Structural investigations on coumarins leading to chromeno[4,3-c]pyrazol-4-ones and pyrano[4,3-c]pyrazol-4-ones: New scaffolds for the design of the tumor-associated carbonic anhydrase isoforms IX and XII

Article abstract of DOI:10.1016/j.ejmech.2018.01.033

Human carbonic anhydrases (hCAs, EC 4.2.1.1) IX and XII are overexpressed in a wide variety of cancers and are considered available drug targets for anti-tumor therapy since their inhibition has been shown to reduce tumor growth and metastasis. A set of coumarin derivatives (1–10) and several 1-aryl and 2-aryl-substituted chromeno[4,3-c]pyrazol-4-ones (11–37) and pyrano[4,3-c]pyrazol-4-ones (38–39) were synthesized and tested against the tumor-associated hCAs IX and XII and the cytosolic isoforms hCAs I and II. Several compounds were potent (K_i < 41 nM) and selective inhibitors of the hCA IX (13, 14, 19, 21, 25, 31, 33, 37 and 39), some derivatives (6, 11 and 17) were active against both hCA IX and XII isoforms (K_i = 5.6–9.6 nM), while none were effective against the off-target cytosolic hCAs I and II. Some selected inhibitors (6, 11, 13, 19, 21, 25, 31 and 39) showed activity as antiproliferative agents on HT-29 colon cancer cell lines both in normoxic and hypoxic conditions. This finding led us to hypothesize for these derivatives more than one mechanism of action, involving hCAs IX and XII inhibition in hypoxia and other not identified target(s) in normoxia.

Full text of DOI:10.1016/j.ejmech.2018.01.033

Accepted Manuscript
Structural investigations on coumarins leading to chromeno[4,3-c]pyrazol-4-ones and
pyrano[4,3-c]pyrazol-4-ones: New scaffolds for the design of the tumor-associated
carbonic anhydrase isoforms IX and XII
Alessandro Bonardi, Matteo Falsini, Daniela Catarzi, Flavia Varano, Lorenzo
Di Cesare Mannelli, Barbara Tenci, Carla Ghelardini, Andrea Angeli, Claudiu T.
Supuran, Vittoria Colotta
PII:
S0223-5234(18)30046-1
10.1016/j.ejmech.2018.01.033
EJMECH 10110
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 September 2017
Revised Date: 9 January 2018
Accepted Date: 11 January 2018
Please cite this article as: A. Bonardi, M. Falsini, D. Catarzi, F. Varano, L. Di Cesare Mannelli, B. Tenci,
C. Ghelardini, A. Angeli, C.T. Supuran, V. Colotta, Structural investigations on coumarins leading to
chromeno[4,3-c]pyrazol-4-ones and pyrano[4,3-c]pyrazol-4-ones: New scaffolds for the design of the
tumor-associated carbonic anhydrase isoforms IX and XII, European Journal of Medicinal Chemistry
(2018), doi: 10.1016/j.ejmech.2018.01.033.
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ACCEPTED MANUSCRIPT
Graphical Abstract
New potent inhibitors of the tumor-associated
hCAs IX and XII

ACCEPTED MANUSCRIPT
Structural investigations on coumarins leading to chromeno[4,3-c]pyrazol-4-
ones and pyrano[4,3-c]pyrazol-4-ones: new scaffolds for the design of the tumor-
associated carbonic anhydrase isoforms IX and XII
Alessandro Bonardi,a Matteo Falsini,a Daniela Catarzi,a Flavia Varano,a Lorenzo Di Cesare
Mannelli,b Barbara Tenci,b Carla Ghelardini,b Andrea Angeli,a Claudiu T. Supuran,a Vittoria
Colottaa*
aDipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino, Sezione di
Farmaceutica e Nutraceutica, Università degli Studi di Firenze, Via Ugo Schiff, 6, 50019 Sesto
Fiorentino, Italy.
bDipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino, Sezione di
Farmacologia e Tossicologia, Università degli Studi di Firenze, Viale Pieraccini 6, 50139 Firenze,
Italy.
1

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Abstract
Human carbonic anhydrases (hCAs, EC 4.2.1.1) IX and XII are overexpressed in a wide variety of
cancers and are considered available drug targets for anti-tumor therapy since their inhibition has
been shown to reduce tumor growth and metastasis. A set of coumarin derivatives (1-10) and
several 1-aryl and 2-aryl-substituted chromeno[4,3-c]pyrazol-4-ones (11-37) and pyrano[4,3-
c]pyrazol-4-ones (38-39) were synthesized and tested against the tumor-associated hCAs IX and
XII and the cytosolic isoforms hCAs I and II. Several compounds were potent (Ki < 41 nM) and
selective inhibitors of the hCA IX (13, 14, 19, 21, 25, 31, 33, 37 and 39), some derivatives (6, 11
and 17) were active against both hCA IX and XII isoforms (Ki= 5.6-9.6 nM), while none were
effective against the off-target cytosolic hCAs I and II. Some selected inhibitors (6, 11, 13, 19, 21,
25, 31 and 39) showed activity as antiproliferative agents on HT-29 colon cancer cell lines both in
normoxic and hypoxic conditions. This finding led us to hypothesize for these derivatives more than
one mechanism of action, involving hCAs IX and XII inhibition in hypoxia and other not identified
target(s) in normoxia.
Keywords
Carbonic anhydrase inhibitors, 2H-cromen-2-one, chromeno[4,3-c]pyrazol-4-one, pyrano[4,3-
c]pyrazol-4-one, anticancer agents.
2

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1. Introduction
Human carbonic anhydrases (hCAs, EC 4.2.1.1) are metallo-enzymes that regulate physiological
functions connected to carbon dioxide/bicarbonate transport, pH, electrolyte secretion, and
biosynthetic reactions, thus they influence many metabolic processes [1-2]. hCAs are divided into at
least 15 isoforms differing in their molecular properties, subcellular localization and tissue
distribution. hCA isoforms IX and XII are membrane-bound enzymes whose expression is strongly
induced by hypoxia [3] that occurs in different types of solid tumors, such as glioma, breast cancer
and colon carcinoma. Hypoxia shifts metabolism toward glycolysis and anaerobic fermentation thus
affording a high production of lactic acid [3]. These conditions, together with an excessive
formation of carbon dioxide, induce progressive acidification of the extracellular environment due
to extrusion of protons from the cell and the entrance of bicarbonate ions [1-6]. Protons and
bicarbonate ions are produced by carbon dioxide hydration catalyzed by the transmembrane hCAs
IX and XII. Both these hCA isoforms contribute to tumor progression by maintaining a more
alkaline resting intracellular pH and the acidic extracellular pH. These conditions up-regulate
proteases, angiogenic and cell-growth factors, and impaired immune functions [5-9]. hCA IX
activity has also been associated with chemoresistance which may be due to two mechanisms [10].
