Derivatives of 4,6-diamino-1,2-dihydro-2-phenyl-1,2,4-triazolo[4,3-a] quinoxalin-2H-1-one: Potential antagonist ligands for imaging the A2A adenosine receptor by positron emission tomography (PET)

Article abstract of DOI:10.1016/j.ejmech.2004.12.005

The importance of the brain A_2A adenosine receptor (A _2AAR) in movement disorders urges the development of radiolabeled ligands for imaging those receptors by positron emission tomography (PET). This study evaluated one class of A

Full text of DOI:10.1016/j.ejmech.2004.12.005

European Journal of Medicinal Chemistry 40 (2005) 421–437
www.elsevier.com/locate/ejmech
Original article
Derivatives of 4,6-diamino-1,2-dihydro-2-phenyl-
1,2,4-triazolo[4,3-a]quinoxalin-2H-1-one: potential antagonist ligands for
imaging the A2A adenosine receptor by positron emission tomography
(PET)
Marcus H. Holschbach a, Dirk Bier a, Walter Wutz a, Wiebke Sihver a,
M. Schüller a, Ray A. Olsson a,b,
*
a Institut für Nuklearchemie, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany
b Department of Internal Medicine, MDC Box 19, University of South Florida, Tampa, FL 33612-4799, USA
Received 30 November 2004; received in revised form 20 December 2004; accepted 21 December 2004
Available online 25 February 2005
Abstract
The importance of the brain A2A adenosine receptor (A2AAR) in movement disorders urges the development of radiolabeled ligands for
imaging those receptors by positron emission tomography (PET). This study evaluated one class ofA2AAR antagonists, derivatives of 4-amino-
6-benzylamino-1,2-dihydro-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-2H-1-one, 10a, as agents for imaging brain A2AARs by PET.. Modifi-
cations of a literature synthesis of 10a efficiently generated analogs 10b–s for pharmacological evaluation. Radioligand binding experiments
showed affinities for the rat brain A2AAR in the low nanomolar range but similar affinities for the A1AR and substantial unspecific binding.
Autoradiography employing [3H]10a, showing that high unspecific binding obscured specific binding to both the A1AR and A2AAR. Thus,
compounds 10b–s are unsuitable as ligands for imaging brain A2AARs by PET.
© 2005 Elsevier SAS. All rights reserved.
Keywords: PET; A2A adenosine receptor antagonists; Triazolo[4,3-a]quinoxoline; Radioligands
1. Introduction
carbon-11. The appreciably longer physical half-life of fluo-
rine-18 over that of carbon-11, 110 vs. 20 min, allows
extended syntheses and imaging protocols and obviates the
need to carry out imaging close to a cyclotron. The decay of
fluorine-18 produces positrons having the lowest energy
—and, thereby, the shortest range in tissue and greatest spa-
tial resolution—of all the medically useful positron emitters.
These reasons have guided our program for the synthesis and
evaluation of A2AAR antagonists that might be suitable for
no-carrier-added (n.c.a.) radiofluorination and brain imag-
ing.
Several potent, highly selective antagonists for the A2A
adenosine receptor (A2AAR) have appeared in the past decade
[1–7]. In addition to their potential as drugs [8], those antago-
nists, if radiolabeled, could be useful ligands for medical
imaging by means of positron emission tomography (PET).
Indeed, labeling of several A2AAR antagonists with carbon-
11 has provided promising radioligands for PET [9–12].
Labeling with fluorine-18 offers some advantages over
Of the available A2AAR antagonists, SCH 58261 (Fig. 1)
has many desirable pharmacological properties [13], and a
carbon 11-labeled congener has been developed for PET
[14,15]. However, neither that compound nor its congeners
are particularly well suited for our purposes because the nitro-
gen heterocycle is difficult to prepare, n.c.a. radiofluorina-
tion would necessarily occur several steps before the end of
Abbreviations: CPDPX, 8-cyclopentyl-3,4,5,6-tetrahydro-1,3-dipropyl-
1H-purine-2,6-dione; ZM241385, 4-[2-(7-amino-2-(furan-2-yl)triazolo[1,5-
a][1,3,5]triazin-5-ylamino)ethyl)phenol; CGS21680, 4-[2-[[6-amino-9-(N-
ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-
yl]amino]ethyl]benzenepropanoic acid.
* Corresponding author. Tel.: +1 813 974 4067; fax: +1 813 974 2189.
E-mail address: rolsson@hsc.usf.edu (R.A. Olsson).
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.12.005

422
M.H. Holschbach et al. / European Journal of Medicinal Chemistry 40 (2005) 421–437
Fig. 1
.
*. Structures of the agonist and antagonists mentioned in the text. KF17837 and SCH58261 have been radiolabeled with carbon-11.
the synthesis, and neither of the exocyclic substituents favors
a regioselective nucleophilic fluorination. Similar consider-
ations apply to another high-affinity A2AAR antagonist,
ZM241385. Some other A2AAR antagonists, the 8-styryl-
caffeines [16], have been labeled with carbon-11 for PET [17],
but their photolability [18] and poor penetration into the CNS
are drawbacks. However, it seemed that modifications of the
recently described synthesis [7] of 4-amino-6-benzylamino-
1,2-dihydro-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-
one, 10a, might provide congeners suitable for radiofluorina-
tion.
A three-atom spacer separating an aromatic, alkyl or
cycloalkyl group from C-2 of the adenine (or other heterocy-
clic) base is a motif common to a number of potent A2AAR
agonists and antagonists (Fig. 1) [4–6,19–23]. For example,
that spacer in the agonist WRC 0470 consists of the methyl-
ene residue and two hydrazine nitrogens linking the cyclo-
hexyl group to adenine C-2. Other three-atom linkers in WRC
0470 isosteres include ethoxy, ethylamino and 1-propyn-1-yl
groups. In the case of antagonists such as SCH58261, the
spacer linking the benzene ring consists of a two-carbon ethyl
bridge and pyrazole N-7, and in ZM241385 an ethylamino
group links the aryl moiety to the heterocyclic base. The linker
in 10a consists of the benzylic carbon, the nitrogen of the
6-amino group and C-6 of the quinoxaline moiety.
Here we report the synthesis of analogues of 10a, with
some modifications of the approach used by Colotta et al.
[7]. As in our earlier study of antagonists for the A1AR [24],
the primary objective was to assess the impact of fluorination
on pharmacological activity. Consistent with the intended use,
we designed the synthetic pathways to meet the needs of n.c.a.
radiofluorination. The series also included compounds with
O-methoxy groups to evaluate ligands with the potential for
labeling with carbon-11. Finally, we used in vitro autoradiog-
raphy to assess the suitability of these fluorinated analogues
to image A2AARs in brain by means of PET.
aromatic nucleophilic radiofluorination and the need, in the
production of a radioligand, for fluorination at the latest pos-
sible step to preserve a high radiochemical yield (RCY). The
carbonyl group of benzaldehyde met the first criterion; chang-
ing the order of the individual steps in the Colotta synthesis
met the second.
Scheme 1 shows the synthetic pathway, which employed
the same intermediate, 3, used by Colotta et al. [7] in their
first report, but differed in subsequent steps. Colotta reduced
the nitro group of 3, then formed and reduced the benzaldi-
mine to yield the 6-aralkylamino compound, and, finally,
introduced the 4-amino group by chlorination and reaction
with ammonia. Our strategy called for introducing the
4-amino group, protecting it, reducing the 6-nitro group and
finally introducing the 6-aralkylamino group. We evaluated
two alternative routes for introducing the 4-amino group. Both
began by chlorinating 3 and the displacement of the chloride
of 4 by azide. The azido group of 5 then underwent Staudinger
reduction [25] to form the 4-amino-6-nitro compound, 7. We
reasoned that it would be necessary to protect the 4-amino
group of 7 to insure the chemoselective reaction of the
6-amino group that reduction would generate from the 6-nitro
group in a subsequent step. However, protection proved
unnecessary; 7 failed to react with either Boc2O, trifluoro-
acetic anhydride or benzaldehyde/ZnCl2. The unreactivity of
the 4-amino group of 7 had two important consequences. First,
it insured that the selective derivatization of the 6-amino group
of diamine 8 was feasible. Second, it simplified the synthesis
of 8. The simultaneous reduction [26] of the 4-azido and
6-nitro groups of 5 by SnCl2 gave 8 in a single step. This
diamine represents a versatile intermediate for the synthesis
of variously substituted N-6 derivatives of 8.
Preliminary studies (Table 1) assessed the feasibility of
various approaches to 4-amino-6-aralkylaminoquinazolines
10a–s. Only one method, reductive alkylation of 8 with NaB-
H(OAc)3, gave relatively low yields. The reaction of 8 with
either benzaldehyde or 4-(2-fluoroethoxy)benzaldehyde gen-
erated imines 9a, b, which then underwent reduction by
NaBH4 to benzylamines 10a, b in satisfactory overall yields.
The reductive amination of aldimines by decaborane [27],
2. Chemistry
The design of the synthetic route anticipated the need for
an electron-withdrawing group to activate the phenyl ring for

M.H. Holschbach et al. / European Journal of Medicinal Chemistry 40 (2005) 421–437
423
Scheme 1
.
which avoided the isolation of the intermediate aldimine,
proved the most efficient and convenient path to compounds
10b–s. In keeping with the need to know whether fluorina-
tion would affect pharmacological activity, the panel of alde-
hydes reacted with 8 were mainly fluorobenzaldehydes, but
also included furan-2-carboxaldehyde and its 5-bromo- and
5-iodo derivatives. Neither pyrrole-2-carboxaldehyde nor its
N-methyl derivative reacted with 8.
halides rather than aldehydes to form amides 11a–e, fol-
lowed by reduction of the amides to benzylamines 10a–e.
That approach offered two potential advantages. First, amides
tend to be more stable than aldimines, especially (ar)aliphatic
aldimines, which would make the products easier to handle.
Second, it would broaden the panel by adding aliphatic amino
substituents. Reductants for 11a–e included borane-THF [28],
NaBH4/acetic acid [29], sodium triacetoxyborohydride
[30,31] and borane-dimethyl sulfide complex [32].An attempt
to reduce a carboxamide with NaBH4/titanium isopropoxide
We also explored an alternative way to introduce the
6-benzylamino substituent, namely, the reaction of 8 with acyl
Table 1
Synthesis of 6-aralkylaminoquinoxalines. Comparison of methods
Compound
Method
Time (h)
Yield (%)
10a
NaBH4 reduction of imine 9a
Gabriel synthesis from 11e
BH3-THF reduction of amide 11d
BMS
8a
94
90
91
63
76
60
90
18b
10c
2b
2b
NaBH4/AcOH “
8b
NaBH(OAc)3 reductive alkylation of 8
1.5
B10H14
18
NaBH4/Ti(OiPr)4
No reaction
10d
BMS reduction of amide 11bb
NaBH(OAc)3 reductive alkylation
2
58
48
90
24
18
B
10H14
a Includes time necessary to make 9a.
b Does not include reaction time generating and purifying the carboxamide.

