Derivatisation of parthenolide to address chemoresistant chronic lymphocytic leukaemia

Article abstract of DOI:10.1039/c9md00297a

Parthenolide is a natural product that exhibits anti-leukaemic activity, however, its clinical use is limited by its poor bioavailability. It may be extracted from feverfew and protocols for growing, extracting and derivatising it are reported. A novel parthenolide derivative with good bioavailability and pharmacological properties was identified through a screening cascade based on in vitro anti-leukaemic activity and calculated "drug-likeness" properties, in vitro and in vivo pharmacokinetics studies and hERG liability testing. In vitro studies showed the most promising derivative to have comparable anti-leukaemic activity to DMAPT, a previously described parthenolide derivative. The newly identified compound was shown to have pro-oxidant activity and in silico molecular docking studies indicate a prodrug mode of action. A synthesis scheme is presented for the production of amine 7 used in the generation of 5f.

Full text of DOI:10.1039/c9md00297a

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RESEARCH ARTICLE
Derivatisation of parthenolide to address
chemoresistant chronic lymphocytic leukaemia†
Cite this: DOI: 10.1039/c9md00297a
a
a
b
b
Xingjian Li, ‡ Daniel T. Payne, ‡ Badarinath Ampolu,§ Nicholas Bland,¶
b
a
c
d
Jane T. Brown, Mark J. Dutton, Catherine A. Fitton,
Abigail Gulliver,
d
b
b
b
e
Lee Hale, Daniel Hamza, Geraint Jones, Rebecca Lane, Andrew G. Leach,
Louise Male, Elena G. Merisor, Michael J. Morton,
f
b
g
a
Alex S. Quy,
gh
c
b
c
Ruth Roberts,
Rosanna Scarll, Timothy Schulz-Utermoehl, Tatjana Stankovic,
b
a
c
Brett Stevenson, John S. Fossey ‡* and Angelo Agathanggelou ‡*
Parthenolide is a natural product that exhibits anti-leukaemic activity, however, its clinical use is limited by
its poor bioavailability. It may be extracted from feverfew and protocols for growing, extracting and
derivatising it are reported. A novel parthenolide derivative with good bioavailability and pharmacological
properties was identified through a screening cascade based on in vitro anti-leukaemic activity and calcu-
lated “drug-likeness” properties, in vitro and in vivo pharmacokinetics studies and hERG liability testing. In
vitro studies showed the most promising derivative to have comparable anti-leukaemic activity to DMAPT,
a previously described parthenolide derivative. The newly identified compound was shown to have pro-
oxidant activity and in silico molecular docking studies indicate a prodrug mode of action. A synthesis
scheme is presented for the production of amine 7 used in the generation of 5f.
Received 28th May 2019,
Accepted 3rd July 2019
DOI: 10.1039/c9md00297a
rsc.li/medchemcomm
Introduction
Chronic lymphocytic leukaemia (CLL) is the most common
1
adult leukaemia with >3000 new cases in the UK annually.
Its variable clinical course ranges from stable indolent disease
a
School of Chemistry, University of Birmingham, Edgbaston, Birmingham, West
Midlands, B15 2TT, UK. E-mail: j.s.fossey@bham.ac.uk
that does not require treatment, to rapidly progressive disease
2
b
that necessitates immediate therapeutic intervention. Clini-
Sygnature Discovery, The Discovery Building, BioCity, Pennyfoot Street,
Nottingham, NG1 1GR, UK
cal response rates to current therapies are strongly influenced
by genetic changes including disruption of DNA damage re-
sponse (DDR) genes ATM and p53. Tumours with a DDR de-
c
Institute for Cancer and Genomic Sciences, University of Birmingham, Edgbaston,
Birmingham, West Midlands, B15 2TT, UK. E-mail: a.agathanggelou@bham.ac.uk
3
d
Winterbourne Botanic Garden, University of Birmingham, 58 Edgbaston Park
fect are refractory to chemotherapeutics because they are un-
able to initiate apoptosis in response to therapy-induced DNA
damage. This means tumours with or that develop this DDR
defect are not treatable with typical DNA-damaging chemo-
therapies. Furthermore, since most CLL patients are over the
age of 60 (median age >70) many have comorbidities that
preclude the use of aggressive chemotherapeutic regimens.
