On the reactivity of 6-acetyl-7-(2-dimethylaminovinyl)pyrazolo[1,5-a] pyrimidines with 1,3- and 1,4-bisnucleophiles

Article abstract of DOI:10.1039/b717835b

Reaction of 6-acetyl-7-(2-dimethylaminovinyl)pyrazolo[1,5-a]pyrimidine 1 with 1,3- and 1,4-bisnucleophiles has been investigated; obtainment of new polycyclic heterocyclic derivatives is reported. A convenient procedure leading to new pyrazolo[1,5-a]quina

Full text of DOI:10.1039/b717835b

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
On the reactivity of 6-acetyl-7-(2-dimethylaminovinyl)pyrazolo[1,5-
a]pyrimidines with 1,3- and 1,4-bisnucleophiles
Stefano Chimichi,*a Marco Boccalini,*a Silvia Selleri,b Camilla Costagli,b Gabriella Guerrinib and
Giampietro Violac
Received 19th November 2007, Accepted 13th December 2007
First published as an Advance Article on the web 14th January 2008
DOI: 10.1039/b717835b
Reaction of 6-acetyl-7-(2-dimethylaminovinyl)pyrazolo[1,5-a]pyrimidine 1 with 1,3- and
1,4-bisnucleophiles has been investigated; obtainment of new polycyclic heterocyclic derivatives is
reported. A convenient procedure leading to new pyrazolo[1,5-a]quinazolines is described; a modest
bioactivity of these compounds against two human tumor cell lines was also ascertained.
Introduction
Fused heterocyclic compounds such as pyrazolo[1,5-a]pyrimidine
and its derivatives are known to possess pharmacological activity
and anxiolytic properties.1 Our research group has been involved
for a long time in the chemistry of pyrazolo[1,5-a]pyrimidines
with the aim of synthesizing new tricyclic systems as novel
selective GABAA a1 receptor agonists or benzodiazepine receptor
(BZR) ligands.1–3 We have previously reported on the reactivity of
compounds of type 1 towards some nucleophiles like ammonia,4
hydroxylamine,5 and hydrazine6 to give pyrazolo[1,5-a]pyrido[3,4-
e]pyrimidines A, pyrazolo[1,5-a]pyrido[3,4-e]pyrimidine-7-oxides
B, and 6-(pyrazol-3ꢀ-yl)pyrazolo[1,5-a]pyrimidines C, respectively
(see Fig. 1).
Following these results we decided to investigate the behaviour
of compound 1 towards various 1,3- and 1,4-bisnucleophiles in an
effort to expand the scope of the above reaction.
Results and discussion
Fig. 1 Reactivity of 1 with ammonia, hydroxylamine and hydrazine.
When the dimethylaminovinyl derivative 1 was allowed to react
with 1,3-bisnucleophiles in glacial acetic acid in the presence
of sodium acetate a complex mixture of products is obtained.
Thus, we carried out the reaction in diglyme with the 1,3-
bisnucleophile as free base. The reaction always afforded an unique
product with moderate (ca. 60%) yields, except for when using S-
methylthiourea. In this case the final product was obtained in low
yield (18%) only when using a large excess of the reagent.
Although no intermediates have been isolated, it is possible to
suggest that the reaction might proceed through compound D
via two alternative pathways (Scheme 1): while route a should
give rise to the pyrazolopyrimido[1,3]diazocine system E, route b
rationalizes the formation of the isomeric compounds 2–5 through
a nucleophilic attack of the NH group on the electron poor C-7 of
the pyrimidine ring. In order to resolve this point with the aim to
attribute the right structure to the reaction products, we examined
the NMR spectra of the obtained compounds. 2D NMR spectra
(gHSQC and gHMBC) show C–H connectivities that hold true
for structures E and 2–5. Thus, to distinguish between the two
types of compound we performed some NOE experiments. To
this end, we started assigning the pyrimidine and pyrazolo[1,5-
a]pyrimidine proton signals on the basis of gHSQC and gHMBC
experiments. Then, we noticed (using, for reference, compound 4)
that irradiation of the singlet at d 8.7 ppm (attributed to H-5 of
the unknown product) leads to a significant enhancement of the
proton resonating at d 7.32 ppm that appears as a doublet. Because
at the same time no NOE is observed on the methyl group, we may
attribute this signal to H-5ꢀ of structures 2–5. This attribution
was then confirmed by irradiation of the singlet at d 2.97 ppm
(7-Me in structure 4 or 6-Me in the corresponding structure E)
obtaining a significant enhancement only on the proton appearing
as a doublet and that cannot be attributed to H-10 of structure E.