424
M.H. Holschbach et al. / European Journal of Medicinal Chemistry 40 (2005) 421–437
[33] failed. Despite the success of these “proof of principle”
preparations, reductive amination of an aldimine seemed best
suited to a radiosynthesis, so we did not expand the carboxa-
mide series. Trifluoroacetamide 11e served as the starting
material for a modified Gabriel synthesis [34]; alkylation with
benzyl bromide followed by alkaline workup gave a good
yield of 6-benzylamino compound 10a. Table 2 lists the char-
acteristics of novel compounds.
For the pharmacological evaluation of this series of com-
pounds the known derivative 10a was radiolabeled with tri-
tium. When 9a was reduced with [3H]NaBH4 under carefully
controlled reaction conditions, [3H]10a was obtained with a
RCY of 12.5%, a radiochemical purity of > 98% and a spe-
cific activity of 5.6 Ci/mmol.
hydrophobicity. The compounds competed at both the A1AR
and the A2AAR with Kis in the low-nanomolar range, but
selectivity for theA2AAR was low, at best just 3.6-fold (com-
pound 10s). While this work was in progress Colotta et al.
[35] described the synthesis of compounds 10a, 10c, 10e, 10g,
10j, 10m and 10q and their affinities for the bovine A1AR
and A2AAR. That study showed that 10a exhibited low-
nanomolar affinity for the A2AAR and > 110-fold less affin-
ity at the A1AR. Our measurements on rat brain receptors
confirm that estimate of affinity for theA2AAR, but we found
that 10a had a similar affinity for the A1AR and, so, little
selectivity. Species difference might account for that diver-
gence. However, our results confirm their report of the low
selectivity for the A2AAR of the other compounds. In agree-
ment with their high lipophilicities, the solubilities of 10a–s
in water were low, ~ 200 nM.
Autoradiographic studies employing [3H]10a (Fig. 2) con-
firmed the lack of selectivity and the high unspecific binding
seen in radioligand binding assays (data not shown). The
uptake of [3H]10a did not reflect the typical distribution in
3. Pharmacology
Table 3 summarizes the studies of the affinity of ratA1 and
A
2AARs for 10a–s, and also measurements of an index of
Table 2
Characteristics of new compounds
Compounds
5
6
7
8
R4
R6
Yield (%)
92
Recryst
THF
M.p. (°C)
>300
Analytical formula
N3
NO2
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
15H8N6O3 (C,H,N,O)
33H23N6O3P (C,H,N,O)
15H10N6O3 (C,H,N,O)
15H12N6O (C,H,N,O)
22H16N6O (C,H,N,O)
24H19N6O2 (C,H,N,O)
22H18N6O (C,H,N,O)
24H21FN6O2 (C,H,N,O)
23H20N6O (C,H,N,O)
23H19FN6O (C,H,N,O)
22H17FN6O (C,H,N,O)
22H17FN6O (C,H,N,O)
22H17FN6O (C,H,N,O)
22H18FN6O2 (C,H,N,O)
22H18FN6O2 (C,H,N,O)
22H18FN6O2 (C,H,N,O)
23H20N6O2 (C,H,N,O)
23H20N6O2 (C,H,N,O)
23H20N6O2 (C,H,N,O)
22H17FN6O2 (C,H,N,O)
23H19FN6O2 (C,H,N,O)
23H19FN6O2 (C,H,N,O)
20H16N6O2 (C,H,N,O)
20H15BrN6O2 (C,H,N,O)
20H15IN6O4 (C,H,N,O)
25H20N6O4 (C,H,N,O)
23H17FN6O2 (C,H,N,O)
17H14N6O2 (C,H,N,O)
23H18N6O2 (C,H,N,O)
17H11F3N6O2 (C,H,N,O)
N=PPh3
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NO2
94
THF
>300
NO2
92
THF/water
MeOEtOH
MeCN
THF
290–1
222–3
>300
NH2
100
100
93
9a
9b
PhCH=N
4-(F-EtO)PhCH=N
PhCH2NH
>300
10a
10b
10c
10d
10e
10f
10g
10h
10i
94
EtOH
213
4-(F-EtO)PhCH2NH
Ph(CH2)2NH
PhCHFCH2NH
2-FPhCH2NH
3-FPhCH2NH
4-FPhCH2NH
2-OHPhCH2NH
3-OHPhCH2NH
4-OHPhCH2NH
2-MeOPhCH2NH
3-MeOPhCH2NH
4-MeOPhCH2NH
2-F,4-OHPhCH2NH
2F,4MeOPhCH2NH
2F,5MeOPhCH2NH
2-FurCH2NH
5-Br-2-FurCH2NH
5-I-2-FurCH2NH
74
THF
201–2
208–9
187–9
210–3
207–9
202–4
198–9
201–2
213–5
207–8
190–1
186–7
219–21
179–81
173–5
204–5
193–4
173–4
242
90
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeOH
MeOH
THF/water
THF-water
EtOAc-Hex
90
90
82
90
81
85
10j
10k
10l
83
87
91
10m
10n
10o
10p
10q
10r
10s
11a
11b
11c
11d
11e
90
74
84
71
88
92
90
PhCH(OCOCH3)CONH 95
PhCHFCONH
CH3CONH
91
83
93
87
219–21
207–8
213–4
222
PhCH2CONH
CF3CONH

M.H. Holschbach et al. / European Journal of Medicinal Chemistry 40 (2005) 421–437
425
Table 3
Adenosine receptor binding and hydrophobicity data
Ki (nM)a
Compounds
8
9a
rA1AR
rA2AAR
A
2AAR/A1AR
log k′w
3.37
ND
19 (15–23)
15 (12–19)
20 (10–39)
20 (16–22)
32 (22–47)
33 (24–45)
12 (9–15)
22 (15–33)
20 (15–280
54 (42–69)
12 (9–15)
15 (12–18)
22 (18–26)
18 (13–24)
22 (14–35)
34 (21–55)
33 (25–42)
18 (9–36)
8 (7–10)
8 (7–9)
0.42
ND
ND
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
10q
10r
10s
17 (12–23)
19 (11–33)
28 (21–37)
25 (19–34)
32 (24–42)
24 (14–43)
30 (24–38)
20 (14–29)
20 (13–25)
20 (13–31)
33 (25–45)
38 (20–67)
29 (21–40)
35 (28–45)
87 (49–153)
53 (23–121)
17 (14–21)
25 (18–33)
51 (49–51)
0.83
0.99
0.87
0.76
2.72
1.10
1.47
0.37
1.75
1.34
1.54
2.17
1.30
1.05
2.66
2.90
2.14
1.57
3.65
5.08
5.02
5.43
5.31
5.25
5.17
4.90
4.39
4.46
4.47
5.10
5.02
5.01
4.77
5.29
5.19
4.73
5.22
5.46
16 (12–20)
14 (11–18)
a Mean and 95% confidence limits of three assays, each in triplicate. ND, not determined.
Fig. 2.
Autoradiographs of consecutive horizontal sections of a rat brain incubated with: A, the A1AR antagonist [3H]DPCPX, which shows the expected
distribution of tracer specifically bound to that receptor; B, the A2AAR antagonist [3H]ZM241385, which shows the expected high density of this receptor in
basal ganglia; C, [3H]10a; D, [3H]10a plus theA2AAR agonist CGS21680 (5 µM), and E [3H]10a plus theA1AR antagonist CPDPX (5 µM). The arrow in Panel
A points to the inferior colliculus, an area of low A1AR density. Note that in panels C–E this area is heavily labeled, indicative of a high degree of unspecific
binding. Note also that neither CPDPX nor CGS21680 appreciably displaced [3H]10a from areas of known high A1AR or A2AAR density, respectively,
additional evidence that unspecific binding obscured specific binding. The range of ligand density represented by the color scale at top right differs from figure
to figure. Maximum density is 260 fmol/mg in panel A, 1700 fmol/mg in panel B and 5600 fmol/mg in panels C–E.

426
M.H. Holschbach et al. / European Journal of Medicinal Chemistry 40 (2005) 421–437
brain of either the A1AR [36] or the AA2AR [37]. Rather,
there was a massive tracer accumulation in all brain regions
having a high density of cell bodies. Likewise, neither the
selective AA2AR agonist CGS21680 nor the selective A1AR
antagonist DPCPX significantly displaced bound [3H]10a.
Similar results were obtained by blockingA2AARs orA1ARs
with either CGS21680 or DPCPX before incubation with
[3H]10a.
CGS21680, and CPDPX. GPP(NH)p and adenosine deami-
nase (AD) were from Sigma-Aldrich. Phosphate buffer (No.
82561) was from Fluka. Sep-Pak Plus C18 cartridges, Long
Body, 360 mg, Part No. WAT023635, were obtained from
Millipore Waters, Eschborn, Germany and were precondi-
tioned with acetonitrile (20 ml) and water (20 ml) prior to
use. Other solvents and reagents were used as supplied by the
vendor. [3H]NaBH4 (150 mCi, 85 Ci/mmol) was from
Biotrend, Cologne, Germany. [3H]CPDPX and [3H]CGS
21680 were from New England Nuclear, and [3H]ZM241,385
was from Tocris. The synthesis of 5-iodofuran-2-carboxal-
dehyde was as described [38].
5. Conclusion
Although 2-phenyl-4-amino-6-arylamino-triazoloquino-
xalines 10a–s are potent antagonists at the A2AAR, they lack
selectivity, have poor water solubility and bind strongly to
brain structures other than theA2AAR. Thus, they are unsuited
for imaging of the brain A2AAR by PET. Aside from their
unsuitability as PET ligands, the low selectivity of this class
of compounds suggests that they might have importantA1AR-
mediated side effects that could curtail their usefulness as
drugs.
6.1.1. N-Phenyl-C-ethoxycarbonylformohydrazidoyl
chloride (1)
Sodium acetate trihydrate (26 g, 191 mmol) was added to
a solution of ethyl-2-chloroacetoacetate (27.6 ml, 200 mmol)
in ethanol (500 ml) and the solution was cooled in an ice/salt
bath to between –4 and 0 °C. A parallel reaction consisted of
the addition of an ice-cold aqueous solution of 4 M NaNO2
(13.8 g, 200 mmol), in H2O (50 ml) to a solution of aniline
(18.24 ml, 200 mmol) in 6 N HCl (120 ml), cooled in an
ice/salt bath to –4 to 0 °C. A slow rate of addition kept the
temperature below 0 °C. The cold solution of phenyldiazo-
nium chloride was then added to the ester at a rate that kept
the temperature between –4 and 0 °C. When the addition was
complete, the yellow suspension was refrigerated for 3 h,
diluted with ice/water (1 l) and again refrigerated for 3 h The
product precipitated as glistening white crystals, pure by TLC
(SolventA), Rf (product) 0.65; (Solvent C), Rf (product) 0.91).
Yield 44 g, 98%. M.p. 79–80 °C. Ref. [7] 79 °C.
6. Experimental protocols
6.1. Chemistry
Melting points were measured on an Electrothermal™
apparatus and are uncorrected. The Zentralabteilung für che-
mischeAnalysen, Forschungszentrum Juelich, performed the
elemental analyses, which were within 0.4% of the calcu-
lated composition. Thin layer chromatography (TLC) em-
ployed precoated silica sheets (4 × 8 cm, Polygram™,
Macherey-Nagel, Düren) developed with one of the follow-
ing mixtures (v/v) of ethyl acetate/hexane: 25:75 (SolventA);
35:65 (Solvent B), or 50:50 (Solvent C). Mass spectra (MS),
ESI, were obtained on a Finnigan Automass III mass spec-
trometer (Thermo Quest, Egelsbach). 1H, 13C and 19F NMR
spectra were obtained at 200.13, 50.32 and 188.31 MHz,
respectively, by means of a Bruker DPX-200 spectrometer
(Avance 200) in ≈ 5% solution in DMSO-d6 at 25 °C. Chemi-
cal shifts are given in d ppm using the residual proton and
carbon resonances of DMSO-d6 at 2.52 and 50.32 MHz,
respectively, as references. The multiplicity symbols s, d, t
and m refer to singlet, doublet, triplet and multiplet, respec-
tively.
6.1.2. 8-Nitro-3-(phenyl-hydrazono)-3,4-dihydro-1H-qui-
noxalin-2-one (2)
Compound 1 (22.6 g, 100 mmol) in EtOH (250 ml) was
added to a mixture of 2,3-diamino nitrobenzene- (15.32 g,
100 mmol) and triethylamine (16.8 ml, 120 mmol) in EtOH
(500 ml) and the dark red mixture was stirred at reflux for 4 h
TLC (Solvent C) Rf (product) 0.00, extended spot, Rf (di-
amine) 0.52, Rf (chloride) 0.91. The dark crystals formed dur-
ing storage overnight at –2 °C were filtered off, washed with
water and ethanol and dried at 120 °C. Yield 22.2 g, 75%,
m.p. 204 °C (EtOH/H2O). Ref. [7] 205–7 °C (AcOH).
6.1.3. 6-Nitro-2-phenyl-2H,5H-[1,2,4]triazolo[4,3-a]qui-
noxaline-1,4-dione (3)
Solvents and reagents, which were of the highest grade
available, were from either Sigma-Aldrich, Deisenhofen, Ger-
many or Lancaster Synthesis, Muelheim am Main, Germany.
N,N-Dimethylformamide (DMF) and dimethyl sulfoxide
(DMSO) were distilled under argon and stored in lightproof
containers over 4 Å molecular sieves. Dichloromethane
(DCM) and 1,2-dichloroethane (DCE) were dried over 4 Å
molecular sieves. Dry methanol, n-propanol and DMF
(puriss., abs., over molecular sieves) were from Fluka,
Buchs, Switzerland. Tocris-Cookson provided ZM241385,
Under efficient stirring triphosgene (14.8 g, 50 mmol) was
added in portions to a solution of 2 (14.87 g, 50 mmol) in
anhydrous THF (250 ml). After 2 min the dark solution light-
ened and became turbid. The mixture was refluxed for 3 h,
cooled to room temperature and then was held for 3 h at –2 °C.
Decanting the supernatant into vigorously stirred ice/water
(600 ml) destroyed unreacted triphosgene. The residual dark
yellow solid was mixed with THF/water (50:50, v/v, 100 ml),
filtered by suction, washed with ethanol and dried for 2 h at
120 °C. TLC (Solvent C) Rf (educt) 0.0, Rf (product) 0.72).

