Hypoxia-Activated Prodrug Derivatives of Carbonic Anhydrase Inhibitors in Benzenesulfonamide Series : Synthesis and Biological Evaluation

molecules Article Hypoxia-Activated Prodrug Derivatives of Carbonic Anhydrase Inhibitors in Benzenesulfonamide Series: Synthesis and Biological Evaluation Emilie Anduran 1,2, Ashok Aspatwar 3, Nanda-Kumar Parvathaneni 1,2, Dennis Suylen 4, Silvia Bua 5, Alessio Nocentini 5, Seppo Parkkila 3, Claudiu T. Supuran 5, Ludwig Dubois 2, Philippe Lambin 2,* and Jean-Yves Winum 1,* 1Institut des Biomolécules Max Mousseron (IBMM) UMR 5247 CNRS, ENSCM, Universitéde Montpellier, 34296 Montpellier CEDEX 05, France; [email protected] (E.A.); [email protected] (N.-K.P.) 2The M-Lab, Department of Precision Medicine, GROW–School for Oncology, Maastricht University, 6200 MD Maastricht, The Netherlands; [email protected] 3 Faculty of Medicine and Health Technology and Fimlab Ltd., University of Tampere and Tampere University Hospital, 33520 Tampere, Finland; [email protected] (A.A.); [email protected] (S.P.) 4 Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University Medical Centre, 6200 MD Maastricht, The Netherlands; [email protected] 5Neurofarba Department, Section of Pharmaceutical Sciences, Universitàdegli Studi di Firenze, 50019 Sesto Fiorentino (Florence), Italy; [email protected] (S.B.); [email protected] (A.N.); [email protected] (C.T.S.) * Correspondence: [email protected] (P.L.); [email protected] (J.-Y.W.) Academic Editors: Fabrizio Carta and Andrea Angeli Received: 29 April 2020; Accepted: 14 May 2020; Published: 18 May 2020   Abstract: Hypoxia, a common feature of solid tumours’ microenvironment, is associated with an aggressive phenotype and is known to cause resistance to anticancer chemoand radiotherapies. Tumour-associated carbonic anhydrases isoform IX (hCA IX), which is upregulated under hypoxia in many malignancies participating to the microenvironment acidosis, represents a valuable target for drug strategy against advanced solid tumours. To overcome cancer cell resistance and improve the efficacy of therapeutics, the use of bio-reducible prodrugs also known as Hypoxia-activated prodrugs (HAPs), represents an interesting strategy to be applied to target hCA IX isozyme through the design of selective carbonic anhydrase IX inhibitors (CAIs). Here, we report the design, synthesis and biological evaluations including CA inhibition assays, toxicity assays on zebrafish and viability assays on human cell lines (HT29 and HCT116) of new HAP-CAIs, harboring different bio-reducible moieties in nitroaromatic series and a benzenesulfonamide warhead to target hCA IX. The CA inhibition assays of this compound series showed a slight selectivity against hCA IX versus the cytosolic off-target hCA II and hCA I isozymes. Toxicity and viability assays have highlighted that the compound bearing the 2-nitroimidazole moiety possesses the lowest toxicity (LC 50 of 1400 µ M) and shows interesting results on viability assays. Keywords: hypoxia-activated prodrug; carbonic anhydrase; inhibitors; sulfonamides; hypoxic tumour 1. Introduction Intratumoral heterogeneity, a main feature of solid tumours, is one of the causes of the intractability of cancers [ 1 ]. There are multiple mechanisms driving tumor heterogeneity, including genetic, epigenetic and microenvironmental factors such as hypoxia [ 2 ]. The presence of oxygen deprivation areas (typically less than 1%), defined as hypoxic domains, and resulting from inadequate tumor vascularization, have been identified in a wide variety of human tumours [ 3 ]. The adaptive cellular response to low Molecules 2020,25, 2347; doi:10.3390/molecules25102347 www.mdpi.com/journal/molecules Molecules 2020,25, 2347 2 of 14 oxygen tensions is coordinated by hypoxia inducible transcription factors HIF-1, which activate gene expression programs controlling multiple responses [ 4 ]. Among them are found the change in glucose metabolism towards anaerobic glycolysis (Warburg effect), which causes a decrease in the pH of the tumour microenvironment [ 5 ]. Several key proteins and buffer systems [ 6 ], including monocarboxylate transporters (MCTs), isoforms of anion exchanger, Na + /HCO 3– co-transporters, Na + /H + exchangers, and carbonic anhydrases (CAs) isoforms IX and XII are involved in this pH regulatory process to maintain a physiological intracellular pH accompanied with extracellular acidification [ 7 ]. This acidosis strongly contributes to the malignant progression, aggressive phenotype, and resistance to therapy (chemotherapy and radiation) of the cancer cells, leading to a poor prognosis regardless of treatment [ 8 ]. The hypoxic microenvironment of solid tumors has attracted significant attention as a target which can be exploited in drug design for the development of novel anticancer or imaging agents [ 9 ]. Two approaches have been considered in the literature: the first approach consists of the inhibition of molecular targets necessary for the survival of hypoxic cells, particularly carbonic anhydrase IX and XII. A wealth of research depicts carbonic anhydrases isoform IX (hCA IX) and hCA XII as biomarkers and therapeutic targets for various cancer types, and both of these enzymes are associated with cancer progression, metastasis, and impaired therapeutic response. As examples, head and neck tumours as well as oral cancers, which are highly hypoxic, were reported to overexpress hCA IX [ 