These experimental data hold true for compounds 2–5. Thus, we
aDipartimento di Chimica Organica “U. Schiff” and Laboratorio di Proget-
tazione, Sintesi e Studio di Eterocicli Biologicamente Attivi (HeteroBio-
Lab), University of Florence, via della Lastruccia 13, I 50019 Sesto F.no
(Firenze), Italy. E-mail: stefano.chimichi@unifi.it; Fax: +39 055 4573568;
Tel: +39 055 4573537
bDipartimento di Scienze Farmaceutiche, University of Florence, via U. Schiff
6, I-50019 Sesto F.no (Firenze), Italy
cDipartimento di Scienze Farmaceutiche, University of Padova, via Marzolo
5, I 35131 Padova, Italy
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Scheme 1
established the pyrimidin-4-yl nature of compounds arising from
the reaction of 1 with 1,3-bisnucleophiles, excluding the formation
of the 1,3-diazocine ring system.
products (ca. 2.5 : 1); work-up of this mixture (see Experimental)
allowed us to isolate compounds 6 and 7. The structure of the
predominant compound 7 was derived as follows: first of all,
1
Following these results and being interested in new heterocycle-
substituted pyrazolo[1,5-a]pyrimidines, we turned our attention to
the reactivity of 1 with 1,4-bisnucleophiles. The reaction of 1 with
e.g. ethane-1,2-diamine could give rise to a 1,4-diazepine system
linked to position 6 of the pyrazolo[1,5-a]pyrimidine moiety
according to that observed for 1,3-bisnucleophiles (Scheme 2,
structure F). Reaction of compound 1 with ethane-1,2-diamine
in acetic acid solution afforded a crude, mainly containing two
the H NMR spectrum clearly shows the presence of a terminal
Csp2H2 group (two doublets at d 4.66 and 3.96 ppm with a geminal
coupling constant of 3.4 Hz), then the gHMBC spectrum contains
two diagnostic correlations: (a) the carbon atom resonating at d
=
140.0 ppm, that cannot be assigned to a C N, is connected to
the Csp2 protons and to those of a NCH2 group, (b) the resonance
=
at d 170.0 ppm, clearly attributable to the C O of the acetyl
group, is connected to the exchangeable proton signal at d 8.0 ppm
Scheme 2 Reagents and conditions: (i) ethane-1,2-diamine, AcOH, reflux, then NaOH, rt.
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(NH) (Fig. 2). Moreover, the existence of a strong NOE effect
between H-5 and only one of the Csp2 protons confirms the assigned
pyrazolo[1,5-a]pyrido[3,4-e]pyrimidine structure and allows the
exclusion of the formation of the 1,4-diazepine ring (Scheme 2,
route a). Similarly, reaction of compound 1 with ethanolamine
in the same experimental conditions gives rise to compound 8
(Scheme 3) whose structure was once again established by NMR
experiments.
2-methylpyrazolo[1,5-a]quinazolin-6-ol 11 together with a minor
amount of the corresponding 6-amino derivative 10 (Scheme 4).
Compounds 12 and 13 showed an analogous reactivity, originating
the corresponding derivatives. This procedure allowed us to obtain
new pyrazoloquinazolines functionalized at position 6, starting
from a suitable 6-acetyl-7-(2-dimethylaminovinyl)pyrazolo[1,5-
a]pyrimidine, thus opening a new synthetic pathway to the
pyrazolo[1,5-a]quinazoline system.
Fig. 2 Significant correlations in the gHMBC spectrum and NOE effect
in compound 7.
Scheme 4
In the light of the reported biological properties of some
pyrazolo[1,5-a]quinazolines,7,8 we evaluated the antiproliferative
activity of the new compounds. The cytotoxicity of compounds
10, 11, and 14–17, was investigated on two cell lines of human
tumor such as Jurkat (human lymphoblastoid leukaemia) and
MCF-7 (human breast adenocarcinoma). Table 1 shows the extent
of cell survival expressed as IC50 which is the concentration,
expressed in lM, which induces 50% inhibition of cell growth, after
Scheme 3 Reagents and conditions: (i) ethanolamine, AcOH, reflux, then
NaOH, rt, 85%; (ii) n-propylamine, AcOH, reflux, then NaOH, rt, 81%.
Thus, both ethane-1,2-diamine and ethanolamine do not react
as 1,4-bisnucleophiles and to confirm this behaviour we carried out
the reaction with n-propylamine. As expected, compound 1 reacts
with this nucleophile in glacial acetic acid to give compound 9 in
81% yield.
A careful examination of the NMR spectra of the products
originating from the reaction of 1 with ethane-1,2-diamine in
glacial acetic acid gave evidence of the presence of a very small
quantity of an heteroaromatic side product in the 1H NMR
spectrum of the separated, unpurified, compound 6. In particular
we noticed that the unknown compound possesses only one methyl
group and showed, besides the signals of H-3 and H-5 of the
pyrazolopyrimidine system, a clear pattern of three contiguous
aromatic protons.