M.H. Holschbach et al. / European Journal of Medicinal Chemistry 40 (2005) 421–437
427
Yield 16 g, 100%. Recrystallization from CH3CN/DMF (1/1)
gave an analytical sample. M.p. 251 °C (CH3CN/DMF); Ref.
[7] 250–2 °C (EtOAc).
127.25, 128.57, 129.68, 129.93, 129.96, 130.13, 133.61,
133.82, 136.09, 138.25, 144.88, 145.20, 149.41, 153.47,
153.56. MS Calc. for C33H23N6O3P, molecular weight 582.55:
m/z 582 (M); 583 (M + 1)+.
For conversion to 7, compound 6 was dissolved in THF
(10 ml/mmol) and 3 N HCl (2 ml/mmol) was added. The mix-
ture was stirred at room temperature for 1 h; after 20 min a
yellow precipitate formed. TLC (Solvent B) showed that start-
ing material and product had the same Rf but different colors,
Rf (starting material): 0.57, yellow spot, Rf (product): 0.57,
colorless spot). The mixture was set aside overnight at –30 °C
to give, after filtration and air-drying, 7 in quantitative yield
as orange crystals, pure by TLC, m.p. 290–1 °C.Yield 1.19 g,
6.1.4. 4-Chloro-6-nitro-2-phenyl-2H-[1,2,4]triazolo[4,3-a]-
quinoxalin-1-one (4)
PCl5 (5.2 g, 25 mmol) was added to a suspension of 3 (8 g,
25 mmol) in POCl3 (150 ml) under argon. Pyridine (1.5 ml,
18.75 mmol) was added and the mixture was stirred for 4 h in
a 125–130 °C oil bath. In the first 30 min starting material
completely dissolved and the solution turned light brown. Stir-
ring continued as the mixture cooled to room temperature
and it was set aside at 2 °C overnight. The canary-yellow
crystals of product were filtered off, taken up in ethanol
(200 ml) (CAUTION! exothermic!), filtered and oven-dried
at 120 °C for 2 h TLC showed a single spot (Solvent C), Rf
(starting material) 0.74, Rf (product) 0.82; (Solvent B), Rf
(product) 0.51.Yield 7.6 g, 95%, m.p. > 300 °C. Recrystalli-
zation from acetone gave an analytical sample.
1
92%. H NMR, d: 7.29–7.57, m, 5H, H-9 and 4 phenyl H;
7.74, dd, 1H, H-8; 8.03, d, 1H, phenyl H; 8.29, br s, 2H, –NH2;
8.74, dd, 1H, H-7. 13C NMR, d: 117.65, 120.15. 120.42,
123.21, 125.83, 127.36, 129.60, 130.04, 131.44, 137.84,
145.29, 148.81, 148.98. MS Calc. for C15H10N6O3, molecu-
lar weight 322.28: m/z 322 (M+).
6.1.7. 4-Amino-6-nitro-2-phenyl-2H-[1,2,4]triazolo[4,3-a]-
quinoxalin-1-one (7). Method B, one pot procedure
Triphenylphosphine (1.45 g, 5.5 mmol, 1.1 eq) was added
in one portion to a suspension of compound 5 (1.74 g, 5 mmol)
in THF (50 ml). The canary-yellow suspension was stirred at
room temperature for 2 h and then at 50 °C for 30 min, dur-
ing which the mixture became clear. After cooling to room
temperature 3 N HCl (10 ml) was added. A yellow precipi-
tate formed within 20 min during stirring at room tempera-
ture. Workup as in Method A gave orange crystalline prod-
uct, 1.2 g, 75%, pure by TLC. Recrystallization from THF
gave an analytical sample, m.p. 290–1 °C.
6.1.5. 4-Azido-6-nitro-2-phenyl-2H-[1,2,4]triazolo[4,3-a]-
quinoxalin-1-one (5)
Adding NaN3 (1.6 g, 24 mmol) in one portion to a suspen-
sion of 4 (6.83 g, 20 mmol) in dry DMF (150 ml) and stirring
at room temperature dissolved starting material almost com-
pletely over 30 min. TLC (Solvent B), Rf (starting material)
0.51; Rf (product) 0.35; (Solvent C), Rf (educt) 0.81, Rf (prod-
uct) 0.68). The product spot became yellow on heating. Pour-
ing the reaction mixture into water (200 ml), precipitated the
faintly yellow product, which was filtered off and air-dried,
yield 6.7 g, 92%. Recrystallization from THF gave an ana-
1
lytical sample. M.p. > 300 °C (dec). H NMR, d: 7.47, m,
1H, H-9; 7.65, m, 2H, phenyl H; 8.08, M, 3H, phenyl H; 8.27,
dd, 1H, H-8; 9.24, dd, 1H, H-7. 13C NMR, d: 113.88, 119.21,
120.46, 122.12, 127.05, 128.18, 130.52, 131.90, 132.02,
137.20, 141.02, 142.16, 144.88, 148.26. MS Calc. for
C15H8N8O3, molecular weight 348.28: m/z 348 (M+), 349
(M + 1)+.
6.1.8. Attempted synthesis of 4-(tert-butoxycarbonyl)-
amino-6-nitro-2-phenyl-2H-[1,2,4]triazolo[4,3-a]-
quinoxalin-1-one
Treatment of 7 with 2.2 eq of Boc2O and 10 mol% of
DMAP in THF (6 h, 85 °C) or acetonitrile (20 h, 85 °C) gave
only unreacted starting material.
6.1.6. 4-Amino-6-nitro-2-phenyl-2H- [1,2,4]triazolo[4,3-a]-
quinoxalin-1-one (7). Method A, via an intermediate
iminophosphorane (6)
6.1.9. Attempted synthesis of 4-benzylideneamino-6-nitro-
2-phenyl-2H-[1,2,4]triazolo[4,3-a]quinoxalin-1-one
Refluxing 7 in trifluoroacetic anhydride or the ZnCl2-
catalyzed reaction of 7 with benzaldehyde in refluxing EtOH
gave only starting material.
Triphenylphosphine (1.33 g, 5.06 mmol, 1.1 eq) was added
in one portion to a suspension of compound 5 (1.61 g,
4.6 mmol) in THF (46 ml). Starting material dissolved dur-
ing stirring at room temperature for 2 h and then at 50 °C for
30 min. After cooling to room temperature water (46 ml) was
added, the mixture was stirred for 1.5 h, stored overnight at
–30 °C, the yellow precipitate filtered off and oven-dried for
2 h at 120 °C. The material was pure by TLC (Solvent B), Rf
(starting material) 0.35, Rf (product) 0.57). HRMS indicated
formation of the iminophosphorane 6, MW 582.55 g/mol. The
6.1.10. 4,6-Diamino-2-phenyl-2H-[1,2,4]triazolo[4,3-
a]quinoxalin-1-one (8). Method A. Reduction of 5
Finely ground 5 (8.58 g, 24 mmol) suspended in 95% etha-
nol (200 ml) was stirred during the addition of a solution of
SnCl2·2H2O (60 g, 264 mmol) in ethanol (150 ml). Heating
to 105 °C (oil-bath temperature), gave a clear solution after
15 min. Stirring continued for 1.5 h, completing the reaction,
as monitored by TLC in two systems. For TLC analysis ali-
quots of the reaction mixture were poured into THF. The addi-
tion of 32% NH4OH precipitated Sn(OH)2 as a fluffy white
1
yield was 2.51 g, 94%, m.p. > 300 °C. H NMR, d: 7.25–
7.43, m, 2H, H-8 and H-9; 7.55–7.72, m, 12H, phenyl H;
7.83–7.94, m, 6H, phenyl H; 8.13, m, 2H, phenyl H; 7.79, dd,
1H, H-7. 13C NMR, d: 117.32, 119.84, 120.54, 126.24, 126.56,

