Development, synthesis and biological investigation of a novel class of potent PC-PLC inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.112162

Phospholipases are enzymes that are involved in the hydrolysis of acyl and phosphate esters of phospholipids, generating secondary messengers that have implications in various cellular processes including proliferation, differentiation and motility. As such inhibitors of phospholipases have been widely studied for their use as anti-cancer therapeutics. Phosphatidylcholine-specific phospholipase C (PC-PLC) is implicated in the progression of a number of cancer cell lines including aggressing triple-negative breast cancers. Most current studies on PC-PLC have utilised D609 as the standard inhibitor however it is known to have multiple failings, including poor stability in aqueous media. 2-Morpholinobenzoic acids were recently identified using vHTS as a potential class of lead compounds, with improvements over D609. In this work 129 analogues in this class were prepared and their PC-PLC inhibitory activity was assessed. It was found that the majority of these novel compounds had improved activity when compared to D609 with the most potent inhibitors completely inhibiting enzyme activity. It was determined that the best compound/s contained a morpholino and 2-substituted N-benzyl moieties with these findings explained using molecular modelling. The compounds reported here will allow for improved study of PC-PLC activity.

Full text of DOI:10.1016/j.ejmech.2020.112162

European Journal of Medicinal Chemistry 191 (2020) 112162
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Development, synthesis and biological investigation of a novel class of
potent PC-PLC inhibitors
Lisa I. Pilkington a, Kevin Sparrow a, Shaun W.P. Rees a, Emily K. Paulin a,
Michelle van Rensburg a, Chris Sun Xu b, Ries J. Langley c, Ivanhoe K.H. Leung a,
a
,
, e, *
Johannes Reynisson d, Euphemia Leung b, David Barker a
ꢀ
a School of Chemical Sciences, University of Auckland, Auckland, 1010, New Zealand
b Auckland Cancer Society Research Centre, University of Auckland, Grafton, Auckland, 1023, New Zealand
c School of Medical Sciences, University of Auckland, Auckland, 1023, New Zealand
d School of Pharmacy, Keele University, Hornbeam Building, Staffordshire, ST5 5BG, United Kingdom
e MacDiarmid Institute for Advanced Materials and Nanotechnology, Wellington, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
Phospholipases are enzymes that are involved in the hydrolysis of acyl and phosphate esters of phos-
pholipids, generating secondary messengers that have implications in various cellular processes
including proliferation, differentiation and motility. As such inhibitors of phospholipases have been
widely studied for their use as anti-cancer therapeutics. Phosphatidylcholine-specific phospholipase C
(PC-PLC) is implicated in the progression of a number of cancer cell lines including aggressing triple-
negative breast cancers. Most current studies on PC-PLC have utilised D609 as the standard inhibitor
however it is known to have multiple failings, including poor stability in aqueous media. 2-
Morpholinobenzoic acids were recently identified using vHTS as a potential class of lead compounds,
with improvements over D609. In this work 129 analogues in this class were prepared and their PC-PLC
inhibitory activity was assessed. It was found that the majority of these novel compounds had improved
activity when compared to D609 with the most potent inhibitors completely inhibiting enzyme activity.
It was determined that the best compound/s contained a morpholino and 2-substituted N-benzyl moi-
eties with these findings explained using molecular modelling. The compounds reported here will allow
for improved study of PC-PLC activity.
Received 18 December 2019
Received in revised form
18 February 2020
Accepted 18 February 2020
Available online 19 February 2020
Keywords:
PC-PLC
Enzyme inhibition
D609
SAR
2-Morpholinobenzoic acids
Molecular modelling
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
phosphatidylinositol-specific phospholipase
C
(PI-PLC) and
phosphatidylcholine-specific phospholipase C (PC-PLC), depending
on the substrate they target [3]. PC-PLC hydrolyses the phospho-
lipid, phosphatidylcholine (PC) to produce DAG and phosphocho-
line [1b,4].
Phospholipases are enzymes that are involved in the hydrolysis
of acyl and phosphate esters of phospholipids, generating second-
ary messengers that are capable of signal transduction with im-
plications in various cellular processes including proliferation,
differentiation, motility, apoptosis and gene expression [1]. Phos-
pholipases can be classified into four different classes (A-D), cat-
egorised according to their site of cleavage on the phospholipid
substrate - the phospholipase C (PLC) family of enzymes catalyses
the hydrolysis of glycerophosphate bonds to form diacylglycerol
(DAG) [2]. PLC enzymes can be further classified as either
Both breakdown products of phosphatidylcholine catabolism,
phosphocholine and DAG, act through kinases and transcription
factors to indirectly activate cell proliferation and differentiation,
although DAG holds the most significance due to its prevalence and
action in a variety of responses [4c,5]. DAG has an important
involvement in the signal transduction cascade to activate protein
kinase C enzymes (PKC) to subsequently phosphorylate down-
stream proteins and induce an array of signalling events [5] and
upregulation of these events can lead to the development and
growth of cancer. Furthermore, DAG is additionally able to activate
acidic sphingomyelinases (SMase) linked to various mitogenic
* Corresponding author. School of Chemical Sciences, The University of Auckland,
Private Bag 92019, Auckland, 1142, New Zealand.
E-mail address: d.barker@auckland.ac.nz (D. Barker).
https://doi.org/10.1016/j.ejmech.2020.112162
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
L.I. Pilkington et al. / European Journal of Medicinal Chemistry 191 (2020) 112162
processes [1b,4c].
and with tractable synthetic routes for further development, the
aforementioned vHTS study was carried out, which identified four
distinct structural classes as potential hits, namely benzenesul-
phonamides, pyrido[3,4-b]indoles, morpholinobenzoic acids and
benzamidobenzoic acids (Fig. 2) [11]. Upon assessing the PC-PLC
inhibitory activity of various compounds belonging to these
structural classes, it was discovered that members of the pyrido
[3,4-b]indole and morpholinobenzoic acid classes were very active
As a consequence of its role in cell signalling, PC-PLC shows
promising potential as a therapeutic target in anticancer therapies
and there is an abundance of evidence that supports the idea that
PC-PLC is involved in cancer development. Such evidence includes
the increased expression of PC-PLC in ovarian tumour cells [6] and
highly metastatic triple-negative MDA-MB-231 breast cancer cell
line [7]. Furthermore, a downregulation of the HER2 oncogene has
been observed upon PC-PLC inhibition [8], and there is evidence of
an association between PC-PLC expression and leukaemia and
hepatocarcinoma progression [9].
against PC-PLC, with IC50 values of 3e4
shown that the tested compounds were more active against PC-PLC
than the much-lauded established inhibitor D609 (IC50 8.1 M),
mM. Pleasingly, it was
m
While the structure of eukaryotic PC-PLC has not been obtained,
the structure of prokaryotic PC-PLC enzymes have been charac-
terised and the most-commonly studied PC-PLC structure is that
isolated from Bacillus cereus, first solved in 1989 [10]. Recently, we
have reported the use of virtual high throughput screening (vHTS)
to discover hit compounds against human PC-PLC for potential
anticancer drug development using PC-PLCBc as a model for
mammalian PC-PLC [11]. This approach was justified, as it has been
reported that PC-PLCBc has antigenic similarity to mammalian PC-
PLC and can mimic similar responses to mammalian PLC, e.g.,
enhancement of prostaglandin biosynthesis [12]. Prior to this vHTS,
establishing these classes, and morpholinobenzoic acids in partic-
ular, as drug-like, hit compound classes with a new standard of
potency against PC-PLC [11].
This recently published work details the vHTS process and
establishment of the morpholinobenzoic acid class of compounds
as potent PC-PLC inhibitors, yet it only reports the testing of a small
number of commercially-available analogues. Consequently, it was
decided to design and implement a synthetic procedure that would
allow access to previously-undiscovered analogues of these mor-
pholinobenzoic acids in an attempt to establish a comprehensive
structure-activity relationship (SAR) and discover analogues more
potent against PC-PLC than those already identified.
a
small number of PC-PLC inhibitors were known (2-
aminohydroxamic acids, univalent anions, N,N0-dihydroxyureas,
phospholipid analogues and the acclaimed xanthate, tricyclodecan-
9-yl-xanthogenate, D609; Fig. 1) but unfortunately none of these
compounds could be considered to be drug-like [13]. Of these in-
hibitors, D609 has been considered to be the most effective and has
been the literature standard for PC-PLC inhibition. D609 competes
with phosphatidylcholine and is widely recognised as a competi-
2. Results and discussion
The main priority when designing a synthetic route for the
morpholinobenzoic acids was to create a methodology that would
be easily adapted to the synthesis of a large number of analogues
exhibiting various different aspects of structural variation from the
tive inhibitor of PC-PLC with a Ki of 6.4
mM. In vitro, D609
hit compound, 1 (IC50 3.69 0.23 mM against PC-PLC, Fig. 3). The
demonstrated ability to block proliferation in EOC (epithelial
compounds previously tested [11] were determined by their
availability, this set was neither systematic nor comprehensive and
only allowed a rudimentary SAR to be developed to date. As such, in
the first series of this study, it was decided to synthesise a range of
analogues to complement this non-systematic set of compounds.
