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A novel series of quinazolin-4(3H)-one/Schiff base hybrids was rationally designed and synthesized. The prepared compounds were evaluated for in vitro activity to inhibit phosphodiesterase 4 (PDE4), where several of them showed good-to-moderate activity compared to rolipram. Compound 7 showed potent PDE4 inhibition in this series, with an IC50 of 1.60 µM. Compounds that showed PDE4 inhibition were further assessed for antiproliferative activity using different human tumor cell lines. Compound 10 exhibited significant antiproliferative activity with IC50 values of 140, 79, and 320 nM in breast, lung, and colon tumor cells, respectively. Docking of compound 7 in the active site of PDE4B illustrates its possible binding mode and provides insight for further optimizations of this novel scaffold for inhibiting PDE4.

Analogues with the scaffolds of 3-cyano-4-alkoxyphenyl-6-bromoaryl-2-pyridone and 2-amino-3-cyano-4-alkoxyphenyl-6-bromoarylpyridine were synthesized. Cyclization of the 2-amino derivatives with formic acid and formamide gave the corresponding pyrido[2,3-d]pyrimidin-4(3H)-one and the pyrido[2,3-d]-pyrimidin-4-amine derivatives, respectively. Active phosphodiesterase 3 (PDE3) inhibitors were identified from each of the four aforementioned scaffolds. This is the first report that pyrido[2,3-d]pyrimidin-4(3H)-one and pyrido[2,3-d]pyrimidin-4-amine derivatives can inhibit PDE3. The analogues with the pyridone and pyrido[2,3-d]pyrimidin-4(3H)-one scaffolds inhibited both cAMP and cyclic guanosine monophosphate (cGMP) hydrolysis by PDE3, while the amine containing scaffolds were more selective for cGMP hydrolysis. This observation may set the base for substrate-selective pharmacological modulation of this important class of drug targets and with less side effects, particularly tachcardia. The dual inhibitors of PDE3 were more potent inhibitor towards the growth of HT-29 cancer cell lines.

By studying the co-crystal information of interactions between PDE5 and its inhibitors, forty new tetrahydro-β-carbolines based-analogues were synthesized, and tested for their PDE5 inhibition. Some compounds were as active as tadalafil in inhibiting PDE5 and of better selectivity profile particularly versus PDE11A, the nature of the terminal ring and its nitrogen substituent are the main determinants of selectivity. Ensemble docking confirmed the role of H-loop closed conformer in activity versus its occluded and open forms. Conformational studies showed the effect of bulkiness of the terminal ring N-alkyl substituent on the formation of stable enzyme ligands conformers. The difference in potencies of hydantoin and piperazinedione analogues, together with the necessity of C-5/C-6 R-absolute configuration has been revealed through molecular docking.

Non-steroidal anti-inflammatory drugs (NSAIDs) have been widely reported to display strong efficacy for cancer chemoprevention, although their mechanism of action is poorly understood. The most well-documented effects of NSAIDs include inhibition of tumor cell proliferation and induction of apoptosis, but their effect on tumor cell invasion has not been well studied. Here, we show that the NSAID, sulindac sulfide (SS) can potently inhibit the invasion of human MDA-MB-231 breast and HCT116 colon tumor cells in vitro at concentrations less than those required to inhibit tumor cell growth. To study the molecular basis for this activity, we investigated the involvement of microRNA (miRNA). A total of 132 miRNAs were found to be altered in response to SS treatment, including miR-10b, miR-17, miR-21 and miR-9, which have been previously implicated in tumor invasion and metastasis. We confirmed that these miRNA can stimulate tumor cell invasion and show that SS can attenuate their invasive effects by downregulating their expression. Employing luciferase and chromatin immunoprecipitation assays, NF-κB was found to bind the promoters of all four miRNAs to suppress their expression at the transcriptional level. We show that SS can inhibit the translocation of NF-κB to the nucleus by decreasing the phosphorylation of IKKβ and IκB. Analysis of the promoter sequences of the miRNAs suppressed by SS revealed that 81 of 115 sequences contained NF-κB-binding sites. These results show that SS can inhibit tumor cell invasion by suppressing NF-κB-mediated transcription of miRNAs.

Two closely related series of novel beta-carboline derivatives, electronically similar to tadalafil (CAS 171596-29-5), were synthesized and evaluated for their inhibitory effects upon phosphodiesterase 5 (PDE5) and phosphodiesterase 11 (PDE11) and their in vitro tumor cell growth inhibitory activity versus HT29 colorectal carcinoma cell line. Interestingly, some of the synthesized compounds showed growth inhibitory properties that appear to be associated with their ability to inhibit PDE5. Moreover, the PDE5 inhibition seems relevant to the stereochemical aspects of the compounds.

Starting from tadalafil as a template, a series of functionalized tetrahydro-β-carboline derivatives have been prepared and identified as novel potent and selective PDE5 inhibitors. Replacing the 3,4-methylenedioxyphenyl at position 6 of tadalafil, together with elongation of the N2-methyl substituent and manipulation of the stereochemical aspects of the two chiral carbons led to the identification of compound XXI, a highly potent PDE5 inhibitor (IC(50) = 3 nM). Compound XXI was also highly selective for PDE5 versus PDE3B, PDE4B, and PDE11A, with a selectivity index of 52 and 235 towards PDE5 rather than PDE11 with both cAMP and cGMP as substrate, respectively.

The synthesis of novel tadalafil analogues in which the benzodioxole moiety is replaced by 2-bromophenyl; the chiral carbons swing from R,R to R,S, S,R and S,S; the piperazinedione ring is maintained or reduced to the 5-membered imidazolidinedione or thioxoimidazolinone is described. The prepared analogues were evaluated for their capacity to inhibit the cyclic guanosine monophosphate (cGMP) selective phosphodiesterase 5 (PDE5) isozyme and the growth of human HT-29 colon adenocarcinoma cells. The R absolute configuration of C-5 in the beta-carboline-hydantoin and C-6 in the beta-carboline-piperazinedione derivatives was found to be essential for the PDE5 inhibition. In addition, tadalafil analogues that were synthesized from l-tryptophan were more active than those derived from d-tryptophan, which is of economic value and expands the horizon for the discovery of new carbolines as PDE5 inhibitors. While some analogues displayed potent tumor cell growth inhibitory activity, there was no apparent correlation with their PDE5 inhibitory activity, which leads us to conclude that other PDE isozymes or PDE5 splice variants may be involved.

