PEGylated Bis-Sulfonamide Carbonic Anhydrase Inhibitors Can Efficiently Control the Growth of Several Carbonic Anhydrase IX-Expressing Carcinomas

Article abstract of DOI:10.1021/acs.jmedchem.6b00492

A series of aromatic/heterocyclic bis-sulfonamides were synthesized from three established aminosulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitor pharmacophores, coupled with either ethylene glycol oligomeric or polymeric diamines to yield bis-sulfonamides with short or long (polymeric) linkers. Testing of novel inhibitors and their precursors against a panel of membrane-bound CA isoforms, including tumor-overexpressed CA IX and XII and cytosolic isozymes, identified nanomolar-potent inhibitors against both classes and several compounds with medium isoform selectivity in a detailed structure-activity relationship study. The ability of CA inhibitors to kill tumor cells overexpressing CA IX and XII was tested under normoxic and hypoxic conditions, using 2D and 3D in vitro cellular models. The study identified a nanomolar potent PEGylated bis-sulfonamide CA inhibitor (25) able to significantly reduce the viability of colon HT-29, breast MDA-MB231, and ovarian SKOV-3 cancer cell lines, thus revealing the potential of polymer conjugates in CA inhibition and cancer treatment.

Full text of DOI:10.1021/acs.jmedchem.6b00492

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Article
PEGylated bis-sulfonamide carbonic anhydrase inhibitors can efficiently
control the growth of several carbonic anhydrase IX-expressing carcinomas
Suleyman Akocak, M Raqibul Alam, Ahmed M Shabana, Rajesh Kishore Kumar Sanku,
Daniela Vullo, Harry Thompson, Erik R Swenson, Claudiu T Supuran, and Marc A. Ilies
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00492 • Publication Date (Web): 04 May 2016
Downloaded from http://pubs.acs.org on May 5, 2016
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PEGylated bis-sulfonamide carbonic anhydrase inhibitors can efficiently control the
growth of several carbonic anhydrase IX-expressing carcinomas
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Suleyman Akocak,1,2 M. Raqibul Alam,1 Ahmed M. Shabana,1 Rajesh Kishore Kumar
Sanku,1 Daniela Vullo,3 Harry Thompson,1 Erik R. Swenson,4
Claudiu T. Supuran,3* Marc A. Ilies1*
1Department of Pharmaceutical Sciences and Moulder Center for Drug Discovery Research,
Temple University School of Pharmacy, 3307 N Broad Street, Philadelphia, PAꢀ19140
2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Adiyaman University, 02040
Adiyaman, Turkey
3NEUROFARBA Department, Pharmaceutical Sciences Section, Universita degli Studi di
Firenze, Polo Scientifico, Via Ugo Schiff no. 6, 50019 Sesto Fiorentino (Florence), Italy
4University of Washington ꢀ Medical Service, VA Puget Sound Health Care System, Seattle,
WA, USA
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Abstract
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A series of aromatic/heterocyclic bisꢀsulfonamides were synthesized from three established
aminosulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitor pharmacophores, coupled with
either ethyleneglycol oligomeric or polymeric diamines to yield bisꢀsulfonamides with short or
long (polymeric) linkers. Testing of novel inhibitors and their precursors against a panel of
membraneꢀbound CA isoforms, including tumorꢀoverexpressed CA IX and CA XII and cytosolic
isozymes, identified nanomolarꢀpotent inhibitors against both classes and several compounds
with medium isoform selectivity in a detailed structureꢀactivity relationship study. The ability of
CA inhibitors to kill tumor cells overꢀexpressing CA IX and CA XII was tested under normoxic
and hypoxic conditions, using 2D and 3D in vitro cellular models. The study identified a
nanomolar potent PEGylated bisꢀsulfonamide CA inhibitor (25) able to significantly reduce the
viability of colon HTꢀ29, breast MDAꢀMB231 and ovarian SKOVꢀ3 cancer cell lines, thus
revealing the potential of polymer conjugates in CA inhibition and cancer treatment.
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Introduction
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The dramatic shift in the control mechanisms that govern cell survival, proliferation, and
differentiation, due to genetic and especially epigenetic factors, causes a modification of the
normal cell phenotype and generation of malignant cells.1, 2 Despite major advances in oncology,
cancer remains a leading cause of mortality worldwide and its incidence is expected to increase
dramatically in the next decades. In this context, it was recognized that microtumors generated
from early malignant cells that divide rapidly become quickly hypoxic due to lack of
vascularization. An immediate consequence of hypoxia is the upregulation of glycolysis to
supply ATP in conditions of low oxygen. Large amounts of pyruvic and lactic acid are produced
in the cytoplasm of early hypoxic malignant cells, which overꢀexpress proteins that can help
maintain internal pH and homeostasis through active export of these metabolic products outside
the cells. Hypoxia triggers the expression of HIFꢀ1, which in turn triggers the expression of more
than 500 genes that are translated into H+ pumps, various transporters (glucose transporter
GLUT1, monocarboxylate transporter MCT, anion exchangers), proteins involved in
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angiogenesis and in different metabolic pathways that become overexpressed such as glycolysis,
3, 4
anabolic processes, etc.
Even in the absence of hypoxia, these same metabolic pathways in
tumor cells are activated to supply large amounts of 3ꢀcarbon substrates (e.g. lactate and
pyruvate) for high rates of protein, lipid and nucleic acid synthesis needed for rapid growth; the
wellꢀknown Warburg effect.5 Among the proteins necessary to support malignant cell
metabolism and homeostasis are the isozymes of carbonic anhydrase (CA):CA IX and CA XII. 3,
6ꢀ11
Carbonic anhydrase is a zinc metalloprotein that catalyzes the reversible hydration of
ꢀ
carbon dioxide (CO2 + H2O → HCO3 + H+). As many as of 15 CA isozymes are currently
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known in humans, with different catalytic activities, subcellular localization and tissue
distribution. Thus, one can distinguish cytosolic isozymes (CA IꢀIII, VII, XIII), mitochondrial
isozymes (CA VA, VB), membraneꢀ bound isozymes (CA IV, IX, XII, XIV and XV), one
secreted isozyme (CA VI) and several CAꢀrelated proteins devoid of catalytic activity (CA VIII,
CA X, CA XI). 3, 7 Some of these izozymes are ubiquitously spread throughout the body (e.g. CA
I, CA II), while others like CA IX, CA XII have a relatively restricted tissue distribution, being
found only in the epithelium lining the stomach and the small intestine, which are sites of normal
high rates of cell growth and turnover. However, the CA IX and CA XII membrane–bound
isozymes become highly expressed in tumors, 7, 12ꢀ14 where they act in tandem with the cytosolic
CA isozymes to maintain internal pH within normal limits despite massive H+ production
through aerobic and anaerobic glycolysis (Figure 1).15 Thus, protons produced in glycolysis
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combine with cytoplasmic HCO3 to form carbonic acid, which then is rapidly converted in the
active site of cytosolic CAs (CA I, II) to yield CO2 and H2O. Carbon dioxide diffuses through
the membrane and is rehydrated outside the malignant cell by overꢀexpressed tumor izozymes
CA IX and CA XII. Bicarbonate ion is imported into the cytoplasm via exchange with Clꢀ
through anion exchanger AE2. As a result, tumor cells can maintain their pH within normal
limits despite intense glycolysis while acidifying the external milieu (Figure 1). 15
HCO3- + H+
CO2 + H2O
pHe = 6.8
Clꢀ
CA IX/XII
glucose
HCO3 /Cl- exchanger
-
Glucose
transporter
aquaporin
CO2 + H2O
pHi = 7.2
HCO3- + H+
H+ channel
glucose
Na+
H+
glycolysis
CA II
tumor
piruvateꢀ
cell
HYPOXIA
lactateꢀ
Na+/H+ exchanger
H+-ATPase
H+
MCT
H+
H+
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Figure 1. Tumor cells upregulate glycolysis for making ATP under hypoxic conditions and for
anabolic purposes. The massive amounts of H+ produced via glycolysis are exported outside the
cell via various H+ pumps and especially via the activity of cytosolic (CA II) and membraneꢀ
bound CA isozymes (CA IX, CA XII), which are overꢀexpressed in hypoxic tumors. Selective
inhibition of CA IX and CA XII has thus the potential to kill tumor cells.
