Triazoloquinazolinediones as novel high affinity ligands for the benzodiazepine site of GABAA receptors

Article abstract of DOI:10.1016/j.bmc.2010.11.050

Based on a pharmacophore model of the benzodiazepine-binding site of GABA_A receptors, a series of 2-aryl-2,6-dihydro[1,2,4]triazolo[4,3-c] quinazoline-3,5-diones (structure type I) were designed, synthesized, and identified as high-affinity lig

Full text of DOI:10.1016/j.bmc.2010.11.050

Bioorganic & Medicinal Chemistry 19 (2011) 111–121
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Triazoloquinazolinediones as novel high affinity ligands for the
benzodiazepine site of GABAA receptors
Jakob Nilsson a, Ritha Gidlöf a, Elsebet Østergaard Nielsen b, Tommy Liljefors c, Mogens Nielsen c,
Olov Sterner a,
⇑
a Division of Organic Chemistry, Lund University, PO Box 124, SE-221 00 Lund, Sweden
b NeuroSearch A/S, DK-2750 Ballerup, Denmark
c University of Copenhagen, Faculty of Pharmaceutical Sciences, 2 Universitetsparken, DK-2100 Copenhagen, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on a pharmacophore model of the benzodiazepine-binding site of GABAA receptors, a series of
2-aryl-2,6-dihydro[1,2,4]triazolo[4,3-c]quinazoline-3,5-diones (structure type I) were designed, synthe-
sized, and identified as high-affinity ligands of the binding site. For several compounds, Ki values of
around 0.20 nM were determined. They show a structural resemblance with the previously described
2-phenyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-ones (II) and 2-phenyl-[1,2,4]triazolo[1,5-a]quinoxalin-
4(5H)-one (III). The 9-bromo substituted compounds 8a–d were prepared in an 8-step synthesis in an
overall yield of approximately 40%, and a library of 9-substituted analogues was prepared by cross-
Received 9 August 2010
Accepted 20 November 2010
Available online 25 November 2010
Keywords:
2-Aryl-2,6-dihydro[1,2,4]triazolo-
[4,3-c]quinazoline-3,5-diones
Benzodiazepine binding site
GABAA receptors
GABAA receptor subtypes
Pharmacophore model
coupling reactions. Compound 8e, 21, 22, and 24 were tested on recombinant rat
3b3 2, and 5b3c2 subtypes, and displayed selectivity for the 1b3c2 isoform.
Ó 2010 Elsevier Ltd. All rights reserved.
a1b3c2, a2b3c2,
a
c
a
a
1. Introduction
different physiological effects,
sedative and anterograde amnesic effects,
ing receptors are involved in anxiolytic activity, while
a1-containing receptors mediate
a
2-, and/or
a3-contain-
Most compounds that elicit a pharmacological response by inter-
acting with the benzodiazepine-binding site (BZDR) of the GABAA
receptors are, inspite of thenameof thebinding-site, not benzodiaze-
pines, and examples are the 2-arylpyrazoloquinolines,1,2
b-carbolines,3 pyridodiindoles,4 triazoloqunioxalines,5 pyrimidin-
5(6H)-ones,6 cyclopyrrolones, and quinolines.7 They display a contin-
uous range of effects, from being positive allosteric modulators that
enhance the GABAergic inhibition exhibiting anxiolytic, anticonvul-
sant, sedative-hypnotic and myorelaxant activities, to negative
allostericmodulators that reduce the GABAergicinhibition andthere-
by exhibit anxiogenic, somnolytic, and convulsant activities. Neutral
allosteric modulators elicit no pharmacological response, but may
affect the BZDR by competitively inhibit binding of other ligands.
The GABAA receptors are pentameric ligand gated chloride ion
channels assembled from at least 16 subunits from seven different
a5-contain-
ing receptors might be associated with cognition and memory.11,12
A pharmacophore model of the BZDR based on 10 different com-
pound classes13 has been further developed and refined based on a
SAR study of synthetic flavone derivatives,14,15 adding additional
pharmacophore elements. This model was recently used to identify
and optimize novel 4-quinolones and azaflavone derivatives as
ligands for the BZDR of the GABAA receptors with Ki values down
to 0.05 nM.16–18 In the present study, the ability of 2-aryl-2,6-dihy-
dro[1,2,4]triazolo[4,3-c]quinazoline-3,5-diones (structure type I,
Fig. 1) to interact with the BZDR has been investigated. The basic
structure of 2-aryl-2,6-dihydro[1,2,4]triazolo[4,3-c]quinazolin-
3,5-dione (2-aryltriazoloquinazolinedione) resembles those of
2-phenyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-one (2-phenylpyraz-
oloquinoline, II) and 2-phenyl-[1,2,4]triazolo[1,5-a]quinoxalin-
4(5H)-one (2-phenyltriazoloquinoxalone, III), and 2-arylpyrazolo-
quinolines and 2-aryltriazoloquinoxalones have previously been
described as BZDR ligands.1,2,5 The 2-aryltriazoloquinazolinediones
retains a [6,6,5]-tricyclic heteroaromatic system and combines the
two carbonyl moieties of II and III into a dicarbonyl compound
(Fig. 1). When positioned in the pharmacophore model (Fig. 2), the
2-aryltriazoloquinazolinediones (exemplified by compound 8a)
appear to fulfill the requirements for a strong BZDR affinity, for
example, the NH(6), N(1), and the 3- and 5-carbonyl oxygens can
be expected to interact with the hydrogen bond donors/acceptors
classes (
be located at an interface between an
ligands of the BZDR are believed to mediate their pharmacological
effect predominately through interactions at the 1bxc2 2bxc2
3bx 2, and 5bxc2 subtype assemblies. Studies with transgenic
a1–6, b1–3
,
c
1–3, d,
e
,
p
, and h).8–10 The BZDR is believed to
and a subunit, and
a
c
a
,
a
,
a
c
a
mice and subtype selective compounds clearly suggest that GABAA
receptors with different subtype composition are associated with
⇑
Corresponding author. Tel.: +46 462228213; fax: +46 462228209.
E-mail address: Olov.Sterner@organic.lu.se (O. Sterner).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.050

112
J. Nilsson et al. / Bioorg. Med. Chem. 19 (2011) 111–121
R2
R3
N2
N1
R1
O
3
N
9
6
N
5
O
H
I, 2-aryl-[1,2,4]triazolo[4,3-c]quinazoline-3,5(2H,6H)-dione
N
N
N
N
R1
N
O
O
N
H
N
H
II, 2-phenyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-one
III, 2-phenyl-[1,2,4]triazolo[1,5-a]quinoxalin-4(5H)-one
Figure 1. Design of the 2-aryltriazoloquinazolinediones based on the two previously described BZDR ligands 2-phenylpyrazoloquinolines and 2-phenyl-triazoloquinoxalines.
H1, A2 and H2/A3. The aim of this study was both to develop a
synthetic route to the 2-aryltriazoloquinazolinediones and use that
for the preparation of new derivatives, as well as to further explore
the pharmacophore region called ‘interface’ (see Fig. 2). A common
feature with previously reported potent ligands that reaches into
the interface region, is a flexible methylene (e.g., a benzyl group)
or ethylene bridge at the position corresponding to the R1 group in
the 2-aryltriazoloquinazolinediones (Fig. 1).6,16–18 To facilitate the
investigation of this region our strategy was to prepare derivatives
with R1 = Br, and to use suitable cross-coupling protocols to attach
various groups to the 2-aryltriazoloquinazolinedione scaffold.
inedione scaffold 8a–e was synthesized using two similar synthetic
routes, as outlined in Scheme 1 and 2. The route leading to the
9-bromo-substituted triazoloquinazolinediones 8a–d via the nitro-
triazole 6 facilitated multi-gram scale synthesis, as the purification
of the intermediates by chromatography was not required (most of
the compounds have limited solubility).
The quinazolinone derivatives 3a and 3b were prepared by
addition of ethoxycarbonyl isothiocyanate to an anthranilic acid
followed by cyclization in acetic anhydride. Deprotection and
methylation gave 5a and 5b, which were activated for a subse-
quent SNAr displacement using two different protocols. Treatment
of 5a with 3-nitro-1H-1,2,4-triazole in I2/PPh3/EtN(i-Pr)2/toluene
gave the nitro-triazole derivative 6, and triazole derivatives 7a–d
were subsequently prepared in a one-pot reaction by the treatment
of 6 with 10a–d under solvent-free conditions.19,20 Alternatively,
activation of 5a and 5b may be achieved using phosphorous oxy-
chloride to give compound 11a and 11b, respectively. The reaction
of 11a with 10b gave 7b, while the reaction of 11b with 10b gave
7e in yields that were essentially the same as for the nitro-triazole
pathway. It is likely that the initial step of these reactions is a SNAr
displacement followed by a base promoted cyclization, a sugges-
tion that is supported by the isolation of 12 as an intermediate
from 11b to 7e, using slightly modified conditions. Cyclization of
12 in the presence of lithium hydroxide as base gave 7e in a yield
similar to the one-pot reactions described above.
Treatment of 7a–e with 1 equiv of mCPBA yielded tria-
zoloquinazolinediones 8a–e in a total yield of 40% over eight steps
(for 8a starting from 1a). Compounds 10a–d were prepared utiliz-
ing a copper-catalyzed coupling between ethyl carbazate and
substituted iodobenzenes in presence of 1,10-phenantroline and
cuprous iodide as a catalyst system (Scheme 2).21 Coupling with
4-iodotoluene and iodobenzene gave 10a and b, respectively, in
good yields, whereas coupling with 4-chloro- or 2-fluoro-substi-
tuted iodobenzene resulted in significantly lower yields. The
triazoloquinazolinedione 8a was subjected to various cross-cou-
pling reactions including a copper-free Sonogashira coupling,22
Stille coupling23 and a Heck reaction24 as shown in Scheme 3.
The copper-free Sonogashira coupling was employed for the syn-
thesis of acetylenes 13–20. The yield of these reactions was
generally somewhat hampered by problems associated with
chromatographic purification of the sparingly soluble acetylenes.
2. Results
2.1. Chemical synthesis
All 2-aryltriazoloquinazolinedione derivatives prepared in this
study are to our knowledge new compounds. The triazoloquinazol-
H1
A2
S1
S5
L1
L2
S4
L3
H2/A3
S2
S3
Interface
Figure 2. The proposed binding mode of triazoloquinazoline-3,5-dione 8a in the
pharmacophore model representation. H1 and H2 are hydrogen bond donor sites and
A2 and A3 are hydrogen bond acceptor sites. L1, L2, and L3 represent lipophilic pockets
and S1–S5 denotes regions of steric repulsive ligand–receptor interactions (receptor
essential volume). The interface region is a partly lipophilic region in the vicinity of the
9-position of triazoloquinazolinediones. This region has been suggested to represent
the interface between the a- and c-subunit in the GABAA receptors.