Two series with the general formula of 4,6-diaryl-2-oxo-1,2 dihydropyridine-3-carbonitriles and their isosteric 4,6-diaryl-2-imino-1,2-dihydropyridine-3-carbonitrile were synthesized through one pot reaction of the appropriate acetophenone, aldehyde, ammonium acetate with ethyl cyanoacetate or malononitrile, respectively. The synthesized compounds were evaluated for their tumor cell growth inhibitory activity against the human HT-29 colon tumor cell line, as well as their PDE3 inhibitory activity. Compound 4-(2-Ethoxyphenyl)-2-oxo-6-thiophen-3-yl-1,2-dihydropyridine-3 carbonitrile (21) showed tumor cell growth inhibitory activity with an IC50 value of 1.25 microM. Meanwhile, 4-(4-Ethoxyphenyl)-2-imino-6-(thiophen-3-yl)-1,2-dihydropyridine-3-carbonitrile (26) showed inhibitory effect upon PDE3 using cAMP or cGMP as substrate. No correlation exists between PDE3 inhibition and the tumor cell growth inhibitory activity. Docking compound 21 to other possible molecular targets showed the potential to bind PIM1 Kinase.

ATP-binding cassette (ABC) transpo rters ABCC1 (MRP1), ABCB1 (P-gp), and ABCG2 (BCRP) contribute to chemotherapy failure. The primary goals of this study were to characterize the efficacy and mechanism of the non­steroidal anti-inflammatory drug (NSAID), sulindac sulfide, to reverse ABCC1 mediated resistance to chemother­apeutic drugs and to determine if sulindac sulfide can influence sensitivity to chemotherapeutic drugs independently of drug efflux. Cytotoxicity assays were performed to measure resistance of ABC-expressing cell lines to doxoru­bicin and other chemotherapeutic drugs. NSAIDs were tested for the ability to restore sensitivity to resistance selected tumor cell lines, as well as a large panel of standard tumor cell lines. Other experiments characterized the mechanism by which sulindac sulfide inhibits ABCC1 substrate and co-substrate (GSH) transport in isolated membrane vesicles and intact cells. Selective reversal of multi-drug resistance (MDR), decreased efflux of doxor­ubicin, and fluorescent substrates were demonstrated by sulindac sulfide and a related NSAID, indomethacin, in resistance selected and engineered cell lines expressing ABCC1, but not ABCB1 or ABCG2. Sulindac sulfide also inhibited transport of leukotriene C4 into membrane vesicles. Sulindac sulfide enhanced the sensitivity to doxoru­bicin in 24 of 47 tumor cell lines, including all melanoma lines tested (7-7). Sulindac sulfide also decreased intra­cellular GSH in ABCC1 expressing cells, while the glutathione synthesis inhibitor, BSO, selectively increased sensitivity to sulindac sulfide induced cytotoxicity. Sulindac sulfide potently and selectively reverses ABCC1-mediated MDR at clinically achievable concentrations. ABCC1 expressing tumors may be highly sensitive to the direct cytotoxicity of sulindac sulfide, and in combination with chemotherapeutic drugs that induce oxidative stress.

Title compound (III) (16 examples) are screened for their antiproliferative activity against A 549 (lung), HT 29 (colon), MCF 7, and MDA-MB 231 (breast) cell lines.

Nonsteroidal anti-inflammatory drugs (NSAID) display promising antineoplastic activity for colorectal and other cancers, but toxicity from COX inhibition limits their long-term use for chemoprevention. Previous studies have concluded that the basis for their tumor cell growth inhibitory activity does not require COX inhibition, although the underlying mechanism is poorly understood. Here, we report that the NSAID sulindac sulfide inhibits cyclic guanosine 3′,5′-monophosphate phosphodiesterase (cGMP PDE) activity to increase intracellular cGMP levels and activate cGMP-dependent protein kinase (PKG) at concentrations that inhibit proliferation and induce apoptosis of colon tumor cells. Sulindac sulfide did not activate the cGMP/PKG pathway, nor affect proliferation or apoptosis in normal colonocytes. Knockdown of the cGMP-specific PDE5 isozyme by siRNA and PDE5-specific inhibitors tadalafil and sildenafil also selectively inhibited the growth of colon tumor cells that expressed high levels of PDE5 compared with colonocytes. The mechanism by which sulindac sulfide and the cGMP/PKG pathway inhibits colon tumor cell growth involves the transcriptional suppression of β-catenin to inhibit Wnt/β-catenin T-cell factor transcriptional activity, leading to downregulation of cyclin D1 and survivin. These observations suggest that safer and more efficacious sulindac derivatives can be developed for colorectal cancer chemoprevention by targeting PDE5 and possibly other cGMP-degrading isozymes. Mol Cancer Ther; 12(9); 1848–59. ©2013 AACR.

New derivatives with the tetrahydro-β-carboline-imidazolidinedione and tetrahydro-β-carboline-piperazinedione scaffolds and a pendant bromothienyl moiety at C-5/C-6 were synthesized and tested for their ability to inhibit PDE5 in vitro. The following SAR can be concluded: The tetracyclic scaffold is essential for PDE5 inhibition; the ethyl group is the most suitable among the adopted N-substituents on the terminal ring (hydantoin/piperazinedione); the appropriate stereochemistry of C-5/C-6 derived from the aldehyde rather than C-11a/C-12a derived from tryptophan appears crucial for inhibition of PDE5; surprisingly, derivatives with the hydantoin terminal ring are more active than their analogs with the piperazinedione ring; the selectivity versus PDE5 relative to PDE11 with cGMP as a substrate is mainly a function of the substitution and stereochemistry pattern of the external ring, in other words of the interaction with the H-loop residues of the isozymes. Thirteen derivatives showed PDE5 inhibitory activity with IC50 values in the range of 0.16–5.4 μm. Compound 8 was the most potent PDE5 inhibitor and showed selectivity towards PDE5 versus other PDEs, with a selectivity index of 49 towards PDE5 rather than PDE11 with cGMP as the substrate.

