PEGylated Bis-Sulfonamide Carbonic Anhydrase Inhibitors Can

May 4, 2016 - PEGylated Bis-Sulfonamide Carbonic Anhydrase Inhibitors Can Efficiently Control the Growth of Several Carbonic Anhydrase IX-Expressing C...
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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

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*

1

Department of Pharmaceutical Sciences and Moulder Center for Drug Discovery Research,

Temple University School of Pharmacy, 3307 N Broad Street, Philadelphia, PA-19140 2

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Adiyaman University, 02040

Adiyaman, Turkey 3

NEUROFARBA Department, Pharmaceutical Sciences Section, Universita degli Studi di

Firenze, Polo Scientifico, Via Ugo Schiff no. 6, 50019 Sesto Fiorentino (Florence), Italy 4

University of Washington - Medical Service, VA Puget Sound Health Care System, Seattle,

WA, USA

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Abstract 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 ethyleneglycol 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 CA 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 over-expressing 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 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.

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Introduction 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 over-express proteins that can help maintain internal pH and homeostasis through active export of these metabolic products outside the cells. Hypoxia triggers the expression of HIF-1, 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 angiogenesis and in different metabolic pathways that become overexpressed such as glycolysis, anabolic processes, etc.

3, 4

Even in the absence of hypoxia, these same metabolic pathways in

tumor cells are activated to supply large amounts of 3-carbon substrates (e.g. lactate and pyruvate) for high rates of protein, lipid and nucleic acid synthesis needed for rapid growth; the well-known 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, 6-11

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 I-III, VII, XIII), mitochondrial isozymes (CA VA, VB), membrane- bound isozymes (CA IV, IX, XII, XIV and XV), one secreted isozyme (CA VI) and several CA-related 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, 12-14 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 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 over-expressed tumor izozymes CA IX and CA XII. Bicarbonate ion is imported into the cytoplasm via exchange with Clthrough 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 Cl-

CA IX/XII

glucose HCO3-/Cl- exchanger

aquaporin H+ channel H

Glucose transporter

pHi = 7.2 HCO3-

CO2 + H2O

+

+H

+

glucose glycolysis

CA II

tumor cell

piruvateHYPOXIA lactate-

+

H -ATPase MCT H+

Na+ Na+/H+ exchanger 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 membranebound CA isozymes (CA IX, CA XII), which are over-expressed in hypoxic tumors. Selective inhibition of CA IX and CA XII has thus the potential to kill tumor cells.

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 process and continuous killing of normal tissues surrounding the tumor.

17-19

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 tumor-overexpressed CA isozymes constitutes a viable strategy to fight these aggressive metastatic tumors.

3, 7, 32, 33

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 aminobenzolamide with halogeno moieties to induce isozyme selectivity.

33

Another class of

potent CA inhibitors introduced by us were ureido- and thioureido-sulfonamides

34

and

subsequent halogen decoration of these scaffolds yielded powerful CA IX inhibitors such as 8 (SLC-0111),

35

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 over-express CA IX and/or XII. Many other approaches to produce potent and/or isozyme-selective 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 between different CA isozymes in their active sites.

3, 6, 7, 38-40

Strategies to enhance the

selectivity for membrane-bound izozymes in vivo involve conjugation of CAI pharmacophores with charged groups,41-45 with sugar,46-50 or with polymeric moieties51,

52

that will make the

inhibitors membrane-impermeant and therefore unable to access the intracellular CA isozymes. For example, our team demonstrated the possibility of generating membrane-impermeant inhibitors through conjugation of primary aromatic and heterocyclic sulfonamides with positively charged pyridinium salts.