J. Nilsson et al. / Bioorg. Med. Chem. 19 (2011) 111–121
113
O
R1
O
O
S
O
O
OH
NH
R1
R1
R1
a
b
c
N
O
NH
OH
NH2
O
N
H
N
H
S
S
N
H
OEt
4a,b
3a,b
1a,b
a: R1 = Br
2a,b
b: R1 = Me
NO2
N
O
N
R1
N
N
d
e
NH
SMe
Br
R1 = Br
N
N
SMe
5a,b
6
g
f
R2
R2
R3
Cl
N
N
R3
N
N
N
R1
h
i
R1
R1
N
O
O
N
N
SMe
N
SMe
N
O
H
11a,b
7a-e
8a-e
a: R1 = Br
a: R1=Br, R2=Me, R3=H
b: R1=Br, R2=H, R3=H
c: R1=Br, R2=Cl, R3=H
d: R1=Br, R2=H, R3=F
e: R1=Me, R2=H, R3=H
b: R1 = Me
Ph
N
HN
N
COOEt
SMe
j
k
N
11b
7e
12
Scheme 1. Reagents and conditions: (a) ethoxycarbonyl isothiocyanate, MeCN, 80 °C, 4 h (yield 95% for 2a and 91% for 2b); (b) Ac2O, 60 °C, 1 h (yield 93% for 3a and 100% for
3b); (c) 0.5 M NaOMe in MeOH, THF, reflux, 90 min (yield 100% for 4a and 88% for 4b); (d) DMF, 0.5 M NaOMe in MeOH, then MeI, rt, 10 h (yield 90% for 5a and 77% for 5b); (e)
3-nitro-1H-1,2,4-triazole, PPh3, I2, toluene, DIPEA, 95 °C, 1 h (yield 89%); (f) POCl3, pyridine, 110 °C, 18 h (yield 75% for 11a and 77% for 11b); (g) DIPEA, 10a–d, 110 °C, 60 h
(yields 69% for 7a, 76% for 7b, 70% for 7c, and 51% for 7d); (h) DIPEA, 10b, 110 °C, 60 h (yields 71% for 7b and 75% for 7e); (i) mCPBA (77%), CH2Cl2, rt, 6 h (quantitative yields);
(j) DIPEA, 10a, 1,4-dioxane, reflux, 48 h (quantitative yield); (k) LiOH, THF, rt, 2 h (yield 78%).
NH2
N
turnings and addition of tributyltin chloride to the resultant
Grignard solution25–27 gave the corresponding arylmethyl Stille-
reagents. Reaction with the 3-thienyl Grignard reagent led to
formation of two products, of which the desired 33 was the major
product. The high reactivity of the 2-position in 3-thienylmagne-
sium bromide has previously been recognized in the synthesis of
3-methyl-2-thenoic acid.27 The methylene-linked aryl derivatives
29–31 were then prepared by a Stille coupling with 8a, and carbox-
ylic acid 36 by a Heck reaction with benzyl acrylate followed by a
hydrogen reduction over palladium on charcoal.23
I
OEt
a
O
R2
R3
R2
R3
9a-d
10a-d
a: R2 = Me, R3 = H
b: R2 = H, R3 = H
c: R2 = Cl, R3 = H
d: R2 = H, R3 = F
Scheme 2. Reagents and conditions: CuI, 1,10-phenantrolein, ethyl carbazate,
Cs2CO3, DMF, 80 °C, 16 h (yields 83% for 10a, 76% for 10b, 40% for 10c, and 22% for
10d).
2.2. Receptor binding
Affinities for the BZDR were determined in vitro by displace-
ment of 3H-Flumazenil in rat cortical tissue (Table 1). Subtype
affinity testing was performed with compounds 8e, 21, 22, and
Hydrogen reduction over palladium on charcoal yielded the
desired ethylene-linked aryl derivatives 21–28. Treatment of aryl-
methyl bromides (i.e., benzyl bromide, 3-fluorobenzyl bromide,
and 3-bromomethylthiophene, respectively), with magnesium
24 on recombinant
a1b3c2, a2b3c2, a3b3c2, and a5b3c2 receptor
subtypes (Table 2).

114
J. Nilsson et al. / Bioorg. Med. Chem. 19 (2011) 111–121
N
N
N
N
R
b
a
R
O
O
8a
N
N
N
O
N
H
O
H
13: R = phenyl
21: R = phenyl
14: R = 3-thienyl
22: R = 3-thienyl
23: R = 2-pyridyl
24: R = 3-pyridyl
25: R = 4-pyridyl
15: R = 2-pyridyl
16: R = 3-pyridyl
17: R = 4-pyridyl
18: R = 4-phenoxyphenyl
19: R = 4-biphenyl
20: R = 3-hydroxyphenyl
26: R = 4-phenoxyphenyl
27: R = 4-biphenyl
28: R = 3-hydroxyphenyl
N
N
c
29: R = phenyl
O
8a
30: R = 3-fluorophenyl
31: R = 3-thienyl
Ar
N
N
O
H
Br
Bu3Sn
e
+
SnBu3
S
S
S
32
33
34
(major)
(minor)
N
N
N
O
O
N
b
O
O
d
BnO
N
HO
N
8a
N
H
O
N
O
H
35
36
Scheme 3. Reagents and conditions: (a) Pd(OAc)2, PPh3, RC„CH, NEt3, DMF, 100 °C, 18 h (yields 32–75%); (b) H2 (1 atm) 10% Pd/C, CH2Cl2, MeOH, rt, 18 h (yields 79–98%); (c)
Pd(PPh3)4, NEt3, DMF, PhCH2SnBu3, tributyl(3-fluorobenzyl)tin, or 33, 110 °C, 18 h (yields 65% for 29, 70% for 30, and 62% for 31); (d) Pd(OAc)2, PPh3, benzyl acrylate, NEt3,
DMF, 120 °C, 15 h (yield 47%); (e) Mg, THF, 35 °C, 30 min, then Bu3SnCl, 1 h (yield 36%).
previously been shown that the presence of a hydrogen, a chlorine
or a methoxy group in position 40 as well as a fluorine in position 20
of the 2-arylpyrazoloquinolines (II, Fig. 1) results in subnanomolar
affinities.1,2 Inspired by this, compounds 8a–e were prepared and
were all found to display subnanomolar affinities (Table 1). 8a
(Ki = 0.47 nM) was chosen as the lead structure for the exploration
of various substituents in position 9. Not surprisingly, the
difference between a methyl group (8e) and a bromine (8b) in
position 9 is small.
Among the most potent compounds presented in this study are
the pyridyl substituted compounds 23–25 which inhibit BZDR
binding at concentrations as low as 0.17 nM. The high affinity of
these compounds may be best understood in the light of the sug-
gestion that the interface region located in this area is a water filled
channel-like cavity.15 This hypothesis is also supported by the high
affinity of other polar compounds, such as the carboxylic acid
derivative 36 (1.4 nM). Compared to 21 and 29, which are substi-
tuted with lipophilic side-chains in position 9, the more polar
3. Discussion
The 2-thioxoquinazolin-4(1H)-one 3b was previously identified
as the best virtual BZDR ligand28 in a 3D database search employ-
ing the program CatalystÒ. A structurally similar quinazoline-
2,4(1H,3H)-dione (with oxygen instead of sulfur) was prepared
and found to be inactive, and when 3b now became available it
was also assayed with the same result (Ki value >30,000 nM). Both
compound types fit into the pharmacophore model in a similar
fashion as the triazoloquinazolinediones, with NH(1) interacting
with A2, the 4-carbonyl oxygen interacting with H2, and the 3-car-
bamate carbonyl oxygen interacting with H1. The low affinity of
the quinazoline-2,4(1H,3H)-dione as well as of 3b can most likely
be attributed to the steric repulsion between the 3-carbamate car-
bonyl oxygen and the 2/4 oxygen/sulfur atom, which forces the
molecule to adopt a non-planar conformation and hence disables
the H1 interaction. The 2-aryltriazoloquinazolinediones are
structurally related to the 2-arylpyrazoloquinolines, and it has

J. Nilsson et al. / Bioorg. Med. Chem. 19 (2011) 111–121
115
Table 1
erated non-flexible substituent indicates that the extension of the
channel-like cavity in the interface region is considerable.
Subtype affinity testing was performed with compounds 8e, 21,
Ki values of triazoloquinazolinediones tested on 3H-Flumazenil binding in vitro to rat
cortical membranes
R2
22, and 24 on recombinant
subtypes (Table 2). All compound investigated in this study display
selectivity for 1b3c2 over the other receptor subtypes with a2
Ki ratios ranging from 18 for 21 to 2 for 8e, a3 a1 Ki ratios ranging
1 Ki ratios ranging from 26 for 22
a1b3c2, a2b3c2, a3b3c2, a5b3c2 receptor
a
/a1
/
N
R3
N
from 5 for 22 to 3 for 24, and a5
/a
R1
O
to 13 for 24.
N
N
O
4. Conclusion
H
Triazoloquinazolinediones, a novel class of BZDR ligands, have
been designed using a pharmacophore model, and a small library
of compounds have been synthesized and assayed. This set of com-
pounds has permitted additional characterization of the BZDR, in
particular of the interface region (as defined in the pharmacophore
model), which has been showed to be a highly interesting region
for optimization of BZDR binding ligands. The decreased affinity
displayed by the derivatives with the largest substituents in posi-
tion 9, 26 and 27, indicate a repulsive interaction with the receptor
essential volumes further out in the interface region. The highest
in vitro affinity observed for the set of compound investigated here
was 0.17 nM (24) and a few compounds were selected and assayed
8a-e, 13, 21-31, 36
Compds
R1
R2
R3
Ki valuea (nM)
8a
8b
8c
8d
8e
13
21
22
23
24
25
26
27
28
29
30
31
36
Br-
Br-
Br-
Br-
Me-
H-
H-
H-
F-
0.47 0.09
0.24 0.07
0.6 0.26
0.20 0.07
0.62 0.13
40 11
H-
Cl-
H-
H-
CH3-
Phenyl-C„C–
H-
H-
H-
H-
H-
H-
H-
H-
H-
H-
H-
H-
H-
H-
Me-
Me-
Me-
Me-
Me-
Me-
Me-
Me-
Me-
Me-
Me-
Me-
Me-
Phenyl-CH2CH2–
4.0 1.2
(3-Thienyl)-CH2CH2–
(2-Pyridyl)-CH2CH2–
(3-Pyridyl)-CH2CH2–
(4-Pyridyl)-CH2CH2–
(4-PhOPh)-CH2CH2–
(4-Biphenyl)-CH2CH2–
(3-Hydroxyphenyl)-CH2CH2–
Benzyl-
0.94 0.27
0.53 0.11
0.20 0.04
0.17 0.03
123 12
1570 150
12.4 5.9
1.0
on recombinant
All compound tested displayed selectivity for
a
1b3c2
,
a
2b3c2
,
a
3b3c2
,
a
5b3c2 receptor subtypes.
1b3 2 over the other
a
c
receptor subtypes, and it is reasonable to suggest that this specific
pharmacophore has a certain selectivity for this subtype.