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The acid produced by the tumor impairs the killing mechanisms in host tumor defense16
and allows continuous proliferation of the tumor while killing the normal cells surrounding the
malignant tissue, thus making room for tumor growth. Even after vascularization of tumor mass,
the hypoxic phenotype and the expression of CA IX and CA XII is retained because is allows
fast growth and proliferation, resistance to temporary (local) hypoxia that accompanies this
17ꢀ19
process and continuous killing of normal tissues surrounding the tumor.
Thus, CA IX
expression has been recognized as an endogeneous marker of hypoxia and its elevated
expression was associated with poor prognosis in breast, 12, 20, 21 colorectal, 14, 22, 23, ovarian, 24, 25
bladder, 26 head and neck, 27 cervical 28, 29, brain, 30 pancreatic 31 etc.
Selective inhibition of these tumorꢀoverexpressed CA isozymes constitutes a viable
3, 7, 32, 33
strategy to fight these aggressive metastatic tumors.
However, potent CA inhibitors
available clinically such as aromatic and heterocyclic primary sulfonamides acetazolamide 1
(AAZ), methazolamide 2, ethoxzolamide 3, benzolamide 4 or dichlorphenamide 5 are not ideal
in cancer treatment due to lack of selectivity against CA IX and CA XII, as well as suboptimal
PK and biodistribution profiles – essential features for good anticancer activity. Our team has
designed, synthesized and assessed halogenated aromatic and heterocyclic sulfonamides such as
6, 7 and congeners as the first CA inhibitors (CAIs) partially selective for CA IX through
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decoration of known carbonic anhydrase pharmacophores such as aminobenzenesulfonamide and
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aminobenzolamide with halogeno moieties to induce isozyme selectivity.
Another class of
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potent CA inhibitors introduced by us were ureidoꢀ and thioureidoꢀsulfonamides
and
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subsequent halogen decoration of these scaffolds yielded powerful CA IX inhibitors such as 8
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(SLCꢀ0111), with proved efficiency against metastatic breast cancer via analysis of unique
active site and protein surface characteristics of the tumor associated isozymes.36, 37 Compound 8
was advanced in phase I clinical trials for the treatment of hypoxic tumors that overꢀexpress CA
IX and/or XII. Many other approaches to produce potent and/or isozymeꢀselective inhibitors
followed (Chart 1). 3, 6, 7, 38, 39
Despite substantial synthetic efforts, is is rather difficult to achieve a high isozyme
selectivity in vitro with classical designs incorporating aromatic/heterocyclic primary
sulfonamides and sulfamates due to sequence homology and thus structural similarities that exist
3, 6, 7, 38ꢀ40
between different CA isozymes in their active sites.
Strategies to enhance the
selectivity for membraneꢀbound izozymes in vivo involve conjugation of CAI pharmacophores
with charged groups,41ꢀ45 with sugar,46ꢀ50 or with polymeric moieties51, 52 that will make the
inhibitors membraneꢀimpermeant and therefore unable to access the intracellular CA isozymes.
For example, our team demonstrated the possibility of generating membraneꢀimpermeant
inhibitors through conjugation of primary aromatic and heterocyclic sulfonamides with
41ꢀ44
positively charged pyridinium salts.
Pyridinium sulfonamides such as 9, 10 and 11 display
41ꢀ44
excellent selectivity against membraneꢀbound CA IV
CA XII.45
and also against tumoral CA IX and
As mentioned above, an alternative technology for generating membraneꢀimpermeant
inhibitors with selectivity for these tumoral CA isozymes is to attach known CA pharmacophores
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to waterꢀsoluble polymeric backbones such as dextran, aminoethyldextran and polyethylene
glycol (PEG). 51, 52 The development of this technology, pioneered in the late 80’s and midꢀ90’s,
was limited by rather impure and polydispersed products such as 12, 13 and 14 that generated
unwanted side effects when used in vivo. Particularly interesting was the PEG conjugate F3500
14,52 which, even impure, allowed the separation of the individual contributions of cytosolic CA
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II and membraneꢀbound CA IV on HCO3 reabsorption at the level of the kidneys. Interestingly,
in vivo levels of bicarbonate excretion in rats generated with either pyridinium sulfonamides 9 or
polymer conjugate 14 coincided, which demonstrated the equivalence of the two technologies.42,
52
Chart 1. CA inhibitors used in the clinics, together with some representative CAIs with
selectivity against membraneꢀbound CA isozymes
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Additional benefits arising from polyethylene glycol conjugation (PEGylation) of drugs
include improved drug solubility, stability and the retention time of the drug in the bloodstream,
decreased drug immunogenicity, proteolysis and renal excretion, and optimal drug
pharmacokinetics. 53
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Building on these premises and taking into account that CA IX and CA XII are dimers in
vivo,8ꢀ11 we decided to synthesize bifunctional PEGylated CAIs by attaching known CAI
pharmacophores on a PEG2000 polymeric backbone (PEG2K). Our efforts were also motivated
by the study of Whitesides et al. that showed increased binding of a ditopic CAI on a synthetic
dimeric CA isozyme due to the cooperative binding effect.54 Other multiꢀpronged CAIs recently
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reported by one of us and Winum’s group showed consistently a tight binding with CA IX
and CA XII targets. The strategy was recently exploited by Neri and Scheuerman for the
development of tight binding inhibitors of CA IX using a combinatorial approach.57
Results and discussion
The design of the new inhibitors was based on the use of three established CAI
pharmacophores, namely 4ꢀaminobenzenesulfonamide 15, 5ꢀaminoꢀ1,3,4ꢀthiadiazoleꢀ2ꢀ
sulfonamide
16
and
5ꢀ(4ꢀaminobenzenesulfonamido)ꢀ1,3,4ꢀthiadiazoleꢀ2ꢀsulfonamide
(aminobenzolamide) 17 (Scheme 1). These pharmacophores were selected due to their proven
3
potency against CA isozymes , and because as shown via Xꢀray crystallography they can
provide three different orientations of the corresponding inhibitors in the active site of the
enzyme (Figure 2).58ꢀ60
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a)
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Figure 2. Ribbon diagram (a) and active site detail (b) of the CA II adducts (pdbs: 4ilx,58 3hs4,59
and 2hoc60) with CAIs containing the three main pharmacophores 4ꢀaminobenzene sulfonamide
15, 5ꢀaminoꢀ1,3,4ꢀthiadiazoleꢀ2ꢀsulfonamide 16, 5ꢀ(4ꢀaminobenzenesulfonamido)ꢀ1,3,4ꢀ
thiadiazoleꢀ2ꢀsulfonamide (aminobenzolamide) 17 used in this study (superimposed), revealing
the different orientation of the tail of the inhibitor induced by each scaffold.