This highlights a need to develop alternative, less aggressive,
Road, Edgbaston, Birmingham, West Midlands, B15 2RT, UK
e
School of Pharmacy and Biomolecular Sciences, Liverpool John Moores
University, Byrom Street, Liverpool, L3 3AF, UK
f
X-Ray Crystallography Facility, School of Chemistry, University of Birmingham,
Edgbaston, Birmingham, West Midlands, B15 2TT, UK
g
ApconiX Ltd, Alderly Park, Nether Alderly, Cheshire, SK10 4TG, UK
h
School of Biosciences, University of Birmingham, Edgbaston, Birmingham, West
Midlands, B15 2TT, UK
†
Electronic supplementary information (ESI) available: General experimental
4
details, aspects relating to cultivation of feverfew, the PTL extraction protocol,
synthesis, biological assays computational information and spectrums are de-
tailed in the supporting information. Methods described and references cited
therapies for the treatment of CLL.
An attractive potential therapeutic is parthenolide (PTL,
1), Fig. 1, upper. PTL (1) is a natural product isolated from
feverfew (Tanacetum parthenium) and varietals thereof (Fig. 1,
45
therein, should be referred to in regard of the docking, crystallographic, syn-
thetic and toxicological studies detailed therein. See DOI: 10.1039/c9md00297a
5
‡
AA and JSF are joint last and co-corresponding authors, XL and DTP share
lower), feverfew being so-named due to its use in traditional
6
joint first authorship. Remaining authors are listed alphabetically, author contri-
butions are described later.
remedies. PTL (1) demonstrates effective and selective anti-
7
CLL activity in vitro, and through its pro-oxidant activity, can
§
Present address: BioMedha Limited, Biocity, Innovation Building, Pennyfoot
Street, Nottingham NG1 1GF, UK.
Present address: Excellerate Bioscience Limited, Discovery Building, Biocity,
8
target CLL cells independently of their p53 status. Further-
9
more, co-authors of this report are among those who have
¶
Pennyfoot Street, Nottingham, NG1 IGR, UK.
shown that ATM-deficiency disrupts redox homeostasis,
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ambition of addressing drug-resistant CLL in elderly patients
who may suffer more intensely from adverse effects of other
therapies. Herein a synthesis cascade to explore the SAR of
PTL derivatives is described and findings pertaining to these
aims presented.
Results and discussion
Source of Parthenolide
Since 2013 feverfew and varietals have been cultivated at
2
1
Winterbourne House and Garden, a UK visitor attraction,
museum and heritage centre adjunct to the University of Bir-
mingham campus (UK), located 52.4527° N. Annual crops of
plant material have been obtained (Fig. 2), and PTL extracted.
Plants in the late stage of flowering were found to contain
the highest extractable content of PTL.
Fig. 1 Upper: Structure of parthenolide (PTL, 1) and dimethyl amino-
PTL (DMAPT) (2a) respectively; lower flowers of feverfew and
button-like (Tanacetum parthenium ‘Flore Pleno’) feverfew varietal.
a
An extraction procedure previously reported by co-authors
9
of this report was used (detailed in ESI†) to access
recrystalised, analytically pure, PTL (1), (see ESI†).
increasing further the sensitivity of this DDR-defective
1
0
subtype of CLL to PTL. In addition to inducing oxidative
stress, PTL has been shown to target tumour cells by sup-
pressing pro-survival and proliferation signalling through the
Derivatisation of parthenolide
The addition of nucleophilic primary and secondary amines
to PTL has been reported to proceed with high diastereo-
selectivity via a conjugate addition to the exocyclic Michael
1
1
NF-κB pathway, by inhibiting JAK–STAT kinase activity and
thereby preventing STAT-mediated transcription of anti-
apoptotic genes and by inhibiting DNMT1 and HDACs lead-
ing to activation of epigenetically silenced tumour suppressor
1
4,16a,18f,22
acceptor double bond part of the lactone unit.
