Facile synthesis and in-vitro antitumor activity of some pyrazolo[3,4-b]pyridines and pyrazolo[1,5-a]pyrimidines linked to a thiazolo[3,2-a]benzimidazole moiety

Article abstract of DOI:10.1002/ardp.200900082

The key precursor E-3-(N,N-dimethylamino)-1-(3-methylthiazolo[3,2-a] benzimidazol-2-yl)prop-2-en-1-one 4 was synthesized in good yield using Gold's reagent. The reaction of enaminone 4 with 5-amino-3-aryl-1-phenylpyrazoles 5a, b in refluxing acetic acid in the presence of sulphuric acid, yielded pyrazolo[3,4-b]pyridines 7a, b. Similarly, pyrazolo[1,5-a]pyrimidines 10a, b and 14a-f were prepared by reaction of enaminone 4 with 5-amino-1H-pyrazoles 8a, b and 12a-f, respectively. The structure of pyrazolo[1,5-a]pyrimidine 10b was determined by X-ray diffraction. The synthesized compounds were tested for their in-vitro antitumor activity against the colon cancer cell line CaCo-2; their cytotoxicity against the normal fibroblast cell line BHK was explored as well. Some of the tested compounds exhibited cell growth inhibitory activity. The significant antitumor activity of compound 14f against the CaCo-2 cell line (IC₅₀ = 0.5 μg/mL) was coupled with a lower toxicity against BHK (IC₅₀ = 2.3 μg/mL).

Full text of DOI:10.1002/ardp.200900082

24
Arch. Pharm. Chem. Life Sci. 2010, 343, 24–30
Full Paper
Facile Synthesis and In-Vitro Antitumor Activity of Some
Pyrazolo[3,4-b]pyridines and Pyrazolo[1,5-a]pyrimidines
Linked to a Thiazolo[3,2-a]benzimidazole Moiety
Hatem A. Abdel-Aziz1, Tamer S. Saleh2, and Heba S. A. El-Zahabi3
1 Department of Applied Organic Chemistry, National Research Centre, Dokki, Cairo, Egypt
2 Department of Green Chemistry, National Research Centre, Dokki, Cairo, Egypt
3 Department of Pharmaceutical Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo, Egypt
The key precursor E-3-(N,N-dimethylamino)-1-(3-methylthiazolo[3,2-a]benzimidazol-2-yl)prop-2-en-
1-one 4 was synthesized in good yield using Gold's reagent. The reaction of enaminone 4 with 5-
amino-3-aryl-1-phenylpyrazoles 5a, b in refluxing acetic acid in the presence of sulphuric acid,
yielded pyrazolo[3,4-b]pyridines 7a, b. Similarly, pyrazolo[1,5-a]pyrimidines 10a, b and 14a–f
were prepared by reaction of enaminone 4 with 5-amino-1H-pyrazoles 8a, b and 12a–f, respec-
tively. The structure of pyrazolo[1,5-a]pyrimidine 10b was determined by X-ray diffraction. The
synthesized compounds were tested for their in-vitro antitumor activity against the colon cancer
cell line CaCo-2; their cytotoxicity against the normal fibroblast cell line BHK was explored as
well. Some of the tested compounds exhibited cell growth inhibitory activity. The significant
antitumor activity of compound 14f against the CaCo-2 cell line (IC50 = 0.5 lg/mL) was coupled
with a lower toxicity against BHK (IC50 = 2.3 lg/mL).
Keywords: Antitumor activity / Colon cancer cell CaCo-2 / Enaminones / Fused pyrazoles / X-ray single crystal /
Received: April 19, 2009; accepted: August 21, 2009
DOI 10.1002/ardp.200900082
tors of mammalian collagenase [5]. It is well-known that
Introduction
the process of invasion of malignant tumors depends on
proteolytic enzymes such as collagenase. Thus, when
these proteinase inhibitors are applied to tumor cells,
they inhibited cell invasion. In a previous work, we
reported the synthesis of 2-(thiazoyl)-thiazolo[3,2-a]benzi-
midazole derivative A (Fig. 1) which showed a remarkable
cytotoxic effect against the colon carcinoma HCT-116 [9].
