DOI:
10.1039/C4RA03819C
(Paper)
RSC Adv., 2014,
4, 33029-33038
Novel metronidazole-sulfonamide derivatives as potent and selective carbonic anhydrase inhibitors: design, synthesis and biology analysis
Received
2nd May 2014
, Accepted 23rd June 2014
First published on 23rd June 2014
Abstract
Metronidazole–sulfonamide derivatives 4a–4l, a new class of human carbonic anhydrase inhibitors (hCA), were designed, synthesized, isolated, and evaluated for their ability to inhibit the enzymatic activity of the physiologically dominant isozymes hCA II and the tumor-associated isozyme hCA IX (h = human). Many of these compounds inhibited CA II and IX in the range of 16–137 and 38–169 nM, respectively. Among all the compounds, the most potent inhibitor against hCA II and IX were compounds 4b (IC50 = 16 nM) and 4h (IC50 = 38 nM). Conversely compounds 4e and 4d displayed the most potent growth inhibitory activity against B16-F10 and MCF-7 cancer cell lines in vitro, respectively, with an IC50 value of 150 nM for B16-F10 and 6.5 nM for MCF-7. These metronidazole–sulfonamide derivatives may prove interesting lead candidates to target tumor-associated CA isozymes, wherein the CA domain is located extracellularly. All the new compounds were evaluated for cytotoxicity against human macrophages by MTT assay.
Introduction
In many types of solid tumors, hypoxia is an advantageous condition for the culture of pluripotent stem cells. It induces the activation of a transcription factor, aptly named hypoxia-inducible factor (HIFa).1 High levels of HIF regulate a signaling cascade, which adapts cellular functions to allow solid tumor cells to not only survive hypoxia but also to proliferate and metastasize. Solid tumors are remarkably unregulated in the functioning of some house-keeping enzymes, among which are the carbonic anhydrases (CAs, EC 4.2.1.1) involved in pH homeostasis, ion transport, and biosynthetic processes.2 CAs are Zn(II) metalloenzymes involved in pH buffering of extra- and intra-cellular spaces by catalyzing the reversible hydration of carbon dioxide and water to bicarbonate and a proton: CO2 + H2O ↔ HCO3− + H+.3 This reaction is known to regulate a broad range of physiological functions by respiration and the transport of CO2/HCO3−.4 In humans, the carbonic anhydrase enzymes, specifically isozymes IX (CAs IX), have recently been shown to be druggable targets for imaging and treatment of hypoxic tumors.5 This enzyme is a multidomain protein with the CA subdomain situated outside the cell and possessing a very high CO2 hydrase catalytic activity, which is also inhibited by the classical CA inhibitors belonging to the sulfonamide, sulfamate, and sulfamide classes of compounds.6 CA IX may be functionally linked to the regulation of the tumour pH, because it contributes to extracellular acidification.7
CA IX expression is strongly increased in many types of solid tumors such as mesotheliomas, kidneys, lungs and breast. Furthermore, such hypoxic tumors do not generally respond to the classic chemo- and radio-therapy and the strong acidification produced by CA IX overexpression also triggers the development of metastases.8 As suggested by Svastova et al., the assumption that pHe in different tumor cell cultures recovers more naturally, to an extent along with a remarkably increased apoptosis of the tumor cells, when CA IX is inhibited by potent and selective sulfonamide inhibitors, is being widely accepted.9 For example, Pouyssegur's group showed recently that in hypoxic LS174Tr tumor cells either one or both CA isoforms were expressed, in response to a “CO2 load,” and both enzymes contributed to extracellular acidification and maintained a more alkaline resting intracellular pH (pHi), which is an action that preserves ATP levels and cell survival in a range of acidic outside pH (6.0–6.8) and low bicarbonate medium. In vivo experiments showed that silencing of CA IX alone leads to a 40% reduction in xenograft tumor volume, with up-regulation of the second gene, encoding for CA XII, whereas invalidation of both CAI X and CA XII gives an impressive 85% reduction of tumor growth.10 Correlated with this fact, it has been shown earlier that some CA inhibitors showed anticancer activity in vivo.11 Moreover, CA IX inhibition may compose a fascinating new approach for the management of hypoxic tumors, which generally do not respond to the classical radio- and chemo-therapy.12
One approach aimed at improving the selectivity of tumor cell killing by antitumor drugs is the use of less toxic prodrug forms that can be selectively activated in the tumor tissue (tumor-activated prodrugs; TAP), utilizing some unique aspects of tumor physiology such as selective enzyme expression or hypoxia.13 To characterize the potential effect of inhibition of CA IX and XII in tumor hypoxia, there is an implied need to develop small molecules, which are capable of targeting tumor cells in the further research.14 Since the discovery of E-7010 in 1992,15 sulfonamides have been proven to be an important class of anticancer agents, which interact with various cellular targets. In fact, indisulam (E7070),16 HMN-214,17 T138067 (ref. 18) are in phase II clinical trials as an antitumor sulfonamide with a complex mechanism of action that also involves CA inhibition of several isozymes participating in tumor genesis (Fig. 1).