Table 1 Antiproliferative activity of tested compounds against two
human tumor cell lines
IC50/lMa of cell lines
Compound
Jurkat
MCF-7
10
11
14
15
16
17
>50
>50
>50
>50
42.4 6.5
31.2 4.3
>50
36.3 6.2
>50
35.5 9.8
18.5 6.2
>50
On this basis, we tentatively attributed a pyrazolo[1,5-
a]quinazoline structure to this product that could arise from loss
of dimethylamine owing to the attack by the enolic form of the
acetyl group. To confirm this hypothesis compound 1 was refluxed
in an acetic buffer solution to give, after separation, the expected
a IC50 concentration of the compound required to inhibit the cellular
growth by 50% after 72 hours of drug exposure, as determined by the
MTT assay as described in the Experimental. Values are expressed as
mean SEM of three experiments.
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incubation for 72 h. The results indicate that these compounds
present a modest reduction of the cell growth: the 6-dimethylamino
derivative 16 is the most active compound. From a structure–
activity relationship point of view the presence of a phenyl group
in position 3 seems to be important for the activity in the series of
compounds bearing the dimethylamino group (compounds 10, 14,
and 16) since its presence strongly increases the antiproliferative
activity.
dimethylguanidine sulfate (163.3 mg, 0.6 mmol) or acetamidine
hydrochloride (113.4 mg, 1.2 mmol) or S-methylthiourea sulfate
(167.0 mg, 0.6 mmol) was heated for 4 h at 120 ◦C. After cooling,
the precipitate was filtered, washed with water and crystallised.
4-(2,7-Dimethylpyrazolo[1,5-a]pyrimidin-6-yl)pyrimidin-2-amine 2
Colourless crystals, mp 148–149 ◦C (from isopropanol); dH
(400 MHz, CDCl3) 8.55 (1 H, s, 5-H), 8.40 (1 H, d, 3J = 5.0 Hz,
On the contrary, the same substitution in the series of com-
pounds bearing the OH group in position 6 (11, 15, and 17) reduces
the activity.
3
6ꢀ-H), 6.81 (1 H, d, J = 5.0 Hz, 5ꢀ-H), 6.53 (1 H, s, 3-H), 5.24
(2 H, br s, NH2), 2.96 (3 H, s, 7-CH3), 2.56 (3 H, s, 2-CH3); dC
(100.58 MHz, CDCl3) 163.0 (s, C-2ꢀ and C-4ꢀ), 158.8 (d, C-6ꢀ),
156.0 (s, C-2), 148.9 (s, C-3a), 148.8 (d, C-5), 144.95 (s, C-7), 117.8
(s, C-6), 111.75 (d, C-5ꢀ), 96.9 (d, C-3), 15.1 (q, 7-CH3), 14.8 (q,
2-CH3); m/z (EI) 240 (M+), 225, 198, 120; mmax(KBr)/cm−1 3423,
1601, 1456, 1254. Anal. found: C, 60.1; H, 4.9; N, 35.1%. Calc. for
C12H12N6: C, 60,0; H, 5.0; N, 35.0%.
Conclusions
To summarise, we described the reactivity of 6-acetyl-7-(2-
dimethylaminovinyl)pyrazolo[1,5-a]pyrimidine 1 with 1,3- and
1,4-bisnucleophiles, showing that no pyrazolopyrimido[1,3]-
diazocines or 2,3-dihydro-1H-1,4-diazepin-5-yl 6-substituted
pyrazolo[1,5-a]pyrimidines can be obtained, and the reactions
give rise to the new 6-pyrimidin-4-ylpyrazolo[1,5-a]pyrimidine sys-
tem or to 6-methylidenepyrazolo[1,5-a]pyrido[3,4-e]pyrimidines,
respectively. Furthermore, from the same starting materials, we
established a general synthetic procedure for the obtainment of
variously substituted pyrazolo[1,5-a]quinazolines whose bioactiv-
ity has been tested against two cell lines of human tumor.
4-(2,7-Dimethylpyrazolo[1,5-a]pyrimidin-6-yl)-N,N-
dimethylpyrimidin-2-amine 3
Colourless crystals, mp 218–219 ◦C (from ethanol); dH (400 MHz,
CDCl3) 8.59 (1 H, s, 5-H), 8.41 (1 H, d, 3J = 5.0 Hz, 6ꢀ-H), 6.66 (1
H, d, 3J = 5.0 Hz, 5ꢀ-H), 6.52 (1 H, s, 3-H), 3.24 [6 H, s, N(CH3)2],
2.99 (3 H, s, 7-CH3), 2.56 (3 H, s, 2-CH3); dC (100.58 MHz, CDCl3)
162.2 (s, C-2ꢀ), 162.0 (s, C-4ꢀ), 158.1 (d, C-6ꢀ), 155.7 (s, C-2), 149.2
(d, C-5), 148.9 (s, C-3a), 145.0 (s, C-7), 118.5 (s, C-6), 108.7 (d,
C-5ꢀ), 96.7 (d, C-3), 37.05 [q, N(CH3)2], 15.1 (q, 7-CH3), 14.8 (q,
2-CH3); m/z (EI) 268 (M+), 253, 239, 224, 210; mmax(KBr)/cm−1
2932, 1611, 1600, 1407. Anal. found: C, 62.6; H, 6.1; N, 31.4%.
Calc. for C14H16N6: C, 62.7; H, 6.0; N, 31.3%.
