Fragment-based drug design targeting syntenin PDZ2 domain involved in exosomal release and tumour spread.

Syntenin stimulates exosome production and its expression is upregulated in many cancers and implicated in the spread of metastatic tumor. These effects are supported by syntenin PDZ domains interacting with syndecans. We therefore aimed to develop, through a fragment-based drug design approach, novel inhibitors targeting syntenin-syndecan interactions. We describe here the optimization of a fragment, 'hit' C58, identified by in vitro screening of a PDZ-focused fragment library, which binds specifically to the syntenin-PDZ2 domain at the same binding site as the syndecan-2 peptide. X-ray crystallographic structures and computational docking were used to guide our optimization process and lead to compounds 45 and 57 (IC50 = 33 µM and 47 µM; respectively), two representatives of syntenin-syndecan interactions inhibitors, that selectively affect the syntenin-exosome release. These findings demonstrate that it is possible to identify small molecules inhibiting syntenin-syndecan interaction and exosome release that may be useful for cancer therapy.

A study on MAPK/ERK and CDK2-Cyclin-E signal switch "on and off" in cell proliferation by bis urea derivatives of 1, 4-Diisocyanatobenzene.

A series of novel substituted bisurea 1,4-Diisocyanatobenzene compounds were designed, synthesized and introduced as potent anticancer compounds and screened for their in vitro anti-proliferative activities in human cancer cell lines. The structures of all titled compounds were characterized using Fourier-transform infrared mass spectra, nuclear magnetic resonance spectroscopy, elemental analysis and evaluated their sustainability using biological experiments. A selected group of ten derivatives were apprised for their anti-proliferative activity. The compounds 3d and 3e displayed potent anticancer activity with low IC50 value of 5.40, and 5.89 µM against HeLa cancer cell lines. The observed apoptosis data has demonstrated that compounds 3d and 3e induce the activaties of caspase-9 and caspase-3, the compounds 3d and 3e regulated fungal zone inhibition. Due to promising growth inhibitions, the all synthesized compounds were allowed to campaign includes quantum-polarized-ligand, quantum mechanical and molecular mechanical, docking experiments. The compounds 3d and 3e have exhibited a higher affinity for ERK/MAP kinase and CDK2 proteins. The molecular docking interactions have demonstrated two stage inhibition of cancer cells by binding with ERK/MAP kinase and CDK2 leads to inactivation of cell proliferation,cell cycle progression,cell divisionanddifferentiation, and hypo-phosphorylation of ribosome leading cells to restricts at point boundary of the G1/S phase. The long-range molecular dynamics, 150 ns, simulations were also revealed more consistency by 3d. Our study conclude good binding propensity for active-tunnel of ERK/MAP kinase and CDK2 proteins, by 3d (1,1'-(1,4-phenylene) bis(3-(2-chlorobenzyl)urea)), to suggest that the designed and synthesized 3d is to use as selective novel nuclei in anti-cancer chemotherapeutics.

Cell Cycle Arrest and Apoptosis Induction by a New 2,4-Dinitrobenzenesulfonamide Derivative In Acute Leukemia Cells.

BACKGROUND: Current therapies for acute leukemias (ALs) are associated with severe adverse reactions and high relapse rates, which makes the search for new antileukemic agents a necessity. Therefore, the aim of this study was to evaluate the effects of a new sulfonamide, S1, in AL cells K562 and Jurkat. METHODS: The cytotoxic activity of S1 was assessed using MTT method. The involvement of apoptosis in the mechanism of cell death was assessed by flow cytometry and fluorescence microscopy. RESULTS: Our results demonstrated that S1 induced morphological changes suggestive of apoptosis in both K562 and Jurkat cells. Additionally, S1 was not cytotoxic to normal erythrocytes and mononuclear cells and had a highly selective cytotoxicity for AL lineages. The mechanisms of cell death induced by S1 in K562 cells involves cell cycle arrest at G2/M phase and the activation of both extrinsic and intrinsic apoptosis, with an increased FasR and AIF expression and the loss of mitochondrial potential. As for Jurkat, we observed cell cycle blockade at G0/G1 phase, phosphatidylserine exposure and the involvement of intrinsic apoptosis only, with mitochondrial potential loss and a reduced expression of Survivin.  Although sulfonamide S1 did not altered Bcl-2 and Bax expression in AL cell lines, it was able to activate caspase-3 in K562 cells. CONCLUSION: Our results suggest that sulfonamide S1 may be a promising candidate for the development of new drugs for the treatment of ALs.