It is also known that sulfonamides are found to be an important class of drugs possessing various types of biological properties such as anti-cancer,19 and antibacterial.20 Many of the structurally novel sulfonamide derivatives have recently been reported to show substantial antitumor activity and low toxicities, both in vitro and/or in vivo. Some of these derivatives are currently being evaluated in clinical trials, and there is much optimism that they may lead to novel alternative anticancer drugs, which are devoid of the side effects of the presently available pharmacological agents.21
Moreover, in a previous study, nitroimidazole derivatives have attracted considerable attention as they showed a tendency to penetrate and accumulate in regions of tumors,22 and can undergo bioreduction to yield electrophilic substances which can damage proteins and nucleic acids.23 All these findings encouraged us to explore the synthesis of metronidazole–sulfonamide derivatives as potential CA inhibition agents.
In this paper, we report the synthesis of a series of metronidazole–sulfonamide derivatives, which might exhibit synergistic effect in anticancer activity, as well as the evaluation of their CA inhibition, their anticancer activity and their interaction with CA by docking studies.
 |
| Fig. 1 Known human CA inhibitors including some clinically used drugs. | |
Results and discussion
Thiry et al.24 reported that, from the analysis of the CA active site and the structure of inhibitors described in the literature,25 a general pharmacophore (Fig. 2) for the compounds that acted as carbonic anhydrase inhibitors was identified. In order to make this pharmacophore interact with CA inhibitors, it should include the structural elements that are required to be present in the compounds. A sulfonamide moiety should be included, which coordinates with the zinc ion of the active site of the CA, and it should be attached to a benzene ring scaffold. The side chain, which might interact with the hydrophobic and hydrophilic parts of the CA active site, can substitute an aromatic or heterocyclic sulfonamide scaffold. Therefore, different hydrophobic side chains were incorporated in the indanesulfonamide scaffold with an amide linker that can interact with the hydrophilic part of the active site, and a hydrophobic moiety that can interact with the hydrophobic part of the CA active site.
 |
| Fig. 2 Structural elements of CA inhibitors in the CA enzymatic active site. | |
Fig. 3 illustrates representative examples of the newly synthesized compounds showing compliance with the above-mentioned pharmacophore model. The new compounds were synthesized according to Scheme 1.
 |
| Fig. 3 Representative examples of the synthesized compounds showing compliance to the general pharmacophore of sulfonamide compounds, acting as CA inhibitors. | |
 |
| Scheme 1 Reagents and conditions: (a) 30 equiv. tosyl chloride, CH2Cl2, RT, 12 h, 75%; (b) 15.7 equiv. 3a, p-hydroxybenzaldehyde; 3b, m-hydroxybenzaldehyde, K2CO3, DMF, 80 °C, 20 h, 3a, 71.6%; 3b, 51.3%; (c) 1.0 equiv. p-RNHSO2PhNH2, CH3CH2OH, 50 °C, 8 h, 50.0–84.3%. | |
Chemistry
All novel metronidazole–sulfanilamide derivatives CA inhibitors (4a–4l) described herein were synthesized following the synthetic pathway depicted in Scheme 1. The starting MET-OTs (2-(2-methyl-5-nitro-1H-imidazole-1-yl)-ethyl-4-methylbenzene sulfonate, compound 2) has been previously synthesized from metronidazole using excess of tosyl chloride in the presence of triethylamine in dichloromethane.26 The synthesis of metronidazole–benzaldehyde 3a–3b was performed by a nucleophilic displacement reaction of the corresponding MET-OTs in the presence of o- or p-hydroxybenzaldehyde and K2CO3 in DMF. Subsequently, compounds 4a–4l were prepared from the reaction of 3a, 3b with the corresponding sulfanilamide in ethanol at room temperature. The reactions were monitored by thin layer chromatography (TLC), and the crude products were purified by recrystallization with ethanol, ethyl acetate and acetone (1
:
1
:
0.05). All of the target compounds gave satisfactory analytical and spectroscopic data, i.e. IR, 1H NMR, 13C NMR, ESI MS, which are in accordance with their depicted structures.
Biological activity
Antiproliferation assays and MTT assays. In the present work, twelve of the newly synthesized compounds 4a–4l were evaluated for their in vitro growth inhibitory activities against two cell lines, which are mouse melanoma cells (B16-F10) and breast carcinoma cell lines (MCF-7) in comparison to the known anticancer drugs: doxorubicin and semaxanib as reference drugs. The values obtained for the twelve compounds against the B16-F10 and MCF-7 cell lines were shown in Table 1. As shown in Table 1, it was observed that metronidazole–sulfanilamide derivatives were found to inhibit the growth of two tumor cell lines with moderate IC50 values. As previously shown, for the MCF-7 cell line, which over-expressed CA, metronidazole–sulfanilamide derivatives, in particular, displayed more potent antiproliferative action than in the other tumor cell lines. The IC50 values of most metronidazole–sulfanilamide derivatives ranged from 6.5 nM to 120 nM in MCF-7. Among them, compound 4d displayed the most potent antiproliferative activity with IC50 of 6.5 nM, comparable to the positive control doxorubicin (IC50 = 65.3 nM) and semaxanib (IC50 = 3.1 nM). Nevertheless, compounds 4a–4l displayed a more potent inhibition to MCF-7 than to F10 with 1–10 folds. The more potent antiproliferative activity for MCF-7 together with virtual screening results both indicated that the over-expressed CA might be a potential target with which these metronidazole–sulfanilamide derivatives interacted.
