Synthesis and benzodiazepine receptor binding activity of 2,9-disubstituted quinolino[2′,3′-5,4](3-pyrazolino)[3,2-b]purin-4-ones

Article abstract of DOI:10.1002/1521-4184(200205)335:5<207::AID-ARDP207>3.0.CO;2-5

2,9-Disubstituted quinolino[2′,3′-5,4](3-pyrazolino)pyrimidin-2-ones and purin-4-ones were synthesized and their benzodiazepine receptor activity was evaluated for their ability to displace [³H]R015-1788 from its specific binding in bovine brain membranes. Compound 5 c caused 83 ± 8 % inhibition in [³H]R015-1788 specific benzodiazepine receptor binding followed by compounds 5 f, 5 h, and 5 i while other analogs were inactive at 10 μM concentration.

Full text of DOI:10.1002/1521-4184(200205)335:5<207::AID-ARDP207>3.0.CO;2-5

Arch. Pharm. Pharm. Med. Chem. 2002, 5, 207–212
Quinolino[2Ј,3Ј-5,4](3-pyrazolino)[3,2-b]purin-4-ones 207
Ola A. El-Sayeda,
Synthesis and Benzodiazepine Receptor Binding
Activity of 2,9-Disubstituted
Quinolino[2Ј,3Ј-5,4](3-pyrazolino)[3,2-b]purin-4-ones
Nargues S. Habiba,
Hassan Y. Aboul-Eneinb,
Barbara Costac,
Antonio Lucacchinic,
Claudia Martinic
2,9-Disubstituted quinolino[2Ј,3Ј-5,4](3-pyrazolino)pyrimidin-2-ones and purin-4-
ones were synthesized and their benzodiazepine receptor activity was evaluated for
their ability to displace [3H]R015-1788 from its specific binding in bovine brain mem-
branes.Compound 5 c caused 83 ± 8 % inhibition in [3H]R015-1788 specific benzo-
diazepine receptor binding followed by compounds 5 f, 5 h, and 5 i while other ana-
logs were inactive at 10 µM concentration.
a
Pharmaceutical Chemistry
Department,
Faculty of Pharmacy,
University of Alexandria.
Alexandria, Egypt
b
Pharmaceutical Analysis
Keywords: Quinolino[2Ј,3Ј-5,4](3-pyrazolino)pyrimidines or purines, synthesis;
Benzodiazepine receptor binding
Laboratory, Biological &
Medical Research
(MBC-03), King Faisal
Specialist Hospital and
Research Centre, Riyadh,
Saudi Arabia
Received: December 10, 2001 [FP655]
c
Department of Psychiatry,
Neurobiology, Pharmacology
and Biotechnology, Pisa,
Italy
thienyl as bioisosteric planar group at position 2 to study
their biological effect on BZR affinity.
Introduction
A number of synthetic compounds with different struc-
tures have been found to possess high affinity binding for
the benzodiazepine receptor (BZR) and act either as ag-
onists or antagonists [1–3].The BZR is unique in the way
it responds to three different types of ligands which act
as allosteric modulators of the GABAA receptor complex
[4]. The interrelationships of these three types of BZR
can be explained on the basis of changes in the confor-
mation of the receptor from its unoccupied resting state
[5].Thus all compounds that bind to the BZR should have
certain common characteristics, viz.a planar hetero-aro-
matic system containing at least one nitrogen atom and
a carbonyl group adjacent to the nitrogen atom [4].
Results and discussion
Chemistry
The synthesis of 2,9-disubstituted quinolino[2Ј,3Ј-
5,4](3-pyrazolino)[3,2-b]purin-4-ones is illustrated in
Scheme 1. Thus, the starting 7-substituted 3-amino-
pyrazolo[3,4-b]quinolines (1 b, c) were prepared accord-
ing to our previously reported method for the preparation
of (1 a) [6]. (1 a–c) were subjected to 1,3-dipolar cycliza-
tion reaction using ethyl oximinocyanoacetate to afford
9-substituted 4-amino-3-nitrosoquinolino[2Ј,3Ј-5,4](3-
pyrazolino)[2,3-a]pyrimidin-2-ones (2 a–c) in good
yields. Upon reduction with sodium dithionite/NH4OH
30 % at 70–80 °C, these compounds produced the key
intermediate 9-substituted 3,4-diaminoquinolino[2Ј,3Ј-
5,4](3-pyrazolino)[2,3-a]pyrimidin-2-ones (3 a–c) in
good yields. Compounds 3 a–c were acylated with ap-
propriate carboxylic acid chloride to give the corre-
sponding acyl derivatives 4 a–i.The latter were cyclized
to the target 2,9-disubstituted quinolino[2Ј,3Ј-5,4](3-
pyrazolino)[3,2-b]purin-4-ones using sodium methoxide
in methanol. The structure of the prepared compounds
Non-benzodiazepine compounds with affinity for the
BZR could be of potential importance as tools for study-
ing the receptor itself and eventually for the introduction
into clinical use of new classes of compounds having the
same properties as benzodiazepines [3].