Two series of 2-(3,5-diaryl-4,5-dihydropyrazol-1-yl)-1-methyl-6-oxo-4-phenyl-1,6-dihydropyrimidine-5-carbonitriles 5a–h and 4-(4-chlorophenyl)-2-(3,5-diaryl-4,5-dihydropyrazol-1-yl)-1-methyl-6-oxo-1,6-dihydropyrimidine-5-carbonitriles 6a–h were synthesized via a cyclocondensation reaction of the corresponding 2-hydrazinopyrimidines 3a,b with the appropriate 2-propen-1-ones 4a–h. The target compounds were screened for their antiproliferative activity against A 549 (lung), HT 29 (colon), MCF 7 and MDA-MB 231 (breast) cell lines. The two most susceptible cell lines were the colon (HT 29) and breast (MDA-MB 231). Generally, the 4-unsubstitutedphenylpyrimidine derivatives 5a–h were more active than their 4-chlorophenylpyrimidine analogs 6a–h. Compounds 5e and 5g, showed high activity against three of the cell lines. The most active compound 5c possessed IC50 = 1.76 μM against A 549 cell line.

Phosphodiesterase 10A (PDE10A) is a cGMP and cAMP degrading PDE isozyme that is highly expressed in the brain striatum where it appears to play an important role in cognition and psychomotor activity. PDE10 inhibitors are being developed for the treatment of schizophrenia and Huntington's disease and are generally well tolerated, possibly because of low expression levels in most peripheral tissues. We recently reported high levels of PDE10 in colon tumors and that genetic silencing of PDE10 by siRNA or inhibition with small molecule inhibitors can suppress colon tumor cell growth with a high degree of selectivity over normal colonocytes (Li et al., Oncogene 2015). These observations suggest PDE10 may have an unrecognized role in tumorigenesis. Here we report that the concentration range by which the highly specific PDE10 inhibitor, Pf-2545920 (MP-10), inhibited colon tumor cell growth parallels the concentration range required to increase cGMP and cAMP levels, and activate PKG and PKA, respectively. Moreover, PDE10 knockdown by shRNA reduced the sensitivity of colon tumor cells to the growth inhibitory activity of Pf-2545920. Pf-2545920 also inhibited the translocation of β-catenin to the nucleus, thereby reducing β-catenin mediated transcription of survivin, which resulted in caspase activation and apoptosis. This was determined to be through a PKG mediated pathway through the use of small molecule inhibitors of PKG and PKA. PDE10 mRNA was also found to be elevated in colon tumors compared with normal tissues in a cDNA array. We also report the increase in PDE10 mRNA in 50% of a small collection of human clinical specimens collected at the Mitchell Cancer Institute (n = 13). In addition, novel PDE10 inhibitor, MCI-030, reduced tumor size and activated PDE10 signaling mechanisms in vivo. These findings suggest that PDE10 can be targeted for cancer therapy or prevention whereby inhibition results in cGMP elevation and PKG activation to reduce β-catenin-mediated transcription of survival proteins leading to the selective apoptosis of cancer cells. Citation Format: Kevin J. Lee, Ashley S. Lindsey, Luciana Madeira da Silva, Alisa Trinh, Bernard Gary, Joel Andrews, Veronica Ramirez-Alcantara, Adam B. Keeton, Wen-Chi Chang, Margie Clapper, Gary A. Piazza. Beta-catenin nuclear translocation in colorectal cancer cells is suppressed by PDE10A inhibition, cGMP elevation, and activation of PKG. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 331.

New derivatives with the tetrahydro-β-carboline-imidazolidinedione and tetrahydro-β-carboline-piperazinedione scaffolds and a pendant bromothienyl moiety at C-5/C-6 were synthesized and tested for their ability to inhibit PDE5 in vitro. The following SAR can be concluded: The tetracyclic scaffold is essential for PDE5 inhibition; the ethyl group is the most suitable among the adopted N-substituents on the terminal ring (hydantoin/piperazinedione); the appropriate stereochemistry of C-5/C-6 derived from the aldehyde rather than C-11a/C-12a derived from tryptophan appears crucial for inhibition of PDE5; surprisingly, derivatives with the hydantoin terminal ring are more active than their analogs with the piperazinedione ring; the selectivity versus PDE5 relative to PDE11 with cGMP as a substrate is mainly a function of the substitution and stereochemistry pattern of the external ring, in other words of the interaction with the H-loop residues of the isozymes. Thirteen derivatives showed PDE5 inhibitory activity with IC(50) values in the range of 0.16-5.4 µm. Compound 8 was the most potent PDE5 inhibitor and showed selectivity towards PDE5 versus other PDEs, with a selectivity index of 49 towards PDE5 rather than PDE11 with cGMP as the substrate.

Two series of compounds with the general formula of 4,6-diaryl-2-oxo-1,2 dihydropyridine-3-carbonitriles and their isosteric imino derivatives were synthesized through a one pot reaction of acetophenone, aldehyde and ammonium acetate with ethyl cyanoacetate or malononitrile, respectively. The synthesized compounds were evaluated for tumor cell growth inhibitory using the human HT-29 colon and MDA-MB-231 breast tumor cell lines. Compound 4-(2- Ethoxyphenyl)-2-imino-6-(4-fluorophenyl)-1,2-dihydropyridine-3 carbonitrile (6) showed IC50 value of 0.70 μM versus HT-29. Meanwhile, compound 4-(2-Hydroxyphenyl)-2-imino-6-(4-fluorophenyl)-1,2-dihydropyridine-3-carbonitrile (4) showed IC50 value of 4.6 μM versus MDA-MB-231. Docking compound 10 to possible molecular targets, survivin and PIM1 kinase showed appreciable interactions with both, which suggest possible targets for the antitumor activity of this novel class of anticancer compounds.

