Phenylpyrazolo[1,5-a]quinazolin-5(4 H)-one: A suitable scaffold for the development of noncamptothecin topoisomerase i (Top1) inhibitors

Article abstract of DOI:10.1021/jm400932c

In search for a novel chemotype to develop topoisomerase I (Top1) inhibitors, the pyrazolo[1,5-a]quinazoline nucleus, structurally related to the indenoisoquinoline system precursor of well-known Top1 poisons, was variously decorated (i.e., a substituted

Full text of DOI:10.1021/jm400932c

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Brief Article
Phenylpyrazolo[1,5-a]quinazolin-5(4H)-one: a suitable scaffold for the
development of non-camptothecin Topoisomerase I (Top1) inhibitors.
Sabrina Taliani, Isabella Pugliesi, Elisabetta Barresi, Silvia Salerno, Christophe Marchand, Keli Agama,
Francesca Simorini, Concettina La Motta, Anna Maria Marini, Francesco Saverio Di Leva, Luciana
Marinelli, Sandro Cosconati, Ettore Novellino, Yves Pommier, Roberto Di Santo, and Federico Da Settimo
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400932c • Publication Date (Web): 29 Aug 2013
Downloaded from http://pubs.acs.org on August 31, 2013
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Journal of Medicinal Chemistry
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Phenylpyrazolo[1,5-a]quinazolin-5(4H)-one: a suitable scaffold
for the development of non-camptothecin Topoisomerase I
(Top1) inhibitors.
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Sabrina Taliani,*,§ Isabella Pugliesi,§ Elisabetta Barresi, § Silvia Salerno,§ Christophe
Marchand,‡ Keli Agama,‡ Francesca Simorini,§ Concettina La Motta,§ Anna Maria Marini,§
Francesco Saverio Di Leva,¥ Luciana Marinelli,$ Sandro Cosconati,† Ettore Novellino,$ Yves
Pommier,‡ Roberto Di Santo,£ and Federico Da Settimo.§
§Department of Pharmacy, University of Pisa, via Bonanno, 6ꢀ56126 Pisa, Italy. £ Istituto PasteurꢀFondazione
Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, “Sapienza” Università di Roma, P.le Aldo
Moro 5, Iꢀ00185, Rome, Italy. ‡Laboratory of Molecular Pharmacology, Center for Cancer Research, National
Cancer Institute, Bethesda, Maryland 20892ꢀ4255. ¥Department of Drug Discovery and Development, Istituto
Italiano di Tecnologia (IIT), Via Morego 30, 16163 Genova, Italy. †DiSTABiF, Seconda Università di Napoli, Via G.
Vivaldi 43, 81100 Caserta, Italy. $Department of Pharmacy, University of Naples “Federico II”, Via D. Montesano
49, 80131 Napoli, Italy
KEYWORDS: Topoisomerase I; pyrazoloquinazoline derivatives; structure–activity relationships; medicinal chemistry.
Supporting Information Placeholder
ABSTRACT: In search for a novel chemotype to develop Topoisomerase I (Top1) inhibitors, the pyrazolo[1,5–
a]quinazoline nucleus, structurally related to the indenoisoquinoline system precursor of wellꢀknown Top1 poisons, was
variously decorated (i.e. a substituted phenyl ring at 2– or 3–position, a protonable side chain at 4– or 5–position) afꢀ
fording a number of Top1 inhibitors with cleavage patterns common to CPT and MJ–III–65. SARs data were rationalꢀ
ized by means of an advanced docking protocol.
