Heterocoumarins Are Selective Carbonic Anhydrase IX and XII Inhibitors with Cytotoxic Effects against Cancer Cells Lines

Article abstract of DOI:10.1021/acsmedchemlett.8b00362

We have synthesized a new series of coumarin-based compounds demonstrating high selectivity and potent effects with low nanomolar affinity against the tumor associated carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA IX and XII. A number of these compounds were evaluated ex vivo against human prostate (PC3) and breast (MDA-MB-231) cancer cell lines. Compounds 4b and 15 revealed effective cytotoxic effects after 48 h of incubation in both normoxic and hypoxic conditions with PC3 cancer cell line. However, compound 3 showed selective cytotoxic effects against MDA-MB-231 in hypoxic condition. These results may be of particular importance for the choice of future drug candidates targeting hypoxic tumors and metastases, considering the fact that a selective carbonic anhydrase CA IX inhibitor (SLC-0111) is presently in phase II clinical trials.

Full text of DOI:10.1021/acsmedchemlett.8b00362

Letter
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Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Heterocoumarins Are Selective Carbonic Anhydrase IX and XII
Inhibitors with Cytotoxic Effects against Cancer Cells Lines
Andrea Angeli,† Elena Trallori,‡ Fabrizio Carta,† Lorenzo Di Cesare Mannelli,‡ Carla Ghelardini,‡
and Claudiu T. Supuran*,†
†University of Florence, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino,
Florence, Italy
‡
̀
NEUROFARBA Department, Section of Pharmacology and Toxicology, Universita degli Studi di Firenze, Viale Pieraccini 6, 50139
Florence, Italy
S
* Supporting Information
ABSTRACT: We have synthesized a new series of coumarin-based
compounds demonstrating high selectivity and potent effects with low
nanomolar affinity against the tumor associated carbonic anhydrase (CA, EC
4.2.1.1) isoforms hCA IX and XII. A number of these compounds were
evaluated ex vivo against human prostate (PC3) and breast (MDA-MB-231)
cancer cell lines. Compounds 4b and 15 revealed effective cytotoxic effects
after 48 h of incubation in both normoxic and hypoxic conditions with PC3
cancer cell line. However, compound 3 showed selective cytotoxic effects
against MDA-MB-231 in hypoxic condition. These results may be of particular
importance for the choice of future drug candidates targeting hypoxic tumors
and metastases, considering the fact that a selective carbonic anhydrase CA IX
inhibitor (SLC-0111) is presently in phase II clinical trials.
KEYWORDS: Carbonic anhydrase inhibitors (CAIs), selenium, metalloenzymes, coumarin, tumor, tellurium
he coumarin scaffold is found in various natural products
we report a facile and convenient access to synthetic strategies
Tand bioactive compounds. For this reason, it is a for the replacement of the endocyclic oxygen atom by selenium
provides new heterocoumarins. The selenophenyl cinnamate
(2a−b) needed for the synthesis of selenocoumarins was
prepared by reduction of diselenides 1a−b with NaBH4 to the
corresponding selenolate, which was treated in situ with
cinnamoyl chloride, affording the compounds 2a−b in
excellent yield. Thus, in order to synthesize selenocoumarins
3a−b, derivatives 2a−b were treated with anhydrous AlCl3 in
chlorobenzene at 100 °C for 30 min to afford a red reaction
mixture, which, after workup, furnished compounds 3a−b in
rather good yield,14 as reported in Scheme 1.
In order to access further heterocoumarins, we evaluated the
possibility to replace the carbonyl group with an isosteric
thiocarbonylic moiety to employ an excess of Lawesson’s
reagent (2 equiv) in refluxing toluene for 4 h. Compounds 4a−
b were obtained in excellent yields as reported in Scheme 2.
We continued the possibility to replace exocyclic oxygen
atom by selenium, but this time, our effort did not give the
desired results. Thus, our study has been extended to the
nitrogen endocyclic system. Cinnamoyl amides, differently
substituted, 6a−f were obtained from the corresponding
amines 5a−f with cinnamoyl chloride in dichloromethane at
biologically important and highly privileged structure also in
the pharmaceutical field.1 Coumarin derivatives showed a large
number of biological activities, including anticoagulant,2
antibacterial,3 antiviral,4 and recently many different derivatives
have been reported as antitumor with interesting cytotoxic
activity in vitro and in vivo.5,6,23 The interesting heterocyclic
system of coumarin derivatives, with their wide range of
biological functions, has made them an excellent starting point
for further chemical derivatization to identify novel therapeutic
agents. In this particular contest, recently, several coumarin
derivatives have been shown to constitute a new class of
selective inhibitors against the human tumor-associate carbonic
anhydrase (CA, EC 4.2.1.1) isoforms hCA IX and hCA XII.7,8
These two isoforms are active extracellular enzymes involved in
metabolism of CO2, and they have been implicated in
acidification of extracellular microenvironment and, at the
same time, in protection of cancer cells from the acidosis.9 For
this reason, we developed potent and selective inhibitors from
synthetic source with the aim to identify new CAIs structurally
related to natural products as coumarins. In the last years, we
have been engaged in the development of new synthetic
methodologies involving different seleno functionalization of
privileged structures10,11 including their possible medical
applications.12,13 In continuation of our research on the
preparation of potentially useful chalcogen derivatives, herein
Received: August 10, 2018
Accepted: August 29, 2018
Published: August 29, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.8b00362
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ACS Medicinal Chemistry Letters
Letter
achieve it from cinnamic acid derivative 14, thus avoiding the
synthesis of telluro esters. The aldehyde group of compound 9
was protected by acetalization with triethyl orthoformate in
refluxing ethanolic solution. Diaryl ditelluride 11 was prepared
from aryl bromide 10 via lithiation, tellurium insertion, and
oxidation in analogy with a literature procedure.16 The
ditelluride acetal derivative 11 was then hydrolyzed with
concentrated HCl, and the resulting aldehyde 13 wa
scondensed with a Knoevenagel reaction with malonic acid
to afford the corresponding cinnamoyl acid derivative 14.