Recently, we extended our work and prepared 2-(pyra-
zolyl)-thiazolo[3,2-a]benzimidazole derivative B (Fig. 1)
which exerted potent and promising anticancer activity
against the same cell line HCT-116 [10]. On the other
hand, some pyrazolo[3,4-b]pyridines [11] and pyra-
zolo[1,5-a]pyrimidines [12] analogs of purine have been
reported as potent anticancer agents against a variety of
human cancer cell lines.
Thiazolo[3,2-a]benzimidazole derivatives have been
reported to possess several biological activities [1–4]. Par-
ticularly remarkable is their anticancer activity [5–10],
since the thiazolo[3,2-a]benzimidazole system can be con-
sidered an analog of levamisole (Fig. 1), a well-known
immunomodulator [6–8], which is used for the adjuvant
treatment of colon cancer. The thiazolo[3,2-a]benzimida-
zole derivative tilomisole (Wy-18,251; Fig. 1) has favor-
able biological response effects in vivo, and it is a suitable
alternative to levamisole in cancer treatments [7, 8]. In
addition, many thiazolo[2,3-a]benzimidazoles have been
reported as antineoplastic agents and/or enhancers of
the immune response with a low incidence of side effects.
In addition, these compounds were mentioned as inhibi-
In view of the above mentioned facts and in continua-
tion of our interest in both chemistry and biological
activities of thiazolo[3,2-a]benzimidazole derivatives [9,
10, 13–15], it was of interest to examine the reactivity of
E-3-(N,N-dimethylamino)-1-(3-methylthiazolo[3,2-a]benzi-
Correspondence: Hatem A. Abdel-Aziz, Department of Applied Organic
Chemistry, National Research Centre, Dokki, Cairo 12622, Egypt.
E-mail: hatem_741@yahoo.com
Fax: +20 2 3760-1877
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Arch. Pharm. Chem. Life Sci. 2010, 343, 24–30
Pyrazolo[1,5-a]pyrimidine Derivatives
25
Figure 1. Figure 1. Chemical structure of Levamisole, Tilomi-
sole, A, and B.
midazol-2-yl)prop-2-en-1-one 4 (enaminone) in the synthe-
sis of some thiazolo[3,2-a]benzimidazole derivatives com-
bined with a pyrazolo[3,4-b]pyridine or pyrazolo[1,5-a]
pyrimidine moiety to evaluate their in-vitro antitumor
activity against the colon carcinoma CaCo-2.
Scheme 1. Synthetic route to compounds 4 and 7a, b.
Results and discussion
while their mass spectra revealed, in each case, a peak
corresponding to their molecular ion. The latter reaction
probably involves the formation of the non-isolable inter-
mediate 6a, b which forms the final isolable compound
7a, b through cyclization, respectively (Scheme 1). How-
ever, all attempts to isolate the intermediate 6a, b failed.
Although, in some cases, acetic acid was not preferred as
reaction medium for such reactions in order to prevent
trimerization of enaminones [24], it was reported in
other studies as a successful reaction medium for several
reactions of enaminones [25, 26]. In addition, an AcOH/
HCl mixture (v/v = 1:1) was used for the same purpose in
similar reactions [26]. In the present work, the mixture
AcOH/H2SO4 (v/v = 50:1) gave higher yields and shorter
reaction times compared to AcOH/HCl; this may be attrib-
uted to the dehydrating character of sulphuric acid.