Experimental
General
Melting points were taken on a Bu¨chi 510 apparatus and are
uncorrected. IR spectra were obtained with a Perkin-Elmer 881
spectrophotometer after dispersion in KBr. Elemental analysis
were obtained with Elemental Analyzer Perkin-Elmer 240C
apparatus. Mass spectra were registered with a Carlo Erba
QMD 1000 instrument at 70 eV. Compounds 1, 12 and 13
were obtained as reported in the literature.4,9 Silica gel plates
(Merck F254) and silica gel 60 (Merck 230–400 mesh) were used
for analytical TLC and for column chromatography, respectively.
Solvents were removed under reduced pressure. All 1D and 2D
NMR experiments were performed on a Varian Mercuryplus-
400 spectrometer (399.95 MHz for 1H, 100.57 MHz for 13C),
with a 5 mm indirect detection probe equipped with a gradient
coil, at 298 K. Chemical shifts (d in ppm) were referenced to
the solvent CDCl3 (7.26 for 1H and 77.00 ppm for 13C NMR) or
DMSO-d6 (2.50 for 1H and 39.50 ppm for 13C NMR). All coupling
constants are in Hz. Assignments are made using 1H, 13C, DEPT
and NOESY 1D experiments and gHSQC, gHMBC, gHMQC,
and gCOSY 2D experiments. All pulse sequences were used as
provided by Varian and processing was done using standard
2,7-Dimethyl-6-(2-methylpyrimidin-4-yl)pyrazolo[1,5-
a]pyrimidine 4
Colourless crystals, mp 168–169 ◦C (from ethanol); dH (400 MHz,
CDCl3) 8.74 (1 H, d, 3J = 5.2 Hz, 6ꢀ-H), 8.57 (1 H, s, 5-H), 7.32 (1
H, d, 3J = 5.2 Hz, 5ꢀ-H), 6.54 (1 H, s, 3-H), 2.97 (3 H, s, 7-CH3),
2.82 (3 H, s, 2ꢀ-CH3), 2.56 (3 H, s, 2-CH3); dC (100.58 MHz, CDCl3)
168.5 (s, C-2ꢀ), 161.7 (s, C-4ꢀ), 157.4 (d, C-6ꢀ), 156.0 (s, C-2), 148.7
(s, C-3a), 148.8 (d, C-5), 144.1 (s, C-7), 118.0 (d, C-5ꢀ), 117.35 (s, C-
6), 96.9 (d, C-3), 26.2 (q, 2ꢀ-CH3), 14.9 (q, 7-CH3), 14.8 (q, 2-CH3);
m/z (EI) 239 (M+), 238, 224, 211, 197, 170; mmax(KBr)/cm−1 2928,
1610, 1576, 1557, 1254. Anal. found: C, 65.1; H, 5.3; N, 29.4%.
Calc. for C13H13N5: C, 65.25; H, 5.5; N, 29.3%.
2,7-Dimethyl-6-[2-(methylsulfanyl)pyrimidin-4-yl]pyrazolo[1,5-
a]pyrimidine 5
Yellow needles, mp 167–168 ◦C (from ethanol); dH (400 MHz,
CDCl3) 8.63 (1 H, d, 3J = 5.1 Hz, 6ꢀ-H), 8.60 (1 H, s, 5-H), 7.18 (1
H, d, 3J = 5.1 Hz, 5ꢀ-H), 6.56 (1 H, s, 3-H), 3.00 (3 H, s, 7-CH3),
2.62 (3 H, s, S-CH3), 2.57 (3 H, s, 2-CH3); dC (100.58 MHz, CDCl3)
172.65 (s, C-2ꢀ), 161.4 (s, C-4ꢀ), 156.9 (d, C-6ꢀ), 155.8 (s, C-2), 148.3
(s, C-3a), 148.15 (d, C-5), 144.9 (s, C-7), 115.3 (d, C-5ꢀ), 116.5 (s,
C-6), 96.6 (d, C-3), 14.9 (q, CH3), 14.8 (q, CH3), 14.7 (q, CH3);
m/z (EI) 271 (M+), 256, 224, 197; mmax(KBr)/cm−1 3030, 2924,
1600, 1490. Anal. found: C, 60.5; H, 5.8; N, 24.5; S, 6.4%. Calc.
for C13H13N5S: C, 60.6; H, 5.7; N, 24.45; S, 6.2%.
