nmax cm–1, 3307.4, 3100.3, 2884.6, 2836.1,
1634.4, 1605.1, 1583.1, 1555.8, 1523.9, 1476.4, 1464.8,
1441.2, 1263.9, 1154.9, 1031.2; 1H NMR (250 MHz,
CD3OD): δ 3.34 (1H, s, NH), 3.80 (3H, s, CH3), 6.98–7.06
(3H, m, Ar-H), 7.42–7.48 (3H, m, Ar-H), 7.91–8.03 (4H, m,
Ar-H), 8.21 (2H, d, J = 8.9, Ar-H); 13C NMR (62.5 MHz,
CD3OD): δ 56.1, 111.75, 114.9, 115.1, 118.0, 120.2, 125.3,
126.9, 132.2, 136.9, 141.7, 162.7; ESI-MS m/z: 301 ([M + H]+);
found 301.1334 (C20H17N2O [M + H]+, requires 301.1341).
6.1.1. Acridines
6.1.1.5. Acridin-9-yl-(4-methoxyphenyl)-amine (1e). Condi-
tions were as employed for 1a. p-Anisidine e (154.9 mg,
1.45 mmol), 4 (213.7 mg, 1.00 mmol), reaction time
24 hours gave product 1e (125.3 mg, 37%) as a solid. m.p.,
284 °C; IR (Solid): nmax cm–1, 3463.7, 2894.5, 2838.3,
1633.2, 1584.7, 1555.5, 1524.0, 1505.7, 1475.9, 1464.7,
6.1.1.1. 9-Phenylaminoacridinium (1a). Procedure A. Aniline
7a (106.0 mg, 0.50 mmol), 4 (50.7 mg, 0.55 mmol), reaction
time 1.5 h, gave product 1a (135 mg, 88%). m.p., > 300 °C;
IR (Solid): nmax cm–1, 2765, 1633, 1580, 1550, 1517, 1474,
1440, 1408, 1373, 1343, 1276, 1237 and 1157; 1H NMR
(250 MHz, d6-DMSO): δ 7.37–7.54 (7H, m, Ar-H), 7.93 (2H,
t, J = 8.0, Ar-H), 8.10 (2H, d, J = 8.5, Ar-H), 8.21 (2H, d,
J = 8.6, Ar-H); 13C NMR (62.5 MHz, d6-DMSO): δ 114.2,
119.7, 124.1, 125.0, 126.3, 127.8, 130.4, 135.6, 140.6, 141.7,
155.6; ESI-MS m/z: 271 ([M + H]+); found 271.1237
(C19H15N2 [M + H]+, requires 271.1235).
1
1441.6, 1297.5, 1241.8, 1162.1, 1106.2; H NMR (250 MHz,
d6-DMSO): δ 3.87 (3H, s, CH3), 7.12 (2H, d, J = 6.7, Ar-H),
7.43–7.50 (4H, m, Ar-H), 7.98–8.09 (4H, m, Ar-H), 8.25 (2H,
d, J = 8.5, Ar-H); 13C NMR (62.5 MHz, d6-DMSO): δ 55.5,
113.2, 115.2, 119.1, 123.5, 125.7, 126.4, 125.1, 133.3, 140.0,
155.3, 158.5; ESI-MS m/z: 301 ([M + H]+); found 301.1349
(C20H17N2O [M + H]+, requires 301.1341).
6.1.1.2. Acridin-9-yl-(3-ethylphenyl)-amine (1b). Conditions
were as employed for 1a. 3-Ethylaniline b (152.0 mg,
1.25 mmol), 4 (213.7 mg, 1.00 mmol), reaction time
24 hours gave product 1b (198.5 mg, 59%) as a solid. m.p.,
264 °C; IR (Solid): nmax cm–1, 2864.9, 2729.2, 1634.5, 1581.4,
1559.3, 1524.3, 1477.4, 1464.3, 1438.7, 1415.3, 1376.0,
1345.4, 1262.3, 1175.2, 1158.3, 1114.2, 1033.4; 1H NMR
(250 MHz, d6-DMSO): δ 1.19 (3H, t, J = 7.6, CH3), 2.65
(2H, q, J = 7.6, Ar-H), 7.30 (3H, m, Ar-H), 7.44–7.50 (3H,
m, Ar-H), 7.99–8.11 (4H, m, Ar-H), 8.25 (2H, d, J = 8.6, Ar-
H); 13C NMR (62.5 MHz, d6-DMSO): δ 15.5, 28.0, 106.8,
113.7, 119.2, 121.9, 123.6, 125.8, 126.7, 129.9, 135.2, 140.2,
6.1.1.6. [3-(Acridin-9-ylamino)-phenyl]-methanol (1f). Condi-
tions were as employed for 1a. 3-Aminobenzylalcohol f
(138.6 mg, 1.13 mmol), 4 (213.7 mg, 1.00 mmol), reaction
time 24 hours gave product 1f (142.5 mg, 42%) as a solid.
m.p., 255 °C; IR (Solid): nmax cm–1, 3341.9, 2860.0, 2800.1,
1633.6, 1578.3, 1554.0, 1523.2, 1464.1, 1447.3, 1373.6,
1343.6, 1280.0, 1255.4, 1164.1, 1113.6, 1051.7; 1H NMR
(250 MHz, d6-DMSO): 4.59 (2H, s, CH2), 7.31 (1H, d,
J = 7.6, Ar-H), 7.38–7.5 (5H, m, Ar-H), 8.00–8.11 (4H, m,
Ar-H), 8.26 (2H, d, J = 8.9, Ar-H); 13C NMR (62.5 MHz, d6-
DMSO): δ 62.3, 113.7, 122.3, 122.7, 123.7, 125.8, 126.8,

1136
H. Cope et al. / European Journal of Medicinal Chemistry 41 (2006) 1124–1143
129.6, 135.3, 140.1, 141.2, 144.9, 154.9; ESI-MS m/z: 301
([M + H]+); found 301.1331 (C20H17N2O [M + H]+, requires
301.1341).
(O)CH3), 7.46 (2H, m, Ar-H), 7.64 (2H, m, Ar-H), 8.01 (6H,
m, Ar-H), 8.20 (2H, d, J = 8.9, Ar-H); 13C NMR (62.5 MHz,
d4-MeOH): δ 26.9, 115.7, 120.5, 124.9, 125.6, 126.8, 128.6,
129.9, 131.7, 136.9, 140.2, 142.0, 143.1, 147.2, 157.1; ESI-
MS m/z: 313 ([M + H]+); found 313.1334 (C21H17N2O [M
+ H]+, requires 313.1341).
6.1.1.7. 4-(Acridin-9-ylamino)-phenol (1g). Conditions were as
employed for 1a. 4-Aminophenol g (108.9 mg, 1.00 mmol), 4
(213.7 mg, 1.00 mmol), reaction time 24 hours gave product
1g (149.9 mg, 46%) as a solid. m.p., > 300 °C; IR (Solid):
nmax cm–1, 3101.7, 2929.5, 1632.9, 1587.0, 1560.7, 1511.1,
1472.2, 1438.2, 1415.5, 1365.3, 1345.3, 1265.0, 1225.7,
6.1.1.11. 4-(Acridin-9-ylamino)-benzoic acid (1k). Conditions
were as employed for 1a. 4-Aminobenzoic acid k (77.3 mg,
0.55 mmol) was reacted with 4 (106 mg, 0.5 mmol), reaction
time 1.5 h, gave 1k as a solid. (137 mg, 79%). m.p., > 300 °C;
IR (Solid): nmax cm–1, 2837, 2539, 1707, 1632, 1578, 1547,
1
1165.7, 1154.2, 1099.7; H NMR (250 MHz, d6-DMSO): δ
6.96 (2H, d, J = 8.9, Ar-H), 7.31 (2H, d, J = 8.9, Ar-H), 7.40
(2H, t, J = 7.6, Ar-H), 7.98 (2H, t, J = 7.6, Ar-H), 8.09 (2H, d,
J = 8.5, Ar-H), 8.26 (2H, d, J = 8.9, Ar-H), 10.11 (1H, s, NH);
13C NMR (62.5 MHz, d6-DMSO): δ 113.0, 116.5, 119.0,
123.4, 125.7, 126.6, 131.4, 135.0, 140.0, 155.3, 157.2; ESI-
MS m/z: 287 ([M + H]+); found 287.1186 (C19H15N2O [M
+ H]+, requires 287.1184).
1
1509, 1474, 1376, 1272, 1236, 1215, 1167, 1116, 1018; H
NMR (250 MHz, d6-DMSO): δ 7.48 (2H, d, J = 8.2, Ar-H),
7.50 (2H, t, J = 7.9, Ar-H), 7.98 (2H, d, J = 8.5, Ar-H), 8.03
(2H, m, Ar-H), 8.22 (2H, d, J = 8.5, Ar-H), 8.33 (2H, d,
J = 8.9, Ar-H) 11.7 (1H, bs, NH), 15.4 (1H, bs, NH); 13C
NMR (62.5 MHz, d6-DMSO): δ 115.4, 119.9, 123.4, 124.8,
126.4, 128.6, 131.3, 136.0, 140.7, 146.2, 155.2, 167; ESI-MS
m/z: 315 ([M + H]+); found 315.1120 (C20H15N2O2 [M + H]+,
requires 315.1134).