This would fully explore the N-benzyl moiety and determine the
effect of electron-withdrawing and electron-donating substituents
and their relative substitution positions, on the activity of the
resulting morpholinobenzoic acid (purple, Fig. 3). As part of this
first series, it was also decided to synthesise the corresponding
esters of each of the compounds. In molecular modelling of 1 and
its analogues, it was shown that the carboxylic acid moiety co-
ordinates to the Zn atoms present at the PC-PLC binding site. If
this were an accurate representation of the actual binding mode
ovarian cancer) cells and in MDA-MB-231 cells with 60e80% inhi-
bition using 50 mg/mL, however in vivo studies required the use of
very high doses of the compound in order to achieve the 60e80%
inhibition previously outlined [6]. This may be due to its lack of
drug-like properties (unstable and short half-life in aqueous media)
that makes it unsuitable for distribution in the body and limits its
therapeutic use. Furthermore, D609 has 8 possible stereoisomers
(with different activities) and no selective synthesis of these iso-
mers e this fact, coupled with the knowledge that different com-
mercial sources give different isomeric ratios render D609 an
unsuitable, but currently the best option, standard molecule for PC-
PLC inhibition [14].
Due to the lack of PC-PLC inhibitors with drug-like properties
Fig. 2. The four classes of compounds identified as potential hits against PC-PLC;
benzenesulphonamides, pyrido[3,4-b]indoles, morpholinobenzoic acids and benza-
midobenzoic acids.
Fig. 1. Examples of known PC-PLC inhibitors.

L.I. Pilkington et al. / European Journal of Medicinal Chemistry 191 (2020) 112162
3
different) as demonstrated by the lower measured PC-PLC activity
in the presence of these compounds. As mentioned previously, this
observation was expected based on the premise that the carboxylic
acid should aid binding to the PC-PLC enzyme, through coordina-
tion to the Zn atoms present at the binding pocket. Surprisingly,
when the analogues 10 with no ester or carboxylic acid function-
ality were tested, it was found that in all cases the inhibitory ac-
tivity of these compounds far exceeded that of both the esters 7 and
carboxylic acids 1. It should be noted that every analogue with no
ester or acid functionality, 10, and carboxylic acid 1, had greater
activity against PC-PLC than the literature standard, D609, con-
firming this class of compounds to be potent PC-PLC inhibitors and
far more effectual than the previously-reported best inhibitor.
Assessing the effect of the N-benzyl substituent, it was immediately
clear that the larger substituents (3-OPh w, 4-Ph x and 2,4,6-triMe
y) were not well-tolerated, with these analogues generally exhib-
iting the lowest activities in each of the analogue series. It could
also be seen that the CF3-substituted analogues t, u and v, were less
active. While there was no apparent trend in the effect of the N-
benzyl substituent position for the carboxylic acids 1 and esters 7,
for analogues 10 it was apparent that the ortho-substituted ana-
logues were more active than the meta- and para-substituted
compounds which had comparable activity. It could be seen that
compounds with both electron-donating and electron-
withdrawing substituents exhibited very good activities, with no
clear observable trend. The results from this screen of PC-PLC
inhibitory activity of the derivatives at 10
gated through the measuring of IC50 values of D609
(8.08 0.19 M), the best-performing acid/ester 1r (3.60 0.19 M)
and one of the three most active compounds that completely
inhibited PC-PLC activity at 10 M, 10k (1.06 0.05 M). These IC50
mM was further investi-
Fig. 3. Structure of hit compound 1a and points of variation explored in this study;
Series 1 (red and purple), Series 2 (green and purple). (For interpretation of the ref-
erences to colour in this figure legend, the reader is referred to the Web version of this
article.)
m
m
m
m
values show that the best-performing inhibitors were ~8 times
more effective than the previous literature standard D609.
then conversion of the carboxylic acid to an ester would be ex-
pected to be detrimental to the PC-PLC inhibitory activity.
It was decided to begin the synthesis from commercially-
available 2-chloro-5-nitrobenzoic acid 2, the carboxylic acid func-
tionality of which was esterified using standard conditions [15],
providing ester 3 in 96% yield. Once formed, 3 underwent nucleo-
philic aromatic substitution of the chlorine with morpholine,
providing morpholinobenzoic acid 4, the nitro group of which was
then reduced to give aniline 5. With 5 in hand, a panel of benzal-
dehydes 6a-y with a range of electron-donating (i.e. -Me, -OMe,
eOH) and electron-withdrawing (i.e. halogens, eCF3) at ortho-,
meta- and para-positions were condensed to form an imine, which
was immediately reduced to the secondary amines present in the
desired esters 7a-y. These esters 7a-y were then hydrolysed to give
the corresponding carboxylic acids 1a-y (see Scheme 1).
In addition to esters 7a-y and carboxylic acids 1a-y, it was
decided to also synthesise derivatives with no functionality at this
position. This required the preparation of a new amine, 8, which
was achieved in two steps e substitution to install the morpholine
moiety and reduction of the nitro group - from 1-chloro-4-
nitrobenzene 9 (Scheme 2). Once formed, amine 8 was reacted
with a range of benzaldehydes 6a-y and the resulting imines were
reduced, providing the desired final products 10a-y.
With the successful synthesis of analogues 1, 7 and 10, consti-
tuting Series 1 of this study, it was decided to assess the PC-PLC
inhibitory activity of these 73 compounds. The PC-PLC inhibition
was measured using a well-established, commercial Amplex Red
Assay and the results for Series 1 are summarised in Table 1
(D609 ¼ 59.7% inhibition, i.e. 40.3% relative PC-PLC enzyme
activity).
As can be seen, the carboxylic acids 1 were, in all cases more
active that the respective esters 7 (except for h with a 2-OH sub-
stitution on the N-benzyl ring where they not significantly
Following on from the biological assessment of the first series of
compounds, it was decided to also investigate the impact of
exchanging the morpholine moiety (green in Fig. 3) with other
cyclic amines, i.e. piperidine 11 and (methyl 12, N-Boc 13 and non-
functionalised 14) piperazine variants, comprising a set of Series 2
analogues. The previously-described method utilised to synthesise
Series 1 compounds was able to be implemented for Series 2
(Scheme 3). As for analogues 10, 1-chloro-4-nitrobenzene 9 was the
starting material for all Series 2 compounds e chloride 9 was
reacted with piperidine, 1-methylpiperazine and 1-Boc-piperazine
to give substituted products 15, 16 and 17, respectively. Nitro-
aromatics 15e17 were then reduced using standard reductive
procedures (Pd/C under
a H2 atmosphere) to provide their
respective amines 18e20. For Series 2, it was decided to reduce the
number of benzaldehydes 6, to focus on those that generally
exhibited better activities, i.e. all ortho-substituted variants, in
addition to benzaldehydes with hydroxyl, fluoro and chloro sub-
stituents at all positions. As performed in the synthesis of Series 1
analogues, amines 18e20 were each condensed with 14 different
benzaldehydes 6, giving the corresponding imine which, upon
isolation, was immediately reduced to provide the desired final
compounds 11e13. Finally, to obtain the non-N-substituted piper-
azine analogues 14, the Boc-protected compounds 13 were treated
with TFA in CH2Cl2.
As for Series 1, after Series 2 was successfully synthesised, the
generated analogues 11e14 were tested for their PC-PLC inhibitory
activity using the Amplex Red Assay (Table 2).
Looking at the results from the biological assays for Series 2
compounds, it was apparent that for most of the variations inves-
tigated, the piperidine-substituted compounds 11 were the most
active than the piperazine 14 and piperazine-derived 12 and 13
analogues. However, interestingly the trend of the most active

4
L.I. Pilkington et al. / European Journal of Medicinal Chemistry 191 (2020) 112162
Scheme 1. Synthesis of Series 1 analogues, altering substitution (substituent and position) on the N-benzyl ring, synthesising both carboxylic acids and their corresponding esters.
compounds being the ortho-N-benzyl-substituted compounds did
not hold for this series. Most importantly, it was immediately clear
that the morpholine-substituted derivatives 10 exhibited greater
PC-PLC inhibitory action than their piperidine 11 and (methyl 12, N-
Boc 13 and non-functionalised 14) piperazine counterparts.
With the identification of analogues 10 with no carboxylic acid
or ester groups and possessing a morpholine moiety as having the
most potent activities against PC-PLC, particularly 10a, 10h and 10l
with no, ortho-OH and ortho-F substituents on the N-benzyl ring,
respectively, we wished to further investigate their potency and
how they bind through molecular modelling and docking at the PC-
PLC active site. We also wished to explore if molecular docking
could account for the differences in activity seen between the acids
1, esters 7 and the most active analogues 10, as well as the greater
activity exhibited by morpholine-containing compounds, i.e. pi-
peridines 11.
