Synthesis of new 3-(2-mercapto-4-oxo-4H-quinazolin-3-yl)-benzenesulfonamides with strong inhibition properties against the tumor associated carbonic anhydrases IX and XII

We report a series of novel metanilamide-based derivatives 3a–q bearing the 2-mercapto-4-oxo-4H-quinazolin-3-yl moiety as tail. All compounds were synthesized by means of straightforward condensation procedures and were investigated in vitro for their inhibition potency against the human (h) carbonic anhydrase (CA; EC 4.2.1.1.1) isoforms I, II, IX and XII. Among all compounds tested the 6-iodo 3g and the 7-fluoro 3i derivatives were the most potent inhibitors against the tumor associated CA IX and XII isoform (K_Is 1.5 and 2.7 nM respectively for the hCA IX and K_Is 0.57 and 1.9 nM respectively for the hCA XII).The kinetic data reported here strongly support compounds of this type for their future development as radiotracers in tumor pathologies which are strictly dependent on the enzymatic activity of the hCA IX and XII isoforms.     

Fluorescent sulfonamide carbonic anhydrase inhibitors incorporating 1,2,3-triazole moieties: Kinetic and X-ray crystallographic studies

Fluorescent sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) were essential for demonstrating the role played by the tumor-associated isoform CA IX in acidification of tumors, cancer progression towards metastasis and for the development of imaging and therapeutic strategies for the management of hypoxic tumors which overexpress CA IX. However, the presently available such compounds are poorly water soluble which limits their use. Here we report new fluorescent sulfonamides 7, 8 and 10 with increased water solubility. The new derivatives showed poor hCA I inhibitory properties, but were effective inhibitors against the hCA II (K_Is of 366–127 nM), CA IX (K_Is of 8.1–36.9 nM), CA XII (K_Is of 4.1–20.5 nM) and CA XIV (K_Is of 12.8–53.6 nM). A high resolution X-ray crystal structure of one of these compounds bound to hCA II revealed the factors associated with the good inhibitory properties. Furthermore, this compound showed a three-fold increase of water solubility compared to a similar derivative devoid of the triazole moiety, making it an interesting candidate for ex vivo/in vivo studies.     

Carbonic anhydrase inhibitors. Diazenylbenzenesulfonamides are potent and selective inhibitors of the tumor-associated isozymes IX and XII over the cytosolic isoforms I and II

A series of diazenylbenzenesulfonamides, azo-dye derivatives of sulfanilamide or metanilamide incorporating phenol and amine moieties, were tested for inhibition of the tumor-associated isozymes of carbonic anhydrase (CA, EC 4.2.1.1), CA IX and XII. These compounds showed moderate-low inhibitory activities against the cytosolic isoforms CA I and II (offtargets) and excellent, low nanomolar inhibitory activity against the transmembrane CA IX and XII (K_Is in the range of 3.5–63 nM against CA IX and 5.0–69.4 nM against CA XII, respectively). The selectivity ratio for inhibiting the tumor-associated CA IX over the offtarget CA II was in the range of 15–104 for these diazenylbenzenesulfonamides, making them among the most isoform-selective inhibitors targeting tumor-associated CAs (over the ubiquitous CA II). Since CA IX/XII were recently shown to be both therapeutic and diagnostic targets for hypoxic solid tumors overexpressing these proteins, such compounds held promise for the management of hypoxic tumors, which are largely non-responsible to classical chemo- and radio-therapy.     

Carbonic anhydrase inhibitors. Comparison of chlorthalidone,indapamide, trichloromethiazide,and furosemide X-ray crystal structures in adducts with isozyme II,when several water molecules make the difference

Thiazide and high ceiling diuretics were recently shown to inhibit all mammalian isoforms of carbonic anhydrase (CA, EC 4.2.1.1) with a very different profile as compared to classical inhibitors, such as acetazolamide, methazolamide, and ethoxzolamide. Some of these structurally related compounds have a very different behavior against the widespread isozyme CA II, with chlorthalidone, trichloromethiazide, and furosemide being efficient inhibitors against CA II (K_Is of 65–138 nM), whereas indapamide is a much weaker one (K_I of 2520 nM). Furthermore, some of these diuretics are quite efficient (low nanomolar) inhibitors of other isoforms, for example, chlorthalidone against hCA VB, VII, IX, and XIII; indapamide against CA VII, IX, XII, and XIII, trichloromethiazide against CA VII and IX, and furosemide against CA I and XIV. Examining the four X-ray crystal structures of their CA II adducts, we observed several (2–3) active site water molecules interacting with the chlorthalidone, trichloromethiazide, and furosemide scaffolds which may be responsible for this important difference of activity. Indeed, indapamide bound to CA II has no interactions with active site water molecules. Chlorthalidone bound within the CA II active site is in an enolic (lactimic) tautomeric form, with the enolic OH also participating in two strong hydrogen bonds with Asn67 and a water molecule. The newly evidenced binding modes of these diuretics may be exploited for designing better CA II inhibitors as well as compounds with selectivity/affinity for various isoforms with medicinal chemistry applications.     

