Molecular features of the prazosin molecule required for activation of Transport-P

Article abstract of DOI:10.1016/j.bmc.2008.06.037

Closely related structural analogues of prazosin have been synthesised and tested for inhibition and activation of Transport-P in order to identify the structural features of the prazosin molecule that appear to be necessary for activation of Transport-P. So far, all the compounds tested are less active than prazosin. It is shown that the structure of prazosin appears to be very specific for the activation. Only quinazolines have been found to activate, and the presence of the 6,7-dimethoxy and 4-amino groups appears to be critically important.

Full text of DOI:10.1016/j.bmc.2008.06.037

Bioorganic & Medicinal Chemistry 16 (2008) 7254–7263
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Molecular features of the prazosin molecule required for activation
of Transport-P
Joaquim Fernando Mendes da Silva a,b, Marcus Walters a, Saad Al-Damluji c,z, C. Robin Ganellin a,
*
a Department of Chemistry, Christopher Ingold Laboratories, University College London, 20 Gordon Street, London WC1H 0AJ, UK
b Department of Organic Chemistry, Federal University of Rio de Janeiro, Brazil
c Divisions of Endocrinology and Biochemistry, Royal Free and University College Medical School, Royal Free Campus, Rowland Hill Street, London NW3 2PF, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Closely related structural analogues of prazosin have been synthesised and tested for inhibition and acti-
vation of Transport-P in order to identify the structural features of the prazosin molecule that appear to
be necessary for activation of Transport-P. So far, all the compounds tested are less active than prazosin. It
is shown that the structure of prazosin appears to be very specific for the activation. Only quinazolines
have been found to activate, and the presence of the 6,7-dimethoxy and 4-amino groups appears to be
critically important.
Received 21 February 2008
Revised 19 June 2008
Accepted 19 June 2008
Available online 25 June 2008
Keywords:
Transport-P
Prazosin
Ó 2008 Elsevier Ltd. All rights reserved.
Peptidergic neurones
Quinazoline
Furan
Antidepressant
Amine uptake
1. Introduction
presynaptic neurones or in non-neuronal cells, such as kidneys
cells or muscle cells.2,4 This indicates that Transport-P is a specia-
Transport-P is an uptake process for amines that is located in
post-synaptic peptidergic neurones.1 It accumulates amines in
acidified vesicles in the peptidergic neurones.2,3 Transport-P has
functional properties which have not been described for any other
lised function of peptidergic neurones.
Although prazosin is a potent adrenergic a1 receptor antago-
nist,6 this uptake effect of prazosin does not appear to involve
adrenergic receptors.4 Two types of compounds have so far been
identified to act as ligands for Transport-P. Group A compounds,
exemplified by prazosin, are accumulated in a cooperative manner
and Group B compounds, which include phenylethylamines and
some well-established antidepressant drugs, are accumulated
non-cooperatively by the same uptake process. The structural
properties of Group B have been investigated7 by testing a series
of compounds for their ability to inhibit competitively the uptake
membrane transport system. In particular, the a1 adrenergic ligand
prazosin, which is a substrate for this uptake process, accumulates
in a non-linear manner in the peptidergic neurones that possess
Transport-P. In the concentration range 10ꢀ10 to 10ꢀ7 M, accumu-
lation of prazosin via Transport-P is linear, but at concentrations of
prazosin greater than 10ꢀ7 M uptake becomes exponential. This is
seen as a paradoxical increase1,4 in the accumulation of [3H]prazo-
sin at concentrations of unlabelled prazosin greater than 10ꢀ7 M.
The prazosin paradox is attributable to activation of a cooperative
uptake process, which accumulates prazosin in a sigmoidal man-
ner, that cannot be described by the Michaelis–Menten model.
The paradoxical increase in accumulation of [3H]prazosin and the
exponential increase in prazosin uptake are abolished by antide-
pressants such as desipramine.5 This antidepressant-sensitive in-
crease in accumulation of the radioligand is not seen in
of prazosin (1 lM) in immortalised gonadotrophin-releasing hor-
mone neurones (GT1-1 GnRH cells) as previously described.7 These
compounds presumably bind to a putative transporter protein in-
volved in the uptake process. The special interest in characterising
this transporter is that it may provide an approach for the develop-
ment of a novel type of antidepressant drug.9
Previously, we reported10 our study of partial structures of prazo-
sin as part of a search for the pharmacophore of prazosin for Trans-
port-P. We now describe our studies to determine the structural
features of the prazosin molecule 1 (Fig. 1) that appear to be required
for activation of Transport-P. Four major features, A–D (Fig. 2) of the
prazosin structure have been investigated, namely: A, the furoyl
system (compounds 2–8), B, the 6,7-dimethoxy groups
* Corresponding author. Tel.: +44 20 7679 4624; fax: +44 20 7679 7463.
E-mail address: c.r.ganellin@ucl.ac.uk (C.R. Ganellin).
z
Deceased. This paper is
a tribute to him and his pioneering discovery of
Transport-P.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.06.037

J. F. M. da Silva et al. / Bioorg. Med. Chem. 16 (2008) 7254–7263
7255
in these latter structures the furoyl group has also been removed).
This approach has given rise to the synthesis of a set of compounds
(Table 1) which have been tested for their influence on the uptake
of prazosin.
The 2-furoyl group A has been replaced by 2-thienoyl, 2-tetra-
hydrofuranoyl, benzoyl, acetyl and trifluoroacetyl to give com-
pounds 2, 3, 4, 5 and 6, respectively. In two other examples, it
has been replaced by benzyloxycarbonyl or phenyl giving 7 and
8. In the last example, therefore, the carbonyl group is no longer
present.
In some other compounds, the 6,7-dimethoxy groups B of
prazosin have been fused into a dioxalane ring giving 9 or re-
placed by hydrogen atoms to give the desmethoxy analogue 10
of prazosin or the desmethoxy analogue 11 of the benzoyl com-
pound 4.
The ring 4-amino group C has been acetylated, methylated,
benzylated or phenylated giving compounds 12, 13, 14 and 15,
respectively. Both amino-hydrogen atoms of the 4-amino group
have also been replaced by ethyl (16) or by incorporating the ami-
no group into a morpholine ring (17). The amino group has also
been removed (18), that is, replaced by H at the ring 2-position,
or replaced by hydroxyl to give 19, which tautomerises to the ring
amide.
O
O
N
O
O
N
N
N
NH2
1
Figure 1. The structure of prazosin (1).
B
B
N
D
A
N
C
Figure 2. Structural features (A–D) studied for the prazosin molecule; where (A)
represents the 2-furoyl moiety, (B) represents 6,7-dialkoxy, (C) represents 4-amino
and (D) represents the 2-piperazino group.
Finally, the six-membered 2-(N-piperazino)ring D has been
enlarged to a seven-membered 1,4-diazepane in compound 20.
(compounds 9–11), C, the 4-amino group (compounds 12–19), and
D, the 2-(N-piperazino-linking moiety (compounds 20 and 21–23;
Table 1
Structures and synthesis schemes of prazosin analogues
D
A
B
B
N
C
N
Compound
A
B
B
C
D
Scheme
1
1a
2
2-Furanyl
2-Thienyl
2-Tetrahydro- furanyl
Ph
CH3
CF3
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
NH2
NH2
NH2
NH2
NH2
NH2
N-Piperazino-COb
N-Piperazino-COb
N-Piperazino-COb
N-Piperazino-COb
N-Piperazino-COb
N-Piperazino-COb
3c
4
1
d
d
5
6
1
7
8
9
MeO
MeO
MeO
NH2
NH2
NH2
N-Piperazino-COb
1
OCH2Ph
Ph
d
MeO
N
N
2-Furanyl
N-Piperazino-COb
1
OCH2
CH2O
d
d
10
11
12
13
14
15
16
2-Furanyl
Ph
H
H
H
H
NH2
NH2
NHCOCH3
NHMe
NHCH2Ph
NHPh
N-Piperazino-COb
N-Piperazino-COb
N-Piperazino-COb
N-Piperazino-COb
N-Piperazino-COb
N-Piperazino-COb
N-Piperazino-COb
2-Furanyl
2-Furanyl
2-Furanyl
2-Furanyl
2-Furanyl
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
2
3
3
3
3
NEt2
17
2-Furanyl
MeO
MeO
N-Piperazino-COb
3
N
O
d
d
18
19
2-Furanyl
2-Furanyl
MeO
MeO
MeO
MeO
H
OH
N-Piperazino-COb
N-Piperazino-COb
O
20
2-Furanyl
MeO
MeO
NH2
4
4
N
C
N
21
22
23
2-Furanyl
MeO
MeO
MeO
MeO
MeO
MeO
NH2
NH2
NH2
NHCH2
N-Piperidinyl
4
NHCH2CH2
N
d
CH3
a
Prazosin.