428
M.H. Holschbach et al. / European Journal of Medicinal Chemistry 40 (2005) 421–437
solid; the yellow supernatant contained the product. Analysis
by TLC (Solvent C), Rf (starting material) 0.68, Rf (product)
0.44; (Solvent B), Rf (starting material) 0.35, Rf (product)
0.16. Evaporation of the reaction mixture left an oily residue
that was taken up in THF (250 ml). The addition of 32%
ammonia (100 ml) precipitated Sn(OH)2. The yellow organic
phase was decanted and the inorganic salt was washed with
THF (100 ml). The pooled organic phases were evaporated
to a yellow solid; TLC revealed a trace of UV-absorbing fluo-
rescent impurity of Rf 0.00. Taking up the solid residue in
10 M HCl (50 ml) and ethanol (50 ml) and stirring vigor-
ously for 15 min dissolved the tin salts and precipitated 8~HCl
as a cream-colored solid. TLC (Solvent B), Rf: 0.00, fluores-
cent in UV light). Exposure to air and light changed the color
of the salt from white to red. The precipitate was collected by
centrifugation (10 min at 2600 × g) and suspended in aque-
ous 32% ammonia (150 ml). THF (250 ml) was added, the
mixture was stirred in a beaker for 10 min, the phases were
separated and the organic layer was washed with brine (2 ×
100 ml). The clear yellow organic phase was dried over
Na2SO4, filtered and taken to dryness. Trituration with hot
methanol (40 ml), cooling overnight, filtration and oven dry-
ing for 1 h at 120 °C gave the product as dark yellow crystals
in quantitative yield (6.9 g). Recrystallization from methoxy-
ethanol (ca. 10 ml/g) gave an analytical sample, m.p. 222–
3 °C; Ref. [7] 220–2 °C. 1H NMR, d: 5.37, br s, 2H, –NH2;
6.67, d, 1H, H-9; 6.98, t, 1H, H-8; 7.35, m, 3H, –NH2 and
phenyl H; 7.55, t, 2H, phenyl H; 7.88, d, 1H, H-7; 8.17, m,
2H, phenyl H. 13C NMR, d: 102.67, 111.01, 120.27, 122.92,
124.69, 124.88, 127.07, 129.99, 132.33, 138.26, 143.58,
144.99, 149.39. MS, Calc. for C15H12N6O, molecular weight
292.30: m/z 292 (M+), 293 (M + 1)+.
6.1.13. 4-Amino-6-{[4-(2-fluoro-ethoxy)benzylidene]-
amino}-2-phenyl-2H-[1,2,4]triazolo[4,3-a]quinoxalin-1-
one (9b)
The reaction of 4-(2-fluoro-ethoxy)-benzaldehyde
(370 mg, 2.2 mmol) and 8 (584 mg, 2 mmol) catalyzed by
ZnCl2 (ca. 100 mg) gave product. TLC (Solvent B), Rf (start-
ing material) 0.15, Rf (product) 0.05. The orange solid was
filtered off, suspended in THF and sonicated. Filtration and
air-drying for 1 h gave the product as yellow crystals. Yield
830 mg, 93%, m.p. > 300 °C (dec). 1H NMR, d: 4.24–4.46,
m, 2H, –OCH2–; 4.63–4.98, m, 2H, –CH2F; 7.06, d, 1H, H-9;
7.15, d, 2H, 2 phenyl H; 7.25–7.44, m, 2H, H-8 and phenyl
H; 7.54–7.62, m, 4H, –NH2 and 2 phenyl H; 7.94, m, 2H,
2 phenyl H; 8.09, m, 2H, H-7 and phenyl H; 8.63, m, 2H,
–CHN– and phenyl H. 13C NMR, d: 68.56 (benzylidene),
81.53, 84.69, 102.72, 111.09, 115.58, 115.86, 120.30, 120.35,
122.96, 124.69, 124.89, 125.41, 127.11,130.02, 130.07,
130.77, 132.33, 132.70, 138.19, 138.25, 143.48, 145.02,
149.40, 163.95, 192.22. 19F NMR, d: −222.57, –222.76. MS,
Calc. for C24C19FN6O2, molecular weight 442.45: com-
pound decomposed to 8 during analysis.
6.1.14. 4-Amino-6-benzylamino-2-phenyl-2H-[1,2,4]tria-
zolo[4,3-a]quinoxalin-1-one (10a). Method A. NaBH4
reduction of 9a
A suspension of 9a (190 mg, 0.5 mmol) in dry MeOH
(10 ml) containing NaBH4 (57 mg, 1.5 mmol) was stirred for
5 min in an 80–90 °C oil bath, when TLC showed the reac-
tion was complete (Solvent B), Rf (starting material) 0.05, Rf
(product) 0.55. After cooling to room temperature the mix-
ture was refrigerated for 1 h Filtration of the yellow solid,
washing with MeOH and oven drying for 1 h at 120 °C gave
the product, 180 mg (94%), m.p. 218 °C (EtOH). Ref. [7]
6.1.11. 4,6-Diamino-2-phenyl-2H-[1,2,4]triazolo[4,3-a]-
quinoxalin-1-one (8). Method B. Reduction of 7
1
215–7 °C (ethanol/ethyl acetate). H NMR, d: 4.45, d, 2H,
–CH2–; 6.03, t, 1H, –NH–; 6.56, d, 1H, H-9; 7.06, t, 1H, H-8;
7.26–7.43, m, 8H, –NH2 and 6 phenyl H; 7.57, m, 2H, phe-
nyl; 7.92, d, 1H, H-7; 8.09, d, 2H, phenyl. 13C NMR, d: 47.63
(benzylic), 102.60, 106.94, 120.29, 122.95, 124.40, 124.83,
127.13, 127.83, 128.28, 129.36, 130.02, 132.31, 138.25,
140.62, 143.09, 144.88, 145.34, 149.39. MS, Calc. for
C22H18N6O, molecular weight 382.42: m/z 382 (M+).
Compound 7 (483 mg, 1.5 mmol) and SnCl2·2H2O (1.7 g,
7.5 mmol) in dry EtOH (15 ml) was stirred in an oil bath
heated to 90–100 °C. In 2 min starting material dissolved and
by 5 min TLC (Solvent B), Rf (starting material) 0.57, Rf
(product) 0.15) showed the reaction was over. Evaporation
gave a yellow solid that was taken up in THF (10 ml). Dilu-
tion with water (30 ml) initiated precipitation of product,
which continued overnight in the refrigerator.
6.1.15. 4-Amino-6-benzylamino-2-phenyl-2H-[1,2,4]tria-
zolo[4,3-a]quinoxalin-1-one (10a). Method B. Alkylation
of 11e (modified Gabriel synthesis)
6.1.12. 4-Amino-6-(benzylidene-amino)-2-phenyl-2H-
[1,2,4]triazolo[4,3-a]quinoxalin-1-one (9a)
Benzaldehyde (112 µl, 1.1 mmol) and a catalytic amount
of dry ZnCl2 (ca. 100 mg) were added to a solution of 8
(292 mg, 1 mmol) in dry THF (15 ml). The suspension was
stirred at 95–100 °C for 8 h; after 1 h product began to pre-
cipitate. TLC (Solvent B), Rf (educt) 0.15, Rf (product) 0.05.
Cooling to room temperature gave a yellow solid that was
filtered off, suspended in THF, filtered again and air-dried for
1 h, giving product as a light yellow powder in quantitative
yield (380 mg, 100%, m.p. > 300 °C (dec). The product, which
is a mixture of the E- and Z-isomers, can be recrystallized
from CH3CN.
Under argon, NaH (60%, 21 mg, 0.525 mmol) was sus-
pended in dry DMF (2 ml). A solution of 11e (194 mg,
0.5 mmol) in dry DMF (2 ml) was added dropwise by syringe
and the clear brownish solution was stirred for 1 h at room
temperature. Benzyl bromide (66 µl, 0.55 mmol) in dry DMF
(1 ml) was added dropwise and the mixture was stirred for
18 h at 70–80 °C. MeOH (4 ml) and NaOH (2 M, 1 ml,
2 mmol) was added and the mixture was stirred at 70–80 °C
for 10 min longer. Dropwise addition of water (2–3 ml) to the
hot, well stirred solution caused the precipitation of a yellow
solid. After cooling to room temperature the solid was fil-

M.H. Holschbach et al. / European Journal of Medicinal Chemistry 40 (2005) 421–437
429
tered off, washed with MeOH (2–3 ml) and triturated twice
with hot MeOH (4–5 ml). Filtration, air-drying and crystalli-
zation from CH3CN yielded 172 mg, 90%, of yellow crys-
tals, m.p. 216–8 °C (CH3CN).
bath was removed and saturated Na2CO3 (6 ml) was added
carefully.After cooling to room temperature the reaction mix-
ture was stirred for another 30 min and poured into MeOH
(40 ml). Precipitated product was filtered off. Three tritura-
tions with hot methanol (10 ml) removed a lipophilic yellow
impurity. After filtration the solid was extracted twice into
DMF (15 ml), insoluble impurities filtered off and product
precipitated by the addition of water (20–30 ml). The amine
collected by centrifugation was recrystallized from CH3CN
as yellow crystals. Yield 250 mg, 63%, m.p. 208–10 °C.
6.1.16. 4-Amino-6-[4-(2-fluoro-ethoxy)benzylamino]-2-
phenyl-2H- [1,2,4]triazolo[4,3-a]quinoxalin-1-one (10b)
Reduction of 9b (442 mg, 1 mmol) in dry MeOH (20 ml)
with 3 equiv. NaBH4 for 10 min at 80–90 °C then cooling on
ice gave product. TLC (solvent B) Rf (starting material) 0.05,
1
Rf (product) 0.40. Yield 330 mg, 74%, m.p. 201–2 °C. H
NMR, d: 4.13–4.88, M, 6H, –CH2CH2F and –CH2–; 5.91, t,
1H, –NH–; 6.57, d, 1H, H-9; 6.89–7.10, m, 4H, H-8 and 3 phe-
nyl H; 7.35, m, 5H, –NH2 and H-8 + 2 phenyl H; 7.57, t, 2H,
phenyl H; 7.88, d, 1H, H-7; 8.07, d, 2H, phenyl H.. 13C NMR,
d: 47.09 (benzylic), 67.08, 68.08, 81.40, 84.72, 102.55,
106.90, 115.34, 120.25, 122.90, 124.36, 124.82, 127.09,
129.70, 130.00, 132.28, 132.77, 138.25, 143.09, 144.88,
145.30, 149.36, 158.12. 19F NMR, d: − 222.45. MS, Calc. for
C24H19FN6O2, molecular weight 444.46: m/z 444 (M+).
6.1.19. 4-Amino-6-(2-phenyl-ethylamino)-2-phenyl-2H-
1,2,4-triazolo[4,3-a]quinoxalin-1-one (10c).
Method C. NaBH4/acetic acid reduction of 11d
Under ice cooling acetic acid (148 µl, 2.5 mmol) in dry
THF (1 ml) was added dropwise over 2 min to a slurry of 11d
(205 mg, 0.5 mmol) and sodium borohydride (95 mg,
2.5 mmol) in dry THF (4 ml). The suspension was stirred at
0 °C for 5 min and at reflux (85–95 °C oil bath) for 8 h. After
the reaction mixture cooled to room temperature, poured into
0.1 M NaOH (25 ml) and extracted with DCM (2 × 50 ml).
The organic phase was washed with water (2 × 50 ml), dried
over Na2SO4 and DCM was rotary evaporated. Crystals
formed when the yellow solid residue was triturated with
methanol (5 ml). The solid was triturated twice more with
hot methanol (2 × 5 ml), product was filtered off and air dried
to give 150 mg, 76% of product as yellow crystals, m.p. 208–
10 °C (CH3CN).
6.1.17. 4-Amino-6-(2-phenyl-ethylamino)-2-phenyl-2H-
[1,2,4]triazolo[4,3-a]quinoxalin-1-one (10c).
Method A. Borane-THF reduction of 11d
Under argon and at room temperature, BH3-THF (5.1 ml
of a 1 M solution of BH3 in THF, 5.1 mmol) was added over
5 min to a slurry of 11d (410 mg, 1 mmol) in dry THF. Educt
dissolved during the addition, giving a clear brown solution.
The mixture was stirred for 2 h, when TLC indicated the com-
plete disappearance of educt. Water (600 µl) was carefully
added (gas evolution!) followed by 10% HCl (1100 µl). After
stirring for another 15 min the volatiles were evaporated and
the aqueous residue was made alkaline by the addition of 2 M
NaOH. The mixture was extracted with ethyl acetate (2 ×
30 ml), the pooled organic phases washed with brine (2 ×
30 ml), dried over anhydrous Na2SO4 and evaporated to give
a yellow semisolid residue. Coevaporation with MeOH
(20 ml) left a yellow solid which was triturated twice with
hot MeOH and recrystallized twice from CH3CN (~8 ml) by
refluxing and cooling to –30 °C). Yield 362 mg (91%) of
golden yellow crystals, m.p. 209–10 °C (CH3CN). 1H NMR,
d: 2.93, t, 2H, –CH2–; 3.38, m, 2H, –CH2NH–; 5.66, t, 1H,
–CH2NH–; 7.11–7.61, m, 11 H, –NH2, H-9, H-8 and 8 phe-
nyl; 7.94–8.10, m, 3H, H-7, phenyl H. 13C NMR, d: 45.29
(benzylic), 102.36, 106.55, 119.10, 120.30, 122.80, 124.40,
125.02, 127.01, 127.10, 129.27, 129.60, 130.01, 132.28,
138.25, 140.43, 143.09, 144.88, 145.18, 149.38. MS, Calc.
for C23H20N6O, molecular weight 396.44: m/z 396 (M+).
6.1.20. 4-Amino-6-(2-fluoro-2-phenyl-ethylamino)-2-phe-
nyl-2H-1,2,4-triazolo[4,3-a]quinoxalin-1-one (10d)
A slurry of 11b (428 mg, 1 mmol) in dry toluene (4 ml)
was treated with boron-methyl sulfide complex (10 M, 210 µl,
2.1 mmol, 2.1 eq). The suspension was stirred at 0 °C for
15 min and at reflux (125–130 °C oil bath) for 2 h. The oil
bath was removed and sat. Na2CO3 (6 ml) was added care-
fully. The reaction mixture was cooled to room temperature
and the orange suspension was stirred for 30 min longer. The
mixture was poured into MeOH (60 ml) and precipitated salts
were filtered off. Concentration of the methanolic solution to
dryness gave a solid that was purified by recrystallization from
1
CH3CN. Yield 378 mg, 58%, m.p. 187–8 °C (CH3CN). H
NMR, d: 3.57–3.83, m, 2H, CHFCH2–; 5.69, t, 1H, –NH–;
5.90, t, 1H, –CHF–; 6.67, d, 1H, H-9; 7.12, t, 1H, H-8; 7.37–
7.62, m, 10H, –NH2– + 8 phenyl; 7.96, d, 1H, H-7; 8.09, d,
2H, phenyl H. 13C NMR, d: 60.64 (benzylic), 120.31, 123.07,
124.46, 126.79, 126.92, 129.18, 129.38, 130.03, 138.24,
145.39. 19F NMR, d: − 180.04. MS, Calc. for C23H19FN6O,
molecular weight 414.43: m/z 415 (M + 1)+.
6.1.18. 4-Amino-6-(2-phenyl-ethyamino)-2-phenyl-2H-
[1,2,4]triazolo[4,3-a]quinoxalin-1-one (10c).
6.1.21. Reductive alkylation of 8 using NaBH(OAc)3.
General method
Diamine 8 (292 mg, 1 mmol) was suspended in dry sol-
vent (10 ml), a solution of the aldehyde (1.05 mmol) in sol-
vent (5 ml) was added, followed by solid NaBH(OAc)3
(300 mg, 1 mmol). The mixture was stirred at room tempera-
Method B. Borane-dimethyl sulfide (BMS) reduction of 11d
At 0 °C under argon, BMS (10 M, 210 µl, 2.1 mmol, 2.1 eq)
was added dropwise to a slurry of 11d (410 mg, 1 mmol) in
dry toluene (4 ml). The suspension was stirred at 0 °C for
15 min and at reflux (oil bath 125–130 °C) for 2 h. The oil