Design, synthesis and biological evaluation of novel naphthoquinone-4-aminobenzensulfonamide/carboxamide derivatives as proteasome inhibitors.

A series of novel 4-aminobenzensulfonamide/carboxamide derivatives bearing naphthoquinone pharmacophore were designed, sythesized and evaluated for their proteasome inhibitory and antiproliferative activities against human breast cancer cell line (MCF-7). The structures of the synthesized compounds were confirmed by spectral and elemental analyses. The proteasome inhibitory activity studies were carried out using cell-based assay. The antiproteasomal activity results revealed that most of the compounds exhibited inhibitory activity with different percentages against the caspase-like (C-L, ß1 subunit), trypsin-like (T-L, ß2 subunit) and chymotrypsin-like (ChT-L, ß5 subunit) activities of proteasome. Among the tested compounds, compound 14 bearing 5-chloro-2-pyridyl ring on the nitrogen atom of sulfonamide group is the most active compound in the series and displayed higher inhibition with IC50 values of 9.90 ± 0.61, 44.83 ± 4.23 and 22.27 ± 0.15 µM against ChT-L, C-L and T-L activities of proteasome compared to the lead compound PI-083 (IC50 = 12.47 ± 0.21, 53.12 ± 2.56 and 26.37 ± 0.5 µM), respectively. The antiproliferative activity was also determined by MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay in vitro. According to the antiproliferative activity results, all of the compounds exhibited cell growth inhibitory activity in a range of IC50 = 1.72 ± 0.14-20.8 ± 0.5 µM and compounds 13 and 28 were found to be the most active compounds with IC50 values of 1.79 ± 0.21 and 1.72 ± 0.14 µM, respectively. Furthermore, molecular modeling studies were carried out for the compounds 13, 14 and 28 to investigate the ligand-enzyme binding interactions.

Effect of 4-Fluoro-N-(4-Sulfamoylbenzyl) Benzene Sulfonamide on Acquisition and Expression of Nicotine-Induced Behavioral Sensitization and Striatal Adenosine Levels.

INTRODUCTION: Behavioral sensitization is a phenomenon that develops from intermittent exposure to nicotine and other psychostimulants, which often leads to heightened locomotor activity and then relapse. Sulfonamides that act as carbonic anhydrase inhibitors have a documented role in enhancing dopaminergic tone and normalizing neuroplasticity by stabilizing glutamate release. OBJECTIVE: The aim of the current study was to explore synthetic sulfonamides derivative 4-fluoro-N-(4-sulfamoylbenzyl) benzene-sulfonamide (4-FBS) (with documented carbonic anhydrase inhibitory activity) on acquisition and expression of nicotine-induced behavioral sensitization. METHODS: In the acquisition phase, selected 5 groups of mice were exposed to saline or nicotine 0.5mg/kg intraperitoneal (i.p) for 7 consecutive days. Selected 3 groups were administered with 4-FBS 20, 40, and 60 mg/kg p.o. along with nicotine. After 3 days of the drug-free period, ie, day 11, a challenge dose of nicotine was injected to all groups except saline and locomotor activity was recorded for 30 minutes. In the expression phase, mice were exposed to saline and nicotine only 0.5 mg/kg i.p for 7 consecutive days. After 3 days of the drug-free period, ie, day 11, 4-FBS at 20, 40, and 60 mg/kg were administered to the selected groups, one hour after drug a nicotine challenge dose was administered, and locomotion was recorded. At the end of behavioral experiments, all animals were decapitated and the striatum was excised and screened for changes in adenosine levels, using HPLC-UV. RESULTS: Taken together, our findings showed that 4-FBS in all 3 doses, in both sets of experiments significantly attenuated nicotine-induced behavioral sensitization in mice. Additionally, 4-FBS at 60mg/kg significantly lowered the adenosine level in the striatum. CONCLUSION: The behavioral and adenosine modulation is promising, and more receptors level studies are warranted to explore the exact mechanism of action of 4-FBS.

Biocompatible and Rapid Cyclization of Peptides with 2,4-Difluoro-6-hydroxy-1,3,5-benzenetricarbonitrile for the Development of Cyclic Peptide Libraries.

