3-Hydroxy-1H-quinazoline-2,4-dione as a New Scaffold to Develop Potent and Selective Inhibitors of the Tumor-Associated Carbonic Anhydrases IX and XII

Article abstract of DOI:10.1021/acs.jmedchem.7b00766

In this paper, we describe the discovery of the 3-hydroxyquinazoline-2,4-dione as a useful scaffold to obtain potent inhibitors of the tumor-associated human carbonic anhydrases (hCAs) IX and XII. A set of derivatives (1-29), bearing different substituents on the fused benzo ring (Cl, NO₂, NH₂, CF₃, ureido, amido, heterocycles), were synthesized, and several of them showed nanomolar activity in inhibiting the hCA IX and XII isoforms, while they were ineffective against the cytosolic enzymes hCAs I and II. Some selected compounds were tested for their antiproliferative activity against HT-29 colon cancer cell lines. After 48 h of treatment with the lower dose (30 μM), derivatives 12, 14, 15, and 19 were significantly active, inducing a mortality by about 50% in both normoxia and hypoxia. This finding led us to hypothesize for these compounds more than one mechanism of action involving both CAs IX and XII and other not yet identified target(s).

Full text of DOI:10.1021/acs.jmedchem.7b00766

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Article
3-Hydroxy-1H-quinazoline-2,4-dione as a New Scaffold to Develop Potent and
Selective Inhibitors of the Tumor-associated Carbonic Anhydrases IX and XII
Matteo Falsini, Lucia Squarcialupi, Daniela Catarzi, Flavia Varano, Marco Betti, Lorenzo Di Cesare Mannelli,
Barbara Tenci, Carla Ghelardini, Muhammet Tanc, Andrea Angeli, Claudiu T Supuran, and Vittoria Colotta
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00766 • Publication Date (Web): 28 Jun 2017
Downloaded from http://pubs.acs.org on June 29, 2017
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3-Hydroxy-1H-quinazoline-2,4-dione as a New Scaffold to
Develop Potent and Selective Inhibitors of the Tumor-
associated Carbonic Anhydrases IX and XII
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Matteo Falsini,a Lucia Squarcialupi,a Daniela Catarzi,a Flavia Varano,a Marco Betti,a Lorenzo Di
Cesare Mannelli,b Barbara Tenci,b Carla Ghelardini,b Muhammet Tanc,a 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.
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ABSTRACT
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The paper describes the discovery of the 3ꢀhydroxyquinazolinꢀ2,4ꢀdione as a useful scaffold to
obtain potent inhibitors of the tumorꢀassociated human carbonic anhydrases (hCAs) IX and XII. A
set of derivatives (1-29), bearing different substituents on the fused benzo ring (Cl, NO2, NH2, CF3,
ureido, amido, heterocycles), were synthesized and several of them showed nanomolar activity in
inhibiting the hCA IX and XII isoforms while they were ineffective against the cytosolic enzymes
hCAs I and II. Some selected compounds were tested for their antiproliferative activity on HTꢀ29
colon cancer cell lines. After 48 h treatment with the lower dose (30 µM), derivatives 12, 14, 15 and
19 were significantly active inducing a mortality by about 50% in both normoxia and hypoxia. This
finding let us to hypothesize for these compounds more than one mechanism of action involving
both CAs IXꢀXII and other not yet identified target(s).
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INTRODUCTION
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The human (h) carbonic anhydrases (CAs) are ubiquitous zinc enzymes distinguished in at least 15
isoforms and differing in molecular properties, subcellular localization and tissue distribution. hCA
activities are involved in the regulation of physiological functions related to carbon
dioxide/bicarbonate transport, pH, electrolyte secretion in tissues and organs and to many metabolic
processes.1 The hCA isoforms IX and XII are membraneꢀbound enzymes whose expression is
significantly enhanced in a large variety of cancer cells, being strongly induced by hypoxia, a
common feature of solid tumor, such as glioma, breast cancer and colon carcinoma. Hypoxia, due to
the discrepancy between the oxygen demand of proliferating tumor cells and oxygen supply from
the vasculature, shifts metabolism towards glycolysis and anaerobic fermentation affording a high
production of lactic acid. Oncogenic metabolism also generates excessive formation of carbon
dioxide and protons, thus inducing a progressive acidification of the extracellular environment. This
condition can lead to apoptosis in the surrounding healthy cells thus benefiting the expansion of
tumor cells which in turn develop different mechanisms to counteract acidosis and maintain a
slightly alkaline intracellular pH, favorable for the cell proliferation. Transporters and ion pumps
lead to the extrusion of lactate and protons from the cell and the entrance of bicarbonate ions,1,2,4ꢀ5
these last being produced by carbon dioxide hydration, catalyzed by the transmembrane hCAs IX
and XII. Hence, both hCA isoforms concur to maintain a) a more alkaline resting intracellular pH
and b) the acidic extracellular pH which, in turn, contributes to tumor progression via upꢀregulation
of proteases, angiogenic and cellꢀgrowth factors, and through impaired immune functions.4ꢀ8 CA IX
activity has been associated with chemoresistance which might be due to two mechanisms.9 First,
chemotherapy causes intracellular acidosis, and hCA IX prevent apoptosis by maintaining a normal
intracellular pH in response to chemotherapy. Second, hCA IXꢀinduced extracellular acidosis is
connected to a chemoresistance by a reduction in uptake of weakly basic anticancer drugs.
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Acetazolamide (AZA), a classical hCA inhibitor, was reported to reduce tumor growth and to
delay the development of the tumor, when combined with chemotherapeutic drugs. Nevertheless,
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AZA, as well as other classical aromatic sulfonamides, are nonꢀselective inhibitors, thus leading to
side effects by blocking the cytosolic isoenzymes of physiological relevance, i.e. hCA I and II.
Hence, selective targeting of the tumorꢀassociated hCA isoenzymes is a promising strategy to
obtain effective and safer agents in cancer therapy.1,2,8ꢀ10
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Up to now several classes of inhibitors directly/indirectly interacting with the Zn2+.ion from the
active hCA site have been disclosed, including sulphonamides, metalꢀcomplexing anions, phenols
and hydroxamic acids.11,12 Within the last class, benzohydroxamic acid (Chart 1) inhibited all the
hCA isoforms with potencies spanning the low to medium micromolar range.11 In particular, it
showed Ki values of 45.9 and 9.51 µM at the membraneꢀbound hCA IX and XII isoforms,
respectively, while lower potencies were found for the cytosolic highly abundant hCA I and II
(Ki= 83.1 and 179 µM). Xꢀray crystallographic studies on the adduct that benzohydroxamic acid
forms with the best characterized and easily crystallizable hCA isoform, i.e. the hCA II,
highlighted that the carbonyl and hydroxyl groups coordinate to the zinc ion to form a 5ꢀmember
chelate complex.11
In the search for new selective hCA inhibitors we envisaged the 3ꢀhydroxyꢀ1Hꢀquinazolineꢀ2,4ꢀ
dione 1 (Chart 1) as a cyclic analogue of the benzohydroxamic acid. We hypothesized that the less
flexible structure of the bicyclic scaffold 1 might have led to an increased selectivity toward the
targeted CA IX and XII isoforms. Moreover, the 3ꢀhydroxy group and either the 2ꢀ or 4ꢀcarbonyl
function were thought to be able to coordinate the Zn2+ from the enzyme active site. Anticipating
our results, compound 1, notwithstanding its simple structure, turned out to be an inhibitor of
hCAs IX and XII endowed with higher potencies (Ki= 480 nM and 104 nM, respectively) than
those of benzohydroxamic acid and high selectivity versus hCA I and hCA II isoforms (both Ki>
10000 nM). These interesting findings suggested that 3ꢀhydroxyꢀ1Hꢀquinazolineꢀ2,4ꢀdione 1
could be a versatile scaffold to develop new nonꢀsulfonamide hCA inhibitors and prompted us to
keep on studying this class of compounds. Thus, taken derivative 1 as lead, the set of 3ꢀhydroxyꢀ
1Hꢀquinazolineꢀ2,4ꢀdione derivatives 2-27 (Chart 1), bearing different substituents on the fused
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benzo ring, were synthesized and evaluated as hCA inhibitors. Moreover, the 3ꢀOꢀmodified
quinazolineꢀ2,4ꢀdione derivatives 28 and 29 were synthesized to evaluate the importance of the
free 3 hydroxy group for hCA inhibition. Some selected compounds (3, 5, 6, 12, 14, 15 and 19)
were also pharmacological evaluated to assess their antiproliferative effect on human colorectal
cancer HTꢀ29 cells.
