Bioorganic & Medicinal Chemistry 22 (2014) 756–761
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of nitric oxide-donating analogues of sulindac for prostate cancer treatment Andrew Nortcliffe a, Alexander G. Ekstrom a, James R. Black b, James A. Ross b, Fouad K. Habib c, Nigel P. Botting a, ,⇑, David O’Hagan a,⇑ a b c
EaStCHEM School of Chemistry, Biomolecular Sciences Research Complex, University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST, UK Tissue Injury and Repair Group, Department of Clinical and Surgical Sciences (Surgery), University of Edinburgh, Edinburgh EH16 4SB, UK Prostate Research Group, Edinburgh Cancer Research Centre, University of Edinburgh, Edinburgh EH4 2XR, UK
a r t i c l e
i n f o
Article history: Received 22 September 2013 Revised 2 December 2013 Accepted 6 December 2013 Available online 12 December 2013 Keywords: Nitric oxide Sulindac analogues Furoxan Sydnonimine Prostate cancer
a b s t r a c t A series of analogues of the non-steroidal anti-inflammatory drug (NSAID) sulindac 1 were synthesised tethered to nitric oxide (NO) donating functional groups. Sulindac shows antiproliterative effects against immortal PC3 cell lines. It was previously demonstrated that the effect can be enhanced when tethered to NO releasing groups such as nitrate esters, furoxans and sydnonimines. To explore this approach further, a total of fifty-six sulindac–NO analogues were prepared and they were evaluated as NO-releasing cytotoxic agents against prostate cancer (PCa) cell lines. Compounds 1k and 1n exhibited significant cytotoxic with IC50 values of 6.1 ± 4.1 and 12.1 ± 3.2 lM, respectively, coupled with observed nitric oxide release. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction In the United Kingdom, prostate cancer (PCa) accounts for 13% of male cancer deaths.1 In 2010, 40,975 men were newly diagnosed with prostate cancer, accounting for almost 1 in 4 of all new male cancers diagnosed.1 Both localised and locally advanced prostate cancers are highly responsive to androgen ablation therapies2 including androgen receptor antagonists for example, bicalutamide and flutamide, GnRH agonists for example, leuprolide and more recently GnRH antagonists for example, degarelix. But over the course of 2–5 years most tumours relapse into a castration-resistant stage2 that is subsequently treated with chemotherapy. Recently, research has been focused on the role of hypoxia in the induction of castration-resistant PCa.3 Hypoxia has been shown to correlate with increased metastasis, angiogenesis and resistance to therapy.3 In PCa, hypoxia is associated with poor patient prognosis.4 It was demonstrated by Stewart et al. that an NO-donating analogue of sulindac 1, NCX-1102 2 has a pro-apoptotic and anti-invasive effect on PC3 prostate cancer cells.5 In addition, the des-nitrato analogue NCX-112 3, had increased activity over sulindac 1, but decreased compared to NCX-1102 2. The activity of NCX-1102 2 was conserved under normoxic and hypoxic conditions. The master regulator of oxygen homeostasis in the cell is the transcription factor ⇑ Corresponding authors. Tel.: +44 (0)1334 463800; fax: +44 (0)1334 467171.
E-mail address: [email protected] (D. O’Hagan). Nigel P. Botting passed away on 4th June 2011.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmc.2013.12.014
hypoxia-inducible factor 1 (HIF-1). Under hypoxic conditions HIF-1a expression was down-regulated upon treatment with NCX-1102 2; normal expression was observed when treated with sulindac 1 and NCX-112 3.5 NCX-1102 2 is predicted to function as a nitric oxide (NO) donor, and as such, the observed increase in activity was attributed to ‘NO bioactivity’. In addition to decreased HIF-1a expression, a reduction in Akt phosphorylation was observed suggesting that the PI3K–Akt–mTOR signal transduction pathway is one of the mechanisms operating in the activity of NCX-1102 2, reinforcing earlier reports of interactions between this pathway and HIF-1a expression under hypoxic conditions.6–8 Herein, we report the synthesis of a library of novel sulindac analogues exploring the relationship between sulindac 1 and a range of NO-donating functional groups. Including the identification of a series of furoxans with significant anti-proliferative activity at 50 lM.