3-Fluorophenyl-CH2–
(3-Thienyl)-CH2–
HOOC–CH2CH2–
2.1 0.3
0.23 0.18
1.4 0.15
5. Experimental
a
Each Ki value is the mean SD of three determinations.
Reagents and solvents (except THF) were used from commercial
sources without purification. THF was distilled from sodium/ben-
zophenone prior to use. 1H and 13C NMR were recorded at room
temperature unless otherwise specified with a Bruker DR400 spec-
trometer. The spectra were recorded in CDCl3, DMSO–d6, and C6D6,
and the solvent signals (7.27 and 77.0, 2.50 and 39.5 or 7.18 and
128.1 ppm, respectively) were used as reference. Analytical thin
layer chromatography (TLC) was performed on Kiselgel 60 F254
plates (Merck). Column chromatography was performed on SiO2
(Matrex LC-gel: 60A, 35-70 MY, Grace). Melting points (uncor-
rected) were determined with a Reichert microscope. EI mass spec-
tra were recorded at 70 eV with a Jeol SX102 spectrometer and ESI
spectra were recorded with Micromass Q-TOF Micro.
Table 2
The affinity of selected triazoloquinazolinediones tested on 3H-Flumazenil binding to
a
1b3c2s
,
a
2b3c2s
,
a3b3c2s, and
a5b3c2s GABAA receptor subtypes
a
a
a
a
Compds
Ki a1 (nM)
Ki a2 (nM)
Ki a3 (nM)
Ki a5 (nM)
8e
21
22
24
0.43 0.18
3.6 2.2
0.14 0.05
0.68 0.02
1.0 0.17
65 8.5
1.7 0.7
4.4 1.7
1.3 0.38
9.7 5.0
0.71 0.25
2.3 0.90
n.d.
66 21
3.6 1.5
9.1 4.1
a
Each Ki value is the mean SD of three determinations. n.d., not determined.
22–25 and 31 may more readily participate in a continuous net-
work of hydrogen bonds through their heteroatoms. From our pre-
vious experiences with other classes of BZDR ligands, the change in
5.1. 5-Bromo-2-({[(ethoxycarbonyl)amino]carbonothioyl}-
amino)-benzoic acid (2a)
affinity observed upon introducing
a lipophilic PhCH2- or
PhCH2CH2-group into the interface region, ranges from unaffected
to a 10-fold increase. This indicates that the formation of a favor-
able interaction between the lipophilic side-chain of the ligand
and the partly lipophilic interface region is possible.16–18 For the
2-aryltriazoloquinazolinediones, the same substitutions actually
decrease the affinity, ninefold for 21 compared to 8a and twofold
for 29 compared to 8a. The 2-aryltriazoloquinazolinediones are,
compared to the other BZDR ligands,16–18 larger and more rigid,
and a possible explanation for this observed difference in binding
strength is that smaller and more flexible BZDR ligands more easily
may adapt a conformation in which favorable interactions are
formed between the lipophilic side-chain and the partially lipo-
philic area in the interface region.
To
a solution of 2-amino-5-bromobenzoic acid (8.09 g,
37.4 mmol) in 60 mL of dry MeCN was added ethoxycarbonyl iso-
thiocyanate (4.32 mL, 37.4 mmol) and the mixture was stirred at
reflux for 5 h. Heating was removed and a white precipitate was
filtered off, to give 2a as a white solid (12.3 g, 95%). Mp: 180 °C
(decomp.), 1H NMR (400 MHz, DMSO-d6) d 11.38 (1H, s), 12.25
(1H, s), 8.07 (1H, d, J = 8.8 Hz), 7.99 (1H, d, J = 2.4 Hz), 7.78 (1H,
dd, J = 8.7 and J = 2.4 Hz), 4.20 (2H, q, J = 7.1 Hz), 1.25 (3H, t,
J = 7.1 Hz); 13C NMR (100 MHz, DMSO-d6) d 179.2, 165.9, 152.9,
137.7, 134.5, 132.5, 129.5, 126.4, 117.9, 62.0, 14.1; HRMS (ESI):
for C11H11BrN2O4SNa calcd: 368.9521 [M+H]; found: 368.9523.
5.2. 2-({[(Ethoxycarbonyl)amino]carbonothioyl}amino)-5-met-
hylbenzoic acid (2b)
The larger phenoxyphenyl- (26) and biphenyl- (27) groups are
evidently discriminated, indicating a sterical repulsive interaction
further out in the interface region. Worth noticing is also the large
tolerance for rigid planar substituents such as the phenylacetylene
13, with an affinity of 40 nM. Although not favoured, this still tol-
Compound 2b was prepared and purified according to the pro-
cedure described for 2a, starting from 2-amino-5-methylbenzoic

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J. Nilsson et al. / Bioorg. Med. Chem. 19 (2011) 111–121
acid. The reaction yielded 2b (91%) as a white solid (mp: 197.0 °C).
1H NMR (400 MHz, CDCl3) d 12.43 (1H, s), 8.3 (1H, s), 8.15 (1H, s),
7.91 (1H, d, J = 1.8 Hz), 7.45 (1H, dd, J = 8.38 and 1.8 Hz), 4.32 (2H,
q, J = 7.1 Hz), 2.41 (3H, s), 1.36 (3H, t, J = 7.1 Hz); 13C NMR
(400 MHz, CDCl3) d 178.4, 167.0, 152.0, 137.1, 136.3, 134.4,
131.9, 126.9, 121.3, 63.2, 21.1, 14.4; HRMS (ESI): for C12H15N2O4S
calcd: 283.0753 [M+H]; found 283.0758.
in methanol (65.6 mL) and the mixture was stirred at room tem-
perature for 15 min. Iodomethane (2.04 mL, 32.8 mmol) was added
and the reaction mixture was stirred at room temperature for 22 h.
The solvents were evaporated under reduced pressure and the
remaining solid was dissolved in 500 mL of EtOAc and washed
with 400 mL of a saturated solution of aqueous NaHCO3. The or-
ganic layer was dried over MgSO4, concentrated under reduced
pressure and precipitated from 50 mL of ethyl alcohol, to give 5a
as a white solid (8.03 g, 90%). Mp: 230 °C, 1H NMR (400 MHz,
DMSO-d6) d 12.77 (1H, s), 8.09 (1H, J = 2.4 Hz), 7.89 (1H, dd,
J = 8.7 and 2.4 Hz), 7.48 (1H, d, J = 8.7 Hz), 2.57 (3H, s); 13C NMR
(100 MHz, DMSO-d6) d 160.0, 157.3, 147.4, 137.3, 128.3, 128.1,
121.5, 117.6, 12.8; HRMS (ESI): C9H8N2OSBr calcd: 270.9541
[M+H]; found 270.9539.
5.3. Ethyl 6-bromo-4-oxo-2-thioxo-1,4-dihydroquinazoline-
3(2H)-carboxylate (3a)
Compound 2a (12.3 g, 35.5 mmol) was dissolved in 150 mL of
acetic anhydride and stirred at 60 °C for 4 h. The mixture was
slowly cooled to 4 °C during crystallization and the white crystals
formed were filtered off, washed with cold acetic anhydride and
dried under vacuum, to give 3a (10.8 g, 32.8 mmol, 93%). Mp:
214.0 °C, 1H NMR (400 MHz, DMSO-d6) d 11.87 (1H, s), 8.06 (1H,
d, J = 2.4 Hz), 7.97 (1H, dd, J = 8.7 and J = 2.4 Hz), 7.47 (1H, d,
J = 8.7 Hz), 4.19 (2H, q, J = 7.1 Hz), 1.25 (3H, t, J = 7.1 Hz); 13C
NMR (100 MHz, DMSO-d6) d 183.4, 154.1, 153.3, 146.9, 138.8,
131.1, 126.3, 119.8, 118.9, 62.0, 14.2; HRMS (ESI): for
5.8. 6-Methyl-2-(methylsulfanyl)quinazolin-4(3H)-one (5b)
Compound 5b was prepared and purified according to the pro-
cedure described for 5a, starting from 4a. The reaction yielded 5b
(77%) as a white solid (mp: 206 °C). 1H NMR (400 MHz, CDCl3) d
10.63 (1H, s), 8.05 (1H, s), 7.55 (2H, m), 2.69 (3H, s), 2.47 (3H, s);
13C NMR (100 MHz, CDCl3) d 163.5, 154.4, 147.5, 136.5, 136.1,
126.39, 126.35, 119.7, 21.4, 13.6; HRMS (ESI): for C10H11N2OS
calcd: 207.0592 [M+H]; found 207.0600.
C11H9BrN2O3S calcd: 327.9517 [M+H]; found: 327.9524.
5.4. Ethyl 6-methyl-4-oxo-2-thioxo-1,4-dihydroquinazoline-
3(2H)-carboxylate (3b)
5.9. 6-Bromo-2-(methylsulfanyl)-4-(3-nitro-1H-1,2,4-triazol-1-
Compound 3b was prepared and purified according to the pro-
cedure described for 3a, starting from 2b. The reaction yielded 3b
(100%) as a white solid (mp: 164.0 °C). 1H NMR (400 MHz, CDCl3) d
8.57 (1H, br s), 7.95 (1H, s), 7.54 (1H, dd, J = 8.3 and 2.0 Hz), 7.42
(1H, d, J = 8.3 Hz), 4.30 (2H, q, J = 7.1 Hz), 2.45 (3H, s), 1.34 (3H, t,
J = 7.1 Hz); 13C NMR (100 MHz, CDCl3) d 184.3, 152.8, 152.6,
145.7, 137.4, 137.2, 129.0, 124.8, 119.4, 62.9, 21.3, 14.4; HRMS
(ESI): for C12H13N2O3S calcd: 265.0647 [M+H]; found 265.0647.
yl)quinazoline (6)
Iodine (16.5 g, 65.1 mmol) was added to a suspension of com-
pound 5a (8.03 g, 29.6 mmol), 3-nitro-1H-1,2,4-triazole (11.8 g,
103.6 mmol), triphenylphosphine (18.1 g, 69.1 mmol) in 630 mL
of toluene. The reaction mixture was rapidly heated to 95 °C for
15 min after which 25 mL of N,N-diisopropylethylamine was added
and the mixture was stirred for another 50 min. The reaction was
cooled to room temperature and concentrated under reduced pres-
sure. The crude product was precipitated from ethyl alcohol, to
give 6 as a yellow solid (9.67 g, 89%). Mp: 204 °C, 1H NMR
(400 MHz, DMSO-d6) d 9.87 (1H, s), 8.97 (1H, dd, J = 2.2 and
0.3 Hz), 8.18 (1H, dd, J = 9.0 and 2.2 Hz), 7.88 (1H, dd, J = 9.0 and
0.3 Hz), 2.70 (3H, s); 13C NMR (100 MHz, DMSO-d6) d 167.0,
163.2, 152.3, 151.1, 148.2, 139.1, 129.1, 127.9, 120.5, 114.3, 14.0;
5.5. 6-Bromo-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (4a)
A solution of sodium methoxide (0.5 M, 36.1 mmol) in metha-
nol (72 mL) was added to a solution of 3a (10.8 g, 32.8 mmol) in
150 mL of dry THF and the mixture was heated at reflux for
90 min. The mixture was allowed to reach room temperature and
quenched by addition of acetic acid (2.1 mL, 36.1 mmol). The mix-
ture was concentrated under reduced pressure and 200 mL of ethyl
alcohol and 100 mL of water was added and the mixture was
heated at reflux for 30 min. The slurry was cooled to rt and filtrated
to give 4 white solid (8.43 g, 100%). Mp: 350 °C (decomp.), 1H NMR
(400 MHz, DMSO-d6) d 12.67 (2H, br s), 7.97 (1H, d, J = 2.3 Hz), 7.88
(1H, dd, J = 8.7 and 2.3 Hz), 7.29 (1H, d, J = 8.7 Hz); 13C NMR
(100 MHz, DMSO-d6) d 174.4, 158.6, 139.8, 137.9, 128.7, 118.4,
118.1, 115.9; HRMS (FAB+): C8H6ON2BrS calcd: 256.9382 [M+H];
found 256.9382.