10 – 13 ]. The development of small molecules as specific CA IX inhibitors represents a successful field with several potent inhibitors reported so far [ 14 – 17 ]. The second approach is based on the exploitation of the redox potential, between hypoxic and normoxic areas for the development of prodrugs that activate selectively in a highly reducing hypoxic environment (Hypoxia Activatable Prodrugs, HAP) [ 18 ]. HAPs are hypothesized to improve the therapeutic index of drugs that are ineffective against tumor cells in hypoxic microenvironments. The potential of HAPs is often evaluated clinically in combination with other cancer treatment (chemotherapy or radiotherapy) to affect both the normoxic and hypoxic fraction of the tumour [18–20]. Over the last two decades, many HAPs have been documented [ 18 ]. The molecular motifs used as cleavable entities are all susceptible to bioreduction, mainly by enzymatic processes (e.g., reductases) by a monoor di-electronic process depending on the enzyme involved. The most frequently found in the literature are N-oxide, quinone, or nitroaromatic derivatives, which have been the subject of in-depth studies up to the clinical stage [ 18 – 20 ]. Nitroaromatic HAPs have been described for targets such as poly ADP ribose polymerase (PARP) inhibitors [ 21 ], protein kinase RNA–like endoplasmic reticulum kinase (PERK) inhibitors [ 22 ], tyrosine kinase (TK) inhibitors [ 23 ] and also for different types of nanosystems [ 24 , 25 ]. Few numbers of carbonic anhydrase IX inhibitors (CAIs) have been designed using this HAP approach [ 26 – 30 ]. As part of an effort to discover novel small-molecule inhibitors of hCA to enhance cancer therapy, we report herein a new class of 2and 5-nitroimidazole, nitrofuran, nitrothiophene and nitrogen mustards- (alkylating agents) based bio-reducible drugs harboring a benzenesulfonamide to target hCA IX. 2. Results 2.1. Chemistry 2and 5-nitroimidazole, nitrofuran, nitrothiophene and nitrogen mustard were conjugated with benzene sulfonamides using a carbamate linker. Because of the low reactivity observed, the introduction of the carbamate linker proved to be challenging for some derivatives and two approaches were used to achieve the synthesis of these inhibitors starting from the nitroaromatic alcohols: (i) a reaction with carbonyldiimidazole to access to the carbamoyl imidazole derivatives that reacted, in a one-pot reaction, with aminomethylor aminoethylbenzenesulfonamide, or (ii) a reaction with phosgene to yield to the chloroformates which were then reacted with aminomethylor aminoethylbenzenesulfonamide in the presence of triethylamine (Scheme 1) [ 31 ]. Compounds 1b – 5b and 2c – 3c were isolated with yields Molecules 2020,25, 2347 3 of 14 ranging from 43% to 88% and characterized extensively by spectroscopic and spectrometric methods (see materials and methods part). Molecules 2020, 25, x 3 of 14 1b–5b and 2c–3c were isolated with yields ranging from 43% to 88% and characterized extensively by spectroscopic and spectrometric methods (see materials and methods part). Scheme 1. Synthesis of hypoxia-activated prodrug carbonic anhydrase IX inhibitors (HAP-CAIs) 1b4b and 2c-3c. 2.2. Carbonic Anhydrase Inhibition Assay The hypoxia-activated prodrug carbonic anhydrase IX inhibitors (HAP-CAIs) reported here were assayed using the CO2 hydrase assay against three physiologically relevant human CA isoforms, the cytosolic hCA I and II and the transmembrane, tumor-associated hCA IX (Table 1) [32]. Table 1. Inhibitory activity of compounds 1b–5b, 2c–3c, and the clinically used sulfonamide inhibitor acetazolamide (AAZ), against hCA I, hCA II, and hCA IX using a stopped flow CO2 hydrase assay. Compounds KI (nM) * Selectivity Ratio hCA I hCA II hCA IX KI hCA II / KI hCA IX 1b 166.7 30.6 7.6 1.19 2b 3.7 4.3 12.1 0.35 2c 2.3 4.0 14.1 0.28 3b 83.0 3.1 32.3 0.09 3c 2179.9 83.7 88.7 0.94 4b 83.8 15.5 5.7 2.72 5b 64.7 19.8 15.1 1.31 AAZ 250 12.0 25 0.48 * Mean from 3 different assays (errors in the range of ±5–10% of the reported values). The clinically used sulfonamide acetazolamide (AAZ, 5-acetamido-1, 3, 4thiadiazole-2sulfonamide) has been taken along as standard in these measurements. All compounds acted as inhibitors against the three isoforms hCA I, II and IX, although with variable potency (Table 1). Against the abundant, cytosolic isoform hCA I compound 3c showed weak inhibition potency (2180 nM). Compound 1b showed moderate inhibition activity towards hCA I (166.7 nM) and hCA II (30.6 nM) isoforms and higher inhibition (7.6 nM) towards hCA IX, while compounds 2b and 2c strongly inhibited all CA isoforms (KI ranging from 2.3 to 14.1 nM) with no differences observed in the selectivity ratios hCA II over hCA IX (2b = 0.35, 2c = 0.28). Compounds 3b and 3c showed a moderate inhibition of hCA IX, whilst compound 3b binding to hCA II was very tight (3.1 nM). The large difference in the binding capacity or selectivity ratios (3b = 0.09, 3c = 0.94) of compounds from the same family (nitrogen mustard) supports the substitution effect on the carbamate linker. Compounds 4b and 5b showed moderate inhibition activity towards hCA I, respectively (84 nM) and (64.7 nM), while they strongly inhibited hCA II and hCA IX (KI ranging from 5.7 to 19.8 nM). Nevertheless, R 1 OH R 1 O N H O R 2 or R 3 N N O 2 NN N O 2 N SO 2 NH 2 SO 2 NH 2 NO 2 N ClCl O H N R 2 = R 3 = R 1 = S O 2 N O O 2 N Yields 1b (R 1 =1a, R 2 ) 92% 2b (R 1 =2a, R 2 ) 88% 2c (R 1 =2a, R 3 ) 56% 3b (R 1 =3a, R 2 ) 40% 3c (R 1 =3a, R 3 ) 43% 4b (R 1 =4a, R 2 ) 55% 5b (R 1 =5a, R 2 ) 88% 1a 2a 3a 4a 5a i or ii i: carbonyldiimidazole, THF, then then aminoethyl - or aminomethylbenzene sulfonamide. ii: phosgene, THF, then aminoethylor aminomethylbenzene sulfonamide, NMe 3 . Scheme 1. Synthesis of hypoxia-activated prodrug carbonic anhydrase IX inhibitors (HAP-CAIs) 1b – 4b and 2c–3c. 2.2. Carbonic Anhydrase Inhibition Assay The hypoxia-activated prodrug carbonic anhydrase IX inhibitors (HAP-CAIs) reported here were assayed using the CO 2 hydrase assay against three physiologically relevant human CA isoforms, the cytosolic hCA I and II and the transmembrane, tumor-associated hCA IX (Table 1) [32]. Table 1. Inhibitory activity of compounds 1b – 5b , 2c – 3c , and the clinically used sulfonamide inhibitor acetazolamide (AAZ), against hCA I, hCA II, and hCA IX using a stopped flow CO2hydrase assay. Compounds KI(nM) * Selectivity Ratio hCA I hCA II hCA IX KIhCA II/KIhCA IX 1b 166.7 30.6 7.6 1.19 2b 3.7 4.3 12.1 0.35 2c 2.3 4.0 14.1 0.28 3b 83.0 3.1 32.3 0.09 3c 2179.9 83.7 88.7 0.94 4b 83.8 15.5 5.7 2.72 5b 64.7 19.8 15.1 1.31 AAZ 250 12.0 25 0.48 * Mean from 3 different assays (errors in the range of ±5–10% of the reported values). The clinically used sulfonamide acetazolamide ( AAZ , 5-acetamido-1, 3, 4thiadiazole-2-sulfonamide) has been taken along as standard in these measurements. All compounds acted as inhibitors against the three isoforms hCA I, II and IX, although with variable potency (Table 1). Against the abundant, cytosolic isoform hCA I compound 3c showed weak inhibition potency (2180 nM). Compound 1b showed moderate inhibition activity towards hCA I (166.7 nM) and hCA II (30.6 nM) isoforms and higher inhibition (7.6 nM) towards hCA IX, while compounds 2b and 2c strongly inhibited all CA isoforms (K I ranging from 2.3 to 14.1 nM) with no differences observed in the selectivity ratios hCA II over hCA IX ( 2b =0.35, 2c =0.28). Compounds 3b and 3c showed a moderate inhibition of hCA IX, whilst compound 3b binding to hCA II was very tight (3.1 nM). The large difference in the binding capacity or selectivity ratios ( 3b =0.09, 3c =0.94) of compounds from the same family (nitrogen mustard) supports the substitution effect on the carbamate linker. Compounds 4b and 5b showed moderate inhibition activity towards hCA I, respectively (84 nM) Molecules 2020,25, 2347 4 of 14 and (64.7 nM), while they strongly inhibited hCA II and hCA IX (K I ranging from 5.7 to 19.8 nM). Nevertheless, considering the difficulty of obtaining small compounds with a better affinity for the tumor-associated isozyme (hCA IX) over hCA II, the selectivity obtained for these series is comparable or better than that of the clinically used CA inhibitor acetazolamide AAZ. 2.3. Stability of Carbamate Linker under Acidic Conditions The stability to chemical hydrolysis of compounds 1b – 3c was evaluated by measuring the peak area or retention time of the compound after incubation at varying pH conditions up to 8 h. No degradation was observed at any pH condition for all tested compounds, indicating significant stability. Additionally, all tested compounds were found to be stable for at least 8 h to harsh acid-catalyzed hydrolysis ( pH =2.0 , 37 ◦ C, data not shown). Literature studies have shown that the electron-withdrawing nitro group conjugated with the carbamate linker resulted in a remarkable decrease in stability, which may explain the apparent low inhibitory potency of these compounds toward fatty acid amide hydrolase (FAAH), due to decomposition under the assay conditions [ 33 ]. Of all compounds ( 1b – 3c ), the nitro group was not in direct conjugation with the carbamate linker, thereby showing no stability loss when incubated under various pH conditions. 2.4. Biological Assays 2.4.1. Cell Viability and Clonogenic Assays The cytotoxicity of all compounds ( 1b – 3c ) was determined in a panel of human tumor cell lines. Compound 1b showed an IC 50 of 204.5 µ M (p<0.0001) in HT29 cells under anoxia (IC 50 A) and no detectable cytotoxicity (IC 50 >highest tested concentration) was observed under normoxia (Figure 1). Furthermore, in HCT116 cells, compound 1b resulted in an IC 50 of 148.6 and 59.3 µ M under normoxic and anoxic conditions, respectively, resulting in a hypoxia selectivity cytotoxicity ratio (HCR) of 2.5 (Table 2). All other compounds, except compound 3b in HCT116 cells, did not show any cytotoxicity at the tested concentrations. Compound 3b resulted in a cell-dependent cytotoxicity with an HCR of 2.7 in HCT116 cells, while no cytotoxicity in HT29 cells was observed. Molecules 2020, 25, x 4 of 14 considering the difficulty of obtaining small compounds with a better affinity for the tumorassociated isozyme (hCA IX) over hCA II, the selectivity obtained for these series is comparable or better than that of the clinically used CA inhibitor acetazolamide AAZ. 2.3. Stability of Carbamate Linker under Acidic Conditions The stability to chemical hydrolysis of compounds 1b–3c was evaluated by measuring the peak area or retention time of the compound after incubation at varying pH conditions up to 8 h. No degradation was observed at any pH condition for all tested compounds, indicating significant stability. Additionally, all tested compounds were found to be stable for at least 8 h to harsh acidcatalyzed hydrolysis (pH = 2.0, 37 °C, data not shown). Literature studies have shown that the electron-withdrawing nitro group conjugated with the carbamate linker resulted in a remarkable decrease in stability, which may explain the apparent low inhibitory potency of these compounds toward fatty acid amide hydrolase (FAAH), due to decomposition under the assay conditions [33]. Of all compounds (1b–3c), the nitro group was not in direct conjugation with the carbamate linker, thereby showing no stability loss when incubated under various pH conditions. 