O
b
N-Piperazino-CO:
.
N
N
C
c
Terazosin12
.
d
Zunszain et al.10

7256
J. F. M. da Silva et al. / Bioorg. Med. Chem. 16 (2008) 7254–7263
Otherwise, it has been replaced by 2-furylmethylamino (in 21),
2-(N-piperidinoethyl)amino (in 22) and 4-methyl-N-piperidino
(in 23).
O
O
N
O
O
N
N
N
2. Chemistry
i
1
N
O
The syntheses of 2-(N-piperazinyl)-4-aminoquinazolines 2, 3, 6,
7, 9 are summarised in Scheme 1. Treatment of 6,7-ethylenedioxy-
quinazoline-2,4(1H,3H)-dione (24a) or 6,7-dimethoxy-quinazo-
line-2,4(1H,3H)-dione (24b) with POCl3 in the presence of N,N-
dimethylaniline afforded the corresponding 2,4-dichloro com-
pounds (25a and 25b) which, upon reaction with NH4OH, led
regioselectively to the 4-amino-2-chloro derivatives 26a and 26b.
Nucleophilic substitution by the appropriate N-substituted pipera-
zine was effected in boiling 1-pentanol under reflux to yield the
desired final products.
H
12
Scheme 2. Reagent and condition: (i) Ac2O, reflux, 1,5 h.
3. Biological studies
Compound 12 was obtained from prazosin by base catalysed
condensation with methyl acetate (Scheme 2). The synthesis of
compounds 13–17 is outlined in Scheme 3. Treatment of 2,4-di-
chloro-6,7-dimethoxyquinazoline (25b) with the appropriate
amines gave the corresponding 2-chloro-4-substituted-amino-
6,7-dimethoxyquinazolines (27). These were then subjected to
nucleophilic substitution of the 2-chloro group with N-(2-furoyl-
piperazine) in boiling 1-pentanol under reflux to furnish the corre-
sponding required products.
1,4-Diazepane was condensed with ethyl 2-furoate (Scheme 4)
to yield the corresponding 2-furoic acid amide which was then
treated with 2-chloro-4-amino-6,7-dimethoxyquinazoline (26b)
to furnish compound 20. Compounds 21 and 22 were similarly ob-
tained from 26b and 2-furfurylamine or N-(2-aminoethyl)piperi-
dine, respectively (Scheme 4).
The compounds were examined for their ability to inhibit
competitively the uptake of prazosin (1 M) in immortalised
l
gonadotrophin-releasing hormone neurones (GT1-1 GnRH cells),
as previously described in detail.7 Each compound was tested
for its ability to compete with prazosin (at 10ꢀ6 M; inhibition of
Transport-P). Efficacy was defined from the inhibition of the up-
take of prazosin (at 10ꢀ6 M) when the test compound was used
in a concentration of 10ꢀ4 M, and was expressed as percentage
of the effect of a maximal inhibitory concentration of desipramine
(10ꢀ4 M). IC50 values were calculated for the new compounds
which were clearly not activators. Each experimental point was
carried out in triplicate, and each experiment was performed
twice; the data are mean SEM of six observations for each
experimental point.
HO
HO
O
O
O
O
NO2
O
O
NH2
iv
N
i
iii
ii
CO2H
O
CO2Me
CO2Me
CO2H
H
N
H
N
O
R'O
R"O
N
Cl
O
O
O
H
vi
vi
v
N
H
N
O
O
Cl
O
25a R'R" = CH2CH2
25b R'=R"= CH3
24b
24a
vii
O
N
R'"
R'O
R"O
N
Cl
xii
R'O
N
N
N
O
N
N
N
H
R"O
S
O
NH2
R'"
R'=R"= Me, R'" =
,
, CF3 , PhCH2O
NH2
26a R'R" = CH2CH2
26b R'=R"= CH3
viii or ix or x or xi
2
3
6
7
H
N
N H
O
R',R"=CH2CH2, R'" =
9
Scheme 1. Reagents and conditions: (i), SOCl2, MeOH; (ii) BrCH2CH2Br, MeOH, KOH; (iii) HNO3; (iv) N2H4ꢁH2O, Pd/C, MeOH; (v) KNCO, AcOH, then NaOH; (vi) PhNMe2, POCl3,
reflux; (vii) 35% NH4OH, THF; (viii) 2-thienyl chloride,110 °C, 16 h then 150 °C, 4 h; (ix) methyl 2-tetrahydrofuroate,110 °C, 5 h; (x) ethyl trifluoroacetate, THF; (xi) CbzCl at
pH 4.5 in MeOH/toluene/H2O; (xii) 1-pentanol, reflux.

J. F. M. da Silva et al. / Bioorg. Med. Chem. 16 (2008) 7254–7263
7257
H
N
N
O
O
O
O
N
O
O
N
Cl
O
O
N
Cl
R'R"NH
i-v
O
O
N
N
N
N
vi
N
Cl
NR'R"
NR'R"
25b
13 R'=Me, R"=H
14 R'=PhCH2, R"=H
15 R'=Ph, R"=H
16 R'=R"=Et
17 R', R"=CH2CH2OCH2CH2
Scheme 3. Reagents and conditions: (i) MeNH2, aq EtOH; (ii) PhCH2NH2, MeOH, rt; (iii) PhNH2, KF, 18-C-6, MeCN; (iv) Et3N, EtOH, reflux; (v) morpholine, MeOH, rt; (vi) 1-
pentanol, reflux.
H
CO2Et
N
+
O
N
H
N
i
O
H
N
N
O
O
Cl
O
ii
O
O
O
N
N
+
N
N
N
O
NH2
NH2
26b
20
O
NH2
iii
O
O
N
N
+
H
O
N
NH2
21
N
NH2
O
O
N
N
iii
H
+
N
N
NH2
22
Scheme 4. Reagents and conditions: (i) 115 °C, 5 h; (ii) 3-methyl-1-butanol, reflux, 115 °C, 5 h; (iii) 3-methyl-1-butanol, reflux under N2.
Each compound was also tested for its ability to activate
70 independent experiments). Compounds were tested at three
different concentrations, that is, at 10ꢀ6, 10ꢀ5 and 10ꢀ4 M. Com-
pound structures are in Table 1; biological results are given in
Table 2.
Transport-P (increasing the uptake of
a
tracer dose of
2 ꢂ 10ꢀ10 M [3H]prazosin). Unlabelled prazosin at 10ꢀ6 M in-
creased the uptake of [3H]prazosin by 126.8 6.3% (derived from

7258
J. F. M. da Silva et al. / Bioorg. Med. Chem. 16 (2008) 7254–7263
These results stand in contrast with our previous finding10 that
Table 2
Activities of prazosin analogues on Transport-P
it is not necessary to have a structure resembling prazosin in order
to achieve good inhibition of prazosin binding. Simple aromatic
alkylamines whether primary, secondary or tertiary were shown
to have IC50s in the low micromolar range of concentrations. The
most potent of this series were N-[5-(1,2,3,4-tetrahydronaphtha-
Compounda
Inhibitionb (%)
Activationc
10ꢀ5
10ꢀ6
M
M
10ꢀ4
M
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Insol.
57 20
161 16
0
Insol.
38
47
len-6-yloxy)pentyl]pyrrolidine (IC50 = 0.19
lM) and N-(5-phen-
41
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
6
7
63 5d
37 5d
40
8
oxypropyl)pyrrolidine (IC50 = 0.46 M) (Fig. 3).
l
0
0
61 12
26
More important for our current purpose is to uncover the struc-
tural features that are required for activating uptake. Here, we ap-
pear to require structures close to that of prazosin. Compound 2,
the thienoyl analogue was active at 10ꢀ6 M and showed a dose-re-
lated increase in activation at 10ꢀ5 M; it is not as potent as prazo-
sin itself, which shows 132% activation at 10ꢀ6 M, but was the most
potent of the compounds tested.