430
M.H. Holschbach et al. / European Journal of Medicinal Chemistry 40 (2005) 421–437
ture for the specified time. Reaction monitoring by TLC
employed Solvent B.
6.1.27. 4-Amino-6-[4-(2-fluoro-2-phenylethylamino]-2-
phenyl-2H-[1,2,4]triazolo[4,3-a]quinoxalin-1-one (10d)
The general method reacted 8 with ( )-a-fluoro-
phenylacetaldehyde for 18 h (TLC: Rf 0.54). The solid resi-
due was triturated twice with CH3CN (2 × 5 ml), filtered and
recrystallized from CH3CN. Yield: 372 mg, 90%, m.p. 187–
9 °C.
6.1.22. 4-Amino-6-[4-(2-phenylethylamino]-2-phenyl-2H-
[1,2,4]triazolo[4,3-a]quinoxalin-1-one (10c)
Phenylacetaldehyde reacted with 8 in THF for 1.5 h accord-
ing to the general method. TLC: Rf 0.61. Adding ethanol
(10 ml) to the mixture precipitated an impurity that centrifu-
gation removed. Evaporation gave a solid that was purified
by MPLC (silica, solvent B). Evaporation of the fraction that
contained product gave yellow crystals for recrystallization
from CH3CN. Yield: 240 mg, 60%, m.p. 208–9 °C.
6.1.28. 4-Amino-6-(2-fluorobenzylamino-2-phenyl-2H-
[1,2,4]triazolo[4,3-a]quinoxalin-1-one (10e)
Compound 8 reacted with 2-fluorobenzaldehyde for 3 h
(TLC: Rf 0.48). The crude product was triturated with hot
MeOH (5 ml), cooled to 0 °C for 6 h and the yellow solid
filtered and air-dried.Yield: 360 mg, 90%, m.p. 201–3 °C. 1H
NMR, d: 4.52, d, 2H, –CH2–; 6.01, t, 1H, –NH–, 6.59, d, 1H,
H-9; 7.07–7.61, 10 H, –NH2, H-8, 7 phenyl H; 7.94, d, 1H,
H-7; 8.09, d, 2H, phenyl H. 13C NMR, d: 46.97 (benzylic),
102.77, 106.99, 114.28, 114.47, 114.69, 114.89, 120.30,
6.1.23. 4-Amino-6-[4-(2-fluoro-2-phenylethylamino]-2-
phenyl-2H-[1,2,4]triazolo[4,3-a]quinoxalin-1-one (10d)
The reaction of 8 with ( )-a-fluorophenylacetaldehyde in
1,2-DCE required 24 h TLC: Rf (product) 0.54, Rf (alde-
hydes) 0.69. The turbid yellow reaction mixture was centri-
fuged and the supernatant evaporated. The semi-solid resi-
due was purified by MPLC (silica, solvent B), Evaporation
of the fraction containing the product gave a solid residue for
recrystallization from CH3CN.Yield: 300 mg, 48%, m.p. 187–
9 °C (CH3CN).
124.03, 124.08, 124.81, 127.14, 130.03, 131.20, 131.37. 19
F
NMR, d: − 119.35. MS, Calc. for C22H17FN6O, molecular
weight 400.41: m/z 400 (M+).
6.1.29. 4-Amino-6-(3-fluorobenzylamino)-2-phenyl-2H-
[1,2,4]triazolo[4,3-a]quinoxalin-1-one (10f)
The reaction of 8 with 3-fluorobenzaldehyde required 6 h
(TLC: Rf 0.52).Yield: 328 mg, 82%, m.p. 207–9 °C. 1H NMR,
d: 4.50, d, 2H, –CH2–; 6.12, t, 1H, –NH–, 6.53, t, 1H, H-9;
7.05–7.58, 10 H, –NH2, H-8, 7 phenyl H; 7.92, d, 1H, H-7;
8.09, d, 2H, phenyl H. 13C NMR, d: 46.97 (benzylic), 102.77,
106.99, 114.28, 114.47, 114.69, 114.89, 120.30, 124.03,
124.08, 124.81, 127.14, 130.03, 131.20, 131.37. 19F NMR,
d: 119.35. 19F NMR, d: − 113.80. MS, Calc. for C22H17FN6O,
molecular weight 400.41: m/z 400 (M+), 401 (M + 1)+.
6.1.24. Attempted synthesis of 4-amino-6-[4-(2-phenylethy-
lamino]-2-phenyl-2H-[1,2,4]triazolo[4,3-a]quinoxalin-1-
one (10c) using Ti(OiPr)4/NaBH4
Diamine 8 (72 mg, 0.25 mmol), phenylacetaldehyde (33 µl,
0.25 mmol) and Ti(OiPr)4 (148 µl, 0.5 mmol) in dry EtOH
(2 ml) were stirred under argon at room temperature for 3 h
NaBH4 (28 mg, 0.75 mmol) was added and the mixture was
stirred for 3 h more.After hydrolysis with water TLC showed
only 8.
6.1.30. 4-Amino-6-(4-fluorobenzylamino)-2-phenyl-2H-
[1,2,4]triazolo[4,3-a]quinoxalin-1-one (10g)
6.1.25. Reductive amination of 8 using decaborane in
methanol. General method
Compound 8 reacted with 4-fluorobenzaldehyde in 3 h
(TLC: Rf 0.46). The solid residue was triturated with hot
MeOH (5 ml), cooled to 0 °C for 6 h, the yellow solid filtered
and air-dried.Yield: 360 mg, 90%, m.p. 202–4 °C. 1H NMR,
d: 4.46, s, 2H, –CH2–; 6.07, br s, 1H, –NH–; 6.54, d, 1H,
H-9; 7.12, t, 1 h H-8; 7.21, t, 2H, phenyl H; 7.40, m, 5H,
–NH2 + 3 phenyl H; 7.57, t, 2H, phenyl H; 7.94, d, 1H, H-7;
8.09, d, 2H, phenyl H. 13C NMR, d: 46.76 (benzylic), 102.68,
106.98, 115.86, 116.28, 120,30, 122.98, 124.42, 124.82,
127.15, 130.4, 130.21, 132.30, 136.80, 136.86, 138.24,142.92,
145.35, 149.39. 19F NMR, d: − 116.48. MS, Calc. for
C22H17FN6O, molecular weight 400.41: m/z 400 (M+).
Under argon at room temperature 8 (292 mg, 1 mmol) was
suspended in dry MeOH (15 ml). The aldehyde (1.2 mmol)
in MeOH (1–2 ml) was added followed by solid decaborane
(36 mg, 0.3 mmol). The mixture was stirred at room tempera-
ture for the specified time. In every instance TLC employed
Solvent B (Rf (diamine) 0.16). In order to decompose excess
borane, acetone (5 ml) and water (5 ml) were added and stir-
ring continued until gas evolution stopped (2 h). The solvents
were decanted, the crude product was suspended in MeOH
(10 ml) and the MeOH evaporated. The text below describes
the work-up of individual preparations. Unless noted, MeCN
was the recrystallization solvent.
6.1.31. 4-Amino-6-(2-hydroxybenzylamino)-2-phenyl-2H-
[1,2,4]triazolo[4,3-a]quinoxalin-1-one (10h)
6.1.26. 4-Amino-6-[4-(2-phenylethylamino]-2-phenyl-2H-
[1,2,4]triazolo[4,3-a]quinoxalin-1-one (10c)
The general method reacted 8 with phenylacetaldehyde for
18 h (TLC: Rf 0.61). Product was triturated twice with hot
MeOH and filtered. The yellow solid product was recrystal-
lized from CH3CN. Yield: 356 mg, 90%, m.p. 208–9 °C.
The reaction of 8 with 2-hydroxybenzaldehyde went to
completion in 4 h (TLC: Rf 0.31). Yield: 323 mg, 81%, m.p.
1
198–9 °C. H NMR, d: 4.30, m, 2H, –CH2–; 5.89, t, 1H,
–NH–; 6.61, m, 3H, H-9 and 2 phenyl H; 6.77–6.90, m, 3H,
H-8 and 2 phenyl H; 7.03–7.38, m, 7H, –NH2 and 5 phenyl