Finally, cyclization into tellurocoumarin 15 was obtained by
converting compound 14 in situ in the corresponding acyl
chloride and subsequently treated with AlCl3 in dichloromeh-
tyl methyl ether as reported previously in the literature17 and
outlined in Scheme 5.
Scheme 1. Synthesis of Selenocoumarins 3a−b
Scheme 2. Synthesis of Thioseleno Coumarins 4a−b
Scheme 5. Synthesis of Tellurocoumarin 15
room temperature in quantitative yields. Treatment of
compounds 6a−f with anhydrous AlCl3 in chlorobenzene at
100 °C for 5 h afforded the corresponding quinolin-2(1H)-one
derivatives 7a−f with similar mechanistic pathway of
compounds 3a−b. Moreover, the reaction proceeded effi-
ciently with substituents on the nitrogen (7b) or on the aryl
group (7a,c−f) as reported in Scheme 3.15
Scheme 3. Synthesis of Different Quinolin-2(1H)-one
Derivatives 7a−f
All synthesized compounds, 3a−b, 4a-b, 7a-f, 8a-b and 15,
were tested in vitro for their inhibitory properties against the
physiologically relevant hCA isoforms (I, II, IX, and XII) by
means of a stopped-flow carbon dioxide hydration assay18 after
a period of 6 h of incubation of the enzyme and inhibitor
solutions.19−21 Their activities were compared to the standard
carbonic anhydrase inhibitor (CAI) acetazolamide (AAZ)
(Table 1).
In order to access an exocyclic selenium, this time, the
isosteric replacement of oxygen was possible in a one-pot
selenation reaction with Wollins’ reagent. Compounds 7a−b
were readily converted to seleno quinolin-2(1H)-one deriva-
tives 8a−b with an excess of WR (1.5 equiv) in refluxing
toluene for 4 h affording in good yield the desiderate
compounds as outlined in Scheme 4.
As a further investigation, we tried to synthesize with s
similar mechanism of reaction also the endocyclic replacement
of oxygen atom by tellurium; however, it proved to be difficult
because telluro esters were more instable. In order to propose
an alternative way to access tellurocoumarin, we sought to
From the analysis of constants of inhibition values reported
in Table 1, we observed that all compounds, according to
previous reports,19−21 were ineffective inhibitors of two
dominant cytosolic hCA I and hCA II showing high selectivity
against tumor-associated isoforms hCA IX and hCA XII. In
terms of structure−activity relationships, it could be observed
that methyl moiety in position 6 of seleno-coumarins 3a−b
and 4a−b played a crucial role for activity. This moiety
increased near two times the potency against hCA IX and over
three times for hCA XII. Moreover, the isosteric substitution of
carbonyl with a thiocarbonyl group did not influence
significantly the activity. Methyl substituent on nitrogen of
quinolin-2(1H)-one 7b led to an increase in the activity two-
fold against hCA IX compared to compound 7a (Ki 44 nM to
82 nM). However, different substituents in position 8 (7c−e)
increase the potency for both tumor-associated isoforms. The
replacement of carbonyl group with selenocarbonyl moiety
(8a−b), this time, influenced the activity especially for
compound 8b, showing a decrease of near four times that of
compound 7b (Ki 44 nM to 172 nM). Finally, tellurocoumarin
15 showed an interesting inhibition pattern compared to
Scheme 4. Synthesis of Seleno Quinolin-2(1H)-one
Derivatives 8a−b
B
DOI: 10.1021/acsmedchemlett.8b00362
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ACS Medicinal Chemistry Letters
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Table 1. Inhibition Data of Human CA Isoforms I, II, IX,
and XII with Compounds 3a−b, 4a−b, 7a−f, 8a−b, 15, and
AAZ by a Stopped Flow CO2 Hydrase Assay18
a
Ki (nM)
compd
hCA I
hCAII
hCA IX
hCA XII
3a
3b
4a
4b
7a
7b
7c
7d
7e
7f
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
26.3
56.1
39.5
59.9
82.0
44.6
23.0
58.8
23.4
123.1
54.4
172.0
59.0
25.8
22.9
7.6
25.8
7.4
93.0
83.5
6.8
6.8
8.4
8.7
7.6
8a
8b
15
AAZ
86.4
8.2
5.7
250.0
12.1
a
Mean from three different assays, by a stopped flow technique
(errors were in the range of 5−10% of the reported values).