Introduction
antitumor drugs, including the phenanthridines III, and
the indenoisoquinolines IV (Chart I).8,9
DNA topoisomerases (Top) are essential enzymes inꢀ
ducing DNA modification required during cellular proꢀ
cesses such as replication, transcription, repair, etc.1
There are two major families of Top: Type I (Top1) and
Type II (Top2) depending on whether they cleave only
one or two DNA strands.2 Top1 relaxes supercoiled DNA
by forming DNA singleꢀstrand breaks, and religates the
broken strand, to rapidly restore intact duplex DNA.2 At
this stage, the enzyme is particularly vulnerable to a
group of anticancer agents, the Top1 poisons, that reꢀ
versibly trap the Top1ꢀmediated cleavage complex, leadꢀ
ing to irreversible DNA strand breaks, activation of apopꢀ
tosis and cell cycle arrest.2
As part of our program in search for new antiproliferative
agents, in the last decades we extensively studied sevꢀ
eral polyheterocyclic systems.10ꢀ12 In the present study,
we have directed our attention to the pyrazolo[1,5ꢀ
a]quinazoline system V (Chart I),13,14 as a novel scaffold
to develop nonꢀCPT agents acting against Top1. Actualꢀ
ly, the core of V would mimic the A, B and C rings of IV,
while the phenyl hanging from the pyrazole portion of V
could mimic the substituted D ring of IV (Chart I). Comꢀ
pounds bearing a phenyl alternatively at 2– or 3–position
of pyrazolo[1,5–a]quinazoline system were designed (V,
Chart I). Further, as a protonable chain linked at the 5–
or 6–position of the indenoisoquinoline ring or at the 5–
position of the benzophenanthridine system is a common
feature of the most active derivatives,9 we decorated the
pyrazoloquinazoline ring at the 4– or 5–position with
aminoalkyl chains. In particular, we studied the influence
on the Top1 inhibitory activity of: (i) the length of the
chain, (ii) the nature of the linker between the ring and
the chain; (iii) the nature of the terminal basic site. Interꢀ
estingly, these compounds featuring a basic nitrogen in
the side chain could be converted in the corresponding
salts, thus increasing aqueous solubility that might faciliꢀ
tate their formulation.
Top1 inhibitors are a relatively new group of anticancer
agents with a wide range of activity in hematological and
solid tumors. Camptothecin (CPT I, Chart I),3 was the
first small molecule identified as a Top1 inhibitor. Efforts
to improve its toxicity profile and pharmacokinetics led to
the development of two clinical waterꢀsoluble CPT derivꢀ
atives, topotecan II (Chart I) and irinotecan,4 as well as
novel compounds currently under clinical evaluation.5
However, CPTs are not ideal drugs as they display a
number of limitations, including chemical instability,6 and
potential induction of cellular resistance.7 To overcome
the main drawbacks of CPTs, several chemical classes
of non–CPT Top1 poisons were developed as promising
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In this study, derivatives 1–34 were prepared and evaluꢀ ucts (4–34) were purified by conversion into the correꢀ
1
2
3
4
5
6
7
8
ated for their ability to inhibit Top1 (Table 1), and an adꢀ
vanced docking protocol was employed to rationalize the
biological results.
sponding hydrochlorides by treatment with ethanol hyꢀ
drochloride in absolute ethanol.
R
R
2
2
N
R1
N
N
3
for 38-40
DEAD, PPh3
N
3
R2
N
N
O
PhPOCl2
N
R
O
O
N
D
B
N
A
C
N
NH
N
HO(CH2)nN R1
R1
Cl
50-54
O
O
O
R1
O(CH2)nN
E
O
O
R1
37-41
N
HO
R
H2N(CH2)nN
R1
9-11, 16-18, 23-25
Cl(CH2)nN R1
R1
O
R2
for 37, 41
n = 2,3
R = Cl, CF3, OCH3
R1 = CH3, CH2CH3
MW
9
NaH, DMF
Camptothecin I: R1 = R2 = H
Topotecan II: R1 = CH2N(CH3)2 R2 = OH
Phenantrhridines III
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R
2
3
2
N
R5
N
3
N
O
N
D
N
D
R4
N
N
X = ꢀCH2ꢀ, ꢀOꢀ,
ꢀNHꢀ
C
C
R1
R2
R1
R2
N
O(CH2)nN R1
R1
4-6, 30-32
HN(CH2)nN
B
A
B
A
N
N
R1
R3
7, 8, 12-15, 19-22, 26-29, 33, 34
n = 2,3
R = H, Cl, CF3, OCH3
X
(CH2)n N
O
R2
2(3)ꢀPhenyl pyrazolo[1,5ꢀa]quinazolines V
n = 2,3
R1 = CH3, CH2CH3
Indenoisoquinolines IV
MJꢀIIIꢀ65, 55: R1 = R2 = OCH3
NR1R2 = N(CH3)2, N(CH2CH3)2, imidazolyl
R3 = (CH2)3NH(CH2)2OH
R4ꢀR5 = ꢀOCH2CH2Oꢀ
Scheme 2. Synthesis of pyrazoloquinazolines 4–34.
