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 functional-
ities to develop into sulindac-NO hybrids; nitrate esters, furoxans
and sydnonimines. Based on literature examples where the oxida-
tion state of sulfur in sulindac conferred differing biological activ-
ities in vitro,9,10 we first began by preparing the known compounds
sulindac sulfide 4 and sulindac sulfone 5. To this effect, commer-
cially available sulindac 1 (sulfoxide) was reduced to the corre-
sponding 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 bu-
tyl linked nitrate ester alcohol 6 (Scheme 2),12,13 analogous to NCX-
1102 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 EDCIÁHCl 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 pre-
pared, analogous to NCX-112 3. Ester formation with mono-
TBDSMS 1,4-butanediol followed by deprotection with camphor-
sulfonic acid furnished the required alcohols 1g, 4g and 5g
(Scheme 4).
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, À10 °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 EDCIÁHCl and
DMAP provided the sydnonimine amides 1q, 4q and 5q (Scheme 8).
The use of acetonitrile as a cosolvent was required to aid dissolu-
tion of 21 in the reaction. Acylation of 21 with p-nitrophenylchlo-
roformate 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 mono-
acylation 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).
2.1.2. Furoxans
Furoxan alcohols 19a–i were readily prepared from bis(phenyl-
sulfonyl)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 pre-
viously established EDCI conditions provided esters 1h–p, 4h–p
and 5h–p (Scheme 6).
2.2. Biological results
2.2.1. Cytotoxicity results
PC3 cells (hormone insensitive human prostate cancer cells) ob-
tained from the European Collection of Cell Cultures (ECACC), Salis-
bury, 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
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
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-Nitrooxybu-
tyl 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 inves-
tigate 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
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.