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RESULTS AND DISCUSSION
Chemistry. The 3ꢀhydroxyquinazolineꢀ2,4ꢀdione derivatives 1-29 were prepared as outlined in
Schemes 1ꢀ5. The synthesis of the lead compound 113 and derivatives 2-8, featuring simple
substituents (Cl, NO2, NH2, CF3) on different positions of the fused benzo ring, is depicted in
Scheme 1. The synthetic pathway to prepare the 3ꢀhydroxyꢀ1Hꢀquinazolineꢀ2,4ꢀdione derivatives
4-7 was already described by us,14,15 but neither the experimental procedures nor the
characterization data of 4-6 were previously reported. Hence, they are described herein in detail.
Derivatives 1-8 were prepared starting from the suitably substituted 1,2ꢀdihydroꢀ3,1ꢀbenzoxazineꢀ
2,4ꢀdiones 30-36, commercially available (30-33) or synthesized following reported procedures
(34-36).14ꢀ16 The 7ꢀtrifluoromethylꢀ3ꢀhydroxyquinazolineꢀdione 7 was obtained as previously
described14 from 36. To prepare 1-6 and 8, derivatives 30-35 were reacted with Oꢀ
benzylhydroxylamine in refluxing ethanol to give the 2ꢀaminoꢀNꢀbenzyloxybenzamides 37-4217
which were cyclized with triphosgene to afford the 3ꢀbenzyloxyquinazolineꢀ2,4ꢀdione derivatives
43-48. Reduction of the 6ꢀnitroquinazoline derivative 45 with stannous chloride led to the
corresponding aminoꢀsubstituted compound 49. Debenzylation of 43-49 with 48% hydrobromic
acid in glacial acetic acid gave the desired compounds 1ꢀ8.13,14,15
The 3ꢀhydroxyquinazolineꢀ2,4ꢀdione derivatives 9-13, characterized by the presence of different
groups (1,2,4ꢀtriazolꢀ4ꢀyl, amido and ureido) at position 6, were synthesized starting from the 6ꢀ
aminoquinazoline derivative 49 (Scheme 2). Treatment of 49 with diformylhydrazine and
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trimethylchlorosilane in anhydrous pyridine afforded the 6ꢀ(1,2,4ꢀtriazolꢀ4ꢀyl)ꢀderivative 50.
Reaction of 49 with succinic anhydride in glacial acetic acid at 60 °C and in the presence of
sodium acetate gave, after acidification, the 6ꢀsuccinamidoꢀ compound 51. The same reaction with
phthalic anhydride, afforded the 6ꢀphthalamidoꢀ derivative 52 which was obtained through a
different workꢀup (see Experimental procedure for details). When derivative 49 was reacted with
benzylꢀ and phenylꢀisocyanate in anhydrous boiling tetrahydrofuran, the related 6ꢀureido
derivatives 53 and 54 were obtained. Derivatives 50-54 were 3ꢀOꢀdebenzylated through catalytic
hydrogenation giving rise to the desired 3ꢀhydroxy derivatives 10-13.
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The 3ꢀhydroxyquinazolineꢀ2,4ꢀdiones 14-26 were prepared as outlined in Scheme 3. These
compounds are featured by the presence of a chlorine atom or a trifluoromethyl group at position 7
combined with different group at position 6, i.e. nitro (14, 15), amino (16), heterocyclic moieties
(17-21), acylaminoꢀ (22-25) and benzylureidoꢀ (26) substituents. The 7ꢀtrifluoromethylꢀ
derivatives 15 and 21 were prepared as previously described.14 Within the 7ꢀchloroꢀsubstituted
compounds, derivative 20 was a new chemical entity while the others (14, 16-19, 22-26) were
previously reported by us.15,19 Nevertheless, for derivatives 14 and 16, as well as for their
precursor 55-57, no experimental data were provided.15 Thus, the synthesis of 14 and 16 is herein
reported in detail, together with that of 20. Briefly, the 7ꢀchloroꢀ3ꢀhydroxyquinazolineꢀ2,4ꢀdione 4
was treated with acetic anhydride to give the corresponding 3ꢀacetoxy derivative 55 whose
nitration afforded the 6ꢀnitro compound 56. This last was either transformed into the 3ꢀhydroxyꢀ6ꢀ
nitro derivative 14 or reduced to the related 6ꢀamino derivative 57. Treatment of 57 with NaOH
solution afforded the corresponding 3ꢀhydroxy derivative 16. Reaction of 57 with formaldehyde
and glyoxal, in the presence of ammonium acetate, gave the (6ꢀimidazolꢀ1ꢀyl)ꢀsubstituted
quinazoline 58 which was desacetylated to yield the desired compound 20. The quinazolineꢀ2,4ꢀ
dione derivatives 27 and 28 were obtained as depicted in Scheme 4. To prepare the 6ꢀ
phenylureidoꢀsubstituted compound 27, the starting 7ꢀchloroꢀ1,2ꢀdihydroꢀ3,1ꢀbenzoxazineꢀ2,4ꢀ
dione 33 was nitrated to yield the 7ꢀchloroꢀ6ꢀnitro derivative 5918 which was reacted with Oꢀ
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benzylhydroxylamine in refluxing ethanol to give the 2ꢀaminoꢀ4ꢀchloroꢀ6ꢀnitroꢀNꢀ
benzyloxybenzamide 60. This compound was cyclized with triphosgene to provide the related
quinazolineꢀ2,4ꢀdione 61 which was transformed into the corresponding amino derivative 62 by
using stannous chloride in refluxing ethanol. Reaction of 62 with phenylisocyanate in refluxing
anhydrous tetrahydrofuran led to the 6ꢀphenylureido derivative 63 which was debenzylated by
catalytic hydrogenation to afford compound 27. The 7ꢀchloroꢀ3ꢀmethoxyquinazolineꢀ2,4ꢀdione 28
was synthesized starting from the 7ꢀchloroꢀbenzoxazineꢀ2,4ꢀdione derivative 33 which was
reacted with Oꢀmethylhydroxylamine in refluxing ethanol to give the 2ꢀaminoꢀ4ꢀchloroꢀNꢀ
methoxybenzamide 64 whose cyclization with triphosgene afforded the desired compound 28.
Finally, derivative 29 was synthesized as outlined in Scheme 5. The 7ꢀchloroquinazoline
derivative 6519 was transformed into the corresponding 7ꢀchloroꢀ6ꢀnitro derivative 66 which was
then hydrolyzed at the ester function to give the related carboxylic acid 29.
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CA Inhibitory Activity. The quinazolineꢀ2,4ꢀdione derivatives 1-29 were tested for their efficacy
toward the targeted hCAs IX and XII and also against the physiologically relevant hCA I and II,
which are cytosolic isoforms abundantly expressed through the body, thus responsible of the side
effects of nonꢀselective hCA inhibitors. The data shown in Table 1 indicate that the 3ꢀhydroxyꢀ1Hꢀ
quinazolineꢀ2,4ꢀdione 1 is a useful scaffold to develop nonꢀclassical inhibitors of the tumorꢀ
associated hCAs. In fact, most of the tested derivatives inhibited both hCA IX and XII at low
nanomolar concentrations, while being inactive at the offꢀtarget hCAs I and II. As anticipated
above, the 3ꢀhydroxyquinazolineꢀ2,4ꢀdione 1 was prepared as cyclic analogue of the nonꢀselective
hCA inhibitor benzohydroxamic acid with the aim that the reduced flexibility of 1 might increase
the compound selectivity and that the NHCO function, inserted to cyclize the lead, might reinforce
interaction of 1 with the enzyme. Very interestingly, compound 1 showed enhanced potency and
selectivity toward the targeted hCAs IX and XII, thus prompting us to continue the investigation of
this class of compounds. Hence, small substituents with different lipophilic and electronic
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properties were singularly introduced at position 6 (Cl, NO2, NH2) and 7 (Cl, CF3) or combined at
the 6,7 (Cl) or 5,7 (Cl) positions. The obtained compounds 2-8 displayed the same selectivity of the
lead 1 versus the hCA I and II isoforms and showed, on the whole, a significant increase of activity
at both the targeted enzymes, most of compounds displaying Ki values in the low nanomolar range
(Ki= 3.4ꢀ8.2 nM). There are only two exceptions, i.e. the 6ꢀchloroꢀ and 6ꢀaminoꢀ derivatives 2 and
8, which are less active than 1 as hCA XII inhibitors.