A. Nortcliffe et al. / Bioorg. Med. Chem. 22 (2014) 756–761
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2. Results and discussion 2.1. Chemistry We identified three classes of nitric oxide-donating functionalities to develop into sulindac-NO hybrids; nitrate esters, furoxans and sydnonimines. Based on literature examples where the oxidation state of sulfur in sulindac conferred differing biological activities in vitro,9,10 we first began by preparing the known compounds sulindac sulfide 4 and sulindac sulfone 5. To this effect, commercially available sulindac 1 (sulfoxide) was reduced to the corresponding sulfide 4 with TiCl4/Zn, and oxidised to the sulfone 6 with OxoneÒ (Scheme 1).11 With these starting materials in hand we turned our attention to a series of nitrate ester hybrids. 2.1.1. Nitrate esters Nitrate ester alcohols 6–10 were readily prepared by halide substitution with silver nitrate. Ring-opening of THF 11 with NaI and TBDMSCl gave TBDMS protected iodobutanol 12, followed by nitrate substitution and subsequent deprotection, provided the butyl linked nitrate ester alcohol 6 (Scheme 2),12,13 analogous to NCX1102 2. Further nitrate ester alcohols 7–10 were prepared from the available bromoalcohols 13–16 in good yields (Scheme 2).14–16 Ester formation of these nitrooxyalcohols 6–10 with sulindac congeners 1, 4 and 5 using EDCIHCl and catalytic DMAP generated the desired esters 1a–e, 4a–e and 5a–e in good yield (Scheme 3). Appropriate hydroxybutyl ester control compounds were also prepared, analogous to NCX-112 3. Ester formation with monoTBDSMS 1,4-butanediol followed by deprotection with camphorsulfonic acid furnished the required alcohols 1g, 4g and 5g (Scheme 4). 2.1.2. Furoxans Furoxan alcohols 19a–i were readily prepared from bis(phenylsulfonyl)furoxan 18.17,18 Treatment of phenyl(sulfonyl)acetic acid 17 with refluxing HNO3/AcOH, generated the desired furoxan by nitrile oxide dimerisation (Scheme 5).17 Selective substitution at the 4-position of furoxan 18 was accomplished by treatment with the corresponding diol and 50% w/w NaOH (Scheme 5).17 The preparation of hydroxyethyl-furoxan 19a and hydroxyethyl(ethoxy)-furoxan 19e required mono-TBDMS protection of the diol to avoid side products. TBDMS deprotection with camphorsulfonic acid provided the furoxans 19a and 19e. Esterification of furoxans 19a–i with acids 1, 4 and 5 using the previously established EDCI conditions provided esters 1h–p, 4h–p and 5h–p (Scheme 6). 2.1.3. Sydnonimines 3-Phenylsydnonimine 21 was prepared from aniline 20 based on the modified procedure reported earlier.19 Alkylation to the aminoacetonitrile and subsequent N-nitrosation and acid catalysed ring closure furnished the required sydnonimine 21 (Scheme 7).19
Scheme 1. Synthesis of sulindac sulfide 4 and sulindac sulfone 6. Reagents and conditions: (a) TiCl4, Zn, THF, rt, 0.5 h, quantitative; (b) OxoneÒ, 1:1 MeOH, H2O, rt, 1 h, quantitative.
Scheme 2. Synthesis of nitrate ester alcohols 6–10. Reagents and conditions: (a) TBDMSCl, NaI, THF, 55 °C, 18 h, quant.; (b) (i) AgNO3, CH3CN, 10 °C to rt, 1 h, (ii) H2O, 1 h, 29%; (c) (a) AgNO3, CH3CN, reflux, 6 h 85–90%; (d.) AgNO3, CH3CN, rt, 7 d, 26%.
Acylation of congeners 1, 4 and 5 with 21 using EDCIHCl and DMAP provided the sydnonimine amides 1q, 4q and 5q (Scheme 8). The use of acetonitrile as a cosolvent was required to aid dissolution of 21 in the reaction. Acylation of 21 with p-nitrophenylchloroformate provided activated sydnonimine 22 for attachment to linkers (Scheme 7).19 To this effect, activation of carboxylic acids 1, 4 and 5 with carbonyl diimidazole and the addition of an excess of ethanolamine or ethylenediamine allowed for selective monoacylation to provide alcohols 1r, 4r, 5r and amines 1s, 4s and 5s (Scheme 9). Reaction of alcohols 1r, 4r, 5r and amines 1s, 4s and 5s with activated carbamate 22 in acetonitrile at 90 °C provided the respective carbamates 1t, 4t, 5t and ureas 1u, 4u and 5u (Scheme 9). 2.2. Biological results 2.2.1. Cytotoxicity results PC3 cells (hormone insensitive human prostate cancer cells) obtained from the European Collection of Cell Cultures (ECACC), Salisbury, UK were used to determine cytotoxicity of the sulindac analogues. The cells were cultured in RPMI 1640 with 5% FCS and seeded in 96 well plates at a concentration of approximately 3000 cells/well. Sulindac-NO analogues were prepared in dimethyl sulfoxide (DMSO) at a final DMSO concentration of 0.05% in the culture media. Initially, the sulindac analogues were incubated at 50 lM with the cells for 72 h and cell growth measured at 24 h intervals using the crystal violet method.20 The inhibitory effect on cell growth was recorded as a percentage of cells remaining compared to medium control (100% cell viability) (Table 1 and Supplementary information, Tables 1–3). As with sulindac (sulfoxide) 1, the corresponding sulfide 4 and sulfone 5 had no effect on PC3 cells at 50 lM (Table 1). In addition, the 4-hydroxybutyl esters 1g, 4g and 5g showed no significant cytotoxicity (Supplementary information, Table 1). 4-Nitrooxybutyl esters 1a, 4a and 5a demonstrated a strongly cytotoxic effect at 50 lM, with 4a showing improved activity over NCX-1102 (Table 1). Further development of the nitrate ester series to investigate increased linker length, and the number of nitrate esters per unit of sulindac, provided a series of compounds which had no cytotoxic activity, but with two exceptions (Supplementary information, Table 2). These were 2-nitrooxyethyl ester 1b and dinitrate ester 1d. They displayed an overall reduction in cell
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Scheme 3. Synthesis of nitrate ester–sulindac analogues. Reagents and conditions: (a) 6–10, EDCIHCl, CH2Cl2, DMAP, rt, 3 h, 44–95%.