M.H. Holschbach et al. / European Journal of Medicinal Chemistry 40 (2005) 421–437
431
H; 7.92, d, 1H, H-7; 8.09, d, 2H, phenyl H. 13C NMR, d:
2H, phenyl H; 7.93, m, 1H, H-7; 809, m, 2H, phenyl H. 13
C
NMR, d: 55.85 (benzylic), 113.05, 113.94, 120.31, 122.97,
124.40, 130.03, 130.45, 138.26, 142.35, 143.11, 145.36,
149.41, 160.32. MS, Calc. for C23H20N6O2, molecular weight
412.44: m/z 412 (M+), 413 (M + 1)+.
63.68 (benzylic), 102.34, 106.76, 115.97, 119.74, 120.27,
122.92, 124.39, 124.60, 124.89, 126.09, 127.09, 128.96,
129.79, 129.99, 130.97, 132.31, 138.13, 138.26, 143.35,
144.88, 145.26, 149.39, 156.26, 157.09. MS, Calc. for
C22H18N6O2, molecular weight 398.42. m/z 398 (M+), 399
(M + 1)+.
6.1.36. 4-Amino-6-(4-methoxybenzylamino)-2-phenyl-2H-
[1,2,4]triazolo[4,3-a]quinoxalin-1-one (10m)
6.1.32. 4-Amino-6-(3-hydroxybenzylamino)-2-phenyl-2H-
[1,2,4]triazolo[4,3-a]quinoxalin-1-one (10i)
Compound 8 required 4 h to react with 4-methoxy-
benzaldehyde (TLC: Rf 0.44). The solid residue was tritu-
rated with hot MeOH (5 ml), cooled to 0 °C for 6 h and the
yellow solid was filtered and air-dried. Yield: 370 mg, 90%,
m.p. 186–7 °C. 1H NMR, d: 3.75, s, 3H, –OCH3; 4.37, t, 2H,
–CH2–; 5.90, t, 1H, –NH–; 6.59, m, 1H, H-9; 6.93, m, 2H,
phenyl H; 7.06, t, 1H, H-8; 7.35, m, 5H, –NH2 and 3 phenyl
H; 7.57, m, 2H, phenyl H; 7.92,d, 1H, H-7, 8.10,d, 2H, phe-
nyl H. 13C NMR, d: 55.92 (benzylic), 102.53, 106.93, 114.75,
120.28, 122.90, 124.37, 124.83, 127.12, 129.66, 130.01,
132.30, 132.31, 138.25, 143.12, 144.88, 145.31, 149.39,
155.30. MS, Calc. for C23H20N6O2, molecular weight 412.44:
m/z 412 (M+), 413 (M + 1)+.
The reaction of 3-hydroxybenzaldehyde with 8 required
4 h (TLC: Rf 0.33). Yield: 338 mg, 85%, m.p. 201–2 °C. 1H
NMR, d: 4.33,t, 2H, –CH2–; 5.97, t, 1H, –NH–; 6.54, d, 1H,
H-9; 6.83, m, 1H, phenyl H; 7.01–7.16, m, 2H, phenyl H,
7.36, m, 3H, H-8 and 2 phenyl H; 7.56, m, 2H, phenyl H;
7.92, d, 1H, H-7; 8.09, d, 2H, phenyl H; 9.40, s, 1H, –OH.
13C NMR, d: 49.49 (benzylic), 102.53, 106.90, 114.80,
114.95, 118.87, 120.28, 122.90, 124.38, 124.84, 127.22,
130.01, 130.35, 132.31, 138.26, 142.10, 143.13, 145.32,
149.39, 158.42. MS, Calc. for C22H18N6O2, molecular weight
398.42: m/z 398 (M+), 399 (M + 1)+.
6.1.33. 4-Amino-6-(4-hydroxybenzylamino)-2-phenyl-2H-
[1,2,4]triazolo[4,3-a]quinoxalin-1-one (10j)
6.1.37. 4-Amino-6-(2-fluoro-4-hydroxybenzylamino)-2-
phenyl-2H-[1,2,4]triazolo[4,3-a]quinoxalin-1-one (10n)
The reaction of 8 and 2-Fluoro-4-hydroxybenzaldehyde
required 7 h (TLC: Rf 0.28). The residue was triturated with
hot MeOH (5 ml), cooled to 0 °C for 6 h and the yellow solid
was filtered and air-dried. Yield: 308 mg, 74%, m.p. 219–
21 °C. 1H NMR, 413, m, 2H, –CH2–; 580, t 1H, –NH–; 661,
m, 3H, H-9 and 2 phenyl H; 709, t, 1H H-8; 733, m, 4H,
–NH2 and 2 phenyl; 757, m, 2H, phenyl H, 791, d, 1H, H-7;
809, d, 2H, phenyl H; 987, s, 1H, –OH. 13C NMR, d: 41.24
(benzylic) 102.70, 103.24, 103.71, 106.72, 112.27, 112.32,
116.73, 117.04, 120.27, 122.94, 124.38, 124.84, 127.10,
129.99, 131.53, 131.66, 132.28, 138.24, 142.87, 145.36,
149.36, 158.96, 159.19, 159.51, 164.35. 19F NMR,
d: − 118.04; MS, Calc. for C22H17FN6O2, molecular weight
416.41: m/z 416 (M+).
The reaction of p-hydroxybenzaldehyde with 8 required
4 h (TLC: Rf 0.28). The solid residue was triturated with hot
MeOH (5 ml), cooled to 0 °C for 6 h and the yellow solid
filtered and air-dried.Yield: 330 mg, 83%, m.p. 213–5 °C. 1H
NMR, d: 4.29, d, 2H, –CH2–; 5.80, t, 1H, –NH–; 6.58, d, 1H,
H-9; 6.76, m, 2H, H-8; 7.03–7.61, m, 11 H, –NH2 + 9 phenyl
H; 8.09, d, 1H, H-7; 9.38, s, 1H, –OH. 13C NMR, d: 47.37
(benzylic), 102.46, 106.88, 115.80, 116.12, 120.31, 124.85,
127.14, 129.83, 130.03,130.25. MS, Calc. for C22H18N6O2,
molecular weight 398.42: m/z 398 (M+), 399 (M + 1)+.
6.1.34. 4-Amino-6-(2-methoxybenzylamino)-2-phenyl-2H-
[1,2,4]triazolo[4,3-a]quinoxalin-1-one (10k)
The reaction of 2-methoxybenzaldehyde with 8 required
4 h (TLC: Rf 0.48). Yield: 359 mg, 87%, m.p. 207–8 °C. 1H
NMR, d: 3.87, s, 3H, –OCH3; 4.40, t, 2H, –CH2–; 5.96, br s,
1H, –NH–; 6.55, d, 2H, H9 and phenyl H; 6.90, t, 1H, H-8;
7.06, t, 2H, phenyl H; 7.23–7.40, m, 5H, –NH2 and 3 phenyl
H; 7.57, t, 2H, phenyl H; 7.92, d, 1H, H-7; 8.09, d, 2H, phe-
nyl H. 13C NMR, d: 56.28 (benzylic), 102.47, 106.84, 111.67,
120.29, 121.10, 122.99, 124.41 12487, 127.12, 127.76,
129.19, 129.36, 130.02, 132.32, 138.26, 143.21, 144.88,
145.30, 149.40, 158.03. MS, Calc. for C23H20N6O2, molecu-
lar weight 412.44: m/z 412 (M+), 413 (M + 1)+.
6.1.38. 4-Amino-6-(2-fluoro-4-methoxybenzylamino)-2-
phenyl-2H- [1,2,4]triazolo[4,3-a]quinoxalin-1-one (10o)
Compound 8 reacted with 2-fluoro-4-methoxybenzal-
dehyde in 4 h (TLC: Rf 0.46). The solid residue was triturated
with hot MeOH (5 ml), cooled to 0 °C for 6 h, the yellow
solid filtered and air-dried. Product was recrystallized from
CH3CN. Yield: 362 mg, 84%, m.p. 179–81 °C. 1H NMR, d:
3.25, s, 3H, –OCH3; 4.39, t, 2H, –CH2–; 5.88, t, 1H, –NH–;
6.60, d, 1H, H-9; 6.73, m. 2H, phenyl H; 7.07, t, 1H, H-8;
7.35, m, 4H, –NH2 and 2 phenyl H; 7.55, m, 2H, phenyl H;
7.92, d, 1H, H-7; 8.09, d, 2H, phenyl H. 13C NMR, d: 56.43
(benzylic), 102.18, 102.69, 102.75, 106.76, 110.95, 111.01,
118.52, 118.83, 120.26, 120.40, 124.82, 127.11, 130.00,
131.32, 131.45, 132.25, 132.28, 138.24, 124.79, 145.33,
14540, 149.37, 159.49, 160.60, 160.82, 164.33. 19F NMR,
d: − 117.26. MS, Calc. for C23H19FN6O2, molecular weight
430.43: m/z 430 (M+), 431 (M + 1)+.
6.1.35. 4-Amino-6-(3-methoxybenzylamino)-2-phenyl-2H-
[1,2,4]triazolo[4,3-a]quinoxalin-1-one (10l)
The reaction of 3-methoxybenzaldehyde with 8 required
2 h (TLC: Rf 0.51). Yield: 375 mg, 91%, m.p. 190–1 °C. 1H
NMR, d: 3.71, s, 3H, –OCH3; 4.44, m, 2H, –CH2–; 6.03, t,
1H, –NH–; 6.58, d, 1H, H-9; 6.88, m, 1H, H-8; 7.03, m, 3H,
phenyl H; 7.24–7.40, m, 4H, –NH2 and 2 phenyl H; 7.57, m,