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It has to be noted that the presence of the 7ꢀchloro substituent (compound 4) gave rise to a potent
hCA XII inhibitor (Ki= 3.4 nM), 20ꢀfold selective versus the hCA IX isoform, while introduction of
a second chlorine atom, either at the 6ꢀ or 5ꢀposition (compounds 5 and 6, respectively) made the
compound equally potent at the two targeted hCAs. Also the 6ꢀnitro group was particularly
advantageous, being derivative 3 one of the most potent dual hCAs IX and XII inhibitor, among
those herein reported.
Subsequently, the 6ꢀamino group of 8 was exploited to be transformed into the 1,2,4ꢀtriazolꢀ1ꢀyl
moiety (compound 9) or used as linker for acyl groups (compounds 10, 11) and carbamoyl residues
(derivatives 12-13). All these transformations were carried out to probe groups with different
properties and, consequently, ability to interact with the catalytic site of hCAs. The above
mentioned substituents made compounds 9-13 more active than the unsubstituted compound 1
against the hCA IX while worsened ability to inhibit the isoform XII. Within this set of compounds,
the 6ꢀbenzylureido derivative 12 displayed the highest inhibitor activity against the hCA IX isoform
(Ki= 26.7 nM). Instead, its inferior homologue, the 6ꢀphenylureido derivative 13, was about 10ꢀfold
less potent at this isoform.
When the 6ꢀnitro substituent was combined with either a chlorine atom or a trifluoromethyl residue
at position 7 (compounds 14 and 15, respectively) two new dual potent hCA IXꢀXII endowed with
similar activity were obtained. The trifluoromethyl and chloro substituents elicited a similar effect
also when they were combined with the 6ꢀ(1,2,4ꢀtriazolꢀ4ꢀyl) moiety (compare compound 17 to 21).
The 6ꢀaminoꢀ7ꢀchloro substitution turned out profitable for the hCA IX inhibition, being
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derivative 16 more active (Ki= 43.1 nM) than 7ꢀchloro derivative 4 (Ki= 69.7 nM) but especially
than the 6ꢀamino derivative 8 (Ki= 272 nM). This finding evidenced the profitable role of the 7ꢀ
chlorine atom that was confirmed when this substituent was introduced on derivatives 9-13 to give
compounds 17, 23, 25-27. These latter inhibitors, in fact, were significantly more potent than the
former (9-13) and showed Ki values in the low nanomolar range (6.9ꢀ42.8 nM) on both the
targeted hCA isoforms, the only exception being the 6ꢀphenylureido derivative 27, scarcely active
at the hCA XII.
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Compounds 18-20 were synthetized as analogues of 17 whose 6ꢀ(1,2,4ꢀtriazolꢀ4ꢀyl) group was
replaced with other fiveꢀmembered heterocyclic rings. The best group proved to be the pyrrolꢀ1ꢀyl,
in fact compound 18 showed an increased activity on the hCA IX, with respect to 17. Instead, the
imidazolꢀ1ꢀyl substituent afforded the lowest activity against the hCA IX and especially the hCA
XII (compound 20).
Comparing the data of derivatives 22-24, it can be observed the beneficial role of the acidic
carboxylic function on the 6ꢀsubstituent. In fact, the 6ꢀ(carboxybenzoylamino)ꢀsubstituted
compounds 23 and 24 are significantly more potent as hCA IXꢀXII inhibitors than the 6ꢀ
benzoylaminoꢀderivative 22. Derivative 25, whose 6ꢀ(3ꢀcarboxypropanoylamino) chain is more
flexible and less lipophilic than the 6ꢀsubstituent of compounds 23 and 24, maintained a similar
activity profile, being a potent inhibitor of both the hCA isoforms (Ki= 7 and 8.9 nM). Concerning
the effects of the 6ꢀbenzylureido (26) and 6ꢀphenylureido (27) moieties, they resemble those
discussed above for the corresponding compounds 12-13, lacking the 7ꢀchloro substituent. In fact,
the 6ꢀbenzylureido derivative 26 is a potent dual inhibitor of the targeted hCAs and it is more active
than the 6ꢀphenylureido 25. The latter, in any case, showed a good hCA IX potency (Ki= 42.8 nM)
and 17ꢀfold selectivity versus the hCA XII isoform.
The 3ꢀmethoxyꢀquinazoline derivatives 28 was synthesized to evaluate the importance of the 3ꢀ
hydroxy group in this series of hCA inhibitors as we hypothesized that this group might be involved
in the coordination of the zinc ion in the catalytic site. The complete inactivity of 28 at the hCA XII
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and the significantly reduced potency against the hCA IX, with respect to the 3ꢀhydroxy derivative
4, point out the important role of the hydroxy substituent and seems to confirm the hypothesis that
this group, partially deprotonated at physiological pH, together with the 2ꢀ or 4ꢀcarbonyl function of
the inhibitor, might coordinate the zinc ion. Actually, also the 3ꢀmethoxy group might interact with
the metal ion but with lower strength than that of the 3ꢀhydroxyl, due to both electronic and steric
factors. When the 3ꢀhydroxy group of derivative 14 was alkylated with an acetic chain (derivative
29) the ability to inhibit the targeted enzymes remained almost the same, compound 29 and the
respective 3ꢀhydroxy derivative 14 being equally active towards both the isoforms.
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Anti-proliferative assays. Some selected compounds (3, 5, 6, 12, 14, 15 and 19), chosen among
those possessing the best inhibitory profile, were tested (30 – 300 µM) to evaluated their effects on
viability of human colon cancer HTꢀ29 cells, over the time (16 – 72 h) in both normoxic and
hypoxic conditions. The efficacy of the tested compounds at the lower concentration (30 µM) was
reassumed in Figure 1. Cell viability was compared after 48 h treatment of each compound.
Derivatives 12, 14, 15 and 19 were significantly active inducing a mortality by about 50%.
Interestingly, no differences were highlighted between normoxic and hypoxic environments. Table
2 shows the detailed results obtained over time. The 72 h long incubation of cells with the tested
derivatives highlighted the activity of 12, 14, 15 and 19 at 30 µM, while 5 and 6 were effective
starting from 100 and 300 µM, respectively. Hypoxia did not modify activities. To note, a decrease
of efficacy was measured at high concentrations after long incubation. This phenomena could be
related to a reduced stability of the tested solutions rather than an intrinsic variation of efficacy.
After 16 h incubation, a significant decrease of cell viability was induced by 12 only
(Supplementary Table S1). It should be mentioned that it is difficult to explain why some of these
compounds showed such significant 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 not
improbable that, besides interference with the hCA IX/XII activity in hypoxia, they may exert their
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cytotoxic effects through other mechanisms of action, not yet identified, involving pathways in
normoxia. This behavior was in fact observed also for some sulfocoumarins, hCA inhibitors
possessing a diverse inhibition mechanism compared to the zinc binders, as they anchor to the zincꢀ
coordinated water molecule.10a,20
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CONCLUSION
This study has led to the discovery of the 3ꢀhydroxyquinazolinꢀ2,4ꢀdione as a new scaffold for the
development of potent inhibitors of the tumorꢀassociated hCAs IX and XII. Several herein reported
derivatives showed high inhibitory activity against these two hCA isoforms with Ki values in the
low nanomolar range while they were ineffective in inhibiting the cytosolic enzymes hCAs I and II.
Some compounds proved to be effective as antiproliferative agents on HTꢀ29 colon cancer cell
lines, both in normoxic and hypoxic conditions. This finding let 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.
EXPERIMENTAL PROCEDURES
Chemistry. All the commercially available reagents and solvents were used as purchased from
SigmaꢀAldrich and Alfa Aesar (Italy) without further purification. Analytical silica gel plates
(Merck F254) and silica gel 60 (Merck, 70ꢀ230 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. 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 PerkinꢀElmer Spectrum RX I spectrometer in Nujol mulls and are expressed in cmꢀ
1. NMR spectra were recorded on a Bruker Avance 400 spectrometer (400 MHz for 1H NMR). The
chemical shifts are reported in δ (ppm) and are relative to the central peak of the solvent which was
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CDCl3 or DMSOd6. The following abbreviations are used: s= singlet, d= doublet, t= triplet, q=
quartet, m= multiplet, br= broad and ar= aromatic protons.