Comparing the binding modes of acid 1a, ester 7a and analogue
10a, it was immediately clear that they all display similar binding
modes within the PC-PLC binding pocket (Fig. 4). The morpholine
moiety occupies the negatively-charged space to the right of the
binding pocket, while the N-benzyl group is situated in the lipo-
philic area to the left. It can also be seen that the secondary amine
linking the aromatic rings is situated in close proximity to the
partially-positive cleft near the centre of the binding cavity. Both
acid 1a and ester 7a are positioned with these titular functionalities
buried deep within the binding site, however the absence of these
moieties in 10a does not greatly affect its positioning. Furthermore,
comparing 10a with no substitution on the N-benzyl ring and 10k,
with an ortho-fluoro substituent, it appears that small substituents
to not greatly impact on the positioning of the structure within the
site. Interestingly, the effect of the morpholine group is apparent
when looking at the binding mode of the piperidine derivative, 11a,
which shows the structure to be completely flipped in the binding
pocket, potentially offering justification for its reduced activity e
while the oxygen in the morpholine moiety coordinates to the
amino acid residues to the right of the pocket, coaxing it into the
depicted position, the absence of this functionality as an anchor-
point leads to a significant change in binding mode which seems
to be responsible for a significant loss in inhibitory activity.
3. Conclusion
PC-PLC is implicated in the progression of a number of cancer
cell lines and the current inhibitor of choice for study of this
enzyme, D609, is known to have multiple failings, including poor
stability in aqueous media. Our recent study identified 2-
morpholinobenzoic acids as a potential class of lead compounds
which could have greater activity than D609, whilst also having
more drug-like characteristics. In this work 129 analogues in this
class were prepared and their PC-PLC inhibitory activity was
assessed. It was found that the majority of these novel compounds
had improved activity compared to D609 and the most potent in-
hibitors completely inhibited enzyme activity. Unlike D609 which
is difficult to prepare and is only available commercially as an un-
defined mixture of stereoisomers, the compounds introduced here
are easily prepared at scale and are achiral. It was determined that

L.I. Pilkington et al. / European Journal of Medicinal Chemistry 191 (2020) 112162
5
separated and the aqueous layer further extracted with diethyl
ether (2 ꢀ 30 mL). The combined organic extracts were washed
with water (25 mL),1 M NaOH (2 ꢀ 25 mL) and water (25 mL), dried
(MgSO4) and the solvent removed in vacuo to give the title product 3
(7.68 g, 96%) as a white solid. m.p.: 69e71 ꢁC (Lit [16]. 68e69 ꢁC). RF:
0.87 (9:1 CH2Cl2eMeOH). dH (400 MHz; CDCl3; Me4Si) 4.00 (3H, s,
OCH3), 7.66 (1H, d, J ¼ 8.0 Hz, H-3), 8.27 (1H, dd, J ¼ 2.0, 8.0 Hz, H-
4), 8.71 (1H, d, J ¼ 2.0 Hz, H-6). dC (100 MHz; CDCl3) 53.1 (OCH3),
126.7 (C-6), 126.8 (C-4), 131.0 (C-1), 132.3 (C-3), 140.8 (C-2), 146.1
(C-5), 164.0 (C]O). The 1H NMR data was consistent with literature
values [15].
Methyl 2-morpholino-5-nitrobenzoate 4; To a solution of methyl
2-chloro-5-nitrobenzoate 3 (7.60 g, 0.035 mol) in MeCN (50 mL)
was added morpholine (9.13 mL, 0.106 mol) and the mixture stirred
under an atmosphere of nitrogen for 24 h. The mixture was then
combined with ice-cold water (75 mL) and extracted with diethyl
ether (3 ꢀ 75 mL). The combined organic extracts were washed
with brine (100 mL), dried (MgSO4) and the solvent removed in
vacuo to give the title product 4 (9.37 g, 99%) as a bright yellow solid.
m.p.: 117e118 ꢁC (Lit [17]. 110 ꢁC). RF: 0.63 (1:1 petroleum ether-
ethyl acetate). dH (400 MHz; CDCl3; Me4Si) 3.25 (4H, t, J ¼ 4.0 Hz,
N(CH2CH2)2O), 3.87 (4H, t, J ¼ 4.0 Hz, N(CH2CH2)2O), 3.93 (3H, s,
OCH3), 6.99 (1H, d, J ¼ 8.0 Hz, H-3), 8.23 (1H, dd, J ¼ 2.0, 8.0 Hz, H-
4), 8.64 (1H, d, J ¼ 2.0 Hz, H-6). dC (100 MHz; CDCl3) 51.7
(N(CH2CH2)2O), 52.5 (OCH3), 66.4 (N(CH2CH2)2O), 117.5 (C-3), 120.4
(C-1), 128.0 (C-4), 128.7 (C-6), 139.7 (C-5), 156.1 (C-2), 166.3 (C]O).
IR: nmax(ATR)/cm-1; 2986 (CH aromatic), 2870, 2849 (CH alkane),
1722 (C]O ester), 1600, 1571, 1503 (C]C aromatic), 1489, 1326 (CH
alkane), 1226, 1105 (CeO ether), 926, 822, 714 (CH aromatic). m/z
(ESIþ): 289 (MNaþ, 100%). HRMS (ESIþ) Found (MNaþ):
289.0805C12H14N2NaO5 requires 289.0795.
Methyl 5-amino-2-morpholinobenzoate 5; To a solution of methyl
2-morpholino-5-nitrobenzoate 4 (9.30 g, 0.035 mol) in EtOH
(50 mL) was added Pd on carbon (10 wt %, 0.93 g) and the reaction
stirred under an atmosphere of hydrogen for 48 h. The reaction
mixture was then filtered through a plug of Celite and the solvent
removed in vacuo to give the title product 5 (8.04 g, 97%) as a pale
yellow solid. m.p.: 119e121 ꢁC (Lit [18]. 121e122 ꢁC). RF: 0.39 (1:1
petroleum ether-ethyl acetate). dH (400 MHz; CDCl3; Me4Si) 2.93
(4H, t, J ¼ 4.0 Hz, N(CH2CH2)2O), 3.59 (2H, br s, NH2), 3.82 (4H, t,
J ¼ 4.0 Hz, N(CH2CH2)2O), 3.87 (3H, s, OCH3), 6.77 (1H, dd, J ¼ 2.0,
8.0 Hz, H-4), 6.95 (1H, d, J ¼ 8.0 Hz, H-3), 7.04 (1H, d, J ¼ 2.0 Hz, H-
6). dC (100 MHz; CDCl3) 52.0 (OCH3), 53.6 (N(CH2CH2)2O), 67.3
(N(CH2CH2)2O),117.5 (C-6), 119.4 (C-4), 121.1 (C-3), 127.4 (C-1), 141.4
(C-5), 144.2 (C-2), 168.1 (C]O). IR: nmax(ATR)/cmꢂ1; 3452, 3365
(NH), 2960 (CH aromatic), 2842 (CH alkane), 1700 (C]O ester),
1628, 1498 (C]C aromatic), 1445, 1324 (CH alkane), 1243, 1112
(CeO ether), 935, 825 (CH aromatic). m/z (ESIþ): 259 (MNaþ, 10%),
237 (MHþ, 50), 205 (100). HRMS (ESIþ) Found (MNaþ):
259.1051C12H16N2NaO3 requires 259.1053. Found (MHþ):
237.1235C12H17N2O3 requires 237.1234. The 1H NMR data was
consistent literature values [18].
Scheme 2. Synthesis of non-ester/carboxylic Series 1 analogues, altering substitution
(substituent and position) on the N-benzyl ring.
the best compound/s contained a morpholino and 2-substituted N-
benzyl moieties, with these findings explained using molecular
modelling. The compounds reported here will allow for improved
study of PC-PLC activity and samples of these inhibitors are easily
prepared using the reported methods or can be supplied upon
request.
4. Experimental procedures and methodology
4.1. General experimental details
NMR spectra were recorded on a 400 MHz spectrometer.
Chemical shifts are reported relative to the solvent peak of chlo-
roform (
d
7.26 for 1H and
d
77.0 for 13C) or DMSO‑d6
(d
2.50 for 1H
), relative
and
d
39.5 for 13C). 1H NMR data is reported as position (
d
integral, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet; br, broad peak; qd, quartet of doublets), coupling con-
stant (J, Hz), and the assignment of the atom. Broadband proton-
decoupled 13C NMR data are reported as position (
d) and assign-
ment of the atom. NMR assignments were performed using HSQC
and HMBC experiments. High-resolution mass spectroscopy
(HRMS) was carried out by electrospray ionization (ESI) on a
MicroTOF-Q mass spectrometer. Unless noted, chemical reagents
were used as purchased.