On the other hand, the reaction of compound 4 with
aminopyrazoles 8a, b under the above described reaction
condition afforded, in each case, only one isolable prod-
uct which has three possible structures: pyrazolo[1,5-a]
pyrimidine 10, pyrazolo[3,4-b]pyridine 11, or pyra-
zolo[2,3-a]pyridine 11A (Scheme 2). IR and1H-NMR spectra
of the reaction products revealed, in each case, no bands
Chemistry
Enaminones were found to be useful intermediates in
the construction of heterocycles [16, 17]; as active syn-
thons, they occupy an important part in our research
work [18–22]. Recently, we reported the synthesis of
enaminone 4 in 62% yield using dimethylformamide/
dimethylacetal (DMF/DMA) [13]; yet, it is moisture- and
heat-sensitive [23]. On the other hand, some enaminones
have been prepared with good yields in simple, efficient,
and economical procedures using Gold's reagent 3
([3-(dimethylamino)-2-azaprop-2-en-1-ylidene]dimethyl-
ammonium chloride), which should be handled under
moisture-free conditions [23]. Herein, we report an alter-
native synthesis of enaminone 4 using Gold's reagent 3
by preparing it in situ overcoming its handling difficul-
ties. Thus, [3-(dimethylamino)-2-azaprop-2-en-1-ylidene]-
dimethylammonium chloride (3, Gold's reagent) was pre-
pared from cyanuric chloride 2, then it was reacted suc-
cessfully with ketone 1 in sodium methoxide to produce
the enaminone 4 in 78% yield (Scheme 1).
Next, the reaction of enaminone 4 with 5-amino-3-aryl-
1-phenylpyrazole 5a, b in refluxing glacial acetic acid in
the presence of sulphuric acid, yielded the corresponding
pyrazolo[3,4-b]pyridine derivatives 7a, b (Scheme 1). The
1H-NMR spectra of compound 7a, b revealed, in each case,
two duplets in the regions d = 7.43 to 7.52 and d = 8.64 to
8.66 with J = 4.5 Hz assigned to the adjacent protons of
the pyridine ring in the pyrazolo[3,4-b]pyridine system,
1
due to a NH function. In addition, their H-NMR spectra
exhibited, in each case, a singlet signal of the pyrazole
proton in the region d = 6.98 to 7.01, and showed also
two downfield duplets in the regions d = 7.22 to 7.28 and
d = 8.61 to 8.63, respectively, assigned to the protons of
the pyrimidine ring with J = 4.2 Hz. These spectral data
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H. A. Abdel-Aziz et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 24–30
Table 1. Selected intramolecular bond lengths (ꢀ), bond angles
(8), and torsion angles (8) of 10b.
bond lengths (ꢀ)
S1-C14
S1-C23
N2-C8
N2-C10
N2-C14
N3-N6
N3-C12
N3-C13
N4-C11
1.726 (3)
1.763 (3)
1.400 (3)
1.397 (4)
1.371 (3)
1.372 (3)
1.394 (4)
1.367 (4)
1.404 (4)
C9-C17
N4-C14
O5-C21
O5-C30
N6-C17
N7-C12
N7-C28
C8-C11
C13-C23
1.490 (4)
1.308 (4)
1.381 (3)
1.417 (4)
1.355 (4)
1.364 (4)
1.306 (4)
1.410 (4)
1.454 (4)
bond angles (8)
C14-S1-C23
C8-N2-C10
C8-N2-C14
C10-N2-C14
N6-N3-C12
N6-N3-C13
90.3 (2)
137.9 (3)
106.1 (2)
115.9 (2)
111.7 (3)
125.6 (3)
C12-N3-C13
122.7 (3)
102.6 (3)
117.2 (3)
102.9 (2)
115.6 (3)
119.3 (2)
C11-N4-C14
C21-O5-C30
N3-N6-C17
C12-N7-C28
S1-C23-C13
torsion angles (8)
Scheme 2. Synthesis of compounds 10a, b.
S1-C23-C13-N3 –131.0 (7)
S1-C23-C13-C26 49.1 (5)
C10-C23-C13-N3
C10-C23-C13-C26 –121.9 (9)
58.0 (7)
Figure 2. X-ray structure of compound 10b.
Scheme 3. Synthesis of compounds 14a–f.
support the assigned structures 10a, b and rule out the
other possible structure 11, but they are not sufficient to
exclude structure 11A. However, an X-ray diffraction of
reaction condition via the loss of a water molecule to
10b (Figure 2) added clear evidence for the proposed afford the final isolable products 10a, b as depicted in
structures 10a, b following the determination of the cyc- Scheme 2.
lization pathway of intermediate 9 to produce com-
pound 10 (Scheme 2). The selected intramolecular bond
In a similar manner, treatment of compound 4 with 5-
amino-3-methyl-4-phenyl-1H-pyrazole 12a, 4-bromo-5-
lengths (ꢀ), bond angles (8), and torsion angles (8) of 10b amino-3-phenyl-1H-pyrazole 12b, or 4-(arylhydrazono)-
are listed in Table 1.
