Structure-based design and in vivo anti-arthritic activity evaluation of a potent dipeptidyl cyclopropyl nitrile inhibitor of cathepsin C

Article abstract of DOI:10.1016/j.bcp.2019.04.006

Cathepsin C (CatC)is a dipeptidyl-exopeptidase which activates neutrophil serine protease precursors (elastase, proteinase 3, cathepsin G and NSP4)by removing their N-terminal propeptide in bone marrow cells at the promyelocytic stage of neutrophil differ

Full text of DOI:10.1016/j.bcp.2019.04.006

Accepted Manuscript
Structure-based design and in vivo anti-arthritic activity evaluation of a potent
dipeptidyl cyclopropyl nitrile inhibitor of cathepsin C
Brice Korkmaz, Adam Lesner, Magdalena Wysocka, Artur Gieldon, Maria
Håkansson, Francis Gauthier, Derek T. Logan, Dieter Jenne, Conni Lauritzen,
John Pedersen
PII:
S0006-2952(19)30141-8
https://doi.org/10.1016/j.bcp.2019.04.006
BCP 13489
DOI:
Reference:
To appear in:
Biochemical Pharmacology
Received Date:
Accepted Date:
22 February 2019
7 April 2019
Please cite this article as: B. Korkmaz, A. Lesner, M. Wysocka, A. Gieldon, M. Håkansson, F. Gauthier, D.T. Logan,
D. Jenne, C. Lauritzen, J. Pedersen, Structure-based design and in vivo anti-arthritic activity evaluation of a potent
dipeptidyl cyclopropyl nitrile inhibitor of cathepsin C, Biochemical Pharmacology (2019), doi: https://doi.org/
1
0.1016/j.bcp.2019.04.006
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Structure-based design and in vivo anti-arthritic activity evaluation of a potent
dipeptidyl cyclopropyl nitrile inhibitor of cathepsin C
1
,2
3
3
3
Brice Korkmaz , Adam Lesner , Magdalena Wysocka , Artur Gieldon , Maria
4
1,2
4
5,6
7
Håkansson , Francis Gauthier , Derek T. Logan , Dieter Jenne , Conni Lauritzen
7
and John Pedersen
1
INSERM, UMR 1100, “Centre d’Etude des Pathologies Respiratoires”, 37032 Tours, France;
2
3
Université de Tours, 37032 Tours, France; Faculty of Chemistry, University of Gdansk, 80-
4
5
3
08 Gdansk, Poland; SARomics Biostructures, 223 63 Lund,Sweden; Comprehensive
Pneumology Center, Institute of Lung Biology and Disease, German Center for Lung
6
Research (DZL), 81377 Munich, Germany; Max Planck Institute of Neurobiology, 82152
7
Planegg-Martinsried, Germany; Neuprozyme Therapeutics A/S, 2970 Hörsholm, Denmark.
*
Corresponding author: Brice Korkmaz (PhD)
INSERM U-1100 “Centre d’Etude des Pathologies Respiratoires (CEPR)”,
Université François Rabelais, Faculté de Médecine
1
0 Bld. Tonnellé, 37032, Tours, France
e-mail: brice.korkmaz@inserm.fr
Tel: 0033 2 47 36 63 86
Fax: 0033 2 47 36 60 46
1