Following these findings we have prepared some
quinolino[2Ј,3Ј-5,4](3-pyrazolino)[3,2-b]purin-4-ones
bearing an alkyl substituent at position 9 and phenyl or
Correspondence: Hassan Y. Aboul-Enein, Pharmaceutical
Analysis Laboratory, Biological & Medical Research (MBC-03),
King Faisal Specialist Hospital
1
was confirmed by IR and H-NMR.
&
Research Centre,
P.O. Box 3354, Riyadh 11211, Saudi Arabia. Phone:
+966-1-442 78 59, Fax +966-1-442 78 58, e-mail:
enein@kfshrc.edu.sa
The IR spectra of 4-amino-3-nitrosoquinolino-
[2Ј,3Ј-5,4](3-pyrazolino)[2,3-a]pyrimidin-2-ones (2 a–c)
© WILEY-VCH Verlag GmbH, 69451 Weinheim, 2002
0365-6233/05/0207 $ 17.50+.50/0

208 El-Sayed et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 207–212
Scheme 1.
showed NH2, N=O, and C=O at 3390–3392, 3320–3327,
1549–1550, 1635–1640 cm–1, respectively; their 1H-
NMR (400 MHz, DMSO-d6) showed, beside the signals
characteristic for the ring protons and the substituents at
position 9 (either CH3 or OCH3), two singlets – one due to
NH at 11.67–13.57 ppm and one due to NH2 either at
8.95 ppm (compound 2 a) or about 5.8 ppm (compounds
2 b, c).
2 NH and NH2 at about 13, 11, and 5.8 ppm respectively,
beside the signals characterizing the protons of the ring
and substituents at their expected chemical shifts.
The IR spectra of the purine derivatives 5 a–i showed on-
1
ly one C=O band at about 1693–1648 cm–1. Their H-
NMR showed two deuterium exchangeable NH, one of
the purine ring and the other of the pyrazoline ring, these
beside the protons of the ring and substituents at their
expected chemical shifts (see experimental section).
The IR spectra of 3,4-diamino derivatives (3 a–c)
showed the disappearance of N=O band at 1549–1550
and the appearance of NH and C=O bands at 3220,
1
3125, and 1630 cm–1, respectively. The H-NMR of 3 a
showed three singlets due to NH and 2 NH2 at 13.55,
8.54, and 8.3 ppm.
Pharmacological activity
The acylamino derivatives showed in their IR spectra two
C=O bands at 1663, 1648 cm–1 due to the acyl group and
the pyrimidone C=O, respectively.The 1H-NMR of 4 a–c
showed three deuterium exchangeable singlets due to
The results of the benzodiazepine receptor binding stud-
ies for the nine synthesized compounds 5 a–i are shown
in Table 1. The data indicate that compound 5 g caused
83 ± 8 % inhibition in the binding of [3H]Ro15-1788 lig-

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 207–212 Quinolino[2Ј,3Ј-5,4](3-pyrazolino)[3,2-b]purin-4-ones 209
Table 1. Central benzodiazepine receptor binding affinity of compounds 5 a–i.