432
M.H. Holschbach et al. / European Journal of Medicinal Chemistry 40 (2005) 421–437
6.1.39. 4-Amino-6-(2-fluoro-5-methoxybenzylamino)-2-
phenyl-2H-[1,2,4]triazolo[4,3-a]quinoxalin-1-one (10p)
Compound 8 reacted in 6 h with 2-fluoro-5-methoxy-
benzaldehyde (TLC: Rf 0.46).Yield: 305 mg, 71%, m.p. 173–
5 °C. 1H NMR, d: 3.68, s, 3H, –OCH3; 4.46, t, 2H, 1-CH2–;
6.00, t, 1H, –NH–; 6.58, d, 1H, H-9; 6.56–7.14, m, 5H, –NH2
and 3 phenyl H; 7.34–7.59, m, 4H, H-8 and 3 phenyl H; 7.93,
d, 1H, H-7; 8.09, d, 2H, phenyl H. 13C NMR, d: 56.20 (ben-
zylic), 102.91, 106.84, 113.72, 113.88, 115.77, 115.86,
116.44, 116.90, 120.24, 123.09, 124.43, 124.81, 127.08,
127.96, 128.29, 129.98, 132.28, 138.24, 142.74, 145.35,
145.40, 149.35, 153.23, 156.28, 156.32, 157.92. 19F NMR,
d: − 130.24, MS, Calc. for C23H19FN6O2, molecular weight
430.43: m/z 430 (M+), 431 (M + 1)+.
6.1.43. Acetic acid (4-amino-1-oxo-2-phenyl-1,2-dihydro-
[1,2,4]triazolo[4,3-a]quinoxalin-6-ylcarbamoyl)-1-phenyl-
methyl ester (11a). General method C
O-Acetyl mandelic acid chloride (226 µl, 1 mmol) in dry
THF (2 ml) was added over 5 min to an ice-cold slurry of 8
(292 mg, 1 mmol) in dry THF (10 ml) containing triethy-
lamine (237 µl, 1.575 mmol). After stirring at 0 °C for 5 min
and room temperature for 10 min, TLC (Solvent B), Rf (educt)
0.20, Rf (product) 0.25 showed a quantitative reaction. Water
(50 ml) was added, the aqueous solution extracted with ethyl
acetate (2 × 50 ml), the pooled organic phases washed with
1 M NaHCO3 (2 × 50 ml) and brine (2 × 50 ml), dried over
anhydrous Na2SO4 and the solvent evaporated. Boiling the
solid yellow residue in methanol (10 ml) and cooling gave an
analytically pure product that was collected by filtration and
dried at 120 °C. Yield 440 mg, 95%, m.p. 242 °C. 1H NMR,
d: 2.34, t, 3H, –COCH3; 6.18, s, 1H, PhCH(OAc)CO–; 7.23–
8.37, m, 15H, –NH2, H-7, H-8, H-9 and 10 phenyl H; 10.00,
s, 1H, –CONH–. 13C NMR, d: 21.67, 76.52, 110.05, 115.71,
120.31, 123.89, 124.54, 126.07, 127.27, 128.22, 129.64,
129.80, 130.07, 131.77, 132.11, 136.34, 138.10, 146.69,
149.25, 167.08, 170.54. MS, Calc. for C25H20N6O4, molecu-
lar weight 468.46: m/z 468 (M+).
6.1.40. 4-Amino-6-furan-2-ylmethylamino-2-phenyl-2H-
[1,2,4]triazolo[4,3-a]quinoxalin-1-one (10q)
The reaction of furan-2-carboxaldehyde with 8 required
2 h (TLC: Rf 0.50). Yield: 328 mg, 88%, m.p. 204–5 °C. 1H
NMR, d: 4.46, m, 2H, –CH2–; 5.88, t, 1H, –NH–; 6.22–6.43,
M, 2H, H-9 and furyl H; 6.72, d, 1H, furyl H-; 7.10, t, 1H,
H-8; 7.34, m, 3H, furyl H and 2 phenyl H; 7.68,m, 3H, –NH2
and phenyl H; 7.94, d, 1H, H-7; 8.09, d, 2H, phenyl. 13C NMR,
d: 40.75 (benzylic); 102.94, 107.06, 108.20, 111.32, 120.30,
123.09, 124.40, 124.76, 127.13, 130.14, 132.29, 138.24,
142.65, 143.20, 145.41, 149.38, 153.64. MS, Calc. for
C20H16N6O2, molecular weight 372.38: m/z 372 (M+).
6.1.44. N-(4-Amino-1-oxo-2-phenyl-1,2dihydro-[1,2,4]tria-
zolo[4,3-a]quinoxalin-6-yl)-2-fluoro-2-phenyl-acetamide
(11b)
Method C applied to 8 (1.02 g, 3.5 mmol) in dry THF
(30 ml), triethylamine (700 µl, 5 mmol) and ( )-a-
fluorophenylacetic acid chloride (634 mg, 3.675 mmol) in
dry THF (2 ml), with stirring at 0 °C for 5 min and then at
room temperature for 20 min, gave a quantitative yield of
product by TLC (ethyl acetate/hexane 35:65, v/v), Rf (start-
ing material) 0.20, Rf (product) 0.51. Workup gave analyti-
6.1.41. 4-Amino-6-(5-bromofuran-2-ylmethylamino)-2-
phenyl-2H-[1,2,4]triazolo[4,3-a]quinoxalin-1-one (10r)
The reaction of 5-bromofuran-2-carboxaldehyde with 8
required 2 h (TLC: Rf 0.53). Yield: 415 g, 92%, m.p. 193–
4 °C. 1H NMR, d: 4.46, t 2H, –CH2–; 5.92, br s, 1H, –NH–;
6.45, m, 2H, H-9 and furyl H; 6.69, d, 1H. furyl H; 708, t, 1H,
H-8; 7.35, m, 3H, –NH2 and phenyl H; 7.58, m, 2H, phenyl;
7.94, d, 1H, H-7; 8.08, d, 2H, phenyl H. 13C NMR, d: 40.61
(benzylic), 103.08, 107.04, 111.27, 113.18, 120.28, 120.89,
123.13, 124.43, 124.72, 127.11, 130.00, 132.27, 138.23,
142.36, 144.88, 145.42, 149.35, 156.25. MS, Calc. for
C20H15BrN6O2, molecular weight 451.28: m/z 451 (M+).
1
cally pure product. Yield 1.36 g, 91%, m.p. 219–21 °C. H
NMR, d: 6.28, d, 1H, –CH(F)–, J (H,F) 47 Hz; 7.18–7.60, m,
11 H, –NH2, –NH–, H-8, H-9, –CH2–, 4 phenyl H; 787, S,
2H, phenyl H; 8.08, d, 2H, phenyl H; 8.31, m, 2H, phenyl,
10.16, d, 1H, H-7. 13C NMR, d: 110.28, 115.75, 120.25,
123.81, 124.51, 126.25, 127.24, 128.05, 128.16, 129.70,
130.04, 131.40, 132.05, 135.82, 138.10, 146.78, 149.20,
166.76. 19F NMR, d: − 172.9. MS, Calc. for C23H17FN6O2,
molecular weight 428.42: m/z 428 (M+).
6.1.42. 4-Amino-6-(5-iodofuran-2-ylmethylamino)-2-phe-
nyl-2H-[1,2,4]triazolo[4,3-a]quinoxalin-1-one (10s)
6.1.45. N-(4-Amino-1-oxo-2-phenyl-1,2-dihydro-
[1,2,4]triazolo[4,3-a]quinoxalin-6-yl)- acetamide (11c)
Method C applied to 8 (584 mg, 2 mmol), triethylamine
(420 µl, 3 mmol) and acetyl chloride (149 µl, 2.1 mmol) in
dry THF (2 ml) gave a quantitative reaction by TLC (Solvent
B), Rf (starting material) 0.20, Rf (product) 0.00, blue fluo-
rescence in UV light). The addition of water (20 ml) and cen-
trifugation (10 min at 2600 × g) separated a pale yellow solid
that was dried at 120 °C overnight. Yield 550 mg, 83%, m.p.
The reaction of 5-iodofuran-2-carboxaldehyde [38] with
8 required 2 h (TLC: Rf 0.55).Yield: 448 mg, 90%, m.p. 173–
4 °C. 1H NMR, d: 4.48, m, 2H, –CH2–; 5.92, br s, 1H, –NH–;
6.35, t, 1H, furyl H; 6.65, d, 2H, H-9 and furyl H; 7.09, t, 1H,
H-8; 7.38, m, 3H, –NH2 and phenyl H; 7.67, m, 2H, phenyl
H; 7.93, d, 1H, H-7, 8.09, d, 2H, phenyl H. 13C NMR, d:
40.61 (benzylic), 91.11, 103.03, 107.01, 111.43, 120.28,
121.45, 123.09, 124.42, 124.77, 127.11, 130.00, 132.27,
138.22, 142.40, 145.41, 149.35, 156.24, 159.15. MS, Calc.
for C20H15IN6O2, molecular weight 498.28: m/z 498 (M+).
1
207–8 °C. H NMR, d: 2.20, s, 3H, –COCH3; 7.24, t, 1H,
H-9; 7.40, m, 1H, H-8; 7.57, m, 2H, phenyl H; 7.72, br s, 2H,
–NH2; 809, d, 2H, phenyl H; 8.23–8.46, m, 2H, H-7 and phe-