We selected a succinyl linker to attach the pharmacophore to the PEG backbone due to its
simplicity, ease of insertion, biodegradability and excellent biocompatibility. Succinyl
derivatives 18ꢀ20 were generated from the amino sulfonamides 15ꢀ17 through reaction with
succinic anhydride in acetonitrile. Their carboxy group was subsequently activated with 2ꢀ
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chloroꢀ4,6ꢀdimethoxyꢀ1,3,5ꢀtriazine (CDMT)/Nꢀmethylmorpholine (NMM)
and was
condensed with diamino(oligo/poly)ethylene glycol to yield the bisꢀsulfonamides 21ꢀ26. We
have selected two linker lengths between the succinyl CAIs: a short one (n = 2) in the bisꢀ
sulfonamide CAIs 21ꢀ23 and a long one (n = 45) in the PEGylated bissulfonamide CAIs 24ꢀ26
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(Scheme 1). They were purified by flash chromatography and characterized by standard
analytical methods (see Materials and Methods section).
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Scheme 1. Synthetic strategy for the generation of bisꢀsulfonamide CA inhibitors.
The two sets of bisꢀsulfonamides were expected to interact in different ways with the
enzyme in solution. In the case of the bisꢀsulfonamides 21ꢀ23 the short backbone was expected
to interact strongly with the surface of the protein and to modulate the potency and selectivity of
the inhibitors, similarly with the case of dendritic CAIs or multimeric CAI supported on
55, 56
nanoparticles.
For polymeric compounds 24ꢀ26 we expected they would have a rather
limited contribution to the potency and selectivity of inhibitors due to the limited interaction of
the strongly hydrated PEG backbone with the surface of CA. However, the membrane
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permeability of the two sets of compounds is expected to be different: while the small MW bisꢀ
sulfonamides 21ꢀ23 are expected to permeate the membranes easily, the polymeric inhibitors 24ꢀ
26 were designed to be membraneꢀimpermeable due to their size and highly hydrated polymeric
backbone. When administered in vivo, compounds 24ꢀ26 are expected to inhibit selectively the
mebraneꢀbound CA isozymes, including the tumorꢀoverꢀexpressed CA IX and CA XII, in a
similar manner with the reported F3500 CA inhibitor.52 Mention must be made that due to the
ionization of carboxylic acid moiety, succinyl aminosulfonamides 18ꢀ20 are negatively charged
at physiologic pH and therefore expected to be partial (but not completely) membraneꢀ
impermeant, similarly to the case of benzolamide.62 Compounds 18ꢀ20 can be generated in the
body as part of the hydrolytic processing or metabolism of the bisꢀsulfonamides 21ꢀ26.
Therefore, all bisꢀsulfonamides 21ꢀ26 and their succinyl precursors 18ꢀ20 were tested for their
ability to inhibit the membraneꢀbound isozymes CA IV, CA IX, CA XII and CA XIV, and also
against cytosolic CA I and CA II (that can be reached in vivo by compounds 18ꢀ20), in a
comparative manner (Table 1).
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Data from Table 1 revealed an excellent inhibitory profile of compounds 18ꢀ26 against
all membraneꢀbound isozymes CA IV, IX, XII, XIV, with nanomolar potency of most
representatives against these targets. Their inhibitory potency matched or surpassed the potency
of acetazolamide 1, thus validating the proposed design. The most susceptible to inhibition with
compounds 18ꢀ26 were the tumor overꢀexpressed isozymes CA IX and XII, followed closely by
CA XIV and CA IV. Cytosolic isozyme CA II was less susceptible to inhibition by compounds
18ꢀ26 than CA IV, although nanomolar inhibitors for this target were identified. The proposed
CAIs displayed only moderate inhibitory power against the second cytosolic isozyme, CA I,
similarly to acetazolamide (Table 1). Sulfonamides 19, 20 and bisꢀsulfonamide 22 were
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identified as potent panꢀinhibitors with nanomolar potency against all isozymes tested except CA
I. We also identified selective inhibitors for membraneꢀbound isozymes versus cytosolic ones,
with either medium selectivity (compounds 21 and 25) or high selectivity – sulfonamide 24.
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Table 1: Inhibition data of human CA isoforms hCA I, II, IV, IX, XII and XIV with derivatives
1-9 reported here and the standard sulfonamide inhibitor acetazolamide 1 by a stopped flow CO2
hydrase assay.17
KI (nM)*
Comp hCA I
hCA II
hCA IV
hCA IX
hCA XII
hCA XIV
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21
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23
24
25
26
1
249 ± 21
245 ± 20
181 ± 13
221 ± 16
180 ± 12
192 ± 9
18.2 ± 1.1
8.1 ± 0.7
9.3 ± 0.8
37.1 ± 0.2
8.7 ± 0.5
8.6 ± 0.4
1764 ± 45
66.6 ± 3.1
12.9 ± 1.0
12 ± 0.8
486 ± 42
6.7 ± 0.3
5.5 ± 0.2
7.8 ± 0.5
5.0 ± 0.3
6.4 ± 0.15
9.5 ± 0.4
5.8 ± 0.5
4.4 ± 0.3
74 ± 5
12.8 ± 0.9
2.7 ± 0.09
3.0 ± 0.3
2.9 ± 0.1
9.6 ± 0.1
24.1 ± 0.2
4.8 ± 0.1
2.5 ± 0.2
1.7 ± 0.1
25 ± 1.7
0.52 ± 0.02 6.1 ± 0.3
3.2 ± 0.1
3.9 ± 0.2
2.6 ± 0.2
5.9 ± 0.4
2.8 ± 0.1
3.2 ± 0.2
5.4 ± 0.3
3.7 ± 0.1
5.7 ± 0.3
4.0 ± 0.2
4.4 ± 0.4
6.2 ± 0.5
4.6 ± 0.3
4.6 ± 0.4
7.3 ± 0.5
6.8 ± 0.15
4.4 ± 0.3
41 ± 2.9
272 ± 25
225 ± 21
181 ± 9
250 ± 12
* Mean ± from standard error, from 3 different assays, by a stopped flow technique. Monomeric
(recombinant) human enzymes used in all cases.
