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Experimental Carbonic Anhydrase Inhibitors for

the Treatment of Hypoxic Tumors
This article was published in the following Dove Press journal:
Journal of Experimental Pharmacology

Claudiu T Supuran Abstract: Carbonic anhydrase (CA, EC 4.2.1.1) isoforms IX and XII are overexpressed in
many hypoxic tumors as a consequence of the hypoxia inducible factor (HIF) activation
Neurofarba Department, Pharmaceutical
and Nutraceutical Section, University of cascade, being present in limited amounts in normal tissues. These enzymes together with
Florence, Florence 50019, Italy many others are involved in the pH regulation and metabolism of hypoxic cancer cells, and
were validated as antitumor targets recently. A multitude of targeting strategies against these
enzymes have been proposed and are reviewed in this article. The small molecule inhibitors,
small molecule drug conjugates (SMDCs), antibody-drug conjugates (ADACs) or cytokine-
drug conjugates but not the monoclonal antibodies against CA IX/XII will be discussed.
Relevant synthetic chemistry efforts, coupled with a multitude of preclinical studies, demon­
strated that CA IX/XII inhibition leads to the inhibition of growth of primary tumors and
metastases and depletes cancer stem cell populations, all factors highly relevant in clinical
settings. One small molecule inhibitor, sulfonamide SLC-0111, is the most advanced candi­
date, having completed Phase I and being now in Phase Ib/II clinical trials for the treatment
of advanced hypoxic solid tumors.
Keywords: carbonic anhydrase, hypoxia, inhibitor, small molecule drug conjugates,
anticancer drug, SLC-0111

Introduction
Tumor cells have many features which make them different from normal cells,
among which are a more acidic pH outside the cell (pHe in the range of 6.5–6.8),
connected with a slightly alkaline cytosolic pH (pHi values around 7.2–7.5), as well
as lower than normal levels of O2 (hypoxia): these and other irregularities are
known as the Warburg effect,1 from the name of the German physiologist who
discovered them and who was awarded a Nobel prize in 1931, for this and similar
breakthrough discoveries related to metabolism. In 2019, another Nobel prize for
medicine was awarded to three scientists who explained the Warburg effect at the
molecular level: Kaelin, Ratcliffe and Semenza, who discovered the transcription
factors involved in tumor oxygen levels sensing, i.e., the hypoxia inducible factors
1 and 2 (HIF-1/2).2–5 HIF-1/2 are also involved in the regulation of genes impli­
cated in the metabolism/homeostasis of cancer cells, such as the glucose transpor­
ters (GLUT1-4), pH regulation (e.g., carbonic anhydrases, CAs; Na+/H+
Correspondence: Claudiu T Supuran exchangers; vacuolar ATPase, etc.), transport of anions, such as the monocarbox­
Neurofarba Department, Pharmaceutical
and Nutraceutical Section, University of ylate transporters (MCTs), the sodium bicarbonate co-transporters, as well as
Florence, Via Ugo Schiff 6, Sesto angiogenesis (vascular endothelial growth factor VEGF).6–18 These abnormalities
Fiorentino, Florence 50019, Italy
Email claudiu.supuran@unifi.it of tumor microenvironment (lower levels of oxygen, disturbed pH balance,

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upregulated glucose metabolisms, etc.) were considered as diuretics, anti-glaucoma, anti-epileptic and anti-obesity
potential opportunities to develop anti-tumor drugs, which drugs.29,33–39 More recent applications of these as well as
might specifically target tumor cells without affecting nor­ newer pharmacological agents belonging to the CAIs,
mal ones, more than one decade ago.18,19 In fact, the demonstrated their potential in the management of neuro­
Warburg effect started to be exploited for diagnostic pur­ pathic pain,40 cerebral ischemia,41 rheumatoid
42,43
poses several decades ago, in the 1980s, through the use of arthritis, or as anti-infective agents (for the treatment
18
F-fluoro-deoxyglucose (FDG) for positron emission of bacterial, fungal and protozoan infections).44–50
tomography (PET) imaging, as this glucose analog is However, the use of CAIs as antitumor agents51–57 con­
a substrate for several GLUT isoforms and has a rapid stituted the subject of very intense research over the last
uptake in cancer cells, thus making possible precise tumor two decades, with significant progress being achieved, and
imaging by means of computer tomography (CT) these derivatives will be reviewed here.
scans.20–23 However, the development of novel antitumor Two of the 15 known human (h) CA isoforms, hCA
drugs based on this approach was more difficult than IX and XII59 are predominantly found in tumor cells and
58