Table 1 In vitro anticancer activities (IC50, nM) against two human tumor cell lines
Compounds |
IC50a (nM) |
IC50a (nM) |
B16-F10b |
MCF-7b |
Antiproliferation activity was measured using the MTT assay. Values were the average of three independent experiments run in triplicate. Variation was generally 5–10%. Cancer cells kindly supplied by State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University. |
4a |
270 |
87 |
4b |
310 |
6.8 |
4c |
200 |
9.5 |
4d |
190 |
6.5 |
4e |
220 |
7.2 |
4f |
220 |
120 |
4g |
230 |
93 |
4h |
150 |
47 |
4i |
300 |
58 |
4j |
160 |
30 |
4k |
270 |
7.8 |
4l |
180 |
90 |
Doxorubicin |
72.3 |
65.3 |
Semaxanib |
3.6 |
3.1 |
CA II and IX enzyme assay. Inhibition of two physiologically relevant CA isoforms (hCAs II and hCAs IX) with compounds 4a–4l AAZ and SA, tow clinically used drugs were presented in Table 2.27 This study has included hCAs II (cytosolic, widespread enzymes) and hCAs IX (transmembrane, tumor-associated CAs) account of their relevance as targets/off targets when developing CAIs. In fact, CA II is the drug target for developing antiglaucoma CAIs.28
Table 2 In vitro anticancer activities (IC50, nM) against two human tumor cell lines
Compounds |
IC50a (nM) |
IC50a (nM) |
hCA IIb |
hCA IXb |
Errors were in the range of 5–10% of the reported values from three different assays. Two different human recombinant enzymes, by the esterase assay (4-nitrophenylacetate as substrate). |
4a |
137 |
169 |
4b |
16 |
93 |
4c |
70 |
135 |
4d |
48 |
110 |
4e |
22 |
81 |
4f |
33 |
112 |
4g |
53 |
42 |
4h |
89 |
38 |
4i |
114 |
69 |
4j |
81 |
61 |
4k |
86 |
126 |
4l |
52 |
56 |
AAZ |
16 |
28 |
SA |
256 |
298 |
CA IX is overexpressed in breast cancer, and studies have shown that CA IX and CA XII are variably expressed in breast cancer cell lines. Moreover, CA IX expression is associated with poor survival in an unselected cohort of patients with invasive breast carcinoma, and it is significantly associated with distant metastasis (Lou et al., unpublished results).10 Therefore, we chose breast cancer as a malignancy model for in vivo testing, and the antimetastatic profile of some of the CA IX inhibitors. The newly described metronidazole–sulfanilamide derivatives 4a–4l were tested in an in vitro carbonic anhydrase assay to evaluate their potential as antitumor drugs. The results are summarized in Table 2 as IC50 values.
The following structure–activity relationship (SAR) can be observed from the data of Table 2. CA activities of these compounds were tested against the standard clinically used inhibitors AAZ and SA. The physiologically dominant and highly active cytosolic isoform hCA II was inhibited by compounds 4a–4l with IC50 in the range of 16–137 nM. Compound 4b was the most active having an IC50 value of 16 nM, whereas compound 4a was the least active with IC50 value of 137 nM. We simplified the situation by only treating steric complexities as the single factor to deal with. Most of these compounds with p-substituted metronidazole residues on the benzene ring and the same sulfanilamide derivatives residues were more effective as hCA II inhibitors compared to the p-substituted metronidazole residues on the benzene ring. The opposite effect was noticed for hCA IX; moreover, the tumor-associated isoform hCA IX was inhibited by compounds 4a–4l with IC50 in the range of 38–169 nM (Table 2). The results indicated that the sulfonamides incorporating the metronidazole led to highly effective hCA IX inhibitors. Compound 4h showed the most potent inhibition to hCA IX with an IC50 value of 38 nM, whereas compound 4a was the least active with an IC50 value of 169 nM among compounds 4a–4l. Thus, we can conclude that compounds 4b and 4h have been identified as the most potent inhibitors.
Molecular modeling
To gain better understanding on the potency of the 12 compounds, we examined the interaction of these compounds with hCA II by molecular docking, which was performed by simulation of the 12 compounds into the ATP binding site in hCA II. The protein structure of the hCA II was downloaded from the PDB (3N4B.pdb)29 and was preprocessed using the Schrodinger Protein Preparation Guide; moreover, hydrogen atoms were added to the structure, and H-bonds within the protein were optimized, and the protein was minimized to an rmsd of 0.3 Å. A 9.9 Å sphere of water molecules was added around the ligand and a short (3 ps) dynamics run was carried out, followed by several cycles of minimization using Quanta/CHARMm. The entire protein–ligand–water complex was allowed to move during calculations.30
The predicted binding interaction energy was used as the criterion for ranking. The estimated interaction energy of other synthesized compounds was ranging from −53.72 to −40.84 kcal mol−1. The selected compounds of 4c and 4f had an estimated binding free energy of −53.72 kcal mol−1 and −42.04 kcal mol−1, respectively. The binding model of compounds 4c, 4f and hCA II was depicted in Fig. 4. The amino acid residues that had interaction with hCA II were labeled. In the binding mode, compound 4c was nicely bound to the ATP binding site of hCA II by a hydrophobic interaction and binding was stabilized by one hydrogen bond. The hydrogen atom of THR199 formed one hydrogen bond interaction with the oxygen atom of N
O bonds of compound 4c (angle N HD22 = 172.3°, distance = 2.42 Å). Moreover, compound 4f was also nicely bound to the ATP binding site of hCA II by a hydrophobic interaction and binding was stabilized by four hydrogen bonds (angle TRP5:HE1 4f:O29 = 119.7°, distance = 2.23 Å; ASN67:HD22 4f:N17 = 154.3°, distance = 2.23 Å; THR199:HN 4f:O32 = 112.5°, distance = 2.42 Å and 4f:H53 A:THR199:OG1 = 145.9°, distance = 1.97 Å), and a π-cation interaction (4f A:HIS94). This molecular docking result, along with the biological assay data, suggested that compounds 4c and 4f were potential inhibitors of CA.
 |
| Fig. 4 Docking of compounds 4c and 4f in the ATP binding site of hCA II: (a) left: 2D model of the interaction between compounds 4c, 4f and the ATP binding site. Right: 3D model of the interaction between compounds 4c, 4f and the ATP binding site, respectively. | |
Cytotoxicity
All compounds were evaluated for their toxicity against human macrophages with the median cytotoxic concentration (CC50) data of tested compounds by the MTT assay, as shown in Table 3. These compounds were tested at multiple doses to study the viability of macrophages.