The formation of compounds 10a, b is therefore
3,5-diamino-1H-pyrazole 12c–f resulted in the formation
of the pyrazolo[1,5-a]pyrimidines 14a, 14b, and 14c–f,
assumed to take place via the nucleophilic addition of respectively (Scheme 3). The IR spectra of 14c–f showed
the exocyclic amino group in pyrazoles 8a, b to the acti- the appearance of NH2 bands in the region 3312 to 3154
cm– 1, while their 1H-NMR spectra revealed a D2O-
exchangeable broad singlet of NH2 group in the region d
vated double bond in the enaminone 4 to give the acyclic
non-isolable intermediates 9a, b followed by cyclization
and aromatization of the latter intermediates, under the = 4.78 to 5.15 in addition to the characteristic duplet sig-
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Arch. Pharm. Chem. Life Sci. 2010, 343, 24–30
Pyrazolo[1,5-a]pyrimidine Derivatives
27
Table 2. IC50 of the tested compounds against of colon cancer
cell line CaCo-2 and normal BHK fibroblasts.
Compound
IC50 (lg/mL)
CaCo-2
BHK
4
7a
7b
10a
10b
14a
14b
14c
8.05
2.15
2.35
1.21
1.14
3.62
0.94
2.35
1.07
1.14
0.5
0.58
0.87
0.54
0.81
0.74
0.6
0.54
1.54
1.14
1.01
2.3
14d
14e
14f
Doxorubicin
6.1
0.4
Figure 3. Dose-response profiles for compounds 4, 7b, 14b,
14f, and doxorubicin.
with a lower toxicity towards the normal cell line BHK
(IC50 = 2.3 lg/mL). The structure-activity relationship of
the tested compounds revealed that the nature of the
substituent at C2 of the thiazolo[3,2-a]benzimidazole
moiety has a diverse impact on their in-vitro antitumor
activity.
nals of pyrimidine protons in the regions d = 7.28 to 7.31
and 8.21 to 8.61 with J = 4.2 Hz. Compounds 14a–f
formed in a reaction similar to that proposed for com-
pounds 10a, b as outlined in Scheme 3.
Antitumor activity
The authors have declared no conflict of interest.
The cytotoxicity of the tested compounds was evaluated
in the National Institute of Cancer, Cairo, Egypt. Com-
pounds 4, 7a, b, 10a, b, and 14a–f were tested for their in-
vitro antitumor activity against the colon cancer cell line
CaCo-2. We also explored their toxicity against the nor-
mal fibroblast cell line BHK. Doxorubicin was used as
reference with IC50 = 6.1 and 0.4 lg/mL against CaCo-2
and BHK, respectively. All the tested compounds showed
a certain antitumor effect as illustrated in Table 2. The
dose-dependent inhibitory effect of compounds 4, 7b,
14b, 14f, and doxorubicin is shown in Fig. 3.
Replacement o the N,N-dimethylenaminone side chain
in compound 4 with a pyrazolo[3,4-b]pyridine or pyra-
zolo[1,5-a]pyrimidine moiety in 7a, b, 10a, b, and 14a–f
increased the antitumor activity against the CaCo-2 cell
line with a remarkable decrease in their cytotoxicity
against the normal fibroblast cell line BHK. Pyrazolo[1,5-a]
pyrimidine derivatives 10a, b and 14a–f displayed an anti-
tumor activity higher than that of the pyrazolo[3,4-b]pyri-
dines 7a, b. On the other hand, the substituted phenylazo
group in 14d–f enhanced the antitumor activity com-
pared tothe phenyazo derivative 14c.