Other acyl groups for A (Table 1) such as tetrahydrofuranyl (3),
acetyl (5) and trifluoroacetyl (6) were some 100-fold less effective
than 2, and benzoate (7) was not active. The benzoyl analogue 4
appeared to be 10 times more potent than these (activation at
10ꢀ5 M) but had a rather shallow dose–response relationship.
Removing the carbonyl group from the piperazine ring (8) also re-
moved the activating activity relative to benzoyl (4).
Removing the 6,7-dimethoxy groups from prazosin (1) or from
the benzoyl analogue (4), in 10 and 11, respectively, removed the
ability of the compounds to activate uptake. On the other hand,
fusing the two methoxy groups into a dioxalane ring (9) gave a
compound that was able to activate uptake, but was more than
10 times less potent than prazosin (10ꢀ4 to 10ꢀ5 M).
As mentioned above, the N-substituted prazosin derivatives with
N-acetyl (12) or N-methyl (13) were able to activate prazosin uptake
at 10ꢀ4 to 10ꢀ5 M. Although weaker than prazosin in activating Trans-
port-P, these compounds appear to retain the full maximum ability to
activate the uptake process. For example, at 10ꢀ4 M, 13 increased the
uptake of [3H]prazosin by 229%, which is similar to the maximal acti-
vating effect of prazosin (which is exerted at 3 ꢂ 10ꢀ6 M). In contrast,
N-substituted derivatives having a larger N-substituent such as ben-
zyl (14) or phenyl (15) were not active. Similarly, the tertiary amines
diethylamino (16) and morpholino (17) were not active.
Enlarging the piperazine ring D (Table 1) of prazosin to 1,4-diaze-
pane (20) also has a profound effect on ability to activate Transport-
P, and 20 has only weak activity. Surprisingly, replacing the carbonyl
piperazine by methylamino (21), or replacing furoylpiperazine by 2-
(N-piperidino)ethylamino (22), provides compounds that retain
some activating effect. Methylpiperidine on its own (23), that is
without a group A, showed very weak activity (at 10ꢀ5 M).
Compound 21 appeared to have a bell-shaped concentration–re-
sponse relationship for activation of Transport-P, since it was apparently
inactive at 10ꢀ4 M yet showed concentration-dependent activation at
10ꢀ6 to 10ꢀ5 M. The precise reason for this is not known, but it is prob-
ably related to the inhibitory effect which was evident at 10ꢀ4 M.
Three other compounds (8, 20, and 23), which were able to acti-
vate Transport-P at 10ꢀ5 M but unable to activate at 10ꢀ4 M also
demonstrated inhibition at 10ꢀ4 M. The situation is complex, since
some compounds (3–6, 9, 10 and 22) are able to both activate and
inhibit at 10ꢀ4 M; 22 has a surprisingly shallow concentration–re-
sponse relationship. On the other hand, compounds 12 and 13,
which demonstrated a high level of activation at 10ꢀ4 M, did not
inhibit Transport-P.
31
75
6
2
8
6
13
4
5
0
0
93 2d,e
42
9
74 14
0
0
163 33
22 14
0
206 22
229 43
0
0
0
0
0
0
0
0
64 5d
73 7d
0
35
8
0
55 16
69 3f
80 8g
55 5h
61 3i
0
0
0
0
0
0
5
10d
0d
82
71
52
3
3
3
31
43
5
7
26
13
0
3
8
29 10
13
37 10
0
85 3d
5
a
Compound structures are in Table 1.
b
Inhibition of prazosin uptake by 10ꢀ4 M concentration of the test compound
expressed as percentage of the effect of
a maximal inhibitory concentration
(10ꢀ4 M) of desipramine. Values are shown with SEM of three replicates.
c
% activation of [3H]prazosin uptake (at 2 ꢂ 10ꢀ10 M) at the indicated concen-
trations of compound.
d
Zunszain et al.10
IC50 = 5.2 2.0
IC50 = 5.4 0.8
IC50 = 4.5 2.0
IC50 = 80
IC50 = 81
l
l
l
Md.
M.
M.
e
f
g
h
i
lM.
7 lM.
4. Results and discussion
Our studies have previously shown that compounds that possess
a basic amino group, which would be protonated at the physiolog-
ical neutral pH of 7.4, are able to inhibit the uptake of prazosin.
Most of the compounds in the current series bind to the uptake sys-
tem to some extent. The thienoyl analogue 2 was too insoluble to be
tested at 10ꢀ4 M. Two compounds lacking a 4-amino group 18 and
19 did not bind, as has been previously reported; it is believed that
this is a consequence of their having low basicities, as has been pre-
viously argued. Two other compounds, in the range of concentra-
tions used, also did not appear to bind; these are the N-acetyl
derivative 12 of prazosin, and N-methylprazosin (13). This is pre-
sumably because they activate uptake rather than inhibiting it.
Other N-monosubstituted compounds such as N-benzyl (14)
and N-phenyl (15) prazosin bind well, having IC50s of 5.4 0.8
and 4.5 2.0
lM, respectively. The tertiary amines diethylamino
(16) and morpholino (17) bind less well than the latter two, having
IC50s of 80 and 81 lM, respectively. Some of the other close struc-
tural analogues showed less than 50% binding at 10ꢀ4 M. These are
compounds 3, 5 and 6, where the furoyl moiety in prazosin is re-
placed by tetrahydrofuranoyl, acetyl or trifluoroacetyl, respec-
tively, and 9 where the 6,7-dimethoxy group is replaced by
ethylenedioxy.
5. Conclusions
O
N
O
N
( )
5
( )
5
1. The structure of prazosin appears to be very specific for the acti-
vation of uptake-P and to date no compound has been found to
be more potent than prazosin. The most active compound so far
Figure 3. Most active compounds in Ref. 8, inhibiting the uptake of prazosin.

J. F. M. da Silva et al. / Bioorg. Med. Chem. 16 (2008) 7254–7263
7259
is the thienoyl analogue. Compounds with other acyl groups for
A were much less effective, but it is of interest to note that a
benzoyl group afforded a better analogue than did tetrahydrof-
uranoyl, acetyl or trifluoroacetyl.
Under a nitrogen atmosphere a mixture of 4-amino-2-chloro-
6,7-dimethoxy-quinazoline (26b), (0.27 g, 1.11 mmol) prepared
as previously described,11 N-(thiophene-2-carbonyl)piperazine
(0.22 g, 1.11 mmol) and 3-methyl-1-butanol (5 mL) was stirred
and boiled under reflux (ꢃ170 °C) for 5 h. The mixture was cooled
to 0–5 °C and left to rest for approximately 30 min. The cream pre-
cipitate was then collected by filtration, washed with diethyl ether
(2ꢂ 10 mL), recrystallised twice from methanol by precipitation
with diethyl ether to yield an off white crystalline product 2
(0.21 g, 48%); mp = 265–267 °C; Rf = 0.68 (EtOAc); HPLC I (MeOH/
H2O 50:50) tR 6.87 min (100%); 1H NMR (300 MHz, DMSO-d6): d
3.84 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), behind methoxy 3.84 (br s,
4H, pip-3,30 + 5,50H), 3.97 (m, 4H, pip-2,20 + 6,60H), 7.17 (t,
J = 4.4 Hz, 1H, thi-4H), 7.52 (d, J = 3.2 Hz, 1H, thi-3H), 7.55 (s, 1H,
Ar-5H), 7.77 (s, 1H, Ar-8H), 7.81 (d, J = 4.7 Hz, 1H, thi-5H); MS
(FAB) m/z 400 (M+ 100%). Anal. Calcd for C19H21N5O3SꢁHClꢁH2O:
C, 50.27; H, 5.33; N, 15.93; Cl, 7.81. Found: C, 50.14; H, 4.94; N,
15.52; Cl, 8.17.
2. The presence of the 6,7-dimethoxy groups B appears to be very
necessary for activation of uptake-P and it will be of importance
to explore alternatives.
3. The4-aminogroupC can accept a smallsubstituentsuch asacetyl
or methyl but with a substantial loss in activity. Larger groups
such as diethyl, benzyl or phenyl cannot be accommodated.
4. The piperazine ring D seems to have a critical geometry in so far
as enlarging it to a 1,4-diazepane greatly reduces activating
activity. Surprisingly, however, the furoylpiperazine can be
replaced by furfurylamine or 2-(N-piperidinyl)ethylamine to
give compounds which retain some ability to activate Trans-
port-P at 10ꢀ5 to 10ꢀ6 M.