Compound
R1
R2
Inhibition (%)a
Ki (µM)b
5 a
5 b
5 c
5 d
5 e
5 f
5 g
5 h
5 i
H
CH3
CH3
CH3
C6H5
C6H5
C6H5
2-thienyl
2-thienyl
2-thienyl
0
0
0
0
0
CH3
OCH3
H
CH3
OCH3
H
25 ± 2
1.02 ± 0.1
83 ± 8
16 ± 2
27 ± 3
CH3
OCH3
Clonazepam
Ro 15-1788
Diazepam
Zolpidem
0.00085 ± 0.00001
0.00090 ± 0.00001
0.00500 ± 0.00043
0.05100 ± 0.00380
a
Percent of inhibition of specific [3H]Ro15-1788 binding at 10 µM concentration are means ± SEM of five determina-
tions (the tests were carried out using DMSO as solvent).
Ki value is the mean ± SEM of five determinations.
b
and in bovine brain membranes, while compounds 5 h,
3-Amino-7-methylpyrazolo[3,4-b]quinoline (1 b)
5 f, and 5 i showed inhibitory activity ranging from
16 ± 2 % to 27 ± 3 %. All the other analogs were inac-
tive. However the Ki value of compound 5 g was
1.02 ± 0.1 µM which is considered high as compared to
other reference drugs such as clonazepam, diazepam,
and zolpidem.It is of interest to mention that the 2-thienyl
substituted analogs showed better inhibitory activity
compared to their 2-phenyl and 2-methyl counterparts.
Further studies will be carried out using combinatorial
chemistry techniques in order to improve on the activity
of the lead compound 5 g.
Mp 209–210 °C; yield 70 %; IR ν cm–1: 3331, 3190 (NH); 1623
(C=N); 1585, 1503 (C=C); 1549 (δ NH).
3-Amino-7-methoxypyrazolo[3,4-b]quinoline (1 c)
Mp 142–143 °C; yield 65 %; IR ν cm–1: 3299, 3185 (NH); 1621
(C=N); 1551 (δ NH); 1504 (C=C); 1224, 1027 (C–O–C).
9-Substituted 4-amino-3-nitrosoquinolino[2Ј,3Ј-5,4](3-pyrazo-
lino)[2,3-a]pyrimidin-2-one (2 a–c): General procedure
To a stirred solution of sodium ethoxide (sodium 13.8 g,
0.6 mol) in anhydrous ethanol (400 mL) at 5–10 °C, ethyl oximi-
nocyanoacetate [7] (28.4 g, 0.2 mol) and the appropriate amine
(1 a–c) (0.2 mol) were added.The reaction mixture was reflux-
ed for 3 h, cooled, and the solid sodium salt was filtered off, dis-
solved in H2O (100 mL), and the pH of the resulting solution was
adjusted to 6 with dil. HCl. The obtained nitroso derivatives
(2 a–c) were filtered off, washed with H2O, and crystallized from
DMF/EtOH (1 : 1).
Experimental
Chemistry
4-Amino-3-nitrosoquinolino[2Ј,3Ј-5,4](3-pyrazolino)[2,3-a]pyr-
imidin-2-one (2 a)
Melting points were determined in open glass capillaries using
a Gallenkamp apparatus and are uncorrected. IR spectra
(KBr): Perkin-Elmer FTIR spectroscopy 1000. 1H-NMR spectra
were recorded on a Jeol-NMR spectrometer 400 MHz (DMSO-
d6).Chemical shifts are expressed in δ (ppm) downfield from in-
ternal TMS as reference. Mass spectra: Mat-711 spectrometer,
inlet temp.ca 200 °C, ionization energy 70 eV.Elemental analy-
ses were performed at the central laboratory of the College of
Pharmacy, King Saud University and the found values were
within ± 0.4 % of the theoretical values.
Mp 207–209 °C; yield 62 %; IR ν cm–1 3393, 3327 (NH); 1635
(C=O);1573 (δ NH);1549 (N=O);1508 (C=C).1H-NMR δ ppm =
13.57 (s, 1 H, NH, D2O exchangeable); 8.95 (s, 2 H, NH2, D2O
exchangeable); 8.44 (s, 1 H, C12-H); 8.14 (d, 1 H, C8-H, J =
8 Hz); 8.01 (d, 1 H, C11-H, J = 8.8 Hz); 7.79 (dd, 1 H, C10-H, J =
8.8, 7.7 Hz); 7.49 (dd, 1 H, C9-H, J = 8.0, 7.7 Hz); Anal.
C13H8N6O2 (C, H, N).