selenocoumarin 3a. The biggest chalcogen atom for compound
15 led to a decrease in potency near two times 3a for hCA IX
(Ki 59 nM to 26.3 nM), but for the other membrane isoform
hCA XII, the activity increased near three times that of
selenocoumarin (Ki 8.2 nM to 22.9 nM), thus showing
tellurocoumarin is more selective against hCA XII.
The high isoform selectivity and upregulated expression of
hCA IX and XII in a wide selection of hypoxic tumors make
these compounds a desirable feature for compounds designed
to target the tumor-associated enzymes. We focused our
attention on the ex vivo activity of compounds 3a, 4b, 8a, and
15, which were evaluated for their effects on cell viability
against the human prostate (PC3) and breast (MDA-MB-231)
cancer cell lines. All compounds were highly selective hCA IX
and XII inhibitors and were used at different concentrations,
being incubated for 48 h in both normoxic and hypoxic
conditions, when overexpression of high amounts of CA IX
occurs.22 In PC3 cells, seleno quinolin-2(1H)-one 8a did not
show any cytotoxic activity in normoxic and hypoxic
conditions. However, selenocoumarin 3a started to reduce
the cell viability only at higher concentration (300 μM) to 68%
in both conditions. Its efficacy increased significantly when
selenium endocyclic was replaced by tellurium; this isosteric
substitution, in normoxic condition, led to a reduced viability
of 54% already at 30 μM and increased to 19% at higher
concentrations (Figure 1). In the hypoxic condition, indeed,
compound 15 showed less effect on cytotoxicity, which
reached 71% at 30 μM and 38% at 100−300 μM. Moreover,
the thioselenocoumarin derivative 4b showed at 30 μM similar
cytotoxicity effect to compound 15 in normoxic and hypoxic
conditions (51% and 79%, respectively).
Figure 1. Effects of newly synthesized compounds 3a, 4b, 8a, and 15
on viability of the human prostatic cancer cell line PC3 following 48 h
treatment in normoxic and hypoxic (1% O2) conditions. *p < 0.05,
**p < 0.01, ***p < 0.001 versus control.
cancer cell line in hypoxic condition. Also this time,
tellurocoumarin 15 exhibited a strong cytotoxicity in normoxic
conditions, already at 30 μM (15.9%). The potency decreased
over three times in hypoxic condition, reducing the cell
viability to 22% with a concentration of 300 μM. A reduced
cell viability (61%) was observed also for compound 4b only at
30 μM. In the hypoxic condition, this compound did not show
any significant activity.
In conclusion, we report a new series of different chalcogen-
coumarins and quinolin-1(2H)-one. These compounds were
evaluated for their inhibitory properties against the two
dominant cytosolic isoforms hCA I and II and the tumor
associated isoforms hCA IX and XII. All derivatives, here
investigated, were ineffective against the off-target cytosolic
hCA I and II, whereas they showed interesting selective and
potent inhibition profiles (in the low nanomolar range) against
the tumor associated isoforms hCA IX and XII. For this reason,
different compounds were evaluated ex vivo against two cancer
cell lines PC3 and MDA-MB231. In particular, compounds 3a
and 15 revealed an interesting cytotoxic effect after 48 h of
incubation. These results may be of particular importance for
the choice of future drug candidates targeting hypoxic tumors.
Derivative 8a also in the MDA-MB231 cell line did not show
any activity in these ex vivo normoxia and hypoxia assays.
However, seleno-coumarin 3a showed only at 300 μM a good
cytotoxic activity in normoxic condition (28%). The potency
against this cancer cell line increased drastically when
compound 3a was used in hypoxic condition. Indeed, this
compound reduced cell viability already by more than 50% at
30 μM and arrived to kill over 90% at 300 μM (Figure 2),
showing, thus, an interesting selectivity against this specific
C
DOI: 10.1021/acsmedchemlett.8b00362
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ACS Medicinal Chemistry Letters
Letter
ABBREVIATIONS
■
CAs, carbonic anhydrases; AAZ, acetazolamide
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Figure 2. Effects of the newly synthesized compounds 3a, 4b, 8a, and
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.8b00362.
■
S
Synthetic procedures, characterization of compounds, in
vitro kinetic procedure, and biological assay (PDF)
AUTHOR INFORMATION
Corresponding Author
*(C.T.S.) Tel/Fax: +39-055-4573729. E-mail: claudiu.
supuran@unifi.it.
■
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ORCID
Fabrizio Carta: 0000-0002-1141-6146
Claudiu T. Supuran: 0000-0003-4262-0323
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
D
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