Table 3 The median cytotoxic concentration (CC50) data of tested compounds
Compounds |
CC50a, μmol |
Compounds |
CC50a, μmol |
Minimum cytotoxic concentration required to cause a microscopically detectable alteration of normal cell morphology. |
4a |
0.53 |
4g |
0.63 |
4b |
0.46 |
4h |
0.49 |
4c |
0.58 |
4i |
0.52 |
4d |
0.72 |
4j |
0.62 |
4e |
0.64 |
4k |
0.56 |
4f |
0.68 |
4l |
0.68 |
As shown in Table 3, all compounds showed that they almost did not exhibit cytotoxicity.
Analysis of apoptosis by Annexin V-PE fluorescence-activated cell sorting (FACS)
To test whether the inhibition of cell growth of A549 was related to cell apoptosis, A549 cell apoptosis induced by compound 4d was determined using flow cytometry. The uptake of Annexin V-PE was significantly increased, and the uptake of normal cells was significantly decreased in a time-dependent manner. Finally, the percentage of early apoptotic cells was markedly elevated in a density-dependent manner from 6.28% to 19.6% after 48 h (Fig. 5).
 |
| Fig. 5 Compound 4d induced apoptosis in A549 cells with the density of 2.5, 10, 40, 160 μg mL−1. A549 cells were treated for 48 h. Values represent the mean, n = 3. P < 0.05 versus control. The percentage of cells in each part was indicated. | |
Conclusion
In summary, CA is an emerging target for the development of novel antitumour chemotherapeutics. We had designed and synthesized novel series of metronidazole–sulfanilamide derivatives, which were tested for their inhibitory activities against B16-F10 and MCF7. These compounds showed a very interesting profile for the inhibition of hCAs II (cytosolic, off-target isoform) and hCAs IX (transmembrane, tumor-associated enzyme). Most of them exhibited CA inhibitory activity and did not show any toxicity. Docking simulation was performed to decipher the probable binding models and poses. The results indicate that metronidazole–sulfanilamide derivatives, which acted as potent antibacterials and also as novel and potent antitumour agents. Given the unforeseen structural differences within the active site of some pathogenic enzymes, the key to discovering inhibitors with broad-spectrum antitumour activity lies in a detailed understanding of the CA active sites. Further studies on the CA inhibition ability of these compounds, and further modifications of the current series, with the hope of improving both enzymatic inhibition and physical properties, are currently underway.
Experiments
Materials and measurements
All chemicals used were purchased from Aldrich (USA). All reagents used in the current study were of analytical grade. Thin layer chromatography (TLC) was performed on silica gel plates with fluorescent indicator. All analytical samples were homogeneous on TLC in at least two different solvent systems. Melting points (uncorrected) were determined on a X-4 MP apparatus (Taike Corp, Beijing, China). All the 1H NMR and 13C NMR spectra were recorded on a Bruker DPX 300 model Spectrometer in DMSO-d6, and chemical shifts (δ) were reported as parts per million (ppm). ESI-MS spectra were recorded on a Mariner System 5304 Mass spectrometer. FT-IR spectra on KBr pellets were recorded on a Thermo Nicolet NEXUS870 model spectrometer.
General procedure for the synthesis of compounds 3a, 3b
Anhydrous K2CO3 (8.0 g, 58 mmol) was added to a stirred solution of 2-(2-methyl-5-nitro-1H-imidazole-1-yl)-ethyl-4-methylbenzenesulfonate (2) (4.0 g, 12.1 mmol) in anhydrous DMF (150 mL) at 80 °C. Subsequently, p- or m-hydroxybenzaldehyde (1.9 g, 15.7 mmol) was added to the reaction mixture and stirring continued for 20 h. The mixture was poured into water (200 mL) and extracted with EtOAc (3 × 250 mL). The combined organic extracts were washed with saturated Na2CO3 solution, brine (200 mL), dried MgSO4, and concentrated in vacuo. The crude product was purified by column chromatography (silica gel, EtOAc/PE 1
:
4) to give compounds 3a, 3b.
3a. Yellow solid, yield 71.6%, Rf = 0.44 (EtOAc/PE 1
:
1); m.p. 41–43 °C; 1H NMR (300 MHz, DMSO-d6) δ: 9.95 (s, 1H, CHO), 7.91 (s, 1H, CH), 7.47–7.05 (m, 4H, ArH), 4.75 (t, J = 4.7 Hz, 2H, –CH2), 4.39 (t, J = 5.0 Hz, 2H, –CH2), 2.64 (s, 3H, CH3). 13C NMR (75.4 MHz, DMSO-d6): δ 190.46, 151.81, 142.24, 138.63, 136.56, 133.23, 128.35, 127.38, 62.51, 44.96. ESI-MS: 292.1 [M + H]+.