HRMS (FAB+):
366.9615.
C11H8N6O2BrS calcd: 366.9613 [M+H]; found
5.10. 9-Bromo-2-(4-methylphenyl)-5-(methylsulfanyl)[1,2,4]-
triazolo[4,3-c]quinazolin-3(2H)-one (7a)
N,N-Diisopropylethylamine (0.95 mL, 5.45 mmol) was added to
a mixture of compound 6 (2.0 g, 5.447 mmol) and 10a (1.164 g,
5.99 mmol) and the reaction mixture were heated at 110 °C for
60 h. The mixture was cooled to rt, concentrated under reduced
pressure and precipitated from ethyl alcohol to give 7a as a white
solid (1.51 g, 69%). Mp: 213 °C, 1H NMR (400 MHz, DMSO-d6) d 8.28
(1H, d, J = 2.2 Hz), 7.94 (2H, dd, J = 8.6 and 1.9 Hz), 7.70 (1H, dd,
J = 8.6 and J = 2.2 Hz), 7.50 (1H, d, J = 8.6 Hz), 7.28 (2H, d,
J = 8.6 Hz), 2.65 (3H, s), 2.40 (3H, s); 13C NMR (100 MHz, DMSO-
d6) d 150.9, 147.1, 141.7, 138.5, 136.5, 135.7, 134.9, 129.9, 129.9,
128.8, 125.3, 120.6, 119.5, 119.5, 116.1, 21.2, 13.6; HRMS (FAB+):
5.6. 6-Methyl-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (4b)
Compound 4b was prepared and purified according to the proce-
duredescribedfor 4a, startingfrom3b. Thereactionyielded 4b (88%)
as a white solid [mp: 312 °C, (decomp.)]; 1H NMR (400 MHz, DMSO-
d6) d 12.65 (1H, s), 12.40 (1H, s), 7.75 (1H, s), 7.55 (1H, d, J = 8.4 Hz),
7.27 (1H, d, J = 8.4 Hz), 2.35 (3H, s); 13C NMR (100 MHz, DMSO-d6) d
174.6, 160.5, 139.3, 137.3, 134.8, 127.0, 116.9, 116.7, 21.3; C9H8ON2S
calcd: 192.0357 [M+H]; found 192.0354.
C17H14ON4BrS calcd: 401.0072 [M+H]; found 401.0073.
5.11. 9-Bromo-2-phenyl-5-(methylsulfanyl)[1,2,4]triazolo[4,3-c]-
quinazolin-3(2H)-one (7b)
5.7. 6-Bromo-2-(methylsulfanyl)quinazolin-4(3H)-one (5a)
Compound 7b was prepared and purified according to the pro-
cedure described for 7a, starting from 10b. The reaction yielded 7b
(76%) as a white solid (mp: 222 °C). 1H NMR (400 MHz, CDCl3) d
To a solution of compound 4a (8.43 g, 32.8 mmol) in 130 mL of
DMF was added a solution sodium methoxide (0.50 M, 32.8 mmol)

J. Nilsson et al. / Bioorg. Med. Chem. 19 (2011) 111–121
117
8.26 (1H, d, J = 1.6 Hz), 8.07 (2H, d, J = 7.9 Hz), 7.69 (1H, dd, J = 8.5
and 1.6 Hz), 7.48 (3H, m), 7.30 (1H, t, J = 7.4 Hz), 2.64 (3H, s); 13C
NMR (100 MHz, DMSO-d6) d 150.9, 147.1, 141.7, 138.6, 137.3,
135.8, 129.3, 129.3, 128.8, 126.6, 125.3, 120.7, 119.5, 119.5,
115.9, 13.6; HRMS (ESI): C16H12ON4BrS calcd: 386.9915 [M+H];
found 386.9912.
(100 MHz, DMSO-d6) d 146.6, 143.7, 139.4, 136,6 135.2, 135.1,
134.7, 129.6, 129.6, 124.5, 118.9, 117.9, 117.9, 114.9, 111.3, 20.5;
HRMS (ESI):
371.0143.
C16H12N4O2Br calcd: 371.0144 [M+H]; found
5.16. 9-Bromo-2-phenyl-2,6-dihydro[1,2,4]triazolo[4,3-c]qui-
nazoline-3,5-dione (8b)
5.12. 9-Bromo-2-(4-chlorophenyl)-5-(methylsulfanyl)[1,2,4]-
triazolo[4,3-c]quinazolin-3(2H)-one (7c)
Compound 8b was prepared and purified according to the pro-
cedure described for 8a, starting from 7b. The reaction yielded 8b
(100%) as a white solid (mp: 405 °C). 1H NMR (400 MHz, DMSO-d6)
d 11.55 (1H, s), 8.06 (1H, d, J = 2.2 Hz), 7.97 (2H, d, J = 7.7 Hz), 7.73
(1H, dd, J = 8.7 and 2.2 Hz), 7.52 (1H, t, J = 8.0 Hz), 7.34 (2H, t,
J = 7.4 Hz), 7.13 (1H, d, J = 8.7 Hz); 13C NMR (100 MHz, DMSO-d6)
d 146.6, 143.6, 139.6, 137.1, 136.6, 135.2, 129.2, 129.2, 125.8,
124.6, 118.8, 118.8, 117.9, 114.9, 111.2; HRMS (ESI): C15H10N4O2Br
calcd: 356.9987 [M+H]; found 356.9993.
Compound 7c was prepared and purified according to the pro-
cedure described for 7a, starting from 10c. The reaction yielded
7c (70%) as a white solid (mp: 247 °C). 1H NMR (400 MHz, CDCl3)
d 8.27 (1H, d, J = 2.2 Hz), 8.06 (2H, dt, J = 9.0 and 2.0 Hz), 7.72
(1H, dd, J = 8.7 and 2.2 Hz), 7.51 (1H, d, J = 8.7 Hz), 7.44 (2H, dt,
J = 9.0 and 2.0 Hz), 2.65 (3H, s); 13C NMR (100 MHz, DMSO-d6) d
150.8, 147.0, 141.8, 138.9, 136.0, 135.9, 132.0, 129.5, 129.5,
128.9, 125.4, 120.8, 120.6, 120.6, 115.8, 13.7; HRMS (ESI):
C16H11ON4BrClS calcd: 420.9525 [M+H]; found 420.9525.
5.17. 9-Bromo-2-(4-chlorophenyl)-2,6-dihydro[1,2,4]triazolo-
[4,3-c]quinazoline-3,5-dione (8c)
5.13. 9-Bromo-2-(2-fluorophenyl)-5-(methylsulfanyl)[1,2,4]-
triazolo[4,3-c]quinazolin-3(2H)-one (7d)
Compound 8c was prepared and purified according to the pro-
cedure described for 8a, starting from 7c. The reaction yielded 8c
Compound 7d was prepared and purified according to the pro-
cedure described for 7a, starting from 10d. The reaction yielded 7d
(51%) as a white solid (mp: 256 °C). NMR (500 MHz, DMSO-d6) d
8.11 (1H, d, J = 2.1 Hz), 7.83 (1H, dd, J = 8.7 and 2.1 Hz), 7.67 (1H,
dt, J = 7.7 and 1.2 Hz), 7.56 (2H, m), 7.43 (1H, t, J = 9.6 Hz), 7.39
(1H, t, J = 7.7 Hz), 2.64 (3H, s); 13C NMR (125 MHz, DMSO-d6) d
155.9 (d, J = 251.4 Hz), 150.5, 146.8, 140.9, 138.8, 135.1, 130.6 (d,
J = 7.9 Hz), 128.3, 127.9, 124.6 (d, J = 3.7 Hz), 123.8, 123.3 6 (d,
J = 11.8 Hz), 119.1, 116.3 (d, J = 19.3 Hz), 115.6, 12.3; HRMS (ESI):
(100%) as a
white solid [mp: 387 °C (decomp.)]. 1H NMR
(400 MHz, DMSO-d6) d 8.07 (1H, br s), 8.02 (2H, d, J = 7.4 Hz),
7.74 (1H, br d, J = 7.8 Hz), 7.59 (2H, d, J = 7.4 Hz), 7.13 (1H, d,
J = 7.8 Hz); 13C NMR (100 MHz, DMSO-d6) d 146.6, 143.6, 139.9,
136.8, 136.0, 135.3, 129.7, 129.2, 129.2, 124.7, 120.2, 120.2,
118.1, 114.9, 111.1; HRMS (ESI): C15H9N4O2BrCl calcd: 390.9597
[M+H]; found 390.9599.
5.18. 9-Bromo-2-(2-fluorophenyl)-2,6-dihydro[1,2,4]triazolo-
C16H11ON4BrFS calcd: 404.9821 [M+H]; found 404.9820.
[4,3-c]quinazoline-3,5-dione (8d)
5.14. 9-Methyl-5-(methylthio)-2-phenyl[1,2,4]triazolo[4,3-c]-
quinazolin-3(2H)-one (7e)
Compound 8d was prepared and purified according to the pro-
cedure described for 8a, starting from 7d. The reaction yielded 8d
(100%) as a white solid [mp: 347 °C (decomp.)]. 1H NMR (400 MHz,
DMSO-d6) d 7.89 (1H, s), 7.63 (2H, m), 7.54 (1H, m), 7.47 (1H, t,
J = 8.7 Hz), 7.38 (1H, t, J = 8.7 Hz), 7.07 (2H, d, J = 8.7 Hz); 13C
NMR (100 MHz, DMSO-d6) d 156.3 (d, J = 250.6 Hz), 147.8, 145.3,
141.7, 141.1, 134.7, 130.7 (d, J = 7.8 Hz), 128.5, 125.0 (d,
J = 3.5 Hz), 124.1, 123.9 (d, J = 11.7 Hz), 120.6, 116.7 (d,
A suspension of 12 (0.25 g, 0.68 mmol) and lithium hydroxide
(0.057 g, 1.35 mmol) in 15 mL of dry THF was stirred at room tem-
perature for 2 h. The mixture was poured onto 100 mL of a satu-
rated solution of aqueous NaHCO3 and extracted twice with
200 mL of EtOAc. The combined organic layers were dried over
Na2SO4 and concentrated under reduced pressure to afford 7e as
a white solid (0.17 g, 0.5 mmol, 78%). Mp >400 °C; 1H NMR
(400 MHz, CDCl3) d 8.09 (2H, dd, J = 8.8 and 1.14 Hz), 7.49 (3H,
m), 7.91 (1H, s), 7.41 (1H, dd, J = 8.3 and 1.6 Hz), 7.29 (1H, t,
J = 7.4 Hz), 2.63 (3H, s), 2.46 (3H, s); 13C NMR (100 MHz, CDCl3) d
149.0, 147.3, 140.9, 139.9, 137.6, 137.5, 134.0, 129.2, 129.2,
126.9, 126.3, 122.3, 119.5, 119.5, 114.1, 21.5, 13.5; HRMS (ESI):
for C17H15N4OS calcd: 323.0967 [M+H]; found 323.0962.