First, hCA IX inhibits apoptosis by maintaining a normal intracellular pH in response to
chemotherapy-induced acidosis. Second, hCA IX-induced extracellular acidosis negatively impacts
efficacy of weakly basic anticancer drugs by reducing their cellular uptake.
Acetazolamide (AZA) and other sulfonamide-containing hCA inhibitors were reported to reduce
tumor growth and to delay tumor development. However, AZA and the classical aromatic
sulfonamides are non-selective inhibitors, thus leading to side effects by blocking the
physiological relevant cytosolic isozymes hCA I and II. Hence, several studies are being carried
out to identify new selective inhibitors for the tumor-associated hCA isoforms which are
considered promising targets for effective and safer agents in cancer therapy [8-13].
Up to now several classes of inhibitors have been disclosed [1, 11, 13, 15], for example
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sulfonamides, metal-complexing anions, phenols and 2H-chromen-2-one derivatives, simply
referred to as coumarins [16-20]. Within the latter class, the unsubstituted coumarin possesses
potencies spanning the medium to high micromolar range. In particular, it showed Ki values of 3.1
and 9.2 µM at the highly abundant hCAs I and II, respectively, while lower potencies were found
for membrane-bound hCA IX and XII isoforms (Ki> 500 µM) [16]. Insertion of substituents on
the coumarin scaffold significantly enhanced inhibitory activity. In particular, the presence of ester
functions or a carboxylic group at position 3, alone or combined with small substituents on the
fused benzo ring (Fig. 1, 3-carboxy-substituted coumarin series), ameliorated the potency toward
the tumor-associated hCA IX and XII, shiftinh the Ki values to the low micromolar range (0.047-
9.9 µM) [16]. These data suggested that further decoration of coumarin might enhance potency
and selectivity for the two hCA isoforms. Therefore, to continue our study in the field of hCA IX
and XII inhibitors [21] we chose the coumarin scaffold for investigation. Thus, we synthesized
compounds 1-10 (Fig. 1) which bear at position 3 the acetyl group, in place of the less stable ester
function present in the previously reported 3-carboxycoumarins [16], and diverse substituents at
the 4-position (R4) and on the fused benzo ring (X).
O
O
coumarin
R
N 2
N
1N
N
O
O
R4
O
O
8
X
7
R3
6
Me
6
X
7
OR
Me
X
O
O
7
Me
O
O
O
O
38-39
11-31 1-substituted
32-37 2-substituted
3-Carboxycoumarins
1-10
R= H, Me, Et
X= H, Me, OMe
OH, CH2OH
R4= H, OH, OMe
R= Aryl, Me, CH2Ph
R3= H, Me, CH2Ph
X= H, 8-Br, 8-NO2, 7-Cl,
X= H, 6-Br, 6-NO2,
7-Cl, 7-Cl-6-NO2
7-Cl-8-NO2, 8-NH2, 8-NHR'
Fig. 1. Design of 2H-chromen-2-one derivatives 1-10 and their analogs chromeno[4,3-c]pyrazol-4-
ones 11-37 and pyrano[4,3-c]pyrazoles 38-39 as inhibitors of the human carbonic anhydrases IX
and XII.
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Next, several chromeno[4,3-c]pyrazol-4-one derivatives (11-37) were synthesized and biologically
tested. To explore the structural requirements of different areas around the tricyclic core, different
substituents with diverse steric bulk and lipophilicity were placed at position 1 or 2 (R= Ar, CH2Ph,
Me) and 3 (R3= H, Me, CH2Ph), and on the fused benzo ring (X= H, Br, NO2, Cl, NH-R’) (Fig. 1).
Finally, the pyrano[4,3-c]pyrazoles 38 and 39 were designed to evaluate how replacement of the
fused benzo ring with the smaller methyl group affected complementarity with the hCAs.
2. Results and discussion
2.1.Chemistry
3-Acetylchromen-2-one (1) was purchased from Sigma-Aldrich while the other target compounds
2-39 were prepared as drawn in Schemes 1-6. The 3-acetylchromen-2-ones 2 [22] and 3 (Scheme 1)
ensued from a Knoevenagel condensation between 5-substituted 2-hydroxybenzaldehydes and ethyl
acetoacetate, carried out in the presence of piperidine and under microwave irradiation. Nitration of
the 7-chloro-derivative 3 furnished the 7-chloro-6-nitro-substituted compound 4 in high yield.
Scheme 1. Reagents and conditions: a) piperidine, mw, 120 °C; b) 90% HNO3, -10 °C.
The synthesis of the 4-substituted 3-acetylchromen-2-one derivatives 5-10 and 3-methyl-1-aryl-
chromeno[4,3-c]pyrazol-4-one derivatives 11-15 and 17 is shown in Scheme 2.
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Scheme 2. Reagents and conditions: a) ethyl acetoacetate sodium salt, anhydrous Et2O (X= H) or
toluene (X= Br), reflux; b) KNO3, 96% H2SO4, 0 °C; c) (MeO)2SO2, anhydrous acetone, K2CO3,
mw, 125 °C; d) EtOH, reflux; e) Ar-NHNH2.HCl, NEt3, EtOH, reflux; f) p-TsOH, xylene, reflux.
The 3-acetyl-4-hydroxychromen-2-one 5 and its 6-bromo derivative 6 were synthesized as
previously reported, i.e. by reacting the suitable acetylsalicyloyl choride with the sodium salt of
ethyl acetoacetate [23, 24]. This method was not employed to prepare the 6-nitro-derivative 7 [24]
which was obtained by nitration of derivative 5. The 4-hydroxy group of 5 and 6 was then alkylated
with dimethylsulfate in anhydrous acetone and in the presence of potassium carbonate, under
microwave-assisted conditions, to yield the 4-methoxy derivatives 8 and 9. The latter afforded the
4-ethoxy derivative 10 after heating in refluxing absolute ethanol. The 3-acetyl-4-hydroxy-
chromen-2-ones 5-7 were used as starting material to prepare the 1-aryl-3-methylchromeno[4,3-
c]pyrazol-4-one 11-14 [23] and 17 [24]. Briefly, compounds 5-7 were reacted with suitable
arylhydrazines in boiling ethanol to give the corresponding arylhydrazones [23, 24] which were
cyclized in refluxing xylene and in the presence of para-toluensulfonic acid to provide derivatives
6

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11-14, 17. The same synthetic pathway was used to prepare the 1-(2-carboxyphenyl)-substituted
compound 15 obtained starting from 5 and through the formation of the hydrazone derivative 40.
H
N
R
O
O
N
O
R1
R2
R1
R2
Me
a
Me
O
O
41-44
1-4
b
R
R1
H
H
R2
H
Cl
H
R
N
O
N
COOH
16, 41
18, 42
19, 43
20, 44
21
8
7
R1
R2
Me
c
H
H
H
H
NO2
NO2 Cl
NH2
O
16, 18- 20
21
H
Scheme 3. Reagents and conditions: a) Ar-NHNH2, EtOH, reflux; b) Cu(AcO)2, CuSO4, EtOH,
reflux; c) from 19, H2, Pd/C, Parr apparatus 33 psi.