41-44

Pyridinium sulfonamides such as 9, 10 and 11 display

excellent selectivity against membrane-bound CA IV

41-44

and also against tumoral CA IX and

CA XII.45 As mentioned above, an alternative technology for generating membrane-impermeant inhibitors with selectivity for these tumoral CA isozymes is to attach known CA pharmacophores

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to water-soluble polymeric backbones such as dextran, aminoethyldextran and polyethylene glycol (PEG). 51, 52 The development of this technology, pioneered in the late 80’s and mid-90’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 II and membrane-bound 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 membrane-bound 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 Building on these premises and taking into account that CA IX and CA XII are dimers in vivo,8-11 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 multi-pronged CAIs recently reported by one of us

55

and Winum’s group

56

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, sulfonamide

namely

16

and

4-aminobenzenesulfonamide

15,

5-amino-1,3,4-thiadiazole-2-

5-(4-aminobenzenesulfonamido)-1,3,4-thiadiazole-2-sulfonamide

(aminobenzolamide) 17 (Scheme 1). These pharmacophores were selected due to their proven potency against CA isozymes 3, and because as shown via X-ray crystallography they can provide three different orientations of the corresponding inhibitors in the active site of the enzyme (Figure 2).58-60

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a)

b)

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 4-aminobenzene sulfonamide 15, 5-amino-1,3,4-thiadiazole-2-sulfonamide 16, 5-(4-aminobenzenesulfonamido)-1,3,4thiadiazole-2-sulfonamide (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 18-20 were generated from the amino sulfonamides 15-17 through reaction with succinic anhydride in acetonitrile. Their carboxy group was subsequently activated with 2chloro-4,6-dimethoxy-1,3,5-triazine

(CDMT)/N-methylmorpholine

(NMM)

61

and

was

condensed with diamino(oligo/poly)ethylene glycol to yield the bis-sulfonamides 21-26. We have selected two linker lengths between the succinyl CAIs: a short one (n = 2) in the bissulfonamide CAIs 21-23 and a long one (n = 45) in the PEGylated bissulfonamide CAIs 24-26

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(Scheme 1). They were purified by flash chromatography and characterized by standard analytical methods (see Materials and Methods section).

Scheme 1. Synthetic strategy for the generation of bis-sulfonamide CA inhibitors.

The two sets of bis-sulfonamides were expected to interact in different ways with the enzyme in solution. In the case of the bis-sulfonamides 21-23 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 nanoparticles.

55, 56

For polymeric compounds 24-26 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 bissulfonamides 21-23 are expected to permeate the membranes easily, the polymeric inhibitors 2426 were designed to be membrane-impermeable due to their size and highly hydrated polymeric backbone. When administered in vivo, compounds 24-26 are expected to inhibit selectively the mebrane-bound CA isozymes, including the tumor-over-expressed 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 18-20 are negatively charged at physiologic pH and therefore expected to be partial (but not completely) membraneimpermeant, similarly to the case of benzolamide.62 Compounds 18-20 can be generated in the body as part of the hydrolytic processing or metabolism of the bis-sulfonamides 21-26. Therefore, all bis-sulfonamides 21-26 and their succinyl precursors 18-20 were tested for their ability to inhibit the membrane-bound 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 18-20), in a comparative manner (Table 1). Data from Table 1 revealed an excellent inhibitory profile of compounds 18-26 against all membrane-bound 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 18-26 were the tumor over-expressed isozymes CA IX and XII, followed closely by CA XIV and CA IV. Cytosolic isozyme CA II was less susceptible to inhibition by compounds 18-26 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 bis-sulfonamide 22 were

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identified as potent pan-inhibitors with nanomolar potency against all isozymes tested except CA I. We also identified selective inhibitors for membrane-bound isozymes versus cytosolic ones, with either medium selectivity (compounds 21 and 25) or high selectivity – sulfonamide 24.