Starting from D- or L-tryptophane methyl ester all possible diastereoisomers with the skeleton of (III)/(IV), (VI), and (X) are prepared and tested as PDE5 inhibitors.

Nonsteroidal anti-inflammatory drugs (NSAID) such as sulindac sulfide (SS) display promising antineoplastic properties, but toxicities resulting from COX inhibition limit their clinical use. Although COX inhibition is responsible for the anti-inflammatory activity of SS, recent studies suggest that phosphodiesterase (PDE) 5 inhibition and activation of cyclic guanosine monophosphate (cGMP) signaling are closely associated with its ability to induce apoptosis of tumor cells. However, the underlying mechanisms responsible for apoptosis induction, factors that influence sensitivity of tumor cells to SS, and the importance of PDE5 for breast tumor cell growth have not been established. Here we show that SS can induce apoptosis of breast tumor cells, which predominantly rely on PDE5 for cGMP hydrolysis but not normal mammary epithelial cells, which rely on PDE isozymes other than PDE5 for cGMP hydrolysis. Inhibition of PDE5 and activation of protein kinase G (PKG) by SS was associated with increased β-catenin phosphorylation, decreased β-catenin mRNA and protein levels, reduced β-catenin nuclear localization, decreased T-cell factor/lymphoid enhancer factor (Tcf/Lef) promoter activity, and decreased expression of Wnt/β-catenin–regulated proteins. Suppression of PDE5 with siRNA or known PDE5 inhibitors was sufficient to selectively induce apoptosis and attenuate β-catenin–mediated transcription in breast tumor cells with minimal effects on normal mammary epithelial cells. These findings provide evidence that SS induces apoptosis of breast tumor cells through a mechanism involving inhibition of PDE5 and attenuation of oncogenic Wnt/β-catenin–mediated transcription. We conclude that PDE5 represents a novel molecular target for the discovery of safer and more efficacious drugs for breast cancer chemoprevention. Cancer Prev Res; 4(8); 1275–84. ©2011 AACR.

New derivatives based upon the tetrahydro-β-carboline-hydantoin and tetrahydro-β-carboline-piperazinedione scaffolds were synthesized. All compounds were evaluated for their ability to inhibit PDE5 in vitro, and numerous compounds with IC(50) values in the low nanomolar range were identified including compounds derived from l-tryptophan. Compounds with high potency versus PDE5 were then evaluated for inhibitory activity against other PDEs to assess isozyme selectivity. Compound 5R,11aS-5-(3,4-dichlorophenyl)-2-ethyl-5,6,11,11a-tetrahydro-1H-imidazo[1',5':1,6]pyrido[3,4-b]indole-1,3(2H)dione 14 showed a selectivity index of >200 for cGMP hydrolysis by PDE5 versus PDE11. Meanwhile, 6R,12aR-6-(2,4-dichlorophenyl)-2-ethyl-2,3,6,7,12,12a-hexahydropyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4dione 45 demonstrated strong potency for inhibition of PDE11 with an IC(50) value of 11 nM, representing the most potent PDE11 inhibitor thus far reported. Docking experiments differentiated between active and inactive analogues and revealing the conformational, steric, and lipophilic necessities for potent PDE5 inhibition. Many derivatives, including potent PDE5 inhibitors, were able to inhibit the growth of the MDA-MB-231 breast tumor cell line with low micromolar potency.

Starting from a previously reported lead compound GR30040X (a hydantoin tetrahydro-β-carboline derivative with a 4- pyridinyl ring at C- 5), a series of structurally related tetrahydro-β-carboline derivatives were prepared. The tetrahydro-β-carboline skeleton was fused either to a hydantoin or to a piperazindione ring, the pendant aryl group attached to C-5 or C-6 was changed to a 3, 4-dimethoxyphenyl or a 3-pyridinyl ring; different N-substituents on the terminal ring were introduced, a straight chain ethyl group, a branched tert. butyl and P-chlorophenyl group rather than n-butyl group of the lead compound. All four possible diastereomers of target tetrahydro-β-carboline derivatives were prepared, separated by column chromatography and the significance of these stereochemical manipulations were studied. Synthesized compounds were evaluated for their inhibitory effect versus PDE5. Seven hits were obtained with appreciable inhibitory activity versus PDE5 with IC₅₀s 0.14 - 4.99 µM.

The synthesis of novel tadalafil analogues in which the benzodioxole moiety is replaced by 2-bromophenyl; the chiral carbons swing from R,R to R,S, S,R and S,S; the piperazinedione ring is maintained or reduced to the 5-membered imidazolidinedione or thioxoimidazolinone is described. The prepared analogues were evaluated for their capacity to inhibit the cyclic guanosine monophosphate (cGMP) selective phosphodiesterase 5 (PDE5) isozyme and the growth of human HT-29 colon adenocarcinoma cells. The R absolute configuration of C-5 in the beta-carboline-hydantoin and C-6 in the beta-carboline-piperazinedione derivatives was found to be essential for the PDE5 inhibition. In addition, tadalafil analogues that were synthesized from l-tryptophan were more active than those derived from d-tryptophan, which is of economic value and expands the horizon for the discovery of new carbolines as PDE5 inhibitors. While some analogues displayed potent tumor cell growth inhibitory activity, there was no apparent correlation with their PDE5 inhibitory activity, which leads us to conclude that other PDE isozymes or PDE5 splice variants may be involved.