Scheme 4. Synthesis of sulindac control alcohols. Reagents and conditions: (a) mono-TBDMS 1,4-butanediol, EDCIHCl, CH2Cl2, DMAP, rt, 3 h, 85–98%; (b) 10-CSA, CH2Cl2/ CH3OH, rt, 3 h, 63–82%.
R1
R1
a F
F
O
O
O O R2
OH
1: R1 = S(O)CH 3 4: R1 = SCH 3 5: R1 = SO2CH 3 Scheme 5. Synthesis of furoxan alcohols 19a–i. Reagents and conditions: (a) HNO3, AcOH, reflux, 1.5 h, 27%; (b) diol or monoprotected diol, 50% w/w NaOH, THF, rt, 3 h, 33–82%.
growth to 35.5% ± 4.23 and 30.2% ± 4.49, respectively. However, given the overall poor activity of this series, none of the compounds emerged as candidates for further testing. The cytotoxicity of the furoxan analogues 1, 4 and 5 h–p is summarised in Table 1. In contrast to the nitrate ester analogues, the furoxans were a much more promising series of compounds. At 50 lM, 13 of the 27 compounds tested showed complete cell death after 24 h. A further 11 compounds displayed a significant reduction in cell growth
O O S N N O
O
1h-p: R1 = S(O)CH 3 4h-p: R1 = SCH 3 5h-p: R1 = SO2CH 3
h: R 2 =
m: R 2 =
i: R 2 =
n: R 2 =
j: R 2 =
o: R 2 =
k: R1 = l: R1 =
O
p: R1 =
Scheme 6. Synthesis of furoxan–sulindac analogues. Reagents and conditions: (a) 19a–i, EDCIHCl, CH2Cl2, DMAP, rt, 3 h, 25–89%.
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A. Nortcliffe et al. / Bioorg. Med. Chem. 22 (2014) 756–761 Table 1 Cytotoxicity results of furoxan–sulindac analogues produced via Scheme 6 Entry
Scheme 7. Synthesis of sydnonimines 21 and 22. Reagents and conditions: (a) (i) BrCH2CN, K2CO3, NaI, CH3CN, rt, 18 h, 87% (ii) isopentyl nitrite, Et2O, 18 h, excluded from light, (iii) HCl (g), Et2O, rt, 15 min, 94%.
Scheme 8. Synthesis of sydnonimine–sulindac analogues. Reagents and conditions: (a) 21, EDCIHCl, CH2Cl2/CH3CN (9:1), DMAP, rt, 3 h, 70–85%.
after 72 h. Only three compounds had no significant activity. Encouraged by the increased activity of this series a sub-set of the library was selected for lower dose screening and NO release. Seven furoxan analogues, 1h, 1k,1l, 1n, 4h, 4i, 4j were tested at lower concentrations to determine their cytotoxic potential (Fig. 1 and Figure 2). Analogues 4j and 4h showed significant cytotoxicity at 500 nM; resulting in a reduction in cell growth to 9.17% ± 2.80 and 11.90 ± 1.70, respectively (Fig. 1). Furoxan–sulindac analogues 1k and 1n, with hexyl and cis-butenyl linkers, provided compounds which were highly cytotoxic at 50 nM (Fig. 2); a concentration at which NCX-1102 2 has a proliferative effect on cells. Inhibition concentrations for compounds 1h, 1k, 1l, 1m, 4h, 4i and 4j were determined using the crystal violet method and are reported in Table 2, with the most active compounds 1k and 1n displaying IC50 values of 6.1 ± 4.1 and 12.1 ± 3.2 lm, respectively. In the sydnonimine series statistically significant cytotoxicity was observed for seven of the nine analogues (Supplementary information, Table 3). With the amide 1q, and sulfide carbamate 4t and urea 4u showing comparable activity to NCX-1102 2 at
DMSO 0.05% NCX-1102 2 1h 4h 5h 1i 4i 5i 1j 4j 5j 1k 4k 5k 1l 4l 4l 1m 4m 5m 1n 4n 5n 1o 4o 5o 1p 4p 5p
% PC3 cells alive at intervals after treatment at 50 lM 24 h
48 h
72 h
99.09 ± 9.87 58.76 ± 8.16 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 34.94 ± 4.70 0.00 ± 0.00 32.33 ± 4.90 0.00 ± 0.00 67.87 ± 15.37 0.00 ± 0.00 57.23 ± 5.15 42.37 ± 2.43 0.00 ± 0.00 55.22 ± 6.04 0.00 ± 0.00 0.00 ± 0.00 25.40 ± 5.15 36.54 ± 5.12 0.00 ± 0.00 30.55 ± 2.72 0.00 ± 0.00 71.77 ± 4.12 101.87 ± 3.96 88.27 ± 3.19 73.98 ± 3.57 81.12 ± 3.10 0.00 ± 0.00