J = 19.1 Hz), 113.0, 111.5; HRMS (ESI):
C15H9N4O2BrF calcd:
374.9893 [M+H]; found 374.9889.
5.19. 9-Methyl-2-phenyl-2,6-dihydro[1,2,4]triazolo[4,3-c]qui-
nazoline-3,5-dione (8e)
Compound 8e was prepared and purified according to the pro-
cedure described for 8a, starting from 7e. The reaction yielded 8e
(100%) as
a
white solid [mp. 330 °C (decomp.)]; 1H NMR
5.15. 9-Bromo-2-(4-methylphenyl)-2,6-dihydro[1,2,4]triazolo-
[4,3-c]quinazoline-3,5-dione (8a)
(500 MHz, DMSO-d6, 323 K) d 7.96 (1H, d, J = 7.7 Hz), 7.77 (1H, s),
7.50 (2H, t, J = 7.7 Hz), 7.35 (3H, m), 7.21 (1H, d, J = 8.36 Hz), 2.33
(3H, s); 13C NMR (125 MHz, DMSO-d6, 323 K) d 146.5, 143.3,
140.3, 136.9, 135.0, 133.0, 132.4, 128.6, 128.6, 125.3, 121.7,
118.7, 118.7, 115.5, 108.6, 19.8; HRMS (ESI): for C16H13N4O2 calcd:
293.1039 [M+H]; found 293.1033.
To a solution of 7a (0.534 g, 1.33 mmol) in 60 mL of CH2Cl2 was
added (77%) mCPBA (0.597 g, 2.67 mmol) and the mixture was stir-
red at rt for 6 h. A white precipitate was slowly formed. Saturated
solutions of aqueous Na2S2O3 (20 mL) and NaHCO3 (20 mL) were
added, subsequently, and the mixture was stirred vigorously for
30 min. The precipitate was filtered off, washed several times with
water and heated in a mixture of 25 mL ethyl alcohol and 25 mL of
water at reflux for 30 min. The mixture was cooled to rt and fil-
tered to give 8a as a white solid (0.488 g, 99%). Mp: 369 °C; 1H
NMR (400 MHz, DMSO-d6) d 11.52 (1H, s), 7.96 (1H, d, J = 2.2 Hz),
7.86 (2H, d, J = 8.4 Hz), 7.58 (1H, dd, J = 8.8 and 2.2 Hz), 7.30 (2H,
d, J = 8.4 Hz), 7.02 (1H, d, J = 8.8 Hz), 2.33 (3H, s); 13C NMR
5.20. Ethyl 1-(4-methylphenyl)hydrazinecarboxylate 10a
N,N-Dimethylformamide (20 mL) was added to a mixture of 4-
iodotoluene (3.0 g, 13.8 mmol), ethyl carbazate (1.72 g,
16.5 mmol), 1,10-phenanthroline (0.50 g, 2.76 mmol), copper(I) io-
dide (0.131 g, 0.69 mmol) and cesium carbonate (6.3 g, 19.3 mmol)
and the mixture was heated under N2 at 80 °C for 18 h. The crude
mixture was concentrated in vacuo and purified by chromatogra-

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J. Nilsson et al. / Bioorg. Med. Chem. 19 (2011) 111–121
phy on a silica gel column. Elution with n-heptane/EtOAc (4:1) as
eluent afforded 10a as a white solid (2.22 g, 83%). Mp. 35 °C, 1H
NMR (400 MHz, C6D6) d 7.53 (2H, d, J = 8.3 Hz), 6.98 (2H, d,
J = 8.3 Hz), 4.00 (2H, q, J = 7.1 Hz), 3.96 (2H, br s), 2.08 (3H, s),
0.94 (3H, t, J = 7.1 Hz), 13C NMR (100 MHz, C6D6) d 155.9, 141.2,
134.0, 129.0, 129.0, 123.4, 123.4, 62.1, 20.8, 14.6; HRMS (ESI):
d 166.7, 161.2, 150.6, 137.5, 137.5, 127.0, 124.9, 121.0, 21.8, 14.5;
HRMS (ESI): for
225.0256.
C10H10N2SCl calcd: 225.0253 [M+H]; found
5.26. Ethyl 2-[6-methyl-2-(methylthio)quinazolin-4(3H)-yli-
dene]-1-phenylhydrazinecarboxylate (12)
C10H15N2O2 calcd: 195.1134 [M+H]; found 195.1129.
N,N-Diisopropylethylamine (0.104 mL, 0.6 mmol) was added to
a solution of 11b (0.071 g, 0.3 mmol) and 10a (0.057 g, 0.3 mmol)
in 5 mL of dioxane and the solution was heated at reflux for 48 h,
during which a white precipitate fell out. The white slurry was fil-
tered and the precipitate was washed with dioxane to give 12 as a
white solid (0.110 g, 100%). Mp. 223 °C; 1H NMR (500 MHz, DMSO-
d6, 323 K) d 8.48 (1H, s), 7.78 (2H, s), 7.59 (2H, d, J = 7.8 Hz), 7.37
(2H, t, J = 7.7 Hz), 7.22 (1H, t, J = 7.4 Hz), 4.19 (2H, q, J = 7.1 Hz),
2.56 (3H, s), 2.46 (3H, s), 1.13 (3H, t, J = 7.1 Hz); 13C NMR
(125 MHz, DMSO-d6, 323 K) d 165.2, 158.3, 153.1, 141.6, 140.8,
136.8, 136.2, 128.2, 125.6, 123.3, 122.8, 120.5, 109.7, 62.0, 20.6,
13.9, 13.1; HRMS (ESI): for C19H21N4O2S calcd: 369.1385 [M+H];
found 369.1380.
5.21. Ethyl 1-phenylhydrazinecarboxylate (10b)
Compound 10b was prepared and purified according to the pro-
cedure described for 10a, starting from iodobenzene. The reaction
yielded 10b (76%) as a white solid (mp: 25 °C). 1H NMR (400 MHz,
C6D6) d 7.66 (2H, br d, J = 7.55 Hz), 7.18 (2H, m), 6.94 (1H, tt, J = 7.4
and 1.1 Hz), 3.99 (2H, q, J = 7.1 Hz), 3.93 (2H, br s), 0.95 (3H, t,
J = 7.1 Hz); 13C NMR (100 MHz, C6D6) d 155.8, 143.6, 128.4, 124.5,
124.5, 123.2, 123.2, 62.2, 14.5; HRMS (ESI): C9H13N2O2 calcd:
181.0977 [M+H]; found 181.0980.
5.22. Ethyl 1-(4-chlorophenyl)-hydrazinecarboxylate (10c)
Compound 10c was prepared and purified according to the pro-
cedure described for 10a, starting from 1-chloro-4-iodobenzene.
The reaction yielded 10c (40%) as a white semisolid. 1H NMR
(400 MHz, C6D6) d 7.43 (2H, d, J = 7.0 Hz), 7.11 (2H, dt, J = 7.0 and
2.2 Hz), 3.92 (2H, q, J = 7.1 Hz), 3.79 (2H, br s), 0.91 (3H, t,
J = 7.1 Hz); 13C NMR (100 MHz, C6D6) d 155.3, 142.1, 129.5, 128.4,
128.4, 124.0, 124.0, 62.3, 14.5; HRMS (ESI): C9H12N2O2Cl calcd:
215.0587 [M+H]; found 215.0586.
5.27. 2-(4-Methylphenyl)-9-phenylethynyl-2,6-dihydro[1,2,4]-
triazolo[4,3-c]-quinazoline-3,5-dione (13)
To a stirred solution of phenylacetylene (15.0
and 8a (25.3 mg, 0.0682 mmol) in a mixture of 2 mL of DMF and
1 mL of NEt3, was added Pd(OAc)2 (1.53 mg, 6.82 mol) and tri-
phenylphosphine (3.57 mg, 13.6 mol) in a seal tube. The tube
lL, 0.137 mmol)
l
l
was sealed under argon and the mixture was stirred at 100 °C for
18 h. The mixture was cooled and concentrated to dryness in vacuo
and the crude product was purified by chromatography on a silica
gel column. Elution with n-heptane/EtOAc (2:1) as eluent yielded
13 as a white solid (14 mg, 70%). Mp: >300 °C (decomp.), 1H NMR
(400 MHz, DMSO-d6) d 11.61 (1H, s), 8.06 (1H, d, J = 1.9 Hz), 7.85
(2H, d, J = 8.5 Hz), 7.70 (1H, dd, J = 8.6 and 1.9 Hz), 7.58 (2H, m),
7.44 (3H, m), 7.31 (2H, d, J = 8.5 Hz), 7.20 (1H, d, J = 8.6 Hz), 2.34
(3H, s); 13C NMR (100 MHz, DMSO-d6) d 146.5, 143.7, 139.7,
137.2, 135.1, 134.7, 131.4, 131.4, 129.5, 129.5, 128.9, 128.8,
128.8, 125.2, 122.0, 118.8, 118.8, 117.0, 116.3, 109.8, 89.5, 88.1,
20.5; HRMS (ESI): for C24H17N4O2 calcd: 393.1352 [M+H]; found:
393.1350.
5.23. Ethyl 1-(2-fluorophenyl)-hydrazinecarboxylate (10d)
Compound 10d was prepared and purified according to the pro-
cedure described for 10a, starting from 1-fluoro-2-iodobenzene.
The reaction yielded 10d (22%) as a white semisolid. 1H NMR
(400 MHz, C6D6) d 7.22 (1H, m), 6.80–6.91 (3H, m). 4.27 (2H, br
s), 4.11 (2H, q, J = 7.1 Hz), 1.12 (3H, t, J = 7.1 Hz); 13C NMR
(100 MHz, C6D6)
d 157.9 (d, J = 248 Hz), 156.8, 131.8 (d,
J = 12 Hz), 128.8, 128.0 (d, J = 23.6 Hz), 124.1 (d, J = 3.8 Hz), 116.2
(d, J = 20.3 Hz), 62.5, 14.5; HRMS (ESI): C9H12N2O2F calcd:
199.0885 [M+H]; found 199.0888.