6. Experimental
6.1.2. rac-4-Amino-6,7-dimethoxy-4-(2-tetrahydrofuranoyl-
piperazin-1-yl)quinazoline hydrochloride monohydrate (3)
To a mixture of tetrahydro-2-furoic acid (5 mL, 0.05 mol) and
methanol (10 mL, 0.21 mol), sulfuric acid (1 mL) was added slowly
down the walls of the flask and the contents swirled gently. The
mixture was heated under reflux for 40 min and left to cool. The
mixture was quenched with ice (22 g) and stirred until all the ice
has melted. The organic product was extracted with ether
(35 mL) and washed successively with water (25 mL), saturated so-
dium carbonate solution (25 mL) and brine (25 mL), dried over
MgSO4 and concentrated in vacuo, to give methyl tetrahydrofu-
ran-2-carboxylate as a sweet smelling transparent oil (1.52 g,
23% yield).
Under a nitrogen atmosphere, methyl tetrahydrofuran-2-car-
boxylate (1.03 g, 7.90 mmol) and piperazine (0.82 g, 9.48 mmol)
were heated at 110 °C for 5 h. The mixture was cooled to room
temperature and partitioned between chloroform (20 mL) and sat-
urated aqueous sodium bicarbonate (20 mL). The organic layer was
washed with water (2ꢂ 10 mL) and dried over Na2SO4. The extract
was concentrated and chromatographed on a silica gel column
(EtOAc/MeOH, 4:1). When the first set of spots emerged the col-
umn was flushed with 100% MeOH to give N-(tetrahydrofuran-2-
carbonyl)piperazine (0.24 g, 16%).
6.1. Chemistry (general)
Melting points were determined on an ElectrothermalÒ melting
point apparatus and are uncorrected. 1H NMR spectra were re-
corded at 300 and 500 MHz on a Bruker AMX-300 and a Bruker
AVANCE-500, respectively. The NMR experiments were carried
out in CDCl3, CD3OD or DMSO-d6 with tetramethylsilane as an
internal reference. Chemical shifts are quoted in parts per million
(ppm) and the coupling constants are reported in Hertz (Hz). Ana-
lytical HPLC was carried out on a Shimadzu HPLC apparatus with a
Kromasil C18
lm reversed column (250 ꢂ 4.6 mm) at a flow rate of
1 mL/min and detected at 254 nm. For HPLC I the mobile phases
were mixtures of A = methanol + 0.1% trifluoroacetic acid, and
B = water + 0.1% trifluoroacetic acid, and are indicated as the ratio
A:B; for HPLC II the mobile phases were mixtures of A = acetoni-
trile + 0.1% trifluoroacetic acid, and B = water + 0.1% trifluoroacetic
acid; eluent gradient: 10:90 to 100:0 in 13 min at 1.5 mL/min.
Mass spectra were recorded on a VG 7070H Double Focusing Mass
Spectrometer or a VG ZAB-SE Double Focusing Mass Spectrometer,
using atmospheric pressure chemical ionization (APCI), electro-
spray (ES) or fast atom bombardment (FAB). Elemental analyses
were determined on a Perkin-Elmer 2400 CHN elemental analyzer
and were carried out at UCL Departmental Microanalysis Service.
Column chromatography was done using Merck silica gel 60 (70–
230 mesh). TLC was carried out using Merck Kieselgel 60 F254 alu-
minum sheets, visualized at 254 nm and stained with potassium
iodoplatinate (KIP) or 5% H2SO4 in ethanol prior to heating.
Under
a nitrogen atmosphere, 4-amino-2-chloro-6,7-dime-
thoxy-quinazoline (0.14 g, 0.59 mmol) and N-(tetrahydrofuran-2-
carbonyl)piperazine (0.11 g, 0.59 mmol) in 3-methyl-1-butanol
(5 mL) were heated under reflux for 4 12 h with stirring. The mixture
was cooled to 0–5 °C and left to stand for approximately 30 min.
The white precipitate was collected by filtration and washed with
acetone (2ꢂ 10 mL), before being recrystallised twice from metha-
nol and precipitated using diethyl ether, to yield the product 3 as a
white crystalline solid (0.17 g, 72%); mp = 276–280 °C; lit.12 mp
278–279 °C, for the hydrochloride dihydrate. Rf = 0.095 (EtOAc) ;
6.1.1. 4-Amino-6,7-dimethoxy-2-(4-(2-thiophene-carbonyl)-
piperazin-1-yl)quinazoline hydrochloride hydrate (2)
Under a nitrogen atmosphere, ethyl thiophene-2-carboxylate
(1.34 mL, 9.96 mmol) and piperazine (1.72 g, 20 mmol) were stir-
red at 110 °C for 25 h. The temperature was then raised to 150 °C
for a further 5 h, and the mixture then left to cool to room temper-
ature. The residue was partitioned between CH2Cl2 (20 mL) and
0.5 N HCl (20 mL), and the aqueous layer was adjusted to pH 10
with saturated aqueous K2CO3 and the free base extracted with
CH2Cl2 (20 mL). The organic layer was then washed with water
(20 mL), dried over Na2SO4 and concentrated in vacuo to yield a
brown coloured oil (0.274 g, 14% yield) of N-(thiophene-2-car-
bonyl)piperazine. Rf = 0.05 (EtOAc, KIP); 1H NMR (300 MHz,
CDCl3): d 2.84 (t, J = 5.1 Hz, 4H, pip), 3.65 (t, J = 5.1 Hz, 4H, pip),
6.97 (t, J = 4.3 Hz, 1H, thi-4H), 7.21 (dd, J = 2.3, 1.38 Hz, 1H, thi-
3H), 7.37 (dd, J = 1.1, 3.9 Hz, 1H, thi-5H); MS (FAB) m/z 197 (M+
35%) 111 (M+ꢀC4H9N2 100%).
HPLC
I
(MeOH/H2O 50 :50), tR 5.47 min (100%); 1H NMR
(500 MHz, CD3OD): d 1.98 (m, 2H, thf-4,40H), 2.08 (m, 1H, thf-
3H), 2.25 (m, 1H, thf-3H), 3.66–4.02 (m, 10H, thf-5,50H and pip-
H), 3.92 (s, 3H, OCH3), 3.98 (s, 3H, OCH3), 4.80 (m, 1H, thf-2H),
7.16 (s, 1H, Ar-5H), 7.55 (s, 1H, Ar-8H); MS (FAB) m/z 388 (M+
100%). Anal. Calcd for C19H25N5O4ꢁ0.9HClꢁ0.8H2O: C, 52.5; H,
6.38; N, 16.11; Cl, 7.34. Found: C, 52.14; H, 6.21; N, 15.94; Cl, 7.60.
6.1.3. 4-Amino-6,7-dimethoxy-2-(4-trifluoroacetylpiperazin-1-
yl)quinazoline hydrochloride (6)
A
mixture of 4-amino-2-chloro-6,7-dimethoxyquinazoline11
(0.45 g, 1.9 mmol) and 1-trifluoroacetylpiperazine (prepared
according to the method of Xu et al.13) (0.35 g, 1.9 mmol) in 1-

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J. F. M. da Silva et al. / Bioorg. Med. Chem. 16 (2008) 7254–7263
pentanol (10 mL) was heated under reflux for 1 h. After cooling, the
solid formed was filtered and recrystallised twice from methanol,
yielding 4-amino-6,7-dimethoxy-2-(4-trifluoroacetylpiperazin-1-
yl)quinazoline hydrochloride (6) as a white solid (0.52 g, 66%);
mp = 287–288 °C; HPLC II tR = 6.52 min (98.4%); 1H NMR
(300 MHz, DMSO-d6): d 3.62 (br s, 4H, pip-H), 3.75 (s, 3H, OCH3),
3.87 (s, 3H, OCH3), 4.08 (br s, 4H, pip-H), 7.54 (s, 1H, Ar-H5), 7.66
(s, 1H, Ar-H8), 8.70 (br s, 1H, NH), 8.95 (br s, 1H, NH); MS (CI+,
(300 MHz, DMSO-d6): d 3.85 (br s, 4H, pip-H), 3.94 (br s, 4H, pip-
H), 4.32 (d, 2H, OCH2), 4.40 (d, 2H, OCH2), 6.65 (dd, 1H, fur-H4),
7.08 (d, 1H, fur-H3), 7.36 (s, 1H, Ar-H5), 7.80 (s, 1H, Ar-H8); 7.87
(d, 1H, fur-H5), 8.76 (br s, 1H, NH), 8.80 (br s, 1H, NH); MS
(FAB+): 382 (M+1, <5%), base peak: 154; Anal. Calcd for
C19H19N5O4ꢁHClꢁ1.5H2O: C, 51.25; H, 5.18; N, 15.73; Cl, 7.98.