9-Methyl-4-amino-3-nitrosoquinolino[2Ј,3Ј-5,4](3-pyrazolino)-
[2,3-a]pyrimidin-2-one (2 b)
7-Substituted 3-aminopyrazolo[3,4-b]quinolines
These compounds were prepared according to the previously
published procedure [6].
Mp 185–187 °C; yield 51 %; IR ν cm–1 3390, 3325 (NH); 1640
(C=O);1570 (δ NH);1550 (N=O);1508 (C=C).1H-NMR δ ppm =

210 El-Sayed et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 207–212
8.44 (d, 1 H, C8-H, J = 8 Hz); 8.3–7.19 (m, 3 H, C9,10,11-H); 2.61
(s, 3 H, CH3). Anal. C15H12N6O2 (C, H, N).
11.67 (s, 1H, NH, D2O exchangeable);8.68 (s, 1 H, C12-H);7.88
(d, 1 H, C11-H, J = 8.54 Hz);7.62 (s, 1 H, C8-H);7.21 (d, 1 H, C10-
H, J = 8.54 Hz); 5.86 (s, 2 H, NH2, D2O exchangeable); 2.50 (s,
3 H, CH3). Anal. C14H10N6O2 (C, H, N).
9-Methyl-3-acetyl-4-aminoquinolino[2Ј,3Ј-5,4](3-pyrazolino)-
[2,3-a]pyrimidin-2-one (4 b)
9-Methoxy-4-amino-3-nitrosoquinolino[2Ј,3Ј-5,4](3-pyrazo-
lino)[2,3-a]pyrimidin-2-one (2 c)
Mp 195–196 °C;yield 72 %; 1H-NMR δ ppm = 13.47, 11.69 (2 s,
2 H, 2 NH, D2O exchangeable);8.85 (s, 1 H, C12-H);8.37 (s, 1 H,
C8-H); 7.84 (d, 1 H, C11-H, J = 8.56 Hz); 7.31 (d, 1 H, C10-H, J =
8.55 Hz); 5.88 (s, 2 H, NH2, D2O exchangeable); 2.53 (s, 6 H,
CH3 and COCH3). Anal. C16H14N6O2 (C, H, N).
Mp 240–241 °C; yield 70 %; IR ν cm–1: 3392, 3320 NH); 1644
(C=O);1570 (δ NH);1550 (N=O);1508 (C=C).1H-NMR δ ppm =
11.68 (s, 1 H, NH, D2O exchangeable);8.63 (s, 1 H, C12-H);7.85
(d, 1 H, C11-H, J = 8.2 Hz); 7.18 (s, 1 H, C8-H); 7.00 (d, 1 H, C10-
H, J = 8.2 Hz); 5.84 (s, 2 H, NH2, D2O exchangeable); 3.88 (s,
3 H, OCH3). Anal. C14H10N6O3 (C, H, N).
9-Methoxy-3-acetylamino-4-aminoquinolino[2Ј,3Ј-5,4]-
(3-pyrazolino)[2,3-a]pyrimidin-2-one (4 c)
Mp 185–186 °C;yield 54 %; 1H-NMR δ ppm = 13.21, 11.66 (2 s,
2 H, 2 NH, D2O exchangeable);8.63 (s, 1 H, C12-H);7.87 (d, 1 H,
C11-H, J = 8.5 Hz); 7.17 (s, 1 H, C8-H); 7.01 (d, 1 H, C10-H, J =
8.5 Hz); 5.83 (s, 2 H, NH2, D2O exchangeable); 3.97 (s, 3 H,
OCH3); 2.17 (s, 3 H, COCH3). Anal. C16H14N6O3 (C, H, N).
9-Substituted 3,4-diaminoquinolino[2Ј,3Ј-5,4](3-pyrazolino)-
[2,3-a]pyrimidin-2-one (3 a–c): General procedure
To a stirred solution of (2 a–c) (20 mol) in aqueous 30 % NH4OH
(250 mL) heated at 70 °C a solution of sodium dithionite (10.5 g,
60 mmol) in H2O (200 mL) was added dropwise at such a rate
as to maintain the internal temperature at 70–80 °C. Heating
and stirring were continued for 30 min; the mixture was then
concentrated in vacuo to 150 mL and the slow-forming precipi-
tate was filtered off after an hour to give compounds (3 a–c)
which were used without further purification. Samples for ana-
lytical purposes were crystallized from DMF/EtOH (2 : 1)
(3 a & b), DMF (3 c).