The
high selectivity for reaction of nucleophilic amines at the
α,β-unsaturated lactone provides evidence of the compatibil-
ity of the epoxide motif contained within PTL and its deriva-
tives thus obtained within a drug discovery programme.
That is, epoxides display unwanted reactivity towards nucleo-
philes, and when this reactivity is unleashed in vivo off-
target effects can render them undesirable motifs in medici-
1
2
genes. Furthermore, through inhibition of NF-κB, PTL is
able to reverse resistance to a number of chemotherapeutics
including paclitaxel, oxaliplatin, doxorubicin and metoxantr-
1
3
one. Despite numerous beneficial anti-tumour activities
demonstrated in vitro, the clinical utility of PTL is limited by
1
4
its poor bioavailability and pharmacokinetics.
2
3
Crooks and co-workers pioneered the derivatisation of PTL
and identified a promising candidate for the treatment of
nal chemistry. In related research, Long et al. reported the
semi-synthesis of PTL and its cyclopropyl (in place of the
1
5
24
acute myeloid leukaemia (AML), a dimethyl amino-PTL
epoxide part) analogue,
the epoxide-free analogue was
1
4,16
(DMAPT) derivative, compound 2a depicted in Fig. 1.
more stable to acid-mediated degradation, showing there
may be scope to improve the drug-like properties of PTL
This derivative of PTL (1), DMAPT (2a) and related com-
pounds, have found utility across different diseases with
1
6u
activities against a range of cancer-types reported.
PTL has
1
7
been shown to induce oxidative stress in cancer cells, and
recently DMAPT (2a) was confirmed to induce oxidative stress
9
in CLL cells. There are a number of reports on the use and
derivatisation of PTL, with a view to addressing various can-
1
6z,18
cer types,
but the only PTL derivative to be applied to
1
9
CLL remains compound 2a. Previous studies have used mo-
lecular docking to investigate 1 and 2a which have suggested
that interaction with IKKβ is a plausible mechanism for their
1
8g
action against haematological and solid tumours.
It is reasoned that retro-Michael-type chemistry, that re-
veals PTL at the site of interest, may be at the origin of the
activity of amino-PTL derivatives, meaning amino-PTL deriva-
tives (such as 2a) serve as excellent prodrugs for PTL
1
6ac,ad
delivery.
Our interest in drug-like nitrogen-containing
2
0
motifs prompted us to investigate analogues of DMAPT (2a)
in the search for increased activity, favourable pharmacoki-
netic properties and minimal toxicological burdens, with the
Fig. 2 Feverfew cultivation effort, Winterbourne Botanic Garden, prior
to harvesting feverfew; (inset) recrystallised PTL 1.
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3
0
analogues by further scaffold manipulation (although that
was not probed herein).