3b. Faint yellow solid, yield 51.3%, Rf = 0.43 (EtOAc/PE 1
:
1); m.p. 68–70 °C; 1H NMR (DMSO-d6, 300 MHz) δ: 9.95 (s, 1H, CHO), 7.91 (s, 1H, CH), 7.96 (s, 1H, ArH), 7.47–7.05 (m, 3H, ArH), 4.75 (t, J = 4.7 Hz, 2H, –CH2), 4.39 (t, J = 5.0 Hz, 2H, –CH2), 2.64 (s, 3H, CH3). 13C NMR (75.4 MHz, DMSO-d6): δ 190.44, 151.76, 142.24, 138.63, 136.56, 133.23, 127.38, 126.98, 62.51, 44.96. ESI-MS: 292.1 [M + H]+.
General procedure for the synthesis of compounds 4a–4l
To a solution of compound 3a or 3b (7.6 mmol) in ethanol (15 mL), the requisite substituted sulfonamide (7.6 mmol) and acetic acid (0.5 mL) were added. The mixture was heated at 55 °C for 3 h. A solid product was immediately formed, which was filtered and washed with water. The crude products were purified by recrystallization with ethanol, ethyl acetate and acetone (1
:
1
:
0.5), and then washed with ice-water (25 mL) three times and dried to give the compounds 4a–4l as yellow solids.
4-(4-(2-(2-Methyl-5-nitro-1H-imidazole-1-yl)ethoxy)benzylideneamino)benzenesulfonamide (4a). Yellow crystals, yield 66.5%. Rf = 0.25 (EtOAc/PE 1
:
1); m.p. 195–197 °C; 1H NMR (DMSO-d6, 300 MHz) δ: 8.52 (s, 1H, CH), 8.03 (s, 1H, CH), 7.88–7.03 (m, 8H, ArH), 7.04 (s, 1H, NH), 7.02 (s, 1H, NH), 4.75 (t, J = 4.7 Hz, 2H, –CH2), 4.39 (t, J = 5.0 Hz, 2H, –CH2), 2.48 (s, 3H, CH3). 13C NMR (75.4 MHz, DMSO-d6): δ 191.03, 157.82, 151.21, 137.54, 131.98, 129.76, 127.68, 123.58, 121.08, 113.23, 112.15, 66.31, 45.16, 30.31, 14.06. IR (KBr, ν, cm−1): 3334, 3222, 3200 (NH, NH2), 1596 (C
N), 1576, 1374 (NO2), 1343, 113 6 (SO2). ESI-MS: 430.5 [M + H]+.
4-(4-(2-(2-Methyl-5-nitro-1H-imidazole-1-yl)ethoxy)benzylideneamino)-N-(thiazol-2-yl)benzenesulfonamide (4b). Yellow crystals, yield 50.0%. Rf = 0.32 (EtOAc/PE 1
:
1); m.p. 258–259 °C; 1H NMR (DMSO-d6, 300 MHz) δ: 8.49 (s, 1H, CH), 8.02 (s, 1H, CH), 7.85–7.77 (m, 4H, ArH), 7.29 (d, J = 5.0 Hz, 2H, ArH), 7.23 (d, J = 2.7 Hz, 1H, CH), 7.02 (d, J = 5.2 Hz, 2H, ArH), 6.81 (d, J = 2.7 Hz, 1H, CH), 4.75 (t, J = 4.7 Hz, 2H, –CH2), 4.39 (t, J = 5.0 Hz, 2H, –CH2), 2.48 (s, 3H, CH3). 13C NMR (75.4 MHz, DMSO-d6): δ 168.36, 158.12, 152.01, 149.23, 131.36, 129.86, 126.46, 125.02, 121.38, 112.68, 107.36, 14.08. IR (KBr, ν, cm−1): 3220 (NH), 1596 (C
N), 1583, 1379 (NO2), 1348, 1139 (SO2). ESI-MS: 513.4 [M + H]+.
N-Carbamimidoyl-4-(4-(2-(2-methyl-5-nitro-1H-imidazole-1-yl)ethoxy)benzylideneamino)benzenesulfonamide (4c). Orange crystals, yield 84.3%. Rf = 0.28 (EtOAc/PE 1
:
1); m.p. 235–237 °C; 1H NMR (DMSO-d6, 300 MHz) δ: 11.43 (s, 1H, SO2NH), 8.49 (s, 1H, CH), 7.85–7.77 (m, 4H, ArH), 7.29 (d, J = 5.0 Hz, 2H, ArH), 7.23 (d, J = 2.7 Hz, 1H, CH), 7.02 (d, J = 5.2 Hz, 2H, ArH), 6.80 (s, 3H, NH), 4.75 (t, J = 4.7 Hz, 2H, –CH2), 4.39 (t, J = 5.0 Hz, 2H, –CH2), 2.48 (s, 3H, CH3). 13C NMR (75.4 MHz, DMSO-d6): δ 190.23, 162.37, 160.52, 157.77, 154.03, 150.87, 141.15, 132.49, 131.44, 130.47, 127.01, 126.57, 120.39, 114.48, 114.32, 112.41, 66.61, 45.17, 14.06. IR (KBr, ν, cm−1): 3396, 3236, 3110 (NH, NH2), 1588 (C
N), 1570, 1368 (NO2), 1341, 1144 (SO2).
