Synthesis of some novel pyrazolo[3,4-d]pyrimidine derivatives as potential antimicrobial agents

Article abstract of DOI:10.1016/j.bmc.2005.10.053

The reaction of 4-hydrazino-8-(trifluoromethyl)quinoline (2) with ethoxymethylenecyanoacetate afforded ethyl 5-amino-1-[8-(trifluoromethyl) quinolin-4-yl]-1H-pyrazole-4-carboxylate (3) and that with ethoxymethylenemalononitrile afforded 5-amino-1-[8-(trifluoromethyl)quinolin-4- yl]-1H-pyrazole-4-carbonitrile (5). Compounds 3 and 5 were hydrolyzed to get 5-amino-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-pyrazole-4-carboxylic acid and then reacted with acetic anhydride to afford 6-methyl-1-[8-(trifluoromethyl) quinolin-4-yl]pyrazolo[3,4-d]oxazin-4-one (6), which was condensed with different aromatic amines to give a series of 5-substituted 6-methyl-1-[8- (trifluoromethyl)quinolin-4-yl]-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-ones (7). Compounds 3 and 5 also reacted with formamide, urea, and thiourea affording the corresponding pyrazolo[3,4-d]pyrimidines (8-13), respectively. Structures of the products have been determined by chemical reactions and spectral studies. All compounds of the series have been screened for their antibacterial and antifungal activity studies. The results are summarized in Tables 1 and 2.

Full text of DOI:10.1016/j.bmc.2005.10.053

Bioorganic & Medicinal Chemistry 14 (2006) 2040–2047
Synthesis of some novel pyrazolo[3,4-d]pyrimidine derivatives
as potential antimicrobial agents
a,
a
a
*
Bantwal Shivarama Holla, Manjathuru Mahalinga, Mari Sitambaram Karthikeyan,
b
c
Padiyath Mohamed Akberali and Nalilu Sucheta Shetty
a
Department of Chemistry, Mangalore University, Mangalagangothri 574199, India
Strides Arcolab Limited, 120 A&B, Industrial Area, Baikampady, New Mangalore, India
b
c
Department of Biochemistry, Justice K. S. Hegde Medical Academy, Deralakatte, India
Received 6 August 2005; revised 27 October 2005; accepted 28 October 2005
Available online 28 November 2005
Abstract—The reaction of 4-hydrazino-8-(trifluoromethyl)quinoline (2) with ethoxymethylenecyanoacetate afforded ethyl 5-ami-
no-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-pyrazole-4-carboxylate (3) and that with ethoxymethylenemalononitrile afforded
5
-amino-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-pyrazole-4-carbonitrile (5). Compounds 3 and 5 were hydrolyzed to get 5-ami-
no-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-pyrazole-4-carboxylic acid and then reacted with acetic anhydride to afford 6-meth-
yl-1-[8-(trifluoromethyl)quinolin-4-yl]pyrazolo[3,4-d]oxazin-4-one (6), which was condensed with different aromatic amines to
give a series of 5-substituted 6-methyl-1-[8-(trifluoromethyl)quinolin-4-yl]-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-ones (7).
Compounds 3 and 5 also reacted with formamide, urea, and thiourea affording the corresponding pyrazolo[3,4-d]pyrimidines (8–
13), respectively. Structures of the products have been determined by chemical reactions and spectral studies. All compounds of the
series have been screened for their antibacterial and antifungal activity studies. The results are summarized in Tables 1 and 2.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
pyrimidine derivatives bearing a fluorinated heterocy-
clic moiety, particularly 8-trifluoromethylquinoline.
Pyrazole and pyrimidine derivatives attracted organic
chemists very much due to their biological and chemo-
therapeutic importance. Pyrazolopyrimidines and relat-
ed fused heterocycles are of interest as potential
bioactive molecules. They are known to exhibit phar-
Moreover, in recent years, fluorinated compounds find
6
much importance in the pharmaceutical field. Fluori-
nated compounds in general, fluorinated heterocycles
in particular, are those focused much in modern-day
medicinal chemistry. Incorporation of a fluorine atom
instead of a hydrogen one can alter the course of the
1
macological activities such as CNS depressant, neuro-
3
2
leptic , and tuberculostatic. Pyrazolo[3,4-d]pyrimidines
were identified as a general class of adenosine recep-
7
–9
reaction as well as biological activities. Further intro-
duction of a fluorine atom as the CF group provides a
4
,5
tors.
There is not much difference in the basic
3
structures of pyrazolopyrimidines and purines. In the
literature, we have found that the replacement of 1H
of pyrazole of pyrazolo[3,4-d]pyrimidine ring system
by some other bioactive moiety drastically alters its
pharmacological properties. Keeping this in mind, we
have contemplated on the synthesis of pyrazolo[3,4-d]-
more lipophilically and pharmacologically interesting
compound compared to their non-fluorinated ana-
logues. The trifluoromethyl substituted compounds have
been reported to possess biological activities as herbi-
1
0
11
12
13
cides,
inhibitors for platelet aggregation.
fungicides,
analgesic,
antipyretic , and
1
4
Prompted by the varied biological activities of pyraz-
olo[3,4-d]pyrimidine derivatives and trifluoromethyl-
bearing compounds, we envisioned our approach
toward the synthesis of a novel series of pyrazolo-
[3,4-d]pyrimidine derivatives bearing a trifluoromethyl
Keywords: 4-Hydrazino-8-(trifluoromethyl)quinoline; Pyrazolopyrimi-
dines; Antibacterial; Antifungal activity studies.