Experimental
Chemistry
Melting points were measured with a Gallenkamp apparatus
(Weiss-Gallenkamp, London, UK) and are uncorrected. IR spectra
were recorded on Shimadzu FT-IR 8101 PC infrared spectropho-
tometer (Shimadzu, Tokyo, Japan). The NMR spectra were
recorded on a Varian Mercury VX-300 NMR spectrometer (Varian
Inc., Palo Alto, CA, USA). 1H spectra were run at 300 MHz in deu-
terated dimethylsulphoxide (DMSO-d6). Chemical shifts are
quoted in d and were related to that of the solvents. Mass spectra
were measured on a GCMS-QP1000 EX spectrometer (Shimadzu)
at 70 eV. Elemental analyses were carried out at the Microanalyt-
ical center of Cairo University. X-ray crystallography was carried
out on a Kappa CCD FR 590 diffractometer (Enraf Nonius),
National Research Center, Dokki, Cairo, Egypt. 1-(3-Methylthia-
zolo[3,2-a]benzimidazol-2-yl)ethanone 1 [27], 5-amino-3-aryl-1-
phenylpyrazole 5a, b [28], 5-amino-3-methyl-1H-pyrazole 8a [28],
5-amino-3-methyl-4-phenyl-1H-pyrazole 12a [29], 4-bromo-5-
amino-3-phenyl-1H-pyrazole 12b [30], and 4-(arylhydrazono)-3,5-
diamino-1H-pyrazole 12c–f [28] were prepared by the reported
methods. Cyanuric chloride 2 and 5-amino-3-(4-methoxyphenyl)-
1H-pyrazole 8b were used as obtained commercially.
Pyrazolo[1,5-a]pyrimidine derivatives 14b and 14f
showed a significant antitumor activity against the CaCo-
2 cell line while pyrazolo[3,4-b]pyridine derivative 7b
revealed a characteristic inhibitory effect at higher con-
centrations. The antitumor activity of compound 14f
against the CaCo-2 cell line (IC50 = 0.5 lg/mL) is coupled
E-3-(N,N-Dimethylamino)-1-(3-methylthiazolo[3,2-a]
benzimidazol-2-yl)prop-2-en-1-one 4
To a mixture of cyanuric chloride 2 (12.91 g, 0.07 mol) in dry 1,4-
dioxane (20 mL), N,N-dimethylformamide (30.7 g, 0.42 mol) was
added. The resulting solution was gently refluxed for 1.5 h while
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H. A. Abdel-Aziz et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 24–30
a considerable amount of carbon dioxide evolved. [3-(dimethyla-
mino)-2-azaprop-2-en-1-ylidene]dimethylammonium chloride 3
rapidly solidified and the reaction mixture was allowed to cool
to room temperature. A mixture of metallic sodium (4.6 g, 0.2
mol) dissolved in absolute methanol (200 mL) was added in small
portions followed by the addition of 1-(3-methylthiazolo[3,2-a]
benzimidazol-2-yl)ethanone 1 (23.3 g, 0.1 mol). The resulting
mixture was refluxed for 3 h through which the non-isolable
salt and the ketone 1 were dissolved and yellow crystals and
sodium chloride were formed. The reaction mixture was cooled
to room temperature, then, water (200 mL) was added. The yel-
low crystals were collected by filtration, washed thoroughly
with water, dried, and finally crystallized from EtOH/DMF to
afford the enaminone 4 as yellow crystals in 78% yield; m.p.:
240–2418C [13].
3-Methyl-2-(2-methylpyrazolo[1,5-a]pyrimidin-7-yl)
thiazolo[3,2-a]benzimidazole 10a
Yellow needles in 66% yield; m.p.: 201–2038C; IR (KBr) mmax
[cm–1]: 3083 (aromatic C-H), 2959 (aliphatic C-H), 1590 (C=N); 1H-
NMR (DMSO-d6) d [ppm]: 2.28 (s, 3H, CH3), 3.04 (s, 3H, CH3), 6.98 (s,
1H, pyrazole), 7.04–8.49 (m, 4H, Ar-H), 7.22 (d, 1H, J = 4.2 Hz, pyr-
imidine), 8.61 (d, 1H, J = 4.2 Hz, pyrimidine); MS m/z [%]: 320 [M+ +
1] (31.6), 319 [M+] (100), 262 (16.4), 187 (4.5), 145 (12.5). Anal.
calcd. for C17H13N5S: C, 63.93; H, 4.10; N, 21.93; S, 10.04. Found:
C, 64.22; H, 4.12; N, 22.15; S, 10.18.