M.H. Holschbach et al. / European Journal of Medicinal Chemistry 40 (2005) 421–437
433
nyl H; 9.34, s 1H, –CONH–. 13C NMR, d: 109.41, 120.38,
123.83, 124.50, 127.29, 130.07, 132.05, 133.03, 138.13,
144.88, 146.47, 149.30, 168.93. MS, Calc. for C17H14N6O2,
molecular weight 334.33: m/z 334 (M+).
Semi-preparative radio-HPLC used a C-18 column (Mul-
tospher 120 5 µm, 250 × 8 mm) eluted with acetonitrile/water,
30:70 (v/v) at a flow rate of 4 ml/min. In this system the
k′-values of the precursor Schiff base and the tritiated ligand
were 2.61 and 5.83, respectively. For continuous measure-
ment of radioactivity the outlet of the UV detector was con-
nected to a flow scintillation analyzer (Radiomatic™ 515 TR
Series, Packard, Dreieich, Germany) and the collected data
were processed by an integrated software system.
Quality control of the product employed a Supersphere
Si-60 column (250 × 4 mm) eluted with ethyl acetate/hexane,
50/50 (v/v) containing 0.6% triethylamine at a flow rate of
1 ml/min. Under these conditions the k′-value for the tritiated
ligand was 2.88 and that of the educt 4.22. Measurement of
the specific activity of the product used HPLC with UV moni-
toring at 254 nm. Integrating the UV absorption of the prod-
uct peak and referring that area to a standard curve measured
the mass of product. The product peak was collected and an
aliquot counted in a liquid scintillation counter. The RCY of
tritiated ligand was 12.5%, radiochemical purity was > 98%
and specific activity was 5.6 Ci/mmol. When stored at –20 °C
the product was stable for over 6 months.
6.1.46. N-(4-Amino-1-oxo-2-phenyl-1,2-dihydro-
[1,2,4]triazolo[4,3-a]quinoxalin-6-yl)-2-phenyl-acetamide
(11d)
Method C employed 8 (584 mg, 2 mmol) in dry THF
(20 ml), triethylamine (420 µl, 3 mmol) and phenylacetic acid
chloride (278 µl, 2.1 mmol) in dry THF (2 ml). TLC showed
a quantitative reaction (Solvent B), Rf (starting material) 0.20,
Rf (product) 0.35, blue fluorescence in UV light). Workup
1
yielded 804 mg, 93%, of 11d, m.p. 213–4 °C. H NMR, d:
3.83, t, 2H, PhCH2–; 7.16–7.68, m, 10 H, –NH2, C-9, C-8,
6 phenyl H; 8.06–8.33, m, 5H, H-7 and 4 phenyl H; 9.50, s,
1H, –CONH–. 13C NMR, d: 44.79, 109.55, 116.25, 120.36,
123.87, 124.51, 125.97, 127.29, 127.64, 129.44, 130.07,
130.26, 132.04, 132.82, 136.45, 138.12, 146.42, 149.27,
169.69. MS, Calc. for C23H18N6O2, molecular weight 410.43,
m/z 410 (M+).
6.1.47. N-(4-Amino-1-oxo-2-phenyl-1,2-dihydro-
[1,2,4]triazolo[4,3-a]quinoxalin-6-yl)-2,2,2-trifluoro-
acetamide (11e)
7. Biological evaluations
Method C employed 8 (292 mg, 1 mmol) in dry THF
(10 ml), triethylamine (210 µl, 1.5 mmol) and trifluoroacetic
acid anhydride (149 µl, 1.05 mmol) in dry THF (2 ml). TLC
(Solvent B): Rf (starting material) 0.20, Rf (product) 0.59,
fluorescent in UV light). Workup gave, after air-drying, ana-
lytically pure product as pale cream-colored crystals, 340 mg,
7.1. Radioligand binding assays using rat brain
membranes
Corpora striata (for A2AAR assays) and frontal cortices
(for A1AR assays) from rat brains were homogenized for
1 min in 20 volumes of ice-cold 50 mM Tris–HCl buffer, pH
7.4 containing 10 mM MgCl2, soybean trypsin inhibitor
(20 µg/ml), bacitracin (200 µg/ml), and benzamidine HCl
(160 µg/ml) by means of an Ultra Turrax at 20,000 rpm. The
homogenate was centrifuged at 48,000 × g for 10 min at 4 °C
(Beckmann Optima L, SW41Ti rotor). The pellet was sus-
pended in 20 volumes of Tris–HCl, pH 7.4, containing AD
(2 U/ml) and Trypsin Inhibitor (20 µg/ml), and then was incu-
bated for 30 min at 37 °C. After centrifugation at 48,000 × g
for 10 min at 4 °C the resulting pellet was diluted in 20 vol-
umes of 50 mM Tris–HCl, pH 7.4, containing 10 mM MgCl2.
Aliquots of the homogenate (1 ml) were stored at –80 °C.
The assays were performed in triplicate by incubating ali-
quots of the membrane fractions (65–120 µg protein per assay)
in Tris–HCl, pH 7.4, containing AD (2 U/ml), [3H]CPDPX
(2 nM) and cortical homogenates for the A1AR or striatal
homogenates for A2AAR, in a total assay volume of 200 µl.
Incubation was carried out at 20 °C for 90 min. In saturation
experiments the non-specific binding was defined as the bind-
ing in the presence of either 10 µM CPDPX or 50 µM
2-chloroadenosine for studies of the A1AR and A2AAR,
1
87%, m.p. 222 °C. H NMR, d: 7.30, m, 2H, H-9 and H-8;
7.56, m, 2H, phenyl H, 7.76–8.08, m, 4H, –NH2, H-7 and
phenyl H; 8.42, d, 2H, phenyl H; 10.37, s, 1H, CF3CONH–.
13C NMR, d: 112.20, 118.20, 120.30, 123.71, 124.76, 127.33,
128.10, 129.54, 130.07, 131.95, 138.05, 147.16, 149.19,
171.23. 19F NMR, d: − 74.90. MS, Calc. for C17H11F3N6O2,
molecular weight 388.37, m/z 388 (M+).
6.1.48. Radiosynthesis of [3H]10a
[3H]NaBH4 reduction of the Schiff base 9a generated
[3H]10a.A 2 ml vial containing a magnetic stirring bar sealed
with a rubber septum was charged with a solution of
[3H]NaBH4 (150 mCi, 85 Ci/mmol, 66 µg, 1.8 µmol) in dry
n-propanol (75 µl). To this solution was added under stirring
a mixture of 9a (2 mg, 5.3 µmol) in dry DMF (200 µl) fol-
lowed by dry methanol (500 µl). After stirring for 8 min at
room temperature, the reaction was stopped by the addition
of acetonitrile/water, 30:70 (v/v). The reaction mixture was
transferred to a semipreparative HPLC column in several
400 µl portions, the fractions containing product were col-
lected, pooled (total volume 26 ml) and diluted with water
(150 ml). The aqueous solution was passed through a Sep-
Pak cartridge (flow 5 ml/min), the cartridge was washed
with water (40 ml) and the product was eluted with ethanol
(10 ml).
respectively. In
A2AAR competition experiments
[3H]CGS21680 was used in a concentration of 1.5 nM. Cen-
trifugation at 48,000 × g for 6 min at 8 °C separated bound
from free ligand. Supernatants were discarded, the pellets

434
M.H. Holschbach et al. / European Journal of Medicinal Chemistry 40 (2005) 421–437
washed with 1 ml ice cold buffer and dissolved by incubating
in SolvableTM (500 µl, Canberra-Packard) for 120 min at
50 °C. Aliquots of 450 µl were placed in scintillation vials
with scintillation cocktail (10 ml, Ultima Gold XR, Canberra-
Packard). Radioactivity was measured in a liquid scintilla-
tion counter. Protein estimation was performed with a com-
mercial assay (Bio-Rad DC ProteinAssay) after solubilization
in 15% NH4OH containing 2% SDS (w/v); human serum
albumin served as a standard. A computer-assisted curve-
fitting program (Graph Pad Prism, version 3.0) calculated
IC50, Ki and KD.
2000 employed a non-linear, curve-fitting analysis program
(Graph Pad Prism 3.00).
7.3. Measurement of an index of lipophilicity
A method employing HPLC [39] estimated of the lipophi-
licities of compounds 8, 9a and 10a–s. Briefly, that method
called for measurements of the retention times of the test com-
pounds on a 4.6 × 250 mm column of 5 µm Kromasil
RP18 eluted at a rate of 1 ml/min with each of the following
proportions of water/methanol: 22.5:77.5; 20:80; 15:85, or
10:90, with monitoring the effluent at 254 nm. Converting
each datum to a capacity factor k’ by the formula k’ = (t –
to)/to, where t and to are the retention time and void time,
respectively, and solving the linear regression of log k’ on
methanol: water ratio for methanol = 0 gave the lipophilicity
parameter log k’w.
7.2. Autoradiography
Frozen rat brains (–20 °C) were sectioned at a thickness of
20 µm in the horizontal plane (Leica AG Microsystems, Ger-
many), mounted on gelatine-coated microscope slides (La-
boroptik GmbH, Germany), dried at 4 °C and stored until use
at –80 °C. Tissue sections were preincubated for 15 min at
4 °C in 50 mM Tris–HCl containing 1 mM MgCl2 and 2 U/ml
AD, pH 7.4, and thereafter for 90 min at 23 °C in the same
buffer containing either 0.9 nM [3H]CPDPX, 13 nM [3H]10a
or 2.2 nM [3H]ZM241385, alone or with 200 nM 10a. Some
sections were incubated for 60 min with 5 µM CPDPX or
CGS21680 before [3H]10a incubation (blocking experi-
ment), others were incubated with [3H]10a followed by incu-
bation with 5 µM CPDPX or CGS21680 for 60 min (displace-
ment experiment). The sections were washed twice for 5 min
in 50 mM Tris–HCl at 4 °C, rinsed in deionized water, dried
in a stream of cold air and placed on a phosphor image plate
(Fujifilm) and exposed for 4 days. Laser scanning of the plates
employed a phosphor imager BAS 5000 (Fujifilm) con-
trolled with software provided by the vendor (Version 2.11a,
Raytest Isotopenmeßgeräte, Germany). The advanced image
analyzer program (AIDA 3.10, Raytest Isotopenmeßgeräte,
Germany) analyzed data from regions of interest (ROIs).
Analysis of binding data arrayed in Microsoft Excel
7.4. Determination of water solubility
A saturated solution of the test compounds in water was
prepared. After the undissolved material was separated by
centrifugation (20,000 × g, 10 min), the supernatant was
diluted with water to a defined volume and the UV absorp-
tion of aliquots (n = 3) of the aqueous solution was deter-
mined at kmax. The concentration of the solution was calcu-
lated using a previously determined standard calibration curve
for each compound.
7.5. Data analysis
Radioligand binding data are the mean and 95% confi-
dence limits of three separate experiments, each in triplicate.
Acknowledgements
R.A.O. was supported in part by the Ed C. Wright Chair in
Cardiovascular Research, University of South Florida.

M.H. Holschbach et al. / European Journal of Medicinal Chemistry 40 (2005) 421–437
435
Appendix
Elemental analyses of new compounds
Compounds
Formula
Molecular
weight
C
H
N
O
Calculated
Found
C
H
N
O
5
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
15H8N6O3
348.28
582.55
322.28
292.30
380.40
442.45
382.42
444.46
396.44
414.43
400.41
400.41
400.41
398.42
398.42
398.42
412.44
412.44
412.44
416.41
430.43
430.43
372.38
451.28
498.28
468.46
428.42
334.33
410.43
388.30
51.73
51.71
38.04
37.97
55.90
55.92
61.64
61.65
69.46
69.41
65.15
65.09
69.10
69.08
64.86
64.81
69.68
69.70
66.66
66.68
65.99
65.95
65.99
66.03
65.99
65.96
66.32
66.30
66.32
66.28
66.32
66.33
66.98
67.03
66.98
66.99
66.98
66.95
63.46
63.42
64.18
64.19
64.18
64.20
64.51
64.48
53.23
53.19
8.21
2.32
2.36
3.98
4.04
3.13
3.06
4.14
4.08
4.24
4.27
4.33
4.30
4.74
4.70
4.76
4.79
5.08
5.11
4.62
4.58
4.28
4.31
4.28
4.30
4.28
4.29
4.55
4.53
4.55
4.54
4.55
4.58
4.89
4.84
4.89
4.88
4.89
4.92
4.11
4.14
4.45
4.43
4.45
4.47
4.33
4.36
3.35
3.33
3.03
2.99
4.30
4.31
4.00
4.02
4.22
4.20
4.42
4.41
2.86
2.90
32.17
32.21
14.43
14.39
26.08
26.13
28.75
28.81
22.09
22.14
18.99
19.03
21.98
22.02
18.91
18.88
21.20
21.17
20.28
20.27
20.99
21.03
20.99
20.94
20.99
21.03
21.09
21.13
21.09
21.05
21.09
21.12
20.38
20.41
20.38
20.34
20.38
20.40
20.18
20.20
19.52
19.56
19.52
19.49
22.57
22.61
18.62
18.60
16.87
16.91
17.94
17.91
19.62
19.65
25.14
25.16
20.48
20.51
21.64
21.61
13.78
13.74
8.24
8.20
14.89
14.87
5.47
5.42
4.21
4.18
7.23
7.26
4.18
4.14
7.20
7.24
4.04
4.07
3.86
3.88
4.00
4.04
4.00
3.96
4.00
4.01
8.03
7.99
8.03
8.07
8.03
8.01
7.76
7.72
7.76
7.78
7.76
7.73
7.68
7.66
7.43
7.39
7.43
7.44
8.59
8.60
7.09
7.13
6.42
6.38
13.66
13.69
7.47
7.50
9.57
9.61
7.80
7.83
8.24
8.27
6
33H23N6O3P
15H10N6O3
15H12N6O
7
8
9a
22H16N6O
9b
24H19N6O2
22H18N6O
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
10q
10r
10s
11a
11b
11c
11d
11e
24H21FN6O2
23H20N6O
23H19FN6O
22H17FN6O
22H17FN6O
22H17FN6O
22H18FN6O2
22H18FN6O2
22H18FN6O2
23H20N6O2
23H20N6O2
23H20N6O2
22H17FN6O2
23H19FN6O2
23H19FN6O2
20H16N6O2
20H15BrN6O2
20H15IN6O4
25H20N6O4
23H17FN6O2
17H14N6O2
23H18N6O2
17H11F3N6O2
48.18
64.10
64.11
64.48
64.52
61.07
61.04
67.31
67.28
52.58
52.62

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M.H. Holschbach et al. / European Journal of Medicinal Chemistry 40 (2005) 421–437
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