6
7
8
9
General Procedure for the Synthesis of 3-Hydroxy-1H-quinazoline-2,4(1H,3H)-dione
derivatives 1-6. The title compounds 1-6 were prepared as previously described for 7,14 i.e. by
heating at reflux for about 3ꢀ5 h a suspension of the 3ꢀbenzyloxyꢀ5,7ꢀdione derivatives 43-48 (3.3
mmol) in 48% hydrobromic acid (6 mL) and glacial acetic acid (6 mL). The resulting solution was
cooled at room temperature and diluted with water (50 mL), the obtained solid was collected by
filtration, washed with water and recrystallized.
10
11
12
13
14
15
16
17
18
19
20
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22
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3-Hydroxy-1H-quinazoline-2,4(1H,3H)-dione (1).13 Yield 75%; mp > 300 °C (2ꢀMethoxyethanol).
1H NMR (DMSOd6) 7.20ꢀ7.24 (m, 2H, ar), 7.66 (t, 1H, J = 8.0 Hz), 7.94 (d, 1H, ar, J = 8.0 Hz).
6-Chloro-3-hydroxy-1H-quinazoline-2,4(1H,3H)-dione (2). Yield 95%; mp > 300°C (2ꢀ
Methoxyethanol). 1H NMR (DMSOd6) 7.22 (d, 1H, ar, J = 8.8 Hz), 7.71 (dd, 1H, ar, J = 2.5 and 8.8
Hz), 7.88 (d, 1H, Hꢀ5, J = 2.5 Hz), 10.68 (br s, 1H, OH), 11.68 (br s, 1H, NH). Anal. Calcd for
C8H5ClN2O3.
3-Hydroxy-6-nitro-1H-quinazoline-2,4(1H,3H)-dione (3). Yield 85%; mp 273ꢀ275 °C
1
(Acetonitrile). H NMR (DMSOd6) 7.36 (d, 1H, ar, J = 8.4 Hz), 8.45 (dd, 1H, ar, J = 2.5 and 8.8
Hz), 8.65 (d, 1H, Hꢀ5, J = 2.5 Hz), 10.87 (br s, 1H, OH), 12.17 (br s, 1H, NH). IR 1691, 1714,
3200ꢀ3500. Anal. Calcd for C8H5N3O5.
1
7-Chloro-3-hydroxy-1H-quinazoline-2,4(1H,3H)-dione (4). Yield 80%; mp >300 °C (EtOH). H
NMR (DMSOd6) 7.18 (d, 1H, ar, J = 1.8 Hz), 7.25 (dd, 1H, ar, J = 8.4 and 1.8 Hz), 7.92 (d, 1H, ar,
J= 8.4 Hz), 10.70 (br s, 1H, OH), 11.70 (br s, 1H, NH). Anal. Calcd for C8H5ClN2O3.
6,7-Dichloro-3-hydroxy-1H-quinazoline-2,4(1H,3H)-dione (5). Yield 72%; mp >300 °C (EtOH). 1H
NMR (DMSOd6) 7.34 (s, 1H, ar), 8.03 (s, 1H, ar), 10.75 (br s, 1H, OH), 11.75 (br s, 1H, NH). Anal.
Calcd for C8H4Cl2N2O3.
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5,7-Dichloro-3-hydroxy-1H-quinazoline-2,4(1H,3H)-dione (6). Yield 70%; mp °C (EtOH). H
NMR (DMSOd6) 7.15 (d, 1H, ar, J = 1.0 Hz), 7.38 (d, 1H, ar, J = 1.0 Hz), 10.63 (br s, 1H, OH),
11.75 (br s, 1H; NH). IR 1680, 1750, 3415. Anal. Calcd for C8H4Cl2N2O3.
9
10
11
12
13
14
15
16
17
18
19
20
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59
60
6-Amino-3-hydroxy-1H-quinazoline-2,4(1H,3H)-dione (8). A suspension of the 3ꢀbenzyloxyꢀ
derivative 43 (3.3 mmol) in 48% hydrobromic acid (6 mL) and glacial acetic acid (6 mL) was
heated at reflux for 6 h. The solvent was distilled off at reduced pressure and the resulting residue
was treated with water (30 mL) and neutralized with a NaHCO3 saturated solution. The obtained
solid was collected by filtration, washed with water and recrystallized. Yield 70%; mp > 300 °C
1
(DMF). H NMR (DMSOd6) 5.20 (br s, 2H, NH2), 6.93 (br s, 2H, ar), 7.10 (s, 1H, ar), 10.40 (br s,
1H, OH), 11. 08 (br s, 1H, OH). Anal. Calcd for C8H7N3O3.
General Procedure for the Synthesis of 3-Hydroxy-1H-quinazoline-2,4(1H,3H)-dione
derivatives 9-13. 10% Pd/C was added (10% w/w with respect to the 3ꢀbenzyloxy derivative) to a
solution of 50-52 (0.87 mmol) in hot EtOH (about 250 mL) or 53, 54 (0.87 mmol) in DMF (5 mL).
The mixture was hydrogenated in a Parr apparatus at 40ꢀ45 psi for 4ꢀ6 h (for 9, 10, 12, 13) and 24 h
(for 11), then the catalyst was filtered off. Evaporation of ethanol at reduced pressure or dilution of
the DMF solution with water (about 20 mL) gave a solid which was collected and recrystallized.
3-Hydroxy-6-(1,2,4-triazol-4-y1)-1H-quinazoline-2,4(1H,3H)-dione (9). Yield 55%; mp> 300 °C
1
(AcOH/DMF); H NMR (DMSOd6) 7.33 (d, 1H, ar, J = 8.8 Hz), 7.98 (dd, 1H, ar, J = 2.6 and 8.8
Hz), 8.28 (d, 1H, Hꢀ5, J = 2.6 Hz), 9.17 (s, 2H, s, triazole proton), 10.72 (br s, 1H, OH), 11.76 (br s,
1H, NH). Anal. Calcd for C10H7N5O3.
6-(3-Carboxypropanoylamino)-3-hydroxy-1H-quinazoline-2,4(1H,3H)-dione (10). Yield 87%; mp
1
247ꢀ249 °C (2ꢀMethoxyethanol/DMF). H NMR (DMSOd6) 2.55ꢀ2.58 (m, 4H, 2CH2), 7.14 (d, 1H,
ar, J = 8.8 Hz), 7.77 (dd, 1H, ar, J = 2.3 and 8.8 Hz), 8.28 (d, 1H, Hꢀ5, J = 2.3 Hz), 10.16 (s, 1H,
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NH), 10.54 (br s, 1H, OH), 11.47 (br s, 1H, NH), 12.14 (br s, 1H, OH). Anal. Calcd for
C12H11N3O6.
6
7
8
6-(2-Carboxybenzoylamino)-3-hydroxy-1H-quinazoline-2,4(1H,3H)-dione (11). Yield 72%; mp >
9
1
300 °C (EtOAc/MeOH). H NMR (DMSOd6) 7.18 (d, 1H, ar, J = 8.8 Hz), 7.56ꢀ7.68 (m, 3H, ar),
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11
12
13
14
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7.83ꢀ7.89 (m, 2H, ar), 8.42 (s, 1H, Hꢀ5), 10.57 (br s, 2H, NH + OH), 11.51 (s, 1H, NH), 13.07 (br s,
1H, COOH). Anal. Calcd for C16H11N3O6.
6-(3-Benzylureido)-3-hydroxy-1H-quinazoline-2,4(1H,3H)-dione (12). Yield 80%; mp> 300 °C (2ꢀ
Methoxyethanol). 1H NMR (DMSOd6) 4.30 (d, 2H, CH2, J = 5.8 Hz), 6.66 (t, 1H, NH, J = 5.9 Hz),
7.09 (d, 1H, ar, J = 8.8 Hz), 7.22ꢀ7.35 (m, 5H, ar), 7.60 (dd, 1H, ar, J = 2.4 and 8.8 Hz), 8.11 (d, 1H,
ar, J = 2.4 Hz), 8.80 (s, 1H, NH), 10.51 (br s, 1H, OH), 11.39 (br s, 1H, NH). Anal. Calcd for
C16H14N4O4.