originally expected, although already in 2006 show a rather limited diffusion in normal cells. Both iso­
Pouysségur,18 and later Neri and Supuran,19 proposed forms are multi-domain trans-membrane proteins with an
various potential drug targets, such as the Na+/HCO3− co- extracellular CA domain, and were demonstrated to parti­
transporters, Na+/H+ exchangers, anion exchangers (e.g., cipate in the rather complex machinery of pH
chloride for bicarbonate exchangers), monocarboxylate regulation,57–63 which as mentioned above, is dysregulated
transporters (MCTs), vacuolar ATPase as well as several in cancer cells due to the activation of HIF-1/2. It should
CA isoforms. At present only therapies which target also be noted that these two enzymes are just a part of the
VEGF, such as the monoclonal antibody (Mab) bevacizu­ complex network of proteins/processes which regulate pH
mab and similar biological drugs (known as anti- and metabolism in tumor cells, most of which were men­
angiogenesis therapies), have been highly successful and tioned in the Introduction.18,19 However, proof-of-concept
constitute an important component of antitumor therapies studies from Pastorekova’s60,61 and Harris’62,63 labora­
for a variety of cancers.24–27 Other proteins whose genes tories demonstrated that hCA IX (but presumably hCA
are under the control of HIF-1/2 proved to be less “drug­ XII has a similar role) participates significantly in the
gable” than originally thought.18,19,28 There is just one extracellular acidification of tumor cells, with the conco­
exception, which is constituted by the CA isoforms over­ mitant alkalization of the cytosol.
expressed in tumors as a consequence of the HIF-1/2 Essential for the validation were the fluorescent sulfo­
activation cascade, CA IX and XII, which will be dis­ namides 1 and 2 incorporating fluorescein thioureido
cussed in this article. moieties,60,61 which unlike the classical, clinically used
sulfonamide acetazolamide 3 (Figure 1) showed
a slightly selective inhibition of the transmembrane iso­
Validation of CA IX/XII as forms hCA IX/XII over the cytosolic enzymes hCA I and
Antitumor Drug Targets II, and were also membrane-impermeant due to the pre­
The CAs (EC 4.2.1.1) are a superfamily of sence of the carboxylate moieties.61 The use of these
metalloenzymes widespread in all life kingdoms, that cat­ inhibitors demonstrated on one hand the significant effect
alyze the conversion of CO2 to bicarbonate.29–33 As the of catalytically active hCA IX in the extracellular acidifi­
hydration of CO2 generates a proton, whereas the reversed cation but also the fact that the inhibitor effectively binds
reaction, i.e., bicarbonate dehydration consumes one, these to the enzyme only in hypoxia and not in normoxia, which
enzymes are involved primarily in pH regulation in many was considered as a very promising finding. Indeed, inhi­
cells, tissues and organisms, but also in several metabolic bition of hCA IX with such compounds reverted the acid­
processes.29–33 The field of CAs29–33 and their ification of extracellular pHe in cell cultures, with an
inhibitors34–40 with pharmacological applications has almost normalization of the parameter which from 6.5
been reviewed extensively and will be not discussed in (pHe of the tumor cells) returned to a more normal phy­
detail here. It should however be mentioned that CA siological value of 7.2–7.3 (after the use of inhibitors such
inhibitors (CAIs) of the sulfonamide/sulfamate type have as 1 and 2).60,61 Furthermore, the fact that the fluorescent
been known for decades and are still in clinical use as inhibitors were observed bound only in hypoxia,

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Figure 1 Fluorescent inhibitors 1, 2 used to validate hCA IX/XII as antitumor drug targets60–63 and the pan-inhibitor acetazolamide 3.