*
Corresponding author. Tel.: +91 0824 2287262; fax: +91 0824
287367/2287424; e-mail: hollashivarama@yahoo.co.in
2
0
968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.10.053

B. S. Holla et al. / Bioorg. Med. Chem. 14 (2006) 2040–2047
2041
Cl
N
1
CF
3
H N
2
N
2
H
4
O
N
NH
2
NH
HN
N
N
N
N
N
H
CN
CN
N
H
CN
CO
N
N
N
EtO
EtO
2
Et
2
H N
CF
2
Ethanol, reflux
CN
3
Ethanol, reflux
31
1
2
CO Et
CF
N
38
CF
Formamide
O
O
N
N
N
Formamide
N
H
NH
2
NH
2
N
N
N
N
N
NH
O
N
N
H
NH CONH
2 2
N
N
NH CONH
2
2
5
12
CF
3
H N
2
CF
3
3
CF
3
2 2
NH CSNH
CO
H
2
N
N
NH
2
CSNH
2
O
1. NaOH
9
CF
3
S
N
N
NaOH
NH
1
.
N
H
NH
2
N
N
N
N
2.
HCl
S
2. HCl
N
N
H
N
CF
4
CF
3
3
13
N
Ac O, reflux
2
CF
3
1
0
O
O
R
O
N
CH
3
CH
3
N
N
N
N
N
N
N
R-NH2
POCl , Reflux
3
N
CF
3
6
CF
3
7
Figure 1. Synthesis of pyrazolo[3,4-d]pyrimidines.
quinoline moiety and to study the biological activity
Fig. 1).
ring protons, appeared as four doublets and one triplet
integrating for five protons. In the mass spectrum of
(
+
3), the molecular ion peak (MH ) appeared at m/z 351
(
(
+
MH , 100%), which is in accordance with its molecular
2
. Results and discussion
formula. Condensation of 4-hydrazino-8-(trifluorometh-
yl)quinoline (2) with ethoxymethylenemalononitrile
afforded 5-amino-1-[8-(trifluoromethyl)quinolin-4-yl]-
2
.1. Chemistry
1
(5) was very clear from its spectral data. Thus, H NMR
H-pyrazole-4-carbonitrile (5). Formation of compound
1
Condensation of 4-hydrazino-8-(trifluoromethyl)quino-
line (2) with ethoxymethylenecyanoacetate afforded eth-
yl-5-amino-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-pyra-
zole-4-carboxylate (3). The 400 MHz H NMR of (3)
showed the signal at d 1.4 triplet (3H) and 4.3 quartet
(400 MHz DMSO-d ) showed peaks at d 6.92, a singlet,
6
integrating for two protons indicating the presence of a
NH group in the pyrazole ring (which disappeared in
1
2
D O exchange) and all the aromatic protons exactly
2
(
2H) indicating the presence of an ethyl group of the es-
ter, 5.3 singlet corresponding to two protons indicating
matching the structure. The formation of compound
(5) was also confirmed by recording its mass spectrum.
+
The molecular ion peak m/z appeared at 304 (MH ,
the presence of NH group in the pyrazole ring (which
2
disappeared in D O exchange), and 7.9 singlet, which
2
corresponds to the pyrazole ring proton and quinoline
100%), which is in accordance with its molecular formu-
la. Compounds (3) and (5) underwent basic hydrolysis to

2042
B. S. Holla et al. / Bioorg. Med. Chem. 14 (2006) 2040–2047
give compound (4) in excellent yield. The spectral data
revealed the formation of the compound (4). Compound
was completed. The mass spectrum of the compound
(11) also confirmed the formation of the molecule. The
compounds (9), (10), (12), and (13) also confirmed the
same by recording their spectra. The structural elucida-
tions of all compounds were carried out on the basis
of elemental analysis and spectral data. The spectral
data of all these compounds supported the assigned
structures.
(4), when heated with acetic anhydride, gave a cyclized
product, 6-methyl-1-[8-(trifluoromethyl)quinolin-4-yl]-
1
pyrazolo[3,4-d]oxazin-4-(1H)-one (6). H NMR spec-
trum revealed that the disappearance of both NH and
2
OH peaks, and the appearance of a singlet at d 2.4, which
corresponds to methyl protons, confirm the formation of
the compound (6). Mass spectrum of compound (6)
exhibited a molecular ion peak at m/z 346, which ap-
peared as a base peak and strongly indicated the forma-
tion of the compound. The compound (6) was treated
with substituted aromatic amines in the presence of
phosphorus oxychloride to give title compounds (7a–
m). The newly synthesized compounds were analyzed
for N content and their structures were confirmed by
IR, NMR, and mass spectral data.
2.2. Biological activity
2.2.1. Antibacterial activity. The newly synthesized com-
pounds were screened for their antibacterial activity
against Escherichia coli (ATTC-25922), Staphylococcus
aureus
(ATTC-25923),
Pseudomonas
(ATTC-27853), and Bacillius subtilis (recultured) bacte-
aeruginosa
1
5,16
rial stains by the disk diffusion method.
Disks mea-
suring 6.25 mm in diameter were punched from
Whatman no.1 filter paper. Batches of 100 disks were dis-
pensed to each screw-capped bottles and sterilized by dry
heat at 140 °C for an hour. The test compounds were pre-
pared with different concentrations using N,N-dimethyl-
formamide. 1 ml containing 100 times the amount of
chemical in each disk was added to each bottle, which
contained 100 discs. Disks of each concentration were
placed in triplicate in nutrient agar medium seeded with
fresh bacteria separately. The incubation was carried
out at 37 °C for 24 h. Streptomycin was used as a stan-
dard drug at a concentration of 10 lg/ml. Solvent and
growth controls were kept and zones of inhibition were
noted. The results of such studies are given in Table 1.