We report a biocompatible and rapid reaction between cysteine thiols and 2,4-difluoro-6-hydroxy-1,3,5-benzenetricarbonitrile (DFB), which enables the efficient cyclization of peptides in neutral aqueous solutions. The reaction was further applied to cyclize peptides displayed on the phage surface without reducing phage infectivity, thus affording high-quality cyclic peptide libraries useful for screening of cyclic peptide ligands. Using the DFB-cyclic peptide library, we identified ligands that can distinguish the pro-survival protein Bcl-xl from its close relative Bcl-2. Therefore, this study on one hand reports a useful reaction for the construction of cyclic peptide libraries, and on the other hand presents valuable hits for further design of selective Bcl-xl ligands.

Properly Substituted Cyclic Bis-(2-bromobenzylidene) Compounds Behaved as Dual p300/CARM1 Inhibitors and Induced Apoptosis in Cancer Cells.

Bis-(3-bromo-4-hydroxy)benzylidene cyclic compounds have been reported by us as epigenetic multiple ligands, but different substitutions at the two wings provided analogues with selective inhibition. Since the 1-benzyl-3,5-bis((E)-3-bromobenzylidene)piperidin-4-one 3 displayed dual p300/EZH2 inhibition joined to cancer-selective cell death in a panel of tumor cells and in in vivo xenograft models, we prepared a series of bis((E)-2-bromobenzylidene) cyclic compounds 4a-n to test in biochemical (p300, PCAF, SIRT1/2, EZH2, and CARM1) and cellular (NB4, U937, MCF-7, SH-SY5Y) assays. The majority of 4a-n exhibited potent dual p300 and CARM1 inhibition, sometimes reaching the submicromolar level, and induction of apoptosis mainly in the tested leukemia cell lines. The most effective compounds in both enzyme and cellular assays carried a 4-piperidone moiety and a methyl (4d), benzyl (4e), or acyl (4k-m) substituent at N1 position. Elongation of the benzyl portion to 2-phenylethyl (4f) and 3-phenylpropyl (4g) decreased the potency of compounds at both the enzymatic and cellular levels, but the activity was promptly restored by introduction of a ketone group into the phenylalkyl substituent (4h-j). Western blot analyses performed in NB4 and MCF-7 cells on selected compounds confirmed their inhibition of p300 and CARM1 through decrease of the levels of acetyl-H3 and acetyl-H4, marks for p300 inhibition, and of H3R17me2, mark for CARM1 inhibition.

Bioorthogonal Tetrazine Carbamate Cleavage by Highly Reactive trans-Cyclooctene.

The high rate of the 'click-to-release' reaction between an allylic substituted trans-cyclooctene linker and a tetrazine activator has enabled exceptional control over chemical and biological processes. Here we report the development of a new bioorthogonal cleavage reaction based on trans-cyclooctene and tetrazine, which allows the use of highly reactive trans-cyclooctenes, leading to 3 orders of magnitude higher click rates compared to the parent reaction, and 4 to 6 orders higher than other cleavage reactions. In this new pyridazine elimination mechanism, wherein the roles are reversed, a trans-cyclooctene activator reacts with a tetrazine linker that is substituted with a methylene-linked carbamate, leading to a 1,4-elimination of the carbamate and liberation of a secondary amine. Through a series of mechanistic studies, we identified the 2,5-dihydropyridazine tautomer as the releasing species and found factors that govern its formation and subsequent fragmentation. The bioorthogonal utility was demonstrated by the selective cleavage of a tetrazine-linked antibody-drug conjugate by trans-cyclooctenes, affording efficient drug liberation in plasma and cell culture. Finally, the parent and the new reaction were compared at low concentration, showing that the use of a highly reactive trans-cyclooctene as the activator leads to a complete cycloaddition reaction with the antibody-drug conjugate in seconds vs hours for the parent system. Although the subsequent release from the IEDDA adduct is slower, we believe that this new reaction may allow markedly reduced click-to-release reagent doses in vitro and in vivo and could expand the application scope to conditions wherein the trans-cyclooctene has limited stability.

A covalent organic framework as a nanocarrier for synergistic phototherapy and immunotherapy.