constituted the first evidence that targeting hCA IX/XII for targeting CA IX, to which a water-solubilizing tetra­
may also have applications for imaging, not only for the peptide fragment has been attached by using click chem­
treatment of hypoxic tumors. Indeed, intense research was istry of the alkyne-azide cycloaddition type. The peptide
thereafter initiated in several laboratories for finding more fragment contained the Cys terminal amino acid residue,
effective, drug-like anti-tumor CAIs. which reacts with the SH group of mertansine, forming
a disulfide bond by which the drug conjugate has been
Antitumor CAIs Developed in obtained (derivative 7 in Figure 2). The SMDC 7 showed
Zurich effective antitumor effects in subcutaneous SK-RC-52
The first CAIs which were reported to show antitumor (renal cell carcinoma tumors) xenograft models.66
effects in vivo, were compounds 4 and 5 discovered by The same approach has been thereafter explored and
Neri’s group in 2009 (Figure 2).64 Over the years, this enriched for acetazolamide/benzenesulfonamide CAIs serving
group made significant contributions to the field, with as selective delivery vehicles for radionuclides (99mTc),67
many interesting approaches being proposed for targeting antibodies,68 dipeptide-linked renal cell carcinoma-targeting
CA IX by scientists from this laboratory.65–72 agents,68 cytokines (such as IL-2)70,72 and other small mole­
Derivatives 4 and 5 incorporated the acetazolamide cule toxins (e.g., auristatin methyl ester, cryptophycin)69,71 as
scaffold as binding moiety to the enzyme zinc ion, and drug conjugates. All these approaches were highly successful
tails73 of the fluorescein carboxamide type in 4, similar to in leading to SMDCs, antibody-drug conjugates (ADACs) or
1 and 2 reported earlier,60,61 as well as the albumin- cytokine-drug conjugates with an enhanced antitumor effect
binding moiety in 5.64 Both derivatives showed effective compared with the parent molecules from which they were
in vitro and in vivo binding to hCA IX, and 5 was also obtained. The ETH–Neri group approach is definitely one of
active in vivo, in a SK-RC-52 xenograft model of cancer.64 the most effective and innovative, in generating small mole­
Neri’s group also reported the first DNA-encoded cules or conjugates with a very interesting pharmacological
chemical libraries for obtaining tight binding CA IX profile for targeting CA IX.
inhibitors.65 This approach, apart from being highly
innovative, also afforded sub-micromolar bis- The Maastricht–Montpellier–
sulfonamide CAIs, some of which have been prepared Tampere–Florence Approach
without the DNA-tag. They showed accumulation within Significant contributions to the discovery of interesting
the hypoxic tumor tissue and effective in vivo anti-tumor CA IX/XII inhibitors were made by Winum’s group in
action.65 Subsequently, another pioneering approach has Montpellier, in collaboration with Lambin’s group
been proposed by the same group: small molecule drug in Maastricht, Parkkila’s group in Tampere and our group
conjugates (SMDCs) targeting CA IX and incorporating in Florence.74–79 Only the most relevant papers for the
toxin payloads such as the cytotoxic, DNA-binding present review will be considered here. In fact many
agents duocarmycins or the tubulin inhibitor mertansine other contributions were achieved by these researchers in
(also known as DM1, compound 6 in Figure 2).66 Such other fields connected to CAIs, which have been reviewed
SMDCs again incorporated the acetazolamide scaffold elsewhere.6,8,32–34

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4 5

6 (DM1)

7
Figure 2 Acetazolamide-based CAIs 4, 5 and 7 reported by Neri’s group and the cytotoxin 6 (DM1).64,66

A very interesting idea from Winum’s laboratory was isoform, was also investigated by means of X-ray crystal­
that of coupling the nitroazole chemotypes, present in lography, proving interesting interactions between the
many radio/chemosensitizing agents, with CAIs belonging inhibitor scaffold and the enzyme active site.75
to the sulfonamide, sulfamate or sulfamide type.74,75 Effects on hypoxia-induced extracellular acidification in
Compounds prepared by this approach (derivatives 8–11, the presence of compounds such as 10 and 11 (which are
Figure 3) incorporated 2- or 5-nitroimidazoles and a range low nanomolar hCA IX/XII inhibitors)75 was evaluated
of aromatic scaffolds on which the zinc-binding groups in vitro, in HT29 and HeLa cancer cells, whereas HT-29
(ZBGs) responsible for binding to CA were appended, tumor bearing mice were treated with the compounds alone
particularly the primary sulfonamide, sulfamate and sulfa­ or in combination with radiation.74–76 One compound, sul­
mide moieties (Figure 3). The binding of some of these famide 11, markedly enhanced sensitization towards radio-
compounds (e.g., 10 and 11) to hCA II, an off-target and chemotherapy, when administered alone or in