Novel thiophene derivatives as PTP1B inhibitors with selectivity and cellular activity

A series of novel thiophene derivatives was designed, synthesized and their activities as competitive inhibitors of protein tyrosine phosphatase (PTPs) 1B (PTP1B) inhibitors were evaluated. All the compounds showed inhibitory potencies, and 10 of these exhibited moderate inhibitory activities with IC₅₀ values less than 10 μM. The activity of the most potent compound P28 (IC₅₀ = 2.1 μM) was 15 times higher than that of the hit compound P01. Further, four representative compounds (P19, P22, P28, and P31) demonstrated remarkably high selectivities against other PTPs (e.g., PTPα, LAR, CD45, and TCPTP); P19 exhibited greater than sixfold selectivity over highly homologous TCPTP. More importantly, these compounds are permeable to cell membranes. The treatment of CHO-K1 cells with P28 (10 μM) resulted in increased phosphorylation of AKT, which suggested extensive cellular activity of this compound. The novel chemical entities reported in this study could be used for overcoming the poor selectivity and low cellular activity of PTP1B inhibitors and might represent a starting point for development of therapeutic PTP inhibitors.     

Structural study of the location of the phenyl tail of benzene sulfonamides and the effect on human carbonic anhydrase inhibition

The crystal structure of 4-phenylacetamidomethyl-benzenesulfonamide (4ITP) bound to human carbonic anhydrase (hCA, EC 4.2.1.1) II is reported. 4ITP is a medium potency hCA I and II inhibitor (K_Is of 54–75 nM), a strong mitochondrial CA VA/VB inhibitor (K_Is of 8.3–8.6 nM) and a weak transmembrane CA inhibitor (K_Is of 136–212 nM against hCA IX and XII). This elongated compound binds in an extended conformation to hCA II, with its tail lying towards the hydrophobic half of the active site whereas the sulfonamide moiety coordinates the zinc ion. The present structure was compared to that of structurally related aromatic sulfonamides, such as 4-phenylacetamido-benzene-sulfonamide (3OYS), 4-(2-mercaptophenylacetamido)-benzene-sulfonamide (2HD6) and 4-(3-nitrophenyl)-ureido-benzenesulfonamide (3N2P). Homology models of the hCA I, VA, VB, IX and XII structures were build which afforded an understanding of the amino acids involved in the binding of these compounds to these isoforms. The main conclusion of the study is that the orientation of the tail moiety and the presence of flexible linkers as well polar groups in it, strongly influence the potency and the selectivity of the sulfonamides for the inhibition of cytosolic, mitochondrial or transmembrane CA isoforms.     

Carbonic anhydrase inhibitors: Synthesis and inhibition of the human carbonic anhydrase isoforms I,II, IX and XII with benzene sulfonamides incorporating 4- and 3-nitrophthalimide moieties

A series of 4 and 5 nitro-1,3-dioxoisoindolin-2-yl benzenesulfonamide derivatives (compounds 1–8) was synthesized by reaction of benzenesulfonamide derivatives with 4 and 3-nitrophthalic anhydrides. These new sulfonamides were investigated as inhibitors of the zinc metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and more specifically against the human (h) cytosolic isoforms hCA I and II and the transmembrane, tumor-associated hCA IX and XII. Most of the novel compounds were medium potency-weak hCA I inhibitors (K_is in the range of 295–10,000 nM), but were more effective hCA II inhibitors (K_is of 1.7–887 nM). The tumor-associated hCA IX was also inhibited, with K_is in the micromolar range, whereas against hCA XII the inhibition constants were in the range of 90–3746 nM. The structure–activity relationship (SAR) with this series of sulfonamides is straightforward, with the main features leading to good activity for each isoforms being established. The high sequence hCA alignment homology and molecular docking studies was performed in order to rationalize the activities reported and binding mode to different hCA as inhibitors.     