Biological Results and Discussion
Chart I. Structures of known Top1 inhibitors; design of pyrazoloꢀ
Table 1 lists the relative potencies of the pyrazoloꢀ
quinazolines 1–34 toward the production of Top1–
mediated DNA cleavage, ranked by a systematic visual
analysis of the number of cleavage sites and their reꢀ
spective intensity in each lane as compared to the posiꢀ
tive control lanes containing I or 55 (MJ–III–65, Chart
I)17,18 at 1 ꢁM. A semiꢀquantitative ranking system is then
used to rank the compounds from 0, not active; 0/+,
trace of activity; +, weak activity; ++, moderate activity;
+++, strong activity; to ++++, activity equivalent to 1 µM I
or 55.
quinazolines V
.
Chemistry
The 2–phenyl–5H–pyrazolo[1,5ꢀa][3,1]benzoxazin–5–one
36 was obtained by an improved microwaves (MW)ꢀ
assisted reported procedure15,16 [experimental details,
Scheme 1S, Supporting Information (SI)] and then reactꢀ
ed with the appropriate dialkylaminoalkylamine to obtain
compounds 1–3 through the use of MW (Scheme 1).
N
CH3
H2N(CH2)nN
or
N
Compounds 1–3, that were synthesized due to their
structural similarities with the indenoisoquinolines IV,
showed null Top1 inhibitory activity (Table 1). So we
turned out our attention to 2–phenylpyrazoloquinazolines
bearing different protonable chains at the 5–position (4–
8). Also these substitution patterns did not produce faꢀ
vorable effects on biological activity. Only derivatives 4
and 5 showed a scarce activity as Top1 inhibitors.
CH3
O
N
NH2CH2CH2CH2
N
O
36
150 W
T= 135°C
P= 100 PSI
t ramp= 2 min
t= 2 min
aluminum oxide basis
N
N
N
N
CH3
(CH2)nN
CH3
Compounds 9–34 were then designed by shifting the
pendant phenyl from the 2– to the 3–position, and slightly
expanding the variability at the 5–chain with respect to
1–8. Initially, an electron–donating OCH3 was inserted at
p–position of the 3–phenyl (9–15), due to the well–known
beneficial effect of this substituent on the potency of
Top1 inhibitors.9 Then, variously (CF3, Cl, H) 4'–
substituted compounds 16–34 were developed to exꢀ
pand the SAR. The presence of the 3–phenyl produced
a general improvement in the biological activity (9–34,
Table 1), suggesting a specific arrangement of the moleꢀ
cule that is favorable for Top1 inhibition. The presence of
a 4'–OCH3 does not particularly favor the activity, yieldꢀ
ing poorly active derivatives (9–15), whatever the nature
of the chain at 5–position. An analogous effect is proꢀ
duced by a highly electron–withdrawing CF3 substituent
(16–22). Conversely, the presence of the lipophilic and
electron–withdrawing 4'–Cl (23–29) determined an enꢀ
hancement in the biological activity. In this subclass, the
N
N
N
(CH2)3
N
O
O
3
1: n=2
2: n=3
Scheme 1. Synthesis of pyrazoloquinazolines 1–3.
The phenylpyrazolo[1,5–a]quinazolin–5(4H)–ones 37–41
(Scheme 2) were obtained by improving known synthetic
procedures (experimental details, Schemes 2S and 3S,
SI).13,14 Treatment of 37, 41 with sodium hydride and adꢀ
dition of the appropriate dialkylaminoalkyl chloride gave
derivatives 4–6, 30–34 (Scheme 2). The best yields in
the preparation of 9–11, 16–18, 23–25, were obtained by
a Mitsunobu reaction between 38–40 and the appropriꢀ
ate aminoalcohol (Scheme 2). The synthesis of comꢀ
pounds 7, 8, 12–15, 19–22, 26–29, 33, 34 includes the
transformation of 37–41 in the corresponding 5–chloro
derivatives 50–54 (Scheme 2, see SI for details), that
were then reacted with the proper dialkylaminoalkylaꢀ
mines through the use of MW (Scheme 2). All the prodꢀ
2
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Journal of Medicinal Chemistry
nature of the 5–chain in terms of type (–O– or –NH–) and
length of the linker, and terminal nitrogen containing
group moderately influence Top1 inhibitory activity.
resentative of the whole series, along with those resultꢀ
ing from I and the indenoisoquinoline 55.17,18 It should be
observed that compounds 26–28, differently from the
poorly active 2, 18, 24, and 29 showed cleavage sites
that are common to I and 55.