Methyl 5-(benzylamino)-2-morpholinobenzoate 7a; Formation of
the crude imine was carried out according to General Procedure A-
1, using methyl 5-amino-2-morpholinobenzoate
0.85 mmol) and benzaldehyde 6a (0.31 g, 1.69 mmol) in MeOH
(15 mL) to give methyl (E)-5-(benzylideneamino)-2-
5
(0.2 g,
4.2. Synthesis of compounds
morpholinobenzoate (0.33 g, 97%) as a bright yellow solid that
was used in the next reaction without further purification. RF: 0.70
(1:1 petroleum ether-ethyl acetate). Reduction of the crude imine
was carried out according to General Procedure B-1, using methyl
General procedures can be found in the Supporting Information.
Methyl 2-chloro-5-nitrobenzoate 3; To a solution of 2-chloro-5-
nitrobenzoic acid 2 (7.50 g, 0.037 mol) in MeOH (30 mL) was
added concentrated H2SO4 (1.35 mL) slowly. The reaction was then
heated at reflux for 24 h. The reaction was cooled and the solvent
was then removed in vacuo and ice-water (30 mL) added to the
residue, followed by diethyl ether (30 mL). The organic layer was
(E)-5-(benzylideneamino)-2-morpholinobenzoate
(0.28
g,
0.85 mmol) in THF (8 mL) and NaBH4 (2 ꢀ 96 mg, 2.5 mmol) in THF/
MeOH (2:3, 5 mL) for 20 h, followed by an additional 24 h after the
second addition of NaBH4. The crude product was purified using

6
L.I. Pilkington et al. / European Journal of Medicinal Chemistry 191 (2020) 112162
Table 1
PC-PLC activity upon treatment by Series 1 compounds using an Amplex Red Assay. Testing was performed in triplicate. Results shaded light grade exhibit 80e90% inhibition
whilst results shaded dark grey exhibit >90% inhibition.
Relative PC-PLC activity (% ± s.e.) at 10 μM
N-Benzyl
No carboxylic
substitution
Carboxylic acid, 1
Ester, 7
acid/ester, 10
0 ± 0.5
No substitution/H, a
2-Me, b
3-Me, c
4-Me, d
2-OMe, e
3-OMe, f
4-OMe, g
2-OH, h
3-OH, i
32.5 ± 0.6
35.0 ± 1.0
23.7 ± 2.1
33.4 ± 4.9
32.5 ± 0.7
28.1 ± 2.4
32.3 ± 0.8
40.0 ± 3.8
41.6 ± 2.9
43.8 ± 2.1
29.9 ± 0.8
49.5 ± 7.6
30.3 ± 0.1
40.4 ± 3.5
20.4 ± 9.0
28.7 ± 1.0
11.5 ± 8.0
10.7 ± 1.5
26.7 ± 1.0
34.1 ± 1.6
44.6 ± 7.0
45.5 ± 8.5
47.5 ± 8.5
40.1 ± 4.4
45.3 ± 6.5
57.7 ± 1.9
58.4 ± 0.7
54.0 ± 1.7
54.3 ± 1.8
43.7 ± 5.3
64.6 ± 11.0
58.9 ± 8.0
38.6 ± 3.7
52.8 ± 0.7
48.3 ± 1.1
56.8 ± 0.3
64.5 ± 1.6
48.9 ± 0.8
56.7 ± 0.03
60.9 ± 8.4
77.3 ± 2.3
73.1 ± 5.4
65.6 ± 2.7
87.5 ± 0.9
83.0 ± 21.0
64.1 ± 2.7
78.6 ± 0.8
130.3 ± 26.9
100.4 ± 16.4
96.8 ± 20.6
40.3 ± 3.2
2.2 ± 0.4
8.3 ± 1.4
5.4 ± 0.6
1.2 ± 0.4
5.6 ± 0.1
3.2 ± 0.3
0 ± 0.1
3.4 ± 0.5
6.5 ± 0.4
0 ± 0.2
4-OH, j
2-F, k
3-F, l
6.1 ± 0.9
1.8 ± 0.2
14.1 ± 0.05
11.6 ± 1.8
18.1 ± 1.3
7.9 ± 0.3
9.1 ± 0.9
12.9 ± 0.3
-
4-F, m
2-Br, n
3-Br, o
4-Br, p
2-Cl, q
3-Cl, r
4-Cl, s
2-CF3, t
3-CF3, u
4-CF3, v
3-OPh, w
4-Ph, x
23.4 ± 1.4
27.6 ± 0.1
39.3 ± 0.5
-
2,4,6-triMe, y
D609
28.9 ± 1.0
flash chromatography (2:1 petroleum ether-ethyl acetate) to give
the title product 7a (0.22 g, 81% over two steps) as a light yellow
solid. m.p.: 105e108 ꢁC. RF: 0.77 (2:1 petroleum ether-ethyl ace-
tate). dH (400 MHz; (CD3)2SO; Me4Si) 2.76 (4H, br s, N(CH2CH2)2O),
3.63 (4H, br s, N(CH2CH2)2O), 3.87 (3H, s, COOCH3), 4.24 (2H, d,
J ¼ 6.0 Hz, CH2), 6.25 (1H, t, J ¼ 6.0 Hz, NH), 6.67 (1H, dd, J ¼ 3.0,
9.0 Hz, H-4), 6.79 (1H, d, J ¼ 3.0 Hz, H-6), 6.96 (1H, d, J ¼ 9.0 Hz, H-
3), 7.22 (1H, m, H-40), 7.29e7.34 (4H, m, H-20, H-30). dC (100 MHz;
(CD3)2SO) 46.6 (CH2), 51.7 (COOCH3), 53.3 (N(CH2CH2)2O), 66.6
(N(CH2CH2)2O), 113.1 (C-6), 115.3 (C-4), 121.3 (C-3), 126.6 (C-40),
127.1 (C-20), 128.1 (C-1), 128.3 (C-30), 140.0 (C-10), 140.9 (C-2), 144.5
(C-5), 168.3 (C]O). IR: nmax(ATR)/cmꢂ1; 3304 (NH), 2955 (CH

L.I. Pilkington et al. / European Journal of Medicinal Chemistry 191 (2020) 112162
7
3.37 (4H, t, J ¼ 5.0 Hz, H-30), 3.86 (4H, t, J ¼ 5.0 Hz, H-20), 6.82 (2H, d,
J ¼ 9.4 Hz, H-3), 8.13 (2H, d, J ¼ 9.4 Hz, H-2). The 1H NMR data were
in agreement with literature values [20].
4-Morpholinoaniline 8; To a solution of 4-(4-nitrophenyl)mor-
pholine (8.45 g, 0.041 mol) in EtOH (250 mL) was added 10% Pd/C
(0.843 g, 10% w/w). The resulting mixture was stirred at room
temperature under an atmosphere of hydrogen for 23 h. After
completion, the mixture was filtered through Celite and washed
with EtOH. The solvent was removed in vacuo to give the title
compound 8 (5.25 g, 74%) as a pink solid which was used without
further purification. m.p. 129e130 ꢁC (Lit [21]. 129.5e130.5 ꢁC). dH
(400 MHz; CDCl3) 3.02 (4H, t, J ¼ 4.7 Hz, H-30), 3.44 (2H, s, NH2),
3.85, (4H, t, J ¼ 4.7 Hz, H-20), 6.66, (2H, d, J ¼ 8.8 Hz, H-3), 6.80 (2H,
d, J ¼ 8.8 Hz, H-2). dC (100 MHz; CDCl3) 51.1 (C-30), 67.0 (C-20), 116.2
(C-2), 118.1 (C-3), 140.3 (C-1), 144.4 (C-4); IR nmax(ATR)/cmꢂ1: 3393,
2855, 1641, 1411, 1259, 1107, 825; m/z (ESIþ): 227 (17%), 179 (MHþ,
100%); HRMS (ESIþ) found (MHþ): 179.1178, C10H15N2O requires
179.1179. The 1H NMR data were in agreement with literature
values [22].
N-Benzyl-4-morpholinoaniline 10a; The reaction was carried out
according to General Procedure A-2 with benzaldehyde 6a
(0.20 mL, 1.96 mmol) and 4-morpholinoaniline
8 (0.200 g,
0.96 mmol) for 2 h to afford the crude imine (0.185 g, 0.695 mmol)
as a yellow solid that was reduced according to General Procedure
B-2 with NaBH4 (0.085 g, 2.25 mol) for 4 h to give the title compound
10a (0.130 g, 50%) as a white solid. m.p. 72e74 ꢁC. dH (400 MHz;
CDCl3) 3.01 (4H, t, J ¼ 4.7 Hz, H-30), 3.80 (1H, s, NH), 3.85 (4H, t,
J ¼ 4.7 Hz, H-20), 4.30 (2H, s, CH2), 6.63 (2H, d, J ¼ 8.9 Hz, H-2), 6.83,
(2H, d, J ¼ 8.9 Hz. H-3) 7.27e7.39 (5H, m, H-200, H-300, H-400, H-500, H-
600); dC (100 MHz; CDCl3) 49.2 (CH2), 51.4 (C-30), 67.3 (C-20), 114.1 (C-
2), 118.5 (C-3), 127.3 (C-400), 127.7 (C-200), 128.7 (C-300), 139.8 (C-100),
142.9 (C-1), 143.8 (C-4); IR nmax(ATR)/cmꢂ1: 3309, 2923, 2853, 1613,
1518, 1295, 1107; m/z (ESIþ): 269 (MHþ, 47%), 178 (M-CH2(C6H4),
100%); HRMS (ESIþ) found (MHþ): 269.1643, C17H21N2O2 requires
269.1648. The 1H and 13C NMR data were in agreement with liter-
ature values [23].