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Figure 3 Nitroazole-containing CAIs of types 8–11.74–79

combination with the anticancer agent doxorubicin, thus compound which underwent a thorough investigation
proving the usefulness of this approach.74,75 Furthermore, in vitro and in vivo was 15, also known as S4.80,81 This
these compounds were shown to be non-toxic in vivo in compound was a highly effective in vitro hCA IX (Ki of 7
several animal models77,78 whereas some newer congeners nM) and hCA XII (Ki of 2 nM) inhibitor, and in an ortho­
were also reported recently.79 topic MDA-MB-231 breast carcinoma tumor model in mice
showed a significant reduction of the primary tumor and
University of Manchester– metastases growth at a dose of 10 mg/kg.80 Furthermore,
University of Florence Sulfamate S4 was also active in a small cell lung cancer (SCLC) animal
model, in mice, both alone and in combination with cispla­
CAIs
tin, through synergistic, hypoxia-specific targeting.81
A series of aromatic sulfamates, highly effective as hCA IX/
XII inhibitors, was reported by Williams’ group in
Manchester in collaboration with our group.80,81 These deri­ Brisbane–Florence Glycomimetic
vatives, of types 12–15 (Figure 4), incorporate the sulfamate CAIs
ZBG (present also in derivative 10 discussed above) and The collaboration between Poulsen’s group in Brisbane,
most of them possess aromatic-ureido (12) or thioureido Australia and the Florence group led to a large number of
(13) tails. The sulfamate fluorescent derivative 14, structu­ highly effective hCA IX/XII inhibitors belonging mainly
rally similar to the early sulfonamide derivatives 1 and 2 to the sulfonamide, sulfamate and sulfamide classes.82–90
which allowed the validation of CA IX as an antitumor drug Many such compounds, among which an example of
target, was also reported in the same study. In this rather sulfonamides is presented in Figure 5, incorporate sugar
large series of congeneric ureas/thioureas 12 and 13, the moieties and they were termed as glycomimetics. The

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Figure 4 Sulfamates incorporating ureido (compounds 12, 14 and 15) and thioureido (13) moieties, with effective CA IX/XII inhibitory action and investigated for their
antitumor effects. S4 was the most extensively investigated such derivative.80

Figure 5 Examples of glycomimetic CAIs incorporating benzenesulfonamide, sugar and 1,2,3-triazole moieties, of types 16–22.82–89

presence of sugars is beneficial both for the interaction with as well as their acetylated, propionylated or butylated esters
the enzyme, as many of these CAIs are highly effective were used for generating a remarkable number of CAIs.
inhibitors,82–90 but also for their physico-chemical and phar­ Furthermore, the chemical diversity was enhanced also by
macological properties, as the presence of the sugar moieties using various position of the sugar where the other two
enhances water solubility and bioavailability. The drug design elements were appended (compare for example 16 and 22
was attentively performed, as most of these compounds in Figure 5). These compounds were prepared by so-called
incorporate three fragments: the CA inhibitory fragment, click-tailing, using the cycloaddition reaction between azides
belonging to the aromatic sulfonamide, aromatic/aliphatic and alkynes, also known as “click chemistry”.82–90 In some
sulfamate and sulfamide, linked through a triazole unit to cases, saccharin90 derivatives were also obtained with sugar
the sugar fragment, which can be with free OH or acylated clicked tails, based on the early finding from our and Klebe’s
moieties. A range of sugars (mono-, di- and tri-saccharides), laboratory91 that saccharin is an effective CA IX inhibitor and

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binds to the metal ion from the CA active site as anion,