2.4. Biological Assays 2.4.1. Cell Viability and Clonogenic Assays. The cytotoxicity of all compounds (1b–3c) was determined in a panel of human tumor cell lines. Compound 1b showed an IC50 of 204.5 µM (p < 0.0001) in HT29 cells under anoxia (IC50A) and no detectable cytotoxicity (IC50 > highest tested concentration) was observed under normoxia (Figure 1). Furthermore, in HCT116 cells, compound 1b resulted in an IC50 of 148.6 and 59.3 µM under normoxic and anoxic conditions, respectively, resulting in a hypoxia selectivity cytotoxicity ratio (HCR) of 2.5 (Table 2). All other compounds, except compound 3b in HCT116 cells, did not show any cytotoxicity at the tested concentrations. Compound 3b resulted in a cell-dependent cytotoxicity with an HCR of 2.7 in HCT116 cells, while no cytotoxicity in HT29 cells was observed. Figure 1. Relative cell viability (%) in (A) HT29 and (B) HCT116 cells exposed to increasing concentrations of the derivative 1b under normoxic (green) and anoxic (red) conditions. The data represent the average ± SEM of three independent biological repeats. Table 2. HCR of all compounds IC50N vs. IC50A. Compounds HT29 HCT116 IC50N IC50A HCR (IC50N/IC50A) IC50N IC50A HCR (IC50N/IC50A) 1b > 500 204.5 >2.44 148.6 59.36 2.50 2b > 500 > 500 - > 500 > 500 - 2c > 500 > 500 - > 500 > 500 - 3b > 500 > 500 - 267.1 97.27 2.74 3c > 500 > 500 - > 500 > 500 - Figure 1. Relative cell viability (%) in ( A ) HT29 and ( B ) HCT116 cells exposed to increasing concentrations of the derivative 1b under normoxic (green) and anoxic (red) conditions. The data represent the average ±SEM of three independent biological repeats. Based on its selective cytotoxicity under anoxia in both cell lines, compound 1b was selected for further studies, investigating its effects on cell survival. Compound 1b did not reduce clonogenic cell survival under normoxia or anoxia at the tested concentrations (Figure 2). TH-302, a 2-nitroimidazole based hypoxia-activated prodrug-alkylating agent was used as positive control (data not shown) [ 34 ]. Molecules 2020,25, 2347 5 of 14 Molecules 2020, 25, x 5 of 14 Abbreviations: IC50: Concentration of an inhibitor where the response is reduced by half; Normoxia (N); Anoxia (A); Hypoxia selectivity cytotoxicity ratio (HCR). Based on its selective cytotoxicity under anoxia in both cell lines, compound 1b was selected for further studies, investigating its effects on cell survival. Compound 1b did not reduce clonogenic cell survival under normoxia or anoxia at the tested concentrations (Figure 2). TH-302, a 2-nitroimidazole based hypoxia-activated prodrug-alkylating agent was used as positive control (data not shown) [34]. Figure 2. Clonogenic cell survival of (A) HT29 and (B) HCT116 cells during normoxia (white bars) and anoxia (black bars) when exposed to compound 1b. The data represent the average ± SEM of three independent biological repeats. 2.4.2. Toxicity Evaluation on Zebrafish During development, zebrafish embryos are easily affected by chemical compounds compared to adult zebrafish or other animal models or cell models and are therefore suitable for assessing the subtle toxic effects of chemicals [28,30]. The toxicity of compound 1b, 4b and 5b was determined by using 24-h post-fertilization zebrafish embryos [35]. In these tests, the LC50, zebrafish phenotypic parameters and the swim pattern were analyzed for each compound. Determination of Half Maximal Lethal Concentration 50 (LC50) The lethality of 1b, 4b and 5b on the developing zebrafish embryos was concentration dependent (Figure 3). Among the three compounds tested for the toxicity, 1b was the less toxic and did not cause any mortality of the larvae, even at a 1 mM concentration at the end of 5 day of exposure to the compound. Compound 4b was more toxic compared to the other two compounds and caused significant mortality even at a 500-µM concentration (Figure 3). The LC50 values of the prodrugs at the end of the 5 days of exposure were in the range of 500 (4b), 1000 (5b) and 1400 µM (1b), as shown in Figure 3. The LC50 concentration of the compounds were higher compared to the inhibitors that we screened in our earlier studies [36,37], suggesting that these compounds can be characterized further for developing as drugs. Figure 3. Lethal concentration 50 (LC50) values of the prodrugs. The LC50 doses of the compounds were calculated based on the 50% mortality of the developing larvae at the end of five days after the exposure of embryos to different concentrations of inhibitors. (A) The ranges of LC50 values of compound 4b (< 500µM concentration). (B) The LC50 value for compound 5b was below 1000 µM and (C) compound 1b showed an LC50 value of about 1400 µM. The LC50 doses were determined after three independent experiments with similar experimental conditions (N = 72 larvae). Figure 2. Clonogenic cell survival of ( A ) HT29 and ( B ) HCT116 cells during normoxia (white bars) and anoxia (black bars) when exposed to compound 1b. The data represent the average ± SEM of three independent biological repeats. Table 2. HCR of all compounds IC50Nvs. IC50A. Compounds HT29 HCT116 IC50N IC50A HCR (IC50N/IC50A) IC50N IC50A HCR (IC50N/IC50A) 1b >500 204.5 >2.44 148.6 59.36 2.50 2b >500 >500 - >500 >500 - 2c >500 >500 - >500 >500 - 3b >500 >500 - 267.1 97.27 2.74 3c >500 >500 - >500 >500 - Abbreviations: IC 50 : Concentration of an inhibitor where the response is reduced by half; Normoxia (N); Anoxia (A); Hypoxia selectivity cytotoxicity ratio (HCR). 