6-(3-Phenylureido)-3-hydroxy-1H-quinazoline-2,4(1H,3H)-dione (13). Yield 78%; mp > 300 °C
(DMF/H2O). 1H NMR (DMSOd6) 7.01 (t, 1H, ar, J = 7.4 Hz), 7.19 (d, 1H, ar, J = 8.8 Hz), 7.32 (t,
2H, ar, J = 7.5 Hz), 7.49 (d, 2H, ar, J = 7.5 Hz), 7.67 (d, 1H, ar, J = 8.8 Hz), 8.20 (s, 1H, ar), 8.70
(br s, 1H, NH), 8.87 (br s, 1H, NH), 10.55 (br s, 1H, OH), 11.42 (br s, 1H, NH). Anal. Calcd for
C15H12N4O4.
General Procedure for the Synthesis of 3-Hydroxy-1H-quinazoline-2,4(1H,3H)-dione
derivatives 14, 16 and 20. A suspension of the 3ꢀacetoxy derivatives 56-58 (0.42 mmol) in aqueous
2.5% NaOH (3 mL) was stirred at room temperature for 1 h and then neutralized with 6N HCl. The
resulting solid was collected, washed with water and recrystallized.
7-Chloro-3-hydroxy-6-nitro-1H-quinazoline-2,4(1H,3H)-dione (14). Yield 50%; mp 287ꢀ289 °C
1
(EtOH); H NMR (DMSOd6) 7.34 (s, 1H, ar), 8.55 (s, 1H, ar), 10.9 (s, 1H, OH), 12.22 (br s, 1H,
NH). Anal. Calcd for C8H4ClN3O5.
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6-Amino-7-chloro-3-hydroxy-1H-quinazoline-2,4(1H,3H)-dione (16). Yield 55%; mp > 300 °C
4
5
6
1
(EtOAc); H NMR (DMSOd6) 5.44 (br s, 2H, NH2), 7.06 (s, 1H, ar), 7.34 (s, 1H, ar), 10.52 (br s,
7
8
1H, OH), 11.23 (br s, 1H, NH). Anal. Calcd for C8H6ClN3O3.
9
7-Chloro-3-hydroxy-6-(1-imidazolyl)-1H-quinazoline-2,4(1H,3H)-dione (20). Yield 49%; mp > 300
10
11
12
13
14
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45
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1
°C (2ꢀMethoxyethanol); H NMR (DMSOd6) 7.11 (s, 1H, imidazole proton), 7.41 (s, 1H, ar), 7.45
(s, 1H, imidazole proton), 7.91 (s, 1H, imidazole proton), 8.17 (s, 1H, ar), 10.78 (br s, 1H, OH),
11.89 (br s, 1H, NH). Anal. Calcd for C11H7ClN4O3.
7-Chloro-3-hydroxy-6-(3-phenylureido)-1H-quinazoline-2,4(1H,3H)-dione (27). The title
compound was prepared by catalytic hydrogenation of the 3ꢀbenzyloxy derivative 63 (0.87 mmol)
in DMF solution (2.5 ml), in the same condition described above to prepare compounds 9-13. The
1
crude compound was purified by crystallization. Yield 62%; mp > 300 °C (DMF/ H2O) H NMR
(DMSOd6) 7.01 (t, 1H, ar, J = 7.4 Hz), 7.27 (s, 1H, ar), 7.31 (t, 2H, ar, J = 7.4 Hz), 7.48 (d, 2H, ar, J
= 8.1 Hz), 8.4 (s, 1H, ar), 8.69 (br s, 1H, NH), 9.37 (br s, 1H, NH), 10.63 (br s, 1H, OH), 11.52 (br
s, 1H, NH). Anal. Calcd for C15H11ClN4O4.
7-Chloro-3-methoxy-1H-quinazoline-2,4(1H,3H)-dione (28). Triethylamine (8.75 mmol) was
dropwise added to a solution of derivative 64 (3.5 mmol) and triphosgene (2.45 mmol) in anhydrous
tetrahydrofuran (20 mL). The mixture was stirred at room temperature for about 15 h, then most of
the solvent evaporated at reduced pressure. The residue was treated with water (about 30ꢀ40 mL) to
give a solid which was collected, washed with water and recrystallized. Yield 70%; mp 280ꢀ282 °C
(EtOH) 1H NMR (DMSOd6) 3.88 (s, 3H, OCH3), 7.19 (s, 1H, Hꢀ8), 7.27 (d, 1H, ar, J = 8.5 Hz),
7.94 (d, 1H, ar, J = 8.5 Hz), 11.72 (br s, 1H, NH) Anal. Calcd for C9H7ClN2O3.
(7-Chloro-2,4-dioxo-2,4-dihydro-6-nitro-1H-quinazolin-3-yl)oxyacetic acid (29). A suspension
of the ethyl ester 66 (1.0 mmol) in 2.5% aqueous NaOH (20 mL) was stirred at room temperature
for 1 h. The cooled (0 °C) solution was acidified to pH 1 with 6N HCl and the resulting solid was
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collected, abundantly washed with water and recrystallized. Yield 95%; mp 263ꢀ265 °C (EtOH); 1H
NMR (DMSOd6) 4.64 (s, 2H, CH2), 7.35 (s, 1H, ar), 8.56 (s, 1H, ar), 12.25 (s, 1H, NH), 13.20 (br s,
1H, OH). IR 1680, 1730, 1770, 3300ꢀ3100. Anal. Calcd for C10H6ClN3O7.
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14
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General Procedure for the Synthesis of 2-Amino-N-(benzyloxy)-arylamide derivatives 37-42.
The title compounds were prepared as described for 2ꢀaminoꢀNꢀ(benzyloxy)arylamides 37 and 39.17
Briefly, triethylamine (4.3 mmol) was added to a suspension of Oꢀbenzylhydroxylamine
hydrochloride (4.3 mmol) in ethanol (15 mL). The proper 2,4ꢀdihydroꢀ1Hꢀ3,1ꢀbenzoxazineꢀ2,4ꢀ
dione derivative 30-35 (4.3 mmol), commercially available with the exceptions of the 6,7ꢀdichloroꢀ
3415 and 5,7ꢀdichloroꢀ 35,16 was added to the solution. The mixture was heated at reflux for about 3
h, then cooled at room temperature and diluted with water (about 50 mL) to give a solid that was
collected, washed with water and recrystallized.
2-Amino-N-(benzyloxy)-5-chlorobenzamide
(38).
Yield
35%;
mp
159ꢀ160
°C
(Cyclohexane/EtOAc). 1H NMR (DMSOd6) 4.90 (s, 2H, CH2), 6.43 (br s, 2H, NH2), 6.73 (d, 1H, ar,
J = 8.8 Hz), 7.18 (d, 1H, ar, J = 8.5 Hz), 7.33ꢀ7.46 (m, 6H, ar). 11.55 (br s, 1H, NH). Anal. Calcd
for C14H13ClN2O2.
1
2-Amino-N-(benzyloxy)-4-chlorobenzamide (40). Yield 95%; mp 140ꢀ142 °C (EtOH). H NMR
(DMSOd6) 4.88 (s, 2H, CH2), 6.47 (d, 1H, ar, J = 2.2 Hz), 6.51 (br s, 2H, NH2), 6.75 (d, 1H, ar, J =
2.2 Hz), 7.27ꢀ7.42 (m, 6H, ar), 11.46 (br s, 1H, NH). Anal. Calcd for C14H13ClN2O2.
2-Amino-N-(benzyloxy)-4,5-dichlorobenzamide
(41).
Yield
75%;
mp
158ꢀ160
°C
(Cyclohexane/EtOAc). 1H NMR (DMSOd6) 4.88 (s, 2H, CH2), 6.60 (br s, 2H, NH2), 6.93 (d, 1H, ar,
J= 2.9 Hz), 7.35ꢀ7.48 (m, 6H, ar), 11.60 (br s, 1H, NH). Anal. Calcd for C14H12Cl2N2O2.
2-Amino-N-(benzyloxy)-4,6-dichlorobenzamide
(42).