100.87 ± 4.87 35.52 ± 5.27 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 21.22 ± 4.02 0.00 ± 0.00 21.78 ± 1.84 0.00 ± 0.00 49.89 ± 3.66 0.00 ± 0.00 41.67 ± 8.42 24.22 ± 4.84 0.00 ± 0.00 71.00 ± 5.29 0.00 ± 0.00 0.00 ± 0.00 20.78 ± 1.95 38.00 ± 5.57 0.00 ± 0.00 31.44 ± 6.11 0.00 ± 0.00 56.24 ± 2.26 79.65 ± 4.61 67.69 ± 8.98 67.69 ± 11.14 88.29 ± 5.33 0.00 ± 0.00
95.69 ± 7.66 24.52 ± 3.92 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 15.67 ± 2.48 0.00 ± 0.00 16.52 ± 3.08 0.00 ± 0.00 38.76 ± 3.31 0.00 ± 0.00 29.24 ± 5.27 15.43 ± 1.25 0.00 ± 0.00 50.76 ± 5.65 0.00 ± 0.00 0.00 ± 0.00 14.95 ± 2.86 27.33 ± 6.91 0.00 ± 0.00 20.62 ± 3.32 0.00 ± 0.00 51.90 ± 1.04 82.67 ± 3.75 63.61 ± 0.73 66.26 ± 10.44 80.65 ± 3.02 0.00 ± 0.00
Figure 1. Cytotoxicity results of furoxan–sulindac analogues 1h, 4h, 4i, 4j and 1l at 500 nM
Scheme 9. Synthesis of sydnonimine–sulindac analogues. Reagents and conditions: (a) (i) CDI, CH2Cl2, rt, 3 h (ii) ethanolamine or ethylenediamine, CH2Cl2, rt, 18 h, 81–90%; (b) 22, CH3CN, reflux, 16 h, 49–90%.
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trite (NO2 ) is a reliable proxy measure for nitric oxide, in the presence of thiol.22 To this effect, the Griess test for nitrite was used to determine NO production from analogues 4h, 4j, 1k, 1n relative to sulindac 2. Sulindac analogues (1 mmol, [final] 400 lM) in a solution of DMSO were analysed after incubation with a glutathione (GT) buffer. ([GT] 12 mmol, final [GT] 1.2 mmol). Furoxans 4h, 4j, 1k, 1n exhibited GT dependent NO release, up to 13.6% of total for 1k (Table 3). No observable nitrite/NO release was recorded from sulindac 1 (Table 3). The amount of NO required to induce a change in cellular response is on the pico- to nano-molar scale.23,24 3. Conclusions
Figure 2. Cytotoxicity results of furoxan–sulindac analogues 1k and 1n at 200–50 nM
Table 2 IC50 values for active furoxan–sulindac analogues produced via Scheme 6 Compound
IC50 (lM)
1h 1k 1l 1n 4h 4i 4j
27.9 ± 7.7 6.1 ± 4.1 26.0 ± 10.4 12.1 ± 3.2 41.4 ± 5.8 48.6 ± 7.2 40.3 ± 4.9
In conclusion, a series of analogues of sulindac 1, containing NO-releasing functional groups appended as esters were prepared. The overall synthesis provided fifty-six novel analogues using nitrate esters, furoxans and sydnonimines as tethered NO-release functional groups. The biological activity was evaluated by PC3 proliferation studies, and NO release assays (Griess test). Two lead compounds (1k and 1n) emerged with significant activity at 50 nM compared to sulindac 1 and NCX-1102 2. NO release was determined from these furoxan analogues showing up to 13% NO-release after incubation with GT for one hour. Further studies are underway to determine the ability of these compounds to knockdown HIF-1 alpha mRNA expression in the cell and inhibit tumour adaptation to hypoxia. 4. Experimental 4.1. Cytotoxicity assays Human prostate adenocarcinoma (PC3) cells were obtained from the European Collection of Cell Cultures (ECACC), Salisbury. Cell cultures were grown in RPMI 1640 medium, supplemented with 5% foetal calf serum (100 lL/well). For assay, cells were plated into 96 well cell culture plates (Grenier Bio-One) at a concentration of 3000 cells/well. Cytotoxicity was determined by a crystal violet assay. After incubation with test compounds for the required length of time, the culture medium was decanted from wells and washed with PBS. Crystal violet (CV) solution (0.2%, 50 lL/well) was added and incubated for 10 min at room temperature. Following incubation the culture plates were washed by submersion in hand-warm water (2) and air dried. SDS solution (1%, 100 lL) added. The plates were agitated to distribute the dye, and the absorbance measured at 570 nm.
Figure 3. Cytotoxicity results of sydnonimine–sulindac analogues at 50–500 nM.