6.1.1.8. Acridin-9-yl-(3-fluoro-4-methoxyphenyl)-amine (1h).
Conditions were as employed for 1a. 3-Fluoro-4-methoxy ani-
line h (143 mg, 1.0 mmol) was reacted with 4 (213 mg,
1.0 mmol) in the presence of acid, reaction time 3.5 h, to
6.1.1.12. Acridin-9-yl-(6-methoxypyrimidin-4-yl)-amine (1l).
Procedure B. 6-Methoxypyrimidin-4-ylamine 7l (34.6 mg,
0.28 mmol), 4 (60.9 mg, 0.29 mmol), reaction time 2 h. The
solids obtained were subjected to column chromatography
(ethyl acetate/chloroform 1:1, Rf = 0.76) to yield product 1l
as yellow amorphous solid. (12.3 mg, 14% yield). m.p.,
265–266 °C; IR (Solid): nmax cm–1, 2922, 1584, 1552, 1504,
1474, 1396, 1357, 1284, 1233, 1203, 1162 and 1042; 1H NMR
(250 MHz, CDCl3): δ 3.76 (3H, s, OCH3), 5.48 (1H, bs, NH),
7.19 (1H, s, Ar-H), 7.41 (2H, t, J = 7.6, Ar-H), 7.68 (2H, t,
J = 7.3, Ar-H), 8.05 (2H, d, J = 8.5, Ar-H), 8.16 (2H, d,
J = 8.5, Ar-H), 8.36 (1H, s, Ar-H); 13C NMR (62.5 MHz,
CDCl3): δ 53.9, 118.9, 124.0, 126.2; ESI-MS m/z: 303 ([M
+ H]+); found 303.1237 (C18H15N4O [M + H]+, requires
303.1246).
yield product 1h as
a solid. (314 mg, 89%). m.p.,
257–259 °C; IR (Solid): nmax cm–1, 3461, 2890, 2938, 1636,
1584, 1557, 1509, 1466, 1441, 1379, 1312, 1265, 1226, 1174,
1135, 1103, 1025 and 970; 1H NMR (250 MHz, d6-DMSO): δ
2.42 (3H, s, OCH3), 7.16 (1H, d, J = 7.3, Ar-H), 7.25 (1H, d,
J = 7.9, Ar-H), 7.41 (4H, m, Ar-H), 7.97 (2H, t, J = 7.6, Ar-H),
8.14 (2H, d, J = 8.2, Ar-H), 8.27 (2H, d, J = 8.5, Ar-H); 13C
NMR (62.5 MHz, d6-DMSO): δ 56.7, 113.4, 113.7, 113.9,
114.9, 118.9, 119.6, 121.8, 124.2, 126.2, 133.8, 134.0, 135.6,
140.5, 146.8, 147.0, 149.9, 153.8, 155.7; ESI-MS m/z: 319
([M + H]+); found 319.1247 (C20H16FN2O [M + H]+, requires
319.1258).
6.1.1.9. Acridin-9-yl-(3-fluorophenyl)-amine (1i). Conditions
were as employed for 1a. 3-Fluroaniline i (145.2 mg,
1.31 mmol), 4 (213.7 mg, 1.00 mmol), reaction time 24 h,
gave product 1i (256.1 mg, 79%) as a solid. m.p., 288 °C; IR
(Solid): nmax cm–1, 2766.9, 1632.2, 1580.1, 1551.5, 1520.2,
1475.3, 1434.0, 1410.5, 1376.9, 1341.8, 1259.9, 1162.4,
6.1.1.13. Acridin-9-yl-(3-phenoxyphenyl)-amine (1m). Condi-
tions were as employed for 1a. 3-Phenoxyaniline
m
(181.6 mg, 0.98 mmol), 4 (213.7 mg, 1.00 mmol), reaction
time 24 hours gave 1m (123.8 mg, 31%) as a solid. m.p.,
221 °C; IR (Solid): nmax cm–1, 3421.1, 3081.8, 2883.4,
2833.9, 1631.9, 1588.4, 1578.1, 1548.1, 1513.8, 1488.4,
1474.0, 1444.2, 1434.6, 1411.1, 1376.7, 1345.2, 1262.3,
1
1109.4, 1080.1, 1033.7; H NMR (250 MHz, d6-DMSO): δ
7.23–7.40 (3H, m, Ar-H), 7.50–7.58 (3H, m, Ar-H), 8.06
(2H, t, J = 7.6 Hz, Ar-H), 8.21 (2H, d, J = 8.6, Ar-H), 8.32
(2H, d, J = 8.5, Ar-H); 13C NMR (62.5 MHz, d6-DMSO): δ
106.8, 106.9, 111.4, 114.3, 119.4, 120.1, 124.1, 125.8, 131.5,
135.4, 140.2, 155.1; ESI-MS m/z: 289 ([M + H]+); found
289.1141 (C19H14FN2 [M + H]+, requires 289.1141).
1
1214.0, 1214.0, 1171.3, 1150.4, 1112.2; H NMR (250 MHz,
d6-DMSO): δ 7.08–7.10 (4H, m, Ar-H), 7.17 (1H, t, J = 7.3,
Ar-H), 7.26 (1H, d, J = 8.6, Ar-H), 7.40 (2H, t, J = 7.9, Ar-H),
7.53–7.60 (3H, m, Ar-H), 8.02–8.13 (4H, m, Ar-H), 8.32 (2H,
d, J = 8.5, Ar-H); 13C NMR (62.5 MHz, d6-DMSO): δ 114.1,
114.2, 117.3, 118.7, 119.3, 123.8, 124.0, 125.8, 130.1, 131.3,
135.3, 140.1, 155.1, 145.1, 157.6; ESI-MS m/z: 363 ([M + H]+);
found 363.1491 (C25H19N2O [M + H]+, requires 363.1497).
6.1.1.10. 1-[3-(Acridin-9-ylamino)-phenyl]-ethanone
(1j).
Conditions were as employed for 1a. 3-Aminoacetophenone j
(134 mg, 1.0 mmol) was reacted with 4 (212 mg, 1.0 mmol),
reaction time 1.5 h, gave 1j as a solid. (293 mg, 85%). m.p.,
292–294 °C; IR (Solid): nmax cm–1, 2705, 1676, 1636, 1578,
1559, 1519, 1484, 1471, 1414, 1379, 1360, 1316, 1301, 1268,
6.1.1.14. 3-(Acridin-9-ylamino)-benzonitrile (1n). Conditions
were as employed for 1a. 3-Aminobenzonitrile n (135.7 mg,
1.15 mmol), 4 (213.7 mg, 1.00 mmol), reaction time 24 h,
1
1226, 1164; H NMR (250 MHz, d4-MeOH): δ 2.60 (3H, s, C

H. Cope et al. / European Journal of Medicinal Chemistry 41 (2006) 1124–1143
1137
gave product 1n (274.6 mg, 83%) as a solid. m.p., 291 °C; IR
(Solid): nmax cm–1, 2735.7, 2211.8, 1635.4, 1578.5, 1554.1,
1516.0, 1474.5, 1435.4, 1410.8, 1375.7, 1350.6, 1269.6,
NMR (62.5 MHz, d6-DMSO): δ 19.9, 99.9, 115.8, 119.7,
123.4, 125.5, 126.5, 127.1, 129.9, 133.7, 137.2, 138.1, 154.1,
154.6; ESI-MS m/z: 235 [M + H]+; found 235.1237 (C16H15N2
[M + H]+, requires 235.1235).
1
1158.0, 1029.8; H NMR (250 MHz, d6-DMSO): δ 7.55 (2H,
t, J = 7.3, Ar-H), 7.72 (2H, d, J = 7.0, Ar-H), 7.84 (1H, d,
J = 7.0, Ar-H), 7.92 (1H, s, Ar-H), 8.08–8.13 (4H, m, Ar-H),
8.27 (2H, d, J = 8.9, Ar-H); 13C NMR (62.5 MHz, d6-DMSO):
δ 106.5, 112.4, 114.8, 118.2, 119.5, 124.3, 125.8, 126.7, 128.2,
130.6, 131.0 135.5, 140.3, 154.9; ESI-MS m/z: 296
([M + H]+); found 296.1187 (C20H14N3 [M + H]+, requires
296.1188).
6.1.2.2. (3-Ethylphenyl)-(2-methylquinolin-4-yl)-amine
(2b).
Procedure C. 3-Ethylaniline 7b (250.7 mg, 2.07 mmol), 5
(0.20 ml, 1.00 mmol), reaction time 18 h, gave 2b as a solid.
(96.7 mg, 99%) m.p., 258 °C; IR (Solid): nmax cm–1, 2770.0,
1638.8, 1597.8, 1586.1, 1553.9, 1487.6, 1442.5, 1368.8,
1279.5, 1204.2, 1167.7, 1143.8, and 1045.9; 1H NMR
(250 MHz, d6-DMSO): δ 1.27 (3H, t, J = 7.6, CH3), 2.64
(3H, s, CH3), 2.73 (2H, q, J = 7.6, CH2), 6.71 (1H, s, Ar-H),
7.33 (1H, t, J = 5.8, Ar-H), 7.33 (1H, d, J = 5.2, Ar-H), 7.35
(1H, s, Ar-H), 7.52 (1H, t, J = 8.1, Ar-H), 7.79 (1H, t, J = 6.3,
Ar-H), 7.99–8.09 (2H, m, Ar-H), 8.76 (1H, d, J = 8.2, Ar-H),
10.74 (1H, s, NH); 13C NMR (62.5 MHz, d6-DMSO): δ 15.4,
19.8, 28.0, 99.9, 116.1, 119.7, 122.7, 123.4, 124.7, 126.5,
126.8, 129.8, 133.6, 137.2, 138.4, 145.8, 154.2, 154.5; ESI-
MS m/z: 263 [M + H]+; found 263.1543 (C18H19N2
[M + H]+, requires 263.1548).