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

18

249 ± 21

18.2 ± 1.1

486 ± 42

12.8 ± 0.9

0.52 ± 0.02

6.1 ± 0.3

19

245 ± 20

8.1 ± 0.7

6.7 ± 0.3

2.7 ± 0.09

3.2 ± 0.1

4.0 ± 0.2

20

181 ± 13

9.3 ± 0.8

5.5 ± 0.2

3.0 ± 0.3

3.9 ± 0.2

4.4 ± 0.4

21

221 ± 16

37.1 ± 0.2

7.8 ± 0.5

2.9 ± 0.1

2.6 ± 0.2

6.2 ± 0.5

22

180 ± 12

8.7 ± 0.5

5.0 ± 0.3

9.6 ± 0.1

5.9 ± 0.4

4.6 ± 0.3

23

192 ± 9

8.6 ± 0.4

6.4 ± 0.15

24.1 ± 0.2

2.8 ± 0.1

4.6 ± 0.4

24

272 ± 25

1764 ± 45

9.5 ± 0.4

4.8 ± 0.1

3.2 ± 0.2

7.3 ± 0.5

25

225 ± 21

66.6 ± 3.1

5.8 ± 0.5

2.5 ± 0.2

5.4 ± 0.3

6.8 ± 0.15

26

181 ± 9

12.9 ± 1.0

4.4 ± 0.3

1.7 ± 0.1

3.7 ± 0.1

4.4 ± 0.3

1

250 ± 12

12 ± 0.8

74 ± 5

25 ± 1.7

5.7 ± 0.3

41 ± 2.9

* 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 pan-inhibitors. The inhibition profile of the short bis-sulfonamides 21-23 was similar to the one displayed by their precursors 18-20, 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 bissulfonamide. It must be emphasized that 5-succinylamido-1,3,4-thiadiazole-2-sulfonamide 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 Interestingly, the long PEG linker increased the selectivity of the bis-sulfonamides 24-26 for the membrane-bound isozymes versus the cytosolic ones (especially against CA II), as compared with their oligoethylene glycol congeners 21-23. Bis-sulfonamide 24 displayed the highest selectivity between membrane-bound isozymes and their cytosolic counterparts (Table 1). Considering the membrane-impermeant nature of the PEGylated bis-sulfonamides 24-26, it is expected that this selectivity will increase in vivo. Moreover, since tumor-overexpressed CA IX and CA XII isozymes are dimeric, it should be expected that the bis-sulfonamides 24-26 will have a more pronounced effect on these targets in vivo, due to cooperative binding and bivalent association of the two-pronged inhibitor with the two neighboring active sites, as shown by Whitesides.54 Therefore, we decided to test all CAIs 18-26 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, 20-31, 63

we have selected three cellular models, namely the colon carcinoma HT-29, the

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breast cancer MDA-Mb231, and the ovarian cancer SKOV3 cell models.

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64-66

For 2D testing,

cells were grown subconfluent in 96 well plates in normal conditions (37 oC, 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).

Figure 32. Effect of CAI 18-26 and acetazolamide 1, at different concentrations, on the viability of colon HT-29, breast MDA-MB231 and ovarian SKOV-3 cancer cell lines under normoxic (a) and hypoxic (b) conditions. P values were determined by one-way 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. 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 concentration-dependent manner, as expected, with colon HT-29 and breast MDA-MB231 cancer cells slightly more sensitive to CAI treatment than ovarian cancer cell line SKOV-3. 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 MDA-MB231 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 bis-sulfonamide 25, bearing the classical 1,3,4-thiadiazole-2sulfonamide “warhead”. It displayed robust cell killing, with cell viabilities as low as 30-40% at 1 mM, 50% at 100 µM and 60-70% at 10 µM, being influenced by the cell type and presence of hypoxia. The above-mentioned data revealed the impact of in vitro models of tumor proliferation for the evaluation of potency of CAI as a pre-requisite 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 HT-29, MDA-MB231 and SKOV-3 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

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 HT29, breast MDA-MB231 and ovarian (SKOV-3) cancer cell lines. CA IX appears as a two-band protein at 54 and 58 KDa 19 and can be quantified relative to β-actin 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: MDA-MB231 expressed the most CA IX, followed by HT29 and SKOV-3. These CA IX expression levels under normoxic conditions correlate well with the order of susceptibility of the three cell lines to the CAI 18-26 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 HT-29 and SKOV-3 and only a modest increase in MDA-MB231. 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 18-26 (Figure 3b). The same significant induction of CA IX expression can be found in tumor spheroids derived from HT-29 and SKOV-3 cell lines, while the MDAMB231 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. After quantifying the relative amount of CA IX in tumor spheroids, we assessed their susceptibility to treatment with CAIs 18-26 at 1 mM and 100 µM concentration, for 24 h incubation time. Viability of the spheroids was assessed via a WST-8 assay 69 (Figure 5).

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Figure 54. Effect of CAI 18-26 and acetazolamide 1, at different concentrations, on the viability of colon HT-29, breast MDA-MB231 and ovarian SKOV-3 3D cell cultures (tumor spheroids). P values were determined by one-way 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 HT-29 and SKOV-3 cell lines, with MDA-MB231 cell line being less affected. Polymeric bis-sulfonamide 25 was the most efficient CAI in this assay too, decreasing the viability to about 50%/80% in SKOV-3, 60%/70% in HT-29 and 80%/90% in MDA-MB231 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, cell-dependent inhibition profile.