Graphical abstract
2

Abstract
Cathepsin C (CatC) is a dipeptidyl-exopeptidase which activates neutrophil serine
protease precursors (elastase, proteinase 3, cathepsin G and NSP4) by removing their N-
terminal propeptide in bone marrow cells at the promyelocytic stage of neutrophil
differentiation. The resulting active proteases are implicated in chronic inflammatory and
autoimmune diseases. Hence, inhibition of CatC represents a therapeutic strategy to suppress
excessive protease activities in various neutrophil mediated diseases. We designed and
synthesized a series of dipeptidyl cyclopropyl nitrile compounds as putative CatC inhibitors.
One compound, IcatCXPZ-01 ((S)-2-amino-N-((1R,2R)-1-cyano-2-(4’-(4-methylpiperazin-1-
ylsulfonyl)biphenyl-4-yl)cyclopropyl)butanamide)) was identified as a potent inhibitor of
both human and rodent CatC. In mice, pharmacokinetic studies revealed that IcatCXPZ-01
accumulated in the bone marrow reaching levels suitable for CatC inhibition. Subcutaneous
administration of IcatCXPZ-01 in a monoclonal anti-collagen antibody induced mouse model of
rheumatoid arthritis resulted in statistically significant anti-arthritic activity with persistent
decrease in arthritis scores and paw thickness.
Keywords: cathepsin C, cysteine protease, neutrophil, serine protease, inhibitor.
Abbreviations: CAIA, collagen antibody-induced arthritis; Cat, cathepsin; CYP450, cytochromes
P450; DTT, dithiothreitol; FRET, fluorescence resonance energy transfer; hERG, human ether-a-go-
go-related gene; HPFC, high-performance flash chromatography; HPLC, high performance liquid
hromatography; HRMS, high resolution mass spectrometry; IC , half maximal inhibitory
5
0
concentration; LPS, lipopolysaccharide; mAb, monoclonal antibody; NE, neutrophil elastase; NSP,
neutrophil serine protease; PR3, proteinase 3; PLS, Papillon-Lefèvre syndrome; pNA, para-
nitroaniline; RT, room temperature.
3

1
. Introduction
Dipeptidyl peptidase I (EC 3.4.14.1), also called cathepsin C (CatC), is a papain
1
-3
family lysosomal cysteine peptidase. CatC removes dipeptides from the N-terminal end of
4
peptides and proteins with relatively large specificity. CatC is a key protease in the
2
processing and maturation of granule-associated serine proteases of immune cell lineages
including the lymphocyte granzymes, mast cell chymase and the neutrophil-associated serine
5
-6
7
proteases (neutrophil elastase (NE), proteinase 3 (PR3), cathepsin G (CatG) and NSP4 ).
All four neutrophil serine proteases (NSPs) are synthesized and activated during the
promyelocyte stage of neutrophil differentiation in the bone marrow and are stored mainly in
8
primary granules in the cytoplasm of neutrophils until they are released. The removal of N-
5
-6
terminal dipeptides by CatC converts zymogens into active proteases (Figure 1) following
9
conformation changes. Once activated, unopposed NSPs can cause tissue damage and
10-11
promote inflammation in pathological situations.
Genetic mutations in the CatC gene (gene symbol: CTSC) is responsible for Papillon-
1
2
13-14
Lefèvre syndrome (PLS, OMIM: 245000)
, a rare disease affecting 1-4 people per
1
5-16
million.
PLS is characterized by severe periodontitis and diffuse transgradient palmo-
1
7
plantar keratoderma. CatC deficiency results in almost complete elimination of NSPs in
6
-7, 18-20
blood neutrophils.
We recently obtained the same results in vitro and in vivo after a
18, 20
prolonged pharmacological inhibition of CatC.
Continuous pharmacological blockade of
CatC activity is supposed to lower the NSP load of circulating neutrophils in patients
suffering from chronic inflammatory diseases, including cystic fibrosis, bronchiectasis, alpha-
2
, 10,
1
antitrypsin deficiency and autoimmune diseases especially ANCA-associated vasculitis.
2
1
CatC inhibition may also help fighting rheumatoid arthritis as suggested by the observations
-/-
in CatC mice that are protected against acute arthritis in response to active collagen-type II
5
, 22
immunization.
4

