P3 SAR exploration of biphenyl carbamate based Legumain inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.04.002

This Letter describes the further development and SAR exploration of a novel series of Legumain inhibitors. Based upon a previously identified Legumain inhibitor from our group, we explored the SAR of the carbamate phenyl ring system to probe the P3 pocke

Full text of DOI:10.1016/j.bmcl.2014.04.002

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2521–2524
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
P3 SAR exploration of biphenyl carbamate based Legumain inhibitors
Catherine Higgins a, Samira Bouazzaoui a, Kishore Gaddale a, Zenobia D’Costa a, Amy Templeman a,
Martin O’Rourke b, Andrew Young c, Christopher Scott a,c, Tim Harrison a,b, Paul 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 further development and SAR exploration of a novel series of Legumain inhib-
itors. Based upon a previously identified Legumain inhibitor from our group, we explored the SAR of
the carbamate phenyl ring system to probe the P3 pocket of the enzyme. This led to the identification
of a sub-nanomolar inhibitor of Legumain.
Received 30 January 2014
Revised 31 March 2014
Accepted 1 April 2014
Available online 12 April 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Legumain
Cancer
Cyano warhead
Protease inhibitor
Legumain, or asparaginyl endopeptidase 1 (AEP1), was first
identified as a cysteine protease in leguminous seeds.1 Analysis
of the cleavage substrates of Legumain revealed that this protease
had a requirement for asparagine in the P1 position adjacent to the
cleaved C-terminus.1 Legumain was found to be reasonably well
conserved in mammalian species and is a member of the CD clan
of proteases, which includes caspases, separase and the gingi-
pains.2 Mammalian Legumain has shown to be expressed within
the lysosome and plays an important role in MHC class II-mediated
antigen presentation.3,4 It has also been shown to play a role in reg-
ulating the activity of the papain family cathepsins, specifically B, L
and H.5 Legumain has also been reported to be involved in the con-
version of pro-MMP2 into the active zymogen, suggesting that
Legumain has a role in cancer.6
Recently a number of publications have reported that Legumain
expression can be used as a marker for poor prognosis in a number
of human cancers, including breast, ovarian, glioma, pancreatic and
prostate.7–11 In addition, the localisation of Legumain expression
observed via full face tumour staining has been shown to be a pow-
erful predictor of patient survival outcomes.11 Patients that display
high vesicular Legumain staining are predicted to have lower over-
all survival rates in prostate cancer.11
The cancer specific expression of Legumain has led to the devel-
opment of a number of Legumain targeted nanoparticle delivery
systems.12,13 These nanoparticles have been designed to utilise
an inhibitory sequence developed in the Powers lab (Z-Ala-Ala-
aza-Asn-X) coupled to dioleoyl-L-a-phosphatidylethanolamine
(DOPE). In vivo studies conducted using these nanoparticles have
revealed that cytotoxic agents (e.g. doxorubicin) can be delivered
to tumour sites with no observed toxicity.12 Legumain knockout
animals exhibits impaired liver and kidney function in an age
dependent manner.14 However, several studies with small mole-
cule irreversible inhibitors dosed IV have shown no impact upon
liver and kidney Legumain activity.15
We recently identified compound 1 as a novel Legumain inhib-
itor through an internal hit development program (Fig. 1). The use
of the cyano warhead in 1 was a key part of our inhibitor design in
place of the more reactive irreversible covalent warheads previ-
ously employed.15–17 As the SAR appeared to be somewhat shallow
in the P2 pocket with only small lipophilic amino acids being pre-
ferred (Fig. 1). Our attention became focused on exploring the SAR
of the Cbz phenyl ring to further explore the P3 pocket. Early mod-
eling studies conducted in house revealed that the carbamate phe-
nyl ring was pointing towards a lipophilic P3 caspase like pocket
without any real interaction taking place. To generate a ‘fit’ for this
potential interaction it was proposed that incorporation of a biphe-
nyl group would be able to better align with this pocket. To ascer-
tain whether this result would yield improvements in inhibitory
potency against Legumain an SAR program was undertaken.
⇑
Corresponding author. Tel.: +44 0 2890972791.
E-mail address: rich.williams@qub.ac.uk (R. Williams).