As traditional cancer treatment methods, photodynamic therapy (PDT) and photothermal therapy (PTT) can eliminate primary tumors, but they cannot inhibit extensive tumor metastasis and local recurrence. Herein, in order to prevent intermolecular accumulation and improve photostability, indocyanine green (ICG) is spontaneously adsorbed onto a covalent organic framework (COF) with high affinity through π-π conjugation, and then chicken ovalbumin (OVA) is coated on the surface of COF@ICG via an electrostatic interaction force. The resultant COF@ICG@OVA can ablate primary tumors under 650 nm and 808 nm laser irradiation due to its high photothermal conversion efficiency (η = 35.75%) and ability to produce reactive oxygen species (ROS). Tumor-associated antigens are also produced after combinational PTT/PDT therapy. By further combining with anti-PD-L1 checkpoint blockade therapy, it can effectively eliminate primary tumors and inhibit the metastasis of cancer cells by generating strong immune responses. Taken together, COF@ICG@OVA nanoparticles offer an efficient synergistic therapeutic modality for the treatment of tumor metastasis.

Cyanide Boosting Copper Catalysis: A Mild Approach to Fluorescent Benzazole Derivatives from Nonemissive Schiff Bases in Biological Media.

An application of nucleophilic cyclization and oxidation of nonemissive Schiff bases via cyanide boosting copper catalysis to synthesize fluorescent benzazole derivatives in high conversion yield is disclosed. This approach is highlighted by broad substrate scope, fast reaction time, and mild conditions and can efficiently proceed in living cells or Arabidopsis root tissues. Furthermore, this methodology can be applied for selective detection of Cu2+ and CN-.

A stimuli-responsive anticancer drug delivery system with inherent antibacterial activities.

We describe a novel class of stimuli-sensitive sulfonium-based synthetic lipids, which exhibit several favorable biophysical properties of phospholipids. The potent sulfonium-based lipid was successfully disassembled by glutathione to release the encapsulated drug molecules in a controlled manner. The cationic lipid also showed lower cytotoxicity against mammalian cells and displayed moderate antibacterial activities.

Direct arylation of C60Cl6 and C70Cl8 with carboxylic acids: a synthetic avenue to water-soluble fullerene derivatives with promising antiviral activity.

We report unprecedented Friedel-Crafts arylation of chlorofullerenes C60Cl6 and C70Cl8 with unprotected carboxylic acids as an efficient single-step synthesis of the inherently stable water-soluble fullerene derivatives. Using this method, a series of previously unaccessible compounds was obtained without chromatographic purification in almost quantitative yields. Promising anti-HIV activity comparable to characteristics of commercial drugs was demonstrated for some of these compounds.

Toward a treatment of diabesity: Rational design, synthesis and biological evaluation of benzene-sulfonamide derivatives as a new class of PTP-1B inhibitors.

Targeting of protein tyrosine phosphatase-1B (PTP1B) has emerged as a promising strategy for therapeutic intervention of diabetes and obesity. Investigation of new inhibitors with good bioavailability and high selectivity is the major challenge of drug discovery program targeting PTP1B. Therefore, herein, new neutral benzene-sulfonamide containing compounds were designed, synthesized and biologically evaluated as potent PTP1B inhibitors. New series of thiazolidine, oxazolidine, thiazinan, oxazinan, oxazole, thiazole, tetrazole, cyanopyridine, chromenone, and iminochromene of benzene-sulfonamide derivatives (MSE-1 to MSE-15) were synthesized in a good yield under mild condition using sulfadiazine as a starting material. Among the synthesized compounds, MSE-13 and MSE-14 showed the most in vitro potent PTP-1B inhibitory activity (IC50 of 0.88 µM and 3.33 µM, respectively). Animal treatment by the target compounds significantly improved the insulin resistance, diminished plasma glucose level, decreased initial body weight, and normalized the serum lipid profile compared to pioglitazone, a standard PTP1B inhibitor. The molecular modeling study showed a high affinity and selectivity of our synthesized compounds to the active site and B-site of PTP1B holding hydrogen bonding, hydrophobic, and electrostatic interactions. Furthermore, Electrostatic Surface Potential (ESP) and HOMO/LUMO analysis indicated the importance of sulfamoyl moiety for PTP1B binding. In silico ADME predictions of such compounds also showed the promising pharmacokinetic and physicochemical properties. The proposed compounds could be considered a lead inhibitory scaffold to PTP1B.

Synthesis, crystal structures and anti-inflammatory activity of four 3,5-bis(arylidene)-N-benzenesulfonyl-4-piperidone derivatives.