Two closely related series of novel beta-carboline derivatives, electronically similar to tadalafil (CAS 171596-29-5), were synthesized and evaluated for their inhibitory effects upon phosphodiesterase 5 (PDE5) and phosphodiesterase 11 (PDE11) and their in vitro tumor cell growth inhibitory activity versus HT29 colorectal carcinoma cell line. Interestingly, some of the synthesized compounds showed growth inhibitory properties that appear to be associated with their ability to inhibit PDE5. Moreover, the PDE5 inhibition seems relevant to the stereochemical aspects of the compounds.

Sulindac displays promising antineoplastic activity, but toxicities from cyclooxygenase inhibition limit its use for chemoprevention. Previous reports suggest that its anticancer properties may be attributed to a cyclooxygenase-independent mechanism, although alternative targets have not been well defined. Here, we show that sulindac sulfide (SS) induces apoptosis and inhibits the growth of human breast tumor cells with IC50 values of 60 to 85 μmol/L. Within the same concentration range, SS inhibited cyclic GMP (cGMP) hydrolysis in tumor cell lysates but did not affect cyclic AMP hydrolysis. SS did not induce apoptosis of normal human mammary epithelial cells (HMEC) nor did it inhibit phosphodiesterase (PDE) activity in HMEC lysates. SS increased intracellular cGMP levels and activated protein kinase G in breast tumor cells but not HMEC. The guanylyl cyclase (GC) activator, NOR-3, and cGMP PDE inhibitors, trequinsin and MY5445, displayed similar growth-inhibitory activity as SS, but the adenylyl cyclase activator, forskolin, and other PDE inhibitors had no effect. Moreover, GC activation increased the sensitivity of tumor cells to SS, whereas GC inhibition reduced sensitivity. By comparing PDE isozyme profiles in breast tumor cells with HMEC and determining the sensitivity of recombinant PDE isozymes to SS, PDE5 was found to be overexpressed in breast tumor cells and selectively inhibited by SS. The mechanism of SS binding to the catalytic domain of PDE5 was revealed by molecular modeling. These data suggest that PDE5 inhibition is responsible for the breast tumor cell growth–inhibitory and apoptosis-inducing activity of SS and may contribute to the chemopreventive properties of sulindac. [Mol Cancer Ther 2009;8(12):3331–40]

Nonsteroidal anti-inflammatory drugs such as sulindac have shown promising antineoplastic activity, although toxicity from cyclooxygenase (COX) inhibition and the suppression of prostaglandin synthesis limits their use for chemoprevention. Previous studies have concluded that the mechanism responsible for their antineoplastic activity may be COX independent. To selectively design out the COX inhibitory activity of sulindac sulfide (SS), in silico modeling studies were done that revealed the crucial role of the carboxylate moiety for COX-1 and COX-2 binding. These studies prompted the synthesis of a series of SS derivatives with carboxylate modifications that were screened for tumor cell growth and COX inhibitory activity. A SS amide (SSA) with a N,N-dimethylethyl amine substitution was found to lack COX-1 and COX-2 inhibitory activity, yet potently inhibit the growth of human colon tumor cell lines, HT-29, SW480, and HCT116 with IC50 values of 2 to 5 μmol/L compared with 73 to 85 μmol/L for SS. The mechanism of growth inhibition involved the suppression of DNA synthesis and apoptosis induction. Oral administration of SSA was well-tolerated in mice and generated plasma levels that exceeded its in vitro IC50 for tumor growth inhibition. In the human HT-29 colon tumor xenograft mouse model, SSA significantly inhibited tumor growth at a dosage of 250 mg/kg. Combined treatment of SSA with the chemotherapeutic drug, Camptosar, caused a more sustained suppression of tumor growth compared with Camptosar treatment alone. These results indicate that SSA has potential safety and efficacy advantages for colon cancer chemoprevention as well as utility for treating malignant disease if combined with chemotherapy.

Two series of 4,6-diaryl-2-imino-1,2-dihydropyridine-3-carbonitriles and their isosteric 4,6-diaryl-2-oxo-1,2-dihydropyridine-3-carbonitriles were synthesized through a combinatorial approach. The prepared analogues were evaluated for their in vitro capacity to inhibit PDE3A and the growth of the human HT-29 colon adenocarcinoma tumor cell line. Compound 6-(4-bromophenyl)-4-(2-ethoxyphenyl)-2-imino-1,2-dihydropyridine-3-carbonitrile (Id) exhibited the strongest PDE3 inhibition when cGMP but not cAMP is the substrate with a IC(50)of 27microM, which indicates a highly selective mechanism of enzyme inhibition. On the other hand, compound 6-(1,3-benzodioxol-5-yl)-4-(2-ethoxyphenyl)-2-imino-1,2-dihydropyridine-3-carbonitrile (Ii) was the most active in inhibiting colon tumor cell growth with a IC(50) of 3microM. The electronic effects, steric effects and conformational aspects of Id seem to be the most crucial for the PDE3 inhibition. Meanwhile, steric factors and the H-bonding capability seem to be the most important factors for tumor cell growth inhibitory activity. Conversely, there is no direct correlation between PDE3 inhibition and anticancer activity for the prepared compounds. An in silico docking experiment indicates the potential involvement of other potential molecular targets such as PIM-1 kinase to explain its tumor cell growth inhibitory activity.