3-Benzoylamino-4-aminoquinolino[2Ј,3Ј-5,4](3-pyrazolino)-
[2,3-a]pyrimidin-2-one (4 d)
Mp 144–145 °C;yield 67 %; 1H-NMR δ ppm = 13.26, 11.25 (2 s,
2 H, 2 NH D2O exchangeable) 9.03 (s, 1 H, C12-H); 8.14 (d, 1 H,
C8-H, J = 7.32 Hz); 7.96 (d, 1 H, C11-H, J = 7.32 Hz); 7.79 (dd,
1 H, C10-H, J = 8.32, 7.32 Hz);7.64 (dd, 1 H, C9-H, J = 8.32, 7.32
Hz); 7.59–7.43 (m, 7 H, Ar-H + NH2). Anal. C20H14N6O2 (C, H,
N).
3,4-Diaminoquinolino[2Ј,3Ј-5,4](3-pyrazolino)[2-3-a]pyrimidin-
2-one (3 a)
9-Methyl-3-benzoylamino-4-aminoquinolino[2Ј,3Ј-5,4](3-pyr-
azolino)[2,3-a]pyrimidin-2-one (4 e)
Mp 286–287 °C; yield 66 %; IR ν cm–1: 3220, 3125 (NH); 1630
(C=O); 1585 (δ NH); 1507 (C=C); disappearance of the N=O at
1549. 1H-NMR δ ppm = 13.55 (s, 1 H, NH, D2O exchangeable);
8.94 (s, 2 H, NH2, D2O exchangeable);8.44 (s, 1 H, C12-H);8.13
(d, 1 H, C8-H, J = 8.8 Hz); 8.00 (d, 1 H, C11-H, J = 8.8 Hz); 7.78
(dd, 1 H, C10-H, J = 8.8, 7.7 Hz);7.47 (dd, 1 H, C9-H, J = 8.8, 7.7
Hz); 3.3 (s, 2 H, NH2 under DMSO). Anal. C13H10N6O (C, H, N).
Mp 216–217 °C;yield 83 %; 1H-NMR δ ppm = 13.48, 11.21 (2 s,
2 H, 2 NH, D2O exchangeable);8.85 (s, 1 H, C12-H);8.38 (s, 1 H,
1
C8-H); 8.13 (d, H, C11-H, J = 6.5 Hz); 8.03 (d, 1 H, C10-H, J =
6.5 Hz); 7.76–7.30 (m, 5 H, Ar-H); 5.88 (s, 2 H, NH2, D2O ex-
changeable). Anal. C21H16N6O2 (C, H, N).
9-Methyl-3,4-diaminoquinolino[2Ј,3Ј-5,4](3-pyrazolino)[2,3-a]-
pyrimidin-2-one (3 b)
9-Methoxy-3-benzoylamino-4-aminoquinolino[2Ј,3Ј-5,4]-
(3-pyrazolino)[2,3-a]pyrimidin-2-one (4 f)
Mp 225–226 °C; yield 75 %; IR ν cm–1: 3220, 3100 (NH); 1624
Mp 110–111 °C;yield 60 %; 1H-NMR δ ppm = 13.13, 11.20 (2 s,
2 H, 2 NH, D2O exchangeable); 8.9 (s, 1 H, C12-H); 8.64 (s, 1 H,
C8-H); 8.14 (d, 1 H, C11-H, J = 7.4 Hz); 7.79 (d, 1 H, C10-H, J =
7.4 Hz); 7.61–7.47 (m, 7 H, 5 Ar-H + NH2); 3.97 (s, 3 H, OCH3).
Anal. C21H16N6O3 (C, H, N).
(C=O); 1548 (δ NH); 1506 (C=C). Anal. C14H12N6O (C, H, N).