The tricyclic derivatives 16 and 18-20 (Scheme 3) were synthesized from the 3-acetylchromen-2-
ones 1-4 which were reacted with arylhydrazines to afford the relative arylhydrazones 41-44. The
latter were cyclized by oxidation with a mixture of cupric sulfate and cupric acetate to give the
chromeno[4,3-c]pyrazoles 16, 18-20. Catalytic reduction (Pd/C) of the 8-nitro derivative 19
provided the corresponding 8-amino derivative 21 which, besides being a target compound, was
used as an intermediate for the preparation of the 1-phenylchromenopyrazoles 22-27 (Scheme 4).
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Scheme 4. Reagents and conditions: a) excess of MeI, K2CO3, CH3CN; b) RCOCl, NEt3, anhydrous
CH2Cl2, rt; c) PhNCO, anhydrous THF, reflux; d) PhCHO, anhydrous ZnCl2, anhydrous THF,
reflux, N2; e) NaBH4, MeOH, reflux, N2.
Reaction of 21 with an excess of methyl iodide furnished a mixture of the 8-methylamino- and 8-
dimethylamino derivatives 22 and 23, respectively, which were separated by column
chromatography. When compound 21 was reacted with acyl chlorides or phenylisocyanate, the
corresponding 8-amido- (24, 25) or 8-phenylureido- (26) derivatives were obtained. The 8-
benzylamino- substituted compound 27 was synthesized by treatment of 21 with benzaldehyde and
subsequent reduction of the Schiff’s base 45 with sodium borohydride. The 1-
benzylchromenopyrazol-4-one derivative (28) (Scheme 5) was prepared by reacting 3-acetyl-4-
hydroxychromen-2-one (5) with benzylhydrazine in refluxing ethanol. The 2-phenylchromeno[4,3-
c]pyrazol-4-one derivatives 32-34 (Scheme 5) were obtained starting from the 3-acetyl-4-
hydroxychromen-2-one derivatives 5-7, as previously reported [23, 24].
8

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Scheme 5. Reagents and conditions: a) Ph-CH2NHNH2 dihydrochloride, NEt3, EtOH, reflux; b) see
Ref. 23 and 24.
The synthesis of the tricyclic derivatives 29-31 and 35-37 is shown in Scheme 6. The
chromeno[4,3-c]pyrazol-4-ones featuring a benzyl group at position 3, combined with a methyl and
a benzyl residue at position 1 (29 and 30) [25] and 2 (35 and 36) [25], were obtained from 4-
hydroxy-3-phenacetyl-chromen-2-one (46). The chromeno[4,3-c]pyrazol-4-one derivatives 31 and
37, lacking a substituent at position 3 and bearing a phenyl group at position 1 or 2, were prepared
starting from 4-chloro-2-oxo-2H-chromene-3-carbaldehyde 47 [26] which was transformed into the
corresponding 3-phenylhydrazone 48 [26]. This compound was cyclized by heating in pyridine and
a catalytic amount of piperidine to provide the 1-phenyl regioisomer 31 [26]. To obtain the 2-phenyl
regioisomer 37 we employed different conditions from those described [27] as they afforded a
mixture of the two isomer 31 and 37. In our hands, the intermediate 47 was reacted with an
equimolar amount of phenylhydrazine in refluxing glacial acetic acid and compound 37 was
isolated as the sole isomer in good yields.
The synthesis of the 3,6-dimethylpyrano[4,3-c]pyrazole derivatives 38 [28] and 39 [29] was
achieved, as previously reported, starting from the commercially available dehydroacetic acid.
9

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Scheme 6. Reagents and conditions: a) see Ref. 25; b) PhNHNH2.HCl, H2O/AcOH, MeOH, 60 °C;
c) piperidine, pyridine, 100 °C; d) PhNHNH2, glacial AcOH reflux.
2.2.CA inhibitory activity
All the synthesized derivatives 1-39 were tested to determine their efficacy against the targeted
hCAs IX and XII and also toward the hCAs I and II, which are physiologically relevant cytosolic
isoforms, highly expressed throughout the human body, thus responsible for the side effects of
nonselective hCA inhibitors.
The inhibition data of the synthesized compounds at hCAs are reported in Tables 1-3, together with
those of acetazolamide (AZA) and coumarin, as reference inhibitors. The obtained results indicate
that enhancement of molecular complexity of the coumarin scaffold ameliorated the potency and
selectivity toward the targeted hCAs, thus confirming the concept that a more complex ligand can
present better complementarity for a specific target. None of the tested derivatives was able to
inhibit the off-target hCAs I and II, some compounds (13, 14, 19, 21, 25, 31, 33, 37 and 39) were
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potent (Ki < 41 nM) and selective against the hCA IX, and some (6, 11 and 17) were dually potent
hCAs IX and XII inhibitors (Ki= 5.6-9.6 nM).
Table 1. Inhibition data of the 3-acetyl-2H-chromen-2-one derivatives 1-10 and Acetazolamide
(AZA) and coumarin, as reference compounds, against hCA I, II, IX and XII isoforms, as
determined by a stopped-flow CO2 hydrase assay.
Ki (nM)a
R4
H
X
H
hCAI
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
31000
hCAII
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
92000
hCAIX
151.8
44.9
hCAXII
7.8
1
2
H
6-NO2
7-Cl
695.2
4529.4
6400.0
453.5
5.6
H
809.4
561.2
35.9
3
H
6-NO2-7-Cl
H
4
OH
OH
OH
OMe
OMe
OEt
5
6-Br
6-NO2
H
8.2
6
44.2
357.1
673.5
706.1
737.2
7
28.4
8
6-Br
6-Br
90.7
9
46.4
10
Coumarinb
>500000 >500000
25.4 5.6
250
12.1
AZA
aMean from three different assays, and errors were within 10% of the reported values. bRef. 16.
Concerning the first set of inhibitors, i.e. the 3-acetylchromen-2-one derivatives 1-10 (Table 1),
introduction of the acetyl group at the 3-position of coumarin resulted in a strong increase of
potency at the hCAs IX and XII, compound 1 being active in the nanomolar range (Ki= 151.8 and
7.8 nM, respectively) against the two isoforms. Due to this encouraging result, further modifications
were carried out on derivative 1, such as introduction on the fused benzo ring of small substituents
11

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(6-NO2, 7-Cl or both) which, to the best of our knowledge, have been never probed on coumarin
derivatives designed as hCA inhibitors. Derivatives obtained in this way (2-4) showed lower
potencies, compared to the lead 1, in particular against the hCA XII isoform. The only exception
was the 6-nitro derivative 2 being about three-fold more active than 1 against the hCA IX.