CLL. The MEC1 cell line (obtained from the American type
culture collection (Manassas, VA 20110 USA) expresses a mu-
tated form of p53 and as previously shown, is resistant to
clinically relevant DNA-damaging agents such as cyclophos-
phamide and is therefore representative of treatment-refrac-
tory, progressive CLL for which there is an urgent unmet clin-
Taking advantage of the ease of derivatisation of the lac-
tone part of parthenolide a small initial library of amino (and
related) PTL derivatives was generated using an established
synthetic protocol (Scheme 1 – conditions i and Fig. 3),
resulting in the isolation of compounds 2a–d, 3, 4a–d, 5a–c
and 6a & b. Following the successful synthesis of this small
collection of PTL derivatives (Scheme 1 - conditions i), and in
order to rapidly expand the library of amino-PTL derivatives
available, a more high-throughput approach that allows for a
library of products to be created with a range of functionali-
3
1
ical need. The in vitro antileukaemic activity of the PTL-
derivatives against the MEC1 CLL cell line was determined
using the alamarBlue® (ThermoFischer Scientific) assay and
3
2
the results are summarised in Table S2.† Among the active
compounds 2c, 2d, 2e, 2f, 2g, 2j, 2aa, 5a, 5b, 5d, 5e, 5f, 5h,
5ab and 6b (Table S2,† entries 4 to 8, 11, 12, 29, 62, 63, 65 to
67, 69, 89 and 91 respectively) gave EC50 values <15 μM. Ow-
ing to resource availability a subset of up to ten of these com-
pounds was prioritised for further investigation. Compounds
2d, 2e, 2j, 2aa, 5a, 5d, 5e, 5f, 5ab and 6b were selected on the
basis of reasons including ease of synthetic access, diversity
across the chemical space probed and novelty. Those pre-
pared under conditions (i) Scheme 1 (2d, 5a and 6b) were
available on a scale and in a purity to directly permit follow-
up studies, whereas compounds prepared under conditions
(ii) Scheme 1 were prepared on a small scale so were
resynthesised. These seven compounds (2e, 2j, 2aa, 5d, 5e, 5f
and 5ab) were isolated on >30 mg scale in high purity and
progressed to more following studies.
2
5
ties and desirable drug-like properties was sought.
Under slightly modified conditions from those previously
9
,26
employed,
PTL was reacted with 120 primary or secondary
amines that were selected based on calculated properties of
the potential products. Used as supplied or obtained
(
whether neutral or as salts thereof) the amines reacted with
PTL 1 in ethanol (Scheme 1 – conditions i) or in DMSO (20%)
methanol (80%) in the presence of Hünig's base (Scheme 1 –
/
conditions ii).
The reaction mixtures were filtered and purified by
reverse-phase HPLC of the 120 attempted synthesise, 76 suc-
cessfully delivered products of sufficient purity (ideally >85%
purity; 65 of which were obtained in >95% purity), as judged
by HPLC or proton NMR spectroscopy). The materials
obtained (shown in Fig. 3) were thus deemed suitable to
progress to in vitro activity screening for anti CLL activity. Of
the 76 compounds successfully synthesised and progressed
to in vitro screening eleven have been previously reported
elsewhere in the peer reviewed or patent literature, namely:
In vitro DMPK
Compounds 2d, 2e, 2f, 2g, 2j, 2aa, 5a, 5d, 5e, 5f, 5ab and 6b
were subjected to in vitro DMPK testing. Seven of the ten PTL
derivatives (2e, 5a, 5b, 5d, 5f, 5ab and 6b) exhibited greater
than two hours stability in aqueous solution and were
retained for further investigation. The other three (2d, 2j and
1
6af
14
14,16a
16a,w,27
14
16a
28
14,16w,27–29
2
5
a,
b,
2b, 2c,
3,
4a, 4c,
4d, 5a,
1
4,27–29
27b,29b
16y
5c
and 6a.
2
aa) were not retained for further investigation due to poor
stability (T1/2 at pH 7.4: 13, 107 and 76 min, Table 1, entries
, 3 and 4 respectively). One obvious aspect contributing to
observed stability is the propensity for PTL derivatives of this
type to undergo retro-amination (reverse-Michael-type
mechanism), indeed this reverse reaction is often claimed as
Anti-chronic lymphocytic leukaemia activity
1
Defective p53-signalling increases genomic instability and
drives tumour progression. A high frequency of p53 inactiva-
tion is found in relapsed, treatment-resistant, high risk
a
2
9a,33
a pro-drug mode of action.
Whether poor stability in this
test is as a result of retro-amination or any other process was
not determined, either way the compound of interest does
not survive long enough in solution to be retained for further
study. It is a reasonable assertion that activity observed for
these unstable compounds could well be ascribed to free PTL
generated rapidly in the assay.