9-Methoxy-3,4-diaminoquinolino[2Ј,3Ј-5,4](3-pyrazolino)-
[2,3-a]pyrimidin-2-one (3 c)
Mp 256–257 °C; yield 80 %; IR ν cm–1: 3357, 3308, 3190 (NH);
1645 (C=O); 1620 (C=N); 1590, 1502 (C=C); 1224, 1024
(C–O–C). Anal. C14H12N6O2 (C, H, N)
3-Thienylamino-4-aminoquinolino[2Ј,3Ј-5,4](3-pyrazolino)-
[2,3-a]pyrimidin-2-one (4 g)
Mp 251–252 °C; yield 70 %; IR ν cm–1; 3122 (NH); 1643 (C=O);
1623 (C=N); 1585, 1503 (C=C); 1549 (δ NH); 1278, 1037
(C–S–C). Anal. C18H12N6O2S (C, H, N, S).
3-Acylamino-4-aminoquinolino[2Ј,3Ј-5,4](3-pyrazolino)-
[2,3-a]pyrimidin-2-one (4 a–i): General procedure
The appropriate diamino derivative (3 a–c) (10 mmol) was sus-
pended in a solution of glacial acetic acid (30 mL) and saturated
aqueous sodium acetate (10 mL). The reaction mixture was
stirred at room temperature for 15–20 min, after which time al-
most complete dissolution occurred.The appropriate acyl chlo-
ride (10 mmol) in anhydrous dioxane (2 mL) was then added
dropwise at 0 °C.The reaction mixture was stirred at room tem-
perature for further 4 h, then poured into water (50 mL) and
carefully neutralized with diluted NH4OH.The formed solid was
filtered, washed with ethanol and crystallized from DMF : EtOH
(2 : 1).
9-Methyl-3-thienylamino-4-aminoquinolino[2Ј,3Ј-5,4](3-pyr-
azolino)[2,3-a]pyrimidin-2-one (4 h)
Mp 195–196 °C;yield 70 %; 1H-NMR δ ppm = 13.48, 13.21 (2 s,
2 H, 2 NH, D2O exchangeable); 13.32 (s, 2 H, NH2, D2O
exchangeable).8.95 (s, 1 H, C12-H); 8.85 (s, 1 H, C8-H); 8.21 (d,
1 H, C3-thiophene, J = 3.6 Hz); 8.03 (d, 1 H, C11-H, J = 8.8 Hz);
7.92 (d, 1 H, C5-thiophene, J = 5.16 Hz); 7.75 (d, 1 H, C10-H, J =
8.5 Hz); 7.31 (m, 1 H, C4-thiophene). Anal. C19H14N6O2S (C, H,
N, S).
9-Methoxy-3-thienylamino-4-aminoquinolino[2Ј,3Ј-5,4](3-pyr-
azolino)[2,3-a]pyrimidin-2-one (4 i)
3-Acetylamino-4-aminoquinolino[2Ј,3Ј-5,4](3-pyrazolino)-
[2,3-a]pyrimidin-2-one (4 a)
Mp 231–232 °C; yield 75 %; 1H-NMR δ ppm = 11.96, 9.1, 8.73
Mp 198–199 °C; yield 62 %; IR ν cm–1; 3442, 3378, 3238 (NH);
(3 s, 4 H, 2 NH, NH2, D2O exchangeable); 8.56 (s, 1 H, C12-H);
1663, 1648 (C=O); 1622 (C=N); 1571, 1503 (C=C); 1553, 1535

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 207–212 Quinolino[2Ј,3Ј-5,4](3-pyrazolino)[3,2-b]purin-4-ones 211
8.56 Hz); 7.78 (dd, 1 H, C8-H, J = 8.56, 6.12 Hz); 7.46 (dd, 1 H,
(δ NH); 1277, 1121 (C–S–C); 1222, 1025 (C–OC). Anal.
C9-H, J = 8.56, 6.12 Hz); 7.28 (dd, 1 H, thiophene C4-H, J =
8.56, 6.12 Hz). Anal. C18H10N6OS (C, H, N).
C19H14N6O3S (C, H, N, S).
2-Substituted quinolino[2Ј,3Ј-5,4](3-pyrazolino)[3,2-b]purin-4-
ones (5 a–i): General procedure
9-Methyl-2-(2-thienyl)quinolino[2Ј,3Ј-5,4](3-pyrazolino)[3,2-b]-
purin-4-one (5 h)
A suspension of each compound (4) (10 mmol) in a solution of
sodium (0.7 g, 30 mmol) in methanol (50 mL) was refluxed for
2 h. After cooling to room temperature, water (150 mL) was
added and the solution was adjusted to pH 6 with dil. HCl.The
formed precipitate was filtered off, washed with water, and
crystallized from DMF.