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Thus, the most selective inhibitors could be generated from the benzenesulfonamide
scaffold, while benzolamides generated potent panꢀinhibitors. The inhibition profile of the short
bisꢀsulfonamides 21ꢀ23 was similar to the one displayed by their precursors 18ꢀ20, revealing a
rather small contribution of the secondary sulfonamide moiety to the inhibitory potency. The
only exception was noted in the case of compounds 18 and 21, where a significant increase in
potency was observed against CA IV while switching from the succinyl precursor to the bisꢀ
sulfonamide. It must be emphasized that 5ꢀsuccinylamidoꢀ1,3,4ꢀthiadiazoleꢀ2ꢀsulfonamide 19
was more potent than acetazolamide 1 against all CA targets, with one order of magnitude more
potent against CA IV, IX and XIV, revealing once again the importance of “tail approach” in
CAI designs.3
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Interestingly, the long PEG linker increased the selectivity of the bisꢀsulfonamides 24ꢀ26
for the membraneꢀbound isozymes versus the cytosolic ones (especially against CA II), as
compared with their oligoethylene glycol congeners 21ꢀ23. Bisꢀsulfonamide 24 displayed the
highest selectivity between membraneꢀbound isozymes and their cytosolic counterparts (Table
1). Considering the membraneꢀimpermeant nature of the PEGylated bisꢀsulfonamides 24ꢀ26, it is
expected that this selectivity will increase in vivo. Moreover, since tumorꢀoverexpressed CA IX
and CA XII isozymes are dimeric, it should be expected that the bisꢀsulfonamides 24ꢀ26 will
have a more pronounced effect on these targets in vivo, due to cooperative binding and bivalent
association of the twoꢀpronged inhibitor with the two neighboring active sites, as shown by
Whitesides.54
Therefore, we decided to test all CAIs 18ꢀ26 in 2D and 3D (tumor spheroids) models of
cancer that express these CA isozymes. From the many cancers that express CA IX and CA XII,
12, 14, 20ꢀ31, 63
we have selected three cellular models, namely the colon carcinoma HTꢀ29, the
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breast cancer MDAꢀMb231, and the ovarian cancer SKOV3 cell models.
For 2D testing,
cells were grown subconfluent in 96 well plates in normal conditions (37 C, 5% CO2 in air),
with half of the plates from each cell line incubated in normal (normoxic) conditions and the
other half subjected to a hypoxic conditions (1% O2, 5% CO2 and 94% N2) to induce the
expression of CA isozymes. After 24 h treatment with CAIs solutions in media at 3 different
concentrations (1 mM, 100 µM, and 10 µM) in either normoxic/hypoxic conditions, viability of
cells was measured using an MTT assay 67 (Figure 2).
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Figure 32. Effect of CAI 18ꢀ26 and acetazolamide 1, at different concentrations, on the viability
of colon HTꢀ29, breast MDAꢀMB231 and ovarian SKOVꢀ3 cancer cell lines under normoxic (a)
and hypoxic (b) conditions. P values were determined by oneꢀway ANOVA, comparing the
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value with the acetazolamide 1 (*P < 0.05, **P < 0.01, ***P < 0.001). Only the statistical
significant differences were shown.
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Data from Figure 3 reveal that all inhibitors impacted tumor cell viability, with a greater
impact elicited in hypoxic conditions where CA IX and CA XII are overexpressed. All three cell
lines were affected by the CAI treatment in a concentrationꢀdependent manner, as expected, with
colon HTꢀ29 and breast MDAꢀMB231 cancer cells slightly more sensitive to CAI treatment than
ovarian cancer cell line SKOVꢀ3. Significant decreases in tumor cell viability were obtained with
inhibitors 18, 21, 22 and 25, with the rest of inhibitors being generally less efficient even at high
concentrations. Some exceptions occurred, such as inhibitor 23 that proved efficient only on
MDAꢀMB231 cell line. These data are in contrast with the rather uniform inhibition profile of
the series against CA IX and XII (Table 1) and demonstrate that besides inhibition profile
physicochemical properties of the inhibitor (lipophilicity, solubility, etc) together with the
cellular penetrability and other factors are essential for achieving efficient tumor cell killing. The
interplay of these factors generated CAIs with efficient in vitro cell killing from all three
scaffolds selected. The most efficient CAI against all three cell lines in both normoxia and
hypoxia proved to polymeric bisꢀsulfonamide 25, bearing the classical 1,3,4ꢀthiadiazoleꢀ2ꢀ
sulfonamide “warhead”. It displayed robust cell killing, with cell viabilities as low as 30ꢀ40% at
1 mM, 50% at 100 µM and 60ꢀ70% at 10 µM, being influenced by the cell type and presence of
hypoxia.
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The aboveꢀmentioned data revealed the impact of in vitro models of tumor proliferation
for the evaluation of potency of CAI as a preꢀrequisite for in vivo testing. Along these lines, we
decided to quantify the CAI effects in 3D cell cultures, which take into account the tissue
penetrability of the inhibitor and are thus closer to the environment encountered in vivo. Thus,
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tumor spheroids were grown as reported before,68 using the same HTꢀ29, MDAꢀMB231 and
SKOVꢀ3 cell lines, maintained in normoxic conditions. Tumor spheroids become naturally
hypoxic in about 7 days due to their 3D growth that isolates spheroid core cells from oxygen
perfusion. We quantified the amount of CA IX generated in all growth conditions, 2D and 3D, as
a function of cell type, in order to correlate the viability of the cells with the expression of the
CA IX target. For this purpose, we relied on western blotting and immunofluorescence analysis
of CA IX presence via the M75 antibody that was shown to be specific to the unique
proteoglycan part of this isozyme (Figure 4).19
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Figure 4. Immunofluorescence analysis of CA IX distribution in 2D cell culture under normoxic
(N) and hypoxic (H) conditions, and in 3D cell culture (tumor spheroids, Spher) for colon HTꢀ
29, breast MDAꢀMB231 and ovarian (SKOVꢀ3) cancer cell lines. CA IX appears as a twoꢀband
protein at 54 and 58 KDa 19 and can be quantified relative to βꢀactin ubiquitously present in all
cells (45 KDa).
Data from Figure 4 revealed that CA IX is present in all three cell lines under normoxic
conditions, but in different amounts: MDAꢀMB231 expressed the most CA IX, followed by HTꢀ
29 and SKOVꢀ3. These CA IX expression levels under normoxic conditions correlate well with
the order of susceptibility of the three cell lines to the CAI 18ꢀ26 in similar conditions (see for
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example CAI 18 at 1 mM, Figure 3a). As expected, hypoxia increases the expression level of CA
IX in these cell lines, but to a different extent, with a significant elevation of target protein level
in HTꢀ29 and SKOVꢀ3 and only a modest increase in MDAꢀMB231. The more uniform CA IX
expression level under hypoxia in all three cell lines is reflected in the rather uniform response of
these cell lines to CAIs 18ꢀ26 (Figure 3b). The same significant induction of CA IX expression
can be found in tumor spheroids derived from HTꢀ29 and SKOVꢀ3 cell lines, while the MDAꢀ
MB231 had a lower expression level, which we attribute to a slower growth of the spheroids in
culture (data not shown) which makes their core less hypoxic.