Microsomal and hepatocyte stability assays are in vitro
ADME assays used to determine metabolic stability of com-
pounds through measuring intrinsic clearance (CLint) by liver
microsomes or by liver hepatocytes. Compounds 2e, 5d, 5e
and 5ab (Table 1, entries 2, 6, 7 and 9) exhibited CLint of
Scheme
thioether), 4 (acyclic secondary amines), 5 (cyclic tertiary amines) and
(amino acid derivatives). Two protocols: Conditions (i) – Addition of
1 Synthesis of PTL derivatives 2 (tertiary amines), 3 (a
−
1
−1
>
100 μL min mg in the microsomal stability assay indi-
6
cating rapid clearance and as such, were removed from fur-
ther investigation. Compounds 5a, 5f and 6b displayed the
lowest microsomal intrinsic clearances, 19 μL, 18 μL and 9
nucleophile in ethanol at room temperature; conditions (ii) – addition
of nucleophile in dimethylsulphoxide/methanol (20/80) with Hünig's
1
2
base at room temperature. All product R and R groups shown in
Fig. 3.
−
1
−1
μL min mg , respectively (Table 1, entries 5, 8 and 10) and
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Fig. 3 Summary of PTL derivatives' amine (and one thioether) parts, prepared according to Scheme 1.
3
4
were therefore retained for further study. As a secondary
screen for metabolic stability, compounds 5a, 5f and 6b were
subjected to the hepatocyte stability assay and this confirmed
their suitability for progression to the Caco-2 permeability as-
say (Table 1, entries 5, 8 and 10). In order to determine the
likelihood of the retained compounds being suitable for oral
dosing a Caco-2 permeability assay, which predicts intestinal
permeability and drug efflux, was conducted. Compounds
5a and 5f (Table 1, entries 5 and 8) gave efflux ratios close to
unity (1.0 and 1.1 respectively), whereas compound 6b
3
5
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Table 1 Screening cascade for favourable drug-like properties of the ten most promising compounds ([—] indicates not tested)
Microsomal
stability (mouse)
Hepatocyte stability
(mouse)
Caco-2 pH 7.4 mean Papp
6
−
−1
(10 cm s )
Stability
pH 7.4
−1
EC50
LipE
CLint/μL min
1/2
T /
Entry Compound
1
ave./μM pEC50–clog P
T
1/2/min A to B
A to B
per million cells min A to B B to A Efflux ratio
4.7
5.6
5.6
5.9
7.7
5.4
3.31
1.61
1.48
2.52
2.35
2.48
13
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
1.0
—
2
3
4
5
6
>120
107
828
—
2
—
—
—
—
75
6
—
—
76
—
—
—
>120
>120
19
22.0
—
65.0 15.9
16.2
—
251
—
—
—
—
7
8
9
6.0
7.7
7.6
1.90
3.69
2.29
>120
>120
>120
391
18
4
—
—
—
77
11
22.0
—
64.0 2.7
2.8
9.1
1.1
0.8
128
—
10.9
10
13.5
3.66
>120
9
160
16.0
88.0 1.5
4.2
2.7
(
Table 1, entry 10) gave an efflux ratio of 2.7. Thus, com-
become increasingly useful and important in medicinal
3
6
pounds 5a and 5f were retained and selected for in vivo phar-
macokinetic studies.
The parameter lipophilic ligand efficiency (LipE) allows ac-
tivity of a compound in a given assay to be tensioned against
the lipophilicity of the compound. The LipE parameter has
chemistry drug discovery decision making. LipE allows the
quality of hits to be compared, i.e. deconvolution of activity
arising due to better chemistry away from increased activity
due to enhancement in lipophilicity alone. It has been
claimed that consideration of LipE alongside other
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chemoinformatic parameters in a drug discovery programme
can result in the identification of compounds with superior
concentrations in blood were measured with time (Fig. 4(a)i
and (b)i and Table 2).