1
2
3
4
5
6
7
8
The imidazole–containing chain (29) scored the worst
results, while the best activity was obtained with the diꢀ
methylaminoethylamino, dimethylaminopropylamino, and
diethylaminoethylamino chains (26–28).
Docking studies were performed to rationalize the Top1
inhibitory activity and the relative potencies of our pyraꢀ
zoloquinazolines.
Table 1. Topoisomerase I Inhibitory Activities of 1–34.
9
X
2
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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N
N
3
N
3
N
4
R
N
5
4
N
5
R
O
1-3
4-34
N
Phenyl position Ra
X
Top1 Inhibition
1
2
3
4
5
6
7
8
ꢀ
ꢀ
ꢀ
A
B
C
D
E
F
H
J
D
E
F
G
H
I
ꢀ
ꢀ
ꢀ
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
Cl
0/+
0
0
0/+
0/+
0
0
0
+
0/+
0/+
+
0/+
0/+
0
0/+
0
0
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
Figure 1. Top1ꢀmediated DNA cleavage induced by compounds
24, 26, 28, 27, 29, 2 and 18. Lane 1: DNA alone; lane 2: Top1
alone; lane 3: I, 1 ꢁM:; lane 4: Indenoisoquinoline 55, 1 ꢁM; lane 5–
25: 24, 26, 28, 27, 29, 2 and 18 at 1, 10 and 100 ꢁM respectively
from left to right. Numbers and arrows on the left indicate arbitrary
cleavage site positions.
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16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
J
We selected the high resolution (2.10 Å) crystal structure
of the human Top1 in complex with the poison Topotecꢀ
an (II, Chart 1) and covalent complex with a 22 bp DNA
duplex (PDB code: 1K4T).19 In this structure II establishꢀ
es direct H–bonds with E356, R364, K532 and D533,
and additional water–mediated interactions with N722
and the phosphotyrosine 723 (PꢀY723) (Figure S1a, SI).
Thus, we decided to explicitly consider the waters moleꢀ
cules in docking calculations using the software Autoꢀ
dock4.2 (AD4).20 In this respect, Forli et al. have recently
developed a new AD4 force field and hydration docking
method that allows for the automated prediction of waꢀ
ters mediating ligand binding.21 To evaluate the predicꢀ
tive power of this approach in our system, we first ran
selfꢀdocking calculations on the Top1–II–DNA complex.
AD4 reproduced the experimental binding conformation
as the lowest energy solution, with a ligand RMSD equal
to 1.08 Å, also recapitulating the water–mediated interacꢀ
tions between II and the enzyme (Figure S1b, SI).
D
E
F
G
H
I
+
0/+
0/+
0
+
0/+
+
++
++
++
0
++
+
J
D
E
F
G
H
I
Cl
Cl
Cl
Cl
Cl
Cl
H
H
J
D
E
F
H
J
H
H
H
+
+
0/+
aA: (CH2)2N(CH3)2; B: (CH2)3N(CH3)2; C: (CH2)3–1–ꢀimidazolyl; D:
O(CH2)2N(CH3)2; E: O(CH2)2N(C2H5)2; F: O(CH2)3N(CH3)2; G:
NH(CH2)2N(CH3)2; H: NH(CH2)2N(C2H5)2; I: NH(CH2)3N(CH3)2; J:
NH(CH2)3–1–imidazolyl. The activity of the compounds to produce
Top1ꢀmediated DNA cleavage was expressed semiꢀquantitatively
as follows: 0, not active; 0/+, trace of activity; +, weak activity; ++,
moderate activity; +++, strong activity; ++++, activity equivalent to 1
µM I or 55.
These results encouraged us to apply this protocol on
our pyrazoloquinazolines. Among these, we selected
compound 26 within the subclass displaying the most
interesting pharmacological profile (26–28). According to
docking results, 26 intercalates at the DNA cleavage site
(Figure 2) stacking with its polyaromatic system between
the downstream (–1) T–A and upstream (+1) G–C base
pairs like other CPT–Top1 inhibitors, including II (Figure
S2, SI).19
An analogous trend was observed when the 4'–position
is unsubstituted (30–34), with the presence of a terminal
imidazole nucleus that again results to be detrimental for
the activity, whereas a 5–dimethylaminoethoxy moiety
confers to 30 an increase in Top1 inhibition.