Compounds (3) and (5) were fused with formamide,
urea, and thiourea to afford the corresponding pyrazolo-
[
3,4-d]pyrimidines (8)–(13), respectively. The formation
of fused pyrimidines was confirmed by recording their
1
spectra. Thus, the 400 MHz H NMR spectrum of
compound (8) showed a downfield signal at d 12.56 cor-
responding to the NH peak, and the NH and CH CH
2
2
3
signals were absent in the spectrum. Thus, it confirmed
the formation of a cyclized product (8). Mass spectrum
of the compound (8) showed a molecular ion peak at
+
m/z 332 (MH ) which appeared as a base peak strongly
supporting the formation of the compound (8). Com-
pound (5), on reacting with formamide, afforded fused
pyrazolopyrimidine (11). The formation of the com-
pound (11) was confirmed by its spectral data. Thus,
The above data showed that compound 1-[8-(trifluo-
romethyl)quinolin-4-yl]-1,5-dihydro-4H-pyrazolo[3,4-
d]pyrimidin-4-one (8) was the most potent compound,
which exhibited very good activity against the four
organisms. The compounds 6-thioxo-1-[8-(trifluoro-
1
the 400 MHz H NMR of (11) showed a signal at d
7
.3 corresponding to the NH peak and all other peaks
2
exactly matching its structure. In the IR spectrum, the
absence of a CN peak indicated that the cyclization
Table 1. Antibacterial activity data of prepared compounds
Compound
Escherichia coli (ATTC-25922)
Staphylococcus aureus
ATTC-25923)
Pseudomonas aeruginosa
(ATTC-27853)
Bacillius subtilis
(recultured)
(
7
7
7
7
7
7
7
7
7
7
7
7
7
8
9
1
1
1
1
a
b
c
d
e
f
12
12
9
12
22
—
13
13
13
—
15
—
19
19
18
—
25
22
26
18
19
22
21
13
10
—
8
9
8
—
10
14
14
13
12
—
9
9
14
—
—
18
14
14
—
15
14
28
22
26
20
19
24
20
17
14
13
12
10
9
g
h
i
j
k
l
10
8
—
11
11
26
17
23
18
21
20
24
m
—
24
18
22
17
20
20
24
0
1
2
3
Standard
Streptomycin)
(
Zone of inhibition in millimeters.

B. S. Holla et al. / Bioorg. Med. Chem. 14 (2006) 2040–2047
2043
methyl)quinolin-4-yl]-4H-pyrazolo[3,4-d]pyrimidin-4-
one (10) and 4-amino-1-[8-(trifluoromethyl)quinolin-4-
yl]-1H-pyrazolo[3,4-d]pyrimidine-6-thione (13) exhibited
very good activity against E. coli and S. aureus. The
remaining compounds were found to have slight or
moderate activity against the tested organisms and some
of the compounds were found to be inactive (indicated
good activity toward A. flavus and A. fumigatus. The
remaining compounds were found to have slight or
moderate activity against the tested organisms and
some of the compounds were found to be inactive
(indicated — sign) (Table 2).
—
sign).
3. Conclusion
2
.2.2. Antifungal activity. Newly prepared compounds
We have successfully synthesized a series of 6-methyl-5-
trifluromethyl)quinolin-4-yl]-1,5-dihydro-4H-pyrazolo[3,4-
d]pyrimidin-4-ones and fused pyrimidines containing
C@O/C@S/NH2 functional groups. The antibacterial
and antifungal activity data of the prepared compounds
showed that the fused pyrimidines (8–13) showed very
good antimicrobial activity compared to those of the
compounds (7a–m). Basically pyrazolo[3,4-d]pyrimidines
are analogs of purine nucleus and various structural
changes of the natural purines have resulted in potent
antagonists in biological systems. In our study, we have
replaced the H atom of the pyrazole ring by 8-trifluorom-
ethylquinoline as an active moiety these structural chang-
es made fused pyrimidines (8–13) more active towards
were screened for their antifungal activity against
Aspergillus flavus (NICM No. 524), Aspergillus fumiga-
tus (NCIM No. 902), Candida albicans (NCIM No.
300), Penicillium marneffei (recultured), and Trichophy-
ton mentagrophytes (recultured) in DMSO by the serial
1
7,18
plate dilution method.
SabouraudÕs agar media were
prepared by dissolving peptone (1 g), D-glucose (4 g),
and agar (2 g) in distilled water (100 ml) and adjusting
the pH to 5.7. Normal saline was used to make a suspen-
sion of the spore of fungal strain for lawning. A loopful
of particular fungal strain was transferred to 3 ml saline to
get a suspension of the corresponding species. Agar media
(
20 ml) was poured into each petri dish. Excess suspension
was decanted and the plates were dried by placing in an
incubator at 37 °C for 1 h. Using an agar punch wells were
made into each well labeled. A control was also prepared
in triplicate and maintained at 37 °C for 3–4 days. Anti-
fungal activity was determined by measuring the diameter
of the inhibition zone. Activity of each compound was
compared with that of flucanazole as standard.
tested organisms. Further, incorporation of CF group
3
in the quinoline ring increased lipophilicity as well as bio-
logical activity.
4. Experimental
Melting points were determined by an open capillary
method and are uncorrected. The IR spectra (in KBr
pellets) were recorded on a Perkin-Elmer 157 IR spec-
trophotometer. PMR spectra were recorded in DMSO-
d6 on either a Bruker AC-300F (300 MHz) or an amx
400 (400 MHz) NMR spectrometer using TMS as an
internal standard. Chemical shift values are given in d
scale. The mass spectra of some of the selected com-
pounds were recorded on a MASPEC low resolution
The antifungal data showed that the newly prepared
compounds have moderate to good activity against
the above-mentioned organisms. The compound 4-ami-
no-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-pyrazolo[3,4-
d]pyrimidine (11) exhibited very good activity against
A. flavus, A. fumigatus, and T. mentagrophytes. Com-
pound 4-amino-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-
pyrazolo[3,4-d]pyrimidine-6-thione (13) showed very
Table 2. Antifungal activity data of prepared compounds (6a–m)
Compound
Aspergillus flavus Aspergillus fumigatus Penicillium marneffei Candida Albicans Trichophyton Mentagrophytes
(
NICM No. 524) (NCIM No. 902)
(recultured)
(NCIM No. 300) (recultured)
7
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
—
13
12
20
—
12
15
—
18
—
19
16
15
18
17
20
25
15
25
—
—
15
15
—
10
16
17
17
17
21
18
—
17
20
18
22
18
24
18
18
12
—
18
—
13
21
—
18
—
14
15
—
15
18
17
19
23
20
21
15
10
—
14
—
18
17
—
15
—
16
18
—
20
17
16
18
16
17
20
17
10
9
15
—
—
16
18
19
—
14
19
—
18
17
19
24
17
19
19
g
h
i
j
k
l
m
8
9
0
1
2
3
1
1
1
1
Standard (Flucanazole) 21
Zone of inhibition in millimeters.