2.4.2. Toxicity Evaluation on Zebrafish During development, zebrafish embryos are easily affected by chemical compounds compared to adult zebrafish or other animal models or cell models and are therefore suitable for assessing the subtle toxic effects of chemicals [ 28 , 30 ]. The toxicity of compound 1b , 4b and 5b was determined by using 24-h post-fertilization zebrafish embryos [ 35 ]. In these tests, the LC 50 , zebrafish phenotypic parameters and the swim pattern were analyzed for each compound. Determination of Half Maximal Lethal Concentration 50 (LC50) The lethality of 1b , 4b and 5b on the developing zebrafish embryos was concentration dependent (Figure 3). Among the three compounds tested for the toxicity, 1b was the less toxic and did not cause any mortality of the larvae, even at a 1 mM concentration at the end of 5 day of exposure to the compound. Compound 4b was more toxic compared to the other two compounds and caused significant mortality even at a 500µ M concentration (Figure 3). The LC 50 values of the prodrugs at the end of the 5 days of exposure were in the range of 500 ( 4b ), 1000 ( 5b ) and 1400 µ M ( 1b ), as shown in Figure 3. The LC 50 concentration of the compounds were higher compared to the inhibitors that we screened in our earlier studies [ 36 , 37 ], suggesting that these compounds can be characterized further for developing as drugs. Different phenotypic parameters were also analyzed (hatching, heartbeat, edema, swim bladder development, yolk sac utilization and body shape) from the developing larvae of 1–5 days, after exposure to the compounds. Figure 4shows the representative images of larvae treated with the concentrations considered safe for further characterization. Among the compounds screened, the prodrug 1b was found to be less toxic with no or minimal phenotypic abnormalities, even at 1 mM. In our earlier study, compounds 1b – 3c also showed minimal or no phenotypic abnormalities at 1 mM concentrations. In the present study, compounds 4b and 5b showed phenotypic defects, such as edema and the absence of a swim bladder (arrows) at lower concentrations compared to 1b , as shown in Figure 4. Molecules 2020,25, 2347 6 of 14 Molecules 2020, 25, x 5 of 14 Abbreviations: IC50: Concentration of an inhibitor where the response is reduced by half; Normoxia (N); Anoxia (A); Hypoxia selectivity cytotoxicity ratio (HCR). Based on its selective cytotoxicity under anoxia in both cell lines, compound 1b was selected for further studies, investigating its effects on cell survival. Compound 1b did not reduce clonogenic cell survival under normoxia or anoxia at the tested concentrations (Figure 2). TH-302, a 2-nitroimidazole based hypoxia-activated prodrug-alkylating agent was used as positive control (data not shown) [34]. Figure 2. Clonogenic cell survival of (A) HT29 and (B) HCT116 cells during normoxia (white bars) and anoxia (black bars) when exposed to compound 1b. The data represent the average ± SEM of three independent biological repeats. 2.4.2. Toxicity Evaluation on Zebrafish During development, zebrafish embryos are easily affected by chemical compounds compared to adult zebrafish or other animal models or cell models and are therefore suitable for assessing the subtle toxic effects of chemicals [28,30]. The toxicity of compound 1b, 4b and 5b was determined by using 24-h post-fertilization zebrafish embryos [35]. In these tests, the LC50, zebrafish phenotypic parameters and the swim pattern were analyzed for each compound. Determination of Half Maximal Lethal Concentration 50 (LC50) The lethality of 1b, 4b and 5b on the developing zebrafish embryos was concentration dependent (Figure 3). Among the three compounds tested for the toxicity, 1b was the less toxic and did not cause any mortality of the larvae, even at a 1 mM concentration at the end of 5 day of exposure to the compound. Compound 4b was more toxic compared to the other two compounds and caused significant mortality even at a 500-µM concentration (Figure 3). The LC50 values of the prodrugs at the end of the 5 days of exposure were in the range of 500 (4b), 1000 (5b) and 1400 µM (1b), as shown in Figure 3. The LC50 concentration of the compounds were higher compared to the inhibitors that we screened in our earlier studies [36,37], suggesting that these compounds can be characterized further for developing as drugs. Figure 3. Lethal concentration 50 (LC50) values of the prodrugs. The LC50 doses of the compounds were calculated based on the 50% mortality of the developing larvae at the end of five days after the exposure of embryos to different concentrations of inhibitors. (A) The ranges of LC50 values of compound 4b (< 500µM concentration). (B) The LC50 value for compound 5b was below 1000 µM and (C) compound 1b showed an LC50 value of about 1400 µM. The LC50 doses were determined after