2-[2-(4-Methoxyphenyl)pyrazolo[1,5-a]pyrimidin-7-yl]-3-
methylthiazolo[3,2-a]benzimidazole 10b
Yellow needles in 68% yield; m.p.: 233–2358C; IR (KBr) mmax [cm–1]:
1
3078 (aromatic C-H), 2964 (aliphatic C-H), 1593 (C=N); H-NMR
(DMSO-d6) d [ppm]: 3.05 (s, 3H, CH3), 3.93 (s, 3H, -OCH3), 7.01 (s,
1H, pyrazole), 7.12–8.27 (m, 8H, Ar-H), 7.28 (d, 1H, J = 4.2 Hz, pyr-
imidine), 8.63 (d, 1H, J = 4.2 Hz, pyrimidine); MS m/z [%]: 411 [M+]
(25.3), 303 (6.6), 292 (100), 223 (13.8), 154 (3.5). Anal. calcd. for
C23H17N5OS: C, 67.13; H, 4.16; N, 17.02; S, 7.79. Found: C, 67.52;
H, 4.23; N, 16.85; S, 7.66.
General procedure for the reaction of enaminone 4 with
1-arylpyrazoles 5a, b
To a mixture of enaminone 4 (0.57 g, 2 mmol) and the appropri-
ate N-phenylpyrazole 5a, b (2 mmol) in glacial acetic acid (25
mL), concentrated sulphuric acid was add (0.5 mL); the reaction
mixture was refluxed for 9 h and then left to cool. The solid prod-
uct was filtered off, washed with ethanol, dried, and finally
recrystallized from DMF/H2O to afford the corresponding pyra-
zolo[3,4-b]pyridine derivatives 7a, b, respectively.
3-Methyl-2-(2-methyl-3-phenyl-pyrazolo[1,5-a]pyrimidin-
7-yl)thiazolo[3,2-a]benzimidazole 14a
Yellow needles in 73% yield; m.p.: 244–2468C; IR (KBr) mmax
[cm–1]: 3064 (aromatic C-H), 2970 (aliphatic C-H), 1590 (C=N); 1H-
NMR (DMSO-d6) d [ppm]: 2.52 (s, 3H, CH3), 3.01 (s, 3H, CH3), 7.08–
8.65 (m, 9H, Ar-H), 7.45 (d, 1H, J = 4.5 Hz, pyrimidine), 8.71 (d, 1H,
J = 4.5 Hz, pyrimidine); MS m/z [%]: 396 [M+ + 1] (25.7), 395 [M+]
100), 362 (25.6), 246 (15.2), 187 (16.8), 77 (11.3). Anal. calcd. for
C23H17N5S: C, 69.85; H, 4.33; N, 17.71; S, 8.11. Found: C, 69.57; H,
4.53; N, 17.83; S, 8.19.
2-(1,3-Diphenyl-1H-pyrazolo[3,4-b]pyridin-4-yl)-3-
methylthiazolo[3,2-a]benzimidazole 7a
Yellow crystals in 65% yield; m.p.: 265–2678C; IR (KBr) mmax
[cm–1]: 3067 (aromatic C-H), 2970 (aliphatic C-H), 1590 (C=N); 1H-
NMR (DMSO-d6) d [ppm]: 2.99 (s, 3H, CH3), 7.24–8.15 (m, 14H, Ar-
H), 7.34 (d, 1H, J = 4.5 Hz, pyridine), 8.66 (d, 1H, J = 4.5 Hz, pyri-
dine); MS m/z [%]: 457 [M+] (100), 379 (7.3), 301 (4.7), 276 (25.2),
187 (6.6), 77 (9.1). Anal. calcd. for C28H19N5S: C, 73.50; H, 4.19; N,
15.31; S, 7.01. Found: C, 73.74; H, 4.00; N, 15.33; S, 6.87.