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After quantifying the relative amount of CA IX in tumor spheroids, we assessed their
susceptibility to treatment with CAIs 18ꢀ26 at 1 mM and 100 µM concentration, for 24 h
incubation time. Viability of the spheroids was assessed via a WSTꢀ8 assay 69 (Figure 5).
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Figure 54. Effect of CAI 18ꢀ26 and acetazolamide 1, at different concentrations, on the viability
of colon HTꢀ29, breast MDAꢀMB231 and ovarian SKOVꢀ3 3D cell cultures (tumor spheroids). P
values were determined by oneꢀway ANOVA, comparing the value with the acetazolamide 1 (*P
< 0.05, **P < 0.01, ***P < 0.001). Only the statistical significant differences were shown.
Inhibition data depicted in Figure 5 correlated well with the expression level of CA IX in
tumor spheroids (Figure 4). Thus, the most efficient cell killing by CAIs was observed in tumor
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spheroids derived from HTꢀ29 and SKOVꢀ3 cell lines, with MDAꢀMB231 cell line being less
affected. Polymeric bisꢀsulfonamide 25 was the most efficient CAI in this assay too, decreasing
the viability to about 50%/80% in SKOVꢀ3, 60%/70% in HTꢀ29 and 80%/90% in MDAꢀMB231
at 1 mM/100 µM respectively. Other efficient CAIs in this in vitro 3D cell model were 19, 20, 21
and 23, but with a less consistent, cellꢀdependent inhibition profile.
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Conclusions
A detailed structureꢀactivity relationship study was carried out within a series of new bisꢀ
sulfonamides 21ꢀ26 and their succinylamido sulfonamide precursors 18ꢀ20 derived from three
different established carbonic anhydrase inhibitor “warheads”. These CAIs were profiled against
a set of membraneꢀbound and cytosolic CA isoforms, including the membraneꢀoverexpressed
CA IX and XII. We have identified potent panꢀinhibitors, together with CAIs with selectivity for
membraneꢀbound isozymes. This CAI set was also assessed in vitro, in 2D and 3D tumor cellular
models expressing CA IX and CA XII and we have successfully identified several potent CAIs
that can significantly kill the tumor cells under both normoxic and hypoxic conditions. The
sensitivity of different cancer cells to CA inhibitors was correlated with the amount of CA IX
expressed in each cell line. The most efficient CAI proved to be polymeric bisꢀsulfonamide 25,
which showed nanomolar potency against purified CA IX and CA XII isozymes and consistent
and significant cancer cell killing at concentrations of 10ꢀ100 uM across different tumors
expressing these CA isozymes. As sulfonamide CAI 8 targeting isoforms IX/XII is currently in
Phase I clinical trials as an antitumor/antimetastatic agent,1 the findings presented in this work
may help the identification of other similar candidates, possibly with improved properties.
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Experimental Section
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Materials: The following materials were used as received: succinic anhydride (TCI America,
Portland, OR), 2,2`ꢀ(ethylenedioxy)ꢀdiethylamine (Fluka, St Louis, MO), Nꢀmethylmorpholine
(TCI America, Portland, OR), 2ꢀchloroꢀ4,6ꢀdimethoxyꢀ1,3,5ꢀtriazine (Acros Organics, New
Jersey, NJ), sulfanilamide (Acros Organics, New Jersey, NJ), H2NꢀPEG2000ꢀNH2 (Laysan Bio,
Arab, AL). Other solvents (HPLC quality) were purchased from Fisher Scientific (Pittsburgh,
PA), EMD (Gibbstown, NJ), and VWR International (West Chester, PA). Human colon
adenocarcinoma cell line (HT 29), human ovary cancer cell line (SKOVꢀ3ꢀLuc), human breast
cancer cell line (MDAꢀMBꢀ231ꢀLuc) were purchased from ATCC (Manassas, VA), Dulbecco's
Modified Eagle's medium (DMEM), RPMIꢀ1640, McCoy's 5A media were from Mediatechꢀ
Corning (Manassas, VA), Fetal bovine serum (FBS) was from Fisher Scientific (Pittsburgh, PA),
3ꢀ(4,5ꢀDimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide (MTT), phosphateꢀbuffered saline
(PBS), dimethyl sulfoxide (DMSO) and waterꢀsoluble tetrazolium saltꢀ8 (WSTꢀ8) were
purchased from VWR International (West Chester, PA).
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Techniques: The purity and the structure identity of the intermediary and the final
products were assessed by a combination of techniques that included thinꢀlayer
1
chromatography (TLC), HPLCꢀMS, HꢀNMR, COSY, 13CꢀNMR, high resolution mass
spectrometry (HRꢀMS), gel permeation chromatography (GPC) coupled with MALDIꢀTOF.
TLC was carried out on SiO2ꢀprecoated aluminum plates (silica gel with F254 indicator;
layer thickness 200 µm; pore size 60 Å, from SigmaꢀAldrich.
Melting points were determined using a Thermolyne heating stage microscope (Dubuque,
o
IA), equipped with an Olympus 5X objective, at heating/cooling rate of ~ 4 C/min and
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were uncorrected. The purity of compounds was assessed via LCꢀMS using an Agilent
1200 HPLCꢀDADꢀMS system equipped with a G1315A DAD and a 6130 Quadrupole MS
using a ZORBAX SBꢀC18 column, eluted with H2O (0.1% HCOOH)/MeCN (0.1% HCOOH)
95/5 to 0/100 linear gradient. All compounds reported in the paper were > 98 % pure.
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Analytical GPC was performed for the polymeric compounds using a Shimadzu
prominence UFLC equipped with vacuum degasser, CC20AD pump, CBM 20A controller, CTO
– 20 A column over, UV and RI detectors, under the control of EZ start software. Separation was
performed with Phenomenex Phenogel columns (1 × Phenogel Guard column, 5µ, linear, 50 ×
7.8 mm + 1 × Phenogel 5µ, 100 Å, 300 × 7.8 mm + 1 × Phenogel 5µ, 500 Å, 300 × 7.8 mm)
eluted with DMF, at 50°C, at a flow rate of 1 mL/min. Calibration was done with 10 PEG
standards, ranging from 200 to 5000 MW. MALDIꢀTOF was performed on a Bruker Daltonix
Autoflex TOFꢀTOF mass spectrometer, using αꢀcyanoꢀ4ꢀhydroxycinnamic acid as matrix.
Samples were dissolved in MeCN containing 0.1% TFA at a concentration of 1 mg/mL. Equal
volumes of sample and matrix (5 mg/mL in MeCN) were mixed and the solution was spotted on
the MALDI plate. After drying, the plate was inserted into the instrument and the spectrum was
determined, using a 20000V acceleration voltage and a variable bombardment time.