5.24. 6-Bromo-4-chloro-2-(methylsulfanyl)quinazoline (11a)
5.28. 2-(4-Methylphenyl)-9-(3-thienylethynyl)-2,6-dihydro-
[1,2,4]triazolo[4,3-c]-quinazoline-3,5-dione (14)
To a suspension of compound 5a (0.65 g, 2.4 mmol) in 6.2 mL of
POCl3 was added 10
l
L of pyridine and the mixture was heated at
Compound 14 was prepared and purified according to the pro-
cedure described for 13, starting from 3-ethynylthiophene and 8a.
The reaction yielded 14 (40%) as a white solid (mp: >320 °C, de-
comp). 1H NMR (400 MHz, DMSO-d6) d 11.60 (1H, s), 8.06 (1H, d,
J = 1.8 Hz), 7.93 (1H, dd, J = 2.9 and 1.2 Hz), 7.87 (2H, d,
J = 8.4 Hz), 7.67 (2H, m), 7.34 (2H, d, J = 8.4 Hz), 7.30 (1H, dd,
J = 5.0 and 1.2 Hz), 7.21 (1H, d, J = 8.5 Hz), 2.35 (3H, s); 13C NMR
(100 MHz, DMSO-d6) d 146.6, 143.7, 139.7, 137.2, 135.1, 134.9,
134.8, 130.2, 129.6, 129.6, 129.6, 127.0, 125.0, 120.9, 118.8,
118.8, 117.1, 116.3, 109.8, 87.4, 85.1, 20.5; HRMS (ESI): for
110 °C for 18 h. The reaction mixture was cooled to rt and concen-
trated under reduced pressure. A saturated solution of 50 mL of
aqueous NaHCO3 was added to the crude solid and the mixture
was extracted with 50 mL of EtOAc. The organic layer was dried
over MgSO4, concentrated under reduced pressure and purified
by chromatography on a silica gel column. Elution with n-hep-
tane/EtOAc (4:1) as eluent afforded 11a as a white solid (0.52 g,
75%). Mp: 124 °C, 1H NMR (400 MHz, DMSO-d6) d 8.29 (1H,
J = 2.2 Hz), 7.92 (1H, dd, J = 8.9 and 2.2 Hz), 7.73 (1H, J = 8.9 Hz),
2.66 (3H, s); 13C NMR (100 MHz, DMSO-d6) d 168.5, 160.7, 150.6,
138.9, 129.0, 128.4, 122.2, 120.7, 14.6; HRMS (FAB+): C9H7N2ClBrS
calcd: 288.9202 [M+H]; found 288.9200.
C22H15N4O2S calcd: 399.0916 [M+H]; found: 399.0920.
5.29. 2-(4-Methylphenyl)-9-(pyridin-2-ylethynyl)-2,6-dihydro-
[1,2,4]triazolo[4,3-c]-quinazoline-3,5-dione (15)
5.25. 4-Chloro-6-methyl-2-(methylsulfanyl)quinazoline (11b)
Compound 15 was prepared and purified according to the pro-
cedure described for 13, starting from 2-ethynylpyridine and 8a.
The reaction yielded 15 (75%) as a white solid (mp: 330 °C). 1H
NMR (400 MHz, DMSO-d6) d 11.65 (1H, s), 8.63 (1H, ddd, J = 4.9,
1.8 and 1.0 Hz), 8.14 (1H, br d, J = 1.9 Hz), 7.86 (3H, m), 7.77 (1H,
dd, J = 8.5 and 1.9 Hz), 7.69 (1H, dt, J = 7.8 and 1.0 Hz), 7.43 (1H,
Compound 11b was prepared and purified according to the pro-
cedure described for 11a, starting from 5b. The reaction yielded
11b (77%) as a white solid (mp: 205 °C, decomp). 1H NMR
(400 MHz, CDCl3) d 7.86 (1H, s), 7.73 (1H, d, J = 8.6 Hz), 7.66 (1H,
d, J = 8.6 Hz), 2.65 (3H, s), 2.55 (3H, s); 13C NMR (100 MHz, CDCl3)

J. Nilsson et al. / Bioorg. Med. Chem. 19 (2011) 111–121
119
ddd, J = 7.6, 4.9 and 1.2 Hz), 7.33 (2H, d, J = 8.4 Hz), 7.24 (1H, d,
J = 8.5 Hz), 2.34 (3H, s); 13C NMR (100 MHz, DMSO-d6) d 150.2,
146.6, 143.7, 142.1, 139.7, 137.8, 136.8, 135.4, 135.1, 134.8,
129.6, 129.6, 127.4, 125.7, 125.7, 123.6, 118.8, 116.4, 116.1,
(100 MHz, DMSO-d6, 373 K) d 146.1, 143.0, 140.1, 139.2, 138.8,
136.9, 134.8, 134.5, 134.4, 131.4, 131.4, 128.9, 128.9, 128.4,
128.4, 127.2, 126.3, 126.3, 126.1, 126.1, 124.7, 120.8, 118.8,
118.8, 117.0, 115.9, 109.3, 89.1, 88.4, 19.8; HRMS (ESI): for
109.9, 89.2, 87.2, 20.5; HRMS (ESI): for
394.1304 [M+H]; found: 394.1310.
C
23H16N5O2 calcd:
C30H21N4O2 calcd: 469.1665 [M+H]; found: 469.1665.
5.34. 9-(3-Hydroxyphenyl)ethynyl-2-(4-methylphenyl)-2,6-
dihydro[1,2,4]triazolo[4,3-c]-quinazoline-3,5-dione (20)
5.30. 2-(4-Methylphenyl)-9-(pyridin-3-ylethynyl)-2,6-dihydro-
[1,2,4]triazolo[4,3-c]-quinazoline-3,5-dione (16)
Compound 20 was prepared and purified according to the pro-
cedure described for 13, starting from 3-tert-butyldimethylsilyloxy
phenylacetylene and 8a.29 The reaction yielded 20 (32%) as a white
solid [mp: 300 °C (decomp.)]. 1H NMR (400 MHz, DMSO-d6) d 11.60
(1H, s), 9.72 (1H, s), 8.05 (1H, s), 7.86 (2H, d, J = 8.0 Hz), 7.70 (1H, d,
J = 8.3 Hz), 7.32 (2H, d, J = 8.0 Hz), 7.22 (2H, m), 7.00 (1H, d,
J = 7.5 Hz), 6.93 (1H, s), 6.84 (1H, d, J = 7.8 Hz), 2.34 (3H, s); 13C
NMR (100 MHz, DMSO-d6) d 157.4, 146.6, 143.7, 139.7, 137.2,
135.1, 135.1, 134.8, 129.9, 129.5, 129.5, 125.2, 122.9, 122.3,
118.8, 118.8, 117.8, 117.1, 116.4, 116.3, 109.8, 20.5; HRMS (ESI):
for C24H17N4O3 calcd: 409.1301 [M+H]; found: 409.1299.
Compound 16 was prepared and purified according to the pro-
cedure described for 13, starting from 3-ethynylpyridine and 8a.
The reaction yielded 16 (50%) as a white solid (mp: 339 °C). 1H
NMR (400 MHz, DMSO-d6) d 11.62 (1H, s), 8.76 (1H, br s), 8.59
(1H, dd, J = 4.9 and 1.5 Hz), 8.06 (1H, d, J = 1.7 Hz), 7.97 (1H, dt,
J = 7.8 and 1.5 Hz), 7.83 (2H, d, J = 8.4 Hz), 7.69 (1H, dd, J = 8.5
and 1.7 Hz), 7.46 (1H, dd, J = 7.8 and 4.9 Hz), 7.29 (2H, d,
J = 8.4 Hz), 7.19 (1H, d, 8.5), 2.32 (3H, s); 13C NMR (100 MHz,
DMSO-d6) d 151.7, 149.0, 146.4, 143.6, 139.6, 139.6, 138.5, 137.5,
135.1, 134.7, 129.5, 129.5, 125.4, 123.6, 119.2, 118.7, 118.7,
116.4, 116.3, 109.7, 91.1, 86.4, 20.5; HRMS (ESI): for C23H16N5O2
calcd: 394.1304 [M+H]; found: 394.1305.
5.35. 2-(4-Methylphenyl)-9-(2-phenylethyl)-2,6-dihydro[1,2,4]-
triazolo[4,3-c]-quinazoline-3,5-dione (21)
5.31. 2-(4-Methylphenyl)-9-(pyridin-4-ylethynyl)-2,6-dihydro-
[1,2,4]triazolo[4,3-c]-quinazoline-3,5-dione (17)
A catalytic amount of 10% Pd/C and 13 (20.0 mg, 0.0510 mmol)
in a mixture of 2 mL of dichloromethane and 2 mL of methyl alco-
hol was stirred during 12 h under H2 at atmospheric pressure. The
mixture was filtered through Celite, washed with additionally
methyl alcohol and triturated from ethyl alcohol to afford 21
(16 mg, 0.0404 mmol, 79%) as a white solid. Mp: 303 °C; 1H NMR
(400 MHz, DMSO-d6) d 11.34 (1H, s), 7.85 (2H, d, J = 8.4 Hz), 7.83
(1H, d, J = 1.7 Hz), 7.43 (1H, dd, J = 8.4 and 1.7 Hz), 7.33 (2H, d,
J = 8.4 Hz), 7.27 (3H, m), 7.18 (1H, m), 7.10 (1H, d, J = 8.4 Hz),
2.92 (4H, m), 2.35 (3H, s); 13C NMR (100 MHz, DMSO-d6) d 146.7,
143.9, 141.2, 140.5, 136.9, 135.4, 135.0, 134.8, 133.0, 129.5,
129.5, 128.4, 128.4, 128.2, 128.2, 125.9, 121.9, 118.8, 118.8,
115.6, 109.0, 37.0, 36.2, 20.5; HRMS (ESI): for C24H21N4O2 calcd:
397.1665 [M+H]; found: 397.1669.
Compound 17 was prepared and purified according to the pro-
cedure described for 13, starting from 4-ethynylpyridine hydro-
chloride and 8a. The reaction yielded 17 (39%) as a white solid
(mp: 330 °C). 1H NMR (400 MHz, DMSO-d6) d 11.66 (1H, s), 8.63
(2H, d, J = 5.9 Hz), 8.15 (1H, d, J = 1.7 Hz), 7.85 (2H, d, J = 8.4 Hz),
7.76 (1H, dd, J = 8.5 and 1.8 Hz), 7.55 (2H, d, J = 5.9 Hz), 7.32 (2H,
d, J = 8.4 Hz), 7.23 (1H, d, J = 8.5 Hz), 2.34 (3H, s); 13C NMR
(100 MHz, DMSO-d6) d 149.9, 149.9, 146.5, 143.6, 139.6, 137.9,
135.3, 135.1, 134.7, 130.0, 129.5, 129.5, 125.8, 125.3, 125.3,
118.7, 118.7, 116.4, 115.9, 109.9, 92.4, 86.9, 20.5; HRMS (ESI): for
HRMS (ESI): for
394.1298.