Yield
83%;
mp
150ꢀ152
°C
1
(Cyclohexane/EtOAc). H NMR (DMSOd6) 4.95 (s, 2H, CH2), 5.55 (br s, 2H, NH2), 6.66ꢀ6.68 (m,
2H, ar), 7.33ꢀ7.42 (m, 5H, ar), 11.65 (s, 1H, NH). Anal. Calcd for C14H12Cl2N2O2.
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General Procedure for the Synthesis of 3-Benzyloxy-1H-quinazoline-2,4(1H,3H)-dione
derivatives 43-48. Triethylamine (3.29 mol) was dropwise added to a solution of 2ꢀaminoꢀNꢀ
(benzyloxy)arylamides 37-42 (1.37 mmol) and triphosgene (0.53 mmol) in anhydrous
tetrahydrofuran (20 mL). The mixture was stirred at room temperature for about 1ꢀ4 h, then it was
diluted with water (about 50 mL) to give a solid which was collected, washed with water and
recrystallized.
10
11
12
13
14
15
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3-Benzyloxy-1H-quinazoline-2,4(1H,3H)-dione (43)13 (noto ma fatto per altra via). Yield 75%; mp
1
207ꢀ209 °C (MeOH). H NMR (DMSOd6) 5.10 (s, 2H, CH2), 7.21ꢀ7.26 (m, 2H, ar), 7.40ꢀ7.44 (m,
3H, ar), 7.57ꢀ7.59 (m, 2H, ar), 7.69 (t, 1H, ar, J = 8.4 Hz), 7.97 (d, 1H, ar, J = 7.7 Hz), 11.65 (br s,
1H, NH). Anal. Calcd for C15H12N2O3.
3-Benzyloxy-6-chloro-1H-quinazoline-2,4(1H,3H)-dione (44). Yield 71%; mp 254ꢀ255 °C (EtOAc).
1H NMR (DMSOd6) 5.10 (s, 2H, CH2), 7.23 (d, 1H, ar, J = 8.7 Hz), 7.56ꢀ7.58 (m, 5H, ar), 7.57 (d,
1H, ar, J = 6.7 Hz), 7.74 (dd, 1H, Hꢀ5, J = 2.4 and 8.7 Hz), 11.80 (br s, 1H, NH). Anal. Calcd for
C15H11ClN2O3.
3-Benzyloxy-6-nitro-1H-quinazoline-2,4(1H,3H)-dione (45). Yield 65%; mp 298ꢀ299 °C (CH3NO2).
1H NMR (DMSOd6) 5.13 (s, 2H, CH2), 7.36 (d, 1H, ar, J = 9.0 Hz), 7.42ꢀ7.46 (m, 5H, ar), 7.59 (d,
1H, ar, J = 7.3 Hz), 8.49 (dd, 1H, ar, J = 2.7 and 9.0 Hz), 8.65 (d, 1H, Hꢀ5, J = 2.7 Hz). Anal. Calcd
for C15H11N3O5.
3-Benzyloxy-7-chloro-1H-quinazoline-2,4(1H,3H)-dione (46). Yield 92%; mp 294ꢀ295 °C (EtOH).
1H NMR (DMSOd6) 5.10 (s, 2H, CH2), 7.22 (s, 1H, ar), 7.30 (d, 1H, ar, J = 8.5 Hz), 7.40ꢀ7.43 (m,
3H, ar), 7.56ꢀ7.58 (m, 2H, ar), 7.97 (d, 1H, ar, J = 8.5 Hz), 11.77 (br s, 1H, NH). Anal. Calcd for
C15H11ClN2O3.
3-Benzyloxy-6,7-dichloro-1H-quinazoline-2,4(1H,3H)-dione (47). Yield 85%; mp 245ꢀ246 °C
1
(EtOH) H NMR (DMSOd6) 5.09 (s, 2H, CH2), 7.39ꢀ7.42 (m, 4H, ar), 7.54ꢀ7.56 (m, 2H, ar), 7.95
(d, 1H, ar, J = 8.4 Hz), 11.75 (br s, 1H, NH). Anal. Calcd for C15H10Cl2N2O3.
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3-Benzyloxy-5,7-dichloro-1H-quinazoline-2,4(1H,3H)-dione (48). Yield 87%; mp 264ꢀ265 °C
4
5
6
1
(EtOH) H NMR (DMSOd6) 5.05 (s, 2H, CH2), 7.16 (d, 1H, ar, J= 1.1 Hz), 7.40ꢀ7.42 (m, 4H, ar),
7
8
7.55ꢀ7.57 (m, 2H, ar), 11.90 (br s, 1H, NH). Anal. Calcd for C15H10Cl2N2O3.
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6-Amino-3-benzyloxy-1H-quinazoline-2,4(1H,3H)-dione (49). A suspension of 3ꢀbenzyloxyꢀ6ꢀ
nitroꢀ1Hꢀquinazolineꢀ2,4ꢀdione 45 (2.72 mmol) and SnCl2 (16.3 mmol) in anhydrous EtOH (10 mL)
was heated at reflux for 3 h, then most of the solvent was removed under vacuum. The resulting
mixture was treated with water (15 mL) and neutralized with a NaHCO3 saturated solution. The
obtained solid was collected by filtration, then, to separate the desired compound 49 from inorganic
salts, it was treated with threeꢀfour portions of boiling EtOAc (30 mL). From the collected organic
solutions the solvent was distilled off at reduced pressure to yield a solid which was recrystallized.
1
Yield 65%; mp 231ꢀ233 °C (EtOH). H NMR (DMSOd6) 5.07 (s, 2H, CH2), 5.45 (br s, 2H, NH2),
6.94ꢀ7.00 (m, 2H, ar), 7.15 (s, 1H, Hꢀ5), 7.41ꢀ7.42 (m, 3H, ar), 7.54ꢀ7.56 (m, 2H, ar), 11.26 (br s,
1H, NH) Anal. Calcd for C15H13N3O3.
3-Benzyloxy-6-(1,2,4,triazol-4-y1)-1H-quinazoline-2,4(1H,3H)-dione (50). Diformylhydrazine
(3.72 mmol) and then, drop by drop, trimethylsilylchloride (18.68 mmol) and triethylamine (8.68
mmol) was added to suspension of the 6ꢀamino derivative 49 (1.24 mmol) in anhydrous pyridine
(10 mL). The solution was heated at reflux for 22 h, then most of the solvent was distilled off at
reduced pressure. The residue was added with water (about 20 mL) to yield a solid which was
collected and recrystallized. Yield 84%; mp > 300 °C (EtOAc/EtOH); 1H NMR (DMSOd6) 5.13 (s,
2H, CH2), 7.34 (d, 1H, ar, J = 8.8 Hz), 7.42ꢀ7.45 (m, 3H, ar), 7.58ꢀ7.59 (m, 2H, ar), 8.01 (dd, 1H,
ar, J = 2.5 and 8.8 Hz), 8.23 (d, 1H, Hꢀ5, J = 2.5 Hz), 9.19 (s, 2H, s, triazole proton), 11.87 (br s,
1H, NH). Anal. Calcd for C17H13N5O3.
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General Procedure for the Synthesis of 6-Acylamino--3-benzyloxy-1H-quinazoline-2,4-diones
51-52. A mixture of the 6ꢀamino derivative 49 (1.06 mmol), succinic anhydride (1.06 mmol) or
phthalic anhydride (3.18 mmol) and sodium acetate (1.06 mmol) in glacial acetic acid (5 mL) was
heated at 60° C for about 6 h. After cooling at room temperature, the suspension was diluted with
water (30 mL) and acidified to pH 4 with 2N HCl to give a solid which was collected, washed with
water, dried and recrystallized (compound 51). To obtain compound 52, the reaction mixture was
filtered and the isolated solid was stirred in 2.5 % aqueous NaOH (10 mL) at room temperature for
about 30 min. The suspension was acidified to pH 1 with 6N HCl and the resulting solid was
collected, washed with water and recrystallized.
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3-Benzyloxy-6-(3-carboxypropanoylamino)-1H-quinazoline-2,4(1H,3H)-dione (51). Yield 86%; mp
1
288ꢀ290 °C (EtOH). H NMR (DMSOd6) 2.55ꢀ2.57 (m, 4H, 2CH2), 5.10 (s, 2H, CH2), 7.15 (d, 1H,
ar, J = 8.8 Hz), 7.39ꢀ7.44 (m, 3H, ar), 7.57ꢀ7.59 (m, 2H, ar), 7.81 (dd, 1H, ar, J = 2.2 and 8.8 Hz),
8.28 (d, 1H, Hꢀ5, J = 2.1 Hz), 10.27 (s, 1H, NH), 11.59 (br s, 1H, NH), 12.14 (br s, 1H, OH). Anal.