Found: C, 50.96; H, 4.79; N, 15.98; Cl, 8.05.
CH4):
16H18N5O3F3ꢁHClꢁ1.3H2O: C, 43.15; H, 4.85; N, 15.73; Cl, 7.98.
Found: C, 43.01; H, 4.63; N, 15.54; Cl, 8.01.
m/z
386
(M+1,
100%);
Anal.
Calcd
for
6.1.6. 4-Acetamido-6,7-dimethoxy-2-[4-(2-furoyl)piperazin-1-
yl]quinazoline (12)
C
To
a warm suspension of 4-amino-6,7-dimethoxy-(2-(2-
furoyl)piperazin-1-yl) quinazoline hydrochloride hydrate (prazo-
sin)17 (0.1 g, 0.26 mmol) in propan-2-ol was added 0.019 g,
0.36 mmol of CH3ONa. The solution was cooled, dried (Na2SO4)
and evaporated, and the resulting free base was dried in vacuo.
Acetic anhydride (2 mL) was added and the mixture was heated
under reflux under nitrogen. After 1.5 h, the mixture was treated
with 10 mL of water and basified with 8 mL of 12 N NaOH, which
was added dropwise in 5 min. After cooling the mixture to room
temperature the compound was extracted into CHCl3 (3ꢂ 20 mL).
The organic layers were combined, washed with water and dried
(Na2SO4). Evaporation of the solvent under reduced pressure affor-
ded yellow crystals of product 12 that was then recrystallised from
MeOH; mp 245–247 °C; HPLC I (MeOH/H2O 50:50) tR 5.54 min
(100%); 1H NMR (300 MHz, CDCl3): d 2.61 (s, 3H, CH3), 3.91 (br
m, 8H, pip-H), 3.93 (s, 3H, OCH3), 3.97 (s, 3H, OCH3), 6.49 (dd,
J = 1.8, 3.4 Hz, 1H, fur-H4), 6.89 (s, 1H, Ar-H), 6.90 (s, 1H, Ar-H),
7.04 (d, J = 3.35 Hz, 1H, fur-H3), 7.50 (d, J = 1.8 Hz, 1H, fur-H5),
8.07 (br s, 1H, N-H); MS (FAB+) m/z 426. Anal. Calcd for
6.1.4. 4-(4-Amino-6,7-dimethoxyquinazolin-2-yl)-piperazine-1-
carboxylic acid benzyl ester hydrochloride (7)
A mixture of 4-amino-2-chloro-6,7-dimethoxyquinazoline (1 g.
4.2 mmol) and benzyl 1-piperazinecarboxylate14 (0.92 g,
4.2 mmol) in 10 mL of 1-pentanol was heated under reflux for
3 h. After cooling, the solid formed was filtered, washed with
Et2O and recrystallised from MeOH, yielding 4-(4-amino-6,7-
dimethoxyquinazolin-2-yl)-piperazine-1-carboxylic acid benzyl
ester hydrochloride (7) as a white solid (1.57 g, 86%); mp = 249–
251 °C; HPLC II tR = 7.88 min (98%); MS (FAB+) m/z 424 (M+1,
82%), base peak: m/z 154; 1H NMR (300 MHz, DMSO-d6 + 1 drop
D2O): d 3.48 (br s, 4H, pip-H), 3.81 (br s + s, 7H, pip-H + OCH3),
3.86 (s, 3H, OCH3), 5.10 (s, 2H, CH2Ph), 7.20 (s, 1H, Ar-H5), 7.33
(s, 5H, C6H5), 7.63 (s, 1H, Ar-H8); Anal. Calcd for
C
22H25N5O4ꢁ1.25HCl: C, 56.33; H, 5.60; N 14.93; Cl 9.47. Found: C
56.31; H 5.59; N 14.70; Cl 9.64.
6.1.5. [4-(4-Amino-7,8-dihydro-[1,4]dioxino[2,3g]quinazolin-2-
yl)piperazin-1-yl]-furan-2-yl methanone hydrochloride (9)
A mixture of 7-amino-1,4-benzodioxan-6-carboxylic acid15,16
(0.9 g, 4.6 mmol) and potassium cyanate (1.5 g, 18.5 mmol) in
30 mL of water containing 1.5 mL of glacial acetic acid was stirred
for 4 h. Then, NaOH (11.2 g, 280 mmol) was added and the reaction
was heated under reflux for 2 h. After cooling, the reaction medium
was acidified with concd HCl and the solid formed was filtered and
recrystallised from MeOH, yielding 7,8-dihydro-1H-[1,4]dioxi-
no[2,3g]quinazoline-2,4-dione (24a) as a white solid (1.0 g, 99%),
mp = 364–365 °C (lit.15 364–366 °C), 1H NMR (300 MHz, DMSO-
d6): d 4.23 (br s, OCH2) + 4.31 (br s, OCH2) (4H), 6.58 (s, 1H), 7.23
(s, 1H).
A mixture of 24a (0.65 g, 2.95 mmol), 5 mL of POCl3 and 0.4 mL
of freshly distilled N,N-dimethylaniline was heated under reflux
for 4 h. After cooling, it was poured into ice, stirred vigorously
and the solid formed was filtered and recrystallised twice from
Et2O/MeOH, yielding 2,4-dichloro-7,8-dihydro-1H-[1,4]dioxi-
no[2,3g]quinazoline (25a) (0.4 g, 53%), mp = 215–218 °C (lit.15
221–223 °C).
C21H23N5O5: C, 59.29; H, 5.45; N, 16.46. Found: C, 58.80; H, 5.49;
N, 16.42.
6.1.7. 6,7-Dimethoxy-4-methylamino-2-(4-(2-furoyl)piperazin-
1-yl)quinazoline hydrochloride (13)
A mixture of 2,4-dichloro-6,7-dimethoxyquinazoline18 (25b,
0.3 g, 1.16 mmol) and 1 mL of aq methylamine (40% wt)
(11.6 mmol) in 20 mL of ethanol was heated under reflux for 1 h.
After cooling, the solvent was removed under reduced pressure
and water was added to the remaining oil, yielding a solid that
was filtered, recrystallised from water and air dried, furnishing 2-
chloro-6,7-dimethoxy-4-methylaminoquinazoline as a white solid
(0.2 g, 69%). Mp = 219–220 °C. 1H NMR (DMSO-d6): d 2.96 (d, 3H),
3.80 (s, 3H), 3.84 (s, 3H), 7.03 (s, 1H), 7.42 (s, 1H), 8.36 (br s, 1H).
13C NMR (DMSO-d6): d 27.7, 55.7, 55.9, 102.0, 106.4, 106.9, 146.7,
148.3, 154.2, 155.2, 160.4.
A mixture of 2-chloro-6,7-dimethoxy-4-methylaminoquinazo-
line (0.15 g, 0.52 mmol), 1-(2-furoyl)piperazine (0.1 g, 0.55 mmol)
and 10 mL of 1-pentanol was heated under reflux for 1.5 h. After
cooling, the solid formed was filtered and recrystallised from
A mixture of 2,4-dichloro-7,8-dihydro-1H-[1,4]dioxino[2,3g]qui-
nazoline (0.26 g, 1.01 mmol), 10 mL of THF and 5 mL of aq NH4OH
35% was stirred at room temperature for 16 h. The solvent was then
removed under reduced pressure and water was added to the solid
obtained, which was filtered and recrystallised from MeOH to yield
4-amino-2-chloro-7,8-dihydro-1H-[1,4]dioxino[2,3g]quinazoline (26a)
as a white solid (0.19 g, 79%), mp = 279–280 °C (d); 1H NMR
(300 MHz, DMSO-d6): d 4.33 (m, 4H), 6.99 (s, 1H), 7.71 (s, 1H),
7.99 (br s, 2H).
methanol,
yielding
6,7-dimethoxy-4-methylamino-2-(4-(2-
furoyl)piperazin-1-yl)quinazoline hydrochloride (13) as a white
solid (0.18 g, 75%), mp = 198–200 °C; HPLC II tR = 6.99 min
(99.9%); 1H NMR (300 MHz, DMSO-d6): d 3.03 (s, 3H, NCH3), 3.83
(br s, 4H, pip-H) + 3.85 (2 s, 6H, OCH3), 3.99 (br s, 4H, pip-H),
6.66 (d, 1H, fur-H4), 7.08 (d, 1H, fur-H3), 7.54 (s, 1H, Ar-H5), 7.89
(d, 1H, fur-H5), 8.02 (s, 1H, Ar-H8), 9.53 (br s, 1H, NH); MS (CI+,
CH4): 398 (M+1, 5%), Base peak: 181; Anal. Calcd for
C
20H23N5O4ꢁ1.1HClꢁ1.8H2O: C, 51.11; H, 5.90; N, 14.91; Cl, 8.32.