C23H16N5O2 calcd: 394.1304 [M+H]; found:
5.32. 2-(4-Methylphenyl)-9-[(4-phenoxyphenyl)ethynyl]-2,6-
dihydro[1,2,4] triazolo[4,3-c]-quinazoline-3,5-dione (18)
5.36. 2-(4-Methylphenyl)-9-[2-(3-thienyl)ethyl]-2,6-dihydro-
[1,2,4]triazolo[4,3-c]-quinazoline-3,5-dione (22)
Compound 18 was prepared and purified according to the pro-
cedure described for 13, starting from 1-ethynyl-4-phenoxyben-
zene and 8a. The reaction yielded 18 (60%) as a white solid (mp:
216 °C). 1H NMR (400 MHz, DMSO-d6) d 11.60 (1H, s), 8.06 (1H,
d, J = 1.9 Hz), 7.85 (2H, d, J = 8.5 Hz), 7.69 (1H, dd, J = 8.5 and
1.9 Hz), 7.59 (2H, dt, J = 8.8 and 1.9 Hz), 7.44 (2H, m), 7.32 (2H, d,
J = 8.3 Hz), 7.20 (2H, m), 7.09 (2H, dd, J = 8.7 and 1.1 Hz), 7.02
(2H, dt, J = 8.8 and 1.9 Hz), 2.34 (3H, s); 13C NMR (100 MHz,
DMSO-d6) d 157.5, 155.6, 146.6, 143.7, 139.8, 137.1, 135.1, 135.0,
134.8, 133.4, 133.4, 130.3, 130.3, 129.6, 129.6, 125.1, 124.3,
119.5, 119.5, 118.8, 118.8, 118.2, 118.2, 117.2, 116.6, 116.3,
Compound 22 was prepared and purified according to the pro-
cedure described for 21, starting from 14. The reaction yielded 22
(88%) as a white solid (mp: 281 °C).1H NMR (400 MHz, DMSO-d6) d
11.40 (1H, s), 7.85 (3H, m), 7.44 (2H, m), 7.32 (2H, d, J = 7.9 Hz),
7.18 (1H, s), 7.12 (1H, d, J = 8.3 Hz), 7.05 (1H, d, J = 4.34 Hz); 13C
NMR (100 MHz, DMSO-d6) d 146.7, 143.9, 141.6, 140.5, 136.9,
135.4, 135.0, 134.8, 132.9, 129.5, 129.5, 128.4, 125.8, 121.8,
120.8, 118.8, 118.8, 115.6, 109.0, 35.3, 31.4, 20.5; HRMS (ESI): for
C22H19N4O2S calcd: 403.1229 [M+H]; found: 402.1226.
5.37. 2-(4-Methylphenyl)-9-[2-(2-pyridyl)ethyl]-2,6-dihydro-
1,2,4]triazolo[4,3-c]-quinazoline-3,5-dione (23)
109.8, 89.2, 87.5, 20.5; HRMS (ESI): for
485.1614 [M+H]; found: 485.1614.
C30H21N4O3 calcd:
Compound 23 was prepared and purified according to the pro-
cedure described for 21, starting from 15. The reaction yielded 23
(85%) as a white solid (mp: >300 °C, decomp). 1H NMR (400 MHz,
DMSO-d6) d 11.39 (1H, s), 8.58 (1H, d, J = 4.42 Hz), 7.84 (4H, m),
7.43 (2H, m), 7.34 (3H, m), 7.11 (1H, d, J = 8.4 Hz), 3.10 (4H, m),
2.34 (3H, s); 13C NMR (100 MHz, DMSO-d6) d 159.4, 147.4, 146.7,
143.8, 140.4, 138.5, 136.5, 135.5, 135.0, 134.8, 132.9, 129.5,
129.5, 123.8, 122.1, 121.9, 118.8, 118.8, 115.7, 109.1, 39.1, 34.0,
20.5; HRMS (ESI): for C23H20N5O2 calcd: 398.1617 [M+H]; found:
398.1621.
5.33. 9-(Biphen-4-ylethynyl)-2-(4-methylphenyl)-2,6-dihydro-
[1,2,4]triazolo[4,3-c]-quinazoline-3,5-dione (19)
Compound 19 was prepared and purified according to the pro-
cedure described for 13, starting from 4-ethynylbiphenyl and 8a.
The reaction yielded 19 (69%) as a white solid (mp: >350 °C, de-
comp.). 1H NMR (400 MHz, DMSO-d6, 373 K) d 11.30 (1H, s), 8.12
(1H, br s), 7.86 (2H, d, J = 8.2 Hz), 7.72 (5H, m), 7.67 (2H, dt,
J = 8.2 Hz), 7.49 (2H, t, J = 7.6 Hz), 7.40 (1H, d, J = 7.4 Hz), 7.33
(2H, d, J = 8.1 Hz), 7.28 (2H, d, J = 8.5 Hz), 2.38 (3H, s); 13C NMR

120
J. Nilsson et al. / Bioorg. Med. Chem. 19 (2011) 111–121
5.38. 2-(4-Methylphenyl)-9-[2-(3-pyridyl)ethyl]-2,6-dihydro-
[1,2,4]triazolo[4,3-c]-quinazoline-3,5-dione (24)
J = 8.4 Hz), 7.03 (1H, dd, J = 7.5 and 8.0 Hz), 6.65 (2H, m), 6.59 (1H,
br d, J = 7.0 Hz), 2.84 (4H, m), 2.33 (3H, s); 13C NMR (100 MHz,
DMSO-d6) d 157.5, 146.8, 143.9, 142.6, 140.6, 137.0, 135.1, 134.9,
133.0, 129.6, 129.6, 129.2, 126.9, 121.8, 119.1, 119.0, 119.0,
115.8, 115.5, 113.0, 109.1, 37.1, 36.2, 20.6; HRMS (ESI): for
Compound 24 was prepared and purified according to the pro-
cedure described for 21, starting from 16. The reaction yielded 24
(91%) as a white solid (mp: 292 °C). 1H NMR (400 MHz, DMSO-d6) d
11.40 (1H, s), 8.57 (1H, s), 8.50 (1H, br s), 7.91 (1H, d, J = 7.8 Hz),
7.82 (3H, m), 7.50 (1H, dd, J = 7.8 and 5.0 Hz), 7.41 (1H, dd, J = 8.4
and 1.7 Hz), 7.30 (2H, d, J = 8.4 Hz), 7.11 (1H, d, J = 8.4 Hz), 2.97
(4H, m), 2.32 (3H, s); 13C NMR (100 MHz, DMSO-d6) d 147.3,
146.7, 145.0, 143.8, 140.4, 138.9, 137.9, 136.2, 135.5, 135.0,
134.8, 132.9, 129.5, 129.5, 124.4, 121.9, 118.8, 118.8, 115.7,
C24H21N4O3 calcd: 413.1614 [M+H]; found: 413.1619.
5.43. 9-Benzyl-2-(4-methylphenyl)-2,6-dihydro[1,2,4]triazolo-
[4,3-c]quinazoline-3,5-dione (29)
To a stirred solution of benzyltributyltin (69 mg, 0.184 mmol)
and 8a (19.5 mg, 0.0525 mmol) in a mixture of 2 mL of DMF and
109.0, 35.5, 33.7, 20.5; HRMS (ESI): for
398.1617 [M+H]; found: 398.1620.
C
23H20N5O2 calcd:
1 mL of NEt3, was added Pd(PPh3)4 (6.0 mg, 5.3 lmol). The seal
tube was sealed under argon and the mixture was stirred at
110 °C for 18 h.26 The reaction the mixture was cooled and concen-
trated to dryness in vacuo and the crude product was purified by
chromatography on a silica gel column. Elution with n-heptane/
EtOAc (2:1) as eluent yielded 29 as a white solid (13 mg, 65%).
Mp: 299 °C, 1H NMR (500 MHz, DMSO-d6, 340 K) d 7.82 (3H, m),
7.43 (1H, d, J = 8.0 Hz), 7.29 (5H, m), 7.20 (1H, m), 7.15 (1H, d,
J = 8.0 Hz), 4.01 (2H, s), 2.34 (3H, s); 13C NMR (125 MHz, DMSO-
d6, 340 K) d 146.4, 143.4, 140.5, 140.1, 136.3, 135.3, 134.8, 134.6,
132.8, 129.1, 129.1, 128.3, 128.3, 128.1, 128.1, 125.8, 121.8,
118.9, 118.9, 115.6, 108.9, 39.9, 20.1; HRMS (ESI): for C23H19N4O2
calcd: 383.1508 [M+H]; found: 383.1512.
5.39. 2-(4-Methylphenyl)-9-[2-(4-pyridyl)ethyl]-2,6-dihydro-
[1,2,4]triazolo[4,3-c]-quinazoline-3,5-dione (25)
Compound 25 was prepared and purified according to the pro-
cedure described for 21, starting from 17. The reaction yielded 25
(91%) as a white solid (mp: 294 °C). 1H NMR (400 MHz, DMSO-d6) d
11.37 (1H, s), 8.47 (1H, d, J = 6.0 Hz), 7.85 (3H, m), 7.45 (1H, dd,
J = 8.4 and 1.9 Hz), 7.34 (4H, m), 7.11 (1H, d, J = 8.4 Hz), 2.97 (4H,
m), 2.35 (3H, s); 13C NMR (100 MHz, DMSO-d6) d 151.0, 148.8,
148.8, 146.7, 143.8, 140.4, 136.2, 135.5, 135.0, 134.8, 132.9,
129.5, 129.5, 124.3, 124.3, 121.8, 118.8, 118.8, 115.7, 109.0, 36.0,
34.8, 20.5; HRMS (ESI): for C23H20N5O2 calcd: 398.1617 [M+H];
found: 398.1615.
5.44. 9-(3-Fluorobenzyl)-2-(4-methylphenyl)-2,6-dihydro-
[1,2,4]triazolo[4,3-c] quinazoline-3,5-dione (30)
5.40. 2-(4-Methylphenyl)-9-[2-(4-phenoxyphenyl)ethyl]-2,6-
dihydro[1,2,4]triazolo[4,3-c]-quinazoline-3,5-dione (26)
Compound 30 was prepared and purified according to the pro-
cedure described for 29, starting from 8a and (3-fluorobenzyl)tri-
butyltin.26 The reaction yielded 30 (70%) as a white solid (mp:
324 °C). 1H NMR (400 MHz, DMSO-d6) d 11.37 (1H, s), 7.86 (1H,
s), 7.83 (2H, d, J = 8.2 Hz), 7.46 (d, J = 8.2 Hz), 7.33 (3H, m), 7.13
(3H, m), 7.02 (1H, t, J = 8.3 Hz), 4.04 (2H, s), 2.34 (3H, s); 13C
NMR (100 MHz, DMSO-d6) d 162.2 (d, J = 243.5 Hz), 146.7, 144.0
(d, J = 7.3 Hz), 143.9, 140.4, 136.1, 135.7, 135.0, 134.8, 133.2,
130.4. (d, J = 8.4 Hz), 129.5, 129.5, 124.8 (d, J = 2.6 Hz), 122.2,
118.9, 118.9, 116.0, 115.4 (d, J = 21.1 Hz), 112.9 (d, J = 20.9 Hz),
109.3, 40.4, 20.5; HRMS (ESI): for C23H18N4O2F calcd: 401.1414
[M+H]; found: 401.1417.