three independent experiments with similar experimental conditions (N = 72 larvae). Figure 3. Lethal concentration 50 (LC 50 ) values of the prodrugs. The LC 50 doses of the compounds were calculated based on the 50% mortality of the developing larvae at the end of five days after the exposure of embryos to different concentrations of inhibitors. ( A ) The ranges of LC 50 values of compound 4b (<500 µ M concentration). ( B ) The LC 50 value for compound 5b was below 1000 µ M and ( C ) compound 1b showed an LC 50 value of about 1400 µ M. The LC 50 doses were determined after three independent experiments with similar experimental conditions (N =72 larvae). Molecules 2020, 25, x 6 of 14 Different phenotypic parameters were also analyzed (hatching, heartbeat, edema, swim bladder development, yolk sac utilization and body shape) from the developing larvae of 1–5 days, after exposure to the compounds. Figure 4 shows the representative images of larvae treated with the concentrations considered safe for further characterization. Among the compounds screened, the prodrug 1b was found to be less toxic with no or minimal phenotypic abnormalities, even at 1 mM. In our earlier study, compounds 1b–3c also showed minimal or no phenotypic abnormalities at 1 mM concentrations. In the present study, compounds 4b and 5b showed phenotypic defects, such as edema and the absence of a swim bladder (arrows) at lower concentrations compared to 1b, as shown in Figure 4. Figure 4. Images of zebrafish larvae in the control and prodrug-treated groups. Representative images of 2–5 dpf zebrafish larvae exposed to different concentrations (80 µM–1000µM) of 1b, 4b and 5b. The upper panel shows images for normal development of zebrafish larvae in the control group (not treated with inhibitors) and the 1% dimethyl sulfoxide (DMSO)-treated group. The lower panel shows the larvae treated with concentrations of the compounds at which they induced minimal or no phenotypic defects. Prodrug 4b showed (arrow) an absence of a swim bladder at a 250-µM concentration at 5 days. Prodrug 5b induced edema (arrow) as early as 2 days post exposure to the compound. Compound 1b showed an absence of a swim bladder (arrow) at a 1000-µM concentration at 5 days. Furthermore, the toxic effects of compounds across the concentrations (20 µM–2 mM) were assessed on individual parameters of the 5 days post exposed zebrafish larvae. Figure 5 presents plot graphs of the dose-dependent effects of prodrugs on larvae. The results indicate that the 1b compound showed minimal or no adverse effects on the observable parameters of the larvae (Figure 5A–E). However, prodrugs 4b and 5b exhibited adverse effect on hatching, swim bladder Figure 4. Images of zebrafish larvae in the control and prodrug-treated groups. Representative images of 2–5 dpf zebrafish larvae exposed to different concentrations (80 µ M–1000 µ M) of 1b , 4b and 5b . The upper panel shows images for normal development of zebrafish larvae in the control group (not treated with inhibitors) and the 1% dimethyl sulfoxide (DMSO)-treated group. The lower panel shows the larvae treated with concentrations of the compounds at which they induced minimal or no phenotypic defects. Prodrug 4b showed (arrow) an absence of a swim bladder at a 250µ M concentration at 5 days. Prodrug 5b induced edema (arrow) as early as 2 days post exposure to the compound. Compound 1b showed an absence of a swim bladder (arrow) at a 1000-µM concentration at 5 days. Molecules 2020,25, 2347 7 of 14 Furthermore, the toxic effects of compounds across the concentrations (20 µ M–2 mM) were assessed on individual parameters of the 5 days post exposed zebrafish larvae. Figure 5presents plot graphs of the dose-dependent effects of prodrugs on larvae. The results indicate that the 1b compound showed minimal or no adverse effects on the observable parameters of the larvae (Figure 5A–E). However, prodrugs 4b and 5b exhibited adverse effect on hatching, swim bladder development, the utilization of the yolk sac, and the shape of the body and induced pericardial edema, as shown in Figure 5A–E. Molecules 2020, 25, x 7 of 14 development, the utilization of the yolk sac, and the shape of the body and induced pericardial edema, as shown in Figure 5A–E. Figure 5. Effect of the prodrugs 4b, 5b, and 1b on the phenotypic parameters of zebrafish larvae. The plot graphs show the phenotypic abnormalities in the zebrafish larvae after 5 days of exposure to compounds. (A) Hatching, (B) edema, (C) swim bladder development, (D) yolk sac utilization and (E) body shape for each concentration (N = 72). Swim Pattern Analysis to Assess the Subtle Toxic Effects of the Prodrugs To assess the subtle toxic effects of the prodrugs on the swim patterns of the larvae, they were assessed at the end of 5 days of exposure to the compounds. The swim pattern analysis showed no abnormal or ataxic movement pattern in the larvae exposed to the concentration (160 µM) that did not induce any of the phenotypic defects and is considered to be safe. In our earlier studies, the nitroimidazole-based compounds DTP338 and DTP348 showed an ataxic movement pattern even at a 100-µM concentration due to neurotoxicity [28,30]. Therefore, the prodrug screening in the current study can be considered safe for further preclinical characterization and development as potential drugs. 