through the secondary sulfonamide nitrogen atom. Some of
these compounds showed effective CA IX/XII inhibition
in vitro, and some of them were shown to interfere with cell
proliferation and to induce cell apoptosis in T-cell lymphomas
expressing CA XII.88
In another study, gallium-68 radiolabeled sulfonamides
targeting CA IX were reported, which were investigated in
a mouse xenograft HT29 tumor model.89 An accumulation
of radioactivity within the tumor and a low uptake in
blood, with clearing into the urine has been observed,
which makes them of interest as imaging agents. It should
be mentioned that this approach was in fact reported ear­
lier by the Vancouver group.92–95
Another research line opened by the Poulsen–Supuran
collaboration was on the natural product sulfonamide
Psammaplin C, 23 (Figure 6), which was discovered in
1991 in the marine sponge Pseudoceratina purpurea.96
The derivative comprises a bromotyrosine-oxime function­
ality as well as a functionalized aminoethyl-sulfonamide
fragment, being one of the very few natural products to Figure 6 The natural product sulfonamide Psammaplin C, 23.96
incorporate such a moiety. Psammaplin C was shown to be
a low nanomolar hCA IX inhibitor (Ki of 12.3 nM) and acting as CA IX/XII-selective in vitro CAIs, but also
a subnanomolar hCA XII inhibitor (Ki of 0.79 nM) and its showing very promising in vivo antitumor/antimetastatic
X-ray crystal structure in complex with hCA II (Ki of 88 effects.98,99
nM) was also resolved.96 In a subsequent study, it has been Benzenesulfonamides incorporating carboxamido- and
demonstrated that combining Psammaplin C (as well as secondary sulfonamide linkers were in fact known for
some of its synthetic derivatives), with temozolomide, many years,29,32–34 and, although showing potent inhibi­
a clinically used chemotherapeutic agent, reversed multi­ tory action, no relevant selectivity for isoforms of interest
drug resistance and significantly increased survival in an
was ever evidenced. Thus, it was rather surprising that the
animal model of glioblastoma, a highly aggressive brain
presence of ureido linkers, as in compounds 24 and 25
tumor.97
(Figure 7) as well as many of their congeners, led to many
highly isoform-selective CAIs.98,100 This was explained
Vancouver–Florence – The Winning when McKenna’s group reported the X-ray crystal struc­
Approach ture of several of these derivatives bound to hCA II.98,100
The collaboration between Dedhar’s and Supuran’s groups It was observed that due to the flexibility conferred by the
started in 2010, and led shortly thereafter to the identifica­ ureido linker, the tails of such sulfonamides were able to
tion of a series of ureido-substituted benzenesulfonamides bind in very different regions of the active site, towards its

Figure 7 Ureido-sulfonamides 24 and 25 and 7-glycosyl-substituted coumarins 26, 27.98–101