NMR spectra were recorded at ≈300 K with a Bruker Avance III 400 Plus
spectrometer equipped with a 5 mm indirect detection probe, operating at 400 MHz for 1HꢀNMR,
at 100 MHz for 13CꢀNMR. Chemical shifts are reported as δ values, using tetramethylsilane
(TMS) as internal standard for proton spectra and the solvent resonance for carbon
spectra. Assignments were made based on chemical shifts, signal intensity, COSY, HMQC,
and HMBC sequences. For 1HꢀNMR, data are reported as follows: chemical shift, multiplicity (s
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= singlet, d = doublet, t = triplet, sep = septet, m = multiplet), coupling constants J (Hz) and
integration.
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The starting aminosulfonamides 5ꢀaminoꢀ1,3,4ꢀthiadiazoleꢀ2ꢀsulfonamide 2 and 5ꢀ(4ꢀ
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aminobenzenesulfonamido)ꢀ1,3,4ꢀthiadiazoleꢀ2ꢀsulfonamide (aminobenzolamide) 3
were
synthesized as previously described.41, 70
General procedure for the synthesis of succinyl derivatives 18-20
A mixture of amino sulfonamide 15ꢀ17 (10 mmol) and succinic anhydride (1.10 g, 11 mmol)
was suspended in a minimum amount of anhydrous MeCN or DMF/MeCN mixture (1/4, v/v) (~
10 mL) and was heated to reflux. A white precipitate was forming as the reaction proceeded. The
reaction advancement was checked by TLC and its completion was confirmed by LCꢀMS. The
white precipitate was filtered, washed with MeCN and dried under vacuum. It was found pure
and its purity was confirmed using LCꢀMS and NMR (> 96 %). Additional useful compound can
be obtained by evaporating the mother liquor to dryness and choromatopgraphing the residue on
SiO2 using MeOH/DCM gradients. Useful fractions were grouped, evaporated to dryness and
crystallized from EtOH.
N-(4-Sulfamoyl-phenyl)-succinamic acid 18; mp 238ꢀ241 oC (lit71 mp 208ꢀ210 0C); Yield 88.1
%; 1HꢀNMR (DMSOꢀd6, δ, ppm): 12.16 (s, 1H, ꢀCOOH), 10.31 (s, 1H, ꢀNHꢀ), 7.78ꢀ7.70 (m, 4H,
ꢀPh), 7.24 (s, 2H, ꢀSO2NH2), 2.60 (t, J = 6.2 Hz, 2H, CH2COOH), 2.53 (t, J = 6.2 Hz, 2H,
13
CH2CONH); CꢀNMR (DMSOꢀd6, δ, ppm): 173.7 (ꢀCONH), 170.6 (ꢀCOOH), 142.0 (C1, Ph),
137.9 (C4, Ph), 126.6 (2C, C2,4 Ph), 118.3 (2C, C3,5 Ph), 31.0 (ꢀCH2COOH), 28.5 (ꢀ
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CH2CONH); LCꢀMS: C10H12N2O5S, exact mass: 272.0; Found: 273.0 (MH+); Anal
(C10H12N2O5S) C, H, N. Requires: C 44.11, H 4.44, N 10.29; Found: C 44.21, H 4.56, N 10.34.
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o
N-(5-Sulfamoyl-[1,3,4]thiadiazol-2-yl)-succinamic acid 19; mp 232ꢀ236 C (lit51 mp 211ꢀ212
0C); Yield 85.5 %; HꢀNMR (DMSOꢀd6, δ, ppm): 13.06 (br s, 1H, ꢀNH), 12.27 (br s, 1H , NH),
1
8.32 (s, 2H, ꢀSO2NH2), 2.76 (t, J = 6.5 Hz, 2H, CH2COOH), 2.60 (t, J = 6.5 Hz, 2H, CH2CONH)
; CꢀNMR (DMSOꢀd6), δ (ppm): 173.3 (CONH), 171.4 (COOH), 164.1 (C5 TDA), 161.0 (C2
13
TDA), 29.9 (ꢀCH2COOH), 28.2 (ꢀCH2CONHꢀ); LCꢀMS: C6H8N4O5S2, exact mass: 280.0; Found:
281.0 (MH+); Anal (C6H8N4O5S2) C, H, N. Requires: C 25.71, H 2.88, N 19.99; Found: C 25.84,
H 2.95, N 20.06.
N-[4-(5-Sulfamoyl-[1,3,4]thiadiazol-2-ylsulfamoyl)-phenyl]-succinamic acid 20; mp 200ꢀ204
oC; Yield 84.5 %; HꢀNMR (DMSOꢀd6, δ, ppm): 12.16 (s, 1H, ꢀCOOH), 10.37 (br s, 1H, NH),
1
8.40 (s, 2H, ꢀSO2NH2), 7.79ꢀ7.72 (m, 4H, Ph), 2.60 (t, J = 6.2 Hz, 2H, CH2COOH), 2.53 (t, J =
6.2 Hz, 2H, CH2CONH) ; 13CꢀNMR (DMSOꢀd6, δ, ppm): 173.6 (CONH), 170.7 (COOH), 167.4
(C5 TDA), 157.7 (C2 TDA), 142.9 (C1, Ph), 134.8 (C4, Ph), 127.0 (2C, C2,6 Ph), 118.5 (2C,
C3,5 Ph), 31.0 (ꢀCH2COOH), 28.5 (ꢀCH2CONH); LCꢀMS: C12H13N5O7S3, exact mass: 435.0;
Found: 436.0 (MH+); Anal (C12H13N5O7S3) C, H, N. Requires: C 33.10, H 3.01, N 16.08; Found:
C 33.24, H 3.14, N 16.15.
General procedure for the preparation of bis sulfonamides 21-23
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In a 50 mL round bottom flask succinylamidosulfonamide 18ꢀ20 (1.66 mmol) was suspended in
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10 mL of anhydrous MeCN and was treated at 0 C, under stirring, with Nꢀmethylmorpholine
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(0.25 g, 2.45 mmol) added dropwise. After 5 min, solid 2ꢀchloroꢀ4,6ꢀdimethoxyꢀ1,3,5ꢀtriazine
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(0.29 g, 1.66 mmol) was added in small portions, under stirring, at 0 C. Separately, 2,2`ꢀ
(ethylenedioxy)bis(ethylamine) (0.12 g, 0.79 mmol) was dissolved in 5 mL of DCM and the
0
solution was added over the first one dropwise, under stirring, at 0 C. The reaction was
continued overnight at room temperature. Next day, the solvent was evaporated to dryness, the
residue was dissolved in a minimum amount of DCM (~ 5 mL), and chromatographed on SiO2
using DCM/MeOH gradients. Fractions containing the useful product were combined and
were subsequently purified by reverse phase liquid chromatography using H2O (0.1%
TFA)/ACN (0.1% TFA) gradients (97/3 to 0/100 over 18 min), on a Phenomenex Gemini® 5
µm C18 LC column (110 Å, 150 x 30 mm) and a Gilson GXꢀ281 preparative LC system. Pure
fractions collected were grouped and evaporated to dryness to yield the final product as a
white powder.