3. Discussion We developed here new HAPs incorporating a benzenesulfonamide through a carbamate linker, designed with different bio-reducible moieties for the selective delivery of anticancer drugs on hypoxic tumors. Through this study, we demonstrated that these HAPs can selectively inhibit the hCA IX at the nanomolar level. In contrast to other nitroaromatic drugs, HAPs 1b–3c did not show stability loss when incubated under various pH conditions as the nitro group was not in direct conjugation with the carbamate linker. Compound 1b, harboring a 2-nitroimidazole as a reducible moiety, showed the best cytotoxic action against HT29 and HCT116 cells (IC 50 of 204.5 and 59.36 µM under hypoxia) and an HCR of approximately 2.5. Our data also show that 1b is the less toxic, with no mortality observed on zebrafish larvae even at a 1-mM concentration at the end of 5 days of Figure 5. Effect of the prodrugs 4b , 5b , and 1b on the phenotypic parameters of zebrafish larvae. The plot graphs show the phenotypic abnormalities in the zebrafish larvae after 5 days of exposure to compounds. ( A ) Hatching, ( B ) edema, ( C ) swim bladder development, ( D ) yolk sac utilization and ( E ) body shape for each concentration (N =72). Swim Pattern Analysis to Assess the Subtle Toxic Effects of the Prodrugs To assess the subtle toxic effects of the prodrugs on the swim patterns of the larvae, they were assessed at the end of 5 days of exposure to the compounds. The swim pattern analysis showed no abnormal or ataxic movement pattern in the larvae exposed to the concentration (160 µ M) that did not induce any of the phenotypic defects and is considered to be safe. In our earlier studies, the nitroimidazole-based compounds DTP338 and DTP348 showed an ataxic movement pattern even at a 100µ M concentration due to neurotoxicity [ 28 , 30 ]. Therefore, the prodrug screening in the current study can be considered safe for further preclinical characterization and development as potential drugs. 3. Discussion We developed here new HAPs incorporating a benzenesulfonamide through a carbamate linker, designed with different bio-reducible moieties for the selective delivery of anticancer drugs on hypoxic Molecules 2020,25, 2347 8 of 14 tumors. Through this study, we demonstrated that these HAPs can selectively inhibit the hCA IX at the nanomolar level. In contrast to other nitroaromatic drugs, HAPs 1b–3c did not show stability loss when incubated under various pH conditions as the nitro group was not in direct conjugation with the carbamate linker. Compound 1b , harboring a 2-nitroimidazole as a reducible moiety, showed the best cytotoxic action against HT29 and HCT116 cells (IC 50 of 204.5 and 59.36 µ M under hypoxia) and an HCR of approximately 2.5. Our data also show that 1b is the less toxic, with no mortality observed on zebrafish larvae even at a 1-mM concentration at the end of 5 days of exposure. Of note, prodrug 1b also did not induce phonotypic abnormalities in the larvae as well as abnormal or ataxic swim movement at the concentration that did not induce any of the phenotypic defects and is considered as safe. As other nitroimidazole derivatives, such as DTP338 and DTP348 [ 30 ], induced a neurotoxic effect even at a 100µ M concentration, the data gathered here suggest a potential for derivative 1b to be optimized for the development of new safer HAPs. 4. Materials and Methods 4.1. Chemistry General. All reagents and solvents were of commercial quality and used without further purification unless otherwise specified. All reactions were carried out under an inert atmosphere of nitrogen. TLC analyses were performed on silica gel 60 F254 plates (Merck Art. no. 1.05554). Spots were visualized under 254 nm UV illumination or by ninhydrin solution spraying. 1 Hand 13 C-NMR spectra were recorded on a Bruker DRX-400 spectrometer (Bruker, Hanau, Germany) using dimethyl sulfoxide-d6 (DMSO-d 6 ) as solvent and tetramethylsilane as an internal standard. For 1 Hand 13 C-NMR spectra, chemical shifts are expressed in δ (ppm) downfield from tetramethylsilane, and coupling constants (J) are expressed in hertz. All compounds that were tested in the biological assays were analyzed by high-resolution mass spectra (HRMS) using a Bruker micrOTOF-Q II mass spectrometer (Bruker, Hanau, Germany) fitted with an electrospray ion source General procedure for preparation of chloroformate: The corresponding hydroxy starting compound ( 1a , 2a and 3a ) (2 mmol) in tetrahydrofuran (10 mL) was added to phosgene (4 mL, 8 mmol) and tetrahydrofuran (THF) (15 mL) at 0 ◦ C. The reaction mixture was stirred for 16 h, then the solvent was removed in vacuo. The crude chloroformate was used without further purification. General procedure for synthesis of 1b , 2b , 2c , 3b , and 3c : A suspension of chloroformate (23.4 mmol) in dry THF (100 mL) was treated with trimethylamine (50 mmol). The corresponding amine (23.4 mmol) was added afterwards and the reaction was stirred overnight at room temperature. The solvent was removed in vacuo. The residue was dissolved in chloroform and washed twice with 1.0 N sodium hydroxide and water. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified by chromatography on silica gel using methylene chloride−methanol 98:2 as eluent. General procedure for synthesis of 4b and 5b : To a suspension of carbonyldiimidazole (CDI) (0.31 mmol) in dry THF (5 mL) the corresponding hydroxy starting compound ( 4a and 5a ) (0.28 mmol) was added. The reaction was stirred for 3h at room temperature and the 4-(2-aminoethyl) benzene sulfonamide was added. The reaction mixture was stirred overnight at room temperature and concentrated under reduced pressure. The residue was dissolved in EtOAc (12.5 mL) and washed with water (2 × 7.5 mL) and brine (7.5 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified by chromatography on silica gel using cyclohexane-ethyl acetate 3:7 as eluent. (1-methyl-2-nitro-1H-imidazol-5-yl) methyl (4-sulfamoylphenethyl) carbamate ( 1b ): yield =92%; 1 H-NMR (400 MHz, DMSO-d 6 ) δ 7.73 (d, J=8.3, 2H), 7.48 (t, J=5.6, 1H), 7.38 (d, J=8.3, 2H), 7.30 (s, 2H), 7.21 (s, 1H), 5.12 (s, 2H), 3.89 (s, 3H), 3.26 (dd, J=13.2, 6.7, 2H), 2.79 (t, J=7.1, 2H). 13 C-NMR (101 MHz, DMSO-d 6 ) δ 155.36, 145.99, 143.42, 142.09, 133.83, 129.17, 128.41, 125.68, 54.82, 48.62, 34.69, 34.16–33.11. HRMS (ESI+) [M +H]+calculated for [C14H18N5O6S]+: 384.0978, found: 384.0982. Molecules 2020,25, 2347 9 of 14 2-(2-methyl-5-nitro-1H-imidazol-1-yl) ethyl (4-sulfamoylphenethyl) carbamate ( 2b ): yield =88%; 1 H-NMR (400 MHz, DMSO) δ 8.03 (s, 1H), 7.73 (d, J=8.3, 2H), 7.38–7.32 (m, 3H), 7.31 (s, 2H), 4.48 (t, J=4.9, 2H), 4.30 (t, J=4.9, 2H), 3.17 (dd, J=13.1, 6.7, 2H), 2.72 (t, J=7.1, 2H), 2.39 (s, 3H). 13 C-NMR (101 MHz, DMSO) δ 155.69, 151.86, 143.61, 142.11, 138.56, 133.18, 129.26, 125.81, 61.93, 35.01, 14.02. HRMS (ESI+) [M +H]+calculated for [C15H20N5O6S]+: 398.1134, found: 398.1136. 2-(2-methyl-5-nitro-1H-imidazol-1-yl) ethyl (4-sulfamoylbenzyl) carbamate ( 2c ): yield =56%; 1 H-NMR (400 MHz, DMSO-d 6 ) δ 8.04 (s, 1H), 7.88 (t, J=6.1, 1H), 7.76 (d, J=8.4, 2H), 7.34 (d, J=8.4, 2H), 7.32 (s, 2H), 4.54 (t, J=5.0, 2H), 4.36 (t, J=5.0, 2H), 4.19 (d, J=6.1, 2H), 2.44 (s, 3H). 13 C-NMR (101 MHz, DMSO-d 6 ) δ 155.87, 151.72, 143.53, 142.68, 138.45, 133.07, 127.26, 125.65, 62.11, 45.52, 43.37, 13.98. HRMS (ESI+) [M +H]+calculated for [C14H18N5O6S]+: 384.0978, found: 384.0972. 2-(2-(bis(2-chloroethyl)amino)-5-nitrobenzamido) ethyl (4-sulfamoylphenethyl) carbamate ( 3b ): yield =40%; 1 H-NMR (400 MHz, DMSO-d 6 ) δ 8.72 (t, J=5.3, 1H), 8.15–8.05 (m, 2H), 7.73 (d, J=8.1, 2H), 7.39 (t, J=6.9 , 2H), 7.29 (s, 2H), 7.22 (d, J=8.0, 1H), 4.11 (t, J=5.4, 2H), 3.74 (dt, J=9.9, 4.9, 8H), 3.46 (d, J=5.4 , 3H), 3.25 (dd, J=13.8, 6.5, 2H), 2.80 (t, J=7.3, 2H). 13 C-NMR (101 MHz, DMSO-d 6 ) δ 167.45, 156.11, 151.59, 143.54, 142.05, 138.21, 129.11, 126.33–123.92, 118.08, 62.04, 59.78, 53.12, 41.38, 34.44, 31.23, 30.39. HRMS (ESI+) [M +H]+calculated for [C22H28Cl2N5O7S]+: 575.1008, found: 575.1011. 2-(2-(bis(2-chloroethyl)amino)-5-nitrobenzamido) ethyl (4-sulfamoylbenzyl)carbamate ( 3c ): yield =43%; 1 H-NMR (400 MHz, DMSO) δ 8.75 (t, J=5.5, 1H), 8.17–8.08 (m, 2H), 7.80 (dd, J=12.0, 5.9, 1H), 7.76 (d, J=8.2, 2H), 7.43 (d, J=8.1, 2H), 7.31 (s, 2H), 7.23 (d, J=9.1, 1H), 4.26 (t, J=8.1, 2H), 4.15 (t, J=5.2, 2H), 3.80–3.69 (m, 8H), 3.17 (s, 2H). 13 C-NMR (101 MHz, DMSO) δ 167.56, 156.53, 151.68, 143.90, 142.68, 138.32, 127.41, 125.76, 117.97, 56.19, 53.22, 48.69, 41.33, 21.08. HRMS (ESI+) [M +H] + calculated for [C21H26Cl2N5O7S]+: 562.0930, found: 562.0935. (5-nitrofuran-2-yl)methyl (4-sulfamoylphenethyl) carbamate ( 4b ): yield =55%; 1 H-NMR (400 MHz, DMSO-d 6 ) δ 7.73 (d, J=8.4, 2H), 7.71 (d, J=4.2, 1H), 7.63 (t, J=5.6 Hz, 1H), 7.39 (d, J=8.4 Hz , 2H), 7.37 (s, 2H), 6.84 (d, J=4.2 Hz, 1H), 5.08 (s, 2H), 3.25 (t, J=7.1 Hz, 2H), 2.78 (t, J=7.1 Hz, 2H). 13 C-NMR (400 MHz, DMSO-d 6 ) δ 156.45, 155.10, 145.30, 142.26, 130.52, 127.00, 113.99, 113.78, 58.50, 42.62, 36.22. HRMS (ESI+) [M +H]+calculated for [C14H16N3O7S]+: 370.0709, found: 370.0708. (5-nitrothiophen-2-yl)methyl(4-sulfamoylphenethyl)carbamate ( 5b ): yield =88%; 1 H-NMR (400 MHz, DMSO-d 6 ) δ 8.05 (d, J=4.2 Hz, 1H), 7.74 (d, J=8.4 Hz, 2H), 7.63 (t, J=5.6 Hz, 1H), 7.40 (d, J=8.4 Hz , 2H), 7.33 (s, 2H), 7.22 (d, J=4.2 Hz, 1H), 5.23 (s, 2H), 3.24 (t, J=7.1 Hz, 2H), 2.82 (t, J=7.1 Hz, 2H). 13 C-NMR (400 MHz, DMSO-d 6 ) δ 156.66, 149.35, 145.27, 142.25, 130.53, 127.52, 126.99, 61.48, 42.59, 36.21. HRMS (ESI+) [M +H]+calculated for [C14H16N3O6S2]+: 386.0481, found: 386.0479. 4.2. Carbonic Anhydrase Inhibition Assays An Sx.18Mv-R Applied Photophysics (Oxford, UK) stopped-flow instrument was used for assaying the CA catalyzed CO 2 hydration activity. Phenol red (at a concentration of 0.2 mM) was used as an indicator, working at the absorbance maximum of 557 nm, with 20 mM HEPES (pH 7.5) as buffer, and 20 mM Na 2 SO 4 (for maintaining the constant ionic strength), following the initial rates of the CA-catalyzed CO 2 hydration reaction for a period of 10–100 s. The CO 2 concentrations ranged from 1.7–17 mM for the determination of the kinetic parameters and inhibition constants. In particular, CO 2 was bubbled in distilled deionized water for 30 min so that the water was saturated (the concentration at a specific temperature is known from literature). In addition, a CO 2 assay kit (from Merck, Darmstadt, Germany) was used to measure the concentration in variously diluted solutions obtained from the saturated one (which was kept at the same temperature and a constant bubbling during the experiments). For each inhibitor, at least six traces of the initial 5%–10% of the reaction were used for determining the initial velocity [ 32 ]. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1mM) were prepared in distilled deionized water and dilutions up to 0.01 nM were done thereafter with distilled deionized