Despite the fact that activating mutations in the various forms of Ras were identified as driving factors in oncogenesis over thirty years ago, there are still no effective therapeutics which act on this target. We recently synthesized and characterized a novel class of compounds that show striking potency and selectivity to inhibit the growth of tumor cells with mutant Ras. Through iterative structure-activity studies, we selected for and optimized Ras selectivity to achieve high potency with IC50 values in the low nanomolar range and selectivity indices of nearly 100-fold. For example, ADT-062 inhibited the growth of human HCT116 colon tumor cells that harbor mutant K-Ras with an IC50 value of 8.4 + 3.6 nM,. In contrast, human HT29 colon tumor cells that have wild type Ras displayed an IC50 value of 521 + 253 nM in response to ADT-062 treatment. When surveying a larger panel of colon tumor cell lines, we observed a direct correlation between potency and Ras activation status. Further, ADT-062 induced cell cycle arrest only in the Ras-mutant cell line. To study the mechanism by which this class of compounds selectively inhibits the growth of tumor cells with mutant K-Ras, we profiled lysates from treated HCT116 and HT29 colon tumor cells using high resolution LC-MS/MS. Over 100 ions were identified that were differentially affected by treatment with ADT-062 at its IC50 value in the respective cell line. Among the ions of interest, a component of the purine biosynthetic pathway, 5′-phosphoribosyl-N-formylglycinamide (FGAR), was found to be induced 200-fold (p = .0031) in K-Ras mutant cells compared with Ras wild type cells. Together, these findings suggest that this compound may cause a Ras selective disruption of purine metabolism and cell cycle progression which contributes to the selectivity by which this novel class of compounds inhibit the growth of tumor cells with mutant Ras. These findings may enable us to develop this compound class as a novel therapeutic which targets Ras driven tumors and tumors which may become resistant to other targeted therapeutics through secondary Ras mutations. Citation Format: Joshua C. Canzoneri, Xi Chen, Adam B. Keeton, Kevin Lee, Bernard Gary, Ethan B. Butler, William E. Grizzle, Landon Wilson, Stephen Barnes, Michael R. Boyd, Gary A. Piazza. A novel class of Ras selective inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 701. doi:10.1158/1538-7445.AM2015-701

Background and Significance: Mutations in the Ras family of proto-oncogenes are relatively common in cancer. Activating Ras mutations occur de novo in approximately one third of cancers and may also develop in tumors that become resistant to targeted therapies, especially against receptor tyrosine kinase inhibitors. The NSAID, sulindac and certain non-cyclooxygenase derivatives are known to have chemopreventive activity and have been reported to be particularly effective against tumors and cell lines with Ras mutations. Although Ras mutations have been known for many years, they remain a target that is poorly represented by current cancer therapeutics. In fact, the target has been described as “undruggable” because of the high affinity of Ras for its substrate, GTP. Methods & Results: Using a synthetic lethal approach, we screened a focused collection of compounds chemically related to sulindac. The screen identified a hit compound that potently and selectively inhibited the growth of tumor cell lines expressing mutant Ras (A-549 and MDA-MB231) compared with a tumor cell line bearing the wild-type Ras (HT-29). Follow-up experiments confirmed that tumor cells of several histotypes expressing mutant Ras were sensitive. The selectivity was also observed in a paired isogenic cell model system in which mutant Ras is artificially introduced (NRK, K-NRK). Based on this scaffold, we have synthesized a series of substituted indene compounds with increased selectivity for cancer cells with mutated Ras. Structure-activity relationships for potency and Ras selectivity are being explored within this series. We have identified highly potent compounds which inhibited growth with IC50 values in the low nM range. The compounds exhibited selectivity ranging between 10- to 500-fold for mutant Ras expressing cell lines. Studies demonstrating the mechanism of growth inhibition as well as studies to identify the molecular target of the prototype compound will be presented. Conclusions: We have synthesized a novel series of compounds that potently inhibit the growth of cancer cell lines of several histotypes harboring Ras mutations. In contrast, tumor cells expressing wild-type Ras are relatively insensitive. This series represents a promising, targeted approach to treatment of cancers with this relatively commonly mutated oncogene. This project is supported by NIH grant 5R01CA155638-02. Citation Format: Xi Chen, Kevin J. Lee, Bernard D. Gary, Joshua C. Canzoneri, Alexandra M. Fajardo, Sara C. Sigler, Veronica Ramirez-Alcantara, Bing Zhu, Gary A. Piazza, Adam B. Keeton. A novel series of substituted indene derivatives that potently and selectively inhibit growth of tumor cells harboring mutant Ras. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B39. doi: 10.1158/1557-3125.RASONC14-B39

Previous studies report that induction of intracellular cGMP can selectively inhibit proliferation and induce apoptosis of tumor cells. However, the phosphodiesterase (PDE) isozymes responsible for regulating cGMP in tumor cells or the basis for this selectivity have not been well studied. Here we report that PDE10 is elevated in colon tumor cell lines compared to normal colonocytes. High levels of PDE10 were also measured in colon tumors from human clinical samples and the ApcMin/+ mouse model compared to normal intestinal mucosa. PDE10 inhibitors (PQ-10, Pf-2545920) and siRNA selectively inhibit colon tumor cell growth by inhibiting proliferating and inducing apoptosis, while stable knockdown inhibits colony formation and increases doubling time. Conversely, ectopic expression of PDE10 increases the growth rate of colonocytes. Pf-2545920 inhibits the growth of all lines in the NCI-60 tumor cell panel, indicating a functional role of PDE10 across histologically diverse tumor types. The mechanism by which PDE10 inhibition suppresses growth involves activation of cGMP/PKG signaling to reduce β-catenin and TCF transcriptional activity. Given its potential as a new cancer target, PDE10 was used to design and screen for novel anticancer agents. A group of indene analogs was found to potently and selectively suppress tumor cell growth with IC50 values less than Pf-2545920. A lead compound, MCI-020, displayed attractive oral bioavailability and pharmacokinetic properties in mice with an unusual characteristic of achieving high lung concentrations compared with plasma and other tissues. Because of its unique biodistribution pattern, MCI-020 was evaluated in a lung orthotopic mouse model using human A549 lung tumor cells. Oral administration of MCI-020 was well tolerated up to at least 250 mg bid for 5 weeks without affecting body weight. As summarized below, MCI-020 (150 mg bid) significantly increased the number of mice showing no visible tumors from 15.4% in the vehicle group (n=13) to 75% in the treated group (n=12) and strongly reduced tumor formation among mice that developed tumors. Microscopic examination of lung sections using a 1-4 grading scale to measure the extent of tumor formation revealed a score of 2.8 ± 0.42 (high involvement) for the vehicle group and 0.83 ± 0.29 (low involvement) for the treated group (p Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C182. Citation Format: Nan Li, Kevin Lee, Yaguang Xi, Bing Zhu, Bernard D. Gary, Veronica Ramirez-Alcantara, Evrim Gurpinar, Joshua Canzoneri, Alexandra Fajardo, Sara Sigler, John T. Piazza, Xi Chen, Joel Andrews, Meagan Thomas, Wenyan Lu, Yonghe Li, Danuel J. Laan, Mary P. Moyer, Suzanne Russo, Brian T. Eberhardt, Larry Yet, Adam B. Keeton, William E. Grizzle, Gary A. Piazza. Phosphodiesterase 10: A novel cancer target. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C182.