A
mixture of 4-amino-2-chloro-7,8-dihydro-1H-[1,4]dioxi-
Found: C, 50.85; H, 5.36; N, 14.81; Cl, 8.36.
no[2,3g]quinazoline (26a) (0.05 g, 0.22 mmol) and 1-(2-
furoyl)piperazine11 (0.05 g, 0.28 mmol) in 5 mL of 1-pentanol was
heated under reflux for 1 h. After cooling, the solid formed was fil-
tered and recrystallised from MeOH, yielding [4-(4-amino-7,8-
dihydro-[1,4]dioxino[2,3g]quinazolin-2-yl)piperazin-1-yl]-furan-2-yl
methanone hydrochloride (9) as a white solid (0.08 g, 89%),
mp = 198–200 °C, HPLC II tR = 6.12 min (100%); 1H NMR
6.1.8. 4-Benzylamino-6,7-dimethoxy-2-(4-(2-furoyl)piperazin-
1-yl)quinazoline hydrochloride (14)
A mixture of 2,4-dichloro-6,7-dimethoxyquinazoline15 (25b,
0.26 g, 1 mmol) and benzylamine (0.5 mL, 4.6 mmol) in 5 mL of
methanol was stirred at room temperature for 1 h. After cooling,
diethyl ether (5 mL) was added and the reaction medium was kept

J. F. M. da Silva et al. / Bioorg. Med. Chem. 16 (2008) 7254–7263
7261
in the fridge, leading to the formation of a white solid that was fil-
tered and recrystallised from petroleum ether and ethyl acetate,
yielding 4-benzylamino-2-chloro-6,7-dimethoxyquinazoline as a
white solid (0.26 g, 79%). Mp = 229–231 °C. 1H NMR (DMSO-d6): d
3.86 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 4.73 (d, 2H, NHCH2Ph),
7.08 (s, 1H, Ar-H5), 7.31 (m, 5H, NHCH2C6H5), 7.70 (s, 1H, Ar-H8),
8.92 (t, 1H, NHCH2Ph).
A mixture of 4-benzylamino-2-chloro-6,7-dimethoxyquinazo-
line (0.161 g, 0.52 mmol), 1-(2-furoyl)piperazine (0.094 g,
0.52 mmol) and 10 mL of 1-pentanol was heated under reflux
for 3 h. After cooling, the solid formed was filtered and recrys-
tallised from methanol, yielding 4-benzylamino-6,7-dimethoxy-
2-(4-(2-furoyl)piperazin-1-yl)quinazoline hydrochloride (14) as
oxyquinazoline as a white solid (0.25 g, 84%), mp = 147–148 °C
(lit.19 148–149 °C). 1H NMR (DMSO-d6): d 1.22 (t, 6H, NCH2CH3),
3.65 (q, 4H, NCH2CH3), 3.87 (s, 3H, OCH3), 3.89 (s, 3H, OCH3),
7.10 (s, 1H, Ar-H5), 7.18 (s, 1H, Ar-H8).
A mixture of 2-chloro-4-diethylamino-6,7-dimethoxyquinazo-
line (0.2 g, 0.7 mmol) and 1-(2-furoyl)piperazine11 (0.13 g,
0.7 mmol) in 10 mL of 1-pentanol was heated under reflux for
4 h. After cooling, the solid formed was filtered and recrystallised
from methanol, yielding 4-diethylamino-6,7-dimethoxy-2-(4-(2-
furoyl)piperazin-1-yl)quinazoline hydrochloride (16) as a white
solid (0.15 g, 45%), mp = 223–224 °C; HPLC II tR = 7.69 min
(100%); 1H NMR (300 MHz, CDCl3): d 1.44 (t, 6H, NCH2CH3); 3.76
(q, 2H, NCH2CH3), 3.81 (s, 3H, OCH3), 3.96 (br s, 4H, pip-H), 4.04
(s, 3H, OCH3), 4.20 (br s, 4H, pip-H), 6.48 (dd, 1H, fur-H4), 7.05
(d, 1H, fur-H3), 7.09 (s, 1H, Ar-H5), 7.50 (d, 1H, fur-H5), 8.42 (s,
1H, Ar-H8); 13C NMR (75 MHz, CDCl3): d 12.9, 45.8, 56.1, 57.1,
101.5, 102.4, 105.3, 111.4, 117,4, 144.3, 146.4; MS (CI+, CH4): 440
(M+1, 100%); Anal. Calcd for C23H29N5O4ꢁ1.1HClꢁ0.7H2O: C, 56.12;
H, 6.40; N, 14.23; Cl, 7.94. Found: C, 56.03; H, 6.44; N, 14.25; Cl,
8.11.
a
white solid (0.08 g, 32%), mp = 267–268 °C; HPLC II
tR = 8.03 min (100%); 1H NMR (300 MHz, DMSO-d6): d 3.54 (br
s, 4H, pip-H), 3.78 (s, 6H, OCH3), 3.89 (br s, 4H, pip-H), 4.61
(d, 2H, NCH2Ph), 6.32 (dd, 1H, fur-H4), 6.84 (d, 1H, fur-H3),
7.10 (m, 5H, C6H5), 7.33 (d, 1H, fur-H5), 7.79 (s, 1H, Ar-H5),
8.03 (s, 1H, Ar-H8), 9.49 (t, 1H, NH); MS (FAB+): 474 (M+1,
40%), base peak: 154; Anal. Calcd for C26H27N5O4ꢁ1.18HCl: C,
60.46; H, 5.46; N, 13.56; Cl, 8.12. Found: C, 60.69; H, 5.57; N,
13.60; Cl, 8.32.
6.1.11. 6,7-Dimethoxy-4-(N-morpholino)-2-(4-(2-
furoyl)piperazin-1-yl)quinazoline hydrochloride (17)
6.1.9. 6,7-Dimethoxy-4-phenylamino-2-(4-(2-furoyl)piperazin-
1-yl)quinazoline hydrochloride (15)
A mixture of 2,4-dichloro-6,7-dimethoxyquinazoline (25b,
0.26 g, 1 mmol) and morpholine (1 mL, 11.5 mmol) in 10 mL of
methanol was stirred at room temperature for 1 h. After cooling,
diethyl ether (5 mL) was added and the reaction medium was kept
in the fridge, leading to the formation of a white solid that was fil-
tered and recrystallised from petroleum ether and ethyl acetate,
A
mixture of 2,4-dichloro-6,7-dimethoxyquinazoline (25 b,
0.26 g, 1 mmol), anhydrous KF (0.25 g. 4.3 mmol) and 18-crown-
6 (1.13 g, 4.3 mmol) in 5 mL of anhydrous acetonitrile was stirred
at room temperature and N2 atmosphere for 10 min. Aniline
(0.2 mL, 2.15 mmol) was then added, and the reaction was stirred
for 24 h. The solvent was then removed under reduced pressure,
and the resulting solid was dissolved with ethyl acetate and water
(10 mL of each). The layers were separated and the organic layer
was dried over anhydrous sodium sulphate, filtered and the sol-
vent was removed under reduced pressure. The solid obtained
was recrystallised from AcOEt/MeOH (1:1), yielding 2-chloro-6,7-
dimethoxy-4-phenylamino-quinazoline (0.139 g, 0.44 mmol) as a
white solid, mp = 218–220 °C; 1H NMR (300 MHz, (CD3)2CO): d
3.83 (s, 3H, OCH3), 3.93 (s, 3 H, OCH3), 7.1–7.2 (m, 2H, NHPh-
H4 + Ar-H5), 7.39 (t, 2H, NHPh-H3), 7.73 (s, 1H, NHPh-H2), 7.81 (d,
2H, Ar-H8).
yielding
2-chloro-6,7-dimethoxy-4-(N-morpholino)quinazoline
(0.3 g, 97%) as a white solid, mp = 221–222 °C. 1H NMR (DMSO-
d6): d 3.71–3.75 (m, 8H), 4.00 (d, 6H), 7.1–7.2 (2 s, 2H); Anal. Calcd
for C14H16N3O3Clꢁ0.1HCl (MW = 313.15): C, 53.66; H, 5.46; N,
13.41; Cl, 12.47. Found: C, 53.87; H, 5.25; N, 13.39; Cl, 12.61.