Introduction of a hydroxy group at position 4 of derivatives 1 and 2, to give 5 and 7, elicited
different effects. Compound 5 was more effective than the parent 1 at the hCA IX (Ki= 35.9 nM)
and significantly less potent at the hCA XII (Ki= 453.5 nM). In contrast, derivative 7 was as active
as the parent compound 2. However, compounds 5 and 7 are equally potent against each hCA of
interest. Introduction of the lipophilic and bulky bromine atom at position 6 of compound 5 turned
out to be profitable, compound 6 being one of the most potent dual inhibitors of hCAs IX and XII
among the herein reported derivatives. The effect of 4-hydroxy alkylation carried out on compounds
5 and 6 is difficult to explain as it afforded opposite results (compounds 8-10). Methylation of the
4-hydroxy group of 5 left the inhibition profile unchanged (see derivative 8) while the same
modification on derivative 6 worsened activity about 10-fold against the hCA IX and more
drastically against the hCA XII (see derivative 9). Transformation of the 4-methoxy derivative 9
into its superior homologue 4-ethoxy derivative 10 slightly enhanced the hCA IX potency.
In the second step of the work, we decided to merge a pyrazole nucleus with the bicyclic 3-
acetylchromen-2-one 1 to verify whether the added ring, and other structural elements on it, could
enhance affinity and selectivity for the targets. First, the 1-phenyl-3-methylchromeno[4,3-c]pyrazol-
4-one 11 was synthesized and tested (Table 2). Compound 11 behaved as a balanced dual inhibitor
of hCAs IX and XII, endowed with low nanomolar activity (Ki= 9.6 and 5.8 nM), and was more
potent at the former isoform than the parent derivative 1. These results indicate that compound 11
possesses a better structural complementarity for the hCA IX, thus suggesting that the fused
pyrazole nucleus and the lipophilic area represented by the 1-phenyl ring reinforce the interaction
with the enzyme.
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Table 2. Inhibition data of derivatives 11-27 and Acetazolamide (AZA), as a reference compound,
against hCA I, II, IX and XII isoforms, as determined by a stopped-flow CO2 hydrase assay.
Ki (nM)a
hCAIX hCAXII
R
X
hCAI
hCAII
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
12.1
H
H
H
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
250
9.6
402.8
22.2
40.2
76.8
1960.4
8.5
5.8
>10000
>10000
>10000
27.3
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
4-Cl
4-Me
H
4-OMe
H
2-COOH
H
4-COOH
H
24.7
H
H
H
H
H
H
H
H
H
H
H
8-Br
7.1
7-Cl
57.9
27.1
801.2
34.5
51.3
59.4
26.8
15.3
77.9
37.8
25.4
818.7
>10000
6062.5
>10000
737.2
359.8
646.5
>10000
583.2
359.8
5.6
8-NO2
7-Cl, 8-NO2
8-NH2
8-NHMe
8-N(Me)2
8-NHCOMe
8-NHCOPh
8-NHCONHPh
8-NHCH2Ph
AZA
aMean from three different assays, and errors were within 10% of the reported values.
To preliminarily investigate the structure-affinity relationships (SAR) in this new class of hCA
inhibitors, small substituents with different lipophilic and electronic properties (Cl, OMe, Me,
COOH) were introduced on the 1-phenyl ring (derivatives 12-16). Only the carboxylic residue, both
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at the ortho (15) and para (16) position, afforded potent hCA XII inhibitors (Ki= 27.3 and 24.7 nM,
respectively). The ortho-isomer 15 also showed quite good activity against the hCA IX. The other
substituents yielded to hCA IX selective compounds (12-14) since they significantly reduced hCA
XII inhibition. The 4-methyl- and 4-methoxy-substituted derivatives 13 and 14 are noteworthy,
showing Ki values in the low nanomolar range (22.2 and 40.2 nM).
In the second set of tricyclic derivatives (17-20), structural modifications were carried out on the
fused benzo ring where the same substituents probed on coumarins 1-10 were evaluated. The Ki
values obtained for 17-20 are overall better than those of the relative bicyclic compounds and the
observed SARs are similar. In detail, the 8-bromo derivative 17 stands out as one of the best dual
hCAs IX and XII inhibitors (Ki= 8.5 and 7.1 nM) among those reported here. Also the 8-nitro
derivative 19 is worth noting, being one of the most potent and selective inhibitors at the hCA IX
isoform (Ki= 27.1 nM) of those investigated in this work. The 7-chlorine atom (compound 18)
resulted in a nanomolar potency at the hCA IX (Ki= 57 nM) and a significantly reduced inhibition
of the hCA XII. The 7-chloro-8-nitro substitution made compound 20 scarcely active at both the
targeted hCA isoforms. Reduction of the 8-nitro derivative 19 gave the 8-amino derivative 21
which, similarly to 19, behaves as a selective hCA IX inhibitor endowed with nanomolar potency
(Ki= 34.5 nM).
The 8-amino group of 21 was exploited for subsequent derivatizations. Its linkage to groups with
different steric, lipophilic and electronic properties, such as methyl (22 and 23), acetyl (24),
phenylcarbamoyl (26) or benzyl (27) showed little effect on the potency against the hCA IX, the
obtained compounds being as active as 21 (Ki= 37.8-77.9 nM). These 8-amino-modified derivatives
also showed some ability to inhibit the hCA XII (Ki= 359.9-737.2 nM). Instead, the presence of a
benzoyl moiety on the 8-amino group (25) afforded the most potent (Ki= 15.3 nM) and selective
hCA IX inhibitor of those reported.
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Table 3. Inhibition data of derivatives 28-39 and Acetazolamide (AZA), as a reference compound,
against hCA I, II, IX and XII isoforms, as determined by a stopped-flow CO2 hydrase assay.
Ki (nM)a
R
C6H4-CH2
C6H4-CH2
Me
R3
X
-
hCAI
hCAII
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
12.1
hCAIX hCAXII
Me
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
250
371.1
31.5
19.7
30.1
38.1
25.2
35.8
289.7
555.2
37.6
26.6
41.4
25.4
731.9
818.7
342.9
>10000
593.2
>10000
359.8
671.9
33.3
28
29
30
31
32
33
34
35
36
37
38
39
C6H4-CH2
-
C6H4-CH2
-
C6H5
C6H5
C6H5
C6H5
C6H4-CH2
Me
H
-
Me
H
Br
NO2
H
H
H
-
Me
Me
C6H4-CH2
C6H4-CH2
C6H5
-
H
-
>10000
517.7
>10000
5.6
-
-
-
AZA
aMean from three different assays, and errors were within 10% of the reported values.
