Development of a potent and selective cell penetrant Legumain inhibitor

Article abstract of DOI:10.1016/j.bmcl.2015.10.001

This Letter describes the continued SAR exploration of small molecule Legumain inhibitors with the aim of developing a potent and selective in vitro tool compound. Work continued in this Letter explores the use of alternative P2-P3 linker units and the P3 group SAR which led to the identification of 10t, a potent, selective and cellularly active Legumain inhibitor. We also demonstrate that 10t has activity in both cancer cell viability and colony formation assays.

Full text of DOI:10.1016/j.bmcl.2015.10.001

Bioorganic & Medicinal Chemistry Letters 25 (2015) 5642–5645
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of a potent and selective cell penetrant Legumain
inhibitor
Kerry A. Ness a,y, Sharon L. Eddie a,y, Catherine A. Higgins a, Amy Templeman a, Zenobia D’Costa a,
Kishore K. D. Gaddale a, Samira Bouzzaoui a, Linda Jordan b, Dominic Janssen b, Timothy Harrison b,
Frank Burkamp b, Andrew Young c, Roberta Burden c, Christopher J. Scott c, Paul B. Mullan a, Rich Williams a,
⇑
a CCRCB Drug Discovery Group, Centre for Cancer Research and Cell Biology, Queen’s University Belfast, 97 Lisburn Rd., Belfast BT9 7BL, UK
b Almac Discovery, Almac House, 20 Seagoe Industrial Estate, Craigavon BT63 5QD, UK
c School of Pharmacy, Queen’s University Belfast, 97 Lisburn Rd., Belfast BT9 7BL, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
This Letter describes the continued SAR exploration of small molecule Legumain inhibitors with the aim
of developing a potent and selective in vitro tool compound. Work continued in this Letter explores the
use of alternative P2–P3 linker units and the P3 group SAR which led to the identification of 10t, a potent,
selective and cellularly active Legumain inhibitor. We also demonstrate that 10t has activity in both can-
cer cell viability and colony formation assays.
Received 10 September 2015
Revised 30 September 2015
Accepted 1 October 2015
Available online 9 October 2015
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
Legumain
Cancer
Cyano warhead
Cellular active inhibitor
Legumain, or Asparaginyl endopeptidase (AEP), is a member of
the CD clan of proteases, which includes caspases, separase and the
gingipains.1,2 Mammalian legumain is expressed within the lyso-
some and plays an important role in MHC class II-mediated antigen
presentation and regulation of the activity of the papain family
cathepsins, specifically B, L and H.3–5 In addition, legumain has also
been reported to play a major role in the conversion and activation
of pro-MMP2 into the active enzyme.6
Recently, several publications have highlighted the use of legu-
main as a marker of prognosis in many human cancers including
breast, ovarian, glioma, pancreatic and prostate.7–11 These studies
demonstrate that within the cancer setting higher levels of legu-
main expression directly correlate with poor patient outcome
and aggressive disease.7–11 Furthermore, localisation of active
legumain determined by vesicular staining is correlated with lower
survival and more aggressive disease in prostate cancer patients.11
A transgenic murine model of prostate cancer also revealed an
association of increased legumain expression with disease
progression.12
Our group identified the transcription factor TBX2 to be a
potent regulator of legumain activity in breast cancer, via suppres-
sion of CST6, the endogenous legumain inhibitor.13 CST6 loss leads
to increased cancer cell proliferation and invasion in breast cancer
cells.13 Re-expression of CST6 in prostate cancer cells significantly
impairs tumour growth and metastasis when xenografted.14 In
addition, suppression of TBX2 or legumain, and re-introduction
of CST6, were performed and found to have a potent cytotoxic
effect against a panel of breast cancer cells but not the correspond-
ing normal cell lines13 demonstrating the dependence of tumori-
geneic cells on legumain signalling.
We recently identified compound 1 as a novel legumain inhibi-
tor (Fig. 1).15 Whilst use of the cyano warhead in our initial inhibi-
tor design was beneficial further development was required to
achieve an optimal in vivo tool compound. As the SAR appeared
to be shallow in both the P1 and P2 we focused on exploring the
SAR of the P2–P3 linker group. Holding both P1 and P2 constant
based upon compound 1 the benzamide 2 and benzylamine 3 were
both found to be poor replacements for the carbamate group in
terms of potency (Fig. 1). The use of the sulphonamide group (4)
however afforded a sub-lM inhibitor. The identification of a potent
alternative to the carbamate linker warranted further exploration.
Proline 5 was converted to the methyl ester 6 with thionyl chlo-
ride in methanol under refluxing conditions (Scheme 1). The
resulting amino methyl ester was converted to the corresponding
⇑
Corresponding author.
E-mail address: rich.williams@qub.ac.uk (R. Williams).
Joint first authors who contributed equally to the research conducted in this
y
Letter.
http://dx.doi.org/10.1016/j.bmcl.2015.10.001
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