A
mixture of 2-chloro-6,7-dimethoxy-4-(N-morpholino)qui-
nazoline (0.161 g, 0.52 mmol) and 1-(2-furoyl)piperazine11
(0.094 g, 0.52 mmol) in 10 mL of 1-pentanol was heated under
reflux for 3 h. After cooling, the solid formed was filtered and
recrystallised from methanol, yielding the product 17 (0.08 g,
32%) as a white solid, mp = 235–235.5 °C; HPLC II = 99.7%; 1H
NMR (300 MHz, DMSO-d6): d 3.73 (br s, 4H, NCH2CH2O), 3.88
(2s, 6H, OCH3) + 3.99 (br s, 4H, NCH2CH2O), 4.01 (br s, 8H, pip-
H), 6.65 (dd, 1H, fur-H4), 7.01 (d, 1H, fur-H3), 7.28 (d, 1H,
fur-H5), 7.53 (s, 1H, Ar-H5), 7.88 (s, 1H, Ar-H8); MS (CI+, CH4):
A mixture of 2-chloro-6,7-dimethoxy-4-phenylamino-quinaz-
oline (0.09 g, 0.26 mmol) and 1-(2-furoyl)piperazine (0.05 g,
0.28 mmol) in 5 mL of 1-pentanol was heated under reflux for
1 h. After cooling, the solid formed was filtered and recrystal-
lised twice from methanol, yielding 6,7-dimethoxy-4-phenyla-
454 (M+1, 100%); Anal. Calcd for
(MW = 503.9): C, 54.77; H, 5.87; N, 14.89; Cl, 7.04. Found C,
54.77; H, 5.68; N, 13.84; Cl, 7.10.
C
23H27N5O5ꢁHClꢁ0.8H2O
mino-2-(4-(2-furoyl)piperazin-1-yl)quinazoline
hydrochloride
(15, 0.071 g, 55%) as a white solid, mp = 209–210 °C; HPLC II
tR = 9.09 min (99.1%); 1H NMR (300 MHz, CDCl3 + drops DMSO-
d6): d 3.68 (br s, 4H, pip-H), 3.76 + 3.79 (2s, 6H, OCH3), 3.85
(br s, 4H, pip-H), 6.28 (dd, 1H, fur-H4), 6.80 (d, 1H, fur-H3),
7.17 (t, 1H, NPh-H4), 7.20 (t, 2H, NPh-H3), 7.26 (s, 1H, Ar-H5),
7.31 (d, 1H, fur-H5), 7.36 (d, 2H, NPh-H2), 7.68 (s, 1H, Ar-H8),
7.95 (br s, 1H, NH Ph); MS (FAB+): 460 (M+1, 100%); Anal.
6.1.12. 4-Amino-6,7-dimethoxy-2-[2-furoyl-4-(1,4-diazepan-1-
yl)]quinazoline (20)
Under a nitrogen atmosphere, a mixture of ethyl 2-furoate
(3.309 g, 0.0236 mmol) and 1,4-diazepane (7.096 g, 0.0708 mmol)
was heated at 115 °C for 5 h, to give a golden brown oil. The
product was extracted with CH2Cl2 (20 mL) and 2 M HCl
(20 mL, pH 2). The aqueous layer was then basified to pH 10
with saturated K2CO3, and the product extracted with CH2Cl2
(2ꢂ 20 mL) and dried over Na2SO4. The organic product was fil-
tered and concentrated in vacuo to give a brown coloured oil,
which was purified by column chromatography (CH2Cl2/MeOH
60:1) to yield N-(furan-2-carbonyl)1,4-diazepane as a golden col-
oured oil (1.471 g, 32%); Rf = 0.61 (MeOH, KIP); 1H NMR
(300 MHz CDCl3-D2O shake): d 2.09 (m, 2H, diazep), 3.10 (t,
J = 5.79 Hz, 2H, diazep), 3.22 (br s, 2H, diazep), 3.95 (br s, 2H,
diazep), 4.03 (br s, 2H, diazep), 6.68 (m, 1H, fur-4H), 7.23 (d,
J = 3.3 Hz, 1H, fur-3H), 7.68 (br s, 1H, fur-5H); MS (FAB) 195
(M+ 100%) 95 (M+ꢀC5H11N2 95%).
Calcd for
C
25H25N5O4ꢁ1.1HClꢁ1.7H2O (MW = 529.75): C, 56.63;
H, 5.57; N, 13.21; Cl, 7.37. Found: C, 56.62; H, 5.34; N, 13.14;
Cl, 7.32 .
6.1.10. 4-Diethylamino-6,7-dimethoxy-2-(4-(2-furoyl)piperazin-
1-yl)quinazoline hydrochloride (16)
A
mixture of 2,4-dichloro-6,7-dimethoxyquinazoline (25b,
0.26 g, 1 mmol) and diethylamine (1 mL, 9 mmol) in 10 mL of eth-
anol was heated under reflux for 1 h. After cooling, the solvent was
removed under reduced pressure and water was added to the
remaining oil, yielding a solid that was filtered, recrystallised from
water and air dried, yielding 2-chloro-4-diethylamino-6,7-dimeth-

7262
J. F. M. da Silva et al. / Bioorg. Med. Chem. 16 (2008) 7254–7263
Under a nitrogen atmosphere, a mixture of 2-chloro-4-amino-
humidified atmosphere containing 5% CO2 in air. Culture media
were changed at 48-h intervals. After seven days, the cells were
dispersed in the presence of trypsin, deoxyribonuclease I and eth-
ylenediamine- tetraacetic acid and incubated in Corning or Nunc
12-well plates (approximately 2 ꢂ 106 cells/well). The wells had
6,7-dimethoxy-quinazoline (26b) (0.411 g, 1.72 mmol) and N-
(furan-2-carbonyl)-1,4-diazepane (28) (0.333 g, 1.72 mmol) in
3-methyl-1-butanol (5 mL) was heated under reflux for 5 12 h.