Main author and project leader.
http://dx.doi.org/10.1016/j.bmcl.2014.04.002
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

2522
C. Higgins et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2521–2524
Table 1
Potency values of the bromo and bi-phenyl carbamates
NH2
O
O
O
O
N
N
H
Br
N
NH2
O
NH2
O
1
O
O
O
O
O
O
IC50 = 140 nM
N
N
N
H
N
H
N
N
P3
Benzyl optimal
Benzyl>Ethyl>Boc
5a-c
6a-c
P1
Asparaginyl
optimal
NH2
N-Me-Asp
Aspartic,
Glutamate,
Glutamic were
inactive
O
H
O
N
a
O
O
N
N
Compound
Position
Legumain IC50
(l
M)
H
R
5a
5b
5c
6a
6b
6c
2-Br
3-Br
4-Br
2-Ph
3-Ph
4-Ph
0.192 ( 0.080)
0.108 ( 0.058)
0.282 ( 0.102)
—
0.138 ( 0.079)
0.018 ( 0.022)
P2
Proline=Ala
>Azetidine>>Ser>Phe>Leucine
>Isoleucine
a
Mean IC50 value performed in triplicate against recombinant human Legumain
using Z-Ala-Ala-Asn-AMC fluorogenic substrate.
Figure 1. Previously identified Legumain inhibitor and SAR.
analogues (6b and c) revealed a significant preference for the
para-biphenyl compound, 6c, which had an IC50 of 18 nM in the
assay. Based on this result a more expansive screen of the distal
phenyl ring SAR was conducted. A library of compounds (7a–x)
was generated via Suzuki coupling using the para-bromophenyl
compound 5c and a small library of mono and di-substituted phen-
ylboronic acids (Scheme 2).
The results from this scan revealed that a number of functional-
ities were well tolerated (7a–x, Table 2). Both the meta- and
para-tolyl analogues (7a and b) displaying comparable activity.
Increasing the size of the alkyl substituent from methyl to ethyl
in the para position reveals a 6 fold drop in activity.
The methyl ester of proline was converted to the bromophenyl
carbamates (3a–c) using non-symmetrical carbonate with
a
p-nitrophenylchloroformate (Scheme 1). The ester was cleaved
under basic conditions and the corresponding acid was coupled
to H-Asp(OBzl)–CONH2. The dipeptides were subsequently
dehydrated with cyanuric chloride to install the cyano warhead
(4a–c). Mild basic cleavage conditions were employed to remove
the benzyl ester protecting group subsequent amide coupling with
NH4Cl to afford the desired products (5a–c) in good yield. Each of
these bromo compounds were converted to the biphenyl analogues
using modified Suzuki conditions (Scheme 1).18
This loss in activity is also observed with other larger substitu-
ents (e.g. the tert-butyl compound (7g) hinting at limitations in the
P3 pocket. Moving to the meta position of the distal phenyl, both
the i-propyl and t-butyl analogues have comparable activity to
Screening of the bromophenyl carbamate compounds (5a–c,
Table 1) in a fluorogenic assay employing recombinant Legumain
revealed that all three analogues were active with comparable
activity (Table 1). Screening of the corresponding biphenyl
Br
Br
O
O
O
H
N
a
O
O
b,c,d
O
O
N
O
O
O
N
N
N
H
O
3a-c
2
4a-c
Br
NH2
NH2
O
O
O
e,f
g
O
O
N
O
O
O
N
N
N
N
H
N
H
5a-c
6a-c
Scheme 1. Reagents and conditions. (a) (i) Bromobenzyl alcohol, p-nitrophenylchloroformate, DCM, 18 h; (ii) proline methyl ester, NMM, THF, MeCN, reflux, 3 h; (b) LiOH
(10 equiv), H2O, MeOH, THF (1:1:9), (c) H-Asp(OBzl)–CONH2, HBTU, NMM, DMF, 18 h (86–95%); (d) cyanuric chloride, DMF, 3 h (85–97%); (e) LiOH (4 equiv), H2O, THF (1:9);
(f) NH4Cl, HBTU, NMM, DMF, 18 h, (g) Ar-B(OH)2, 2 M Na2CO3, H2O, DMF, 60 °C.

C. Higgins et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2521–2524
2523
X
Br
NH2
NH2
O
O
O
O
O
a
O
O
O
N
N
N
N
H
N
H
N
7a-x
Scheme 2. Reagents and conditions. (a) Ar-B(OH)2, 2 M Na2CO3, H2O, DMF, 60 °C.
5c
and i-propoxy (7v) displaying low nanomolar IC50’s. The 3-ethoxy
analogue was the most active compound identified from this
screen displaying sub-nanomolar activity (IC50 = 0.9 nM). This
potency was comparable to previously reported irreversible inhib-
itors.16–18 Further studies have been conducted around this substi-
tution pattern and it was found that moving beyond i-propoxy in
terms of increasing the steric size of the alkyl chain led to a drop
off in activity.