1
Varian methods. H NMR spectra were acquired using 4.6 kHz
spectral width with 32k data points (4.5 ls 90◦ pulse width,
0.28 Hz/point digital resolution).
General procedure for the synthesis of compounds 2–5
A
solution of 1-{7-[(E)-2-(dimethylamino)ethenyl]-2-methyl-
pyrazolo[1,5-a]pyrimidin-6-yl}ethanone 1 (245 mg, 1 mmol) in
diglyme (8 mL) containing sodium methoxide (64.8 mg, 1.2
mmol) and guanidine nitrate (146.5 mg, 1.2 mmol) or 1,1-
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Reaction of 1 with ethane-1,2-diamine
96.3 (d, C-3), 86.7 (d, C-9), 77.0 (t, Csp2H2), 54.0 (t, C-1ꢀ), 18.9 (t,
C-2ꢀ), 14.8 (q, 2-CH3), 11.2 (q, 2ꢀ-CH3); m/z (EI) 240 (M+), 225,
198; mmax(KBr)/cm−1 1640, 1553, 1522, 1341, 1126. Anal. found:
C, 70.2; H, 6.5; N, 23.5%. Calc. for C14H16N4: C, 70.0; H, 6.7; N,
23.3%.
To a solution of compound 1 (245 mg, 1 mmol) in acetic acid
(10 mL) was added ethane-1,2-diamine (72.10 mg, 1.2 mmol) and
the mixture was heated under reflux for 3 h, after which it was
cooled, diluted with water (30 mL) and extracted exhaustively with
ethyl acetate. The combined organic extracts were dried (MgSO4),
filtered and concentrated under reduced pressure to give a solid
resulting in a mixture of compounds 6 (25%) and 10 (traces).
An analytical sample of 2,6-dimethylpyrazolo[1,5-a]pyrido[3,4-
e]pyrimidine 6 was obtained by crystallisation: colourless crystals,
Treatment of compounds 1, 12 and 13 with AcOH–AcONa
The dimethylaminovinyl derivative (1 mmol) was heated under
reflux in glacial acetic acid (10 mL) containing anhydrous
sodium acetate (393.8 mg, 4.8 mmol) for 4 h. After cooling,
the solution was diluted with water (30 mL) and extracted
exhaustively with ethyl acetate. The combined organic extracts
were dried (MgSO4), filtered and concentrated under reduced
pressure to give a solid. The obtained material resulted in a
mixture of N,N-dimethylpyrazolo[1,5-a]quinazolin-6-amine and
pyrazolo[1,5-a]quinazolin-6-ol derivatives that were separated by
flash chromatography (CH2Cl2–MeOH = 20 : 1, as eluent).
◦
mp 163–164 C (from ethanol).4 The remaining acidic aqueous
solution was made alkaline (pH 13) with solid NaOH, and
extracted with ethyl acetate. The combined organic extracts were
dried (MgSO4), filtered and concentrated under reduced pressure
to give N-[2-(2-methyl-6-methylidenepyrazolo[1,5-a]pyrido[3,4-
e]pyrimidin-7(6H)-yl)ethyl]acetamide 7: yield 54%; red crystals,
mp 175–176 ◦C (from ethyl acetate); dH (400 MHz, DMSO-d6)
3
8.63 (1 H, s, 5-H), 8.03 (1 H, t, J = 6.0 Hz, NH), 7.18 (1 H, d,
3J = 7.6 Hz, 8-H), 6.36 (1 H, s, 3-H), 5.99 (1 H, d, J = 7.6 Hz,
3
9-H), 4.66 (1 H, d, 2J = 3.4 Hz, Csp2H), 3.96 (1 H, d, 2J = 3.4 Hz,
Csp2H), 3.59 (2 H, m, 2ꢀ-CH2), 3.31 (2 H, m, 1ꢀ-CH2), 2.37 (3 H, s,
2-CH3), 1.79 (3 H, s, COCH3); dC (100.58 MHz, DMSO-d6) 170.2
(s, CO), 154.5 (s, C-2), 148.3 (s, C-3a), 147.5 (d, C-8), 145.5 (d,
C-5), 140.1 (s, C-6/C-9a), 140.0 (s, C-9a/C-6), 107.2 (s, C-5a),
96.3 (d, C-3), 87.1 (d, C-9), 77.1 (t, Csp2H2), 51.7 (t, C-2ꢀ), 34.9 (t,
C-1ꢀ), 23.0 (q, COCH3), 14.8 (q, 2-CH3); m/z (EI) 283 (M+), 225,
198; mmax(KBr)/cm−1 3287, 1596, 1548, 1435, 1347. Anal. found:
C 63.8, H 6.2, N, 24.6%. Calc. for C15H17N5O: C, 63.6, H 6.05, N
24.7%.
N,N,2-Trimethylpyrazolo[1,5-a]quinazolin-6-amine 10
First running band starting from 1: yellow needles, mp 104–105 ◦C
(from ethanol); dH (400 MHz, CDCl3) 9.15 (1 H, s, 5-H), 7.98–7.69
(2 H, m, 8-H and 9-H), 6.98 (1 H, dd, 3J = 8.0 Hz, 4J = 0.8 Hz, 7-
H), 6.54 (1 H, s, 3-H), 2.99 [6 H, s, N(CH3)2], 2.55 (3 H, s, 2-CH3);
dC (100.58 MHz, CDCl3) 153.4 (s, C-6), 152.7 (s, C-2), 149.1 (d,
C-5), 146.2 (s, C-3a), 137.5 (s, C-9a), 134.3 (d, C-8), 112.9 (d, C-7),
112.0 (s, C-5a), 107.8 (d, C-9), 98.6 (d, C-3), 45.6 [q, N(CH3)2],
14.6 (q, 2-CH3); m/z (EI) 226 (M+), 210, 184, 112; mmax(KBr)/cm−1
2864, 1594, 1487, 1430. Anal. found: C, 69.2; H, 6.1; N, 24.85%.