1-(4-Nitrophenyl)piperidine 15; A mixture of piperidine (7.42 mL,
0.075 mol), 4-chloronitrobenzene 9 (11.82 g, 0.075 mol) and K2CO3
(10.37 g, 0.075 mol) in DMSO (50 mL) was heated at 120 ꢁC for 23 h
with stirring. The reaction was cooled to r.t., then 1:1 EtOH:H2O
(200 mL) added. The resulting precipitate was collected by filtration
and dried to give the title compound 15 (14.45 g, 93%) as an orange
solid which was used without further purification. m.p. 99e101 ꢁC
(Lit [24]. 101e102.5 ꢁC) The 1H NMR data were in agreement with
literature values [25]. dH (400 MHz; CDCl3) 1.67e1.70 (6H, m, H-30,
H-40), 3.43 (4H, s, H-20), 6.79 (2H, d, J ¼ 9.5 Hz, H-2), 8.10 (2H, d,
J ¼ 9.5 Hz, H-3).
Scheme 3. Synthesis of Series
2 analogues, varying at the saturated cyclic ring,
substituting morpholine with piperidine 11 and (methyl 12, N-Boc 13 and non-
functionalised 14) piperazine variants and altering the altering substitution (substit-
uent and position) on the N-benzyl ring.
aromatic), 2803 (CH alkane), 1717 (C]O ester), 1612, 1568 (C]C
aromatic), 1496, 1450 (CH alkane), 1309, 1202, 1069 (CeO ether),
926, 817, 743 (CH aromatic). m/z (ESIþ): 327 (MHþ, 100%), 295 (75).
HRMS (ESIþ) Found (MNaþ): 349.1531C19H19N2NaO3 requires
349.1523. Found (MHþ): 327.1711C19H23N2O3 requires 327.1703.
5-(Benzylamino)-2-morpholinobenzoic acid 1a; The reaction was
carried out following General Procedure C using methyl 5-(benzy-
lamino)-2-morpholinobenzoate 7a (0.10 g, 0.3 mmol) and 1M
NaOH (4.4 mL) in MeOH/THF (2:3, 10 mL) for 24 h, to give the title
product 1a (0.07 g, 70%) as a white solid. m.p.: >230 ꢁC. RF: 0.09 (1:1
petroleum ether-ethyl acetate). dH (400 MHz; (CD3)2SO) 2.96 (4H,
br s, N(CH2CH2)2O), 3.76 (4H, br s, N(CH2CH2)2O), 4.29 (2H, br s,
CH2), 6.71 (1H, br s, NH), 6.81 (1H, dd, J ¼ 3.0, 9.0 Hz, H-4), 7.23 (1H,
m, H-40), 7.25 (1H, d, J ¼ 3.0 Hz, H-6), 7.31e7.34 (4H, m, H-20, H-30),
7.43 (1H, d, J ¼ 9.0 Hz, H-3). dC (100 MHz; (CD3)2SO) 46.0 (CH2), 52.9
(N(CH2CH2)2O), 66.3 (N(CH2CH2)2O), 112.9 (C-6), 116.8 (C-4), 123.9
(C-3), 125.2 (C-1), 126.7 (C-40), 127.0 (C-20), 128.3 (C-30), 138.3 (C-2),
139.6 (C-10), 147.6 (C-5), 166.8 (C]O). IR: nmax(ATR)/cmꢂ1; 3322
(OH, NH), 2975 (CH aromatic), 2861 (CH alkane), 1689 (C]O car-
boxylic acid), 1607, 1514 (C]C aromatic), 1493, 1454 (CH alkane),
1331, 1260, 1067 (CeO ether), 976, 895, 654 (CH aromatic). m/z
(ESIþ): 355 (MNaþ, 100%), 313 (MHþ, 10). HRMS (ESIþ) Found
(MNaþ): 335.1372C18H20N2NaO3 requires 355.1366. Found (MHþ):
313.1542C18H21N2O3 requires 313.1542.
4-(Piperidin-10-yl)aniline 18; To a solution of 1-(4-nitrophenyl)
piperidine 15 (5.67 g, 0.028 mol) in EtOH (150 mL) was added 10%
Pd/C (0.586 g, 10% w/w). The resulting mixture was stirred at room
temperature under an atmosphere of hydrogen for 23 h. After
completion, the mixture was filtered through Celite and washed
with EtOH. The solvent was removed in vacuo to afford a red oil
which was extracted with DCM (3 ꢀ 10 mL) and washed with brine.
The combined organic extracts were dried (MgSO4) and the solvent
was removed in vacuo to give the title compound 18 (4.12 g, 85%) as a
red oil which was used without further purification. dH (400 MHz;
CDCl3) 1.51e1.57 (2H, m, H-40), 1.70e1.75 (4H, m, H-30), 3.00 (4H, t,
J ¼ 5.5 Hz, H-20), 3.41 (2H, s, NH2), 6.63 (2H, d, J ¼ 8.8 Hz, H-2), 6.83
(2H, d, J ¼ 8.8 Hz, H-3). dC (100 MHz; CDCl3) 24.2 (C-40), 26.2 (C-30),
52.6 (C-20), 116.2 (C-2), 119.1 (C-3), 139.8 (C-1), 145.8 (C-4); IR
nmax(ATR)/cmꢂ1: 3336, 2930, 2851, 1620, 1509, 1229, 819; m/z
(ESIþ): 259 (71%), 177 (100%); HRMS (ESIþ) found (MHþ): 177.1380,
4’-(4-Nitrophenyl)morpholine; A mixture of morpholine (6.6 mL,
0.075 mol), 4-chloronitrobenzene 9 (11.82 g, 0.075 mol) and K2CO3
(10.35 g, 0.075 mol) in DMSO (50 mL) was heated at 120 ꢁC for 19 h
with stirring. The reaction was cooled to r.t., then 1:1 EtOH:H2O
(200 mL) added. The resulting precipitate was collected by filtration
and dried to give the title compound (15.62 g, quant.) as an orange
solid. m.p. 147e149 ꢁC (Lit [19]. 147e149 ꢁC). dH (400 MHz; CDCl3)
C
11H17N2 requires 177.1386. The 1H NMR data were in agreement
with literature values [26].

8
L.I. Pilkington et al. / European Journal of Medicinal Chemistry 191 (2020) 112162
Table 2
PC-PLC activity upon treatment by Series 2 compounds using an Amplex Red Assay. Testing was performed in triplicate. Results shaded light grade exhibit 80e90% inhibition
whilst results shaded dark grey exhibit >90% inhibition.