2044
B. S. Holla et al. / Bioorg. Med. Chem. 14 (2006) 2040–2047
mass spectrometer operating at 70 eV. The purity of the
compounds was checked by thin layer chromatography
4.4. Synthesis of 5-amino-1-[8-(trifluoromethyl)quinolin-
4-yl]-1H-pyrazole-4-carboxylic acid (4) (method A)
(
TLC) on a silica gel plate.
A mixture of ethyl 5-amino-1-[8-(trifluoromethyl)quino-
lin-4-yl]-1H-pyrazole-4-carboxylate (3) (17 g, 0.05 mol)
and sodium hydroxide (4.2 g, 0.1 mol) was dissolved in
65 ml of methanol and 21 ml water. The contents were
heated to reflux on a water bath for 5 h and the comple-
tion of the reaction was monitored by TLC. Cooled to
room temperature and the reaction mass poured into
340 ml ice water, then adjusting pH of the reaction mass
to four using concentrated hydrochloric acid, the solids
obtained were filtered, washed with water, and crystal-
4
.1. Synthesis of 4-hydrazino-8-(trifluoromethyl)quinoline (2)
To 4-Chloro-8-trifluoromethyl quinoline (1) (25 g,
.1 mol) in 100 ml ethanol was added hydrazine hydrate
0
(
100%) (10 ml). After being stirred at 80 °C for 8 h in the
absence of light, the reaction mixture was diluted with
water, the resulting precipitate collected and recrystal-
lized from ethanol to give compound (2) as light yellow
1
needles (20.2 g, 82.5%); mp 198–200 °C; H NMR
(
400 MHz, DMSO-d ): d 4.54 (br s, 2H, NH ), 7.03 (d,
lized from methanol yielding (4) (14.2 g, 78%); mp
6
2
1
1
J = 7.2 Hz), 8.41 (d, 1H, J = 8.3 Hz), 8.66 (d, 1H,
H, J = 8.1 Hz), 7.46 (t, 1H, J = 7.7 Hz), 7.98 (d, 1H,
234–36 °C. H NMR (400 MHz, DMSO-d ) d: 5.6 (s,
6
2H, NH ), 7.4 (d, 1H, J = 8.4 Hz), 7.6 (t, 1H,
2
J = 4.5 Hz), 8.71 (s, 1H, NH); IR (KBr) m: 3388, 3394
J = 8.0 Hz), 7.7 (s, 1H, pyrazole ring), 8.0 (d, 1H,
J = 7.8 Hz), 8.2 (d, 1H, J = 8.2 Hz), 9.2 (d, 1H,
J = 4.5 Hz); IR (KBr) m: 3410 (OH str), 3382 (NH str),
3035, 2940 (Ar–H str), 1678 (C@O str) 1026 (C–F str);
À1
(
NH str), 3038, 2942 (Ar–H str), 1028 (C–F str) cm
;
+
+
MS: (MH , %) m/z 228 (MH , 100), 227 (50), 192
10), 166 (5), 120 (5), 107 (10).
(
+
+
MS: (MH , %) m/z 323 (M , 65), 322 (15), 307 (40),
4.2. Synthesis of ethyl 5-amino-1-[8-(trifluorometh-
yl)quinolin-4-yl]-1H-pyrazole-4-carboxylate (3)
279 (5), 228 (100), 196 (6), 120 (10), 107 (8).
4.5. Synthesis of 5-amino-1-[8-(trifluoromethyl)quinolin-
4-yl]-1H-pyrazole-4-carboxylic acid (4) (method B)
Ethoxymethylenecyanoacetate (14.8 g, 0.09 mol) was
carefully added in small portions to (20 g, 0.09 mol) of
4
-hydrazino-8-(trifluoromethyl)quinoline. The reaction
5-Amino-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-pyr-
azole-4-carbonitrile (5) (10 g, 0.03 mol) and sodium
hydroxide (4 g, 0.1 mol) in 20 ml water were heated on
a water bath for 15 h. After cooling to 10 °C the reaction
mixture was acidified using acetic acid and the precipi-
tated product was filtered, washed with water, and
recrystallized from methanol to yield (4) (6.5 g, 61%);
mp 234–36 °C. Spectral data showed that the product
obtained in method B is exactly matching the product
obtained in method A.
mass was heated to 80 °C for 1 h on a water bath, the
completion of the reaction monitored by TLC. Cooled
to room temperature and to the reaction mass added
100 ml water, stirred for 2 h, the solid obtained was fil-
tered, washed with water, and suck dried. Crystalliza-
tion from ethanol gave compound (3) as colorless
1
crystals (18 g, 58.4%); mp 138–140 °C; H NMR
(
CH ), 5.3 (s, 2H, NH ), 7.6 (d, 1H, J = 8.0 Hz), 7.7 (t,
400 MHz, DMSO-d ): d 1.4 (t, 3H, CH ), 4.3 (q, 2H,
6 3
2
2
1
J = 7.9 Hz), 8.2 (d, 1H, J = 8.2 Hz), 9.3 (d, 1H,
H, J = 8.8 Hz), 7.9 (s, 1H, pyrazole ring), 8.1 (d, 1H,
4.6. Synthesis of 6-methyl-1-[8-(trifluoromethyl)quinolin-
4-yl]pyrazolo[3,4-d]oxazin-4(1H)-one (6)
J = 4.5 Hz); IR (KBr) m: 3380 (NH str), 3035, 2940
À1
(
(
(
Ar–H str), 1720 (CO str), 1026 (C–F str) cm ; MS:
MH , %) m/z 351 (MH , 100), 350 (20), 323 (8), 305
50), 273 (10), 218 (5), 196 (2), 107 (10).