Introduction of two substituents on the distal phenyl ring was
found to be well tolerated. Both the 2,4-, 2,3-, and 3,4-difluoro sub-
stitution patterns (7k, 7l and 7l) were highly potent. The one out-
lier here was the 3,5-difluoro anagolue 7j which 9–10 fold less
active than 7l. Use of the mono-halogen substituents gave some
intriguing results. Both the meta- and para-fluoro analogues dis-
played good activity with compound 7i being most active. The
Chloro analogues (7n and 7o) gave a switch in the preference to
the meta position. Interestingly, incorporation of a pyridyl nitrogen
into the distal ring system reversed this trend in activity (7n vs 7w;
and 7o vs 7x). This result was somewhat surprising given the pre-
vious results and hints at different modes of binding. We are cur-
rently exploring this result with additional analogues.
A representative cross-section of the more active compounds
where then selected for selectivity screening against a panel of
other cysteine proteases (Table 3). It was observed that the com-
pounds showed good selectivity, which is in line with published
data. Bogyo and co-workers had previously reported that their
inhibitors were highly selective across a panel of proteases.17 The
key driver of this selectivity was the absolute requirement for
the asparaginyl side chain at P1. To date any manipulation of the
asparaginyl side chain in our series of inhibitors has led to a com-
plete loss of activity against Legumain.
Table 2
Potency values of bi-phenylcarbamtes
X
NH2
O
O
O
O
N
N
H
N
7a-v
a
Compound
X
Legimain IC50 (lM)
6a
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
H
0.018 ( 0.022)
0.049 ( 0.037)
0.028 ( 0.018)
0.169 ( 0.077)
0.049 ( 0.033)
0.097 ( 0.037)
0.083 ( 0.026)
0.348 ( 0.118)
0.060 ( 0.045)
0.005 ( 0.0008)
0.079 ( 0.028)
0.029 ( 0.006)
0.011 ( 0.005)
0.041 ( 0.011)
0.009 ( 0.009)
0.124 ( 0.029)
1.640 ( 0.385)
0.065 ( 0.021)
0.015 ( 0.014)
0.041 ( 0.012)
3-Me
4-Me
4-Ethyl
3-i-Pr
4-i-Pr
3-t-Bu
4-t-Bu
3-F
4-F
3,5-DiF
2,3-DiF
3,4-DiF
2,4-DiF
3-Cl
4-Cl
2-CF3
3-CF3
4-CF3
4-OMe
3-OMe
3-OEt
3-OiPr
7s
7t
7u
7v
7w
7x
0.0032 ( 0.0002)
0.0009 ( 0.001)
0.0078 ( 0.002)
0.343 ( 0.097)
0.009 ( 0.003)
A number of breast cancer cells, in our hands, have shown to
have an addiction to Legumain activity via siRNA studies. Treat-
ment of both MCF7 and T47D with all inhibitors displaying sub-
100 nM IC50 values. This study revealed that compounds 7u and
5-Cl-3-Pyr
6-Cl-3-Pyr
a
Mean IC50 determinations performed in triplicate against recombinant human
Legumain using Z-Ala-Ala-Asn-AMC fluorogenic substrate.
7q, at 30 lM drug concentrations, gave >70% reduction in cell
count after 120 h exposure versus vehicle control. Similar studies
against normal cell lines, MCF10a and HME-1, at drug concentra-
meta methyl compound 7a. This result suggests that there is more
depth to the P3 pocket for these substituents. Modelling studies are
currently ongoing to further explore this finding. In the ether series
the meta-methoxy analogue displayed a 12 fold improvement in
potency over the para analogue (7t vs 7s). Increasing steric bulk
at this position was also well tolerated with both the ethoxy (7u)
tions of 30 lM, had no impact upon cell viability. This result con-
firms observations made with Legumain siRNA and endogeneous
inhibitor (CST6) treatment.
In conclusion, we have identified a highly potent Legumain
inhibitor, 7u, that display sub-nanomolar activity. In addition,
counter screening against a panel of cysteine proteases, including
Table 3
Selectivity screening against a panel of Cystiene proteases
CatS%inh@ 30
lM
CatB%inh@ 30
lM
CatK%inh@ 30
lM
CatL%inh@ 30
lM
Casp3%inh@ 30
lM
Casp7%inh@ 30 lM
7u
7d
7l
0
0
0
0
16
6
10
8
22
22
32
20
6
8
5
6
35
40
12
20
13
20
18
19
7q
All IC50 values were an average from three or more experiments.

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C. Higgins et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2521–2524
5. Shirahama-Noda, K.; Yamamoto, A.; Sugihara, K.; Hashimoto, N.; Asano, M.;
Nishimura, M.; Hara-Nishimura, I. J. Biol. Chem. 2003, 278, 33194.
6. Chen, J. M.; Fortunato, M.; Stevens, R. A.; Barrett, A. J. Biol. Chem. 2001, 382, 777.
7. Zhang, J.; Shridhar, R.; Dai, Q.; Song, J.; Barlow, S. C.; Yin, L.; Sloane, B. F.; Miller,
F. R.; Meschonant, C.; Li, B. D. L.; Abreo, F.; Keppler, D. Cancer Res. 2004, 64,
6957.