6.1.1.15. N-acridin-9-yl-benzene-1,4-diamine (1o). Conditions
were as employed for 1a. 1,3-Phenyldiamine o (118.8 mg,
1.10 mmol), 4 (213.7 mg, 1.00 mmol), reaction time 24 h,
gave product 1o (236.2 mg, 73%) as a solid. m.p., 266 °C
(decomposed); IR (Solid): nmax cm–1, 2791.5, 2586.2, 1633.4,
1577.8, 1552.3, 1517.8, 1485.4, 1472.3, 1434.6, 1412.3,
1375.1, 1347.4, 1266.1, 1156.4, 1114.3, 1032.3; 1H NMR
(250 MHz, d6-DMSO): δ 6.85 (2H, d, J = 7.9, Ar-H), 7.29
(1H, t, J = 7.8, Ar-H), 7.47 (1H, d, J = 9.1, Ar-H), 7.56–7.66
(2H, m, Ar-H), 8.07, (2H, t, J = 7.5, Ar-H), 8.20 (2H, d,
J = 8.6, Ar-H), 8.44 (2H, d, J = 8.6, Ar-H); 13C NMR
(62.5 MHz, d6-DMSO): δ 106.8, 114.5, 118.9, 122.9, 123.5,
125.8, 130.0, 135.1, 140.2, 155.2; ESI-MS m/z: 286
([M + H]+); found 286.1344 (C17H16N2O2 [M + H]+, requires
286.1332).
6.1.2.3. (2-Methylquinolin-4-yl)-(3-methylsulphanylphenyl)-
amine (2c). Conditions were as employed for 2b. 3-(Methyl-
mercapto) aniline c (142.2 mg, 1.02 mmol), 5 (94.6 mg,
0.53 mmol), reaction time 18 h, gave 2c as a solid. (115 mg,
68%). m.p., 160 °C; IR (Solid): nmax cm–1, 1596, 1570, 1485,
1
1412, 1377, 1343, 1208; H NMR (250 MHz, d6-DMSO): δ
6.1.1.16. Acridin-9-yl-methylphenyl-amine (1x). Conditions
were as employed for 1a. N-Methylaniline x (173.7 mg,
1.62 mmol), 4 (213.7 mg, 1.00 mmol), reaction time
24 hours gave product 1x (194.9 mg, 61%) as a solid. m.p.,
249 °C; IR (Solid): nmax cm–1, 2365.0, 1878.1, 1631.4,
1575.7, 1489.4, 1470.0, 1433.9, 1408.1, 1380.2, 1291.9,
2.65 (3H, s, CH3), 3.18 (3H, s CH3), 6.76 (1H, s, Ar-H),
7.27–7.37 (3H, m, Ar-H), 7.50 (1H, dd, J = 9.2, J = 8.3, Ar-
H), 7.78 (1H, t, J = 7.6, Ar-H), 7.99–8.11 (2H, m, Ar-H),
8.77 (1H, d, J = 8.5, Ar-H), 10.81 (1H, s, NH); 13C NMR
(62.5 MHz, d6-DMSO): δ 14.4, 19.8, 106.8, 116.2, 119.7,
121.6, 122.2, 123.4, 124.6, 127.9, 130.2, 133.7, 137.9, 138.4,
140.3, 154.2, 154.7; ESI-MS m/z: 281 [M + H]+; found
281.1108 (C17H17N2S [M + H]+, requires 281.1112).
1
1209.9, 1155.3 and 1100.4; H NMR (250 MHz, d6-DMSO):
δ 3.84 (3H, s, CH3), 6.97–7.01 (3H, m, Ar-H), 7.30 (2H, t,
J = 7.8, Ar-H), 7.73 (1H, d, J = 8.5, Ar-H), 7.76 (1H, d,
J = 7.6, Ar-H), 8.15–8.22 (4H, m, Ar-H), 8.53 (2H, d,
J = 8.9, Ar-H); 13C NMR (62.5 MHz, d6-DMSO): δ 42.2,
106.1, 108.9, 116.4, 120.2, 123.6, 126.0, 127.0, 129.5, 135.5,
148.7; ESI-MS m/z: 285 ([M + H]+); found 285.1387
(C20H17N2 [M + H]+, requires 285.1392).
6.1.2.4. (3-Methoxyphenyl)-(2-methylquinolin-4-yl)-amine
(2d). Conditions were as employed for 2b. m-Anisidine d
(248.1 mg, 2.01 mmol), 5 (0.20 ml, 1.00 mmol), reaction
time 18 h, gave product 2d (108.5 mg, 36%) as a solid. m.p.,
237 °C; IR (Solid): nmax cm–1, 2683.7, 1638.2, 1586.9, 1553.7,
1488.6, 1390.1, 1287.9, 1259.9, 1200.6, 1158.5, 1099.0,
1035.5; 1H NMR (250 MHz, d6-DMSO): δ 2.68 (3H, s,
CH3), 3.85 (3H, s, OCH3), 6.78 (1H, s, Ar-H), 7.01–7.11
(3H, m, Ar-H), 7.51 (1H, t, J = 7.9, Ar-H), 7.77 (1H, t,
J = 6.9, Ar-H), 8.02 (1H, t, J = 5.5, Ar-H), 8.19 (1H, d,
J = 8.2, Ar-H), 8.88 (1H, d, J = 7.9, Ar-H), 10.94 (1H, s, NH);
13C NMR (62.5 MHz, d6-DMSO) 19.7, 55.4, 100.2, 106.8,
111.1, 112.8, 116.2, 117.3, 119.6, 123.6, 126.4, 130.6, 133.5,
138.4, 154.2, 154.5; ESI-MS m/z: 265 [M + H]+; found
265.1353 (C17H17N2O [M + H]+, requires 265.1341).
6.1.2. 2-Methylquinolines
6.1.2.1. (2-Methylquinolin-4-yl)-phenylamine (2a). Procedure
C. Aniline 7a (93 mg, 1.0 mmol), 5 (89 mg, 0.5 mmol), reac-
tion time 16 h, gave 2a as a solid (102 mg, 85%). Alternatively
an identical procedure as that employed for 1a was used. 7a
(100 mg, 1.1 mmol), 5 (218 mg, 1.0 mmol), reaction time
1.5 h, gave 2a, after basic extraction, as a solid (free base).
(388 mg, 36%). m.p., 185–187 °C; IR (Solid): nmax cm–1,
3473, 2901, 1637, 1608, 1592, 1555, 1492, 1450, 1370,
1
1278, 1144, 860, 753, 700, 636 and 524; H NMR (250 MHz,
6.1.2.5. (4-Methoxyphenyl)-(2-methylquinolin-4-yl)-amine (2e).
Conditions were as employed for 2b. p-Anisidine e (246.7 mg,
2.00 mmol), 5 (0.20 ml, 1.00 mmol), reaction time 18 h, gave
product 2e (230.7 mg, 77%) as a solid. m.p., 295 °C; IR
d6-DMSO): δ 2.65 (3H, s, CH3), 6.73 (1H, s, Ar-H), 7.44–7.65
(4H, m, Ar-H), 7.79 (1H, t, J = 7.5, Ar-H), 8.00–8.12 (2H, m,
Ar-H), 8.79 (1H, d, J = 8.5, NH), 10.83 (1H, bs, NH); 13C

1138
H. Cope et al. / European Journal of Medicinal Chemistry 41 (2006) 1124–1143
(Solid): nmax cm–1, 2686.0, 1638.4, 1607.5, 1588.6, 1553.6,
154.9, 164.5; ESI-MS m/z: 253 [M + H]+; found 253.1145
1489.2, 1447.5, 1367.8, 1288.2, 1260.4, 1201.4, 1159.0,
(C16H14FN2 [M + H]+, requires 253.1141).
1
1143.3, 1099.5 and 1034.3; H NMR (250 MHz, d6-DMSO):
δ 2.61 (3H, s, CH3), 3.87 (3H, s, CH3), 6.57 (1H, s, Ar-H),
7.17 (2H, d, J = 8.9 Hz, Ar-H), 7.42 (2H, d, J = 8.9 Hz, Ar-
H), 7.75–7.82 (1H, m, Ar-H), 8.02 (2H, d, J = 3.4 Hz, Ar-H),
8.71 (1H, d, J = 8.5 Hz, Ar-H), 10.67 (1H, s, NH); 13C NMR
(62.5 MHz, d6-DMSO): δ 19.9, 55.4, 99.6, 115.1, 119.6,
123.3, 126.4, 127.3, 129.6, 133.6, 154.3, 154.9, 158.3; ESI-
MS m/z: 265 [M + H]+; found 265.1345 (C17H17N2O
[M + H]+, requires 265.1341).
6.1.2.9. 1-[3-(2-Methylquinolin-4-ylamino)-phenyl]-ethanone
(2j). Conditions were as employed for 2b. 3-Aminoacetophe-
none j (347 mg, 2.6 mmol), 2 (229 mg, 1.3 mmol), reaction
time 18 h, gave 2j as a solid. (348 mg, 82%). m.p., 210 °C
(dec); IR (Solid): nmax cm–1, 1686, 1636, 1585, 1556, 1447,
1
1360, 1294, 1260, 1204; H NMR (250 MHz, d6-DMSO): δ
2.67 (3H, s, CH3), 2.68 (3H, s, CH3), 6.81 (1H, s, Ar-H),
7.72–7.84 (3H, m, Ar-H), 8.00–8.13 (4H, m, Ar-H), 8.81
(1H, d, J = 8.2, Ar-H), 10.94 (1H, s, NH); 13C NMR
(62.5 MHz, d6-DMSO): δ 19.8, 26.9, 106.8, 155.6, 116.3,
119.8, 123.5, 124.9, 126.7, 126.8, 125.9, 130.1, 130.4, 130.9,
130.9, 134.1, 154.1, 155.4; ESI-MS m/z: 277 [M + H]+; found
277.1344 (C18H17N2O2 [M + H]+, requires 277.1341).