Relative PC-PLC activity (% ± s.e.) at 10 μM
N-Benzyl
N-Methyl
piperazine 12
31.5 ± 7.1
40.9 ± 9.7
29.3 ± 4.3
13.4 ± 6.4
12.7 ± 6.2
9.4 ± 4.6
N-Boc
piperazine 13
42.3 ± 16.3
32.2 ± 4.8
19.9 ± 0.9
26.5 ± 7.5
18.4 ± 6.3
23.9 ± 8.6
29.4 ± 4.2
33.5 ± 6.5
68.3 ± 15.9
35.6 ± 3.6
43.2 ± 6.7
36.8 ± 1.4
53.3 ± 16.1
41.3 ± 3.4
substitution
Piperidine 11
Piperazine 14
No substitution/H, a
2-Me, b
2-OMe, e
2-OH, h
3-OH, i
4-OH, j
2-F, k
8.1 ± 1.3
14.7 ± 0.9
9.9 ± 0.9
26.3 ± 2.9
29.3 ± 4.3
27.7 ± 0.6
37.9 ± 10.6
30.9 ± 4.8
51.3 ± 3.7
24.1 ± 1.4
26.0 ± 4.9
21.1 ± 3.3
26.8 ± 0.8
31.0 ± 4.3
27.4 ± 0.4
31.3 ± 6.3
30.4 ± 4.2
14.8 ± 0.5
7.6 ± 0.8
11.0 ± 0.1
9.0 ± 1.9
42.7 ± 11.5
44.2 ± 12.8
23.9 ± 2.6
20.0 ± 7.5
29.7 ± 4.6
19.5 ± 5.9
16.6 ± 2.9
25.4 ± 7.9
3-F, l
10.7 ± 2.0
6.6 ± 2.8
4-F, m
2-Br, n
28.0 ± 0.4
22.6 ± 2.6
24.7 ± 5.1
23.0 ± 1.8
49.3 ± 10.6
2-Cl, q
3-Cl, r
4-Cl, s
2-CF3, t
D609
40.3 ± 3.2
N-Benzyl-4-(piperidin-10-yl)aniline 11a; The reaction was carried
out according to General Procedure A-3, with benzaldehyde 6a
(0.27 mL, 2.65 mmol) and 4-(piperidin-10-yl)aniline 15 (0.2332 g,
1.32 mmol) for 7 d to afford the crude imine (0.073 g, 0.276 mmol)
as a yellow solid that was reduced according to General Procedure
B-3 with NaBH4 (0.038 g, 0.991 mmol) for 1 h to give the title
compound 11a (0.073 g, 21%) as a white solid. m.p. 59e61 ꢁC. dH
(400 MHz; CDCl3) 1.50e1.56 (2H, m, H-40), 1.69e1.74 (4H, m, H-30),
2.98 (4H, t, J ¼ 5.4 Hz, H-20), 3.75 (1H, s, NH), 4.29 (2H, d, J ¼ 5.5 Hz,
CH2), 6.61 (2H, d, J ¼ 8.8 Hz, H-2), 6.86 (2H, d, J ¼ 8.8 Hz, H-3),
7.25e7.39 (5H, m, H-200, H-300, H-400); dC (100 MHz; CDCl3) 24.4 (C-
40), 26.4 (C-30), 49.3 (CH2), 52.8 (C-20), 114.0 (C-2), 119.4 (C-3), 127.3
(C-400), 127.8 (C-200), 128.7 (C-300), 140.0 (C-100), 142.5 (C-1), 145.3 (C-
4); IR nmax(ATR)/cmꢂ1: 3396, 2929, 1613, 1512, 1268, 818; m/z
(ESIþ): 267 (MHþ, 100%); HRMS (ESIþ) found (MHþ): 267.1859,
(400 MHz, CDCl3) 2.35 (s, 3H, CH3), 2.55 (4H, t, J ¼ 5.2 Hz, H-2, H-6),
3.43 (4H, t, J ¼ 5.2 Hz, H-3, H-5), 6.82 (2H, d, J ¼ 9.6 Hz, H-20, H-60),
8.11 (2H, d, J ¼ 9.6 Hz, H-30, H-50). The spectroscopic values were in
agreement with literature [27].
4-(40-Methylpiperazin-10-yl)aniline 19; To a stirred solution of 10-
methyl-4’-(4-nitrophenyl)piperazine (5.75 g, 26.0 mmol) in 1:1
MeOH:EtOAc (60 mL) was added 10% palladium on carbon (0.058 g,
10% w/w) slowly. The mixture was then stirred under an atmo-
sphere of hydrogen for 24 h before being filtered through Celite and
washed with further MeOH. The solvent was removed in vacuo to
give the title compound 19 (4.84 g, 97%) as a maroon solid. m.p.
85e87 ꢁC. dH (400 MHz, CDCl3) 2.33 (3H, s, CH3), 2.57 (4H, t,
J ¼ 4.8 Hz, H-30, H-50), 3.06 (4H, t, J ¼ 4.8 Hz, H-20, H-60), 3.41 (2H, br
s, NH2), 6.64 (2H, d, J ¼ 8.6 Hz, H-2, H-6), 6.81 (2H, d, J ¼ 8.6 Hz, H-3,
H-5). The spectroscopic values were in agreement with literature
[27].
C
18H23N2 requires 267.1856.
1-Methyl-4-(40-nitrophenyl)piperazine 16; A mixture of 1-chloro-
tert-Butyl 4-(40-nitrophenyl)piperazine-1-carboxylate 17;
A
4-nitrobenzene 9 (5.0 g, 31.7 mmol) and K2CO3 (5.26 g, 38.1 mmol)
in DMSO (10 mL) was stirred at r.t. for 30 min before 1-
methylpiperazine (3.52 mL, 31.7 mmol) was added dropwise. The
mixture was then heated to 90 ꢁC and stirred for 24 h before being
poured into cold water and the resultant solid filtered and
collected. The solid was dissolved in EtOAc and washed with acid
(aq. 2M HCl, 3 ꢀ 10 mL). The combined aqueous layers were then
neutralised with base (1M NaOH) and extracted with EtOAc
(3 ꢀ 10 mL). The solvent was removed in vacuo to give the title
compound 16 (5.75 g, 82%) as an orange solid. m.p. 104e106 ꢁC. dH
mixture of 1-chloro-4-nitrobenzene 9 (5.0 g, 31.7 mmol) and K2CO3
(5.26 g, 38.1 mmol) in DMSO (10 mL) was stirred at r.t. for 30 min
before tert-butyl piperazine-1-carboxylate (5.91 g, 31.7 mmol) was
added dropwise. The mixture was then heated to 90 ꢁC and stirred
for 24 h before being poured into cold water and the resultant solid
filtered and collected. The solid was recrystallized from petroleum
ether, filtered while hot and precipitated to give the title compound
17 (6.74 g, 69%) as a yellow solid. m.p. 144e145 ꢁC. dH (400 MHz,
CDCl3) 1.49 (9H, s, t-Bu), 3.42 (4H, t, J ¼ 5.6 Hz, H-3, H-5), 3.60 (4H, t,
J ¼ 5.6 Hz, H-2, H-6), 6.82 (2H, d, J ¼ 9.4 Hz, H-20, H-60), 8.14 (2H, d,

L.I. Pilkington et al. / European Journal of Medicinal Chemistry 191 (2020) 112162
9
Fig. 4. The docked configuration of acid 1a (top left), ester 7a (top right), analogues 10a (middle left) and 10k (middle right) and piperidine 11a (bottom middle) in the PC-PLCBc
binding site. The protein surface is rendered. Red and blue surfaces depicts negative and positive partial charge whereas grey shows neutral lipophilic areas. (For interpretation of
the references to colour in this figure legend, the reader is referred to the Web version of this article.)
J ¼ 9.4 Hz, H-30, H-50). dC (100 MHz, CDCl3) 28.4 (CH3), 46.9 (C-2, C-
3, C-5, C-6), 80.4 (t-Bu), 112.9 (C-20, C-60), 126.0 (C-30, C-50), 138.8 (C-
40), 154.55 (C]O), 154.63 (C-10). IR nmax(ATR)/cmꢂ1 2971, 1672,
1585, 1319, 1239, 830. m/z (ESIþ): 330 (MNaþ, 100%); HRMS (ESIþ)
found (MNaþ): 330.1428, C15H21N3NaO4 requires 330.1424. The 1H
NMR data was in accordance with literature [28].
from petroleum ether. Reduction with NaBH4 afforded the title
compound as a beige solid without further purification (0.22 g, 75%).
m.p. 117e119 ꢁC. dH (400 MHz, CDCl3) 2.37 (3H, s, CH3), 2.62 (4H, t,
J ¼ 4.8 Hz, H-30, H-50), 3.09 (4H, t, J ¼ 4.8 Hz, H-20, H-60), 4.29 (2H, s,
CH2), 6.61 (2H, d, J ¼ 8.8 Hz, H-2, H-6), 6.85 (2H, d, J ¼ 8.8 Hz, H-3,
H-5), 7.22e7.30 (1H, m, H-400), 7.30e7.40 (4H, m, H-200, H-300, H-500,
H-600). dC (100 MHz, CDCl3) 45.8 (CH3), 49.0 (CH2), 50.7 (C-20, C-60),
55.2 (C-30, C-60), 113.8 (C-2, C-6), 118.9 (C-3, C-5), 127.1 (C-400), 127.5
(C-200, C-600), 128.6 (C-300, C-500), 139.7 (C-100), 142.8 (C-1), 143.5 (C-4).