+
+
5-Amino-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-pyr-
azole-4-carboxylic acid (4) (13 g 0.04 mol) taken in 26 ml
acetic anhydride was heated to reflux on an oil bath for
8 h. It was then cooled to room temperature and poured
into ice-cold water, stirring for 30 min. The solid was fil-
tered, washed with water, and then crystallized from
4
4
.3. Synthesis of 5-amino-1-[8-(trifluoromethyl)quinolin-
-yl]-1H-pyrazole-4-carbonitrile (5)
Ethoxymethylenemalononitrile (10.8 g, 0.09 mol) was
carefully added in small portions to (20 g, 0.09 mol)
of 4-hydrazino-8-(trifluoromethyl)quinoline. The reac-
tion mass was heated to 80 °C for 1 h on a water
bath, the completion of the reaction monitored by
TLC. Cooled to room temperature and to the reaction
mass added 100 ml of water, stirred for 2 h, the solid
obtained was filtered and washed with water. Crystal-
lization from ethanol gave, compound (5) as colorless
ethyl acetate to obtain (11.3 g, 81.2%) pure product
1
(6); mp 220–222 °C; H NMR (400 MHz, DMSO-d )
6
d: 2.4 (s, 3H, CH ), 7.65 (d, 1H, J = 8.1 Hz), 7.69 (t,
3
1H, J = 8.8 Hz), 8.14 (d, 1H, J = 8.5 Hz), 8.19 (d, 1H,
J = 7.4 Hz), 8.31 (s, 1H, pyrazole ring), 9.26 (d, 1H,
J = 4.8 Hz); IR (KBr) m: 3035, 2940 (Ar–H str), 1710
À1
+
(C@O str), 1026 (C–F str) cm ; MS: (M , %) m/z 346
(M , 100), 303 (80), 276 (40), 210 (10), 177 (40), 149
(35), 77 (12), 60 (30), 52 (20).
+
1
crystals (15.5 g, 58%); mp 238–240 °C; H NMR
(
400 MHz DMSO-d ) d: 6.98 (s, 2H, NH ), 7.79 (d,
4.7. Synthesis of 5-(5-chloro-2-methylphenyl)-6-methyl-1-
[8-(trifluoromethyl)quinolin-4-yl]-1,5-dihydro-4H-pyraz-
olo[3,4-d]pyrimidin-4-one (7a)
6
2
1H, J = 8.2 Hz), 7.82 (t, 1H, J = 8.0 Hz), 7.98 (s, 1H,
pyrazole ring), 8.02 (d, 1H, J = 7 Hz), 8.30 (d, 1H,
J = 8.5 Hz), 9.20 (d, 1H, J = 4.6 Hz); IR (KBr) m:
3
038, 2950 (Ar–H str), 2240 (CN str), 1022 (C–F
To the compound (6) (1.0 g, 0.003 mol) taken in 10 ml
phosphorus oxychloride was added one molar equivalent
of 5-chloro-2-methyl aniline and heated to 100–105 °C for
À1
+
+
str) cm ; MS: (MH , %) m/z 304 (MH , 100), 303
10), 277 (5), 218 (5), 196 (6), 107 (8).
(

B. S. Holla et al. / Bioorg. Med. Chem. 14 (2006) 2040–2047
2045
8
h on an oil bath. It was then cooled to room temperature
2955 (Ar–H str), 1708 (C@O str), 1021 (C–F str)
+ +
À1
and poured into ice-cold water, neutralized with sodium
bicarbonate solution, the solid filtered, and washed with
water. The crude product was recrystallized from ethyl
cm ; MS: (M , %) m/z 421 (M , 5), 378 (100), 335
(10), 312 (5), 244 (10), 184 (5), 118 (15); Anal. Calcd
for N: 16.55. Found: 16.58.
acetate to obtain 0.8 g, 57.1 % of pure product; mp 291–
1
2
CH ), 2.16 (s, 3H, CH ), 7.21 (s, 1H), 7.38 (d, 1H,
93 °C; H NMR (400 MHz, DMSO-d ) d: 2.12 (s, 3H,
4.12. Synthesis of 6-methyl-5-(4-methyl-2-nitrophenyl)-1-
[8-(trifluoromethyl)quinolin-4-yl]-1,5-dihydro-4H-pyraz-
olo[3,4-d]pyrimidin-4-one (7f)
6
3
3
J = 8.2 Hz), 7.44 (d, 1H, J = 8.1 Hz), 7.69 (t, 1H,
J = 7.9 Hz), 7.81 (d, 1H, J = 8.5 Hz), 8.19 (d, 1H,
J = 7.1 Hz), 8.33 (d, 1H, J = 8.5 Hz), 8.4 (s, 1H, pyrazole
ring), 9.26 (d, 1H, J = 4.7 Hz); IR (KBr) m: 3035, 2940
Obtained from compounds (6) and 4-methyl-2-nitro
aniline; yellow solid (0.80 g, 54%); mp 267-269 °C;
1
(
Ar–H str), 1710 (C@O str), 1026 (C–F str), 816 (C–Cl
H NMR (400 MHz, DMSO-d ) d: 1.43 (s, 3H,
6
À1
+
str) cm ; MS: (M , %) m/z 471 (M , 2), 469 (M , 5)
2+
+
CH ), 2.12 (s, 3H, CH ), 6.89–77.69, (m, 7H, Ar–H),
8.1 (s, 1H, pyrazole ring), 9.19–9.21 (d, 1H,
3
3
4
6
54 (100), 370 (35), 305 (5), 227 (30), 196 (20), 149 (20),
0 (16). Anal. Calcd for N: 14.88. Found: 14.90.