3,5-Bis(arylidene)-4-piperidone (BAP) derivatives display good antitumour and anti-inflammatory activities because of their double α,ß-unsaturated ketone structural characteristics. If N-benzenesulfonyl substituents are introduced into BAPs, the configuration of the BAPs would change significantly and their anti-inflammatory activities should improve. Four N-benzenesulfonyl BAPs, namely (3E,5E)-1-(4-methylbenzenesulfonyl)-3,5-bis[4-(trifluoromethyl)benzylidene]piperidin-4-one dichloromethane monosolvate, C28H21F6NO3S·CH2Cl2, (4), (3E,5E)-1-(4-fluorobenzenesulfonyl)-3,5-bis[4-(trifluoromethyl)benzylidene]piperidin-4-one, C27H18F7NO3S, (5), (3E,5E)-1-(4-nitrobenzenesulfonyl)-3,5-bis[4-(trifluoromethyl)benzylidene]piperidin-4-one, C27H18F6N2O5S, (6), and (3E,5E)-1-(4-cyanobenzenesulfonyl)-3,5-bis[4-(trifluoromethyl)benzylidene]piperidin-4-one dichloromethane monosolvate, C28H18F6N2O3S·CH2Cl2, (7), were prepared by Claisen-Schmidt condensation and N-sulfonylation. They were characterized by NMR, FT-IR and HRMS (high resolution mass spectrometry). Single-crystal structure analysis reveals that the two 4-(trifluoromethyl)phenyl rings on both sides of the piperidone ring in (4)-(7) adopt an E stereochemistry of the olefinic double bonds. Molecules of both (4) and (6) are connected by hydrogen bonds into one-dimensional chains. In (5) and (7), pairs of adjacent molecules embrace through intermolecular hydrogen bonds to form a bimolecular combination, which are further extended into a two-dimensional sheet. The anti-inflammatory activity data reveal that (4)-(7) significantly inhibit LPS-induced interleukin (IL-6) and tumour necrosis factor (TNF-α) secretion. Most importantly, (6) and (7), with strong electron-withdrawing substituents, display more potential inhibitory effects than (4) and (5).

Synthesis and mechanistic studies of quinolin-chlorobenzothioate derivatives with proteasome inhibitory activity in pancreatic cancer cell lines.

Inhibition of proteasome activity blocks the degradation of dysfunctional proteins and induces cancer cell death due to cellular stress. Thus, proteasome inhibitors represent an attractive class of anticancer agents, and bortezomib, carfilzomib and ixazomib have been FDA-approved to treat multiple myeloma. However, cancer cells acquire resistance to these inhibitors through point mutations in the proteasome catalytic subunit or induction of alternative compensatory mechanisms. In this study, we identified a quinolin-chlorobenzothioate, QCBT7, as a new proteasome inhibitor showing cytotoxicity in a panel of cancer cell lines. QCBT7 is a more stable derivative of quinoline-8-thiol that targets the regulatory subunit instead of the catalytic subunit of the proteasome. QCBT7 caused the accumulation of ubiquitylated proteins in the cancer cells, indicating its proteasome inhibitory activity. Additionally, QCBT7 increased the expression of a set of genes (PFKFB4, CHOP, HMOX1 and SLC7A11) at both nascent RNA and protein levels, similarly to the known proteasome inhibitors MG132 and ixazomib. Together, QCBT7 induces proteasome inhibition, hypoxic response, endoplasmic reticulum stress and glycolysis, finally leading to cell death. Importantly, we have identified PFKFB4 as a potential biomarker of proteasome inhibitors that can be used to monitor treatment response.

A new triazine based π-conjugated mesoporous 2D covalent organic framework: its in vitro anticancer activities.

We report a new highly crystalline 2D π-conjugated hexagonal mesoporous covalent organic framework material, TrzCOF, through the solvothermal polycondensation of 1,3,5-tri(4-formylbiphenyl) benzene [Ph7(CHO)3, TFBPB] with 2,4,6-tris(4-aminophenyl)-1,3,5-triazine (TAPT) and it displayed excellent anticancer activity for human colorectal carcinoma HCT-116 cells.

Nickel-Catalyzed Stereoselective Diarylation of Alkenylarenes.

A three-component coupling of aryl bromides, arylboron reagents, and alkenylarenes is presented. The method tolerates a variety of substitution patterns on all of the components. In particular, 1,2-disubstituted alkenylarenes are suitable and undergo highly diastereoselective diarylation.