Conclusions A detailed structure-activity relationship study was carried out within a series of new bissulfonamides 21-26 and their succinylamido sulfonamide precursors 18-20 derived from three different established carbonic anhydrase inhibitor “warheads”. These CAIs were profiled against a set of membrane-bound and cytosolic CA isoforms, including the membrane-overexpressed CA IX and XII. We have identified potent pan-inhibitors, together with CAIs with selectivity for membrane-bound 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 bis-sulfonamide 25, which showed nanomolar potency against purified CA IX and CA XII isozymes and consistent and significant cancer cell killing at concentrations of 10-100 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 Materials: The following materials were used as received: succinic anhydride (TCI America, Portland, OR), 2,2`-(ethylenedioxy)-diethylamine (Fluka, St Louis, MO), N-methylmorpholine (TCI America, Portland, OR), 2-chloro-4,6-dimethoxy-1,3,5-triazine (Acros Organics, New Jersey, NJ), sulfanilamide (Acros Organics, New Jersey, NJ), H2N-PEG2000-NH2 (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 (SKOV-3-Luc), human breast cancer cell line (MDA-MB-231-Luc) were purchased from ATCC (Manassas, VA), Dulbecco's Modified Eagle's medium (DMEM), RPMI-1640, McCoy's 5A media were from MediatechCorning (Manassas, VA), Fetal bovine serum (FBS) was from Fisher Scientific (Pittsburgh, PA), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), phosphate-buffered saline (PBS), dimethyl sulfoxide (DMSO) and

water-soluble tetrazolium salt-8 (WST-8) were

purchased from VWR International (West Chester, PA).

Techniques: The purity and the structure identity of the intermediary and the final products were assessed by a combination of techniques that included thin-layer chromatography (TLC), HPLC-MS, 1H-NMR, COSY,

13

C-NMR, high resolution mass

spectrometry (HR-MS), gel permeation chromatography (GPC) coupled with MALDI-TOF. TLC was carried out on SiO2-precoated aluminum plates (silica gel with F254 indicator; layer thickness 200 µm; pore size 60 Å, from Sigma-Aldrich. Melting points were determined using a Thermolyne heating stage microscope (Dubuque, IA), equipped with an Olympus 5X objective, at heating/cooling rate of ~ 4 oC/min and

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were uncorrected. The purity of compounds was assessed via LC-MS using an Agilent 1200 HPLC-DAD-MS system equipped with a G1315A DAD and a 6130 Quadrupole MS using a ZORBAX SB-C18 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. 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. MALDI-TOF was performed on a Bruker Daltonix Autoflex TOF-TOF mass spectrometer, using α-cyano-4-hydroxycinnamic 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 1H-NMR, at 100 MHz for 13C-NMR. 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 1H-NMR, 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. The

starting

aminosulfonamides

5-amino-1,3,4-thiadiazole-2-sulfonamide

2

and

aminobenzenesulfonamido)-1,3,4-thiadiazole-2-sulfonamide (aminobenzolamide) 3

5-(4were

synthesized as previously described.41, 70

General procedure for the synthesis of succinyl derivatives 18-20

A mixture of amino sulfonamide 15-17 (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 LC-MS. The white precipitate was filtered, washed with MeCN and dried under vacuum. It was found pure and its purity was confirmed using LC-MS 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 238-241 oC (lit71 mp 208-210 0C); Yield 88.1 %; 1H-NMR (DMSO-d6, δ, ppm): 12.16 (s, 1H, -COOH), 10.31 (s, 1H, -NH-), 7.78-7.70 (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, CH2CONH);

13

C-NMR (DMSO-d6, 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); LC-MS: 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.