Mp 209–210 °C;yield 56 %; 1H-NMR δ ppm = 13.38, 11.40 (2 s,
2 H, 2 NH D2O exchangeable); 9.07 (s, 1 H, C6-H); 8.22 (d, 1 H,
C7-H, J = 8.7 Hz); 8.10 (d, 1 H, thiophene C5-H, J = 8.56 Hz);
7.94 (d, 1 H, thiophene C5-H, J = 8.56 Hz)); 7.78 (s, 1 H, C10-H);
7.36 (d, 1 H, C8-H, J = 8.7 Hz); 7.28 (dd, 1 H, thiophene C4-H, J
= 8.56 Hz); 2.56 (s, 3 H, CH3). Anal. C19H12N6OS (C, H, N).
2-Methylquinolino [2Ј,3Ј-5,4](3-pyrazolino)[3,2-b]purin-4-ones
(5 a)
9-Methoxy-2-(2-thienyl)quinolino[2Ј,3Ј-5,4](3-pyrazolino)-
[3,2-b]purin-4-one (5 i)
Mp 135–136 °C;yield 60 %; 1H-NMR δ ppm = 13.54, 11.77 (2 s,
2 H, 2 NH, D2O exchangeable); 9.90 (s, 1 H, C6-H); 8.08 (d, 1 H,
C10-H, J = 8 Hz); 8.02–7.21 (m, 3 H, Ar-H, C7,8,9-H); 2.50 (s,
3 H, CH3). Anal. C15H10N6O (C, H, N).
Mp 219–220 °C; yield 52 %; 1H-NMR δ ppm = 11.54, 9.20 (2 s,
2 H, 2 NH, D2O exchangeable); 8.24 (s, 1 H, C6-H); 8.13 (d, 1 H,
C7-H, J = 8.7 Hz); 7.92 (d, 1 H, thiophene C5-H, J = 8.56 Hz);
7.37 (d, 1 H thiophene C3-H, J = 8.56 Hz); 7.30 (s, 1 H, C10-H);
7.26 (d, 1 H, C8-H, J = 8.7 Hz); 7.18 (dd, 1 H, thiophene C4-H, J
= 8.56 Hz); 3.90 (s, 3 H, CH3). Anal. C19H12N6O2S (C, H, N).
2,9-Dimethylquinolino[2Ј,3Ј-5,4](3-pyrazolino)[3,2-b]purin-
4-one (5 b)
Mp 241–242 °C; yield 65 %; 1H-NMR δ ppm = 9.30, 8.87 (2 s,
2 H, 2 NH, D2O exchangeable); 8.38 (s, 1 H, C6-H); 8.11 (d, 1 H,
C7-H, J = 8.8 Hz); 7.81 (s, 1 H, C10-H); 7.35 (d, 1 H, C8-H, J =
7.36 Hz); 2.65, 2.56 (2 s, each 3 H, 2 CH3). Anal. C16H12N6O (C,
H, N).
Radioligand and binding assay
[3H]Ro15-1788 (sp. act. 83.2 Ci/mmol) chemically known as
9-fluoro-3-carboethoxy-5,6-dihydro-s-methyl-6-oxo-4H-imid-
azole [1,5-a]-1,4-benzodiazepine was purchased from Dupont-
New England Nuclear (Boston, MA, U.S.A.). All other reagents
were obtained from commercial suppliers.