Table 3 Griess test results for furoxan analogues Compound
NO2 production (lM)
%NO
4h 4j 1k 1n Sulindac 1 DMSO
41.1 ± 13.5 33.0 ± 6.8 54.4 ± 2.9 51.1 ± 1.3 1.1 ± 2.2 3.3 ± 1.2
10.3 8.2 13.6 12.8
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of nitric oxide-donating analogues of sulindac for prostate cancer treatment Andrew Nortcliffe a, Alexander G. Ekstrom a, James R. Black b, James A. Ross b, Fouad K. Habib c, Nigel P. Botting a, ,⇑, David O’Hagan a,⇑ a b c
EaStCHEM School of Chemistry, Biomolecular Sciences Research Complex, University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST, UK Tissue Injury and Repair Group, Department of Clinical and Surgical Sciences (Surgery), University of Edinburgh, Edinburgh EH16 4SB, UK Prostate Research Group, Edinburgh Cancer Research Centre, University of Edinburgh, Edinburgh EH4 2XR, UK
a r t i c l e
i n f o
Article history: Received 22 September 2013 Revised 2 December 2013 Accepted 6 December 2013 Available online 12 December 2013 Keywords: Nitric oxide Sulindac analogues Furoxan Sydnonimine Prostate cancer
a b s t r a c t A series of analogues of the non-steroidal anti-inflammatory drug (NSAID) sulindac 1 were synthesised tethered to nitric oxide (NO) donating functional groups. Sulindac shows antiproliterative effects against immortal PC3 cell lines. It was previously demonstrated that the effect can be enhanced when tethered to NO releasing groups such as nitrate esters, furoxans and sydnonimines. To explore this approach further, a total of fifty-six sulindac–NO analogues were prepared and they were evaluated as NO-releasing cytotoxic agents against prostate cancer (PCa) cell lines. Compounds 1k and 1n exhibited significant cytotoxic with IC50 values of 6.1 ± 4.1 and 12.1 ± 3.2 lM, respectively, coupled with observed nitric oxide release. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction In the United Kingdom, prostate cancer (PCa) accounts for 13% of male cancer deaths.1 In 2010, 40,975 men were newly diagnosed with prostate cancer, accounting for almost 1 in 4 of all new male cancers diagnosed.1 Both localised and locally advanced prostate cancers are highly responsive to androgen ablation therapies2 including androgen receptor antagonists for example, bicalutamide and flutamide, GnRH agonists for example, leuprolide and more recently GnRH antagonists for example, degarelix. But over the course of 2–5 years most tumours relapse into a castration-resistant stage2 that is subsequently treated with chemotherapy. Recently, research has been focused on the role of hypoxia in the induction of castration-resistant PCa.3 Hypoxia has been shown to correlate with increased metastasis, angiogenesis and resistance to therapy.3 In PCa, hypoxia is associated with poor patient prognosis.4 It was demonstrated by Stewart et al. that an NO-donating analogue of sulindac 1, NCX-1102 2 has a pro-apoptotic and anti-invasive effect on PC3 prostate cancer cells.5 In addition, the des-nitrato analogue NCX-112 3, had increased activity over sulindac 1, but decreased compared to NCX-1102 2. The activity of NCX-1102 2 was conserved under normoxic and hypoxic conditions. The master regulator of oxygen homeostasis in the cell is the transcription factor ⇑ Corresponding authors. Tel.: +44 (0)1334 463800; fax: +44 (0)1334 467171.
E-mail address: [email protected] (D. O’Hagan). Nigel P. Botting passed away on 4th June 2011.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmc.2013.12.014
hypoxia-inducible factor 1 (HIF-1). Under hypoxic conditions HIF-1a expression was down-regulated upon treatment with NCX-1102 2; normal expression was observed when treated with sulindac 1 and NCX-112 3.5 NCX-1102 2 is predicted to function as a nitric oxide (NO) donor, and as such, the observed increase in activity was attributed to ‘NO bioactivity’. In addition to decreased HIF-1a expression, a reduction in Akt phosphorylation was observed suggesting that the PI3K–Akt–mTOR signal transduction pathway is one of the mechanisms operating in the activity of NCX-1102 2, reinforcing earlier reports of interactions between this pathway and HIF-1a expression under hypoxic conditions.6–8 Herein, we report the synthesis of a library of novel sulindac analogues exploring the relationship between sulindac 1 and a range of NO-donating functional groups. Including the identification of a series of furoxans with significant anti-proliferative activity at 50 lM.