N-(4,6-Dimethylpyrimidin-2-yl)-4-(4-(2-(2-methyl-5-nitro-1H-imidazole-1-yl)ethoxy) benzylideneamino)benzenesulfonamide (4d). Yellow crystals, yield 64.3%. Rf = 0.32 (EtOAc/PE 1
:
1); m.p. 235–237 °C; 1H NMR (DMSO-d6, 300 MHz) δ: 9.02 (s, 1H, CH), 8.04 (s, 1H, CH), 8.00–6.75 (m, 8H, ArH), 6.55 (s, 1H, CH), 5.95 (s, 1H, SO2NH), 4.74 (t, J = 4.9 Hz, 2H, –CH2), 4.45 (t, J = 4.9 Hz, 2H, –CH2), 2.53 (s, 3H, CH3), 2.25 (s, 6H, CH3). 13C NMR (75.4 MHz, DMSO-d6): δ 171.28, 159.01, 156.78, 153.06, 151.68, 141.96, 137.48, 132.76, 129.86, 129.56, 125.86, 124.75, 119.36, 118.43, 116.54, 95.35, 14.08, 12.02. IR (KBr, ν, cm−1): 3187 (NH), 1591 (C
N), 1581, 1376 (NO2), 1345, 1140 (SO2). ESI-MS: 536.4 [M + H]+.
4-(4-(2-(2-Methyl-5-nitro-1H-imidazole-1-yl)ethoxy)benzylideneamino)-N-(4-methylpyrimidin-2-yl)benzenesulfonamide (4e). Yellow crystals, yield 63.2%. Rf = 0.26 (EtOAc/PE 1
:
1); m.p. 207–209 °C; 1H NMR (DMSO-d6, 300 MHz) δ: 11.12 (s, 1H, SO2NH), 9.02 (s, 1H, CH), 8.18 (s, 1H, CH), 8.04 (d, J = 1.95 Hz, 1H, CH), 8.00–6.75 (m, 8H, ArH), 6.55 (d, J = 2.0 Hz, 1H, CH), 4.74 (t, J = 4.9 Hz, 2H, CH2), 4.45 (t, J = 4.9 Hz, 2H, –CH2), 2.53 (s, 3H, CH3), 2.28 (s, 3H, CH3). 13C NMR (75.4 MHz, DMSO-d6): δ 190.06, 167.66, 157.00, 156.77, 151.96, 132.47, 131.40, 129.91, 125.64, 114.30, 114.18, 112.16, 66.49, 45.10, 23.32, 14.06. IR (KBr, ν, cm−1): 3218 (NH), 1592 (C
N), 1576, 1365 (NO2), 1344, 1139 (SO2). ESI-MS: 522.4 [M + H]+.
4-(4-(2-(2-Methyl-5-nitro-1H-imidazole-1-yl)ethoxy)benzylideneamino)-N-(5-methylisoxazol-3-yl)benzenesulfonamide (4f). Yellow crystals, yield 61.8%. Rf = 0.30 (EtOAc/PE 1
:
1); m.p. 285–287 °C; 1H NMR (DMSO-d6, 300 MHz) δ: 11.78 (s, 1H, SO2NH), 9.02 (s, 1H, CH), 8.18 (s, 1H, CH), 8.97–7.44 (m, 8H, ArH), 6.21 (s, 1H, CH), 4.74 (t, J = 4.9 Hz, 2H, –CH2), 4.45 (t, J = 4.9 Hz, 2H, –CH2), 2.53 (s, 3H, CH3), 2.23 (s, 3H, CH3). 13C NMR (75.4 MHz, DMSO-d6): δ 169.69, 159.64, 157.06, 152.56, 151.37, 142.42, 137.68, 132.96, 130.37, 129.93, 129.11, 125.79, 125.03, 124.70, 119.00, 118.69, 116.94, 95.15, 12.02. IR (KBr, ν, cm−1): 3221 (NH), 1588 (C
N), 1585, 1376 (NO2), 1346, 1139 (SO2). ESI-MS: 511.6 [M + H]+.
4-(3-(2-(2-Methyl-5-nitro-1H-imidazole-1-yl)ethoxy)benzylideneamino)benzenesulfonamide (4g). Yellow crystals, yield 64.3%. Rf = 0.29 (EtOAc/PE 1
:
1); m.p. 193–195 °C; 1H NMR (DMSO-d6, 300 MHz) δ: 8.52 (s, 1H, CH), 8.03 (s, 1H, CH), 7.93–7.08 (m, 8H, ArH), 7.04 (s, 1H, NH), 7.02 (s, 1H, NH), 4.75 (t, J = 4.7 Hz, 2H, –CH2), 4.39 (t, J = 5.0 Hz, 2H, –CH2), 2.48 (s, 3H, CH3). 13C NMR (75.4 MHz, DMSO-d6): δ 191.17, 157.84, 151.16, 137.79, 132.52, 129.82, 127.43, 123.50, 120.78, 113.34, 112.33, 66.36, 45.21, 30.32, 14.06. IR (KBr, ν, cm−1): 3338, 3221, 3208, 3115 (NH, NH2), 1594 (C
N), 1577, 1374 (NO2), 1345, 1133 (SO2). ESI-MS: 430.3 [M + H]+.