N-(5-Sulfamoyl-[1,3,4]thiadiazol-2-yl)-succinamic acid 19; mp 232-236 oC (lit51 mp 211-212 0

C); Yield 85.5 %; 1H-NMR (DMSO-d6, δ, ppm): 13.06 (br s, 1H, -NH), 12.27 (br s, 1H , NH),

8.32 (s, 2H, -SO2NH2), 2.76 (t, J = 6.5 Hz, 2H, CH2COOH), 2.60 (t, J = 6.5 Hz, 2H, CH2CONH) 13

;

C-NMR (DMSO-d6), δ (ppm): 173.3 (CONH), 171.4 (COOH), 164.1 (C5 TDA), 161.0 (C2

TDA), 29.9 (-CH2COOH), 28.2 (-CH2CONH-); LC-MS: 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 200-204 o

C; Yield 84.5 %; 1H-NMR (DMSO-d6, δ, ppm): 12.16 (s, 1H, -COOH), 10.37 (br s, 1H, NH),

8.40 (s, 2H, -SO2NH2), 7.79-7.72 (m, 4H, Ph), 2.60 (t, J = 6.2 Hz, 2H, CH2COOH), 2.53 (t, J = 6.2 Hz, 2H, CH2CONH) ; 13C-NMR (DMSO-d6, 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); LC-MS: 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 18-20 (1.66 mmol) was suspended in 10 mL of anhydrous MeCN and was treated at 0 0C, under stirring, with N-methylmorpholine (0.25 g, 2.45 mmol) added dropwise. After 5 min, solid 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.29 g, 1.66 mmol) was added in small portions, under stirring, at 0 0C. Separately, 2,2`(ethylenedioxy)bis(ethylamine) (0.12 g, 0.79 mmol) was dissolved in 5 mL of DCM and the solution was added over the first one dropwise, under stirring, at 0 0C. 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 GX-281 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]ethoxy}-ethoxy)-ethyl]-succinamide 21: mp 252-256 oC ; Yield 52.6 %; 1H-NMR (DMSO-d6, δ, ppm); 10.29 (s, 2H, 2 -PhNHCO-), 7.96 (t, J = 5.6 Hz, 2H, 2 -NHCO-), 7.73 (br s, 8H, 2 Ph), 7.24 (s, 4H, 2 -SO2NH2), 3.50 (br s, 4H, 2 -OCH2CH2O-), 3.39 (t, J = 5.9 Hz, 4H,

-

OCH2CH2NH-), 3.19 (q, J = 5.9 Hz, 4H, 2 -OCH2CH2NH-), 2.58 (q, J = 6.9 Hz, 4H, 2 OCCH2CH2CO-), 2.42 (t, J = 7 Hz, 4H, 2 -OCCH2CH2CO-) ;

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C-NMR (DMSO-d6, δ, ppm):

171.1 (-NHCO-), 171.0 (-CONH), 142.1 (C1, Ph), 137.9 (C4, Ph), 126.5 (C2,6, Ph), 118.2 (C3,5, Ph), 69.4 (-OCH2CH2O-), 69.0 (-OCH2CH2NH-), 38.5 (-OCH2CH2NH-), 31.5 (-OCCH2CH2CO), 29.8 (-OCCH2CH2CO-),; LC-MS 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.

N-(5-Sulfamoyl-[1,3,4]thiadiazol-2-yl)-N'-[2-(2-{2-[3-(5-sulfamoyl-[1,3,4]thiadiazol-2ylcarbamoyl)-propionylamino]-ethoxy}-ethoxy)-ethyl]-succinamide 22; mp 240-243

o

C;

Yield 55.6 %; 1H-NMR (DMSO-d6, δ, ppm): 12.99 (s, 2H, 2 -NH-), 8.29 (s, 4H, 2 -SO2NH2), 7.99 (t, J = 5.7 Hz, 2H, 2 -CONHCH2-), 3.49 (br s, 4H, 2 -OCH2CH2O-), 3.39 (t, J = 5.9 Hz, 4H, 2 -OCH2CH2NH-), 3.20 (t, J = 5.9 Hz, 4H, 2 -OCH2CH2NH-), 2.73 (t, J = 6.4 Hz, 4H, COCH2CH2CO-), 2.48 (m, 4H, -CH2CH2-) ;

13

C-NMR (DMSO-d6, δ, ppm): 171.7 (-NHCO-),

171.0 (-CONH-), 164.1 (C5 TDA), 161.0 (C2 TDA), 69.4 (-OCH2CH2O-), 68.0 (-OCH2CH2NH-), 38.5

(-OCH2CH2NH-),

30.2

(-COCH2CH2CO-),

29.3

(-COCH2CH2CO-),;