IR nmax(ATR)/cmꢂ1 3235, 2932, 2802,1619, 1517, 1450, 1231, 816. m/z
(ESIþ): 282 (100), 223 (7), 197 (2), 191 (5), 176 (1), 145 (2), 132 (3);
tert-Butyl 4-(40-aminophenyl)piperazine-1-carboxylate 20; To a
stirred solution of tert-butyl 4-(4-nitrophenyl)piperazine-1-
carboxylate (3.00 g, 9.76 mmol) in MeOH (30 mL) was added 10%
palladium on carbon (0.30 g, 10% w/w) slowly. The mixture was
then stirred under an atmosphere of hydrogen for 24 h before being
filtered through Celite and washed with further MeOH. The solvent
was removed in vacuo to give the title compound 20 (2.85 g, quant.)
as a purple solid. m.p. 89e91 ꢁC. dH (400 MHz, CDCl3) 1.48 (9H, s, t-
Bu), 2.96 (4H, t, J ¼ 4.8 Hz, H-3, H-5), 3.45 (2H, br s, NH2), 3.56 (4H, t,
J ¼ 4.8 Hz, H-2, H-6), 6.65 (2H, d, J ¼ 8.6 Hz, H-30, H-50), 6.80 (2H, d,
J ¼ 8.6 Hz, H-20, H-60). dC (100 MHz, CDCl3) 28.5 (CH3), 51.2 (C-2, C-3,
C-5, C-6), 79.8 (t-Bu), 116.2 (C-20), 119.2 (C-30), 140.7 (C-40), 144.5 (C-
10), 154.8 (C]O). IR nmax(ATR)/cmꢂ1 3446, 3345, 2965, 1673, 1627,
1512, 1432, 1128, 819. m/z (ESIþ): 300 (MNaþ, 24%), 222
(C11H16N3Oþ2 , 48%), 178 (C10H16N3þ, 100%); HRMS (ESIþ) found
(MNaþ): 300.1678, C15H23N3NaO2 requires 300.1682. The 1H NMR
data was in accordance with literature [29].
HRMS (ESIþ) found (MHþ): 282.1963,
C
18H24N3þ requires
282.1965.
tert-Butyl
4-(4’-(benzylamino)phenyl)piperazine-1-carboxylate
13a; The reaction was carried out according to General Procedure
AB-4, with tert-butyl 4-(4-aminophenyl)piperazine-1-carboxylate
20 (0.20 g, 0.72 mmol) and benzaldehyde 6a (73 mL, 0.72 mmol).
The crude imine was filtered and washed with MeOH. Reduction
with NaBH4 afforded the title compound 13a as a light-tan solid
without further purification (0.16 g, 56%). m.p. 148e150 ꢁC. dH
(400 MHz, CDCl3) 1.48 (9H, s, t-Bu), 2.95e2.97 (4H, m, H-20, H-60),
3.55e37 (4H, m, H-30, H-50), 4.29 (2H, s, CH2), 6.61 (2H, d, J ¼ 8.8 Hz,
H-3, H-5), 6.84 (2H, d, J ¼ 8.8 Hz, H-2, H-6), 7.24e7.29 (1H, m, H-400),
7.31e7.39 (4H, m, H-200, H-300, H-500, H-600). dC (100 MHz, CDCl3) 28.4
(CH3), 43.4 (C-2, C-6), 48.9 (CH2), 51.8 (C-3, C-5), 79.9 (t-Bu), 113.9
(C-3, C-5), 119.7 (C-2, C-6), 127.3 (C-400), 127.6 (C-200, C-600), 128.6 (C-
300, C-500), 139.3 (C-100), 144.4 (C-40), 154.7 (C]O). C-10 not observed.
N-Benzyl-4-(40-methylpiperazin-10-yl)aniline 12a; The reaction
was carried out according to General Procedure AB-4, with 4-(40-
methylpiperazin-10-yl)aniline 19 (0.20 g, 1.05 mmol) and benzal-
dehyde 6a (0.10 mL, 1.05 mmol). The crude imine was triturated

10
L.I. Pilkington et al. / European Journal of Medicinal Chemistry 191 (2020) 112162
IR nmax(ATR)/cmꢂ1: 3369, 2971, 2856, 1684, 1516, 1415, 1225, 1156,
815; m/z (ESIþ): 368 (100), 312 (45), 277 (36), 268 (23), 221 (18);
HRMS (ESIþ) found (MHþ): 368.2331, C22H30N3O2þ requires
368.2333.
Society of New Zealand (Ref: 17.01) and the Breast Cancer Part-
nership, Health Research Council, New Zealand (Ref: 17/671).
Appendix A. Supplementary data
N-Benzyl-4-(piperazin-10-yl)aniline 14a; The reaction was carried
out according to General Procedure D, with tert-butyl 4-(4’-(ben-
zylamino)phenyl)piperazine-1-carboxylate 13a (0.1 g, 0.27 mmol)
to afford the title compound 14a as a green solid (48 mg, 66%). m.p.
100e102 ꢁC. dH (500 MHz, CDCl3) 2.96e3.05 (8H, m, H-20, H-30, H-
50, H-60), 4.28 (2H, s, CH2), 6.61 (2H, d, J ¼ 8.7 Hz, H-2, H-6), 6.84
(2H, d, J ¼ 8.7 Hz, H-3, H-5), 7.24e7.28 (1H, m, H-400), 7.30e7.38 (4H,
m, H-200, H-300, H-500, H-600). dC (100 MHz, CDCl3) 46.2 (C-30, C-50),
49.0 (CH2), 52.3 (C-20, C-60), 113.8 (C-2), 118.8 (C-3), 127.1 (C-400),
127.5 (C-200, C-600), 128.5 (C-300, C-500), 139.7 (C-100), 142.7 (C-1), 144.3
(C-4). IR nmamx/(zATR)/cmꢂ1: 3293, 3209, 2939, 2795, 1616, 1509, 1452,
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112162.
References
[1] (a) C.M. Vines, C.A. Bill, Phospholipases, in: eLS, John Wiley & Sons, Ltd, 2001;
(b) Z. Chen, Y. Yu, D. Fu, Z. Li, X. Niu, M. Liao, S. Lu, Cell Biochem. Funct. 28
(2010) 249e257;
(c) E.A. Dennis, J. Cao, Y.-H. Hsu, V. Magrioti, G. Kokotos, Chem. Rev. 111
(2011) 6130e6185.
[2] (a) M. Waite, Chapter 8 - phospholipases. In new comprehensive biochem-
istry, in: D.E. Vance, J.E. Vance (Eds.), Biochemistry of Lipids, Lipoproteins and
Membranes, vol. 31, Elsevier, 1996, pp. 211e236;
1227, 812;
(ESIþ): 268 (20), 237 (15), 223 (100), 180 (73), 123
(23); HRMS (ESIþ) found (MHþ): 268.1800, C17H22N3þ requires
(b) J.W. Pelley, 11 - metabolism of steroids and other lipids, in: Elsevier’s
Integrated Review Biochemistry, second ed., W.B. Saunders, Philadelphia,
2012, pp. 89e98.
268.1808.
[3] J. Collignon, L. Lousberg, H. Schroeder, G. Jerusalem, Breast Cancer Targets
Ther 8 (2016) 93e107.
4.3. Molecular modelling
[4] (a) R.M. Adibhatla, Biochem. Biophys. Res. Commun. 419 (2) (2012) 447,
author reply 448-449;
The crystal structure was obtained from Protein Data Bank (PDB)
[30] ID: 1AH710 with resolution 1.50 Å. The Scigress v2.6 program
[31] was used to prepare the crystal structure for docking, i.e.,
hydrogen atoms were added and crystallographic water molecules
removed. The centre of PC-PLCBc binding pocket was defined as the
position of Zn2þ ion (x ¼ 42.4820, y ¼ 22.996, z ¼ 8.556) with 10 Å
radius. The basic amino acids lysine and arginine were defined as
protonated. Furthermore, aspartic and glutamic acids were
assumed to be deprotonated. The GoldScore (GS) [32], ChemScore
(CS) [33,34], Piecewise Linear Potential (ChemPLP) [35] and Astex
Statistical Potential (ASP) [36] scoring functions were implemented
to validate the predicted binding modes and relative energies of the
ligands using the Genetic Optimisation for Ligand Docking software
package (GOLD) version 5.4.
ꢀ
(b) I. Plo, D. Lautier, T. Levade, H. Sekouri, J.P. Jaffrezou, G. Laurent, A. Bettaïeb,
Biochem. J. 351 (2) (2000) 459e467;
€
(c) R.A. Heller, M. Kronke, J. Cell Biol. 126 (1994) 5e9;
(d) M.C.M. van Dijk, F.J.G. Muriana, P.C.J. van der Hoeven, J. de Widt, D. Schaap,
W.H. Moolenaar, W.J. van Blitterswijk, Diacylglycerol generated by exogenous
phospholipase C activates the mitogen-activated protein kinase pathway in-
dependent of ras- and phorbol ester-sensitive protein kinase C: dependence
on protein kinase C-z, Biochem. J. 323 (1997) 693e699.