Compounds 28-31 (Table 3) ensued from the chromeno[4,3-c]pyrazol-4-one 11 whose 1-phenyl and
3-methyl substituents were replaced by a benzyl, a methyl or a hydrogen atom to evaluate the steric
and lipophilic requirements of the molecule in proximity of the two positions. None of these
modifications ameliorated the inhibitory profile of the lead. In fact, the presence of a benzyl
pendant at position 1 (28) caused a drop of potency at both hCAs of interest. Compound 29, more
15

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hindered than 28 since it features a benzyl group at both the 1- and 2-positions, showed a restored
activity at the hCA IX, being active at low nanomolar concentration (Ki= 31.5 nM). A comparable
inhibitory profile was obtained when the 1-benzyl moiety of 29 was replaced with the less lipophilic
methyl group to give compound 30. An interesting result, although difficult to explain, was found
for compound 31, where a hydrogen atom was present at position 3 in the place of the 3-methyl
group of the potent dual hCA IX/XII inhibitor 11. This small change made compound 31 inactive at
the hCA XII isoform while it preserved activity against the hCA IX (Ki= 30.1 nM), thus suggesting
that the methyl substituent of 11, although being a small group, plays a crucial role for interaction
with the hCA XII enzymatic site.
The set of derivatives 32-37 (Table 3) was synthesized to evaluate the effect of moving the
lipophilic area (Ph, CH2Ph, Me) from the 1- to the 2-position of the best inhibitors identified in the
meanwhile, i.e. compounds 11, 17, 19, 29-31. This modification, although it significantly changed
the shape of the molecules, gave rise to some potent hCA IX inhibitors (32-34, 37). The compounds
showed in fact nanomolar activities (Ki= 25.2-38.1 nM) even if they were in general less active than
the 1-substituted counterparts (11, 17, 19 and 31) on both the isoforms. It is worth noting that the 8-
bromo-derivative 33 was hCA IX selective while its regioisomer 17 was active on both the targeted
enzymes. Derivatives 35 and 36, featuring a benzyl moiety at position 3, displayed scarce potencies
on the hCAs of interest, with the exception of the hCA XII inhibitor activity of 36 in the low
nanomolar range.
Finally, the fused benzo ring of the 1-phenyl- and 2-phenyl- substituted chromenopyrazole
derivatives 11 and 32 was replaced with a methyl residue to give the pyrano[4,3-c]pyrazol-4-one
derivatives 38 and 39. Both compounds maintained the capability of blocking the hCA IX with high
potency (Ki= 26.6 and 41.4 nM) while they were less active against the hCA XII. As observed in
the tricyclic series, the 1-phenyl derivative 38 showed a higher potency with respect to the 2-
substituted regioisomer 39.
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2.3. Cell toxicity assays
Some selected compounds, belonging to the 3-acetylcoumarin set (6) and to the chromeno[4,3-
c]pyrazol-4-one (11, 13, 19, 21, 25, 31) and pyrano[4,3-c]pyrazol-4-one (39) series, were chosen
among those possessing the best inhibitory profile and tested (30 – 300 µM) to evaluate their effects
on viability of human colon cancer HT-29 cells. Tests were performed over time (16 and 48 h)
under normoxic and hypoxic conditions. The efficacy of the compounds is summarized in Figures 2
(6, 11, 13 and 19) and 3 (21, 25, 31 and 39) in comparison to untreated control (100% viability).
As a general trend, after 16 h incubation, the tested derivatives significantly inhibited cell viability
in normoxic conditions while most of them did not have any effect in hypoxia. A similar behavior
was also observed after 48 h incubation, with the exception of compounds 19, 31 and 39 which
turned out to be more active under hypoxic conditions.
Analyzing the results in greater detail, in normoxic conditions, compounds 6, 13, 21 and 25 showed
a concentration- and time-dependent efficacy in reducing cancer cell viability. It was not possible to
test 6 and 13 at the higher concentration (300 µM) for their low solubility in the assay medium.
Derivatives 6 and 25 (100 µM, 48 h) reduced the number of living cells up to about 35 and 50%,
respectively. Compounds 11, 19 and 39 showed the peculiarity of a dramatic concentration-
dependent effect after 16 h incubation. The lower activity after 48 h could be related to a reduced
stability of the solutions rather than an intrinsic variation of efficacy. In hypoxic conditions, the
longer incubation time favors activity: after 48 h compounds 6, 11, 19, 21, 31 and 39 were
significantly effective. In particular, the 2-phenylchromeno-pyrazol-4-one 31 (30 µM, 48 h,
hypoxia) showed the best profile reducing cell viability up to 70%.
Finally, a long incubation time (72 h) was experimented. Only the activities of the most interesting
derivatives (selected by solution stability and effectiveness) are reported in Figure 4. Compound 6
maintained efficacy as shown also for shorter times. With regard to compounds 21, 25 and 39, a
longer time of incubation promoted activity in hypoxic conditions. In particular, compound 25 (100
µM) decreased cancer cell viability to 40%.
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It is difficult to explain why some compounds showed cytotoxic effects not only in hypoxia (when
hCA IX is overexpressed) but also in normoxia, when the enzyme is not present in these cells. Thus,
it is possible that, besides interfering with the hCA IX/XII activity in hypoxia, they may exert their
cytotoxic effects through other mechanisms of action, involving pathways in normoxia. A similar
behavior was observed also for other hCA inhibitors, such as sulfocoumarins [13], 3-
hydroxyquinazoline-2,4-diones [21] and selenoureido-benzensulfonamides [31].
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Fig. 2. HT-29 cells (1.104/well) were treated with compounds 6 (A), 11 (B), 13 (C) and 19 (D) (0 -
300 µM). Incubation was allowed for 16 or 48 h in normoxic (20% O2) and hypoxic conditions
(0.1% O2). Cell viability was measured by MTT assay. The control condition was arbitrarily set as
100% and values are expressed as the mean ± SEM of three experiments. *P<0.05, **P<0.01 and
***P<0.001 in comparison to control (0 µM).
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Fig. 3. HT-29 cells (1.104/well) were treated with compounds 21 (E), 25 (F), 31 (G), and 39 (H) (0 -
300 µM). Incubation was allowed for 16 or 48 h in normoxic (20% O2) and hypoxic conditions
(0.1% O2). Cell viability was measured by MTT assay. The control condition was arbitrarily set as
100% and values are expressed as the mean ± SEM of three experiments. *P<0.05, **P<0.01 and
***P<0.001 in comparison to control (0 µM).
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Fig. 4. HT-29 cells (1.104/well) were treated with compounds 6 (A), 21 (B), 25 (C), and 39 (D) (0 -
300 µM). Incubation was allowed for 72 h in normoxic (20% O2) and hypoxic conditions (0.1%
O2). Cell viability was measured by MTT assay. The control condition was arbitrarily set as 100%
and values are expressed as the mean ± SEM of three experiments. *P<0.05, **P<0.01 and
***P<0.001 in comparison to control (0 µM).
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3. Conclusions
In this work, different structural modifications were carried out on the coumarin scaffold leading to
a set of 3-acetyl-coumarin derivatives and, more interestingly, to new classes of inhibitors of the
tumor-associated hCAs IX and XII: the chromeno[4,3-c]pyrazol-4-ones and the pyrano[4,3-
c]pyrazol-4-ones. Several synthesized derivatives showed Ki values in the low nanomolar range,
being either selective against the hCA IX or dual inhibitors on both the hCA isoforms of interest.