LC-MS:

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-1,3,4thiadiazol-2-yl)sulfamoyl)phenyl)succinamide) 23 mp 155-160oC; Yield 40.4 %; 1H-NMR (DMSO-d6, δ, ppm): 10.35 (s, 2H, 2 -NH-), 8.45 (s, 4H, 2 -SO2NH2), 7.95 (t, J = 5.6 Hz, 2H, 2 CONHCH2-), 7.75 (m, 8H, 2 Ph), 3.80 (m, 4H, 2 -OCH2CH2O-), 3.40 (t, J = 5.9 Hz, 4H, 2 OCH2CH2NH-), 3.18 (m, 4H, 2 -OCH2CH2NH-), 2.55 (m, 4H, -COCH2CH2CO-), 2.45 (m, 4H, COCH2CH2CO-) ; 13C-NMR (DMSO-d6, δ, ppm): 171.1 (-NHCO-), 171.0 (-CONH-), 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 (-OCH2CH2O-), 68.0 (-OCH2CH2NH-), 388.6 (-OCH2CH2NH-), 31.6 (-COCH2CH2CO-), 29.8 (COCH2CH2CO-); LC-MS: 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.

General procedure for the preparation of long bis sulfonamides 24-26 In a 50 mL round bottom flask succinylamidosulfonamide 18-20 (0.26 mmol) was suspended in 10 mL of anhydrous MeCN and was treated at 0 0C, under stirring with Nmethylmorpholine (0.26 g, 0.262 mmol) added dropwise. After 5 min, solid 2-chloro-4,6dimethoxy-1,3,5-triazine (0.46 g, 0.262 mmol) was added in small portions, under stirring, at 0 0

C. Separately, NH2-PEG2000-NH2 (0.25 g, 0.125 mmol) was dissolved in 5 mL of DCM and

the solution was added over the first one dropwise, under stirring, at 0 0C. 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%; 1HNMR (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), 3.80-3.58 (m, 176 H, 44 OCH2CH2O), 3.54 (m, -OCH2CH2NH-), 3.31 (t, J = 4.7 Hz, 4H, OCH2CH2NH-), 2.69 (t, J = 7 Hz, 4H, -OCCH2CH2CO-), 2.55 (t, J = 7 Hz, 4H,

-

OCCH2CH2CO). GPC: 96%+, tR = 15.11 min, PDI = 1.15; MALDI-TOF: C112H208N6O53S2, exact mass: 2549.3; Found: 2413.0.

Bis-(5-Sulfamoyl-[1,3,4]thiadiazol-2-yl)-amidosuccinyl)-polyethyleneglycol2000diamide 25 Yield 56.7%; 1H-NMR (D2O, δ, ppm): 3.80-3.58 (m, 176 H, 44 OCH2CH2O), 3.53 (t, J = 5.8 Hz,

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4H, -OCH2CH2NH-), 3.31 (t, J = 5.4 Hz, 4H, -OCH2CH2NH-), 2.85 (t, J = 6.6 Hz, 4H, COCH2CH2CO-), 2.61 (t, J = 6.6 Hz, 4H, -OCCH2CH2CO-). GPC: 96%+, tR = 15.09 min, PDI = 1.13; MALDI-TOF: C104H200N10O53S4, exact mass: 2565.2; Found: 2534.4.

Bis-(N4-(4-(N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)aminosulfonyl)phenyl)amidosuccinyl)

-

polyethyleneglycol2000diamide 26 Yield 81.6 %; 1H-NMR (D2O, δ, ppm): 7.78 (d, J = 8.5 Hz, 2H, HAA’ Ph), 7.58 (d, J = 8.5 Hz, 2H, HBB’ Ph), 3.82-3.57 (m, 176 H, 44 OCH2CH2O-), 3.54 (t, J = 5.7 Hz, 4H, -OCH2CH2NH-), 3.31 (t, J = 5.3 Hz, 4H, -OCH2CH2NH-), 2.67 (t, J = 6.5 Hz, 4H, -OCCH2CH2CO-), 2.53 (t, J = 6.5 Hz, 4H, -OCCH2CH2CO-). GPC: 96%+, tR = 14.01 min, PDI = 1.19; MALDI-TOF: C116H210N12O57S6, exact mass: 2875.2; Found: 2844.1.