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entrance, which is the most variable part of the different effects are a general feature of the potent, CA IX/XII –
CA isoforms. This in fact may explain the observed selec­ selective compounds.
tivity of many ureido sulfonamides to diverse CA iso­ Subsequent studies from the Vancouver group evidenced
forms. For example 25 (later known as SLC-0111, Figure the synergistic effects of SLC-0111 in combination with
7) had a Ki of 4.5 nM for hCA XII, of 45 nM for hCA IX other anticancer drugs in various animal models, such as
and > 400 nM for all other 10 catalytically active human pancreatic cancer (combination with gemcitabine);108 com­
isoforms.98–100 Initial in vivo experiments were done with bination with immune check point inhibitors;109 combination
the nitro-derivative 24 and thereafter with SLC-0111 with temozolomide for glioblastoma treatment,110 etc.
25,98,99 as well as the coumarin CAIs 26 and 27,101 in Other research groups apart from the Vancouver one
orthotopic breast cancer models expressing CA IX (the were also active in employing SLC-0111, alone or in
4T1 cell line) or without any CA IX expression (67NR combination with other agents/procedures, observing inter­
cell line). A marked decrease of the primary tumor growth esting antitumor effects. They include the observation that
was observed only for the 4T1 line, irrespective of the CAI interference with pH regulation in hepatocellular carcino­
used (all these compounds 24–27 are low nanomolar CA mas, by using CA IX inhibitors, has a potent antitumor
IX/XII inhibitors and poorly inhibit the cytosolic off-target effect,111 which was also evident when CA IX inhibitors
isoforms CA I and II). Furthermore, mice harboring the were administered together with radiation,112 proton pump
4T1 tumors also spontaneously develop lung metastases. inhibitors (lansoprazole, omeprazole),113 cisplatin,81
After treatment with the CAIs of type 24–27 a strong antimetabolites,114 apurinic/apyrimidinic endonuclease 1
reduction in the formation of the lung metastases or even (APE1) inhibitors,115 histone deacetylase inhibitors,116
their total lack (at higher doses of inhibitor) was also etc. Another study showed the lack of endothelial toxicity
observed proving that the inhibitors are effective in inhi­ of SLC-0111,117 whereas 24 (also known as U104) was
biting the growth of the primary tumors and shown to reduce prostate cancer118 and breast cancer119
metastases.98–101 cell growth by research groups which did not participate in
Regarding the coumarin CAIs, it should be mentioned the discovery of these drugs. Indeed, SLC-0111 was
that they were discovered in 2009 by a collaboration with proved to be useful in diverse biomedical studies not
Poulsen’s group in Brisbane,102 and they represented related to carcinogenesis, when selective CAIs were
a completely new CAI chemotype with an unprecedented needed, such as the demonstration that CA IX is involved
inhibition mechanism. The coumarins act as prodrug inhi­ in the pH regulation of pulmonary microvascular endothe­
bitors, being hydrolyzed by the esterase CA activity at the lial cells,120,121 or in proving that CA VB is involved in
lactone ring, and the formed hydroxyl-cinnamic acid there­ mitochondrial biogenesis and production of lactate by
after binds at the entrance of the active site cavity, human Sertoli cells.122
obstructing its entrance.102,103 SLC-0111 entered in Phase I clinical studies as an anti­
Apart the dual effect on the primary tumors and tumor/antimetastatic agent in 2014 and the quite positive
metastases, subsequent work from Dedhar’s group104,105 report of the study was recently published.123 In 2017
with CAIs of the type mentioned above as well as a Phase Ib/II study was initiated which is still in progress,
genetic depletion of CA IX with small hairpin RNAs, for assessing the efficacy of SLC-0111 in combination with
showed a third beneficial effect of this class of com­ other therapeutic agents for the management of pancreatic
pounds: depletion of the cancer stem cells population, cancer (ClinicalTrials.gov Identifier: NCT03450018).
which is considered a clinically significant phenomenon.
In another paper from the same group106 it has also been
shown that CA IX activates a matrix metalloproteinase Various Other Approaches for
isoform (MMP-14) for initiating the invasion which is Designing CA IX/XII Inhibitors
essential for tumor cell migration from the primary The interesting results observed with SLC-0111 and its
tumor to other organs for the formation of metastases. congeners as antitumor/antimetastatic agents, fostered
Similar effects were thereafter observed with structurally a large number of research studies in the design of CAIs
related sulfonamides to SLC-0111, which possess the which used this compound as the lead molecule. For lack
sulfonamide moiety in meta to the ureido of space we will be unable to mention all of them, but
functionality,107 proving that the antitumor/antimetastatic some relevant examples will be presented.

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As shown in Figure 8, a large number of compounds The main approaches for obtaining these compounds
have been designed and often also investigated in antic­ were: (i) modification of ZBG which in SLC-0111 is of
ancer studies for efficacy, using SLC-0111 as the lead the primary sulfonamide type, but it has been changed
compound.107,124–134 to sulfamate in compound 28;80 (ii) modification of the

Figure 8 Sulfonamides 28–43 designed as anti-cancer derivatives using SLC-0111 as lead molecule.