A. Nortcliffe et al. / Bioorg. Med. Chem. 22 (2014) 756–761
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2. Results and discussion 2.1. Chemistry We identified three classes of nitric oxide-donating functionalities to develop into sulindac-NO hybrids; nitrate esters, furoxans and sydnonimines. Based on literature examples where the oxidation state of sulfur in sulindac conferred differing biological activities in vitro,9,10 we first began by preparing the known compounds sulindac sulfide 4 and sulindac sulfone 5. To this effect, commercially available sulindac 1 (sulfoxide) was reduced to the corresponding sulfide 4 with TiCl4/Zn, and oxidised to the sulfone 6 with OxoneÒ (Scheme 1).11 With these starting materials in hand we turned our attention to a series of nitrate ester hybrids. 2.1.1. Nitrate esters Nitrate ester alcohols 6–10 were readily prepared by halide substitution with silver nitrate. Ring-opening of THF 11 with NaI and TBDMSCl gave TBDMS protected iodobutanol 12, followed by nitrate substitution and subsequent deprotection, provided the butyl linked nitrate ester alcohol 6 (Scheme 2),12,13 analogous to NCX1102 2. Further nitrate ester alcohols 7–10 were prepared from the available bromoalcohols 13–16 in good yields (Scheme 2).14–16 Ester formation of these nitrooxyalcohols 6–10 with sulindac congeners 1, 4 and 5 using EDCIHCl and catalytic DMAP generated the desired esters 1a–e, 4a–e and 5a–e in good yield (Scheme 3). Appropriate hydroxybutyl ester control compounds were also prepared, analogous to NCX-112 3. Ester formation with monoTBDSMS 1,4-butanediol followed by deprotection with camphorsulfonic acid furnished the required alcohols 1g, 4g and 5g (Scheme 4). 2.1.2. Furoxans Furoxan alcohols 19a–i were readily prepared from bis(phenylsulfonyl)furoxan 18.17,18 Treatment of phenyl(sulfonyl)acetic acid 17 with refluxing HNO3/AcOH, generated the desired furoxan by nitrile oxide dimerisation (Scheme 5).17 Selective substitution at the 4-position of furoxan 18 was accomplished by treatment with the corresponding diol and 50% w/w NaOH (Scheme 5).17 The preparation of hydroxyethyl-furoxan 19a and hydroxyethyl(ethoxy)-furoxan 19e required mono-TBDMS protection of the diol to avoid side products. TBDMS deprotection with camphorsulfonic acid provided the furoxans 19a and 19e. Esterification of furoxans 19a–i with acids 1, 4 and 5 using the previously established EDCI conditions provided esters 1h–p, 4h–p and 5h–p (Scheme 6). 2.1.3. Sydnonimines 3-Phenylsydnonimine 21 was prepared from aniline 20 based on the modified procedure reported earlier.19 Alkylation to the aminoacetonitrile and subsequent N-nitrosation and acid catalysed ring closure furnished the required sydnonimine 21 (Scheme 7).19
Scheme 1. Synthesis of sulindac sulfide 4 and sulindac sulfone 6. Reagents and conditions: (a) TiCl4, Zn, THF, rt, 0.5 h, quantitative; (b) OxoneÒ, 1:1 MeOH, H2O, rt, 1 h, quantitative.
Scheme 2. Synthesis of nitrate ester alcohols 6–10. Reagents and conditions: (a) TBDMSCl, NaI, THF, 55 °C, 18 h, quant.; (b) (i) AgNO3, CH3CN, 10 °C to rt, 1 h, (ii) H2O, 1 h, 29%; (c) (a) AgNO3, CH3CN, reflux, 6 h 85–90%; (d.) AgNO3, CH3CN, rt, 7 d, 26%.
Acylation of congeners 1, 4 and 5 with 21 using EDCIHCl and DMAP provided the sydnonimine amides 1q, 4q and 5q (Scheme 8). The use of acetonitrile as a cosolvent was required to aid dissolution of 21 in the reaction. Acylation of 21 with p-nitrophenylchloroformate provided activated sydnonimine 22 for attachment to linkers (Scheme 7).19 To this effect, activation of carboxylic acids 1, 4 and 5 with carbonyl diimidazole and the addition of an excess of ethanolamine or ethylenediamine allowed for selective monoacylation to provide alcohols 1r, 4r, 5r and amines 1s, 4s and 5s (Scheme 9). Reaction of alcohols 1r, 4r, 5r and amines 1s, 4s and 5s with activated carbamate 22 in acetonitrile at 90 °C provided the respective carbamates 1t, 4t, 5t and ureas 1u, 4u and 5u (Scheme 9). 2.2. Biological results 2.2.1. Cytotoxicity results PC3 cells (hormone insensitive human prostate cancer cells) obtained from the European Collection of Cell Cultures (ECACC), Salisbury, UK were used to determine cytotoxicity of the sulindac analogues. The cells were cultured in RPMI 1640 with 5% FCS and seeded in 96 well plates at a concentration of approximately 3000 cells/well. Sulindac-NO analogues were prepared in dimethyl sulfoxide (DMSO) at a final DMSO concentration of 0.05% in the culture media. Initially, the sulindac analogues were incubated at 50 lM with the cells for 72 h and cell growth measured at 24 h intervals using the crystal violet method.20 The inhibitory effect on cell growth was recorded as a percentage of cells remaining compared to medium control (100% cell viability) (Table 1 and Supplementary information, Tables 1–3). As with sulindac (sulfoxide) 1, the corresponding sulfide 4 and sulfone 5 had no effect on PC3 cells at 50 lM (Table 1). In addition, the 4-hydroxybutyl esters 1g, 4g and 5g showed no significant cytotoxicity (Supplementary information, Table 1). 4-Nitrooxybutyl esters 1a, 4a and 5a demonstrated a strongly cytotoxic effect at 50 lM, with 4a showing improved activity over NCX-1102 (Table 1). Further development of the nitrate ester series to investigate increased linker length, and the number of nitrate esters per unit of sulindac, provided a series of compounds which had no cytotoxic activity, but with two exceptions (Supplementary information, Table 2). These were 2-nitrooxyethyl ester 1b and dinitrate ester 1d. They displayed an overall reduction in cell
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A. Nortcliffe et al. / Bioorg. Med. Chem. 22 (2014) 756–761
Scheme 3. Synthesis of nitrate ester–sulindac analogues. Reagents and conditions: (a) 6–10, EDCIHCl, CH2Cl2, DMAP, rt, 3 h, 44–95%.