None of the compounds was effective in inhibiting the cytosolic enzymes hCAs I and II. Some
inhibitors proved to be active as antiproliferative agents on HT-29 colon cancer cell lines, both in
normoxic and hypoxic conditions. This finding led us to hypothesize for these derivatives more than
one mechanism of action, involving hCAs IX and XII in hypoxia and other not identified target(s)
in normoxia.
4. Experimental section
4.1 Chemistry.
All the commercially available reagents and solvents were used as purchased from Sigma-Aldrich
and Alfa Aesar (Italy) without further purification. Analytical silica gel plates (Merck F254) and
silica gel 60 (Merck, 70-230 mesh) were used for analytical TLC and for column chromatography,
respectively. All melting points were determined on a Gallenkamp melting point apparatus and are
uncorrected. Compounds were named following IUPAC rules as applied by ChemDrawUtra 9.0.
Elemental analyses were performed with a Flash E1112 Thermofinnigan elemental analyzer for C,
H, N and the results were within ± 0.4% of the theoretical values. All final compounds revealed
purity not less than 95%. The IR spectra were recorded with a Perkin-Elmer Spectrum RX I
spectrometer in Nujol mulls and are expressed in cm-1. NMR spectra were recorded on a Bruker
Avance 400 spectrometer (400 MHz for 1H NMR and 100 MHz for 13C NMR). The chemical shifts
are reported in δ (ppm) and are relative to the central peak of the solvent which was CDCl3 or
22

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DMSOd6. The following abbreviations are used: s= singlet, d= doublet, t= triplet, q= quartet, m=
multiplet, br= broad and ar= aromatic protons.
Compounds 2 [22], 5 [23], 6 [24], 11-14 [23], 17 [24], 29-30 [25], 32 [23], 33 and 34 [24], 35 and
36 [25], 38 [28], 39 [29] were prepared according previously reported procedures.
4.1.1. 3-Acetyl-7-chloro-2H-chromen-2-one (3)
A mixture of 4-chloro-2-hydroxybenzaldeyde (8.98 mmol), ethyl acetoacetate (22.4 mmol) and
catalytic amount of piperidine (0.22 mmol) was stirred at room temperature for 10 min, then it was
diluted with EtOH (10 mL). The solid which precipitated was collected by filtration and
1
recrystallized. Yield 90%; mp 170-171 °C (2-Methoxyethanol); H NMR (CDCl3): δ 2.74 (s, 3H,
CH3), 8.34 (dd, 1H, H-6, J = 8.4, 1.9 Hz), 7.35 (d, 1H, H-8, J = 1.7 Hz), 8.01 (d, 1H, H-5, J = 8.3
Hz), 8.49 (s, 1H, H-4). 13C NMR (DMSO-d6): δ 30.92 (CH3), 117.27, 118.18, 125.33, 126.26,
133.02, 139.74, 147.25, 155.92, 158.88, 195.80. IR: 1678, 1741. Anal. C11H7ClO3 (C, H, N).
4.1.2. 3-Acetyl-7-chloro-6-nitro-2H-chromen-2-one (4)
3-Acetyl-7-chloro-2H-chromen-2-one 3 (2.25 mmol) was added portionwise to cooled HNO3 (90
%, -10 °C). When the addition was complete, the mixture was stirred for about 2h at -10 °C, then
the solution was poured onto ice (about 100 g) and the solid collected, washed with abundant water
(about 100 mL), dried and recrystallized. Yield 88%; mp 210-212 °C (EtOH); 1H NMR (CDCl3): δ
2.75 (s, 3H, CH3), 7.58 (s, 1H, H-8), 8.31 (s, 1H, ar), 8.49 (s, 1H, ar). 13C NMR (DMSO-d6):
δ 30.36 (CH3), 118.16, 120.01, 126.67, 128.61, 130.97, 143.92, 145.33, 156.66, 157.58, 194.95. IR:
1686, 1737. Anal. C11H6ClNO5 (C, H, N).
4.1.3.
3-Acetyl-4-hydroxy-6-nitro-2H-chromen-2-one (7)
3-Acetyl-4-hydroxy-2H-chromen-2-one 5 (2.45 mmol) [23] was portionwise added to a cooled
solution (0 °C) of KNO3 (2.45 mmol) in H2SO4 (10 mL). When the addition was complete, the
23

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mixture was stirred for about 1h at 0 °C, then the solution was poured onto ice (about 50 g) and the
solid collected, washed with abundant water (about 50 mL), dried and recrystallized. Yield 98%;
mp 230-231 °C (EtOH); 1H NMR (CDCl3): δ 2.83 (s, 3H, CH3), 7.48 (d, 1H, H-8, J = 9.0 Hz), 8.55
(dd, 1H, H-7, J = 9.2, 2.5 Hz), 8.98 (d, 1H, H-5, J = 2.2 Hz).
4.1.4. General procedure for the synthesis of 3-acetil-4-metossi-2H-chromen-2-one derivatives
8, 9
A mixture of 3-acetil-4-hydroxy-2H-chromen-2-one derivatives 5 [23] or 6 [24] (0.98 mmol),
K2CO3 (2.94 mmol) and dimethylsulfate (1.17 mmol) in anhydrous acetone (3 mL), was microwave
irradiated at 125 °C for 6 min. The suspension was poured onto iced water (50 mL) and neutralized
with 10% HCl solution. The mixture was extracted with Et2O (30 mL x 3), then the organic phase
was washed with a NaHCO3 saturated solution (50 mL) and brine (50 mL). The dried (Na2SO4)
organic phase was evaporated at reduced pressure to give an oil residue which solidified upon
treatment with Et2O (2-3 mL). The crude compound was purified by column chromatography
(cyclohexane/EtOAc 6:4).
1
4.1.4.1. 3-Acetyl-4-methoxy-2H-chromen-2-one (8). Yield 74%; mp 81-83 °C (MeOH); H NMR
(CDCl3): δ 2.72 (s, 3H, CH3), 4.02 (s, 3H, OCH3), 7.30-7.34 (m, 2H, ar), 7.61 (t, 1H, ar, J = 7.2 Hz),
7.91 (d, 1H, H-5, J = 7.9 Hz). IR 1699, 1713. Anal. C12H10O4 (C, H, N).