Compound 26 was prepared and purified according to the pro-
cedure described for 21, starting from 18. The reaction yielded 26
(80%) as a white solid (mp: 213 °C). 1H NMR (400 MHz, DMSO-d6) d
11.40 (1H, s), 7.83 (3H, m), 7.44 (1H, d, J = 8.5 Hz), 7.33 (3H, m),
7.27 (2H, d, J = 8.2 Hz), 7.11 (3H, m), 6.94 (4H, m), 2.35 (3H, s);
13C NMR (100 MHz, DMSO-d6) d 157.1, 154.6, 146.8, 143.9, 140.5,
136.9, 136.5, 135.4, 135.0, 134.8, 133.0, 130.0, 130.0, 130.0,
130.0, 129.5, 129.5, 123.1, 121.9, 118.9, 118.9, 118.8, 118.8,
118.2, 118.2, 115.7, 109.0, 36.4, 36.3, 20.5; HRMS (ESI): for
C30H25N4O3 calcd: 489.1927 [M+H]; found: 489.1929.
5.45. 2-(4-Methylphenyl)-9-(3-thienylmethyl)-2,6-dihydro-
5.41. 9-[2-(Biphen-4-yl)ethyl)]-2-(4-methylphenyl)-2,6-
[1,2,4]triazolo[4,3c] quinazoline-3,5-dione (31)
dihydro[1,2,4]triazolo[4,3-c]-quinazoline-3,5-dione (27)
Compound 31 was prepared and purified according to the pro-
cedure described for 29, starting from 8a and 33. The reaction
yielded 31 (62%) as
(400 MHz, DMSO-d6) d 11.37 (1H, s), 7.82 (3H, m), 7.46 (2H, m),
7.32 (2H, d, J = 8.3 Hz), 7.25 (1H, br s), 7.12 (1H, d, J = 8.3 Hz),
6.98 (1H, d, J = 4.5 Hz), 4.01 (2H, s), 2.34 (3H, s); 13C NMR
(100 MHz, DMSO-d6) d 146.7, 143.9, 141.1, 140.5, 136.3, 135.5,
135.0, 134.8, 133.1, 129.5, 129.5, 128.4, 126.4, 122.0, 121.6,
121.6, 118.9, 115.9, 109.2, 34.8, 20.5; HRMS (ESI): for C21H17N4O2S
calcd: 389.1072 [M+H]; found: 389.1074.
Compound 27 was prepared and purified according to the pro-
cedure described for 21, starting from 19. The reaction yielded 27
(98%) as a white solid (mp: 345 °C). 1H NMR (500 MHz, DMSO-d6) d
11.07 (1H, s), 7.82 (3H, m), 7.62 (2H, d, J = 6.3 Hz), 7.56 (2H, d,
J = 6.5 Hz), 7.43 (3H, m), 7.31 (5H, m), 7.18 (1H, d, J = 7.9 Hz),
3.01 (4H, m), 2.36 (3H, s); 13C NMR (125 MHz, DMSO-d6) d 146.2,
143.1, 139.9, 139.8, 139.7, 137.5, 136.4, 135.0, 134.6, 134.5,
132.3, 128.8, 128.8, 128.3, 128.3, 128.1, 128.1, 126.4, 125.9,
125.9, 125.8, 125.8, 121.3, 118.8, 118.8, 115.2, 108.5, 35.7, 35.3,
19.8; HRMS (ESI): for C30H25N4O2 calcd: 473.1978 [M+H]; found:
473.1981.
a
white solid (mp: 287 °C). 1H NMR
5.46. Tributyl-(methyl-3-thienyl)tin (33)
5.42. 9-[2-(3-Hydroxyphenyl)ethyl]-2-(4-methylphenyl)-2,6-
dihydro[1,2,4]triazolo[4,3-c]-quinazoline-3,5-dione (28)
A solution of 32 (620 mg, 3.5 mmol) in 3 mL of tetrahydrofuran
were added to magnesium turnings (170 mg, 7.0 mmol) and the
mixture was heated at 35 °C for 1 h.25,27 The mixture was cooled
Compound 28 was prepared and purified according to the pro-
cedure described for 21, starting from 20. The reaction yielded 28
(96%) as a white solid [mp: 300 °C (decomp.)]. 1H NMR (400 MHz,
DMSO-d6) d 11.48 (1H, s), 9.41 (1H, s), 7.83 (2H, d, J = 8.3 Hz), 7.79
(1H, s), 7.41 (1H, d, J = 8.4 Hz), 7.31 (2H, d, J = 8.3 Hz), 7.18 (1H, d,
to room temperature and a solution of tributyltin chloride
(0.760 mL, 2.8 mmol) in 3 mL of tetrahydrofuran was added to
the Grignard solution. The mixture was stirred at rt for 1 h and
was then poured onto a saturated aqueous NH4Cl solution and
the mixture was extracted twice with EtOAc. The organic layers

J. Nilsson et al. / Bioorg. Med. Chem. 19 (2011) 111–121
121
were washed with brine, dried over MgSO4, concentrated and puri-
fied by chromatography on a silica gel column. Elution with n-hep-
tane/EtOAc (100:1) as eluent yielded 33 as a clear oil (490 mg,
36%). 1H NMR (400 MHz, CDCl3) d 7.13 (1H, dd, J = 4.9 and
2.95 Hz), 6.77 (1H, dd, J = 4.9 and 1.33 Hz), 6.62 (1H, m), 2.33
(2H, s), 1.44 (6H, m), 1.27 (6H, m), 0.88 (9H, m), 0.83 (6H, m),
13C NMR (100 MHz, CDCl3) d 142.9, 128.7, 125.1, 115.8, 29.2,
27.5, 17.7, 13.9, 9.6.
bated at an ice-bath (0–4 °C) for 40 min. The incubated samples
were added 5 mL ice-cold buffer (Tris–citrate, 50 mM pH 7.1),
poured directly onto Whatman GF/C glass fiber filters under suc-
tion and immediately washed with 5 mL ice-cold buffer. Non-
specific binding was determined by adding Clonazepam (1 lM fi-
nal concentration) to separate samples. Protein was estimated by
conventional protein assay method using Bovine serum albumin
as standard.
IC50 values were determined using 4–6 different concentrations
5.47. Benzyl (2E)-3-[2-(4-methylphenyl)-3,5-dioxo-2,6-dihydro-
[1,2,4]triazolo[4,3-c]quinazolin-9-yl]acrylate (35)
of test substance. Ki values were calculated according to Ki = IC50/
(1 + (L)/KD), (L) is the concentration (nM) of 3H-Flumazenil; KD is
binding affinity constant of 3H-Flumazenil (1.6 nM).
To a stirred solution of benzyl acrylate (44
8a (50.6 mg, 0.137 mmol) in a mixture of 8 mL of DMF and 4 mL of
NEt3, was added Pd(OAc)2 (3.06 mg, 13.7 mol) and triphenylphos-
phine (7.14 mg, 27.4 mol) in a sealed tube. The tube was sealed
lg, 0.274 mmol) and
Acknowledgments
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Financial support from the Swedish board for Scientific research
(V.R.), the Knut and Alice Wallenberg Foundation, the research
School for Pharmaceutical Sciences at Lund University, Carlsberg
Foundation, Denmark, and the NeuroScience PharmaBiotec
Research Center, Denmark, are gratefully acknowledged.
under argon and the mixture was stirred at 120 °C for 15 h. The
mixture was cooled and concentrated to dryness in vacuo and
the crude product was precipitated from acetone to give 35 as a
white solid (29 mg, 47%). Mp: 242 °C. 1H NMR (400 MHz, DMSO-
d6) d 11.58 (1H, br s), 8.29 (1H, s), 7.95 (1H, br d, J = 8.5 Hz), 7.87
(2H, d, J = 8.3 Hz), 7.78 (1H, d, J = 16.0 Hz), 7.35–7.46 (5H, m),
7.32 (2H, d, J = 8.3 Hz), 7.19 (1H, d, J = 8.5 Hz), 6.74 (1H, d,
J = 16.0 Hz), 5.23 (2H, s,), 2.35 (3H, s); 13C NMR (100 MHz,
DMSO-d6) d 166.0, 146.7, 144.0, 143.5, 140.2, 139.5, 136.2, 135.0,
134.8, 131.7, 131.5, 129.5, 129.5, 129.0, 128.5, 128.5, 128.1,
128.1, 123.2, 118.8, 118.8, 117.5, 116.7, 109.8, 65.6, 20.5; HRMS
(ESI): for C26H21N4O4 calcd: 453.1563 [M+H]; found: 453.1559.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.11.050.
References and notes
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Compound 36 was prepared and purified according to the pro-
cedure described for 21, starting from 35. The reaction yielded 36
(71%) as a white solid (mp: 275 °C). 1H NMR (400 MHz, DMSO-d6) d
11.43 (1H, br s), 7.85 (2H, d, J = 8.4 Hz), 7.82 (1H, br s), 7.44 (1H, br
d, J = 8.2 Hz), 7.32 (2H, d, J = 8.4 Hz), 7.14 (1H, d, J = 8.2 Hz), 3.58
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118.9, 115.8, 109.1, 34.7, 29.5, 20.5; HRMS (ESI): for C19H17N4O4
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human
a1b3c2, a2b3c2, a3b3c2, or a5b3c2 GABAA receptors was
done following methods previously described by Kahnberg
et al.28 In brief: Tissue is homogenized in 20 mL Tris–HCl
(30 mM, pH 7.4) using an Ultra-Turrax homogenizer. The suspen-
sions are centrifuged at 27,000g for 15 min followed by three cen-
trifugations resuspension cycles. The washed pellet is resuspended
in 20 mL buffer, incubated at 37 °C for 30 min and then centrifuged
for 10 min (27,000g). The pellet is washed once and the final pellet
is resuspended in 30 mL Tris–HCl buffer (50 mM, pH 7.1) and
stored at -20 °C until use. For binding studies frozen membrane
suspensions were thawed and centrifuged (27,000g, 10 min). The
pellet was resuspended into Tris–citrate buffer (50 mM, pH 7.1)
at a tissue concentration: cortex preparation ca. 50
0.55 mL assay (1 mg original tissue/0.55 mL assay) and HEK cells
ca. 25 g protein per 0.55 mL assay. Aliquots of 0.5 mL membrane
preparation are added to 25
L of 3H-Flumazenil solution (1 nM
final concentration) and 25 L containing test substance and incu-
lg protein/
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