8. Wang, L.; Chen, S.; Zhang, M.; Li, N.; Chen, Y.; Su, W.; Liu, Y.; Lu, D.; Li, S.; Yang,
Y.; Li, Z.; Stupack, D.; Qu, P.; Hu, H.; Xiang, R. J. Cell. Biochem. 2012, 113, 2679.
9. Qiu, J.; Ai, L.; Ramachandran, C.; Yao, B.; Gopalakrishnan, S.; Fields, C. R.;
Delmas, A. L.; Dyer, L. M.; Melnick, S. J.; Yachnis, A. T.; Schwartz, P. H.; Fine, H.
A.; Brown, K. D.; Robertson, K. D. Lab. Invest. 2008, 88, 910.
10. Loukopoulos, P.; Shibata, T.; Katoh, H.; Kokubu, A.; Sakamoto, M.; Yamazaki, K.;
Kosuge, T.; Kanai, Y.; Hosoda, F.; Imoto, I.; Ohki, M.; Inazawa, J.; Hirohashi, S.
Cancer Sci. 2007, 98, 392.
11. Ohno, Y.; Nakashima, J.; Izumi, M.; Ohori, M.; Hashimoto, T.; Tachibana, M.
World J. Urol. 2013.
the closely homologous caspase 3, has revealed these inhibitors are
highly selective for Legumain. Further studies are currently under-
way to develop a suitable in vivo tool compound from this series to
explore the role of Legumain in a number of preclinical cancer
models.
Acknowledgments
We would like to acknowledge Northern Ireland Department of
Education and Learning (DEL), Invest Northern Ireland and the
Heather Clarke Breast Cancer Foundation in their support of this
research.
12. Liao, D.; Liu, Z.; Wrasidlo, W.; Chen, T.; Luo, Y.; Xiang, R.; Reisfeld, R. A.
Nanomed.: Nanotechnol., Biol., Med. 2007, 7, 665.
13. Liao, D.; Liu, Z.; Wrasidlo, W. J.; Luo, Y.; Nguyen, G.; Chen, T.; Xiang, R.; Reisfeld,
R. A. Cancer Res. 2011, 71, 5688.
Supplementary data
14. Chan, C.-B.; Abe, M.; Hashimoto, N.; Hao, C.; Williams, I. R.; Liu, X.; Nakao, S.;
Yamamoto, A.; Zheng, C.; Henter, J.-I.; Meeths, M.; Nordenskjold, M.; Li, S.-Y.;
Hara-Nishimura, I.; Asano, M.; Ye, K. PNAS 2009, 106, 468.
15. (a) Liu, C. WO 2007/064759; (b) Liu, C. U.S. Army Medical Research and
Material Command Final report, Grant award number W81XWH-05-1-0318,
2008.
16. Gotz, M. G.; James, K. E.; Hansell, E.; Dvorak, J.; Seshaadri, A.; Sojka, D.;
Kapacek, P.; McKerrow, J. H.; Caffrey, C. R.; Powers, J. C. J. Med. Chem. 2008, 51,
2816.
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
04.002.
References and notes
1. Kembhavi, A. A.; Buttle, D. J.; Knight, C. G.; Barrett, A. J. Arch. Biochem. Biophys.
1993, 303, 208.
17. Lee, J.; Bogyo, M. ACS Chem. Biol. 2010, 19, 233.
18. Gauthier, J. Y.; Chauret, N.; Cromlish, W.; Desmarais, S.; Duong, L. T.;
Falgueyret, J.-P.; Kimmel, D. B.; Lamontagne, S.; Leger, S.; LeRiche, T.; Sing Li,
C. S.; Masse, F.; Mckay, D. J.; Nicoll-Griffith, D. A.; Oballa, R. M.; Palmer, J. T.;
Percival, M. D.; Riendeau, D.; Robichaud, J.; Rodan, G. A.; Rodan, S. B.; Seto, C.;
Therien, M.; Truong, V.-L.; Venuti, M. C.; Wesolowski, G.; Young, R. N.;
Zamboni, R.; Black, W. C. BMCL 2008, 18, 923.
2. Chen, J. M.; Rawlings, N. D.; Stevens, R. A.; Barrett, A. J. FEBS Lett. 1998, 441, 361.
3. Manoury, B.; Mazzeo, D.; Li, D. N.; Billson, J.; Loak, K.; Benaroch, P.; Watts, C.
Immunity 2003, 18, 489.
4. Maehr, R.; Hang, H. C.; Mintern, J. D.; Kim, Y. M.; Cuvillier, A.; Nishimura, M.;
Yamada, K.; Shirahama-Noda, K.; Hara-Nishimura, I.; Ploegh, H. L. J. Immunol.
2005, 174, 7066.