4.1.4.2. 3-Acetyl-6-bromo-4-methoxy-2H-chromen-2-one (9). Yield 70%; mp 147-149 °C
(MeOH); 1H NMR (CDCl3): δ 2.71 (s, 3H, CH3), 4.01 (s, 3H, OCH3), 7.22 (d, 1H, H-8, J = 7.8 Hz),
13
7.69 (dd, 1H, H-7, J = 8.4, 2.2 Hz), 8.03 (d, 1H, H-5, J =2.2 Hz). C NMR (DMSO-d6): δ 32.95
(CH3), 62.67 (OCH3), 110.63, 117.50, 118.96, 119.80, 127.40, 136.82, 151.93, 161.22, 162.02,
200.74. IR: 1693, 1710. Anal. C12H9ClBrO4 (C, H, N).
4.1.5. 3-Acetyl-4-ethoxy-6-bromo-2H-chromen-2-one (10)
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A suspension of the 4-methoxy-2H-chromen-2-one derivative 9 (0.3 mmol) in absolute EtOH (25
mL) was refluxed for 6 h. The insoluble solid was collected by filtration and recrystallized. Yield
75%; mp 116-118 °C (EtOH); 1H NMR (CDCl3): δ 1.52 (t, 3H, CH3, J = 6.9 Hz), 2.69 (s, 3H, CH3),
4.15 (q, 2H, CH2, J = 6.9 Hz), 7.22 (d, 1H, H-8, J = 8.8 Hz), 7.68 (dd, 1H, H-7, J = 8.4, 2.2 Hz), 8.01
(d, 1H, H-5, J = 2.2 Hz). 13C NMR (CDCl3): δ 15.36 (CH3), 32.16 (CH3), 70.97 (CH2), 117.21,
118.37, 127.05, 135.95, 147.34, 149.04, 151.42, 153.07, 160.81, 161.63. IR: 1699, 1712. Anal.
C13H11ClBrO4 (C, H, N).
4.1.6. 2-(2-(1-(2-Oxo-2H-chromen-3-yl)ethylidene)hydrazinyl)benzoic acid (40)
A mixture of the suitable 2-hydrazinobenzoic acid hydrochloride [30] (2.58 mmol) and
triethylamine (2.58 mmol) in EtOH (30 mL) was stirred at reflux for 5-10 min, then the 3-acetyl-4-
hydroxy-2H-chromen-2-one 5 (1.29 mmol) was added and the mixture was heated for an additional
30 min. After cooling at room temperature, the solid was collected, washed with water (about 10-20
1
mL) and recrystallized. Yield 92%; mp 245-246 °C (EtOH); H NMR (DMSOd6): δ 2.69 (s, 3H,
CH3), 6.97 (t, 1H, ar, J = 7.6 Hz), 7.02 (d, 1H, ar, J = 8.5 Hz), 7.32−7.37 (m, 2H, ar), 7.57 (t, 1H,
ar, J = 8.5 Hz), 7.66 (t, 1H, J = 7.8 Hz), 7.96 (d, 1H, ar, J = 7.8 Hz), 8.00 (d, 1H, ar, J = 7.6
Hz), 10.28 (s, 1H, NH), 13.37 (br s, 1H, OH) Anal. C18H14N2O5 (C, H, N).
4.1.7. 2-(3-Methyl-4-oxochromeno[4,3-c]pyrazol-1(4H)yl)benzoic acid (15)
A mixture of arylhydrazone 40 (2.96 mmol) and a catalytic amount of p-toluensulfonic acid in
anhydrous xylene (50 mL) was refluxed for about 1h, while water was distilled off using a Dean-
Stark apparatus. The mixture was concentrated by removing about half of the solvent, then cooled
at rt and filtered to remove the tarry residue. After addition of ligroin to the filtrate, a solid was
obtained which was collected by filtration and recrystallized. Yield 48%; mp > 300 °C (EtOH). 1H
NMR (DMSOd6): δ 2.55 (s, 3Η, CH3), 6.79 (d, 1H, ar, J = 7.3 Hz), 7.17 (t, 1H, ar, J = 8.0 Hz), 7.52-
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ACCEPTED MANUSCRIPT
7.57 (m, 2H, ar), 7.77 (d, 1H, ar, J = 7.6 Hz), 7.83-7.89 (m, 2H, ar), 8.15 (d, 1H, ar, J = 7.4 Hz). 13C
NMR (DMSOd6): δ 13.49 (CH3), 105.93, 112.55, 118.64, 122.69, 125.38, 130.33, 130.57; 132.00,
132.31, 132.45, 134.56, 138.97, 143.31, 149.94, 153.48, 158.02, 166.50. IR 1713, 1747. Anal.
C18H12N2O4 (C, H, N).
4.1.8 General procedure for the synthesis of arylhydrazones of 3-acetyl-2H-chromen-2-ones 41-
44
A mixture of the suitable arylhydrazine (1.42 mmol) and 3-acetyl-derivatives 1-4 (1.29 mmol) in
EtOH (5 mL) was heated at reflux for about 1-2 h. After cooling at room temperature, the solid was
collected and recrystallized.
4.1.8.1. 4-(2-(1-(2-Oxo-2H-chromen-3-yl)ethylidene)hydrazinyl)benzoic acid (41) Yield 87%; mp
1
227-229 °C (2-methoxyethanol); H NMR (DMSOd6): δ 2.26 (s, 3H, CH3), 7.29 (d, 2H, ar, J = 8.8
Hz), 7.42 (t, 1H, ar, J = 8.2 Hz), 7.62 (t, 1H, ar, J = 7.2 Hz), 7.82 (d, 2H, ar, J = 8.8 Hz), 7.86 (d,
1H, ar, J = 6.7 Hz), 8.25 (s, 1H, H-4), 9.89 (s, 1H, NH), 12.35 (br s, 1H, OH). IR 1682, 1720, 3290.
Anal. C18H14N2O4 (C, H, N).
4.1.8.2. 7-Cloro-3-(1-(2-phenyldrazono)ethyl)-2H-chromen-2-one (42). Yield 58%; mp 153-155 °C
(EtOH); 1H NMR (CDCl3): δ 2.31 (s, 3H, CH3), 6.94 (t, 1H, ar, J = 7.2 Hz), 7.18 (d, 2H, ar, J = 7.6
Hz), 7.31 (m, 3H, ar), 7.38 (d, 1H, H-8, J = 1.6 Hz), 7.52 (d, 1H, H-5, J = 8.4 Hz), 7.58 (s, 1H, NH),
8.05 (s, 1H, H-4). IR: 1720, 3331. Anal. C17H13ClN2O2 (C, H, N).
4.1.8.3. 3-(1-(2-Phenyldrazono)ethyl)-8-nitro-2H-chromen-2-one (43). Yield 52%; mp 204-206 °C
(EtOH); 1H NMR (CDCl3): δ 2.33 (s, 3H, CH3), 6.97 (t, 1H, ar, J = 7.6 Hz), 7.20 (d, 2H, ar, J = 7.6
Hz), 7.34 (t, 2H, ar, J = 7.6 Hz), 7.48 (d, 1H, H-8, J = 9.2 Hz), 7.68 (s, 1H, NH), 8.16 (s, 1H, H-4),
8.39 (dd, 1H, H-7, J = 8.8, 2.8 Hz), 8.53 (d, 1H, H-5, J = 2.4 Hz). IR: 1340, 1516, 1726, 3328. Anal.