6.1.2.6. [4-(2-Methylquinolin-4-ylamino)-phenyl]-methanol
(2f). Conditions were as employed for 2b. 3-Aminobenzylalco-
hol f (246.2 mg, 2.00 mmol), 2 (0.20 ml, 1.00 mmol), reaction
time 18 h, gave product 2f (222.3 mg, 74%) as a solid. m.p.,
259 °C; IR (Solid): nmax cm–1, 2789.6, 1640.6, 1601.1, 1584.3,
1556.8, 1446.3, 1365.9, 1279.6, 1207.1, 1169.9, 1140.2 and
1046.3; 1H NMR (250 MHz, d6-DMSO): δ 2.65 (3H, s,
CH3), 3.20 (2H, s, CH2), 6.70 (1H, s, Ar-H), 7.38 (2H, d,
J = 7.6, Ar-H), 7.51–7.59 (2H, m, Ar-H), 7.76 (1H, t, J = 7.8,
Ar-H), 8.01 (1H, t, J = 7.6, Ar-H), 8.15 (1H, d, J = 8.2, Ar-H),
8.83 (1H, d, J = 7.5, Ar-H) 10.90 (1H, s, NH); 13C NMR
(62.5 MHz, d6-DMSO): δ 19.8, 62.3, 99.8, 116.1, 119.6,
123.2, 123.5, 123.6, 125.1, 126.5, 129.6, 133.6, 137.1, 138.1,
144.7, 154.3, 154.5; ESI-MS m/z: 265 [M + H]+; found
265.1329 (C17H17N2O [M + H]+, requires 265.1341).
6.1.2.10. 4-(2-Methylquinolin-4-ylamino)-benzoic acid (2k).
Conditions were as employed for 1a. 4-Aminobenzoic acid k
(152 mg, 1.1 mmol), 5 (208 mg, 1.2 mmol), reaction time 5 h,
gave 2k after basic extraction as a solid. (107 mg, 37%). m.p.,
> 300 °C; IR (Solid): nmax cm–1, 1692, 1639, 1588, 1524,
1439, 1369, 1277, 1220, 1169; 1H NMR (250 MHz, d6-
DMSO): δ 2.70 (3H , s, CH3), 7.01 (1H, s, Ar-H), 7.67 (2H,
d, J = 8.6, Ar-H), 7.84 (1H, t, J = 6.7, Ar-H), 8.02 (1H, t,
J = 7.9, Ar-H), 8.11–8.17 (3H, m, Ar-H), 8.85 (1H, d,
J = 8.5, Ar-H), 11.0 (1H, bs, NH); 12C NMR (62.5 MHz, d6-
DMSO): δ 19.1, 101.0, 116.6, 119.8, 123.7, 124.5, 126.8,
128.7, 131.0, 133.8, 138.8, 141.8, 153.6, 155.1, 166.7; ESI-
MS m/z: 279 [M + H]+; found 279.1140 (C17H15N2O2
[M + H]+, requires 279.1134).
6.1.2.7. (3-Fluoro-4-methoxyphenyl)-(2-methylquinolin-4-yl)-
amine (2h). Conditions were as employed for 2b. 3-Fluoro-4-
methoxy aniline h (148 mg, 1.1 mmol), was reacted with 5
(96 mg, 0.5 mmol), reaction time 23 h, gave 2h as a solid.
(208 mg, 95%). m.p., 160 °C (dec); IR (Solid): nmax cm–1,
1605, 1509, 1450, 1371, 1284, 1221, 1162, 1129, 1095, 1018;
1H NMR (250 MHz, d6-DMSO): δ 2.64 (3H, s, CH3), 3.94
(3H, s, OCH3), 6.67 (1H, s, Ar-H), 7.05–7.50 (4H, m, Ar-H),
7.75 (1H, t, J = 6.7, Ar-H), 8.04 (1H, d, J = 8.6, Ar-H), 8.05
(1H, t, J = 9.0, Ar-H), 8.74 (1H, d, J = 8.9, Ar-H), 10.76 (1H,
s, NH); 13C NMR (62.5 MHz, d6-DMSO): δ 19.6, 56.2, 99.9,
113.8, 114.2, 114.4, 114.5, 116.0, 119.5, 122.3, 123.6, 126.4,
129.8, 133.5, 138.3, 146.2, 154.5; ESI-MS m/z: 283 [M + H]+;
found 238.1238 (C17H16FN2O [M + H]+, requires 283.1247).
6.1.2.11. (2-Methylquinolin-4-yl)-(3-phenoxyphenyl)-amine
(2m). Conditions were as employed for 2b. 3-Phenoxyaniline
m (367.6 mg, 1.98 mmol), 5 (0.20 ml, 1.00 mmol), reaction
time 18 h, gave product 2m (222.3 mg, 61%) as a solid. m.p.,
229 °C; IR (Solid): nmax cm–1, 3013.9, 2672.0, 1640.8, 1605.9,
1582.4, 1554.3, 1533.6, 1482.7, 1440.1, 1367.4, 1293.7,
1
1253.2, 1208.6, 1153.0, 1142.6, 1099.2, 1068.4, 1020.7; H
NMR (250 MHz, d6-DMSO): δ 2.69 (3H, s, CH3), 6.87 (1H,
s, Ar-H), 6.87–7.33 (7H, m, Ar-H), 7.60 (2H, t, J = 7.8, Ar-H),
7.77 (1H, t, J = 8.1, Ar-H), 8.02 (1H, t, J = 7.6, Ar-H), 8.16
(1H, d, J = 8.2, Ar-H), 8.81 (1H, d, J = 8.5, Ar-H), 10.90
(1H, s, NH); 13C NMR (62.5 MHz, d6-DMSO): δ 19.8,
100.4, 106.9, 114.8, 116.3, 116.9, 119.2, 119.7, 123.6, 124.1,
126.5, 130.2, 131.2, 133.6, 138.4, 138.9, 154.0, 154.7, 156.0,
156.0; ESI-MS m/z: 327 [M + H]+; found 327.1490
(C22H19N2O [M + H]+, requires 327.1497).
6.1.2.8. (3-Fluoro-phenyl)-(2-methylquinolin-4-yl)-amine (2i).
Conditions were as employed for 2b. 3-Fluroaniline 2i
(219.5 mg, 1.97 mmol), 5 (0.20 ml, 1.00 mmol), reaction
time 18 h, gave product 2i (195.8 mg, 68%) as a solid. m.p.,
190 °C; IR (Solid): nmax cm–1, 2901.1, 1638.4, 1591.2, 1556.2,
1487.5, 1442.8, 1385.7, 1370.0, 1256.3, 1207.5, 1141.8,
1099.4; 1H NMR (250 MHz, d6-DMSO): δ 2.70 (3H, s,
CH3), 6.88 (1H, s, Ar-H), 7.30 (1H, t, J = 7.3, Ar-H), 7.43
(1H, d, J = 8.6, Ar-H), 7.48 (1H, s, Ar-H), 7.66 (1H, d,
J = 7.0, Ar-H), 7.76 (1H, t, J = 7.3, Ar-H), 8.04 (1H, t,
J = 7.3, Ar-H), 8.18 (1H, d, J = 8.5, Ar-H), 8.88 (1H, d,
J = 8.2, Ar-H), 11.01 (1H, s, NH); 13C NMR (62.5 MHz, d6-
DMSO): δ 19.7, 100.5, 112.2, 112.6, 113.7, 114.0, 116.3,
119.7, 121.6, 123.6, 126.6, 131.4, 131.5, 138.4, 139.1, 153.9,
6.1.2.12. 3-(2-Methylquinolin-4-ylamino)-benzonitrile
(2n).
Conditions were as employed for 2b. 3-Aminobenzonitrile n
(243.0 mg, 2.06 mmol), 5 (0.20 ml, 1.00 mmol), reaction
time 18 h, gave product 2n (135.7 mg, 46%) as a solid. m.p.,
298 °C; IR (Solid): nmax cm–1, 2849.2, 2230.4, 1639.1, 1583.4,
1557.2, 1486.1, 1444.2, 1393.1, 1368.7, 1280.6, 1246.0,
1
1208.0 and 1143.3; H NMR (250 MHz, d6-DMSO): δ 1.96
(3H, s, CH3), 6.07 (1H, s, Ar-H), 6.79 (7H, m, Ar-H), 7.24

H. Cope et al. / European Journal of Medicinal Chemistry 41 (2006) 1124–1143
1139
(1H, d, J = 8.5); 13C NMR (62.5 MHz, d6-DMSO): δ 19.8,
100.7, 112.7, 116.4, 118.2, 118.9, 121.7, 123.7, 126.7, 128.6,
130.3, 130.4, 131.2, 133.8, 138.4, 153.9, 155.2; ESI-MS m/z:
260 [M + H]+; found 260.1194 (C17H14N3 [M + H]+, requires
260.1188).
0.5 mmol, reaction time 22 h, gave 3c as a solid. (148 mg,
78%). m.p., 171–175 °C; IR (Solid) nmax cm–1, 3047, 2628,
1
1598, 1557, 1356; H NMR (250 MHz, d6-DMSO): δ 2.53
(3H, s, SCH3), 7.24 (1H, d, J = 7.9, Ar-H), 7.48 (1H, t,
J = 7.9, Ar-H), 7.60–7.77 (5H, m, Ar-H), 7.83 (1H, t, J = 7.6,
Ar-H), 7.87 (1H, s, Ar-H), 8.11 (1H, t, J = 7.9, Ar-H), 8.42
(1H, s, Ar-H), 8.44 (2H, d, J = 7.3, Ar-H), 9.03 (1H, d,
J = 8.2, Ar-H), 11.76 (1H, s, NH); 13C NMR (62.5 MHz, d6-
DMSO): δ 14.7, 106.8, 112.8, 120.8, 121.5, 123.6, 124.6,
126.0, 128.1, 129.0, 129.2, 129.2, 131.0, 133.3, 135.9, 137.6,
138.7, 157.2, 159.0; ESI-MS m/z: 344 [M + H]+; found
344.1210 (C21H18N3S [M + H]+, requires 344.1221).