3
7
in vivo properties. As such the calculated log P values (see
Table S2†) were in determination of the LipE for the active
compounds detailed in Table 1. Notably, the two compounds
identified as superior through the conducted screening cas-
cade of this manuscript (5a and 5f) have the same 7.7 μM
EC50 (pEC50 5.11) values against the MEC1 cell line, whereas
the calculated log P values differ by more than one log unit
Unlike the similarity between in vitro intrinsic clearance
between 5a and 5f (Table 1), the in vivo clearance of 5a was
−
1
−1
nearly four times that of 5f (207 versus 58 mL min kg ,
Table 2). The plasma half-life (T1/2) of 5f was approximately
twice that of 5a (1.14 versus 0.66 hours, Table 2), and the to-
tal body exposure to 5f was more than three times greater
−
1
than 5a (289 versus 80.5 ng h mL , Table 2).
2
.76 versus 1.42 (respectively). Thus, the corresponding LipE
The in vivo oral bioavailability of compounds 5a and 5f
−
1
values of 2.35 versus 3.69 for 5a and 5f respectively in the
probed MEC1 cell line assay (Table 1, entries 5 and 8) reveal
that whilst compound 5f has an equivalent activity to 5a, its
activity may arise from advantageous chemical features and
was compared in a murine PK study at 10 mg kg (Fig. 4(a)ii
and (b)ii and Table 2). In keeping with the intravenous find-
ings, the murine PO half-life of 5f was twice as long as 5a
(1.0 versus 0.5 hours, Table 2) and the Cmax attained by 5f
was approximately 40% higher than that achieved by 5a (404
3
8
not from lipophilicity alone.
Coupled with the data
−
1
presented in Table S2,† consideration of the LipE indicates
compound 5f to offer a potential advantage in a drug discov-
ery programme.
versus 283 ng mL ). The total body exposure was over two
−
1
times greater for 5f than 5a (1696 versus 711 ng h mL ,
Table 2), indicating superior bioavailability. Compound 5f
was therefore identified as a promising potential new agent
with desirable pharmacological properties towards a therapy
for drug-resistant CLL.
In vivo pharmacokinetics
In order to determine 5a and 5f are suitable for use as phar-
maceutical agents their pharmacokinetic (PK) parameters
were next probed. A murine intravenous PK study of com-
Comparison of the activity of 5f with 1 (PTL) and 2a (DMAPT)
Following the identification of 5f as the most promising
drug-like derivative of PTL from the aforementioned parallel/
−
1
pounds 5a and 5f at 1.0 mg kg was conducted and their
−
1
−1
Fig. 4 Graphical representation of murine in vivo pharmacokinetic study, concentrations of 5a (a) and 5f (b) i. IV (1 mg kg ); ii. PO (10 mg kg ).
Data summarised in Table 2.
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−1
−1
Table 2 Data obtained from murine in vivo pharmacokinetic study, concentrations of 5a and 5f (IV (1 mg kg ) and PO (10 mg kg )). Data represented
graphically in Fig. 4
Mouse PK (IV)
Cl/mL
min kg
Mouse PK (PO)
EC50
ave./μM
T1/2/h
UC (0-inf)/ng
h mL
C
mL
max/ng
UC (0-inf)/ng
h mL
−1
−1
−1
−1
−1
−1
Entry
1
Compound
VSS/L kg
11.6
Tmax/h
F%
7.7
207
0.66
80.5
0.5
283
95.8
711
2
7.7
58
1.14
4.62
289
1
404
58.5
1696
high-throughput screening, the anti-leukaemic activity of 5f
was tested once more and compared directly with that of PTL
test concentration). At said top (100 μM) test concentration
the novel compound 5f elicited only a 24% inhibition,
whereas compounds 2a and 5a performed slightly less well
with inhibitions of 40 and 45% (Fig. S6 and Table S4,† entries
1 and 2) respectively. Thus, hERG liability study, confirms
PTL derivatives tested to represent a minimal risk according
to results of the in vitro tests conducted. Among the
(
1) and DMAPT (2a), which serve as comparators and positive
controls. Fig. 5(a) shows that the activity of 5f (n = 5) (EC50
.5 μM) was not significantly different to the activity of PTL
n = 5) or DMAPT (n = 5) with EC50 values of 6.2 μM and 5.6
=
4
(
μM, respectively (Table S2,† entries 1 and 2) indicating that it
has comparable activity with the parent and literature com-
pounds (1 and 2a respectively). Pre-treatment with
N-acetylcysteine (NAC) abrogated activity demonstrating that
similar to PTL and other derivatives, 5f is a pro-oxidant and
induces cell death through induction of oxidative stress,
Fig. 5(b). Furthermore, in silico modelling suggests that 5f is
likely to function as a pro-drug and deliver PTL to also in-
hibit IKKβ and NF-κB, Fig. S3 and S4.†
In vitro hERG liability testing
Early toxicological screening is an important step in
derisking drug discovery programmes and is increasingly ac-
3
9
cessible. Since an unacceptable human ether a go-go re-
lated gene (hERG) liability is an all too common reason for
failure at the candidate selection stage, a hERG assay was
conducted to ensure the compounds being developed did not
4
0
carry this liability.