J = 4.7 Hz); IR (KBr) m: 3035, 2932 (Ar–H str), 1723
À1
(
C@O str), 1076 (C–F str) cm . Anal. Calcd for N:
4
.8. Synthesis of 5-(3-chloro-4-fluorophenyl)-6-methyl-1-
8-(trifluoromethyl)quinolin-4-yl]-1,5-dihydro-4H-pyraz-
18.74. Found: 18.74.
[
olo[3,4-d]pyrimidin-4-one (7b)
4.13. Synthesis of 5-(2,5-dichlorophenyl)-6-methyl-1-[8-
(trifluoromethyl)quinolin-4-yl]-1,5-dihydro-4H-pyrazolo-
[3,4-d]pyrimidin-4-one (7g)
Obtained from compounds (6) and 2-chloro-4-fluoro
aniline; light yellow needles (0.75 g, 55%); mp 249–
1
2
3
50 °C; H NMR (400 MHz, DMSO-d ) d: 2.13 (s,
Obtained from compounds (6) and 2,5 dichloro aniline;
6
1
H, CH ), 7.22–8.00 (m, 7H, Ar–H), 8.2 (s, 1H, pyrazole
colorless crystals (0.78 g, 52.7%); mp 280–282 °C; H
3
ring), 9.25–9.26 (d, 1H, J = 4.7 Hz); IR (KBr) m: 3030,
942 (Ar–H str), 1713 (C@O str), 1026 (C–F str), 876
C–Cl str) cm ; MS: (M , %) m/z 473 (M , 5), 455
5), 377 (6), 340 (40), 304 (11), 278 (10), 236 (10), 170
35), 149 (100), 109 (10), 6 (17); Anal. Calcd for N:
NMR (400 MHz, DMSO-d ) d: 2.21 (s, 3H, CH ), 7.39
6
3
2
(s, 1H), 7.51 (d, 1H, J = 8.6 Hz), 7.60 (d, 1H,
J = 8.7 Hz), 7.70 (t, 1H, J = 7.9 Hz), 7.81 (d, 1H,
J = 4.7 Hz), 8.19 (d, 1H, J = 7.2 Hz), 8.32 (d, 1H,
J = 8.3 Hz), 8.43 (s, 1H, pyrazole ring), 9.27 (d, 1H,
À1
+
+
(
(
(
1
4.75. Found: 14.78.
J = 4.7 Hz). IR (KBr) m: 3030, 2952 (Ar–H str), 1713
À1
(
C@O str), 1106 (C–F str), 845 (C–Cl str) cm ; MS:
+
+
4
.9. Synthesis of 6-methyl-5-(4-methylphenyl)-1-[8-(tri-
(M , %) m/z 490 (M , 100), 454 (10), 412 (15), 342 (5),
321 (10), 279 (5), 186 (15); Anal. Calcd for N: 14.26.
Found: 14.28.
fluoromethyl)quinolin-4-yl]-1,5-dihydro-4H-pyrazolo[3,4-
d]pyrimidin-4-one (7c)
Obtained from compounds (6) and 4-methyl aniline;
H
4.14. Synthesis of 6-methyl-5-(2,4,5-trichlorophenyl)-1-[8-
(trifluoromethyl)quinolin-4-yl]-1,5-dihydro-4H-pyrazolo-
[3,4-d]pyrimidin-4-one (7h)
1
light yellow needles (0.8 g, 64%); mp 245-48 °C;
NMR (400 MHz, DMSO-d ) d: 1.7 (s, 3H, CH ), 2.15
6
3
(
1
s, 3H, CH ), 7.25–8.20 (m, 9H, Ar–H), 9.27–9.29 (d,
H, J = 4.5 Hz); IR (KBr) m: 3033, 2945 (Ar–H str),
3
Obtained from compounds (6) and 2,4,5 trichloro ani-
line; colorless crystals (0.78 g, 49%); mp 272–274 °C;
IR (KBr) m: 3035, 2955 (Ar–H str), 1716 (C@O str),
À1
+
1
m/z 435 (M , 15), 379 (6), 342 (10), 304 (10), 235 (10),
703 (C@O str), 1021 (C–F str) cm ; MS: (M , %)
+
À1
1
1
47 (30), 57 (100); Anal. Calcd for N: 16.05. Found:
5.88.
1126 (C–F str), 845, 855 (C–Cl str) cm . Anal. Calcd
for N: 13.37. Found: 13.34.
4
1
.10. Synthesis of 6-methyl-5-[2-(trifluoromethyl)phenyl]-
-[8-(trifluoromethyl)quinolin-4-yl]-1,5-dihydro-4H-pyr-
4.15. Synthesis of 6-methyl-5-(2-methylphenyl)-1-[8-(tri-
fluoromethyl)quinolin-4-yl]-1,5-dihydro-4H-pyrazolo[3,4-
d]pyrimidin-4-one (7i)
azolo[3,4-d]pyrimidin-4-one (7d)
Obtained from compounds (6) and 2-trifluoromethyl
aniline; amorphous powder (0.78 g, 52%); mp 240–
Obtained from compounds (6) and 2-methyl aniline; col-
orless solid (0.88 g, 66%); mp 260–262 °C; IR (KBr) m:
3043, 2965 (Ar–H str), 1736 (C@O str), 1128 (C–F
1
4
CH ), 7.26–8.20 (m, 9H, Ar–H), 9.25–9.27 (d, 1H,
2 °C; H NMR (400 MHz, DMSO-d ) d: 2.16 (s, 3H,
6
À1
str), cm . Anal. Calcd for N: 16.05. Found: 16.08.
3
J = 4.6 Hz); IR (KBr) m: 3039, 2935 (Ar–H str), 1713
(C@O str), 1021, 1049 (C–F str) cm ; Anal. Calcd for
N: 14.30. Found: 14.31.
À1
4.16. Synthesis of 5-(4-chlorophenyl)-6-methyl-1-[8-(tri-
fluoromethyl)quinolin-4-yl]-1,5-dihydro-4H-pyrazolo[3,4-
d]pyrimidin-4-one (7j)
4.11. Synthesis of 6-methyl-5-phenyl-1-[8-(trifluorometh-
yl)quinolin-4-yl]-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimi-
din-4-one (7e)
Obtained from compounds (6) and 4-chloro aniline; col-
orless solid (0.85 g, 60%); mp 280–282 °C; IR (KBr) m:
3
040, 2960 (Ar–H str), 1730 (C@O str), 1118 (C–F
À1
Obtained from compounds (6) and aniline; colorless
solid (0.65 g, 50%); mp 255–257 °C; IR (KBr) m: 3038,
str), 824 (C–Cl str) cm . Anal. Calcd for N: 15.35.
Found: 15.36.

