S.W.P. Rees et al.
Bioorganic Chemistry 114 (2021) 105152
activity than D609, while also showcasing potent anti-proliferative ac-
tivity not achieved by D609. Anti-proliferative activity was obtained by
replacing the carboxylic acid motif of the recently-discovered class of
PC-PLC inhibitors, 2-morpholinobenzoic acids, with a hydroxamic acid,
likely enhancing the membrane permeability of these compounds while
maintaining strong chelation to catalytic Zn2 ions found in the PC-PLC
active site. The reported compounds set a new benchmark for combining
PC-PLC inhibitory activity with potent antiproliferative activity. Also,
the compounds can be easily prepared at scale using the reported
methods, while samples are available upon request.
4.5. General procedure for synthesis of o-(tetrahydro-2H-pyran-2-yl)
benzamides 8a-m
To a solution of corresponding acid 7a-m (1 equiv.), O-(tetrahydro-
2H-pyran-2-yl)hydroxylamine (1.5 equiv.) and DIPEA (3 equiv.) in dry
DMF (3 mL) was added a solution of COMU (1.3 equiv.) in dry DMF (3
mL) dropwise over 15 min. This mixture was allowed to stir for 2 d at
room temperature under nitrogen, then subsequently diluted with ethyl
+
acetate (20 mL) and washed with NaHCO
3
(2 × 15 mL), followed by
brine (15 mL), dried with Na SO and filtered. The resulting organic
2
4
filtrate was evaporated in vacuo and the crude product was purified by
flash chromatography to afford 8a-m.
4
. Experimental procedures and methodology
4
.6. General procedure for synthesis of hydroxamic acids 9a-m
4
.1. Synthesis of compounds 2–4
To a solution of amide 8a-m (1 equiv.) in MeOH/THF (3:1, 4 mL) was
Compounds 2 – 4 were synthesised according to previously reported
methods [17].
added 2 M HCl (0.5 equiv.) dropwise, the mixture was then allowed to
stir at room temperature overnight. The mixture was concentrated in
vacuo forming a white residue, which was redissolved in minimal water
3
and neutralised with sat. aqueous NaHCO forming a pink precipitate.
4
.2. General procedure for synthesis of esters 6a, 6 h, 6i, 6 k, 6 l
This solid was collected by extraction with dichloromethane (3 × 20
[
17]
mL), the combined organic layers were washed with brine (15 mL),
4
dried with MgSO and the solvent removed to give a crude product.
To a solution of methyl 5-amino-2-morpholinobenzoate 4 (1 equiv.)
Subsequent trituration with cold diethyl ether/petroleum ether (4:1)
afforded pure 9a-m, which was collected by vacuum filtration and
required no further purification.
in MeOH (10 mL) was added the corresponding benzaldehyde (2 equiv.)
and the mixture heated at reflux overnight. The reaction was then cooled
and the solvent removed in vacuo to give the required imine as a brown
residue, which was used in the next step without further purification.
The synthesised imine was dissolved in dry THF (6 mL) and a solution of
4.7. Amplex-Red PC-PLC inhibition assay
NaBH
4
(3 equiv.) in dry MeOH (9 mL) was added dropwise, the subse-
◦
Relative PC-PLC inhibitory activities for the synthesised compound
library was determined using previously reported methods, using an
Amplex-Red assay kit (Molecular Probe, Inc.) [17]. 0.01U PC-PLCBC was
added to a reaction mixture of 10 µM of PC-PLC inhibitor, 4 U/mL
alkaline phosphatase, 1 U/mL horseradish peroxidase, 0.1 U/mL choline
oxidase, 0.5 mM phosphatidylcholine, and 0.4 mM Amplex-Red reagent
in 1X Reaction buffer (50 mM Tris-HCl, pH 7.4, 0.14 M NaCl, 10 mM
quent mixture stirred at 35 C for 24 h. A further portion of NaBH
4
(3
◦
equiv.) was added and the reaction stirred at 35 C until completion on
TLC. The reaction was then cooled and CH
mixture was then washed with water (3 × 20 mL), followed by brine (15
mL), dried (MgSO ) and the solvent removed in vacuo to give the crude
product which was purified with flash chromatography to give 6a, 6 h,
j, 6 k, 6 l.