[5] (a) N. Isakov, Protein kinase C (PKC) isoforms in cancer, tumor promotion and
tumor suppression, Semin. Canc. Biol. 48 (2018) 36e52;
(b) C.E. Antal, A.M. Hudson, E. Kang, C. Zanca, C. Wirth, N.L. Stephenson,
E.W. Trotter, L.L. Gallegos, C.J. Miller, F.B. Furnari, et al., Cell 160 (2015)
489e502;
(c) C.H. Wilson, E.S. Ali, N. Scrimgeour, A.M. Martin, J. Hua, G.A. Tallis,
G.Y. Rychkov, G.J. Barritt, Biochem. J. 466 (2015) 379e390;
(d) S.L. Pelech, D.E. Vance, Trends Biochem. Sci. 14 (1989) 28e30;
(e) F. Spadaro, C. Ramoni, D. Mezzanzanica, S. Miotti, P. Alberti, S. Cecchetti,
E. Iorio, V. Dolo, S. Canevari, F. Podo, Canc. Res. 68 (2008) 6541e6549.
[6] L. Abalsamo, F. Spadaro, G. Bozzuto, L. Paris, S. Cecchetti, L. Lugini, E. Iorio,
A. Molinari, C. Ramoni, F. Podo, Breast Cancer Res. 14 (2012) R50.
[7] F. Podo, F. Sardanelli, E. Iorio, R. Canese, G. Carpinelli, A.F. Canevari,
S. Abnormal choline phospholipid metabolism in breast and ovary cancer:
molecular bases for noninvasive imaging approaches (accessed Jan 12, 2018),
http://www.eurekaselect.com/59183/article.
[8] L. Paris, S. Cecchetti, F. Spadaro, L. Abalsamo, L. Lugini, M.E. Pisanu, E. Iorio,
P.G. Natali, C. Ramoni, F. Podo, Breast Cancer Res. 12 (2010) R27.
[9] (a) X. Wu, H. Lu, L. Zhou, Y. Huang, H. Chen, Cell Biol. Int. 21 (1997) 375e381;
(b) I. Plo, D. Lautier, T. Levade, H. Sekouri, J.P. Jaffrezou, G. Laurent, A. Bettaieb,
Biochem. J. 351 (2000) 459e467.
4.4. Biological testing
Relative changes of PC-PLC activities were determined using the
Amplex Red assay kit (Molecular Probe, Inc.), as described by the
manufacturer. Briefly, PC-PLC activity was determined by adding
the 0.01U PC-PLC protein to a reaction mixture containing 0.4 mM
Amplex Red, 1 unit/mL horseradish peroxidase, 4 unit/mL alkaline
phosphatase, 0.1 unit/mL choline oxidase, and 0.5 mM phosphoti-
dylcholine (PtdCho) in 1X Reaction Buffer (50 mM Tris-HCl, pH 7.4,
0.14 M NaCl, 10 mM dimethylglutarate, 2 mM CaCl2). Phos-
phocholine released from PtdCho by PC-PLC was converted to
choline by alkaline phosphatase, which choline was oxidized to
form H2O2. In the presence of horseradish peroxidase, the H2O2
reacts with Amplex Red to generate the fluorescent product,
ꢀ
ꢀ
[10] P. Gonzalez-Bulnes, A. Gonzalez-Roura, D. Canals, A. Delgado, J. Casas,
A. Llebaria, Biorg. Med. Chem. 18 (2010) 8549e8555.
[11] C. Eurtivong, L.I. Pilkington, M. van Rensburg, R.M. White, H. Kaur Brar,
S. Rees, E.K. Paulin, C.S. Xu, N. Sharma, I.K.H. Leung, E. Leung, D. Barker,
J. Reynisson, Identification of novel phosphatidylcholine - specific phospho-
lipase C inhibitors using virtual high throughput screening, Eur. J. Med. Chem.
(2020) 187, https://doi.org/10.1016/j.ejmech.2019.111919.
[12] (a) S.E. Aakre, C. Little, Biochem. J. 203 (1982) 799e801;
(b) S.F. Martin, B.C. Follows, P.J. Hergenrother, C.L. Franklin, J. Org. Chem. 65
(2000) 4509e4514.
resorufin, which was detected using lex
¼
560 nm and
lem ¼ 590 nm on the EnSpire multimode plate reader. Inhibition
concentration at 50% (IC50) are the averages of five replicates using
the Amplex Red biochemical assay. Six different concentrations
[13] (a) S.F. Martin, Y.-L. Wong, A.S. Wagman, J. Org. Chem. 59 (1994) 4821e4831;
(b) R. Adibhatla, J.F. Hatcher, A. Gusain, Neurochem. Res. 37 (2012) 671e679;
(c) E. Amtmann, Drugs Exp. Clin. Res. 22 (1996) 287e294;
(d) E. Hough, L.K. Hansen, B. Birknes, K. Jynge, S. Hansen, A. Hordvik, C. Little,
E. Dodson, Z. Derewenda, Nature 338 (1989) 357e360;
were used to derive the IC50 values: 0, 0.1, 0.4, 1.1, 3.3 and 10.0 mM.
(e) S. Subramaniam, M. Mehrotra, D. Gupta, Virtual high throughput screening
(vHTS)–a perspective, Bioinformation 3 (2008) 14e17;
Declaration of competing interest
(f) R.V. Guido, G. Oliva, A.D. Andricopulo, Curr. Med. Chem. 15 (2008) 37e46.
[14] M. Kato, M.A.S. Hammam, T. Taniguchi, Y. Suga, K. Monde, Org. Lett. 18 (2016)
768e771.
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
€
[15] L.M. Peschel, F. Belaj, J.A. Schachner, N.C. Mosch-Zanetti, Eur. J. Inorg. Chem.
(2017) 2808e2817.
[16] J. Srogl, J. Hyvl, A. Revesz, D. Schroder, Chem. Commun. 23 (2009) 3463e3465
(Cambridge, U. K.).
[17] J. Kosary, I.K. Szabo, E. Kasztreiner, Pharmazie 37 (1982) 484e486.
[18] B.C. Chenna, J.R. King, B.A. Shinkre, A.L. Glover, A.L. Lucius, S.E. Velu, Eur. J.
Med. Chem. 45 (2010) 3752e3761.
Acknowledgements
We are very grateful for the financial support from the Cancer
[19] K.H. Hoi, S. Calimsiz, R.D.J. Froese, A.C. Hopkinson, M.G. Organ, Chem. Eur J. 17

L.I. Pilkington et al. / European Journal of Medicinal Chemistry 191 (2020) 112162
11
(2011) 3086e3090. S3086/1-S3086/391.
[20] Y. Monguchi, K. Kitamoto, T. Ikawa, T. Maegawa, H. Sajiki, Adv. Synth. Catal.
350 (2008) 2767e2777.
[21] R. Adams, K.A. Schowalter, J. Am. Chem. Soc. 74 (1952) 2597e2602.
[22] M.-U. Hung, S.-T. Yang, M. Ramanathan, S.-T. Liu, Appl. Organomet. Chem. 32
(2018).
[23] C.M. Wong, M.B. Peterson, I. Pernik, R.T. McBurney, B.A. Messerle, Inorg.
Chem. 56 (2017) 14682e14687.
S. Kaku, T. Kawasaki, Y. Matsumoto, S. Sakamoto, S.-i. Tsukamoto, Bioorg. Med.
Chem. 12 (2004) 2179e2191.
[29] Mizojiri, R.; Hirashima, S.; Oka, T.; Aoki, K.; Noji, S.; Ando, I.; Sato, T.; Niwa, Y.
WO2005049622A1, 2005
[30] H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig,
I.N. Shindyalov, P.E. Bourne, The protein Data Bank, Nucl.Acids Res. 28 (2000)
235e242.
[31] Fujitsu Scigress, v2.6, Fijitsu Limited, 2016.
[24] H. Kotsuki, S. Kobayashi, H. Suenaga, H. Nishizawa, High pressure organic
chemistry. XI. A new convenient synthesis of aromatic amines from activated
phenols, Synthesis (12) (1990) 1145e1147.
[25] D. Wang, Y. Zheng, M. Yang, F. Zhang, F. Mao, J. Yu, X. Xia, Org. Biomol. Chem.
15 (2017) 8009e8012.
[26] J. Mou, A. Park, Y. Cai, J. Yuan, C. Yuan, Bioorg. Med. Chem. Lett 25 (2015)
3057e3061.
[27] C.M. Park, J.I. Choi, J.H. Choi, S.Y. Kim, W.K. Park, C.M. Seong, Bioorg. Med.
Chem. Lett 21 (2011) 698e703.
[32] G. Jones, P. Willet, R.C. Glen, A.R. Leach, R. Taylor, J. Mol. Biol. 267 (1997)
727e748.
[33] M.D. Eldridge, C. Murray, T.R. Auton, G.V. Paolini, P.M. Mee, J. Comput. Aided
Mol. Des. 11 (1997) 425e445.
[34] M.L. Verdonk, J.C. Cole, M.J. Hartshorn, C.W. Murray, R.D. Taylor, Proteins 52
(4) (2003) 609e623.
[35] O. Korb, T. Stützle, T.E. Exner, J. Chem. Inf. Model. 49 (2009) 84e96.
[36] M.L. Verdonk, G. Chessari, J.C. Cole, M. Hartshorn, C.W. Murray, J.W.M. Nissink,
R.D. Taylor, R. Taylor, J. Med. Chem. 48 (2005) 6504e6515.
[28] H. Koshio, F. Hirayama, T. Ishihara, Y. Taniuchi, K. Sato, Y. Sakai-Moritani,