Design,synthesis and evaluation of N-substituted saccharin derivatives as selective inhibitors of tumor-associated carbonic anhydrase XII

A series of N-alkylated saccharin derivatives were synthesized and tested for the inhibition of four different isoforms of human carbonic anhydrase (CA, EC 4. 2.1.1): the transmembrane tumor-associated CA IX and XII, and the cytosolic CA I and II. Most of the reported derivatives inhibited CA XII in the nanomolar/low micromolar range, hCA IX with K_Is ranging between 11 and 390 nM, whereas they were inactive against both CA I (K_Is >50 μM) and II (K_Is ranging between 39.1 nM and 50 μM). Since CA I and II are off-targets of antitumor carbonic anhydrase inhibitors (CAIs), the obtained results represent an encouraging achievement for the development of new anticancer candidates without the common side effects of non-selective CAIs. Moreover, the lack of an explicit zinc binding function on these inhibitors opens the way towards the exploration of novel mechanisms of inhibition that could explain the high selectivity of these compounds for the inhibition of the transmembrane, tumor-associated isoforms over the cytosolic ones.     

Carbonic anhydrase inhibitors; Fluorinated phenyl sulfamates show strong inhibitory activity and selectivity for the inhibition of the tumor-associated isozymes IX and XII over the cytosolic ones I and II

A series of fluorinated-phenylsulfamates have been prepared by sulfamoylation of the corresponding phenols and the inhibition of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic CA I and II (off-targets), and the transmembrane, tumor-associated CA IX and XII is investigated. Unlike the lead molecule (phenylsulfamate), a very potent CA I and II inhibitor and a modest CA IX/XII inhibitor, the fluorinated sulfamates were stronger inhibitors of CA IX (K_Is of 2.8–47 nM) and CA XII (K_Is of 1.9–35 nM) than of CA I (K_Is of 53–415 nM) and CA II (K_Is of 20–113 nM). Some of these compounds were selective CA IX over CA II inhibitors, with selectivity ratios in the range of 11.4–12.1, making them interesting candidates for targeting hypoxic tumors overexpressing CA IX and/or XII.     

Carbonic anhydrase inhibitors: Synthesis and inhibition of the human carbonic anhydrase isoforms I,II, VII,IX and XII with benzene sulfonamides incorporating 4,5,6,7-tetrabromophthalimide moiety

A series of 4,5,6,7-tetrabromo-1,3-dioxoisoindolin-2-yl benzenesulfonamide derivatives (compounds 1–8) was synthesized by reaction of benzene sulfonamide derivatives with 4,5,6,7-tetrabromophthalic anhydride moiety. These new sulfonamides were investigated as inhibitors of the zinc metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) and more specifically against the human (h) cytosolic isoforms hCA I, II and VII and the transmembrane tumor-associated isoform hCA IX and XII. The new compounds were good hCA I inhibitors (Kis in the range of 143 to >10,000 nM), but were moderately effective, as hCA II inhibitors (Kis of 47–190 nM) and poor hCA VII inhibitors (Kis in the range of 54–175 nM) compared to acetazolamide. The tumor-associated hCA IX was effectively inhibited with Kis ranging between 8.5 and 234 nM and hCA XII with inhibition constants in the range of 6.1–197 nM with high selectivity ratio. The structure–activity relationship (SAR) with this series of sulfonamides is straightforward, with the main features leading to good activity for each isoforms being established. The high sequence hCA alignment homology and molecular docking study of compounds was performed to rationalize the SAR reported over here.     

Benzenesulfonamide bearing 1,2,4-triazole scaffolds as potent inhibitors of tumor associated carbonic anhydrase isoforms hCA IX and hCA XII