Sulindac displays promising antineoplastic activity, but toxicity from cyclooxygenase (COX) inhibition limits its long-term use for chemoprevention. Previous studies have concluded that the tumor cell growth inhibitory activity of sulindac is COX-independent and suggest that safer and more efficacious drugs can be developed by targeting the underlying mechanism. Here we report that sulindac sulfide (SS) inhibits cyclic guanosine monophosphate phosphodiesterase (cGMP PDE) activity, increases intracellular cGMP levels and activates protein kinase G at concentrations that suppress proliferation and induce apoptosis of colon tumor cells. Normal colonocytes were refractory to the anti-proliferative and pro-apoptotic effects of SS as well as its ability to induce cGMP signaling. PDE5-specific inhibitors such as tadalafil also inhibited the growth of colon tumor cells that expressed high levels of the cGMP-specific PDE5 isozyme compared with colonocytes. RNAi knockdown of PDE5 selectively inhibited proliferation and induced apoptosis of colon tumor cells as did SS. SS, 8-bromo-cGMP, and PDE5 siRNA reduced β-catenin levels, as well as the expression of cyclin D and survivin that are regulated by β-catenin-dependent TCF transcriptional activity by a mechanism involving the transcriptional repression of β-catenin. The significance of these findings are highlighted by studies in the FCCC Min mouse model of intestinal tumorigenesis showing that a novel non-COX inhibitory derivative of SS can strongly inhibit colon tumor formation by a mechanism that appears to involve cGMP PDE inhibition. These observations suggest that safer and more efficacious drugs can be developed for colorectal cancer chemoprevention by targeting PDE5 and possibly other cGMP degrading isozymes. Citation Format: Nan Li, Yaguang Xi, Heather N. Tinsley, Evrim Gurpinar, Bernard D. Gary, Bing Zhu, Yonghe Li, Adam B. Keeton, William E. Grizzle, Gary A. Piazza. Next generation sulindac. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3659. doi:10.1158/1538-7445.AM2013-3659

Nitric oxide-releasing NSAIDs (NO-NSAIDs) were originally developed as prodrugs to reduce the gastrointestinal toxicity of conventional NSAIDs and were found to have improved potency to inhibit tumor growth compared with the parent NSAID. Although different mechanisms have been proposed for their potent anticancer properties, the role of the NO-moiety has not been clearly defined. We have investigated whether the NO group contributes to the anticancer properties of NO-NSAIDs by inducing soluble guanylyl cyclase and elevating intracellular cGMP levels, effects that are associated with the activity of NO donors. We confirmed the increased potency of the NO-derivatives of sulindac, aspirin (o-, p- and m- isomers), exisulind and naproxen with respect to the parent compounds for inhibiting growth of HCT116, SW480 and COLO741 human colon tumor cells. The two derivatives with the lowest IC50 values, NO-sulindac (16.50 µM) and NO-ASA(o) (8 µM) were also tested for cGMP elevation using a novel cGMP biosensor assay in live cells. In brief, HEK293 cells were transfected with a biosensor construct containing cGMP binding domains fused to the modified firefly luciferase gene (Promega) which produces bioluminescence in the cGMP-bound or ‘activated’ state. Cells were treated with NO-Sulindac, NO-ASA(o) and known NO-donors, NOR-3 and SNP as positive controls. Chemiluminescence was measured over a period of 1h. A strong and sustained signal was obtained with the NO-donors, SNP and NOR-3 (50 µM), although no induction was observed with NO-NSAID treatment (50 µM). In addition, SW480 colon tumor cells were treated with 50 µM NO-Sulindac, NO-ASA(o) or NOR-3 and levels of phosphorylated vasoactivator stimulated phosphoproteins (p-VASP), an intracellular marker of cGMP signaling activity, were measured by Western Blotting. NOR-3 caused a time-dependent increase in p-VASP expression, whereas the NO-NSAIDs had no such effect. Since sulindac and certain derivatives have previously been reported to inhibit cGMP-phosphodiesterase (PDE) and inhibit tumor cell growth by a mechanism involving cGMP elevation, we also determined if NO-NSAIDS can inhibit cGMP PDE activity. NO-NSAIDs did not inhibit cGMP hydrolysis by either recombinant PDE5 or in lysates from colon tumor cells, although sulindac sulfide was an effective inhibitor as previously reported. These results suggest that mechanisms other than cGMP elevation explain the anticancer properties of NO-NSAIDs. Funding provided by NIH/NCI grants CA131378 and CA148817. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4610. doi:10.1158/1538-7445.AM2011-4610