2046
B. S. Holla et al. / Bioorg. Med. Chem. 14 (2006) 2040–2047
4
.17. Synthesis of 6-methyl-5-(4-nitrophenyl)-1-[8-(triflu-
cipitation from dilute sodium hydroxide solution with
acetic acid. Yield, 1.8 g (36.7%); mp > 300 °C; MS:
(m/z, %) 347 (M , 100), 325 (55), 177 (35), 152 (40),
oromethyl)quinolin-4-yl]-1,5-dihydro-4H-pyrazolo[3,4-
d]pyrimidin-4-one (7k)
+
1
20 (5). IR (KBr) m: 3389, 3396, (NH str), 1675, 1680,
À1
Obtained from compounds (6) and 4-nitro aniline; yel-
low solid (0.75 g, 52%); mp 244–246 °C; IR (KBr) m:
(C@O str) cm Anal. Calcd for N: 20.17 Found: 20.20.
3
str), cm ; MS: (M , %) m/z 467 (MH , 100), 466
044, 2966 (Ar–H str), 1730 (C@O str), 1138 (C–F
4.22. Synthesis of 6-thioxo-1-[8-(trifluoromethyl)quinolin-
4-yl]-4H-pyrazolo[3,4-d]pyrimidin-4-one (10)
À1
+
+
+
(
(
M , 35), 451 (10), 421 (18), 365 (20), 279 (10), 163
25), 149 (60); Anal. Calcd for N: 18.00. Found: 18.02.
The reaction was done in the same manner as in the
preparation of (9). The product was isolated as in the
preparation of (9) to yield 34% after purification; mp
280–282 °C; IR (KBr) m: 3385 (NH), (1690) C@O
4
(
[
.18. Synthesis of 5-(3,4-dichlorophenyl)-6-methyl-1-[8-
trifluoromethyl)quinolin-4-yl]-1,5-dihydro-4H-pyrazolo-
3,4-d]pyrimidin-4-one (7l)
À1
cm . Anal. Calcd for N: 19.27. Found: 19.20.
Obtained from compounds (6) and 3, 4-dichloro aniline;
colorless solid (0.85 g, 56%); mp 278–280 °C; IR (KBr)
m: 3033, 2944 (Ar–H str), 1736 (C@O str), 1148 (C–F
4.23. Synthesis of 4-amino-1-[8-(trifluoromethyl)quinolin-
4-yl]-1H-pyrazolo[3,4-d]pyrimidine (11)
À1
str), 834 (C–Cl str) cm . Anal. Calcd for N: 14.24.
Five grams of 5-amino-1-[8-(trifluoromethyl)quinolin-4-
yl]-1H-pyrazole-4-carbonitrile (5) and 10 ml formamide
were heated at 180–190 °C for 30 min. The cooled solu-
tion was diluted with 100 ml ice-cold water and the pre-
cipitate obtained was filtered to yield 2.2 g of the crude
product. The product was purified by dissolving it in
Found: 14.28.
4
.19. Synthesis of 5-(4-fluorophenyl)-6-methyl-1-[8-(tri-
fluoromethyl)quinolin-4-yl]-1,5-dihydro-4H-pyrazolo[3,4-
d]pyrimidin-4-one (7m)
1
0% HCl, the insolubles removed by filtration. The fil-
Obtained from compounds (6) and 4-fluoro aniline; col-
orless solid (0.81 g, 60%); mp 270–272 °C; IR (KBr)
trate was taken and pH was adjusted to 8 with concen-
trated ammonium hydroxide. This solution was allowed
to cool to 10–15 °C and then filtered, washed with water
m: 3055, 2946 (Ar–H str), 1716 (C@O str), 1138, 1234
À1
(
C–F str)cm . Anal. Calcd for N: 15.96. Found: 15.94.
to give pure compound (11), yield 1.5 g (32.6%); mp
1
2
(s, 2H, NH ), 7.83 (d, 1H, J = 7.8 Hz), 7.98 (t, 1H,
80–282 °C; H NMR (400 MHz, DMSO-d ): d 5.63
6
4
1
.20. Synthesis of 1-[8-(trifluoromethyl)quinolin-4-yl]-
,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (8)
2
J = 7.6 Hz), 8.19 (s, 1H, proton on of pyrazole ring),
8.22 (d, 1H, J = 8.5 Hz), 8.35 (d, 1H, J = 6.9 Hz), 8.39
(s, 1H, proton on C-1 of pyrimidine ring), 9.24 (d, 1H,
Five grams of ethyl 5-amino-1-[8-(trifluoromethyl)quin-
olin-4-yl]-1H-pyrazole-4-carboxylate (3) and 10 ml
formamide were heated at 180–190 °C for 1.5 h. The
cooled solution was diluted with 100 ml of ice-cold
water and the precipitate obtained was filtered to yield
À1
+
J = 4.5 Hz); IR (KBr) m: 3385 (NH) cm ; MS: (M ,
%) m/z 331 (MH , 100), 330 (10), 307 (40), 242 (5),
+
196 (9), 165 (13); Anal. Calcd for N: 25.44. Found:
25.38.
2.8 g of the crude product. The product was purified
by dissolving it in 10% sodium hydroxide solution and
reprecipitating using acetic acid to obtain pure product
4.24. Synthesis of 4-amino-1-[8-(trifluoromethyl)quinolin-
4-yl]-1,7-dihydro-6H-pyrazolo[3,4-d]pyrimidin-6-one (12)
1
(
(
8). Yield 2.1 g (44.6%); mp 270–273 °C dec; H NMR
400 MHz, DMSO-d ): d 7.82 (d, 1H, J = 7.84 Hz),
Five grams of 5-amino-1-[8-(trifluoromethyl)quinolin-4-
yl]-1H-pyrazole-4-carbonitrile (5) and 10 g urea were
heated together at 180–185 °C for 10 min. The resulting
solid was dissolved in dilute sodium hydroxide and then
carefully acidified with acetic acid to obtain 4.2 g of
crude product (12). Further purification was accom-
plished by reprecipitation from dilute sodium hydroxide
6
7
.97 (t, 1H, J = 7.5 Hz), 8.18 (s, 1H, proton on of pyra-
zole ring), 8.22 (d, 1H, J = 8.4 Hz), 8.33 (d, 1H,
J = 6.9 Hz), 8.57 (s, 1H, proton on C-1 of pyrimidine
ring), 9.28 (d, 1H, J = 4.5 Hz) and 12.56 (br s, 1H,
+
NH, of pyrimidine ring); MS: (MH , %) m/z, 332
+
MH , 100), 331 (25), 251 (70), 175 (35), 136 (60), 120
(
(
1
5), 107 (25). IR (KBr) m: 3385 (NH str), 1690 (C@O
with acetic acid. Yield 2.5 g (43.8%); mp > 300 °C; H
À1
str) cm . Anal. Calcd for N: 21.14. Found: 21.12.