Calc. for C13H14N4: C, 69.0; H, 6.2; N, 24.8%.
2-(2-Methyl-6-methylidene-6,7-dihydropyrazolo[1,5-a]pyrido[3,4-
e]pyrimidine)ethanol 8
2-Methylpyrazolo[1,5-a]quinazoline-6-ol 11
This compound was obtained from compound
1 with
ethanolamine as described for 7. Red crystals, mp 155–156 ◦C
(from ethyl acetate); dH (400 MHz, DMSO-d6) 8.62 (1 H, s, 5-H),
7.25 (1 H, d, 3J = 7.6 Hz, 8-H), 6.35 (1 H, s, 3-H), 5.98 (1 H, d, 3J
= 7.6 Hz, 9-H), 4.92 (1 H, t, OH), 4.62 (1 H, d, 2J = 3.0 Hz, Csp2H),
3.81 (1 H, d, 2J = 3.0 Hz, Csp2H), 3.66–3.58 (4 H, m, 1ꢀ-CH2 and
2ꢀ-CH2), 2.37 (3 H, s, 2-CH3); dC (100.58 MHz, DMSO-d6) 154.5
(C-2), 148.3 (C-3a), 148.2 (C-8), 145.5 (C-5), 140.4 (C-6), 140.2
(C-9a), 107.0 (C-5a), 96.2 (C-3), 86.5 (C-9), 76.8 (Csp2H2), 56.0
(C-2ꢀ), 54.9 (C-1ꢀ), 14.8 (2-CH3); m/z (EI) 242 (M+), 227, 198, 183;
mmax(KBr)/cm−1 3500–3000, 1637, 1556, 1525, 1347, 1123. Anal.
found: C, 64.6; H, 6.0; N, 23.0%. Calc. for C13H14N4O: C, 64.45;
H, 5.8; N, 23.1%.
Second running band starting from 1: yellow needles, mp 272–
273 ◦C (from ethanol); dH (400 MHz, DMSO-d6) 11.16 (1 H, br s,
OH), 9.09 (1 H, s, 5-H), 7.76–7.63 (2 H, m, 8-H and 9-H), 6.92
(1 H, dd, 3J = 8.0 Hz, 4J = 1.2 Hz, 7-H), 6.59 (1 H, s, 3-H), 2.43
(3 H, s, 2-CH3); dC (100.58 MHz, DMSO-d6) 156.9 (s, C-6), 152.3
(s, C-2), 147.4 (d, C-5), 146.4 (s, C-3a), 136.6 (s, C-9a), 136.2 (d,
C-8), 110.2 (d, C-7), 108.6 (s, C-5a), 104.25 (d, C-9), 98.9 (d, C-3),
14.8 (q, 2-CH3); m/z (EI) 199 (M+), 198, 146, 76; mmax(KBr)/cm−1
3100–2000, 1595, 1480. Anal. found: C, 66.5; H, 4.65; N, 21.0%.
Calc. for C11H9N3O: C, 66.3; H, 4.55; N, 21.1%.
N,N-Dimethyl-2-phenylpyrazolo[1,5-a]quinazolin-6-amine 14
First running band starting from 12: yellow needles, mp 120–
121 ◦C (from ethanol); dH (400 MHz, CDCl3) 9.22 (1 H, s, 5-H),
8.08–8.03 (2 H, m, 2ꢀ-H), 8.14 (1 H, d, 3J = 8.0 Hz, 9-H), 7.75 (1
2-Methyl-6-methylidene-7-propyl-6,7-dihydropyrazolo[1,5-
a]pyrido[3,4-e]pyrimidine 9
3
This compound was obtained from compound 1 with n-
propylamine as described for 7. Red crystals, mp 125–126 ◦C (from
ethyl acetate); dH (400 MHz, DMSO-d6) 8.62 (1 H, s, 5-H), 7.35
H, pt, J = 8.0 Hz, 8-H), 7.51–7.45 (2 H, m, 3ꢀ-H), 7.42–7.36 (1
H, m, 4ꢀ-H), 7.08 (1 H, s, 3-H), 7.04 (1 H, dd, 3J = 8.0 Hz, 7-H),
3.03 [6 H, s, N(CH3)2]; dC (100.58 MHz, CDCl3) 154.5 (s, C-2),
153.2 (s, C-6), 148.5 (d, C-5), 145.1 (s, C-3a), 137.8 (s, C-9a), 135.3
(d, C-8), 132.9 (s, C-1ꢀ), 128.8 (d, C-3ꢀ and C-4ꢀ), 126.4 (d, C-2ꢀ),
113.6 (d, C-7), 111.6 (s, C-5a), 108.3 (d, C-9), 95.7 (d, C-3), 45.7
[q, N(CH3)2]; m/z (EI) 288 (M+), 144, 77; mmax(KBr)/cm−1 2839,
1605, 1594, 1469, 1321. Anal. found: C, 75.2; H, 5.7; N, 19.5%.