4-(3-(2-(2-Methyl-5-nitro-1H-imidazole-1-yl)ethoxy)benzylideneamino)-N-(thiazol-2-yl)benzenesulfonamide (4h). Yellow crystals, yield 68.9%. Rf = 0.23 (EtOAc/PE 1
:
1); m.p. 148–150 °C; 1H NMR (DMSO-d6, 300 MHz) δ: 12.43 (s, 1H, SO2NH), 9.95 (s, 1H, CH), 8.04 (s, 1H, CH), 7.44–6.54 (m, 7.40, 4H, ArH), 7.19 (d, J = 5.0 Hz, 2H, ArH), 7.17 (s, 1H, CH), 7.02 (d, J = 5.2 Hz, 2H, ArH), 6.58–6.54 (m, 4H, ArH), 4.75 (t, J = 4.7 Hz, 2H, –CH2), 4.39 (t, J = 5.0 Hz, 2H, –CH2), 2.48 (s, 3H, CH3). 13C NMR (75.4 MHz, DMSO-d6): δ 167.98, 158.32, 151.96, 149.03, 130.78, 129.32, 127.46, 124.42, 120.98, 112.48, 108.96, 14.06. IR (KBr, ν, cm−1): 3221 (NH), 1594 (C
N), 1585, 1378 (NO2), 1348, 1140 (SO2). ESI-MS: 513.5 [M + H]+.
N-Carbamimidoyl-4-(3-(2-(2-methyl-5-nitro-1H-imidazole-1-yl)ethoxy)benzylideneamino)benzenesulfonamide (4i). Light yellow crystals, yield 65.9%. Rf = 0.25 (EtOAc/PE 1
:
1); m.p. 229–231 °C; 1H NMR (DMSO-d6, 300 MHz) δ: 11.43 (s, 1H, SO2NH), 8.49 (s, 1H, CH), 7.85–7.77 (m, 4H, ArH), 7.29 (d, J = 5.0 Hz, 2H, ArH), 7.23 (s, 1H, CH), 7.02 (d, J = 5.2 Hz, 2H, ArH), 6.80 (s, 3H, NH), 4.75 (t, J = 4.7 Hz, 2H, –CH2), 4.39 (t, J = 5.0 Hz, 2H, –CH2), 2.48 (s, 3H, CH3). 13C NMR (75.4 MHz, DMSO-d6): δ 189.26, 162.31, 160.42, 156.07, 153.95, 150.27, 141.11, 133.06, 131.61, 130.53, 127.31, 125.97, 120.29, 114.08, 114.23, 112.36, 66.58, 45.22, 14.08. IR (KBr, ν, cm−1): 3390, 3233, 3111 (NH, NH2), 1593 (C
N), 1573, 1370 (NO2), 1343, 1138 (SO2). ESI-MS: 472.6 [M + H]+.
N-(4,6-Dimethylpyrimidin-2-yl)-4-(3-(2-(2-methyl-5-nitro-1H-imidazole-1-yl)ethoxy)benzylideneamino)benzenesulfonamide (4j). Yellow crystals, yield 71.2%. Rf = 0.26 (EtOAc/PE 1
:
1); m.p. 226–228 °C; 1H NMR (DMSO-d6, 300 MHz) δ: 9.02 (s, 1H, CH), 8.04 (s, 1H, CH), 8.00–6.75 (m, 8H, ArH), 6.55 (s, 1H, CH), 5.95 (s, 1H, SO2NH), 4.74 (t, J = 4.9 Hz, 2H, CH2), 4.45 (t, J = 4.9 Hz, 2H, CH2), 2.53 (s, 3H, CH3), 2.25 (s, 6H, CH3). 13C NMR (75.4 MHz, DMSO-d6): δ 171.18, 159.86, 157.08, 152.86, 151.28, 141.78, 138.02, 132.56, 130.14, 129.58, 125.46, 124.70, 119.36, 118.23, 116.54, 95.35, 14.08, 12.02. IR (KBr, ν, cm−1): 3228 (NH), 1587 (C
N), 1585, 1368 (NO2), 1346, 1139 (SO2). ESI-MS: 536.7 [M + H]+.
4-(3-(2-(2-Methyl-5-nitro-1H-imidazole-1-yl)ethoxy)benzylideneamino)-N-(4-methylpyrimidin-2-yl)benzenesulfonamide (4k). Light yellow crystals, yield 62%. Rf = 0.28 (EtOAc/PE 1
:
1); m.p. 128–130 °C; 1H NMR (DMSO-d6, 300 MHz) δ: 9.95 (s, 1H, CH), 8.31 (d, J = 5.1 Hz, 2H, ArH), 8.03 (s, 1H, CH), 7.66–7.51 (m, 6H, ArH), 7.34 (s, 1H, SO2NH), 6.95 (s, 1H, CH), 6.88 (s, 1H, CH), 4.74 (t, J = 4.9 Hz, 2H, CH2), 4.45 (t, J = 4.9 Hz, 2H, –CH2), 2.55 (s, 3H, CH3), 2.31 (s, 3H, CH3). 13C NMR (75.4 MHz, DMSO-d6): δ 191.37, 157.03, 156.81, 152.08, 132.44, 129.89, 129.58, 129.15, 123.13, 120.80, 120.10, 114.20, 112.80, 112.12, 66.44, 45.18, 23.32, 14.08. IR (KBr, ν, cm−1): 3220 (NH), 1592 (C
N), 1585, 1380 (NO2), 1347, 1139 (SO2). ESI-MS: 522.5 [M + H]+.