Three series of novel heterocyclic compounds (3a–3g, 4a–4g and 5a–5g) containing benzenesulfonamide moiety and incorporating a 1,2,4-triazole ring, have been synthesized and investigated as inhibitors against four isomers of the α-class carbonic anhydrases (CAs, EC 4.2.1.1), comprising hCAs I and II (cytosolic, ubiquitous isozymes) and hCAs IX and XII (transmembrane, tumor associated isozymes). Against the human isozymes hCA I and II, compounds of two series (3a–3g and 4a–4g) showed K_i values in the range of 84–868 nM and 5.6–390 nM, respectively whereas compounds of series 5a–5g were found to be poor inhibitors (K_i values exceeding 10,000 nM in some cases). Against hCA IX and XII, all the tested compounds exhibited excellent to moderate inhibitory potential with K_i values in the range of 2.8–431 nM and 1.3–63 nM, respectively. Compounds 3d, 3f and 4f exhibited excellent inhibitory potential against all of the four isozymes hCA I, II, IX and XII, even better than the standard drug acetazolamide (AZA) whereas compound of the series 5a–5g were comparatively less potent but more selective towards hCA IX and XII.     

Aminoquinoline derivatives with antiproliferative activity against melanoma cell line

The synthesis of a novel series of aminoquinoline derivatives 1a–p and their antiproliferative activities against A375 human melanoma cell line were described. Most compounds showed superior antiproliferative activities to Sorafenib as a reference compound. Among them, quinolinyloxymethylphenyl compounds 1k and 1l exhibited potent activities (IC₅₀ = 0.77 and 0.79 μM, respectively) and excellent selectivity against melanoma and fibroblast cell lines.     

Design,synthesis and biological evaluation of benzyloxyphenyl-methylaminophenol derivatives as STAT3 signaling pathway inhibitors

STAT3 signaling pathway has been validated as a vital therapeutic target for cancer therapy. Based on the novel STAT3 inhibitor of a benzyloxyphenyl-methylaminophenol scaffold hit (1) discovered through virtual screening, a series of analogues had been designed and synthesized for more potent inhibitors. The preliminary SAR had been discussed and the unique binding site in SH2 domain was predicted by molecular docking. Among them, compounds 4a and 4b exhibited superior activities than hit compound (1) against IL-6/STAT3 signaling pathway with IC₅₀ values as low as 7.71 μM and 1.38 μM, respectively. Compound 4a also displayed potent antiproliferative activity against MDA-MB-468 cell line with an IC₅₀ value of 9.61 μM. We believe that these benzyloxyphenyl-methylaminophenol derivatives represent a unique mechanism for interrogating STAT3 as well as a potential structure type for further exploration.     

Design and synthesis of novel androgen receptor antagonists via molecular modeling

Several androgen receptor (AR) antagonists are clinically prescribed to treat prostate cancer. Unfortunately, many patients become resistant to the existing AR antagonists. To overcome this, a novel AR antagonist candidate called DIMN was discovered by our research group in 2013. In order to develop compounds with improved potency, we designed novel DIMN derivatives based on a docking study and substituted carbons with heteroatom moieties. Encouraging in vitro results for compounds 1b, 1c, 1e, 3c, and 4c proved that the new design was successful. Among the newly synthesized compounds, 1e exhibited the strongest inhibitory effect on LNCaP cell growth (IC₅₀ = 0.35 μM) and also acted as a competitive AR antagonist with selectivity over the estrogen receptor (ER) and the glucocorticoid receptor (GR). A docking study of compound 1e fully supported these biological results. Compound 1e is considered to be a novel, potent and AR-specific antagonist for treating prostate cancer. Thus, our study successfully applied molecular modeling and bioisosteric replacement for hit optimization. The methods here provide a guide for future development of drug candidates through structure-based drug discovery and chemical modifications.     

Synthesis and biological evaluation of 5-aryloxypyrazole derivatives bearing a rhodanine-3-aromatic acid as potential antimicrobial agents

Three novel series of 5-aryloxypyrazole derivatives have been synthesized and tested for their antibacterial activity. The majority of the synthesized compounds showed potent inhibitory activity against Gram-positive bacteria Staphylococcus aureus 4220, especially against the strains of multidrug-resistant clinical isolates (MRSA3167/3506 and QRSA3505/3519). Among which compounds IIIb, IIIg and IIIm showed the most potent levels of activity (MIC = 1 μg/mL) against the multidrug-resistant strains. And cytotoxic activity assay showed that the compounds tested did not affect cell viability on the Human cervical (HeLa) cells at their MICs. The current study therefore suggests that 5-aryloxypyrazoles bearing a rhodanine-3-aromatic acid moiety are promising scaffolds for the development of novel Gram-positive antibacterial agents.     