Calc. for C18H16N4: C, 75.0; H, 5.6; N, 19.4%.
3
3
(1 H, d, J = 7.5 Hz, 8-H), 6.35 (1 H, s, 3-H), 5.99 (1 H, d, J
2
= 7.5 Hz, 9-H), 4.64 (1 H, d, J = 3.0 Hz, Csp2H), 3.81 (1 H, d,
2J = 3.0 Hz, Csp2H), 3.50 (2 H, m, 1ꢀ-CH2), 2.37 (3 H, s, 2-CH3),
1.65 (2 H, m, 2ꢀ-CH2), 0.89 (3 H, s, 3ꢀ-CH3); dC (100.58 MHz,
DMSO-d6) 154.5 (s, C-2), 148.3 (s, C-3a), 147.4 (d, C-8), 145.5
(d, C-5), 140.2 (s, C-6/C-9a), 140.0 (s, C-9a/C-6), 107.0 (s, C-5a),
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2-Phenylpyrazolo[1,5-a]quinazoline-6-ol 15
adenocarcinoma (MCF-7) were grown in DMEM medium (Sigma
Co., MO, USA), all supplemented with 115 units mL−1 of penicillin
G (Invitrogen, Milano, Italy), 115 lg mL−1 streptomycin (Invitro-
gen, Milano, Italy) and 10% fetal calf serum (Invitrogen, Milano,
Italy). Individual wells of a 96-well tissue culture microtiter plate
were inoculated with 100 lL of complete medium containing
8 × 103 Jurkat cells or 5 × 103 MCF-7 cells. The plates were
Second running band starting from 12: yellow needles, mp 299–
300 ◦C (from ethanol); dH (400 MHz, DMSO-d6) 11.20 (1 H, br s,
OH), 9.15 (1 H, s, H-5), 8.12–8.02 (2 H, m, 2ꢀ-H), 7.85–7.74 (2
H, m, 8-H and 9-H), 7.55–7.44 (2 H, m, 3ꢀ-H), 7.44–7.36 (1 H,
3
4
m, 4ꢀ-H), 6.98 (1 H, dd, J = 7.6 Hz, J = 1.0 Hz, 7-H), 7.30
(1 H, s, 3-H); dC (100.58 MHz, DMSO-d6) 157.0 (s, C-6), 153.5
(s, C-2), 148.0 (d, C-5), 147.0 (s, C-3a), 136.7 (s, C-9a), 136.4 (d,
C-8), 133.1 (s, C-1ꢀ), 129.3 (d, C-3ꢀ), 129.2 (d, C-4ꢀ), 126.4 (d, C-2ꢀ),
110.8 (d, C-7), 109.0 (s, C-5a), 104.5 (d, C-9), 96.6 (d, C-3); m/z
(EI) 261 (M+), 232, 130, 103, 77; mmax(KBr)/cm−1 3100–2000, 1621,
1465, 1372, 1307. Anal. found: C, 73.6; H, 4.2; N, 16.2%. Calc. for
C16H11N3O: C, 73.55; H, 4.2; N, 16.1%.
◦
incubated at 37 C in a humidified 5% incubator for 18 h prior
to the experiments. After medium removal, 100 lL of the drug
solution, dissolved in DMSO and diluted with complete medium,
were added to each well and incubated at 37 ◦C for 72 h. Cell
viability was assayed by the MTT [3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazolium bromide] test as described previously.10
Cell growth at each drug concentration was expressed as a
percentage of untreated controls and the concentration resulting in
50% (IC50) growth inhibition was determined by linear regression
analysis.
N,N-Dimethyl-3-phenylpyrazolo[1,5-a]quinazolin-6-amine 16
First running band starting from 13: yellow needles; mp 115–
◦
116 C (ethanol); dH (400 MHz, CDCl3) 9.35 (1 H, s, 5-H), 8.38
(1 H, s, 2-H), 8.10 (1 H, d, 3J = 8.4 Hz, 9-H), 8.08–8.04 (2 H, m,
2ꢀ-H), 7.76 (1 H, pt, 3J = 8.4 Hz, 8-H), 7.51–7.44 (2 H, m, 3ꢀ-H),
7.33–7.27 (1 H, m, 4ꢀ-H), 7.09 (1 H, d, 3J = 8.4 Hz, 7-H), 3.06 [6
H, s, N(CH3)2]; dC (100.58 MHz, CDCl3) 152.95 (s, C-6), 149.25
(d, C-5), 141.8 (s, C-3a), 140.7 (d, C-2), 137.8 (s, C-9a), 134.55
(d, C-8), 132.2 (s, C-1ꢀ), 128.8 (d, C-3ꢀ), 126.65 (d, C-2ꢀ), 126.4 (d,
C-4ꢀ), 113.6 (d, C-7), 113.1 (s, C-3), 112.3 (s, C-5a), 108.05 (d, C-
9), 45.6 [q, N(CH3)2]; m/z (EI) 288 (M+), 144, 77; mmax(KBr)/cm−1
2840, 1595, 1538, 1420. Anal. found: C, 74.8; H, 5.7; N, 19.3%.