2 2
Cl (25 mL) was added. The
4
6
2
dimethylglutarate, 2 mM CaCl ). Phosphatidylcholine is enzymatically
cleaved by PC-PLC forming phosphocholine, which is converted to
choline by alkaline phosphatase. Choline is then oxidised by choline
4
.3. General procedure for synthesis of esters 6b-g, 6j, 6 m
2 2
oxidase resulting in the formation of H O , which is used by horseradish
To a solution of methyl 5-amino-2-morpholinobenzoate 4 (1 equiv.)
peroxidase to convert the Amplex-Red reagent to the fluorescent prod-
uct, resorufin. Resorufin is excited at a wavelength of 560 nm and emits
fluorescence at 590 nm, which is measured using an EnSpire multimode
plate reader. All results reported are the average of triplicate testing.
in MeOH (10 mL) was added the corresponding benzaldehyde (2 equiv.)
and the reaction heated at reflux overnight. The reaction was then
cooled to room temperature and the solvent removed in vacuo to give the
required imine as a brown residue, which was stirred in cold petroleum
ether (10 mL) to form a yellow precipitate. The imine intermediate was
collected by vacuum filtration, then redissolved in dry THF (6 mL) and a
4.8. Cell proliferation assay
solution of NaBH
The subsequent mixture was stirred at 35 C for 24 h, then cooled and
CH Cl
(25 mL) was added. The mixture was washed with water (3 × 20
mL), followed by brine (15 mL), dried (MgSO ) and the solvent removed
4
(3 equiv.) in dry MeOH (9 mL) was added dropwise.
Two cancer cell lines HCT116 (colon) and MDA-MB-231 (breast)
were used to test the potency of 2-morpholinobenzoic acids and its de-
rivatives using a thymidine incorporation assay. Cell proliferation assays
were conducted according to literature methods [29,30]. In brief, 3000
cells per well were seeded in 96 well plates that were tissue culture-
treated for monolayer culture and incubated in the presence of 10 µM
◦
2
2
4
in vacuo to give the crude product which was purified with flash chro-
matography to give 6b-g, 6j, 6 m.
3
PC-PLC inhibitor for 3 days. Prior to harvest, 0.04 µCi of H-thymidine
was added to each well and incubated for 5 h, after which the cells were
transferred using an automated TomTec harvester onto glass fiber filters.
Filters were incubated in Betaplate Scint, then a Trilux/Betaplate
counter was used to measure incorporation of radiolabelled thymidine.
All experiments were performed using triplicate wells and repeated
three times. The human breast cancer cell line MDA-MB-231 and colo-
rectal cancer cell line HCT-116 was purchased from the American Type
Culture Collection (ATCC).
4
.4. General procedure for synthesis of carboxylic acids 7a-m [17]
1
M NaOH (2 mL) was added to a solution of corresponding ester 6a-
◦
m in THF/MeOH (1:1) and stirred for 2 hr at 80 C. The mixture was
allowed to cool to room temperature, the solvent was then removed in
vacuo to afford a white/pink precipitate. This solid was redissolved in
2
minimal H O and adjusted to pH 3 using 2 M HCl, the resulting sus-
pension was extracted using ethyl acetate (3 × 15 mL). The organic
layers were combined and washed once with brine (15 mL), dried with
4.9. Molecular modelling
MgSO
4
and filtered. The filtrate was collected and solvent removed in
vacuo to give 7a-m, no further purification was required.
The crystal structure of PC-PLCBC was obtained from PDB (ID:
7
Rees, Shaun W.P.