Three PTL derivatives (2a, 5a and 5f) were tested for inhi-
+
bition of the hERG K channel (against cisapride as a positive
control) using IonWorks patch clamp electrophysiology. In
assessment of any hERG liability, compound 5a was com-
pared to compound 5f which had already been identified as a
superior pre-candidate compound; whilst 2a (DMAPT) is spe-
cifically not the focus of this manuscript it was deemed ap-
propriate to contrast any hERG liability of this the literature-
1
6af
reported anti-AML compound.
Eight-point concentration-
response curves were generated using three-fold serial dilu-
tions from a maximum final test concentration of 100 μM, re-
sults are summarised in Fig. S6.†
The three PTL derivatives compared (2a, 5a and 5f) in this
hERG liability assay all performed well displaying IC50 values
Fig.
5 Concentration versus activity plots (alamarBlue® assay)
showing (a) anti-leukaemic activity of 5f versus PTL and DMAPT in
MEC1 cells and (b) the effect of NAC pretreatment on the activity of 5f.
>100 μM (i.e. inhibition of less than 50% at the top 100 μM
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lem. Whilst it was possible to synthesise compound 5f in
83% isolated yield in high purity via a hybrid of the earlier
mentioned protocols (Scheme 2a), the availability of amine 7
caused some issues, namely during the course of this work
resupply of amine 7 (or salts thereof) became challenging.
This uncertain supply chain led us to probe the synthesis
and set about synthesising our own material. Despite the
compound being listed in the catalogues of numerous sup-
pliers, a literature search (SciFinder) revealed very few in-
4
1
stances of the motif occurring in the peer reviewed or pat-
4
2
ent literature. The synthesis of amine 7 had not, to the best
of our knowledge at the time of writing this report, been
reported in the peer reviewed literature. As such a synthetic
protocol was proposed (Scheme 2b).
Amine
7
was successfully prepared as follows:
4
9
-Piperidinecarboxylic acid was N-Boc protected to deliver 8 in
4
3
2% yield. In order to confidently assign the NMR spectra
1
1
of compound 8 J-MOD, HSQC and H– H COSY were helpful,
particularly in establishing that a broad resonance in the
1
3
proton-decoupled C NMR spectrum, centred on 42.8 ppm,
corresponds to the ring-carbons bonded to nitrogen. The car-
boxylic acid of 8 was converted to the corresponding ethyl-3-
keto-propanoate 9 in 24% yield. Conversion of 9 to the N-Boc
protected congener 10, was achieved by treatment with
acetamidine hydrochloride and potassium carbonate, fur-
nishing 10 in 77% yield. Treatment with trifluoroacetic acid
delivered desired compound 7 in 99% yield for the Boc-
deprotection step (scale 200 mg, 17% over four linear steps).