Discovery of novel purine derivatives with potent and selective inhibitory activity against c-Src tyrosine kinase

We report here the discovery of novel purine derivatives with potent and selective inhibitory activity against c-Src tyrosine kinase by adopting a strategy integrating focused combinatorial library design, virtual screening, chemical synthesis, and bioassay. Thirty two compounds were selected and synthesized. All compounds showed potent inhibitory activity against c-Src kinase with IC₅₀ values ranging from 3.14 μM to 0.02 μM. Compound 5i was identified as one of the most potent agent with an IC₅₀ 120 times lower than those of the hits. The high hit rate (100%) and the potency of the new Src kinase inhibitors demonstrated the efficiency of the strategy for the focused library design and virtual screening. The novel active chemical entities reported here should be good leads for further development of purine-based anticancer drugs targeting Src tyrosine kinase.     

Synthesis and anticancer activity of novel 4-morpholino-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine derivatives bearing chromone moiety

Herein, we designed and synthesized of a novel series of 7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidine derivatives bearing chromone moiety (10a–j, 13a–j). All the compounds were evaluated for the IC₅₀ values against five cancer cell lines (A549, PC-3, MCF-7, Hela and HepG2). Seven of the target compounds exhibited moderate to excellent cytotoxicity. For these compounds, we tested their inhibitory activities against mTOR kinase, and four of them were tested their inhibitory activities against PI3Kα kinase in further. The results indicated that the optimized compound 10j showed excellent inhibitory activity and cytotoxicity against mTOR kinase, PI3Kα kinase and five cancer cell lines with IC₅₀ values of 1.1 μM, 0.92 μM and 8.77–14.3 μM. Structure–activity relationships (SARs) and docking studies indicated that the thiopyrano[4,3-d]pyrimidine scaffolds exerted little effect on antitumor activities of target compounds. Substitutions of chromone moiety at C-6 position with carboxyl were benefit to the antitumor activities.     

4-Functionalized 1,3-diarylpyrazoles bearing 6-aminosulfonylbenzothiazole moiety as potent inhibitors of carbonic anhydrase isoforms hCA I,II, IX and XII

A series of 24 novel heterocyclic compounds—functionalized at position 4 with aldehyde (5a–5f), carboxylic acid (6a–6f), nitrile (7a–7f) and oxime (8a–8f) functional groups—bearing 6-aminosulfonybenzothiazole moiety at position 1 of pyrazole has been synthesized and investigated for the inhibition of four isoforms of the α-class carbonic anhydrases (CAs, EC 4.2.1.1), comprising hCAs I and II (cytosolic, ubiquitous isozymes) and hCAs IX and XII (transmembrane, tumor associated isozymes). Against the human isozyme hCA I, compounds 6a–6f showed medium-weak inhibitory potential with K_i values in the range of 157–690 nM with 6a showing better potential than the standard drug acetazolamide (AZA). Against hCA II, all the compounds showed excellent to moderate inhibition with K_i values of compounds 5a, 5d, 5f, 6a–6f, 8d and 8f lower than 12 nM (K_i of AZA). Against hCA IX, all the compounds showed moderate inhibition with the exception of 6e which showed nearly 9 fold a better profile compared to AZA, whereas against hCA XII, four compounds 6e, 7a, 7b and 7d showed K_i in the same order as that of AZA. Carboxylic acid 6e was found to be an excellent inhibitor of both hCA IX and XII, with K_i values of 2.8 nM and 5.5 nM, respectively.     

Coumarins incorporating hydroxy- and chloro-moieties selectively inhibit the transmembrane,tumor-associated carbonic anhydrase isoforms IX and XII over the cytosolic ones I and II

A series of coumarins incorporating hydroxy-, chloro- and/or chloromethyl-moieties in positions 3-, 4-, 6- and 7- of the heterocyclic ring were investigated for the inhibition of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). These coumarins were very weak or ineffective as inhibitors of the house-keeping, offtarget isoforms CA I and II, but showed effective, submicromolar inhibition of the transmembrane, tumor-associated isoforms CA IX and XII. The nature and position of the groups substituting the coumarin ring greatly influenced CA inhibitory properties. 6-Hydroxycoumarin showed K_Is >100 μM against CA I and II, of 0.198 μM against CA IX and of 0.683 μM against CA XII, being thus a selective, efficient inhibitor for the tumor-associated over cytosolic isoforms. These compounds are also excellent leads for designing isoform-selective enzyme inhibitors.