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position of the sulfamoyl moiety, as in compounds 29 kinetics,53 native mass spectrometry measurements,147–149
and 30, which have the ZBG in meta, not in para to the and X-ray crystallography strongly support the Ki values
ureido functionality;107 (iii) modification of the ureido obtained in our laboratory using the stopped-flow kinetic
linker, as in compounds 30–36, which incorporate method for a range of different types of CAIs and demon­
thioureido, selenoureido, N cyanoguanidino, triazene, strate that this provides sufficient accuracy levels for drug
enaminone, piperazino-carboxamide and piperidine- discovery and development applications. It should also be
carboxamide functionalities;124–130 (iv) modification of noted that more than 10,000 different CAIs were synthe­
the tail, as in compounds 37–41, in which the 4-fluor­ sized and assayed in our laboratory and a number of
ophenyl moiety has been changed to various other moi­ >15,000 were assayed through collaborations with >100
eties, similar or much more different to the original academic research groups and 15 pharmaceutical compa­
compound;131–133 (v) more drastic changes of both the nies worldwide during the three decades of activity in CA
tail and the linker present in the lead, as in derivatives research. I have not mentioned in detail here, but we have
42 and 43.134 It should be mentioned that polyamino­ reported more than 80% of the new chemotypes acting as
carboxylate-polycyclic moieties of the DOTA type have CAIs and discovered at least three innovative CA inhibi­
also been introduced in the SLC-0111 scaffold (in place tion mechanisms, i.e., anchoring to zinc coordinated water,
of the fluorine atom) for obtaining compounds able to occlusion of the active site entrance and binding out of the
complexate PET emitting isotopes such as111 In- and active site.146,150 Liljas is a crystallographer who only
90
Y-, with such labeled ureidosulfonamides being useful reported the X-ray crystal structure of just one isoform
for SPECT imaging.135 For lack of space it is impossi­ (hCA II) in complex with three sulfonamides and five
ble to mention many other highly valuable studies of inorganic anions, and never worked in anticancer drug
drug design of various other classes of CAIs. design.145 We thus consider the statements from the
Essential for these studies were also the X-ray above-mentioned paper as totally erroneous and motivated
crystallography data obtained by many groups, but in by non-scientific issues.
which McKenna’s was undoubtedly the most relevant,
with a huge number of compounds belonging to a variety Conclusions
of classes crystallized in complex with hCA II, hCA IX (or CA IX/XII are activated through the HIF-1/2 cascade in
one of its mimics) as well as other isoforms.98,100,136–144 hypoxic tumors and were validated as antitumor/antime­
This led to a thorough understanding of the favorable tastatic targets in recent years. CA IX and XII play
interactions between the inhibitor scaffold and the enzyme crucial roles in regulating the extracellular pH in tumor
and paved the way towards more efficient and isoform- cells, and were shown to be abundantly expressed in
selective inhibitors. many types of advanced solid metastatic tumors present
in a variety of organs (kidneys, lung, breast, colon, liver,
prostate, melanoma, etc). As a result of careful studies
A Recent Controversy on involving chemical synthesis, discovery of new chemo­
Antitumor CAIs types and CA inhibition mechanisms, stopped-flow
Recently, in a controversial paper, Jonsson and Liljas145 kinetic measurements, X-ray crystallography, mass spec­
queried the validity of CA IX/XII as antitumor drug targets trometry and other techniques, involving the screening of
as well as the inhibition data generated in my laboratory, tens of thousands of potential inhibitors, many key lead
with a direct attack on the most advanced drug candidate compounds targeting CA IX and XII emerged, including
available to date, SLC-0111. They state that “it is doubtful the sulfonamide CA inhibitor SLC-0111, which is pre­
that SLC-0111 has the required Ki to advance to clinical sently in Phase Ib/II clinical trials. A variety of pre-
trials” and that the “commercial interests of pharmaceuti­ clinical cancer models were developed, all demonstrating
cal companies and patients may be hurt”.145 As shown in that these two enzymes are promising cancer therapeutic
this review, and elsewhere, as a reply to this attack,53,146 targets in advanced, hypoxic solid tumors, due to the fact
a multitude of studies form various laboratories all over that they possess at least three beneficial effects: they
the world, through an effort of more than 20 years, reduce the growth of the primary tumor, inhibit the
allowed the validation of the two enzymes as drug targets. formation of metastases, and deplete the number of can­
Furthermore, very diverse techniques such as stopped-flow cer stem cells. We anticipate that the use of selective CA

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IX/XII inhibitors, such as SLC-0111 or many of the 14. Turner KJ, Crew JP, Wykoff CC, et al. The hypoxia-inducible
genes VEGF and CA9 are differentially regulated in superficial vs
interesting compounds developed in other laboratories,
invasive bladder cancer. Br J Cancer. 2002;86:1276–1282.
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Disclosure 90:1429–1436.
Claudiu T Supuran reports Welichem Biotech Inc. is 17. Chia SK, Wykoff CC, Watson PH, et al. Prognostic significance
developing the drug that I have discovered, SLC-0111, of a novel hypoxia-regulated marker, carbonic anhydrase IX, in
invasive breast carcinoma. J Clin Oncol. 2001;19:3660–3668.
during the conduct of the study; in addition, Claudiu 18. Pouysségur J, Dayan F, Mazure NM. Hypoxia signalling in cancer
T Supuran has a patent WO2012/021963 issued to and approaches to enforce tumour regression. Nature. 2006;44
Wellichem. The author reports no other potential conflicts 1:437–443.
19. Neri D, Supuran CT. Interfering with pH regulation in tumours as
of interest for this work. a therapeutic strategy. Nat Rev Drug Discov. 2011;10:767–777.
20. Beaney RP. Positron emission tomography in the study of human
tumors. Semin Nucl Med. 1984;14(4):324–341.
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