Calcd for C19H17N3O6.
3-Benzyloxy-6-(2-carboxybenzoylamino)-1H-quinazoline-2,4(1H,3H)-dione (52). Yield 83%; mp >
1
300 °C (EtOH) H NMR (DMSOd6) 5.12 (s, 2H, CH2), 7.19 (d, 1H, ar, J = 8.8 Hz), 7.35ꢀ7.45 (m,
3H, ar), 7.57ꢀ7.69 (m, 4H, ar), 7.89 (d, 1H, ar, J = 6.9 Hz), 8.40 (d, 2H, ar, J = 2.2 Hz), 8.42 (d, 1H,
Hꢀ5, J= 2.2 Hz), 10.57 (br s, 1H, NH), 11.63 (br s, 1H, NH), 13.09 (br s, 1H, COOH). Anal. Calcd
for C23H17N3O6.
General Procedure for the Synthesis of 3-Benzyloxy-6-ureido-1H-quinazoline-2,4(1H,3H)-
dione derivatives 53-54. A mixture of the 6ꢀamino derivative 49 (1.59 mmol) and benzylꢀ or
phenylꢀisocyanate (2.54 mmol) in anhydrous tetrahydrofuran (10 mL) was refluxed for 3 h. The
suspension was cooled at room temperature and the solvent evaporated at reduced pressure. The
residue was treated with Et2O (20 mL) and the solid obtained was collected by filtration and
recrystallized.
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3-Benzyloxy-6-(3-benzylureido)-1H-quinazoline-2,4(1H,3H)-dione derivative (53). Yield 68%; mp
4
5
6
1
> 300 °C (2ꢀMethoxyethanol). H NMR (DMSOd6) 4.30 (d, 2H, CH2, J = 5.8 Hz), 5.09 (s, 2H,
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8
9
CH2O), 6.66 (t, 1H, NH, J = 5.8 Hz), 7.11 (d, 1H, J = 8.8 Hz), 7.24ꢀ7.44 (m, 8H, 7ar + NH), 7.56ꢀ
7.58 (m, 2H, ar), 7.65 (dd, 1H, ar, J = 2.4 and 8.8 Hz), 8.11 (d, 1H, J = 2.4 Hz), 8.80 (s, 1H, NH),
11.50 (br s, 1H, NH). Anal. Calcd for C23H20N4O4.
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3-Benzyloxy-6-(3-phenylureido)-1H-quinazoline-2,4(1H,3H)-dione derivative (54). Yield 77%; mp
> 300 °C (EtOH/DMF). 1H NMR (DMSOd6) 5.11 (s, 2H, CH2), 6.97 (t, 1H, ar, J = 7. 2 Hz), 7.17
(d, 1H, ar, J = 8.7 Hz), 7.29 (t, 2H, ar, J = 7.6 Hz), 7.42ꢀ7.48 (m, 5H, ar), 7.57ꢀ7.59 (m, 2H, ar, J =
7.4 Hz), 7.67 (d, 1H, ar, J = 8.7 Hz), 8.16 (s, 1H, ar), 8.70 (br s, 1H, NH), 8.90 (br s, 1H, NH),
11.55 (br s, 1H, NH). Anal. Calcd for C22H18N4O4.
3-Acetoxy-7-chloro-1H-quinazoline-2,4(1H,3H)-dione (55). A suspension of derivative 4 (1.4
mmol) in acetic anhydride (15 mL) was refluxed for 2 h. Evaporation of the excess of acetic
anhydride at reduced pressure gave a residue that was stirred with water (40 mL). The solid
obtained was collected, washed with water and recrystallized. Yield 85%; mp 266ꢀ268 °C (EtOH);
1H NMR (DMSOd6) 2.39 (s, 3H, CH3), 7.27 (s, 1H, Hꢀ8), 7.35 (d, 1H, ar, J = 8.5 Hz), 7.95 (d, 1H,
ar, J = 8.5 Hz), 12.07 (br s, 1H, NH). Anal. Calcd for C10H7ClN2O4.
3-Acetoxy-7-chloro-6-nitro-1H-quinazoline-2,4(1H,3H)-dione (56). Compound 55 (7.9 mmol)
was portion wise added to a cold (ꢀ10 °C) solution of 90% HNO3 (25 mL). After the addition was
completed, the solution was stirred at ꢀ10 °C for about 30 min, then it was poured onto iced water
(about 30ꢀ50 mL) and the solid obtained was collected by filtration, abundantly washed with water,
1
dried and recrystallized. Yield 85 %; mp 264ꢀ266 °C (EtOH); H NMR (DMSOd6) 2.41 (s, 3H,
CH3), 7.41 (s, 1H, ar), 8.59 (s, 1H, ar), 12.6 (br s, 1H, NH). Anal. Calcd for C10H6ClN3O6.
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3-Acetoxy-6-amino-7-chloro-1H-quinazoline-2,4(1H,3H)-dione (57). A suspension of the 6ꢀnitro
derivative 56 (3.34 mmol) and SnCl2 (20 mmol) in anhydrous EtOH (20ꢀ30 mL) was heated at
reflux for 3 h, then most of the solvent was removed under vacuum. The resulting mixture was
treated with water (15 mL) and neutralized with a NaHCO3 saturated solution. The obtained solid
was collected by filtration and then, to separate the desired compound 57 from inorganic salts, it
was treated with threeꢀfour portions of boiling EtOAc (about 30 mL). From the collected organic
solutions the solvent was distilled off at reduced pressure to yield a solid which was recrystallized.
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60
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Yield 54%; mp 246ꢀ248 °C (EtOH); H NMR (DMSOd6) 2.37 (s, 3H, CH3), 5.58 (br s, 2H, NH2),
7.12 (s, 1H, ar), 7.35 (s, 1H, ar), 11.60 (s, 1H, NH). Anal. Calcd for C10H8ClN3O4.
3-Acetoxy-7-chloro-6-(1-imidazolyl)-1H-quinazoline-2,4(1H,3H)-dione
(58).
Equimolar
amounts (2.96 mmol) of formaldehyde, glyoxal and ammoniun acetate were added to a suspension
of the 6ꢀaminoꢀderivative 57 (1.18 mmol). The mixture was stirred at 50°C for 4h, then cooled at
room temperature, diluted with water (about 20 mL) and neutralized with a NaHCO3 saturated
solution. The mixture was extracted with EtOAc (30 mL x 4) and the organic phase was washed
with water (50 mL) and anhydrified (Na2SO4). Evaporation of the solvent at reduced pressure gave
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a solid which was used as such for the next step. Yield 60%; H NMR (DMSOd6) 2.09 (s, 3H,
CH3), 7.11 (s, 1H, imidazole proton), 7.41ꢀ7.46 (m, 2H, 1 ar + 1 imidazole proton), 7.91ꢀ7.95 (m,
2H, 1 ar + 1 imidazole proton), 10.81 (br s, 1H, NH). Anal. Calcd for C13H9ClN4O4.
2-Amino-N-(benzyloxy)-4-chloro-5-nitrobenzamide (60). The title compound was prepared by
reacting 7ꢀchloroꢀ1,4ꢀdihydroꢀ6ꢀnitroꢀ2Hꢀ3,1ꢀbenzoxazineꢀ2,4ꢀdione 59 (4.3 mmol),18 prepared by
nitration of 33 (4.3 mmol), and the Oꢀbenzylhydroxylamine (4.3 mmol) in the experimental
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condition described above to prepare 37-42. Yield 85%; mp 168ꢀ170 °C (cyclohexane/EtOAc) H
NMR (DMSOd6) 4.92 (s, 2H, CH2), 6.94 (s, 1H, ar), 7.39ꢀ7.45 (m, 5H, ar), 7.63 (br s, 2H, NH2),
8.28 (s, 1H, ar), 11.93 (br s, 1H, NH). Anal. Calcd for C14H12ClN3O4.