N-(4-Sulfamoyl-phenyl)-N'-[2-(2-{2-[3-(4-sulfamoyl-phenylcarbamoyl)-propionylamino]-
o
1
ethoxy}-ethoxy)-ethyl]-succinamide 21: mp 252ꢀ256 C ; Yield 52.6 %; HꢀNMR (DMSOꢀd6,
δ, ppm); 10.29 (s, 2H, 2 ꢀPhNHCOꢀ), 7.96 (t, J = 5.6 Hz, 2H, 2 ꢀNHCOꢀ), 7.73 (br s, 8H, 2 Ph),
7.24 (s, 4H, 2 ꢀSO2NH2), 3.50 (br s, 4H, 2 ꢀOCH2CH2Oꢀ), 3.39 (t, J = 5.9 Hz, 4H,
ꢀ
OCH2CH2NHꢀ), 3.19 (q, J = 5.9 Hz, 4H, 2 ꢀOCH2CH2NHꢀ), 2.58 (q, J = 6.9 Hz, 4H, 2 ꢀ
OCCH2CH2COꢀ), 2.42 (t, J = 7 Hz, 4H, 2 ꢀOCCH2CH2COꢀ) ; CꢀNMR (DMSOꢀd6, δ, ppm):
13
171.1 (ꢀNHCOꢀ), 171.0 (ꢀCONH), 142.1 (C1, Ph), 137.9 (C4, Ph), 126.5 (C2,6, Ph), 118.2 (C3,5,
Ph), 69.4 (ꢀOCH2CH2Oꢀ), 69.0 (ꢀOCH2CH2NHꢀ), 38.5 (ꢀOCH2CH2NHꢀ), 31.5 (ꢀOCCH2CH2COꢀ
), 29.8 (ꢀOCCH2CH2COꢀ),; LCꢀMS C26H36N6O10S2, exact mass: 656.7; found: 657.2 (MH+);
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Anal (C26H36N6O10S2) C, H, N, O, S. Requires: C 47.55, H 5.53, N 12.80, O 24.36, S 9.77;
Found: C 47.63, H 5.71, N 12.83.
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N-(5-Sulfamoyl-[1,3,4]thiadiazol-2-yl)-N'-[2-(2-{2-[3-(5-sulfamoyl-[1,3,4]thiadiazol-2-
ylcarbamoyl)-propionylamino]-ethoxy}-ethoxy)-ethyl]-succinamide 22; mp 240ꢀ243 oC;
1
Yield 55.6 %; HꢀNMR (DMSOꢀd6, δ, ppm): 12.99 (s, 2H, 2 ꢀNHꢀ), 8.29 (s, 4H, 2 ꢀSO2NH2),
7.99 (t, J = 5.7 Hz, 2H, 2 ꢀCONHCH2ꢀ), 3.49 (br s, 4H, 2 ꢀOCH2CH2Oꢀ), 3.39 (t, J = 5.9 Hz, 4H,
2 ꢀOCH2CH2NHꢀ), 3.20 (t, J = 5.9 Hz, 4H, 2 ꢀOCH2CH2NHꢀ), 2.73 (t, J = 6.4 Hz, 4H, ꢀ
COCH2CH2COꢀ), 2.48 (m, 4H, ꢀCH2CH2ꢀ) ; 13CꢀNMR (DMSOꢀd6, δ, ppm): 171.7 (ꢀNHCOꢀ),
171.0 (ꢀCONHꢀ), 164.1 (C5 TDA), 161.0 (C2 TDA), 69.4 (ꢀOCH2CH2Oꢀ), 68.0 (ꢀOCH2CH2NHꢀ),
38.5 (ꢀOCH2CH2NHꢀ), 30.2 (ꢀCOCH2CH2COꢀ), 29.3 (ꢀCOCH2CH2COꢀ),; LCꢀMS:
C18H28N10O10S4, exact mass: 672.7; Found: 673.0; Anal (C18H28N10O10S4) C, H, N. Requires: C
32.14, H 4.20, N 20.82; Found: C 32.25, H 4.43, N 20.91.
N1,N1'-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis(N4-(4-(N-(5-sulfamoyl-1,3,4-
1
thiadiazol-2-yl)sulfamoyl)phenyl)succinamide) 23 mp 155ꢀ160oC; Yield 40.4 %; HꢀNMR
(DMSOꢀd6, δ, ppm): 10.35 (s, 2H, 2 ꢀNHꢀ), 8.45 (s, 4H, 2 ꢀSO2NH2), 7.95 (t, J = 5.6 Hz, 2H, 2 ꢀ
CONHCH2ꢀ), 7.75 (m, 8H, 2 Ph), 3.80 (m, 4H, 2 ꢀOCH2CH2Oꢀ), 3.40 (t, J = 5.9 Hz, 4H, 2 ꢀ
OCH2CH2NHꢀ), 3.18 (m, 4H, 2 ꢀOCH2CH2NHꢀ), 2.55 (m, 4H, ꢀCOCH2CH2COꢀ), 2.45 (m, 4H, ꢀ
COCH2CH2COꢀ) ; 13CꢀNMR (DMSOꢀd6, δ, ppm): 171.1 (ꢀNHCOꢀ), 171.0 (ꢀCONHꢀ), 167.3 (C5
TDA), 157.8 (C2 TDA), 143.1 (C1, Ph), 134.6 (C4, Ph), 127.0 (C2,6, Ph), 118.5 (C3,5, Ph), 69.4
(ꢀOCH2CH2Oꢀ), 68.0 (ꢀOCH2CH2NHꢀ), 388.6 (ꢀOCH2CH2NHꢀ), 31.6 (ꢀCOCH2CH2COꢀ), 29.8 (ꢀ
COCH2CH2COꢀ); LCꢀMS: C18H28N10O10S4, exact mass: 982.1; Found: 983.0; Anal
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(C30H38N12O14S6) C, H, N. Requires: C 36.65, H 3.90, N 17.10; Found: C 36.74, H 4.03, N
17.15.
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General procedure for the preparation of long bis sulfonamides 24-26
In a 50 mL round bottom flask succinylamidosulfonamide 18ꢀ20 (0.26 mmol) was
0
suspended in 10 mL of anhydrous MeCN and was treated at 0 C, under stirring with Nꢀ
methylmorpholine (0.26 g, 0.262 mmol) added dropwise. After 5 min, solid 2ꢀchloroꢀ4,6ꢀ
dimethoxyꢀ1,3,5ꢀtriazine (0.46 g, 0.262 mmol) was added in small portions, under stirring, at 0
0C. Separately, NH2ꢀPEG2000ꢀNH2 (0.25 g, 0.125 mmol) was dissolved in 5 mL of DCM and
0
the solution was added over the first one dropwise, under stirring, at 0 C. The reaction was
continued overnight at room temperature. Next day, the solvent was evaporated to dryness. The
residue was dissolved in a minimum amount of DCM (~ 5 mL), and chromatographed on SiO2
using DCM/MeOH gradients. Pure fractions collected were grouped and evaporated to dryness
to yield the final product, which was dried under high vacuum for 24 h.