Scheme 4. Synthesis of sulindac control alcohols. Reagents and conditions: (a) mono-TBDMS 1,4-butanediol, EDCIHCl, CH2Cl2, DMAP, rt, 3 h, 85–98%; (b) 10-CSA, CH2Cl2/ CH3OH, rt, 3 h, 63–82%.
R1
R1
a F
F
O
O
O O R2
OH
1: R1 = S(O)CH 3 4: R1 = SCH 3 5: R1 = SO2CH 3 Scheme 5. Synthesis of furoxan alcohols 19a–i. Reagents and conditions: (a) HNO3, AcOH, reflux, 1.5 h, 27%; (b) diol or monoprotected diol, 50% w/w NaOH, THF, rt, 3 h, 33–82%.
growth to 35.5% ± 4.23 and 30.2% ± 4.49, respectively. However, given the overall poor activity of this series, none of the compounds emerged as candidates for further testing. The cytotoxicity of the furoxan analogues 1, 4 and 5 h–p is summarised in Table 1. In contrast to the nitrate ester analogues, the furoxans were a much more promising series of compounds. At 50 lM, 13 of the 27 compounds tested showed complete cell death after 24 h. A further 11 compounds displayed a significant reduction in cell growth
O O S N N O
O
1h-p: R1 = S(O)CH 3 4h-p: R1 = SCH 3 5h-p: R1 = SO2CH 3
h: R 2 =
m: R 2 =
i: R 2 =
n: R 2 =
j: R 2 =
o: R 2 =
k: R1 = l: R1 =
O
p: R1 =
Scheme 6. Synthesis of furoxan–sulindac analogues. Reagents and conditions: (a) 19a–i, EDCIHCl, CH2Cl2, DMAP, rt, 3 h, 25–89%.
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A. Nortcliffe et al. / Bioorg. Med. Chem. 22 (2014) 756–761 Table 1 Cytotoxicity results of furoxan–sulindac analogues produced via Scheme 6 Entry
Scheme 7. Synthesis of sydnonimines 21 and 22. Reagents and conditions: (a) (i) BrCH2CN, K2CO3, NaI, CH3CN, rt, 18 h, 87% (ii) isopentyl nitrite, Et2O, 18 h, excluded from light, (iii) HCl (g), Et2O, rt, 15 min, 94%.
Scheme 8. Synthesis of sydnonimine–sulindac analogues. Reagents and conditions: (a) 21, EDCIHCl, CH2Cl2/CH3CN (9:1), DMAP, rt, 3 h, 70–85%.
after 72 h. Only three compounds had no significant activity. Encouraged by the increased activity of this series a sub-set of the library was selected for lower dose screening and NO release. Seven furoxan analogues, 1h, 1k,1l, 1n, 4h, 4i, 4j were tested at lower concentrations to determine their cytotoxic potential (Fig. 1 and Figure 2). Analogues 4j and 4h showed significant cytotoxicity at 500 nM; resulting in a reduction in cell growth to 9.17% ± 2.80 and 11.90 ± 1.70, respectively (Fig. 1). Furoxan–sulindac analogues 1k and 1n, with hexyl and cis-butenyl linkers, provided compounds which were highly cytotoxic at 50 nM (Fig. 2); a concentration at which NCX-1102 2 has a proliferative effect on cells. Inhibition concentrations for compounds 1h, 1k, 1l, 1m, 4h, 4i and 4j were determined using the crystal violet method and are reported in Table 2, with the most active compounds 1k and 1n displaying IC50 values of 6.1 ± 4.1 and 12.1 ± 3.2 lm, respectively. In the sydnonimine series statistically significant cytotoxicity was observed for seven of the nine analogues (Supplementary information, Table 3). With the amide 1q, and sulfide carbamate 4t and urea 4u showing comparable activity to NCX-1102 2 at
DMSO 0.05% NCX-1102 2 1h 4h 5h 1i 4i 5i 1j 4j 5j 1k 4k 5k 1l 4l 4l 1m 4m 5m 1n 4n 5n 1o 4o 5o 1p 4p 5p
% PC3 cells alive at intervals after treatment at 50 lM 24 h
48 h
72 h
99.09 ± 9.87 58.76 ± 8.16 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 34.94 ± 4.70 0.00 ± 0.00 32.33 ± 4.90 0.00 ± 0.00 67.87 ± 15.37 0.00 ± 0.00 57.23 ± 5.15 42.37 ± 2.43 0.00 ± 0.00 55.22 ± 6.04 0.00 ± 0.00 0.00 ± 0.00 25.40 ± 5.15 36.54 ± 5.12 0.00 ± 0.00 30.55 ± 2.72 0.00 ± 0.00 71.77 ± 4.12 101.87 ± 3.96 88.27 ± 3.19 73.98 ± 3.57 81.12 ± 3.10 0.00 ± 0.00
100.87 ± 4.87 35.52 ± 5.27 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 21.22 ± 4.02 0.00 ± 0.00 21.78 ± 1.84 0.00 ± 0.00 49.89 ± 3.66 0.00 ± 0.00 41.67 ± 8.42 24.22 ± 4.84 0.00 ± 0.00 71.00 ± 5.29 0.00 ± 0.00 0.00 ± 0.00 20.78 ± 1.95 38.00 ± 5.57 0.00 ± 0.00 31.44 ± 6.11 0.00 ± 0.00 56.24 ± 2.26 79.65 ± 4.61 67.69 ± 8.98 67.69 ± 11.14 88.29 ± 5.33 0.00 ± 0.00