6.1.2.13. 2-Methyl-4-phenoxyquinoline (2y). Procedure similar
to that employed for 1l. 4-Amino-6-methoxypyrimidine l
(69 mg, 0.5 mmol), 5 (101 mg, 0.6 mmol) and phenol (1 g).
2y was isolated after column chromatography (ethyl acetate/
hexane 1:4) as a solid. (77 mg, 55%). m.p., 77 °C; IR (Solid):
nmax cm–1, 3309, 3244, 2926, 1619, 1590, 1509, 1486, 1415,
1
1378, 1344, 1232, 1209, 1178 and 1069; H NMR (250 MHz,
CDCl3): δ 2.6 (3H, s, CH3), 6.4 (1H, s, Ar-H), 7.1–7.2 (2H, m,
Ar-H), 7.3 (1H, d, J = 6.9, Ar-H), 7.4–7.5 (3H, m, Ar-H), 7.7
(1H, t, J = 6.3, Ar-H), 8.0 (1H, d, J = 7.5, Ar-H), 8.3 (1H, d,
J = 7.5, Ar-H); 13C NMR (62.5 MHz, CDCl3): δ 104.8, 121.1,
121.9, 125.4, 125.6, 130.3; ESI-MS m/z: 236 [M + H]+; found
236.106 (C16H13ON.HCl [M + H]+, requires 236.107).
6.1.3.4. (3-Methoxyphenyl)-(2-phenylquinazolin-4-yl)-amine
(3d). Conditions were as employed for 3a. m-Anisidine d
(496.1 mg, 2.01 mmol), 6 (269.9 mg, 1.12 mmol), reaction
time 18 h, gave product 3d (402.9 mg, 99%) as a solid. m.p.,
232 °C; IR (Solid) nmax cm–1, 2889.9, 2835.0, 1632.8, 1599.7,
1555.9, 1504.2, 1456.6, 1358.4, 1331.4, 1326.9, 1296.9,
1
1243.6, 1168.2, 1026.2; H NMR (250 MHz, d6-DMSO): δ
6.1.3. 2-Phenylquinazolines
3.86 (3H, s, OCH3), 7.13 (2H, d, J = 9.2, Ar-H), 7.64–7.87
(6H, m, Ar-H), 8.11 (1H, t, J = 7.6, Ar-H), 8.40 (3H, d,
J = 7.6, Ar-H), 8.97 (1H, d, J = 8.2, Ar-H), 11.69 (1H, s, NH);
13C NMR (62.5 MHz, d6-DMSO): δ 55.4, 109.9, 112.2, 112.9,
116.5, 124.6, 129.0, 129.2, 129.6, 135.8, 138.3, 157.4, 157.6,
159.0, 159.5; ESI-MS m/z: 328 [M + H]+; found 328.1435
(C21H18N3O [M + H]+, requires 328.1450).
6.1.3.1. Phenyl-(2-phenylquinazolin-4-yl)-amine (3a). Proce-
dure D. Aniline 7a (97 mg, 1.0 mmol), 6 (121 mg,
0.5 mmol), reaction time 4 h, gave 3a as a solid. (134 mg,
80%). m.p., 239–242 °C; IR (Solid): nmax cm–1, 3046, 2332,
1
1608, 1546; H NMR (250 MHz, d6-DMSO): δ 7.40 (1H, t,
J = 7.3, Ar-H), 7.59 (2H, t, J = 7.9, Ar-H), 7.61–7.80 (2H, m,
Ar-H), 7.69 (2H, d, J = 7.6, Ar-H), 7.84 (1H, s, NH), 7.90 (2H,
d, J = 7.9, Ar-H), 8.13 (1H, t, J = 7.6, Ar-H), 8.42 (1H, d,
J = 7.0, Ar-H), 8.43 (2H, d, J = 7.0, Ar-H), 9.00 (1H, d,
J = 7.9, Ar-H), 11.71 (1H, s, NH); 13C NMR (62.5 MHz, d6-
DMSO): δ 106.7, 112.8, 124.5, 124.6, 126.4, 128.1, 128.2,
128.7, 129.0, 129.2, 133.2, 135.8, 137.0, 157.3, 159.0; ESI-
MS m/z: 298 [M + H]+; found 298.1345 (C20H16N3
[M + H]+, requires 298.1344).
6.1.3.5. (4-Methoxyphenyl)-(2-phenylquinazolin-4-yl)-amine
(3e). Conditions were as employed for 3a. p-Anisidine e
(496.1 mg, 2.01 mmol), 6 (234.3 mg, 0.97 mmol), reaction
time 18 h, gave product 3e (303.1 mg, 86%) as a solid. m.p.,
248 °C; IR (Solid) nmax cm–1, 2836.5, 2656.5, 1627.6, 1627.6,
1610.2, 1559.8, 1495.9, 1457.1, 1435.9, 1421.4, 1370.6,
1
1358.7, 1329.6, 1294.4, 1264.1, 1186.7, 1152.5, 1032.1; H
NMR (250 MHz, d6-DMSO): δ 3.86 (3H, s, OCH3), 6.96
(1H, d t, J = 6.7, J = 2.5, Ar-H), 7.44–7.49 (2H, m, Ar-H),
7.59–7.83 (4H, m, Ar-H), 7.90 (1H, t, J = 7.7, Ar-H), 8.13
(1H, t, J = 7.6, Ar-H), 8.38–8.46 (3H, m, Ar-H), 8.97 (1H, d,
J = 8.24, Ar-H), 11.55 (1H, s, NH); 13C NMR (62.5 MHz, d6-
DMSO): δ 55.4, 109.9, 112.2, 112.9, 124.6, 129.0, 129.2,
129.6, 135.8, 138.4, 157.32, 159.0, 159.5; ESI-MS m/z: 328
[M + H]+; found 328.1456 (C22H18N3O [M + H]+, requires
328.1450).
6.1.3.2. (3-Ethylphenyl)-(2-phenylquinazolin-4-yl)-amine (3b).
Conditions were as employed for 3a. 3-Ethylaniline b
(250.6 mg, 2.06 mmol), 6 (232.5 mg, 0.96 mmol), reaction
time 5 h, gave product 3b (321.6 mg, 92%) as a solid. m.p.,
158 °C; IR (Solid): nmax cm–1, 2827.8, 2341.5, 2228.8, 2030.3,
1628.2, 1609.5, 1550.4, 1503.2, 1487.4, 1457.3, 1414.9,
1
1361.4, 1329.4, 1147.0; H NMR (250 MHz, d6-DMSO): δ
1.28 (3H, t, J = 7.5, CH3), 2.71 (2H, q, J = 7.5, CH2), 7.21
(1H, d J = 7.6, Ar-H), 7.45 (1H, t, J = 7.8, Ar-H), 7.59–7.85
(6H, m, Ar-H), 8.10 (1H, t, J = 7.9, Ar-H), 8.46–8.55 (3H, m,
Ar-H), 9.07 (1H, d, J = 8.2, Ar-H), 11.87 (1H, s, NH); 13C
NMR (62.5 MHz, d6-DMSO): δ 15.6, 28.2, 112.6, 120.2,
121.2, 123.9, 124.7, 126.0, 128.0, 128.5, 128.8, 129.3, 131.2,
133.4, 135.5, 136.7, 140.2, 144.2, 156.8, 158.8; ESI-MS m/z:
326 [M + H]+; found 326.1667 (C22H20N3 [M + H]+, requires
326.1657).
6.1.3.6. [4-(2-Phenylquinazolin-4-ylamino)-phenyl]-methanol
(3f). Conditions were as employed for 3a. 3-Aminobenzylalco-
hol f (246.2 mg, 2.00 mmol), 6 (271.9 mg, 1.16 mmol), reac-
tion time 18 h, gave product 3f (396.6 mg, 96%) as a solid. m.
p., 240 °C; IR (Solid): nmax cm–1, 2828.0, 2230.3, 1971.3,
1590.8, 1551.7, 1502.5, 1458.0, 1419.7, 1367.9, 1328.1,
1
1164.8, 1021.4; H NMR (250 MHz, d6-DMSO): δ 4.65 (2H,
s, CH2OH), 7.27 (1H, d, J = 4.6, Ar-H), 7.52 (1H, t, J = 7.6,
Ar-H), 7.76–7.79 (4H, m, Ar-H), 7.86 (1H, t, J = 7.3, Ar-H),
7.97 (1H, s, Ar-H), 8.12 (1H, t, J = 7.6, Ar-H), 8.24 (1H, d,
J = 7.9, Ar-H), 8.43 (2H, d, J = 7.6, Ar-H), 8.86 (1H, d,
J = 9.5, Ar-H); 13C NMR (62.5 MHz, d6-DMSO): δ 55.3,
6.1.3.3. (3-Methylsulphanylphenyl)-(2-phenylquinazolin-4-yl)-
amine (3c). Conditions were as employed for 3a. 3-(Methyl-
mercapto) aniline c (0.13 ml, 1.0 mmol), 6 (121 mg,

1140
H. Cope et al. / European Journal of Medicinal Chemistry 41 (2006) 1124–1143
111.3, 114.7, 115.0, 115.7, 117.2, 120.5, 120.5, 120.7, 120.9,
122.9, 125.5, 128.1, 128.5; ESI-MS m/z: 328 [M + H]+; found
328.1458 (C21H18N3O [M + H]+, requires 328.1450).
8.44 (1H, d, J = 9.2, Ar-H), 8.48 (2H, d, J = 7.0, Ar-H), 8.62
(1H, s, Ar-H), 9.05 (1H, d, J = 8.2, Ar-H), 11.86 (1H, s, NH);
13C NMR (62.5 MHz, d6-DMSO): δ 106.8, 112.9, 123.6,
124.6, 126.0, 128.1, 128.7, 129.0, 129.1, 129.3, 130.0, 131.1,
133.3, 135.9, 137.2, 137.6, 157.3, 159.0, 197.5; ESI-MS m/z:
340 [M + H]+; found 340.1437 (C22H18N3O [M + H]+,
requires 340.1450).