CA inhibition assay An Applied Photophysics stopped-flow 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 CAcatalyzed CO2 hydration reaction for a period of 10-100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor at least six traces of the initial 5-10% 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 distilleddeionized 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 E-I complex. The inhibition constants were obtained by non-linear leastsquares methods using PRISM 3 and the Cheng-Prusoff equation, as reported earlier,73 and represent the mean from at least three different determinations. All CA isofoms were recombinant ones obtained in-house as reported earlier,20, 33, 74, 75 and their concentrations in the assay system were in the range of 7.8 – 10.6 nM.

Viability assays for measuring the cytotoxicity of novel CAIs in normoxic and hypoxic conditions

Three cell lines derived from colon (HT-29), ovarian (SKOV3) and breast (MDA-MB-231) carcinomas were used in the study. Cells were cultured using RPMI media with 10% fetal bovine serum (FBS) for HT-29 line, McCoy’s media with 10% FBS for SKOV3 line, and DMEM media supplemented with 10% FBS for MDA-MB231 cell line. For tests in 2D cell culture, cells were plated in 96 well plates at density of 10,000 cells/well and were allowed to attach and to grow in normal conditions (37 oC, 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 hypoxia-designated 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, re-purged 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 µL, 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 and 150 µL of DMSO was added to solubilize the blue formazan crystals, at 37 oC 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 HT-29, MDA-MB-231 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 WST-8 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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CA IX profiling using western blotting The HT-29, MDA-MB231 and SKOV-3 cell lines were screened for the expression of carbonic anhydrase IX in 2D and 3D cell culture, under normoxic and hypoxic conditions. Cells were cultured in 60 mm petri dishes at a density of 106 cells under normoxic and hypoxic conditions. The hypoxic conditions were induced as described above, by placing the cells into a hypoxic chamber purged with hypoxia gas mixture containing 1% oxygen for 24 h. The chamber was placed in a 37⁰C incubator. For normoxic conditions cells were kept in regular incubator for 24 h. The next day, all cells were harvested, pelleted and washed with phosphate buffer saline (PBS). Cells were then lysed with RIPA buffer containing protease inhibitors and lysates were collected and stored at -20⁰C. Total protein concentration was determined using bicinchoninic acid method (BCA). Cells were also cultured in rounded bottom 96 well plates at a density of 1000 cells/well and were placed in the incubator for 1-2 week to grow into tumor spheroids. After the spheroids were fully developed, they were harvested and lysed as mentioned previously. Western Blot analysis was performed as described.19 Briefly, cell lysate samples were loaded onto 10% SDS-PAGE precast gels (Bio-rad, 456-8034) and separation was done at 150 V for 2 h. Gels were transblotted onto nitrocellulose membranes for 30 min and membranes were blocked using blocking buffer followed by incubation with a specific primary mouse monoclonal antibody for CA IX (M75, Bioscience Slovakia) at 4⁰C overnight. After washing, membranes were incubated with anti-mouse IgG IRDye800CW secondary antibody (Rockland) for 1 h at room temperature. Finally, membranes were detected using an Odyssey image system (Li-Cor, Lincoln NE). Beta actin was used as a control and was detected using a mouse

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monoclonal beta actin antibody (Genetex) binding followed by detection with the same IgG IRDye800CW secondary antibody.

Ancillary Information Supporting Information. Molecular Formula Strings for compounds, with biological data Corresponding author information *

Correspondence

authors:

Tel:

39-055-4573005;

Fax:

39-055-4573385;

Email:

[email protected] (Claudiu T. Supuran); Tel 215-707-1749, Fax 215-707-5620, E-mail: [email protected] (Marc A. Ilies).

Acknowledgements The financial support of Seattle Foundation (grant 255345), Temple Drug Discovery Initiative, TUSP Dean’s Office, Temple Undergraduate Research Program is gratefully acknowledged. Suleyman Akocak acknowledges the financial support of the Turkish Minister of Education for a PhD scholarship. We would like to acknowledge Dr. Salim Merali and Mr. George Mateo for their help with MALDI-TOF spectra.

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