In particular, the pyrazoloquinazoline scaffold stacks beꢀ
tween the –1T and the +1G of the scissile strand, with
the N4 atom establishing water–mediated H–bonds with
Figure 1 displays the Top1–mediated DNA cleavage patꢀ
terns for compounds 2, 18, 24, 26–29, selected as repꢀ
3
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R364 and the ribose endocyclic oxygen (O5) of the –1
adenosine on the non–scissile strand. The 3–phenyl esꢀ
tablishes well–oriented parallel–displaced interactions
with the –1A and the +1C which should be lost when the
phenyl is moved to position 2, thus explaining the lower
activity of compounds 1–8.
for the 26/Top1/DNA ternary complex provided by hyꢀ
1
2
3
4
5
6
7
8
drated docking calculations, allowed to identify the folꢀ
lowing structural requirements to gain Top1 inhibitory acꢀ
tivity: (i) a properly–substituted 3–phenyl ring; (ii) a proꢀ
tonable dialkylaminoalkylamino chain at 5–position.
Compounds 26–28 are among the most active Top1 inꢀ
hibitors developed in this study showing cleavage patꢀ
terns that are common to I and 55.
As expected, in the 3–phenyl compounds enhancement
of ligand–target charge–transfer interactions, through the
introduction of an electron–withdrawing 4'–Cl (23–29),
resulted in higher inhibitory potencies if compared to the
unsubstitued 30–34. Also, the Cl seems to perfectly fit in
the crevice formed by the –1A and +1C residues of the
non–scissile strand (Figure S3, SI); this is further conꢀ
firmed by the lower potency displayed by analogues feaꢀ
turing bulkier substituents in the same position such as
the p–OCH3 and p–CF3 substituted 9–22.
Taken together, all these findings highlight the pyrazoloꢀ
quinazoline nucleus as a suitable scaffold to further exꢀ
pand the chemical diversity in Top1 inhibitors, and proꢀ
vided SAR data for the optimized design of new derivaꢀ
tives with improved biological activity.
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26
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Experimental Section
Chemistry. General directions are in the SI. Purity of
tested compounds is ≥95% (combustion analysis).
General procedure for the synthesis of 2–
phenylpyrazolo[1,5–a]quinazolin–5(4H)–ones 1–3. A
mixture of 2–phenyl–5H–pyrazolo[1,5–a][3,1]benzoxazin–
5–one 36 (0.262 g, 1 mmol) and the proper alkylamine
(0.9 mmol) in DMF (2 ml) was irradiated at a T=135 °C,
P=100 PSI, power=150 W for 2 min. Products 1–3 (SI)
crystallized from the reaction mixture by dilution with
ice/water in the desired purity degree (≥95%).
General procedure for the synthesis of 5–(N,N–
dialkylaminoalkoxy)–2(3)–phenylpyrazolo[1,5–
a]quinazolines 4–6, and 30–32. Sodium hydride (1.1
mmol, 0.044 g, 60% dispersion in mineral oil) was added
portion–wise to an ice–cooled solution of 37, 41 (1 mmol)
in 10 ml of DMF, and the mixture was stirred at rt for 1 h.
The appropriate dialkylaminoalkyl chloride (1.1 mmol)
was added dropwise, and stirring was continued for 24 h
at rt (TLC analysis). The reaction mixture was evapoꢀ
rated to dryness, and the residue triturated with ice/water
and extracted with CHCl3. Evaporation of the organic
phase yielded products 4–6, 30–32 in the desired purity
degree (≥95%). Samples of 4–6, 30–32 were characterꢀ
ized as hydrochloride salts, obtained by treatment with
ethanol hydrochloride in absolute ethanol (SI).
Figure 2. Binding mode of 26 (yellow sticks) at the Top1 (green
cartoons) DNA (cyan cartoons) cleavage site. Residues important
for ligand binding are highlighted as sticks. H–bonds are dashed
black lines.