6.1.3.7. 4-(2-Phenylquinazolin-4-ylamino)-phenol (3g). Condi-
tions were as employed for 3a. 4-Aminophenol g (220.5 mg,
2.02 mmol), 6 (252.5 mg, 1.05 mmol), reaction time 18 h, gave
product 3g (347.9 mg, 96%) as a solid. m.p., 241 °C decom-
posed; IR(Solid) nmax cm–1, 2911.4, 2591.1, 1627.3, 1601.1,
1557.0, 1501.7, 1450.1, 1421.9, 1367.5, 1267.7, 1218.4,
1187.4, 1170.0, 1107.1, 1030.2; 1H NMR (250 MHz, d6-
DMSO): δ 6.89 (1H, d, J = 8.9, Ar-H), 6.96 (2H, d, J = 8.9,
Ar-H), 7.23 (1H, d, J = 8.9, Ar-H), 7.62–7.78 (4H, m, Ar-H),
7.83 (1H, t, J = 7.6, Ar-H), 8.10 (1H, t, J = 7.8, Ar-H), 8.38
(3H, m, Ar-H), 8.89 (1H, d, J = 7.9, Ar-H), 11.46 (1H, s, NH);
13C NMR (62.5 MHz, d6-DMSO): δ 112.6, 115.2, 116.0,
122.3, 124.3, 126.0, 127.9, 129.0, 129.1, 131.5, 133.8, 134.9,
155.5, 157.2, 159.2; ESI-MS m/z: 314 [M + H]+; found
314.1293 (C20H16N3O [M + H]+, requires 314.1293).
6.1.3.11. 4-(2-Phenylquinazolin-4-ylamino)-benzoic acid (3k).
Conditions were as employed for 3a. 4-Aminobenzoic acid k
(210 mg, 1.0 mmol), 6 (122 mg, 0.5 mmol), reaction time 26 h,
gave 3k as a solid. (188 mg, 98%). m.p., 281–284 °C; IR
(Solid): nmax cm–1, 2904, 1709, 1598, 1542; 1H NMR
(250 MHz, d6-DMSO): δ 7.64–7.80 (4H, m, Ar-H), 7.88 (1H,
t, J = 7.6, Ar-H), 8.05–8.19 (5H, m, Ar-H), 8.46 (2H, d,
J = 8.5, Ar-H), 8.45 (1H, s, OH), 9.06 (1H, d, J = 8.2, Ar-H),
11.87 (1H, s, NH); 13C NMR (62.5 MHz, d6-DMSO): δ 107.9,
113.0, 123.9, 124.7, 127.9, 128.2, 129.1, 129.3, 130.0, 131.1,
132.6, 133.3, 136.0, 141.2, 157.3, 159.1, 166.8; ESI-MS m/z:
342 [M + H]+; found 342.1243 (C21H16N3O2 [M + H]+,
requires 342.1243).
6.1.3.8. (3-Fluoro-4-methoxyphenyl)-(2-phenylquinazolin-4-
yl)-amine (3h). Conditions were as employed for 3a. 3-Fluoro-
4-methoxy aniline h (85 mg, 0.6 mmol) was reacted with 6
(72 mg, 0.3 mmol), reaction time 19 h, gave 3h as a solid.
(114 mg, 99%). m.p., 219–222 °C; IR (Solid): nmax cm–1,
2781, 1632, 1502, 1269, 1026; 1H NMR (250 MHz, d6-
DMSO): δ 4.37 (3H, s, OCH3), 7.60–7.85 (2H, m, Ar-H),
8.01–8.22 (3H, m, Ar-H), 8.22–8.35 (2H, m, Ar-H), 8.54
(1H, t, J = 7.6, Ar-H), 8.78 (1H, s, Ar-H), 8.83 (2H, d,
J = 7.0, Ar-H), 9.40 (1H, d, J = 8.5, Ar-H), 12.12 (1H, s, NH);
13C NMR (62.5 MHz, d6-DMSO): δ 56.2, 102.0, 112.7, 113.5,
118.0, 120.6, 124.5, 128.1, 129.0, 129.1, 129.8, 131.8, 133.2,
135.8, 145.4, 152.5, 157.3, 158.8; ESI-MS m/z: 346 [M + H]+;
found 346.1347 (C21H17FN3O [M + H]+, requires 346.1356).
6.1.3.12. (6-Methoxypyrimidin-4-yl)-(2-phenylquinazolin-4-yl)-
amine (3l). Procedure C. 6-Methoxypyrimidin-4-ylamine 7l
(300.0 mg, 2.40 mmol), 6 (112.0 mg, 0.47 mmol), reaction
time 70 h. Purification by flash column chromatography (1:1
EtOAc/hexane, Rf = 0.43) gave 3l as a yellow crystalline solid
(5 mg, 3% yield). IR (Solid): nmax cm–1 3423, 2952, 2927,
1699, 1597, 1555, 1509, 1471, 1441, 1384, 1350, 1327,
1
1297, 1195, 1160; H NMR (250 MHz, CDCl3): δ 4.0 (3H,
s, OMe), 7.45–7.57 (4H, m, Ar-H), 7.79 (1H, t, J = 7.0, Ar-
H), 7.90–8.02 (2H, m, Ar-H), 8.23 (1H, s, Ar-H), 8.32 (1H,
s, NH), 8.14 (1H, s, Ar-H), 8.48 (2H, d, J = 5.6, Ar-H); ESI-
MS m/z: 330 [M + H]+; found 330.1354 (C19H16N5O [M + H]+
requires 330.1355).
6.1.3.9. (3-Fluorophenyl)-(2-phenylquinazolin-4-yl)-amine
(3i). Conditions were as employed for 3a. 3-Fluroaniline i
(219.5 mg, 1.97 mmol), 6 (232.1 mg, 0.96 mmol), reaction
time 18 h, gave product 3i (299.0 mg, 89%) as a solid. m.p.,
278 °C; IR (Solid): nmax cm–1, 3049.4, 2681.7, 1626.3, 1598.3,
1574.0, 1545.9, 1485.0, 1456.0, 1419.6, 1360.7, 1326.7,
6.1.3.13. (3-Phenoxyphenyl)-(2-phenylquinazolin-4-yl)-amine
(3m). Conditions were as employed for 3a. 3-Phenoxyaniline
m (367.6 mg, 1.98 mmol), 6 (226.1 mg, 0.94 mmol), reaction
time 18 h, gave product 3m (281.5 mg, 70%) as a solid. m.p.,
282 °C decomposed; IR (Solid): nmax cm–1, 2909.6, 2230.0,
1627.6, 1600.2, 1553.9, 1503.4, 1488.5, 1455.8, 1419.7,
1
1256.3, 1139.4; H NMR (250 MHz, d6-DMSO): δ 7.2 (1H,
1
t, J = 7.3, Ar-H), 7.5–8.00 (7H, m, Ar-H), 8.1 (1H, t, J = 7.8,
Ar-H), 8.2 (1H, d, J = 7.9, Ar-H), 8.4 (2H, d, J = 7.5, Ar-H),
8.9 (1H, d, J = 7.6, Ar-H); 1H NMR (62.5 MHz, d6-DMSO): δ
113.0, 124.4, 129.0, 130.4, 130.2, 134.0, 135.7, 157.6, 159.0;
ESI-MS m/z: 316 [M + H]+; found 316.1251 (C20H15N3F [M
+ H]+, requires 316.1250).
1365.2, 1331.3, 1267.6, 1217.1, 1167.1; H NMR (250 MHz,
d6-DMSO): δ 6.25–6.37 (4H, m, Ar-H), 6.51–6.59 (2H, m, Ar-
H), 6.72–6.85 (5H, m, Ar-H), 6.95 (1H, t, J = 7.5, Ar-H), 7.07
(1H, td, J = 8.1, J = 1.4, Ar-H), 7.29 (1H, d, J = 4.0, Ar-H),
7.29 (1H, t, J = 8.3, Ar-H), 7.41 (1H, s, Ar-H), 7.43 (1H, d,
J = 7.3, Ar-H), 7.84 (1H, d, J = 7.9, Ar-H); 13C NMR
(62.5 MHz, d6-DMSO): δ 104.6, 106.5, 109.1, 110.6, 110.7,
111.5, 115.4, 115.6, 120.5, 120.6, 120.9, 121.6, 121.8, 125.4,
128.1, 129.8; ESI-MS m/z: 390 [M + H]+; found 390.1595
(C26H20N3O [M + H]+, requires 390.1606).
6.1.3.10. 1-[3-(2-Phenylquinazolin-4-ylamino)-phenyl]-etha-
none (3j). Conditions were as employed for 3a. 3-Aminoaceto-
phenone j (136 mg, 1.0 mmol), 6 (122 mg, 0.5 mmol), reaction
time 6 h, gave 3j as a solid. (170 mg, 89%). m.p., 199–204 °C;
IR (Solid) nmax cm–1, 2500, 1673, 1636, 1567; 1H NMR
(250 MHz, d6-DMSO): δ 2.68 (3H, s, OCH3), 7.62–7.79 (4H,
m, Ar-H), 7.87 (1H, t, J = 7.6, Ar-H), 7.98 (1H, d, J = 7.9, Ar-
H), 8.14 (1H, t, J = 7.6, Ar-H), 8.19 (1H, d, J = 9.2, Ar-H),
6.1.3.14. 3-(2-Phenylquinazolin-4-ylamino)-benzonitrile (3n).