Differentiating Antiproliferative and Chemopreventive Modes of Activity for Electron-Deficient Aryl Isothiocyanates against Human MCF-7 Cells.

The consumption of Brassica vegetables provides beneficial effects through organic isothiocyanates (ITCs), products of the enzymatic hydrolysis of glucosinolate secondary metabolites. The ITC l-sulforaphane (l-SFN) is the principle agent in broccoli that demonstrates several modes of anticancer action. While the anticancer properties of ITCs like l-SFN have been extensively studied and l-SFN has been the subject of multiple human clinical trials, the scope of this work has largely been limited to those derivatives found in nature. Previous studies have demonstrated that structural changes in an ITC can lead to marked differences in a compound's potency to 1) inhibit the growth of cancer cells, and 2) alter cellular transcriptional profiles. This study describes the preparation of a library of non-natural aryl ITCs and the development of a bifurcated screening approach to evaluate the dose- and time-dependence on antiproliferative and chemopreventive properties against human MCF-7 breast cancer cells. Antiproliferative effects were evaluated using a commercial MTS cell viability assay. Chemopreventive properties were evaluated using an antioxidant response element (ARE)-promoted luciferase reporter assay. The results of this study have led to the identification of 1) several key structure-activity relationships and 2) lead ITCs for continued development.

Sarcodonin G Derivatives Exhibit Distinctive Effects on Neurite Outgrowth by Modulating NGF Signaling in PC12 Cells.

Sarcodonin G, one of the cyathane diterpenoids isolated from the mushroom Sarcodon scabrosus, possesses pronounced neurotrophic activity but ambiguous mechanical understanding. In this work, sarcodonin G was chosen as a lead compound to prepare a series of 19- O-benzoyl derivatives by semisynthesis and their neuritogenic activities were evaluated. 6 and 15 (10 µM) were investigated with opposite effects in PC12 cells. 6 exhibited a superior activity to sarcodonin G by promoting NGF-induced neurite outgrowth, while 15 showed an inhibitory effect. Supportingly, 6 and 15 (20 µM) significantly induced and suppressed neurite extension in primary cultured rat cortical neurons, respectively. In mechanism, the two derivatives were revealed to influence NGF-induced neurite outgrowth in PC12 cells through the regulation of PKC-dependent and -independent ERK/CREB signaling as well as the upstream TrkA receptor phosphorylation. Furthermore, a possible pattern of interaction among NGF, 6/15 and TrkA was presented using molecular simulations. It revealed that 6/15 may contribute to the stabilization of the NGF-TrkAd5 complex by establishing several hydrophobic and hydrogen-bond interactions with NGF and TrkA, respectively. Taken together, 6 and 15 modulate PKC-dependent and -independent ERK/CREB signaling pathways possibly by influencing the binding affinity of NGF to the receptor TrkA, and finally regulate neurite outgrowth in PC12 cells.

Anticancer effects of new dibenzenesulfonamides by inducing apoptosis and autophagy pathways and their carbonic anhydrase inhibitory effects on hCA I, hCA II, hCA IX, hCA XII isoenzymes.

In this study, new dibenzensulfonamides, 7-9, having the chemical structure 4,4'-(5'-chloro-3'-methyl-5-aryl-3,4-dihydro-1'H,H-[3,4'-bipyrazole]-1',2-diyl)dibenzenesulfonamide were synthesized in five steps to develop new anticancer drug candidates. Their chemical structures were confirmed by 1H NMR, 13C NMR and HRMS spectra. Cytotoxicities of the dibenzensulfonamides were investigated towards HCC1937, MCF7, HeLa, A549 as tumor cell lines and towards MRC5 and Vero as non-tumor cells. Carbonic anhydrase (CAs, EC 4.2.1.1) inhibitory effects of the dibenzensulfonamides 7-9 were also evaluated on the cytosolic human (h) hCA I and II and the tumor-associated hCA IX and XII isoenzymes. Results indicate that both 7 and 8 induced cleavage of poly (ADP ribose) polymerase (PARP), activation of caspases -3, -7 and -9 which are the hallmarks of apoptosis. Meanwhile both compounds induced autophagy in HCC1937 cells which is shown by enhanced expression of LC3 and decreased level of p62 protein. The compounds tested were also effectively inhibited tumor-associated hCA IX and hCA XII isoenzymes in the range of 20.7-28.1 nM and 4.5-9.3 nM, respectively.