95.69 ± 7.66 24.52 ± 3.92 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 15.67 ± 2.48 0.00 ± 0.00 16.52 ± 3.08 0.00 ± 0.00 38.76 ± 3.31 0.00 ± 0.00 29.24 ± 5.27 15.43 ± 1.25 0.00 ± 0.00 50.76 ± 5.65 0.00 ± 0.00 0.00 ± 0.00 14.95 ± 2.86 27.33 ± 6.91 0.00 ± 0.00 20.62 ± 3.32 0.00 ± 0.00 51.90 ± 1.04 82.67 ± 3.75 63.61 ± 0.73 66.26 ± 10.44 80.65 ± 3.02 0.00 ± 0.00
Figure 1. Cytotoxicity results of furoxan–sulindac analogues 1h, 4h, 4i, 4j and 1l at 500 nM
Scheme 9. Synthesis of sydnonimine–sulindac analogues. Reagents and conditions: (a) (i) CDI, CH2Cl2, rt, 3 h (ii) ethanolamine or ethylenediamine, CH2Cl2, rt, 18 h, 81–90%; (b) 22, CH3CN, reflux, 16 h, 49–90%.
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trite (NO2 ) is a reliable proxy measure for nitric oxide, in the presence of thiol.22 To this effect, the Griess test for nitrite was used to determine NO production from analogues 4h, 4j, 1k, 1n relative to sulindac 2. Sulindac analogues (1 mmol, [final] 400 lM) in a solution of DMSO were analysed after incubation with a glutathione (GT) buffer. ([GT] 12 mmol, final [GT] 1.2 mmol). Furoxans 4h, 4j, 1k, 1n exhibited GT dependent NO release, up to 13.6% of total for 1k (Table 3). No observable nitrite/NO release was recorded from sulindac 1 (Table 3). The amount of NO required to induce a change in cellular response is on the pico- to nano-molar scale.23,24 3. Conclusions
Figure 2. Cytotoxicity results of furoxan–sulindac analogues 1k and 1n at 200–50 nM
Table 2 IC50 values for active furoxan–sulindac analogues produced via Scheme 6 Compound
IC50 (lM)
1h 1k 1l 1n 4h 4i 4j
27.9 ± 7.7 6.1 ± 4.1 26.0 ± 10.4 12.1 ± 3.2 41.4 ± 5.8 48.6 ± 7.2 40.3 ± 4.9
In conclusion, a series of analogues of sulindac 1, containing NO-releasing functional groups appended as esters were prepared. The overall synthesis provided fifty-six novel analogues using nitrate esters, furoxans and sydnonimines as tethered NO-release functional groups. The biological activity was evaluated by PC3 proliferation studies, and NO release assays (Griess test). Two lead compounds (1k and 1n) emerged with significant activity at 50 nM compared to sulindac 1 and NCX-1102 2. NO release was determined from these furoxan analogues showing up to 13% NO-release after incubation with GT for one hour. Further studies are underway to determine the ability of these compounds to knockdown HIF-1 alpha mRNA expression in the cell and inhibit tumour adaptation to hypoxia. 4. Experimental 4.1. Cytotoxicity assays Human prostate adenocarcinoma (PC3) cells were obtained from the European Collection of Cell Cultures (ECACC), Salisbury. Cell cultures were grown in RPMI 1640 medium, supplemented with 5% foetal calf serum (100 lL/well). For assay, cells were plated into 96 well cell culture plates (Grenier Bio-One) at a concentration of 3000 cells/well. Cytotoxicity was determined by a crystal violet assay. After incubation with test compounds for the required length of time, the culture medium was decanted from wells and washed with PBS. Crystal violet (CV) solution (0.2%, 50 lL/well) was added and incubated for 10 min at room temperature. Following incubation the culture plates were washed by submersion in hand-warm water (2) and air dried. SDS solution (1%, 100 lL) added. The plates were agitated to distribute the dye, and the absorbance measured at 570 nm.
Figure 3. Cytotoxicity results of sydnonimine–sulindac analogues at 50–500 nM.
Table 3 Griess test results for furoxan analogues Compound
NO2 production (lM)
%NO
4h 4j 1k 1n Sulindac 1 DMSO
41.1 ± 13.5 33.0 ± 6.8 54.4 ± 2.9 51.1 ± 1.3 1.1 ± 2.2 3.3 ± 1.2
10.3 8.2 13.6 12.8