2-(3-Bromo-2-phenyl-pyrazolo[1,5-a]pyrimidin-7-yl)-3-
methyl-thiazolo[3,2-a]benzimidazole 14b
Yellow needles in 70% yield; m.p.: 235–2378C; IR (KBr) mmax [cm–1]:
3086 (aromatic C-H), 2965 (aliphatic C-H),1595 (C=N); 1H-NMR
(DMSO-d6) d [ppm]: 2.99 (s, 3H, CH3), 7.11–8.52 (m, 9H, Ar-H), 7.38
(d, 1H, J = 4.2 Hz, pyrimidine), 8.67 (d, 1H, J = 4.2 Hz, pyrimidine);
MS m/z [%]: 463 [M+ + 3] (8.8), 462 [M+ + 2] (16.9), 461 [M+ + 1] (100),
460 [M+] (17.3) ,379 (24.8), 187 (18.5), 173 (7.3), 77 (16.3). Anal.
calcd. for C22H14BrN5S: C, 57.40; H, 3.07; N, 15.21; S, 6.97. Found:
C, 57.64; H, 2.92; N, 15.26; S, 7.21.
2-[3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazolo[3,4-
b]pyridin-4-yl]-3-methylthiazolo[3,2-a]benzimidazole 7b
Yellow crystals in 69% yield; m.p.: 290–2928C; IR (KBr) mmax
[cm–1]: 3059 (aromatic C-H), 2965 (aliphatic C-H), 1592 (C=N); 1H-
NMR (DMSO-d6) d [ppm]: 3.02 (s, 3H, CH3), 3.82 (s, 3H, CH3), 7.21–
8.22 (m, 13H, Ar-H), 7.52 (d, 1H, J = 4.5 Hz, pyridine), 8.64 (d, 1H, J
= 4.5 Hz, pyrimidine); MS m/z [%]: 487 [M+] (14.4), 317 (100), 241
(28.7), 187 (12.9), 77 (12.4). Anal. calcd. for C29H21N5OS: C, 71.44;
H, 4.34; N, 14.36; S, 6.58. Found: C, 71.12; H, 4.55; N, 14.19; S,
6.43.
2-(2-Amino-3-phenylazo-pyrazolo[1,5-a]pyrimidin-7-yl)-3-
methylthiazolo[3,2-a]benzimidazole 14c
Yellow needles in 67% yield; m.p.: A3008C; IR (KBr) mmax [cm– 1]:
3312, 3154 (NH2), 3067 (aromatic C-H), 2918 (aliphatic C-H), 1591
(C=N);1H-NMR (DMSO-d6) d [ppm]: 2.99 (s, 3H, CH3), 4.78 (br. s, 2H,
NH2, D2O-exchangeable), 7.25–8.05 (m, 9H, Ar-H), 7.31 (d, 1H, J =
4.2 Hz, pyrimidine), 8.59 (d, 1H, J = 4.2 Hz, pyrimidine); MS m/z
[%]: 425 [M+ + 1] (8.6), 424 [M+] (100), 396 (11.0), 318 (14.6), 211
(18.2), 77 (6.8). Anal. calcd. for C22H16N8S: C, 62.25; H, 3.80; N,
26.40; S, 7.55. Found: C, 62.07; H, 3.86; N, 26.73; S, 7.39.
General procedure for the reaction of enaminone 4 with
pyrazoles 8a, b and 12a–f
This reaction was carried out by the same procedure as described
in the synthesis of compounds 7a, b using pyrazoles 8a, b and
12a–f instead of 5a, b with 4 to 6 h refluxing time. The solid
product was filtered off, washed with ethanol, dried, and finally
recrystallized from DMF/H2O to afford the corresponding pyra-
zolo[1,5-a]pyrimidine derivatives 10a, b and 14a–f, respectively.
2-[2-Amino-3-(4-tolylazo)-pyrazolo[1,5-a]pyrimidin-7-yl]-
3-methylthiazolo[3,2-a]benzimidazole 14d
Yellow needles in 66% yield; m.p.: 286–2888C; IR (KBr) mmax [cm–1]:
3319, 3122 (NH2), 3072 (aromatic C-H), 2948 (aliphatic C-H), 1586
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Arch. Pharm. Chem. Life Sci. 2010, 343, 24–30
Pyrazolo[1,5-a]pyrimidine Derivatives
29
1
(C=N); H-NMR (DMSO-d6) d [ppm] 2.33 (s, 3H, CH3), 2.96 (s, 3H,
99%) and diluted 1000-fold in the assay. Increasing concentra-
tions of the test compounds 1, 2.5, 5, and 10 lg/mL were added
to the cell monolayer. Monolayer cells were incubated with the
compounds for 48 h at 378C, in an atmosphere of 5% CO2. After
48 h, cells were fixed, washed, and stained with Sulfo-Rhod-
amine-B stain (SRB). Excess stain was washed off with acetic acid.