Thus, evidencing accessibility of amine 7 and with it in hand,
it was timely to probe any activity arising from the amine al-
one. MEC1 sensitivity to 1, 5f and 7 was compared in parallel
using the alamarBlue® assay (Fig. 6). EC50 values were deter-
mined (CalcuSyn Version 2.11, BIOSOFT) for 1 (3.6 μM ± 0.46)
and 5f (4.3 μM ± 0.60). In contrast, an EC50 could not be de-
termined for amine 7 since MEC1 cells were unaffected by
equivalent concentrations of it indicating that 7 is not a
source of cytotoxicity observed in this assay. A pre-peer-
Scheme 2 (a) The optimised synthesis of 5f; (b) the designed and
executed plan for the synthesis of the required amine 7.
compounds tested 5f displayed the lowest hERG liability and
was flagged as inactive (Table S4,† entry 3) confirming com-
pound 5f as the most promising lead from the studies
conducted herein.
Synthetic access to compound 5f
Through identification of compound 5f as the resulting pre-
candidate from the described screening cascade in earlier
sections the requirement to provide more material for this
and onwards studies presented a new and unexpected prob-
Fig. 6 Concentration versus activity plots (alamarBlue® assay) showing anti-leukaemic activity of 1 (PTL), 5f and amine 7 in MEC1 cells.
Med. Chem. Commun.
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viewed elsewhere.
facilities but also via Winterbourne House and Garden.
Their crucial, critical and extremely valuable support in-
cludes the staff who have dedicated time, resources and ef-
fort to the cultivation of feverfew and varieties thereof. The
CASE network has facilitated cooperation, training and net-
4
4
Conclusions
4
6
The cultivation of feverfew, from which PTL 1 was extracted,
was described. The isolation of PTL (1) permitted the synthe-
sis of a library of derivatives, through smooth 1,4-addition to
PTL's exocyclic Michael-acceptor CC double bond. The li-
brary thus synthesised displayed good coverage of medici-
nally relevant chemical space. The compounds synthesised
were tested for activity against the MEC1 CLL cell line. These
compounds showed no particular trend in activity across the
working opportunities. Professor Jonathan Frampton (Uni-
versity of Birmingham) is thanked for help throughout and
providing support that was crucial at the project initiation
stage. The authors thank the ERDF AWM II for support.
DTP thanks The School of Chemistry for a studentship. Huy
Van Nguyen (University of Birmingham) is thanked for assis-
tance with aspects of experiments conducted in the latter
stages. JSF is extremely grateful to the Royal Society Industry
Fellowship scheme (2012/R1) and the EPSRC for funding
(EP/J003220/1). JSF also acknowledges the importance of the
training received through a residential Royal Society of
Chemistry Drug Discovery training workshop. Dr. Chi Tsang
and Dr. Peter Ashton are thanked for helpful discussions
about mass spectrometry. Dr. Cécile S. Le Duff and Dr. Neil
Spencer gave advice on aspects of NMR spectroscopy. The
laboratory of TS is grateful for the support of Bloodwise
window of already acceptable medicinal chemistry parame-
3
ters, such as pK , cLog D, cLog P, TPSA, Fsp and molecular
a
weight. The DMPK screening cascade described identified
compound 5f as the most promising from activity, safety pro-
file and ADME property stand-points. Direct in vitro compari-
son showed 5f exhibited comparable activity to DMAPT
against the MEC1 cell line and pre-treatment with NAC
suggested a pro-oxidant mechanism of action. Molecular
docking studies with components of the NF-κB pathway also
support a pro-drug mode of action of the compounds involv-
ing release of 1 and covalent interaction with one or more
proteins involved in that pathway. This is partially supported
by the lack of activity of the thioether adduct, compound 3,
that is far less prone to undergo a reverse reaction to reveal
(
14031). AGL thanks Liverpool John Moores University for
support. Dr. Sam Butterworth (University of Manchester, for-
merly of University of Birmingham), Dr. David Fox (Vulpine
Science and Learning) and Dr. John Liddle (GSK) are
thanked for advice, mentorship and training through agree-
ments with the University of Birmingham and internal advi-
sory panels. Past and present members of the research
group headed by JSF are thanked where appropriate for ef-
forts in harvesting feverfew and extraction of PTL.
1, although lack of activity cannot be ascribed to this phe-
nomenon alone. Taken together, these findings confirm 5f as
a promising motif for further study and elaboration towards
treatments for drug resistant CLL.
References
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