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3-Benzyloxy-7-chloro-6-nitro-1H-quinazoline-2,4(1H,3H)-dione (61). The title compound was
obtained by cyclizing the 2ꢀaminobenzamide derivative 60 (1.37 mmol) with triphosgene (0.53
mmol) in anhydrous tetrahydrofuran, in the same experimental conditions described above to
prepare compounds 44-50. Yield 90%; mp > 300 °C (2ꢀmethoxyethanol/DMF) 1H NMR (DMSOd6)
5.11 (s, 2H, CH2), 7.39ꢀ7.44 (m, 4H, ar) 7.57ꢀ7.58 (m, 2H, ar), 8.60 (s, 1H, ar), 12.26 (br s, 1H,
NH). Anal. Calcd for C15H10ClN3O5.
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60
6-Amino-3-benzyloxy-7-chloro-1H-quinazoline-2,4(1H,3H)-dione (62). The title compound was
obtained from the corresponding 6ꢀnitro derivative 61 (2.72 mmol) and SnCl2 (16,3 mmol) in
anhydrous EtOH (10 mL) in the same experimental conditions described above to prepare
derivative 49. The crude compound was purified by crystallization. Yield 63%; mp > 300 °C (2ꢀ
1
methoxyethanol) H NMR (DMSOd6) 5.07 (s, 2H, CH2), 5.50 (br s, 2H, NH2), 7.09 (s, 1H, ar),
7.38ꢀ7.42 (m, 4H, ar), 7.55ꢀ7.57 (m, 2H, ar), 11.31 (br s, 1H, NH). Anal. Calcd for C15H12ClN3O3.
3-Benzyloxy-7-chloro-6-(3-phenylureido)-1H-quinazoline-2,4(1H,3H)-dione 63. The title
compound was prepared by reacting the 6ꢀamino derivative 62 (1.59 mmol) with phenylisocyanate
(2.54 mmol) in anhydrous tetrahydrofuran (10 mL) in the same experimental conditions described
above for the preparation of compounds 53 and 54. The crude compound was purified by
1
crystallization. Yield 80%; mp > 300 °C (2ꢀMethoxyethanol) H NMR (DMSOd6) 5.11 (s, 2H,
CH2), 7.02 (t, 1H, ar, J = 7.1 Hz), 7.16ꢀ7.32 (m, 3H, ar), 7.42ꢀ7.59 (m, 7H, ar), 8.44 ( s, 1H, Hꢀ5),
8.73 (br s, 1H, NH), 9.40 (br s, 1H, NH) 11.63 (br s, 1H, NH). Anal. Calcd for C22H17ClN4O4.
2-Amino-4-chloro-N-methoxybenzamide (64). Triethylamine (5.08 mmol) was added to a
suspension of Oꢀmethylhydroxylamine hydrochloride (5.08 mmol) in ethanol (40 mL). The
commercially available 7ꢀchloroꢀ2,4ꢀdihydroꢀ1Hꢀ3,1ꢀbenzoxazineꢀ2,4ꢀdione derivative 33 (4.3
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mmol) was added to the solution and the mixture was heated at reflux for 3 h. After cooling at room
temperature, the solvent was evaporated at reduced pressure and the residue was treated with water
(about 50 mL) to give a mixture that was extracted with EtOAc (30 mL x 3). The organic phase was
anhydrified (Na2SO4) and the solvent evaporated under vacuum to afford an oily residue which was
purified by column chromatography (eluent cyclohexane/EtOAc/MeOH, 6:4:1). Compound 64 was
obtained as an oil that was used as such for the next step. Yield 69%; 1H NMR (DMSOd6) 3.71 (s,
3H, OCH3), 6.57 (d, 1H, ar, J = 7.3 Hz), 6.59 (br s, 2H, NH2), 6.88 (s, 1H, Hꢀ3), 7.68 (d, 1H, ar, J =
7.3 Hz), 11.50 (br s, 1H, NH).
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59
60
Ethyl (7-chloro-2,4-dioxo-2,4-dihydro-6-nitro-1H-quinazolin-3-yl)oxyacetate (66). Compound
6519 (1.0 mmol) was portion wise added to cool (T= ꢀ10 °C) HNO3 (90%, 4 mL). After the addition
was completed, the mixture was stirred for about 30 min, then the solution was poured onto ice
(about 50 g). The obtained solid was collected, abundantly washed with water and recrystallized.
Yield 87%; mp 193ꢀ194 °C (EtOH). 1H NMR (DMSOd6) 1.21 (s, 3H, CH3, J = 7.0 Hz), 4.17 (q, 2H,
CH2, J = 7.0 Hz), 4.72 (s, 2H, CH2), 7.35 (s, 1H, ar), 8.56 (s, 1H, ar), 12.25 (br s, 1H, NH). Anal.
Calcd for C12H10ClN3O7.
CA inhibition. An applied photophysics stoppedꢀflow instrument has been used for assaying the
hCA catalyzed CO2 hydration activity.21 Phenol red (at a concentration of 0.2 mM) has been used as
indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.4) as buffer, and
20 mM Na2SO4 for maintaining constant the ionic strength (this anion is not inhibitory and has a
Ki>200mM against these enzymes), following the initial rates of the CAꢀcatalyzed CO2 hydration
reaction for a period of 10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the
determination of the kinetic parameters and inhibition constants. For each measurement three traces of
the initial 5–10% of the reaction have been used for determining the initial velocity (which was the
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mean of the three traces), working with 10ꢀfold decreasing inhibitor concentrations ranging between
0.1nM and 10–100 mM (depending on the inhibitor potency, but at least five points at different
inhibitor concentrations were employed for determining the inhibition constants). The uncatalyzed
rates were determined in the same manner and subtracted from the total observed rates. Stock
solutions of inhibitor (0.1 mM) were prepared in distilledꢀdeionized water and dilutions up to 0.1 nM
were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together
for 15 min at room temperature prior to assay, in order to allow for the formation of the EꢀI complex.
The inhibition constants were obtained by nonꢀlinear leastꢀsquares methods using the Cheng–Prusoff
equation, and represent the mean from three independent experiments. All human isoforms were
recombinant enzymes produced as described earlier in our laboratory.22
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Cytotoxicity Assay.
Cell culture and treatments. Human colon cancer cell lines HTꢀ29 were obtained from American
Type Culture Collection (Rockville, MD). HTꢀ29 were cultured in DMEM high glucose with 20%
FBS in 5% CO2 atmosphere at 37° C. Media contained 2 mM Lꢀglutamine, 1% essential amino acid
mix, 100 IU mlꢀ1 penicillin and 100 ꢁg mlꢀ1 streptomycin (Sigma, Milan, Italy). HTꢀ29 cells were
plated in 96ꢀwells cell culture (1.104/well) and, 24 h after, treated with the tested compounds (0ꢀ300
µM) for 16, 48 and 72 h. Low oxygen conditions were acquired in a hypoxic workstation (Concept
400 anaerobic incubator, Ruskinn Technology Ltd., Bridgend, UK). The atmosphere in the chamber
consisted of 0.1% O2 (hypoxia), 5% CO2, and residual N2. In parallel, normoxic (20% O2) dishes
were incubated in air with 5% CO2.
Cell viability assay. HTꢀ29 cell viability was evaluated by the reduction of 3ꢀ(4,5ꢀdimethylthiozolꢀ
2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT) as an index of mitochondrial compartment
functionality. Cells were plated and treated as described. Postꢀtreatments, after extensive washing, 1
mg/ml MTT was added into each well and incubated for 30 minutes at 37 °C. After washing, the
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formazan crystals were dissolved in 150 ꢁl DMSO. The absorbance was measured at 550 nm.
Experiments were performed in quadruplicate on at least three different cell batches.
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Supporting information
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ꢀCombustion analysis data of the newly synthesized compounds; effect of some selected hCA
inhibitors on HTꢀ29 cell viability after 16 h incubation.
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ꢀMolecular formula strings and the associated biochemical data (CSV)
AUTHOR INFORMATION
Corresponding author
*Tel: +39 055 4573731. eꢀmail: vittoria.colotta@unifi.it.
ORCID
Vittoria Colotta: 0000ꢀ0002ꢀ9296ꢀ6819
ACKNOWLEDGMENTS. The work was financially supported by the University of Florence.
ABBREVIATIONS USED
CA, carbonic anhydrase; AZA, acetazolamide; MTT, 3ꢀ(4,5ꢀdimethylthiozolꢀ2ꢀyl)ꢀ2,5ꢀ
diphenyltetrazolium bromide
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