C17H13N3O4 (C, H, N).
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4.1.8.4. 7-Chloro-3-(1-(2-phenyldrazono)ethyl)-8-nitro-2H-chromen-2-one (44). Yield 70%; mp
1
220-221 °C (2-Methoxyethanol); H NMR (CDCl3): δ 2.32 (s, 3H, CH3), 6.97 (t, 1H, ar, J = 7.6
Hz), 7.18 (d, 2H, ar, J = 8.0 Hz), 7.33 (t, 2H, ar, J = 8.0 Hz), 7.52 (s, 1H, H-8), 7.69 (s, 1H, NH),
8.08 (s, 1H, H-5), 8.23 (s, 1H, H-4). IR: 1355, 1525, 1741, 3342. Anal. C17H12ClN3O4 (C, H, N).
4.1.9. General procedure for the synthesis of 1-aryl-3-methylchromeno[4,3-c]pyrazol-4-(1H)-one
derivatives 16, 18-20
A mixture of arylhydrazones (1.55 mmol), Cu(AcO)2·monohydrate and CuSO4·pentahydrate,
respectively 20% and 10% w/w with respect to the hydrazone, in EtOH (8 mL) was heated at reflux
for 6-24 h. After cooling, the insoluble solid was collected by filtration, washed with abundant
water (about 20-30 mL), dried, and purified by column chromatography (CH2Cl2/MeCN 9.8:0.2).
To obtain compound 16, the crude solid was suspended in HCl 6N and the mixture was stirred at rt
for about 30 min, then collected, washed with water, dried, and recrystallized.
4.1.9.1. 4-(3-Methyl-4-oxochromeno[4,3-c]pyrazol-1(4H)yl)benzoic acid (16). Yield 25%; mp >300
°C (2-Methoxyethanol) 1H NMR (DMSOd6): δ 2.57 (s, 3H, CH3), 7.08 (d, 1H, ar, J = 7.9 Hz), 7.20
(t, 1H, ar, J = 7.5 Hz), 7.52 (d, 1H, ar, J = 8.1 Hz), 7.58 (t, 1H, ar, J = 7.8 Hz), 7.80 (d, 2H, ar, J =
8.0 Hz), 8.20 (d, 2H, ar, J = 8.0 Hz), 13.39 (br s, 1H, COOH). 13C NMR (DMSOd6): δ 13.43 (CH3),
106.93, 112.22, 118.72, 123.21, 125.34, 128.05, 131.92, 132.55, 133.28, 142.37, 143.35, 150.77,
153.63, 157.93, 167.37. IR: 1690, 1742. Anal. C18H12N2O4 (C, H, N).
4.1.9.2. 7-Chloro-3-methyl-1-phenylchromeno[4,3-c]pyrazol-4-(1H)-one (18). Yield 48%; mp 193-
195 °C (EtOH); 1H NMR (CDCl3): δ 2.71 (s, 3H, CH3), 7.04 (br s, 2H, H-6 + H-8), 7.46 (s, 1H, H-
9), 7.54-7.56 (m, 2H, ar), 7.63-7.65 (m, 3H, ar). 13C NMR (CDCl3): δ 12.84 (CH3), 110.48, 118.31,
118.86, 123.28, 124.45, 126.80, 128.73, 130.06, 130.39, 136.91, 139.16, 141.08, 151.02, 153.61.
IR: 1747. Anal. C17H11ClN2O2 (C, H, N).
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4.1.9.3. 3-Methyl-8-nitro-1-phenylchromeno[4,3-c]pyrazol-4-(1H)-one (19). Yield 80%; mp 241-
1
242 °C (EtOAc); H NMR (CDCl3): δ 2.75 (s, 3H, CH3), 7.55-7.59 (m, 3H, ar), 7.60-7.80 (m, 3H,
ar), 8.05 (d, 1H, H-9, J = 2.4 Hz), 8.33 (dd, 1H, H-7, J = 9.2, 2.4 Hz).
4.1.9.4. 7-Chloro-3-methyl-8-nitro-1-phenylchromeno[4,3-c]pyrazol-4-(1H)-one (20). Yield 77%;
mp 265-267 °C (2-Methoxyethanol); 1H NMR (CDCl3): δ 2.72 (s, 3H, CH3), 7.50-7.60 (m, 2H, ar),
7.61 (s, 1H, ar), 7.65-7.70 (m, 3H, ar), 7.74 (s, 1H, ar). IR: 1377, 1577, 1768. Anal. C17H10ClN3O4
(C, H, N).
4.1.10. 8-Amino-3-methyl-1-phenylchromeno[4,3-c]pyrazol-4-(1H)-one (21).
Pd/C 10% (10% w/w with respect to the nitro derivative) was added to a solution of the nitro
derivative 19 (3.58 mmol) in DMF (5 mL). The mixture was hydrogenated in a Parr apparatus at 35
psi for 14 h. The catalyst was filtered off, and the clear solution was diluted with water (about 50
mL) and the obtained solid was collected by filtration, washed with water and purified by column
1
chromatography (eluent cyclohexane/EtOAc 6:4). Yield 77%; mp 201-204 °C (EtOH); H NMR
(CDCl3): δ 2.73 (s, 3H, CH3), 3.52 (br s, 2H, NH2), 6.33 (d, 1H, H-9, J = 2.4 Hz), 6.81 (dd, 1H, ar, J
= 8.8, 2.4 Hz), 7.22 (d, 1H, ar, J = 8.8 Hz), 7.50-7.70 (m, 5H, ar). 13C NMR (CDCl3): δ 12.92
(CH3), 106.69, 106.86, 112.18, 118.18, 118.88, 127.02, 129.76, 130.07, 139.48, 141.70, 142.38,
146.67, 150.79, 158.39. IR: 1726, 3228-3342. Anal. C17H13N3O2 (C, H, N).
4.1.11. 3-Methyl-8-methylamino-1-phenylchromeno[4,3-c]pyrazol-4-(1H)-one (22) and 8-
dimethylamino-3-methyl-1-phenylchromeno[4,3-c]pyrazol-4-(1H)-one (23)
A mixture of the 8-amino derivative 21 (1.56 mmol), anhydrous K2CO3 (2.34 mmol) and methyl
iodide (15.58 mmol) in anhydrous acetonitrile (10 mL), was stirred at rt for 22 h, then it was diluted
with water (15 mL) and extracted with CH2Cl2 (33 mL x 3). The organic phase was anhydrified
(Na2SO4) and the solvent was evaporated at reduced pressure to yield a solid which was treated with
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