2-Methyl-9-Methoxyquinolino[2Ј,3Ј-5,4](3-pyrazolino)[3,2-b]-
purin-4-one (5 c)
Bovine cerebral cortex membranes were prepared as previ-
ously described [8]. In brief, tissue was homogenized in 10 vol-
umes of ice-cold 0.32 M sucrose, containing protease inhibi-
tors. The homogenate was centrifuged for 5 min at 2000 g at
4 °C, and the supernatant was recentrifuged for 30 min at
30,000 g at 4 °C. The resulting pellet was resuspended in
50 mM Tris-citrate buffer at pH 7.4 (buffer T), homogenized,
and centrifuged for 30 min at 30,000 g at 4 °C.The resulting pel-
let was subjected to washing procedures to remove endog-
enous GABA [9].For this purpose the membranes were frozen,
thawed, resuspended in 32 volumes of T buffer, containing
0.01 % Triton X-100 and protease inhibitors, homogenized, in-
cubated for 60 min at 37 °C, and centrifuged for 30 min at
30,000 g at 4 °C. The pellet was resuspended in buffer T, ho-
mogenized, and recentrifuged.The washing procedure was re-
peated three times. The washed membranes were incubated
with [3H]Ro15-1788 (~0.2 nM, Kd = 0.60 nM) for 90 min at 0 °C
in 500 µL of buffer T.The incubation was terminated by dilution
to 5 mL with ice-cold buffer T, followed immediately by rapid fil-
tration through glass fiber Whatman GF/C filters. The filters
were then washed (2 × 5 mL) with buffer T and the amount of
radioactivity retained on the filters was determined by Packard
1600 TR liquid scintillation counter at 66 % efficiency. Non-spe-
cific binding was estimated in the presence of 10 µM diazepam.
The compounds were dissolved in DMSO, and tested at a con-
centration of 10 µM. For the active compound, the IC50 value
was determined and the Ki value was derived according to the
equation of Cheng and Prusoff [10]. Protein concentration was
assayed by the method of Lowry et al. [11].
Mp 155–156 °C; yield 70 %; IR ν cm–1: 3143 (NH); 1693 (C=O);
1619 (C=N); 1536 (δ NH); 1224, 1026 (C–O–C). Anal.
C16H12N6O2 (C, H, N).
2-Phenylquinolino[2Ј,3Ј-5,4](3-pyrazolino)[3,2-b]purin-4-one
(5 d)
Mp 287–288 °C; yield 70 %; IR ν cm–1: 3266 (NH), 1648 (C=O);
1620 (C=N); 1548, 1509 (C=C); 1547 (δ NH). Anal. C20H12N6O
(C, H, N).
9-Methyl-2-phenylquinolino[2Ј,3Ј-5,4](3-pyrazolino)[3,2-6b]-
purin-4-one (5 e)
Mp 204–205 °C; yield 62 %; 1H-NMR δ ppm = 9.62, 9.31 (2 s,
2 H, 2 NH, D2O exchangeable); 9.10 (s, 1 H, C6-H); 8.82 (s, 1 H,
C10-H); 8.21–7.22 (m, 7 H, Ar-H); 2.59 (s, 3 H, CH3). Anal.
C21H14N6O (C, H, N).
9-Methoxy-2-phenylquinolino[2Ј,3Ј-5,4](3-pyrazolino)[3,2-
b]purin-4-one (5 f)
Mp 120–121 °C;yield 70 %; 1H-NMR δ ppm = 13.21, 11.23 (2 s,
2 H, 2 NH, D2O exchangeable); 8.95 (s, 1 H, C6-H); 8.13 (d, 2 H,
phenyl C2,6-H, J = 7 Hz); 8.06 (d, 1 H, C7-H, J = 8.56 Hz); 7.64–
7.51 (m, 3 H, phenyl C3,4,5-H); 7.29 (s, 1 H, C10-H); 7.12 (d, 1 H,
C8-H, J = 8.56 Hz);3.94 (s, 3 H, OCH3).Anal.C21H14N6O2 (C, H,
N).
2-(2-Thienyl)quinolino[2Ј,3Ј-5,4](3-pyrazolino)[3,2-b]purin-4-
one (5 g)
The BDZ binding affinity of the compounds was evaluated by
their ability to displace [3H]Ro15-1788 from its specific binding
in bovine brain membranes. First a single concentration of the
compounds was examined; for the most potent compound this
was followed by examination of the IC50 value from log-probit
plot. The results are shown in the table.
Mp > 300 °C; yield 70 %; 1H-NMR δ ppm = 13.29, 11.38 (2 s,
2 H, 2 NH, D2O exchangeable); 9.04 (s, 1 H, C6-H); 8.26 (d, 1 H,
C10-H, J = 8.56 Hz);8.16 (d, 1 H, C7-H, J = 8.56 Hz);7.98 (d, 1 H,
thiophene C3-H, J = 8.56 Hz); 7.93 (d, 1 H, thiophene C5-H, J =

212 El-Sayed et al.
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