NMR (400 MHz, DMSO-d ): d 7.4 (s, 2H, NH ), 7.81
6
2
(d, 1H, J = 7.8 Hz), 7.96 (t, 1H, J = 7.5 Hz), 8.15 (s,
4.21. Synthesis of 1-[8-(trifluoromethyl)quinolin-4-yl]-1H-
pyrazolo[3,4-d]pyrimidine-4,6(5H, 7H)-dione (9)
1H, proton on of pyrazole ring), 8.20 (d, 1H,
J = 8.2 Hz), 8.33 (d, 1H, J = 7 Hz), 9.27 (d, 1H,
J = 4.5 Hz), 13.44 (br s, 1H, NH). IR (KBr) m: 3385,
3390 (NH) cm . Anal. Calcd for N: 24.27. Found:
24.30.
À1
Five grams of ethyl 5-amino-1-[8-(trifluoromethyl)quin-
olin-4-yl]-1H-pyrazole-4-carboxylate (3) and 10 g urea
were heated together at 150–160 °C for 30 min. The
clear solution went mushy and heating was continued
for another 10 min at 170 °C. The resulting solid was
dissolved in dilute sodium hydroxide and then carefully
acidified with acetic acid to obtain 5.5 g of crude prod-
uct (9). Further purification was accomplished by repre-
4.25. Synthesis of 4-amino-1-[8-(trifluoromethyl)quinolin-
4-yl]-1H-pyrazolo[3,4-d]pyrimidine-6-thione (13)
Five grams of 5-amino-1-[8-(trifluoromethyl)quinolin-4-
yl]-1H-pyrazole-4-carbonitrile (5) and 10 g thiourea

B. S. Holla et al. / Bioorg. Med. Chem. 14 (2006) 2040–2047
2. Filler, R. Chem. Technol. 1974, 4, 752.
2047
were heated together at 180 °C for 30 min until the clear
3
4
5
6
. Ghorab, M. M.; Ismail, Z. H.; Abdel-Gawad, S. M.;
Abdel Aziem, A. Heteroatom Chemistry 2004, 15, 57.
. Davies, L. P.; Brown, D. J.; Chow, S. C.; Johnston, G. A.
R. Neurosci. Lett. 1983, 41, 189.
. Davies, L. P.; Chow, S. C.; Skerritt, J. H.; Brown, D. J.;
Johnston, G. A. R. Life Sci. 1984, 34, 2117.
solution became mushy. The cooled melt was dissolved
in dilute sodium hydroxide and then carefully acidified
with acetic acid to obtain 3.1 g crude product (13). Fur-
ther purification was accomplished by reprecipitation
from dilute sodium hydroxide with acetic acid. Yield
1
1
DMSO-d ):
.8 g (30%); mp 288–290 °C; H NMR (400 MHz,
. Shivarama Holla, B.; Shivananda, M. K.; Akberali, P. M.;
Shalini Shenoy, M. Indian J. Chem. 2000, 39B, 440–447.
d 7.4 (s, 2H, NH2), 7.82 (d, 1H,
6
J = 7.6 Hz), 7.97 (t, 1H, J = 7.5 Hz), 8.16 (s, 1H, proton
on of pyrazole ring), 8.21 (d, 1H, J = 8.5 Hz), 8.34 (d,
7. Lee, L. F.; Schleppnik, F. M.; Schneider, R. W.; Camp-
bell, D. H. J. Heterocycl. Chem. 1990, 27, 243.
8. Pilgram, K. H.; Skiles, R. D. J. Heterocycl. Chem. 1988,
1
H, J = 7 Hz), 9.28 (d, 1H, J = 4.5 Hz), 13.55 (br s,
H, NH/SH). IR (KBr) m: 3388, 3395 (NH) cm . Anal.
À1
2
5, 129.
. Beck, J. R.; Wright, F. L. J. Heterocycl. Chem. 1987, 24,
39.
0. Bravo, P.; Dillido, D.; Resnati, G. Tetrahedron 1994, 50,
827.
1. Jung, J. C.; Watkins, E. B.; Avery, M. A. Tetrahedron
002, 58, 3639.
2. Kenneth, L. K.; John, J. F.; Kurt, E. S.; James, F. M.;
Brenda, M.; Theresa, T.; Diana, M.; Michael, L. M.
J. Med. Chem. 1996, 39, 3920.
1
Calcd for N: 23.19. Found: 23.15.
9
7
1
1
1
8
Acknowledgment
2
The authors are grateful to Head, R.S.I.C, Punjab
University, Chandigarh, SAIF, IISc Bangalore and
Head, SAIF, I.I.T., Madras, for providing elemental
1
analysis, IR, H NMR and mass spectral data.
13. Gregory, T. P.; Darlene, C. D.; David, M. S. Pestic.
Biochem. Phys. 1998, 60, 177.
1
4. Kucukguzel, S. G.; Rollas, S.; Erdeniz, H.; Kiranz, A.
C.; Ekinci, M.; Vidin, A. Eur. J. Med. Chem. 2000, 35,
Supplementary data
7
61.
1
5. Cruickshank, R.; Duguid, J. P.; Marion, B. P.; Swain, R.
H. A., 12th ed.. In Medicinal Microbiology; Churchil
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Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2005.10.053.
1
1
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