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C. Congiu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1776–1779
Indeed, the key intermediates 2, 3, 5–8 were then reacted with
the tumor-associated isoforms hCA IX and XII should be inhib-
ited.12,13 The new compounds 10–22 investigated here behaved
as weak inhibitors also of hCA II; with KIs in the range of 274–
510 nM, being thus quite different from the strong hCA II inhibitor
AAZ (KI of 12 nM) but similar to the lead SA (KI of 300 nM)—
Table 1.
1,1,1-trifluoro-4-isobutoxybut-3-en-2-one leading to a series of 4-
N,N-disubstituted SAs of types 10–15 (Scheme 1) which incorpo-
rate the lipophilic trifluoromethylcrabonylethenyl fragment.21
The trifluoromethylcarbonylethenyl fragment present in com-
pounds 10–15, was not investigated, as far as we know, for its im-
pact on the CA inhibitory of sulfonamides incorporating it. We
decided to use this fragment due to the increased lipophilicity
which it can induce in some of the prepared new sulfonamides
(Scheme 1).
Some of the key intermediates, such as 5 and 6 were also re-
acted with ethoxycarbonyl isothiocyanate leading to thioureas 16
and 17 which incorporate an additional hydrophilic moiety
(Scheme 1). In order to explore a different chemical space for the
SA derivatives, we also cyclized some of the intermediates, such
as 5, 7 and 9 in the presence of potassium cyanate or potassium
isothiocyanate, obtaining the corresponding imidazol-2(3H)-ones
18, 19 and imidazol-2(3H)-thiones 20–22 (Scheme 1).21 In this
way we obtained a series of 13 novel SAs incorporating either
two different 4-N,N-moieties (compounds 10–17), or possessing
the 4-amino group of SA incorporated in a five-membered hetero-
cyclic ring (compounds 18–22). The choice of the intermediates
which were further derivatized (such as 5–7 and 9) was dictated
by their availability (and yields of their synthesis) and not by any
chemical consideration.
(iii) The tumor associated hCA IX was effectively inhibited by
sulfonamides 10–22, with KIs in the range of 9.5–39 nM (Table 1),
being in many cases more effective as inhibitors compared to acet-
azolamide (KI of 25 nM) and much more effective when compared
to the lead SA (KI of 294 nM). The SAR is rather straightforward: for
the first subseries of compounds 10–17, the presence of acyl in-
stead of aryl moieties as one of the N-4 substituents, leads to more
effective hCA IX inhibitors. Indeed, the benzyl derivatives 10 and
11 were the least effective in the subseries, whereas the benzoylat-
ed compounds 12–17 showed a better inhibition profile against
this isoform. The thioureido fragment present in 16 and 17 led to
slightly less effective inhibitors compared to the compounds incor-
porating the trifluoromethylcarbonylethenyl fragment 12–15. For
the second subseries of derivatives, incorporating the imidazolone
or imidazole-thione rings, of types 18–22, except for 20 which was
a quite potent inhibitor (KI of 13 nM), the remaining compounds
showed a compact behavior of effective hCA IX inhibitors with
inhibition constants ranging between 25 and 37 nM. Thus, the
presence of the endocyclic oxygen or sulfur seems to be of little
importance for the SAR, whereas the nature of the R moiety pres-
ent in the 4-position of the aryl functionality is the main contribu-
tor to the inhibitory power of these derivatives.
(iv) hCA XII was also powerfully inhibited by sulfonamides 10–
22 with inhibition constants ranging between 12 and 38 nM, com-
parable to those of acetazolamide and sulfanilamide (Table 1).
Again SAR was rather straightforward, as all the substitution pat-
terns present in these compounds led to effective hCA XII inhibi-
tors. The best inhibitors were 17, 18 and 21 (KIs of 12–14 nM)
which incorporated thioureido (17), imidazol-2(3H)-one (18) and
imidazol-2(3H)-thione (19) moieties. For the remaining derivatives
the SAR was rather flat (inhibition constants ranging only between
21 and 38 nM, Table 1) being thus difficult to draw detailed conclu-
sions, apart that hCA XII is effectively inhibited by most of these
derivatives.
CA inhibition data with compounds 10–22 reported here as well
as the lead SA and the standard sulfonamide inhibitor acetazola-
mide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide, AAZ) are
shown in Table 1. The following SAR could be observed for the inhi-
bition of the four physiologically significant isoforms hCA I, II, IX
and XII with these compounds:
(i) SA is a weak hCA I inhibitor, with an inhibition constant of
28 lM, whereas AAZ is a medium potency inhibitor, with a KI of
250 nM (Table 1). The new SA derivatives 10–22 reported here
showed a very compact behavior of medium potency—weak hCA
I inhibitors, with inhibition constants ranging between 302 and
593 nM. Thus, they were more effective compared to SA, but
showed weaker inhibitory properties compared to acetazolamide.
As hCA I is basically an offtarget isoform,22 its weak inhibition with
the new sulfonamides reported here may be considered a very po-
sitive finding of this study.
(ii) hCA II is the physiologically dominant isoform, being in-
volved in a variety of physiologic functions, house-keeping fac-
tions, as it is ubiquitous in the vertebrate cells.1–3 It is also a
target for obtaining antiglaucoma CAIs,1,14 but an offtarget when
(v) The new sulfonamides reported here were selective hCA IX/
XII (over hCA I/II), which is highly significant for the design of iso-
form-selective CAIs with potential clinical applications.
In conclusion, we report a series of sulfonamides incorporating
the sulfanilamide scaffold, which were prepared by reaction of the
4-amino moiety of SA with benzyl chlorides or substituted bromo-
acetophenones. The 4-mono-alkylated derivatives were then re-
acted with 1,1,1-trifluoro-4-isobutoxybut-3-en-2-one leading to a
series of 4-N,N-disubstituted SAs, or they were reacted with eth-
oxycarbonyl isothiocyanate leading to thioureas. Their cyclization
in the presence of potassium cyanate/isothiocyanate led to the cor-
responding imidazol-2(3H)-one/thiones. The new compounds
were tested as inhibitors of four CA isoforms, the cytosolic CA I
and II, and the transmembrane, tumor-associated CA IX and XII.
These sulfonamides were ineffective CA I and II inhibitors but were
nanomolar CA IX and XII inhibitors, making them of interest as
clinical candidates for antitumor/antimetastasis applications. The
disubstitution of the 4 amino group of the parent compound sulfa-
nilamide seems to be essential for the observed selectivity of the
sulfonamides reported here acting as CA IX/XII-selective inhibitors.
Table 1
Inhibition data against hCA I, II (cytosolic) and IX, XII (transmembrane, tumor-
associated isoforms) with sulfonmides 10–22 by a stopped-flow, CO2 hydrase assay19
Compound
Ki* (nM)
hCA I
302
405
397
336
433
455
498
593
572
497
521
498
411
28,000
250
hCA II
hCA IX
hCA XII
10
11
12
13
14
15
16
17
18
19
20
21
22
SA
AAZ
276
297
421
275
419
408
415
510
375
336
296
351
274
300
12
39
33
9.5
17
13
18
25
25
34
25
13
37
30
294
25
36
24
29
27
29
28
24
14
12
38
21
12
35
37
5.7
Acknowledgments
This work was funded in part by two EU FP7 projects (Metoxia
and Dynano) to C.T.S. Thanks are addressed to Drs. A. Maresca and
A. Innocenti for technical assistance.
*
Mean from three different assays, errors 10% of the reported value.