Bis-(4-Sulfamoyl-phenyl)amidosuccinyl-polyethyleneglycol2000diamide 24 Yield 53.5%; 1Hꢀ
NMR (D2O, δ, ppm): 7.83 (dd, J = 1.5, 8.7 Hz, 2H, HAA’ Ph), 7.63 (dd, J = 1.5, 8.7 Hz, HBB’ Ph),
3.80ꢀ3.58 (m, 176 H, 44 OCH2CH2O), 3.54 (m, ꢀOCH2CH2NHꢀ), 3.31 (t, J = 4.7 Hz, 4H, ꢀ
OCH2CH2NHꢀ), 2.69 (t, J = 7 Hz, 4H, ꢀOCCH2CH2COꢀ), 2.55 (t, J = 7 Hz, 4H,
ꢀ
OCCH2CH2CO). GPC: 96%+, tR = 15.11 min, PDI = 1.15; MALDIꢀTOF: C112H208N6O53S2,
exact mass: 2549.3; Found: 2413.0.
Bis-(5-Sulfamoyl-[1,3,4]thiadiazol-2-yl)-amidosuccinyl)-polyethyleneglycol2000diamide 25
Yield 56.7%; 1HꢀNMR (D2O, δ, ppm): 3.80ꢀ3.58 (m, 176 H, 44 OCH2CH2O), 3.53 (t, J = 5.8 Hz,
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4H, ꢀOCH2CH2NHꢀ), 3.31 (t, J = 5.4 Hz, 4H, ꢀOCH2CH2NHꢀ), 2.85 (t, J = 6.6 Hz, 4H, ꢀ
COCH2CH2COꢀ), 2.61 (t, J = 6.6 Hz, 4H, ꢀOCCH2CH2COꢀ). GPC: 96%+, tR = 15.09 min, PDI =
1.13; MALDIꢀTOF: C104H200N10O53S4, exact mass: 2565.2; Found: 2534.4.
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Bis-(N4-(4-(N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)aminosulfonyl)phenyl)amidosuccinyl)
-
polyethyleneglycol2000diamide 26 Yield 81.6 %; 1HꢀNMR (D2O, δ, ppm): 7.78 (d, J = 8.5 Hz,
2H, HAA’ Ph), 7.58 (d, J = 8.5 Hz, 2H, HBB’ Ph), 3.82ꢀ3.57 (m, 176 H, 44 OCH2CH2Oꢀ), 3.54 (t,
J = 5.7 Hz, 4H, ꢀOCH2CH2NHꢀ), 3.31 (t, J = 5.3 Hz, 4H, ꢀOCH2CH2NHꢀ), 2.67 (t, J = 6.5 Hz,
4H, ꢀOCCH2CH2COꢀ), 2.53 (t, J = 6.5 Hz, 4H, ꢀOCCH2CH2COꢀ). GPC: 96%+, tR = 14.01 min,
PDI = 1.19; MALDIꢀTOF: C116H210N12O57S6, exact mass: 2875.2; Found: 2844.1.
CA inhibition assay
An Applied Photophysics stoppedꢀflow instrument has been used for assaying the CA catalysed
CO2 hydration activity.72 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 7.5) as buffer, and 20
mM Na2SO4 (for maintaining constant the ionic strength), following the initial rates of the CAꢀ
catalyzed CO2 hydration reaction for a period of 10ꢀ100 s. The CO2 concentrations ranged from
1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each
inhibitor at least six traces of the initial 5ꢀ10% of the reaction have been used for determining the
initial velocity. 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 distilledꢀ
deionized water and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor
and enzyme
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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
leastsquares methods using PRISM 3 and the ChengꢀPrusoff equation, as reported earlier,73 and
represent the mean from at least three different determinations. All CA isofoms were
recombinant ones obtained inꢀhouse as reported earlier,20, 33, 74, 75 and their concentrations in the
assay system were in the range of 7.8 – 10.6 nM.
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Viability assays for measuring the cytotoxicity of novel CAIs in normoxic and hypoxic
conditions
Three cell lines derived from colon (HTꢀ29), ovarian (SKOV3) and breast (MDAꢀMBꢀ231)
carcinomas were used in the study. Cells were cultured using RPMI media with 10% fetal bovine
serum (FBS) for HTꢀ29 line, McCoy’s media with 10% FBS for SKOV3 line, and DMEM media
supplemented with 10% FBS for MDAꢀMB231 cell line.
For tests in 2D cell culture, cells were plated in 96 well plates at density of 10,000
o
cells/well and were allowed to attach and to grow in normal conditions (37 C, 5% CO2 in air).
Two 96 well plates were prepared from each cell line, one to be incubated in normoxic
conditions and another one to be subjected to a hypoxic environment. After 24 h growth in
normal conditions the hypoxiaꢀdesignated plates were placed in a hypoxic chamber, which was
purged and filled with a low oxygen gas mixture (1% O2, 5% CO2 and 94% N2) to induce
hypoxia, and the closed chamber with hypoxic plates was kept in the incubator at 37 oC for 24 h.
Normoxic plates were kept in the same incubator in normal conditions. After 24 h media was
aspirated from all plates and cells were treated with CAIs solutions in media at 3 different
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concentrations (1 mM, 100 µM, and 10 µM). Each experiment was done in triplicate. Eight
wells, used as control, received only media. Hypoxic plates were returned to hypoxic chamber,
reꢀpurged and filled with low oxygen gas mixture, then the closed chamber with plates was
returned to the incubator. Normoxic plates were also returned to the incubator. After another 24
h the normoxic and hypoxic plates were collected, media was aspirated, cells were washed once
with PBS, which was subsequently removed. An MTT solution in media (120 ꢁL, made out from
MTT 5 mg/mL concentration in PBS diluted 1:6 with the corresponding media) was added to
each well and the plates were returned to the incubator for 4 h. The MTT solution was removed
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o
and 150 ꢁL of DMSO was added to solubilize the blue formazan crystals, at 37 C for 5 min.
Analysis of produced formazan was done spectrophotometrically, measuring the absorbance at
570 nm, with a reference absorbance at 690 nm that was subtracted from readings. Data was
reported as the average of three experiments, with one standard deviation from the average value.
Statistical comparisons were performed by analysis of variance (ANOVA) using GraphPad
Prism 6, where *P < 0.05, **P < 0.01 and ***P < 0.001 unless specified otherwise.
For tests in 3D cell culture, tumor spheroids were grown as reported before,68 using the same
HTꢀ29, MDAꢀMBꢀ231 and SKOV3 cell lines, maintained as described above. The plating
density was 103 cells/well within a volume of 100 µL. After a 7 days of incubation in a 5%
humidified incubator at 37 °C, tumor spheroids were treated with 50 µL of inhibitor solution in
media at the specified concentrations. After a 24 h incubation time, spheroids were treated with
15 µL of a WSTꢀ8 solution, the plate returned to the incubator for 4 h and subsequently read at
450 nm with a reference wavelength of 650 nm. Each test was done in quadruplicate and results
were reported as average of the four experiments ± one standard deviation.
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