Conditions were as employed for 3a. 3-Aminobenzonitrile n
(243.0 mg, 2.55 mmol), 6 (253.3 mg, 1.05 mmol), reaction
time 18 h, gave product 3n (300.5 mg, 80%) as a solid. m.p.,

H. Cope et al. / European Journal of Medicinal Chemistry 41 (2006) 1124–1143
1141
260 °C; IR (Solid): nmax cm–1, 2827.7, 2231.6, 1630.5, 1592.5,
8.55–8.64 (2H, d, J = 8.2 Hz, Ar-H); 13C NMR (62.5 MHz,
CDCl3): δ 38.2, 108.0, 110.6, 113.6, 120.6, 125.5, 128.3,
128.4, 128.9, 130.2, 132.7, 138.9, 142.3, 150.6; ESI-MS m/z:
302 [M + H]+; found 302.1283 (C19H16N3O [M + H]+,
requires 302.1293).
1556.7, 1505.4, 1458.3, 1443.8, 1420.0, 1374.0, 1330.0,
1
1164.7; H NMR (250 MHz, d6-DMSO): δ 7.63–7.93 (6H,
m, Ar-H), 8.14 (1H, t, J = 7.6, Ar-H), 9.02 (1H, d, J = 7.6,
Ar), 8.36–8.43 (4H, m, Ar-H), 9.02 (1H, d, J = 8.2, Ar-H),
11.90 (1H, s, NH); 13C NMR (62.5 MHz, d6-DMSO): δ
111.5, 112.9, 118.5, 124.6, 127.5, 128.1, 129.0, 129.1, 130.1,
133.1, 135.9, 138.2, 157.5, 159.1, 209.4; ESI-MS m/z: 323 [M
+ H]+; found 323.1283 (C21H15N4 [M + H]+, requires
323.1297).
6.1.3.18. Cyclohexyl-(2-phenylquinazolin-4-yl)-amine
(3r).
Conditions were as employed for 3l although heated at
98 °C. Cyclohexylamine r (247.9 mg, 2.45 mmol), 6
(120.4 mg, 0.50 mmol), reaction time 2.5 h, gave 3r as a yel-
low crystalline solid (130 mg, 86%). m.p., 154–155 °C; IR
(Solid): nmax cm–1 3325, 3060, 2926, 2846, 1619, 1560,
6.1.3.15. N-(2-Phenylquinazolin-4-yl)-benzene-1,4-diamine
(3o). Conditions were as employed for 3a. 1,3-Phenylenedia-
mine o (217.4 mg, 2.00 mmol), 3 (222.3 mg, 1.08 mmol), reac-
tion time 18 h, gave product 3o, (51.8 mg, 17%) as a solid. m.
p. 201 °C, IR (Solid): nmax cm–1, 2909.6, 2230.0, 1627.6,
1600.2, 1553.9, 1503.4, 1488.5, 1455.8, 1419.7, 1365.2,
1331.3, 1267.6, 1217.1, 1167.1; 1H NMR (250 MHz, d6-
DMSO): δ 6.62 (1H, dt, J = 7.3, J = 2.0, Ar-H), 7.18 (1H, dd,
J = 3.7, J = 1.8, Ar-H), 7.47–7.53 (6H, m, Ar-H), 7.78 (1H, td,
J = 7.6, J = 1.3, Ar-H), 7.86 (1H, d, J = 8.2, Ar-H), 8.0 (1H, d,
J = 8.5, Ar-H), 8.52–8.57 (2H, m, Ar-H); 13C NMR
(62.5 MHz, d6-DMSO): δ 107.6, 108.2, 111.1, 111.5, 114.1,
120.4, 126.2, 128.6, 128.7, 129.4, 130.0, 130.4, 133.0, 139.8,
147.4, 157.5; ESI-MS m/z: 313 [M + H]+; found 313.1462
(C20H17N4 [M + H]+, requires 313.1463).
1
1517, 1420, 1367; H NMR (250 MHz, CDCl3): δ 1.10–1.85
(10H, m, C-CH2-C), 4.34 (1H, dtt, J = 7.3, J = 7.0, J = 4.0, N-
CH), 5.47 (1H, d, J = 7.0, NH), 7.31 (1H, t, J = 7.0, Ar-H),
7.36–7.47 (3H, m, Ar-H), 7.54–7.68 (2H, m, Ar-H), 7.88
(1H, d, J = 7.9, Ar-H), 8.47 (2H, d, J = 7.9, Ar-H); 13C NMR
(62.5 MHz, CDCl3): δ 25.1, 25.8, 33.0, 49.8, 107.5, 113.7,
120.4, 125.2, 128.3, 128.4, 129.0, 130.0, 132.4, 139.1, 150.6,
158.8; ESI-MS m/z: 304 [M + H]+; found 304.813 (C20H22N3
[M + H]+, requires 304.1814).
6.1.3.19. 2-Phenyl-4-piperidin-1-ylquinazoline (3s). Condi-
tions were as employed for 3l. Piperidine s (2.5 mmol), 6
(120 mg, 0.5 mmol), reaction time 0.3 h, gave 3s as a yellow
crystalline solid (123 mg, 84%). m.p., 106 °C; IR (Solid): nmax
cm–1 3062, 2930, 2817, 1611, 1563, 1502, 1433, 1353, 1109;
1H NMR (250 MHz, CDCl3): δ 1.75 (6H, s, C-CH2-C), 3.74
(4H, s, N-CH2-C), 7.32 (1H, t, J = 7.0 Hz, Ar-H), 7.36–7.48
(3H, m, Ar-H), 7.63 (1H, t, J = 7.7 Hz, Ar-H), 7.81 (1H, d,
J = 8.2 Hz, Ar-H), 7.92 (1H, d, J = 8.2 Hz, Ar-H), 8.49 (2H,
d, J = 7.9 Hz, Ar-H); 13C NMR (62.5 MHz, CDCl3): δ 24.9,
26.1, 51.0, 124.6, 125.1, 128.3, 128.4, 128.6, 130.1, 132.3;
ESI-MS m/z: 290 [M + H]+; found 290.1654 (C19H20N3 [M
+ H]+, requires 290.1657).
6.1.3.16. (2-Phenylquinazolin-4-yl)-pyridin-3-ylmethylamine
(3p). Conditions were as employed for 3l although heated at
70 °C. 3-Picolylamine
p (265.0 mg, 2.45 mmol), 6
(121.0 mg, 0.50 mmol), reaction time 3 h, gave 3p as a yellow
crystalline solid (137 mg, 88%). m.p., 216–217 °C; IR (Solid):
nmax cm–1 3253, 3033, 2932, 1563, 1532, 1484, 1424, 1363,
1
1322, 1180, 1031; H NMR (250 MHz, DMSO): δ 4.92 (2H,
d, J = 5.5 Hz, N-CH2), 7.34 (1H, dd, J = 7.6 Hz, J 4.6 Hz, Ar-
H), 7.47 (2H, t, J = 3.7 Hz, Ar-H), 7.48–7.60 (2H, m, Ar-H),
7.78 (2H, d, J = 4.0 Hz, Ar-H), 7.85 (1H, d, J = 7.9 Hz, Ar-H),
8.27 (1H, d, J = 8.2 Hz, Ar-H), 8.44 (2H, d, J = 3.7 Hz, Ar-H),
8.41–8.47 (1H, m, Ar-H), 8.72 (1H, d, J = 1.8 Hz, N-CH) 9.02
(1H, t, J = 5.8 Hz, NH); 13C NMR (62.5 MHz, DMSO): δ
41.6, 113.8, 122.7, 123.5, 125.5, 127.8, 127.9, 128.3, 130.2,
132.9, 135.2, 138.4, 148.1, 149.0, 149.8, 159.1, 159.5; ESI-
MS m/z: 313 [M + H]+; found 313.1467 (C20H17N4
[M + H]+, requires 313.1453).
6.1.3.20. Allyl-(2-phenylquinazolin-4-yl)-amine (3t). Condi-
tions were as employed for 3l although heated at 50 °C. Ally-
lamine t (144.3 mg, 2.53 mmol), 6 (122.4 mg, 0.51 mmol),
reaction time 19 h gave 3t as a yellow crystalline solid
(120 mg, 90%). m.p., 128 °C; IR (Solid): nmax cm–1 3303,
1
3060, 2929, 1618, 1563, 1529, 1436, 1412, 1374, 1322; H
NMR (250 MHz, CDCl3): δ 4.37 (2H, d, J = 5.5, CH2–C=C),
5.16 (1H, dq, Jtrans = 9.9, J = 1.2, C=CH2), 5.28 (1H, dq, Jcis
= 17.4, J = 1.5, C=CH2), 5.72 (1H, s, NH), 6.02 (1H, dtt, ,
J = 5.8, Jcis = 17.1, Jtans = 10.4, HC=CH2), 7.33 (1H, t,
J = 7.0, Ar-H), 7.35–7.46 (3H, m, Ar-H), 7.65 (2H, t, J = 5.5,
Ar-H), 7.85 (1H, d, J = 8.2, Ar-H), 8.49 (2H, m, Ar-H); 13C
NMR (62.5 MHz, CDCl3) 43.8, 117.0, 120.4, 125.5, 128.3,
128.4, 128.9, 130.1, 132.6, 134.5; ESI-MS m/z: 262 [M + H]+;
found 262.1343 (C17H16N3 [M + H]+, requires 262.1344).