In the docking pose predicted for 26, the 5–
dimethylaminoethylamino branch extends outside the
double–helix DNA towards a rather shallow protein
pocket where the exocyclic NH group can donate a H–
bond to the adjacent water molecule. Thus, it can be
predicted that the lower Top1 inhibition rate displayed by
compounds bearing exocyclic ether oxygen (23–25)
might in part be ascribed to the loss of this water–
mediated interaction. In this position, the terminal dimeꢀ
thylamino moiety of 26 establishes a tight salt bridge with
the carboxylate group of D533. In this regard, the length
of the aminoalkyl chain (12 vs 14, 19 vs 21, and 26 vs
28) or the alkyl on the terminal amine group (12 vs 13,
19 vs 20, and 26 vs 27) poorly influence the Top1 inhibiꢀ
tory potency. In this respect, longer but still flexible
chains [the dimethylaminopropylamino (i.e. 14, 21, 28),
the diethylaminoethylamino (i.e. 13, 20, 27) branches]
can rearrange without steric restrictions to preserve the
salt bridge with D533. However, the introduction of a
more rigid and less basic substituent such as the imidꢀ
azole (8, 15, 22, 29 and 34) should result in low or null
Top1 inhibitory activity due to the loss of the ionic interꢀ
action described above.
General procedure for the synthesis of 5–
(substitutedalkylamino)–2(3)–phenylpyrazolo[1,5–a]–
quinazolines 7, 8, 12–15, 19–22, 26–29, 33, and 34. A
mixture of the appropriate 5–chloro derivatives 50–54
(1.2 mmol) and the proper alkylamine (2.4 mmol) were
irradiated at a T=80 °C, P=100 PSI, power=100 W for 2
min, using aluminium oxide basis as solid support. Then,
the mixture was dissolved in ethanol, the aluminium oxꢀ
ide basis was filtered off and the organic solvent was
evaporated yielding compounds 7, 8, 12–15, 19–22, 26–
29, 33, and 34 in the desired purity degree (≥95%).
Samples of 7, 8, 12–15, 19–22, 26–29, 33, and 34 were
characterized as hydrochloride salts, obtained by treatꢀ
ment with ethanol hydrochloride in absolute ethanol (SI).
In conclusion, we designed and synthesized a series of
novel non–CPT Top1 inhibitors based on the phenylpyꢀ
razolo[1,5–a]quinazolin–5(4H)–one scaffold, structurally
related to the indenoisoquinoline nucleus. SARs emergꢀ
ing from this series, together with the theoretical model
General procedure for the synthesis of 5–(N,N–
dialkylaminoalkoxy)–3–(4–
substitutedphenyl)pyrazolo[1,5–a]quinazolines 9–11,
16–18, 23–25. The opportune pyrazolo[1,5–a]quinazolin–
5(4H)–ones 38–40 (0.62 mmol) was stirred with PPh3
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Journal of Medicinal Chemistry
topoisomerase I–mediated DNA damage. Prog. Nucleic Acid
Res. Mol. Biol. 2006, 81, 179–229.
(0.340g, 1.3 mmol) in dry THF under nitrogen atmosꢀ
1
2
3
4
5
6
7
8
phere for 5 min. DEAD (0.21 ml, 1.2 mmol) was then
added dropwise. After 15 min, the appropriate aminoalꢀ
cohol (0.63 mmol) was added and the mixture was
stirred for 16 h at rt (TLC analysis). The reaction mixture
was evaporated to dryness, and the residue was treated
with water and extracted with CHCl3. Evaporation of the
organic phase yielded products 9–11, 16–18 and 23–25
in the desired purity degree (≥ 95%). Samples of 9–11,
16–18 and 23–25 were characterized as hydrochloride
salts, obtained by treatment with ethanol hydrochloride in
absolute ethanol (SI).
8. Pommier, Y. Topoisomerase I inhibitors: camptothecins and
beyond. Nat. Rev. Cancer 2006, 6, 789–802.
9. Beretta, G. L.; Zuco, V.; Perego, P.; Zaffaroni, N. Targeting
DNA topoisomerase I with non–camptothecin poisons. Curr.
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10. Da Settimo, A.; Da Settimo, F.; Marini, A. M.; Primofiore,
G.; Salerno, S.; Viola, G.; Dalla Via, L.; Marciani Magno, S.