K. A. Ness et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5642–5645
5643
Table 1
Potency values of progenitor compound 4 and analogues
NH2
O
F
NH2
O
O
a
Cell Inh @30 l
Mb (%)
O
Compound
X group
Legumain IC50
(lM)
O
O
O
O
4
4-F
2,4-DiCl
4-Cl
4-OMe
4-Br
3,4-DiCl
4-CF3
4-tBu
4-Ph
0.269
0.593
0.073
>10
0.282
>10
0.332
>10
0.012
0
0
0
ND
0
ND
0
ND
47
N
N
H
N
9a
9b
9c
9d
9e
9f
N
H
N
N
1
2
IC50 = 0.9 nM
IC50 = 3.51µM
9g
9h
NH2
O
NH2
O
F
F
O
S
N
O
O
O
a
IC50 performed in triplicate against recombinant human Legumain via an 8
point dose response curve. Z-Ala-Ala-Asn-AMC fluorogenic substrate (R&D Sys-
tems) was used.
N
N
N
H
N
N
H
b
Compound was incubated with MCF7 cells @37 °C for 24 h, assay media
3
4
removed, cells washed with assay buffer and lysed. Inhibition was determined with
IC50 = >30 µM
IC50 = 269 nM
fluorogenic substrate and carried out in triplicate.
Figure 1. Preceding Legumain inhibitor and P2–P3 linker scan results.
To explore the SAR of the bi-aryl sulfonamides the 4-bro-
mophenylsulphonamide analogue (9d) was used as a chemical
handle for a series of Suzuki cross coupling reactions to afford
10a–t (Scheme 2 and Table 2).16
The SAR of the distal phenyl ring (10a–t) was robust when com-
pared to that of the mono-phenyl sulfonamides (9a–h) with a
number a functionalities and substitution patterns displaying low
nanomolar activity (Table 2). All active compounds were tested
to determine the intracellular activity at a single drug concentra-
sulphonamide (7a–h) in good yield. The ester was cleaved under
standard conditions and the corresponding acid was coupled to
H-Asp(OBzl)-CONH2. The dipeptides (8a–h) were subsequently
dehydrated with cyanuric chloride to install the cyano warhead
followed by cleavage of the benzyl ester and coupling with ammo-
nium chloride to afford desired analogues (9a–h) in moderate to
good yield.
Based upon results from compounds that were synthesized for
screening the SAR appeared limited. Increasing the size of the halo-
gen from fluoro (4) to chloro (9b) had a positive effect upon the
potency with a 3.7-fold increase observed (Table 1). This increase
in potency was lost when moving to the para-bromo analogue
(9d). Incorporation of di-halo substitution patterns was poorly tol-
erated with a 8-fold loss in activity observed with the 2,4-DiCl ana-
logue (9a) and the 3,4-DiCl compound (9e) inactive. Additional
functionalities were attempted, however these led to significant
drop in potency with the exception of the para-phenyl analogue
9h. This biphenyl compound, 9h, was highly potent against recom-
binant enzyme and out of this collection of compounds it was the
only analogue to display intracellular activity. After an incubation
period of 2 h compound 9h significantly suppressed intracellular
legumain activity by ꢀ50% (Table 1). Additional time points
revealed that this activity was lost after 24 h with no effect upon
cancer cell.
tion of 30 lM in MCF7 cells 24 h post treatment. With a few excep-
tions (10f, 10p and 10q) all of the analogues displayed varying
levels of intracellular legumain inhibition (Table 2).
Compounds that displayed P70% intracellular legumain inhibi-
tion were assayed for effects on cellular viability against a panel of
breast (MCF7 and T47D) and prostate cancer cells (PC3 and
DU145). Each of the cell lines were treated with 10 and 30
each compound for 5 days. From this study only one compound,
10t, reduced cell survival with >50% cell viability at 30 M across
lM of
l
all cell lines (Fig. 2a). To further understand this finding we
assessed the ability of each of the selected compounds to retain
intracellular activity across the timeline of the cellular experiment
(5 days). This study revealed that only 10t inhibited legumain
activity post 48 h drug treatment suggesting that this compound
has a slower off rate than the other analogues tested.
Legumain has been reported to play a significant number of
roles in cancer biology, including cellular invasion and formation
X
O
O
O
O
S
O
H
N
H
N
b
a
N
O
O
OH
•HCl
5
6
7a-h
X
O
X
NH2
O
O
S
N
O
S
N
O
O
O
O
NH2
c,d
O
e,f,g
N
H
N
H
N
O
8a-h
9a-h
Scheme 1. Reagents and conditions; (a) thionyl chloride (2 equiv), MeOH, reflux, 2 h, 99%; (b) X-benzenesulphonyl chloride, NMM, DCM, 4 h, 72–98%; (c) LiOH (6 equiv),
H2O/MeOH/THF (1:1:9), 1–3 h; (d) H-Asp(OBzl)-CONH2, HBTU, NMM, DMF, 18 h (86–95%); (e) cyanuric chloride, DMF, 3 h (85–97%); (f) LiOH (4 equiv), H2O/THF (1:9), 1–2 h;
(g) NH4Cl, HBTU, NMM, DMF, 18 h (32–67%).