4-(3-(2-(2-Methyl-5-nitro-1H-imidazole-1-yl)ethoxy)benzylideneamino)-N-(5-methylisoxazol-3-yl)benzenesulfonamide (4l). Yellow crystals, yield 65.7%. Rf = 0.24 (EtOAc/PE 1
:
1); m.p. 88–90 °C; 1H NMR (DMSO-d6, 300 MHz) δ: 9.31 (s, 1H, CH), 8.04–7.83 (m, 4H, ArH), 7.63 (s, 1H, CH), 7.33–6.74 (m, 4H, ArH), 6.55 (s, 1H, CH), 5.95 (s, 1H, SO2NH), 4.74 (t, J = 4.92 Hz, 2H, –CH2), 4.45 (t, J = 4.92 Hz, 2H, CH2), 2.53 (s, 3H, CH3), 2.23 (s, 3H, CH3). 13C NMR (75.4 MHz, DMSO-d6): δ 168.96, 157.88, 157.69, 151.60, 137.30, 132.46, 129.84, 128.50, 127.87, 125.83, 123.40, 120.78, 113.02, 112.46, 94.91, 66.38, 45.21, 14.08, 11.97. IR (KBr, ν, cm−1): 3220 (NH), 1593 (C
N), 1582, 1379 (NO2), 1350, 1139 (SO2). ESI-MS: 511.6 [M + H]+.
CA inhibition assay
An Applied Photophysics stopped-flow instrument has been used for assaying the CA-catalyzed CO2 hydration activity.31 Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 10 mM Hepes (pH = 7.5) as buffer and 0.1 M Na2SO4 (for maintaining constant the ionic strength) at 25 °C, followed by the CA-catalyzed CO2 hydration reaction for a period of 10–100 s (the uncatalyzed reaction needs around 60–100 s in the assay conditions, whereas the catalyzed reactions need around 6–10 s). The CO2 concentrations ranged from 1.7 to 17 mM for the determination of kinetic parameters. For each inhibitor, tested in the concentration range between 0.01 nM and 100 μM, 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 (1 mM) were prepared in distilled-deionized water with 10–20% (v/v) DMSO (which is not inhibitory at these concentrations), and dilutions up to 0.01 nM were performed thereafter with distilled-deionized water. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to the assay in order to allow for the formation of the E–I complex. The inhibition constants were obtained by nonlinear least-squares methods using PRISM3. The curve-fitting algorithm allowed us to obtain the IC50 values, working at the lowest concentration of substrate of 1.7 mM, from which Ki values were calculated by using the Cheng–Prusoff equation. The catalytic activity (in the absence of inhibitors) of these enzymes were calculated from Lineweaver–Burk plots, as reported earlier, and represent the mean from at least three different determinations. 38–42 enzyme concentrations in the assay system were 7.3 nM for hCA II and 8.5 nM for hCA IX. Enzymes used here were recombinant ones, and they were prepared and purified as described earlier.32
Cell proliferation assay
MTT is much more convenient and helpful than MTT for analyzing cell proliferation, because it can be reduced to soluble formazan by dehydrogenase in mitochondria and has little toxicity to cells. Cell proliferation was determined using MTT dye (BeyotimeInst Biotech, China) according to manufacturer's instructions. Briefly, 1–5 × 103 cells per well were seeded in a 96-well plate, grown at 37 °C for 12 h. Subsequently, cells were treated with the target compounds at increasing concentrations in the presence of 10% FBS for 24 or 48 h. After 10 μL MTT dye was added to each well, cells were incubated at 37 °C for 1–2 h and plates were read in a Victor-V multilabel counter (Perkin-Elmer) using the default europium detection protocol. Percent inhibition or IC50 values of compounds were calculated by comparison with DMSO-treated control wells.
Flow cytometry
Cells (1.3 × 105 cells per mL) were cultured in the presence or not of novobiocin analogues at 200 μM. Nvb at the same concentration served as reference inhibitor. After treatment for 48 and 72 h, cells were washed and fixed in PBS/ethanol (30/70). For cytofluorometric examination, cells (104 cells per mL) were incubated for 30 min in PBS/Triton X-100, 0.2% EDTA (1 mM), and propidium iodide (PI) (50 μg mL−1) in PBS supplemented by RNase (0.5 mg mL−1). The number of cells in the different phases of the cell cycle was determined, and the percentage of apoptotic cells was quantified. Analyses were performed with a FACS Calibur (Becton Dickinson, Le Pont de Claix, France). Cell Quest software was used for data acquisition and analysis.33
Experimental protocol of docking study
Molecular docking of compound 4c and 4f into the three-dimensional X-ray structure of human hCA II (PDB code: 3N4B) was carried out using the Discovery Studio (version 3.5) as implemented through the graphical user interface DS-CDOCKER protocol. The three-dimensional structures of the aforementioned compounds were constructed using Chem. 3D ultra 12.0 software [Chemical Structure Drawing Standard; Cambridge Soft corporation, USA (2010)], and then they were energetically minimized by using MMFF94 with 5000 iterations and minimum RMS gradient of 0.10. The crystal structures of protein complex was retrieved from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). All bound water molecules and ligands were eliminated from the protein, and the polar hydrogen was added to the proteins. Molecular docking of all 20 compounds was then carried out using the Discovery Studio (version 3.5) as implemented through the graphical user interface CDOCKER protocol. CDOCKER is an implementation of a CHARMm based molecular docking tool using a rigid receptor.
Acknowledgements
The work was financed by a grant from Major Projects on Control and Rectification of Water Body Pollution (no. 2011ZX07204–001–004), and supported by “PCSIRT” (IRT1020).
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Footnote |
† These authors contributed equally to this work. |
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