Synthesis, DNA binding and in vitro antiproliferative activity of
purinoquinazoline,
pyridopyrimidopurine
and
9
pyridopyrimidobenzimidazole derivatives as potential antitumor
agents. Eur. J. Med. Chem. 1998, 33, 685–696.
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11. Dalla Via, L.; Gia, O.; Marciani Magno, S.; Da Settimo, A.;
Primofiore, G.; Da Settimo, F.; Simorini, F.; Marini, A. M.
Dialkylaminoalkylindolonaphthyridines as potential antitumour
agents: synthesis, cytotoxicity and DNA binding properties. Eur.
J. Med. Chem. 2002, 37, 475–486.
12. Dalla Via, L.; Marciani Magno, S.; Gia, O.; Marini, A. M.; Da
Settimo, F.; Salerno, S.; La Motta, C.; Simorini, F.; Taliani, S.;
Lavecchia, A.; Di Giovanni, C.; Brancato, G.; Barone, V.;
Novellino, E. Benzothiopyranoindoleꢀbased antiproliferative
agents: synthesis, cytotoxicity, nucleic acids interaction, and
topoisomerases inhibition properties. J. Med. Chem. 2009, 52,
5429–5441.
13. Vasquez, T. E., Jr.; Nixey, T.; Chenera, B.; Gore, V.; Bartꢀ
berger, M. D.; Sun, Y.; Hulme, C. One–pot microwave assisted
preparation of pyrazoloquinazolinone libraries. Mol. Divers.
2003, 7, 161–164.
14. Penning, T. D.; Thomas, S. A.; Hajduk, P. J.; Sauer, D. R.;
Sarris, K.; Giranda, V. L. Preparation of 4H–pyrazolo[1,5–
a]quinazolin–5–one derivatives as PARP inhibitors. PCT Int.
Appl. WO 2007149907, 2007.
ASSOCIATED CONTENT
Supporting Information. General chemistry directions;
synthesis of compounds 35–41, and 46–54; yields, physꢀ
ical, and spectral data of compounds 1–34; biological
tests; computational studies. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: (+39)0502219547. Fax: (+39)0502219605. Eꢀ
mail: sabrina.taliani@farm.unipi.it.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final verꢀ
sion of the manuscript.
15. Veibel, S.; Arnfred, N. H. Pyrazole Studies. II. Anhydrides of
Funding Sources
1–(2'–carboxyphenyl)–3–phenylpyrazoloneꢀ5
and
1–(2'–
carboxyphenyl)–3–methyl–4–ethyl–pyrazoloneꢀ5. Acta Chem.
Scand. 1948, 921–926.
16. Michaelis, I. A. 1–Phenyl–5– and 3–pyrazolone–o–
carboxylic Anhydrides. Justus Liebigs Annalen der Chemie
1910, 373, 129–212.
This work was financially supported by University of Piꢀ
sa, “Sapienza” University of Rome, and the Center for
Cancer Research, Intramural Program of the NCI, NIH.
ABBREVIATIONS
17. Antony, S.; Jayaraman, M.; Laco, G.; Kohlhagen, G.; Kohn,
K. W.; Cushman, M.; Pommier, Y. Differential induction of
topoisomerase IꢀDNA cleavage complexes by the indenoisoꢀ
quinoline MJ–III–65 (NSC 706744) and camptothecin: base seꢀ
quence analysis and activity against camptothecinꢀresistant
topoisomerases I. Cancer Res. 2003, 63, 7428–7435.
18. Antony, S.; Kohlhagen, G.; Agama, K.; Jayaraman, M.;
Cao, S.; Durrani, F. A.; Rustum, Y. M.; Cushman, M.; Pommier,
Y. Cellular topoisomerase I inhibition and antiproliferative activiꢀ
ty by MJ–III–65 (NSC 706744), an indenoisoquinoline topoisoꢀ
merase I poison. Mol. Pharmacol. 2005, 67, 523–530.
19. Staker, B. L.; Hjerrild, K.; Feese, M. D.; Behnke, C. A.;
Burgin, A. B., Jr.; Stewart, L. The mechanism of topoisomerase
I poisoning by a camptothecin analog. Proc. Natl. Acad. Sci. U.
S. A. 2002, 99, 15387–15392.
CPT, camptothecin; MW, microwaves; SAR, structure–
activity relationship; Top1, Topoisomerase I.
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