6.1.3.17. Furan-2-ylmethyl-(2-phenylquinazolin-4-yl)-amine
(3q). Conditions were as employed for 3a although heated at
98 °C. Furfurylamine q (241.5 mg, 2.49 mmol), 6 (122.0 mg,
0.51 mmol), reaction time 3 h, gave 3q as a yellow crystalline
solid (126 mg, 84%). m.p., 119–121 °C; IR (Solid): nmax cm–1
3360, 3122, 3047, 2908, 1619, 1563, 1530, 1423, 1376, 1342,
1
1205, 1149, 1009, 928; H NMR (250 MHz, CDCl3): δ 4.99
(2H, d, J = 5.2 Hz, CH2), 6.03 (1H, t, J = 4.3 Hz, NH), 6.33–
6.42 (2H, m, Ar-H), 7.39 (2H, t, J = 8.2 Hz, Ar-H), 7.45–7.57
(3H, m, Ar-H), 7.68 (1H, d, J = 9.2 Hz, Ar-H), 7.71 (1H, td,
J = 7.0 Hz, J 1.5 Hz, Ar-H), 7.93 (1H, d, J = 8.2 Hz, Ar-H),
6.1.3.21. N,N-dimethyl-N′-(2-phenylquinazolin-4-yl)-ethane-
1,2-diamine (3u). Conditions were as employed for 3l although
heated at 98 °C. N′,N′-Dimethylethane-1,2-diamine 7u
(120.0 mg, 0.50 mmol), 3 (192.7 mg, 2.19 mmol), reaction

1142
H. Cope et al. / European Journal of Medicinal Chemistry 41 (2006) 1124–1143
time 10 min, gave 3u as a viscous orange oil (121 mg, 83%).
IR (Solid): nmax cm–1 2942, 2823, 1618, 1572, 1530, 1454,
1430, 1413, 1363, 1274, 1219, 1202, 1070; 1H NMR
(250 MHz, CDCl3): δ 2.25 (6H, s, N-(CH3)2), 2.59 (2H, t,
J = 5.8 Hz, CH2-N-Me2), 3.75 (2H, q, J = 5.2 Hz, CH2-NH),
6.50 (1H, br-t, NH), 7.32 (1H, t, J = 7.0 Hz, Ar), 7.35–7.45
(2H, m, Ar-H), 7.62 (1H, t, J = 8.2 Hz, Ar-H), 7.69 (1H, d,
J = 7.9 Hz, Ar-H), 7.82 (1H, d, J = 8.2 Hz, Ar-H), 8.5 (2H,
dd, J = 7.0 Hz, J = 2.4 Hz, Ar-H); 13C NMR (62.5 MHz,
CDCl3): δ 38.3, 45.3, 57.6, 90.3, 107.5, 113.9, 121.0, 125.3,
128.2, 128.4, 128.7, 130.0, 132.4, 139.1, 150.4, 160.6; ESI-
MS m/z: 293 [M + H]+; found 293.1776 (C18H21N4
[M + H]+, requires 293.1772).
25.4, 26.6, 28.4, 36.1, 40.6, 113.6, 121.8, 125.1, 126.3,
127.7, 128.0, 129.7, 132.3, 138.5, 149.2, 160.1, 160.9, 177.4;
ESI-MS m/z: 336 [M + H]+; found 336.1703 (C20H22N3O2 [M
+ H]+, requires 336.1712).
6.1.3.24. Methylphenyl-(2-phenylquinazolin-4-yl)-amine (3x).
Conditions were as employed for 3a. N-methylaniline x
(212.6 mg, 1.98 mmol), 6 (245.9 mg, 1.02 mmol), reaction
time 18 h, gave product 3x (308.1 mg, 87%) as a solid. m.p.,
256 °C; IR (Solid): nmax cm–1 2766.8, 1605.1, 1557.9, 1537.9,
1515.2, 1485.7, 1466.9, 1448.3, 1425.8, 1374.3, 1330.0,
1
1162.4, 1101.4, 1072.5; H NMR (250 MHz, d6-DMSO): δ
3.92 (3H, s, CH3), 6.84 (1H, d, J = 8.5, Ar-H), 7.29 (1H, t,
J = 8.1, Ar-H), 7.57–7.66 (6H, m, Ar-H), 7.70–7.92 (3H, m,
Ar-H), 8.31 (1H, d, J = 8.2, Ar-H), 8.62 (1H, d, J = 8.2, Ar-
H), 8.63 (1H, d, J = 7.9, Ar-H); 13C NMR (62.5 MHz, d6-
DMSO): δ 43.6, 126.3, 127.4, 129.0, 129.2, 130.6, 134.7;
ESI-MS m/z: 312 [M + H]+; found 312.1494 (C21H18N3 [M +
H]+, requires 312.1501).
6.1.3.22. N1-(2-Phenylquinazolin-4-yl)-butane-1,4-diamine
(3v). Conditions for synthesis were as applied to compound 3u.
Butane-1,4-diamine v (219.3 mg, 2.49 mmol), 6 (120.0 mg,
0.50 mmol), reaction time 0.5 h, gave 3v as a yellow crystal-
line solid (89 mg, 61%). m.p., 90–95 °C; IR (Solid): nmax cm–1
2932, 2855, 1592, 1572, 1531, 1489, 1454, 1436, 1372, 1322,
1260, 1166; 1H NMR (250 MHz, CDCl3): δ 1.46–1.60 (4H, m,
CH2), 1.72 (2H, qu, J = 6.7, CH2), 2.70 (2H, t, J = 6.4, CH2),
3.67 (2H, td, J = 6.4, J = 5.2, HN-CH2), 6.8 (1H, s, NH), 7.26
(1H, t, J = 7.0, Ar-H), 7.33–7.45 (3H, m, Ar-H), 7.59 (1H, t,
J = 7.0, Ar-H), 7.68 (1H, d, J = 8.2, Ar-H), 7.81 (1H, d,
J = 7.6, Ar-H), 8.48 (2H, d, J = 7.9, Ar-H); 13C NMR
(62.5 MHz, CDCl3): δ 26.7, 30.8, 41.2, 41.6, 114.0, 121.0,
125.2, 128.2, 128.4, 128.7, 130.0, 132.3, 139.2, 150.5, 159.7,
160.6; ESI-MS m/z: 293 [M + H]+; found 293.1776 (C18H21N4
[M + H]+, requires 293.1766).
6.2. Pharmacology
Evaluation of the activity and cytotoxicity of the com-
pounds was performed in Scrapie-infected mouse brain
(SMB) cell lines with exposure to accurate concentrations of
candidate drug for 7 days. The assay was adapted from the
methods of Rudyk et al. [27]. A persistently infected mouse
cell line (SMB), cloned originally from scrapie infected
mouse brain but of non-neuronal origin, was used for screening
of compounds for their ability to prevent the formation of PrPSc
[28]. The cells were grown in Medium 199 with Earle’s salts
(Containing 2.2 g l–1 sodium bicarbonate and glutamine as per
formula) at 37 °C under 5% CO2 in air at 95% relative humid-
ity. The medium was supplemented with newborn calf serum
(heat inactivated) 10%, foetal calf serum (heat inactivated) 5%,
and penicillin-streptomycin at 10 mg l–1. Every 7 days conflu-
ent cells were passaged using 0.05% trypsin, 0.002% EDTA, at
a split ratio of 4:1. To assess the effects of compounds cells
were distributed into 24-well cluster plates at 30 000 cells per
well and incubated for 24 h to allow for cell attachment. The
medium was changed for that dosed with the desired com-
pounds which were prepared by dilution of a DMSO stock
solution to the concentration required. The final DMSO con-
centration was at ≤ 0.2% (v/v). An equal concentration of
DMSO was added to the medium of the untreated control.
The exposures were carried out in duplicate on two plates to
allow separate viability and activity measurements. Within
each plate the experiments were carried out in triplicate.
6.1.3.23. 6-(2-Phenylquinazolin-4-ylamino)-hexanoic
acid
(3w). To a stirring solution of 4-chloro-2-phenylquinazoline 6
(119 mg, 0.49 mmol) in dioxane (4 ml) was added ε-amino-n-
caproic acid 7w (326 mg, 2.48 mmol) and sodium carbonate
(267 mg, 2.52 mmol). At room temperature one solid was in
suspension. The reaction vessel was fitted with a condenser
and the mixture stirred at 98 °C for 94 hours. The reaction
was followed by TLC and when complete the reaction mixture
was dissolved in methanol (10 ml) to give a cloudy suspension.
Acetic acid was added drop-wise until the precipitate dis-
solved. The solution was reduced under vacuum to yield an
off-yellow solid which was recrystallised from ethyl acetate
(20 ml) to give a white solid which was dissolved in methanol
and toluene (11 ml, 10:1) and reduced to dryness, a procedure
repeated twice. The solids were washed once more in methanol
and toluene (11 ml, 10:1) and dried under high vacuum. 6-(2-
Phenylquinazolin-4-ylamino)-hexanoic acid 3w was isolated as
a white crystalline solid (157 mg, 90%). IR (Solid): nmax cm–1
2939, 2860, 2360, 1570, 1531, 1406, 1322, 1221, 1126, 1013;
1H NMR (250 MHz, d-MeOH): δ 1.50 (2H, q, J = 7.0, CH2),
1.74 (2H, q, J = 7.6, CH2), 1.87 (2H, t, J = 7.3, CH2), 2.29
(2H, qu, J = 7.9, CH2), 3.80 (2H, t, J = 7.0, CH2), 7.48–7.56
(3H, m, Ar-H), 7.77 (1H, t, J = 8.2, Ar-H), 7.85 (1H, d,
J = 8.2, Ar-H), 8.14 (2H, d, J = 8.2, Ar-H), 8.43 (2H, dd,
J = 5.8, J = 2.1, Ar-H); 13C NMR (62.5 MHz, d-MeOH): δ
6.2.1. Cytotoxicity assays in vitro
Viability of the cells following exposure was assessed using
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide
(MTT). MTT stock solution at 5 mg ml–1 was added to each
well of the 24-well culture dish at 10% the original culture
volume and incubated at 37 °C for 2 hours. After exposure
the medium was removed and the formazan crystals formed

H. Cope et al. / European Journal of Medicinal Chemistry 41 (2006) 1124–1143
1143
by mitochondrial dehydrogenase action were solubilised with
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Acknowledgements
The authors would like to thank the Institute of Animal
Health for the supply of PrPC and SMB cells and Jennifer
Louth for technical assistance. This work was funded by the
Department of Health (contract no. DH007/0102).