5644
K. A. Ness et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5642–5645
X
Br
NH2
O
NH2
a
O
O
O
O
O
N
S
N
S
O
O
N
N
H
N
H
N
10a-t
9d
Scheme 2. Reagents and conditions. (a) Ar-B(OH)2, 2 M Na2CO3, H2O, DMF, 60 °C.
of metastasis.14,17 To determine whether 10t, could impact meta-
static ability of cancer cells, a soft agar anchorage-independent
growth assay was performed utilising both metastatic breast
(MCF7) and prostate (PC3) cancer cell lines (Fig. 2b). 10t could
block colony formation in both breast and prostate cancer cell
lines, with ꢀ50% blockade being observed for 10t at
5 lM
(MCF7) and 2.5
l
M (PC3) drug concentrations.18
In conclusion, we have developed a novel, potent and cell active
legumain inhibitor (10t) that displays excellent selectivity (Supple-
mentary data Table 1). Whilst we have identified a potent inhibitor
against the recombinant enzyme intracellular potency is still not
ideal. With legumain being expressed on the cell surface of cancer
cells, as well as intracellularly, it is difficult to attribute this low
potency level to a lack of cell penetration alone. In addition, the
on and off rate kinetics of these compounds have not been
explored and it could be argued that compounds with fast off rate
kinetics will not perform as well in these cell based assays. How-
ever, we have demonstrated that 10t is a highly potent inhibitor
of intracellular legumain which reduced cancer cell survival and
blocked anchorage-independent growth, an early hallmark of
tumorigeneic potential. However due to a lack of metabolic stabil-
ity (data not reported) these compounds are considered sub-opti-
mal for further study in vivo. Further development is ongoing
with scope for use in ex vivo and in vivo models.
Figure 2. (A) Cell viability data derived from MTT assay (n = 6) after 96 h incubation
with 10t or vehicle control (DMSO). Both breast (MCF7 and T47D) and prostate
cancer (PC3 and DU145) cell viability were significantly reduced (B) Soft agar
anchorage-independent colony formation assay (n = 5) in both MCF7 and PC3 cells
post 2 week incubation with 10t. Compounds and media were refreshed every
4 days and quantified compared to vehicle control (DMSO) treated cells. Signifi-
cance determined by t-test. *p <005, **p <0.01 ***p 60.001. Graphs depict
mean SEM.
Table 2
Acknowledgments
Potency and cellular activity of 9 and 10a–t
Cell Inh @30 l
Mb (%)
a
Compound
X group
Legumain IC50
(
lM)
We thank the Heather Clarke Memorial Breast Cancer fund,
Prostate Cancer UK (PG13-021) and Invest Northern Ireland
(RD0212980) for their generous funding. Dr Rich Williams would
also like to express his gratitude and thanks to a true mentor
whom will be sorely missed—Professor Roger Griffin. Without his
support and friendship my transition into academic life in the UK
would not have been successful.
9h
H
3-F
4-F
2-Cl
0.012
0.047
0.033
0.035
0.045
0.067
0.023
0.006
0.169
0.093
0.121
0.006
0.073
0.969
>10
47
42
40
29
64
90
0
76
90
29
52
90
90
49
—
10a
10b
10c
10d
10e
10f
10g
10h
10i
4-Cl
2,4-DiF
3,4-DiF
2,4-DiCl
3,4-DiCl
2-CF3
4-CF3
4-OMe
4-OEt
4-OiPr
4-OCF3
3,4-Dioxolane
4-CN
Supplementary data
10j
10k
10l
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.10.
001.
10m
10n
10o
10p
10q
10r
10s
10t
0.049
0.033
0.032
0.071
0.005
0.071
49
0
0
42
75
90
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