Previous studies have concluded that the mechanism responsible for the antineoplastic properties of NSAIDs is cyclooxygenase (COX) independent, which suggests that more selective drugs can be developed for cancer chemoprevention by targeting such mechanisms. With this paradigm, we are synthesizing novel sulindac derivatives that have reduced COX inhibitory activity, but enhanced tumor cell growth inhibitory activity. A prototypic dimethylethyl amide derivative referred to as sulindac sulfide amide (SSA) was previously reported to lack COX-1 or COX-2 inhibitory activity, yet have improved potency to inhibit tumor cell growth in vitro. For example, SSA inhibited the growth of human HT-29 colon tumor cells with IC50 values of 1-2 µM compared with 70-90 µM for sulindac sulfide (SS), a non-selective COX inhibitor. SSA was also appreciably more effective than SS in inhibiting HT-29 colon tumor cell growth using a 3-dimensional in vitro tumor cell model. SSA induced apoptosis of HT-29 carcinoma cells, but normal human colonocytes were insensitive to treatment, suggesting an element of tumor selectivity. The mechanism of SSA induced apoptosis is associated with cGMP phosphodiesterase (PDE) inhibition, elevation of intracellular cGMP, and activation of protein kinase G, which also appears to be an important off-target effect of SS. SSA inhibited the growth of a variety of histologically diverse tumor cell lines, an effect that is consistent with studies showing the overexpression of PDE5 in various tumor types, including colorectal adenomas and carcinomas. SSA was well tolerated in the FCCC Min mouse model in which oral administration reduced colon tumor multiplicity by approximately 80% and tumor incidence by 50%. SSA also inhibited tumor growth in the human HT-29 colon mouse xenograft model by approximately 60%, while sulindac at its maximum tolerated dose was marginally effective. The antitumor activity of SSA in the HT-29 model was accompanied by decreased numbers of proliferating cells and increased numbers of apoptotic cells as determined by immunohistochemistry. Pharmacokinetic studies in mice bearing HT-29 xenografts revealed that plasma and tumor levels of SSA from a tolerated dose of SSA appreciably exceeded the in vitro IC50 value for growth inhibition. In contrast, sulindac generated plasma and tumor levels of SS that were appreciably less than the IC50 value for tumor cell growth inhibition. These results support the feasibility of chemically modifying sulindac to design out COX associated toxicities, while increasing anticancer efficacy. Newer sulindac derivatives with higher potency and target selectivity, as well as formulations to improve oral bioavailability are being developed for colon cancer chemoprevention. Funding provided by NIH/NCI grants CA131378 and CA148817. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1865. doi:10.1158/1538-7445.AM2011-1865

Many colon cancers harbor APC mutations that prevent the proteosomal degradation of oncogenic β-catenin. Previous studies have found that certain nonsteroidal anti-inflammatory drugs (NSAIDs) have the ability to reduce nuclear levels of β-catenin and inhibit tumor cell growth through a cyclooxygenase (COX)-independent mechanism, which might be responsible for their cancer chemopreventive activity. A potential mechanism may involve cGMP phosphodiesterase (PDE) inhibition and the activation of protein kinase G (PKG). In this study we evaluated over 500 novel sulindac derivatives for colon tumor cell growth inhibitory activity. Select compounds were also evaluated for COX and cGMP PDE inhibitory activity to determine which activity is associated with colon tumor cell growth inhibition and ability to suppress nuclear β-catenin levels. A subset of derivatives was identified that displayed reduced COX-1 and COX-2 inhibitory activity, yet increased potency to inhibit the growth of several colon tumor cell lines (HT29, HCT116, SW480, and COLO741) with IC50 values of 2-10 µM compared with IC50 values of 80-110 µM for the parent compound, sulindac sulfide (SS). Certain derivatives also displayed increased potency to inhibit recombinant phosphodiesterase 5 (PDE5), which appears to be the isozyme responsible for the cGMP PDE inhibitory activity of SS. Treatment of colon tumor cells with such compounds also increased the phosphorylation of vasoactive stimulated protein (VASP), a known PKG substrate. Furthermore, nuclear levels of β-catenin decreased within the same time period as PKG activation, which is consistent with previous reports suggesting that PKG can phosphorylate β-catenin to induce proteosomal degradation. Together, these data indicate that sulindac can be chemically modified to eliminate COX inhibitory activity while enhancing its ability to inhibit colon tumor cell growth. We conclude that the suppression of nuclear β-catenin levels by SS is independent of COX inhibition and that novel derivatives can be developed with improved antineoplastic activity while reducing the toxicities associated with COX inhibition. Funding provided by NIH/NCI grants CA131378 and CA148817. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 837. doi:10.1158/1538-7445.AM2011-837

Sulindac and other nonsteroidal anti-inflammatory drugs have shown promise for chemoprevention of colorectal cancer, but toxicities arising from cyclooxygenase (COX) inhibition limit their clinical use. Previous reports suggest that sulindac may exert its antitumor effects by a COX-independent target involving cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE) inhibition, although the specific isozymes involved and mechanism have not been well defined. In this study, we show that sulindac sulfide (SS) inhibits the growth of human colon tumor cells, HCT116, HT29 and SW480 with IC50 values of 60-80μmol/L. Within this concentration range, SS did not affect the growth of human-derived normal colonic epithelial cells, NCM460. Furthermore, SS induced apoptosis of colon tumor cells but not NCM460 cells. SS inhibited cGMP PDE activity in colon tumor cell lysates with minimal effect on cGMP PDE activity in NCM460 cell lysates. By determining the sensitivity of all eleven PDE isozymes to SS, the cGMP-specific PDE5 was found to be the most sensitive with an IC50 value for enzyme inhibition of 38µmol/L. In addition, PDE5 was found to be overexpressed in colon tumor cell lines compared with NCM460 cells. Knockdown of PDE5 expression in colon tumor cells with siRNA resulted in a significant reduction of tumor cell growth and increased apoptosis. Moreover, SS increased intracellular cGMP levels, activated protein kinase G (PKG) and suppressed the expression of β-catenin in colon tumor cells. SS also inhibited the transcriptional activity of β-catenin and reduced the expression of the β-catenin regulated proteins cyclin D1 and survivin, which are important mediators of tumor cell proliferation and apoptosis, respectively. Our results suggest that the chemopreventive properties of sulindac are closely associated with PDE5 inhibition and involve the activation of cGMP signaling to suppress β-catenin mediated transcriptional activity. Funding provided by NIH/NCI grants CA131378 and CA148817. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3707. doi:10.1158/1538-7445.AM2011-3707