The mixture was then left to cool between 0 and 5 °C for
approximately 30 min. The white solid precipitate was collected
by filtration, washed with diethyl ether and recrystallised twice
from methanol (precipitated with diethyl ether) to yield a white
solid (0.416 g, 61%); mp = 270–271 °C; Rf 0.72 (EtOAc); HPLC I
(MeOH/H2O 1:1) tR 6.05 min (100%); 1H NMR (500 MHz,
DMSO-d6): d 1.98 (m, 2H, diazep), 3.73 (br t, J = 5.43 Hz, 2H, dia-
zep), 3.86 (s 3H, OCH3), 3.89 (s 3H, OCH3) 3.93 (br t, J = 5.43 Hz,
2H, diazep), 3.97 (br t, J = 5.93 Hz, 2H, diazep), 4.09 (br t,
J = 5.58 Hz, 2H, diazep) 6.56 (dd, J = 1.72 and 1.68 Hz, 1H, fur-
4H), 6.92 (d, J = 3.34 Hz, 1H, fur-3H), 7.73 (s and d, 2H, Ar-5H
and fur-5H overlap), 7.75 (s, 1H, Ar-8H). Anal. Calcd for
been coated with poly-
70,000–150,000) and laminin (0.25
D-lysine (2.5 l
g/cm2; Sigma P-6407; MW
l
g/cm2; Sigma L-2020). Drugs
were dissolved in buffer consisting of DMEM with 25 mM Hepes
and 0.5 mM sodium ascorbate, pH 7.4. Uptake studies were per-
formed on the intact cells. After 2–4 days in culture, the cells were
washed twice with buffer at 25 °C then incubated at 37 °C for
60 min in the presence of [3H]prazosin 2 ꢂ 10ꢀ10 M and unlabelled
prazosin 10ꢀ6 M. The test compounds were present in the indi-
cated concentrations. At the end of the incubation period, the cul-
ture plates were placed on ice to inhibit the release of amines.20
After 30 s, the buffer was removed and the cells were washed twice
with buffer at 0 °C. The cold buffer was then removed and the cells
were solubilised with 2 mL of a warm solution of 0.1% sodium
dodecyl sulphate and 0.1 M sodium hydroxide. Fifty microlitre ali-
quots were removed for protein assay and 10 mL of scintillation li-
quid was then added to the cell extract, mixed and radioactivity
was measured in a scintillation spectrometer with an efficiency
of 50%. Protein content was measured by the bicinchoninic acid
modification of the biuret reaction using albumin standards (Per-
bio, Chester, Cheshire, England). Non-specific uptake was defined
as uptake in the presence of the antidepressant desipramine (at
10ꢀ4 M) and specific (desipramine-sensitive) uptake was obtained
by subtracting non-specific from total uptake. Efficacy was defined
from the inhibition of the uptake of prazosin (at 10ꢀ6 M) when the
test compound was used in a concentration of 10ꢀ4 M and ex-
pressed as percentage of the effect of a maximal inhibitory concen-
tration of desipramine (10ꢀ4 M). Half-maximal inhibitory
concentrations (IC50 values) were calculated from the concentra-
tion–response curves. Where IC50 values were not calculated, the
data were expressed only as efficacy (percentage inhibition relative
to desipramine 10ꢀ4 M). The experiments were carried out in trip-
licate and each experiment was performed twice; the data are
therefore presented as the mean SEM of six observations.
C
20H23N5O4ꢁ0.95HClꢁ0.5H2O: C, 54.46; H, 5.7; N, 15.88; Cl, 7.69.
Found: C, 54.54; H, 5.64; N, 15.94; Cl, 7.46.
6.1.13. 4-Amino-6,7-dimethoxy-2-(furfurylamino)quinazoline
hydrochloride (21)
A
mixture of 2-chloro-4-amino-6,7-dimethoxy-quinazoline
(0.211 g, 0.88 mmol), furfuryl amine (0.08 mL, 0.88 mmol) and 3-
methyl-1-butanol (5 mL) was heated at reflux (ꢃ180 °C) for 5 h.
After cooling to 0–5 °C, the mixture was left to rest for approxi-
mately 30 min, and the precipitate was collected by filtration and
washed with acetone. The hydrochloride salt was then recrystal-
lised from methanol twice, and on the second occasion it was pre-
cipitated with ether, to yield white crystals (0.151 g, 57%); mp
249–251 °C; Rf = 0.09 (EtOAc); HPLC
I (MeOH/H2O 1:1) tR
9.09 min (100%); 1H NMR (300 MHz, CD3OD): d 4.07 (s, 3H,
OCH3), 4.11 (s, 3H, OCH3), 4.84 (s, 2H, CH2), 6.52 (m, 2H, furan-
3H, 4H), 7.10 (s, 1H, Ar-5H), 7.60 (t, J = 1.26 Hz, 1H, furan, 5H),
7.69 (s, 1H, Ar-8H); MS (FAB) m/z 301 (M+, 100%). Anal. Calcd for
C
15H16N4O3ꢁHClꢁ0.1H2O: C, 53.21; H, 5.06; N, 16.55; Cl, 10.47.
Found: C, 53.15; H, 5.14; N, 16.71; Cl, 10.04.
6.1.14. 4-Amino-6,7-dimethoxy-2-[N-(2-aminoethyl)piperid-
inyl]quinazoline dihydrochloride (22)
Under a nitrogen atmosphere, a mixture of 2-chloro-4-amino-
6,7-dimethoxy-quinazoline (0.14 g, 0.56 mmol), N-(2-amino-
ethyl)piperidine (0.08 mL, 0.56 mmol) and 3-methyl-1-butanol
(5 mL) was heated under reflux (ꢃ180 °C) for 5 h. The mixture
was then cooled to 0–5 °C and left to rest for approximately
30 min. The white precipitate was collected by filtration and
washed with 3-methyl-1-butanol (5 mL) and acetone (5 mL). The
hydrochloride salt was then recrystallised twice from methanol
(precipitated with diethyl ether) to yield small white (powdery)
crystals (0.04 g, 20% yield); mp = 266–269 °C; Rf = 0.36 (MeOH/
EtOAc 1:1); HPLC I (MeOH/H2O 1:1) tR 8.34 min (100%); 1H NMR
(300 MHz, CD3OD): d 1.69 (br s, 2H, pip), 1.91 (t, J = 5.65 Hz, 4H,
pip), 3.35 (o, 4H, pip), 3.38 (t, J = 5.99 Hz, 2H, CH2), 3.92 (s, 3H,
OCH3), 3.93 (o, 2H, CH2), 3.97 (s, 3H, OCH3), 6.97 (s, 1H, Ar-5H),
7.56 (s, 1H, Ar-8H); MS (FAB) m/z 332 (M+, 100%). Anal. Calcd for
6.3. Pharmacology: activation of uptake
The compounds were tested for their ability to increase the up-
take of a tracer dose of [3H]prazosin in the peptidergic neurones.
The cells were incubated in the presence of [3H]prazosin
2 ꢂ 10ꢀ10 M and concentrations of the test compounds, ranging
from 10ꢀ9 M to 10ꢀ4 M. Each experiment included a positive con-
trol (unlabelled prazosin 10ꢀ6 M). Basal uptake was defined as
the amount of [3H]prazosin accumulated by the cells in the pres-
ence of [3H]prazosin 2 ꢂ 10ꢀ10 M. For the purpose of these experi-
ments, activation of Transport-P was defined as the increase of
uptake of [3H]prazosin which is caused by unlabelled prazosin
10ꢀ6 M; the value for activation of Transport-P was therefore ob-
tained by subtracting basal uptake from uptake in the presence
of unlabelled prazosin 10ꢀ6 M. The increase in uptake of [3H]prazo-
sin was expressed as percentage of the basal value. Each experi-
mental point was carried out in triplicate and each experiment
was carried out twice.
C
17H25N5O2ꢁ2HClꢁ2H2O: C, 46.18; H, 7.11; N, 15.84. Found: C,
45.81; H, 7.12; N, 15.67.
6.2. Pharmacology: inhibition of uptake
The compounds were dissolved in water, uptake/binding buffer
(vide supra) or DMSO. When DMSO was used as a solvent, it was
present in all solutions to 1:1000. In six consecutive experiments,
DMSO had no effect on the accumulation of [3H]prazosin when the
cells were exposed to this radioligand at a concentration of
2 ꢂ 10ꢀ10 M (DMSO: 26.9 1.8 fmol/mg protein; water: 26.5 1.9
fmol/mg protein). However DMSO diminished the activation of
Transport-P, which was caused by unlabelled prazosin 10ꢀ6 M. In
The compounds were tested for their ability to inhibit the up-
take of prazosin (at 10ꢀ6 M) in GT1-1 immortalised gonadotro-
phin-releasing hormone (GnRH) peptidergic neurones, as has
been described in detail.7 Briefly, the cells were grown at 37 °C in
Corning 175 cm2 flasks in culture medium consisting of Dulbecco’s
modified Eagle’s medium (DMEM) and Ham’s F-12 (ratio 1:1) con-
taining 10% fetal bovine serum and sodium bicarbonate 3.7 g/L, in a

J. F. M. da Silva et al. / Bioorg. Med. Chem. 16 (2008) 7254–7263
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six consecutive experiments, the increase in uptake in the presence
of DMSO was 19.8 2.7 fmol/mg protein, representing 76 12% in-
crease above basal; in the absence of DMSO, the increase in uptake
was 34.3 4.0 fmol/mg protein, representing 132 16% increase
above basal. Despite this effect of DMSO, there were no qualitative
differences in the experiments and the conclusions were the same,
regardless of whether DMSO was included or not.
Acknowledgments
We warmly thank Cesare Federico for the synthesis of com-
pound 12 and CAPES (Brazil) for financial assistance to J.F.M.S.
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