Calc. for C18H16N4 : C, 75.0; H, 5.6; N, 19.4%.
Acknowledgements
The authors wish to thank MIUR for financial support and Ente
Cassa di Risparmio di Firenze, Italy, for granting a 400 MHz
NMR spectrometer. Mrs Brunella Innocenti and Mr Maurizio
Passaponti (Dipartimento di Chimica Organica “Ugo Schiff”,
Universita di Firenze) are acknowledged for technical assistance.
`
Notes and references
1 S. Selleri, F. Bruni, C. Costagli, A. Costanzo, G. Guerrini, G. Ciciani,
P. Gratteri, F. Besnad, B. Costa, M. Montali, C. Martini, J. Fohlin,
G. De Siena and P. Malmberg Aiello, J. Med. Chem., 2005, 48, 6756–
6760.
3-Phenylpyrazolo[1,5-a]quinazoline-6-ol 17
2 S. Selleri, F. Bruni, C. Costagli, A. Costanzo, G. Guerrini, G. Ciciani,
P. Gratteri, C. Bonaccini, P. Malmberg Aiello, F. Besnard, S. Renard,
B. Costa and C. Martini, J. Med. Chem., 2003, 46, 310–313.
3 S. Selleri, F. Bruni, C. Costagli, A. Costanzo, G. Guerrini, G. Ciciani,
B. Costa and C. Martini, Bioorg. Med. Chem., 1999, 7, 2705–2711.
4 F. Bruni, S. Chimichi, B. Cosimelli, A. Costanzo, G. Guerrini and S.
Selleri, Heterocycles, 1990, 31, 1141–1149.
Second running band starting from 13: yellow needles, mp
>300 ◦C (from ethanol); dH (400 MHz; DMSO-d6) 11.30 (1 H,
br s, OH), 9.24 (1 H, s, 5-H), 8.66 (1 H, s, 2-H), 8.18–8.12 (2 H,
m, 2ꢀ-H), 7.84–7.74 (2 H, m, 8-H and 9-H), 7.50–7.48 (2 H, m,
3
3ꢀ-H), 7.29–7.21 (1 H, m, 4ꢀ-H), 7.01 (1 H, d, J = 7.5 Hz, 7-H);
dC (100.58 MHz, DMSO-d6) 157.0 (s, C-6), 148.1 (d, C-5), 141.9
(s, C-3a), 141.2 (d, C-2), 137.0 (s, C-9a), 136.6 (d, C-8), 132.4 (s,
C-1ꢀ), 129.1 (d, C-3ꢀ), 126.7 (d, C-4ꢀ), 126.55 (d, C-2ꢀ), 112.3 (s,
C-3), 111.0 (d, C-7), 109.1 (s, C-5a), 104.5 (d, C-9); m/z (EI) 261
(M+), 205, 117, 102; mmax(KBr)/cm−1 3100–2000, 1620, 1604, 1473,
1298. Anal. found: C, 73.4; H, 4.3; N, 16.2%. Calc. for C16H11N3O:
C, 73.5; H, 4.2; N, 16.1%.
5 F. Bruni, S. Chimichi, B. Cosimelli, A. Costanzo, G. Guerrini and S.
Selleri, Heterocycles, 1990, 31, 1635–1640.
6 S. Chimichi, B. Cosimelli, F. Bruni, S. Selleri, A. Costanzo, G. Guerrini
and G. Valle, J. Chem. Soc., Perkin Trans. 2, 1994, 1657–1660.
7 S. M. Siddiqi, X-d. Ji, N. Melman, M. E. Olah, R. Jain, P. Evans, M.
Glashofer, W. L. Padgett, L. A. Cohen, J. W. Daly, G. L. Stiles and
K. A. Jacobson, Nucleosides Nucleotides, 1996, 15, 693–718.
8 S. Schenone, F. Manetti and M. Botta, Curr. Pharm. Des., 2007, 13,
2118–2128.
9 F. Bruni, B. Cosimelli, A. Costanzo, G. Guerrini and S. Selleri,
Heterocycles, 1993, 36, 87–99.
Growth inhibitory activity
10 G. Viola, L. Facciolo, M. Canton, F. Di Lisa, S. Dall’Acqua, D. Vedaldi,
O. Tabarrini, A. Fravolini and V. Cecchetti, Toxicol. in Vitro, 2004, 18,
581–594.
Human lymphoblastoid leukaemia cells (Jurkat) were grown in
RPMI-1640 medium, (Sigma Co., MO, USA) human breast
744 | Org. Biomol. Chem., 2008, 6, 739–744
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The Royal Society of Chemistry 2008
©