The attached stain was recovered with Tris-EDTA buffer. Cell sur-
vival and drug activity were determined by measuring color
intensity using an ELISA reader. Cell proliferation was deter-
mined by measuring the absorbance at 570 nm. Data were repre-
sentative of three individual experiments, performed in three
replicates for each individual dose and measured by SRB assay.
Control values did not exhibit significant changes compared to
the dimethylsulfoxide (DMSO) vehicle. The relations between
survival percentage of the tested compounds and doxorubicin
were recorded. IC50 was determined by using a program Graph-
Pad PRISM version 3. Statistical significance was determined by
using one-way analysis of variance (ANOVA) test. A P value more
than 0.05 was considered insignificant.
CH3), 5.03 (br. s, 2H, NH2, D2O-exchangeable), 7.26 (d, 1H, J = 4.2
Hz, pyrimidine), 7.41–7.93 (m, 8H, Ar-H), 8.21 (d, 1H, J = 4.2 Hz,
pyrimidine); MS m/z [%]: 438 [M+] (100), 347 (23.0), 410 (9.9), 291
(15.6), 225 (12.5), 113 (4.7). Anal. calcd. for C23H18N8S: C, 63.00; H,
4.14; N, 25.55; S, 7.31. Found: C, 63.07; H, 4.03; N, 25.73; S, 7.25.
2-[2-Amino-3-(4-chlorophenylazo)-pyrazolo[1,5-a]-
pyrimidin-7-yl]-3-methylthiazolo[3,2-a]benzimidazole 14e
Yellow needles in 70% yield; m.p.: A3008C; IR (KBr) mmax [cm– 1]:
3395, 3154 (NH2), 3070 (aromatic C-H), 2981 (aliphatic C-H), 1596
(C=N);1H-NMR (DMSO-d6) d [ppm]: 3.02 (s, 3H, CH3), 4.98 (br. s, 2H,
NH2, D2O-exchangeable), 7.28 (d, 1H, J = 4.2 Hz, pyrimidine),
7.34–7.92 (m, 8H, Ar-H), 8.45 (d, 1H, J = 4.2 Hz, pyrimidine); MS
m/z [%]: 459 [M+ + 1] (32.8), 458 [M+] (100), 430 (26.3), 255 (12.9),
186 (16.1), 150 (4.4). Anal. calcd. for C22H15ClN8S: C, 57.58; H,
3.29; N, 24.42; S, 6.99. Found: C, 57.66; H, 3.27; N, 24.53; S, 7.14.
2-[2-Amino-3-(4-fluorophenylazo)-pyrazolo[1,5-a]-
pyrimidin-7-yl]-3-methylthiazolo[3,2-a]benzimidazole 14f
Yellow needles in 65% yield; m.p.: A3008C; IR (KBr) mmax [cm– 1]:
3364, 3151 (NH2), 3077 (aromatic C-H), 2963 (aliphatic C-H),1602
(C=N);1H-NMR (DMSO-d6) d [ppm]: 2.98 (s, 3H, CH3), 5.15 (br. s, 2H,
NH2, D2O-exchangeable), 7.28 (d, 1H, J = 4.2 Hz, pyrimidine),
7.51–8.27 (m, 8H, Ar-H), 8.61 (d, 1H, J = 4.2 Hz, pyrimidine); MS
m/z [%]: 442 [M+] (100), 414 (19.0), 246 (11.7), 186 (5.2), 98 (5.3).
Anal. calcd. for C22H15FN8S: C, 59.72; H, 3.42; N, 25.32; S, 7.25.
Found: C, 59.49; H, 3.54; N, 25.18; S, 7.32.
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