CatC itself is synthesized as an inactive zymogen bearing a removable internal
2
3-24
propeptide.
Evidence has been provided that recombinant CatC zymogen in vitro can be
2
3
activated by CatL and CatS but not autocatalytically. Proteolytically active mature CatC,
2
5-27
characterized by its tetrameric structure, consists of four homo-subunits.
Each subunit
2
5, 27
contains an N-terminal fragment (the exclusion domain), a heavy chain and a light chain.
The two latter adopt the papain fold containing the active site. The exclusion domain, the
specific structural determinant of CatC, confers CatC its exopeptidase specificity by blocking
25
substrate access beyond the S2 subsite , according to the nomenclature of Schechter and
2
8
Berger and allowing the Asp1 residue (conserved in all known CatC sequences) to interact
2
7
with the free amino group of the substrate N-terminal residues excluding arginine or lysine.
The presence of a proline at P1 or P1’, or a P1 isoleucine also precludes substrate hydrolysis
4
29
by CatC. CatC that optimally functions at a slightly acidic pH is activated by chloride
3
0
anions in a pH dependent manner.
31-36
We and others have previously designed and developed CatC inhibitors.
Nitrile-
based compounds with three chemical classes of nitrile (cyanamides, aryl or heteroaryl nitriles
and amino- or amidoacetonitriles), differing in their electrophilic effects are the most
2, 32
investigated group of inhibitors of CatC.
Amidoacetonitrile-based inhibitors are dipeptides
with a nitrile warhead allowing covalent bonding with the cysteine residue (Cys234) of the
CatC active site. However, initially developed dipeptidyl nitrile compounds derived from
Abu-Bip-CN containing an Abu at P2 and biphenyl (Bip) at P1 (Figure 2A) are not stable in
3
1-33
plasma due to the rapid hydrolysis of their amide bond.
Stabilization of the peptide bond
can be obtained through the substitution of the N-terminal amino acid with an amino acid
3
2, 37
displaying a piperidine or cyclohexyl ring as side chain
(Figure 2B). Another strategy to
enhance the metabolic stability is the insertion of a 1,1-cyclopropyl amino nitrile moiety in
5

38-39
P1
(Figure 2C). However, the rigidification of the backbone by cyclopropyl strongly
3
3, 40
reduces the inhibitory potency of these inhibitors.
In this work, we have designed and synthesized a novel series of dipeptidyl
cyclopropyl nitrile compounds as putative CatC inhibitors with characteristics suitable for in
vivo use. Structure-activity relationships analysis of crystallized inhibitors in complex with
CatC and molecular docking trials were used to characterize the interaction of inhibitors with
CatC to optimize efficacy at very low-dose. IcatCXPZ-01, a potent and cell-permeable
compound ((S)-2-amino-N-((1R,2R)-1-cyano-2-(4’-(4-methylpiperazin-1-ylsulfonyl)biphenyl-
4
-yl)cyclopropyl)butanamide)) (Figure 2D), which displayed an excellent PK-profile leading
to high and sustained concentration in the bone marrow, was further evaluated in a mouse
model of rheumatoid arthritis induced by anti-collagen antibodies.
2
. Materials and methods
2
.1. Chemical synthesis of CatC inhibitors
CatC inhibitors were synthesized by PepTech Corporation (Burlington,
Massachusetts, USA). The purity of all synthetized compounds was determined by HPLC and
LCMS (PepTech Corporation (Burlington, Massachusetts, USA)). The purity of all tested
compounds was ≥95%. Calculated and mass spectrometry determined masses of compounds
1
-14 are shown in Table 1.
2
.1.1. Chemical synthesis of compound 8a (IcatCXPZ-01) and compound 8b
(
S)-2-amino-N-((1R,2R)-1-cyano-2-(4’-(4-methylpiperazin-1-ylsulfonyl)biphenyl-4-yl)cyclopro-
pyl)butanamide (compound 8a, IcatCXPZ-01) and (S)-2-amino-N-((1S,2S)-1-cyano-2-(4’-(4-
methylpiperazin-1-ylsulfonyl)biphenyl-4-yl)cyclopropyl)butanamide (compound 8b).
N’-(4-bromobenzylidene)-4-methylbenzenesulfonohydrazide (II).
6

To a solution of 4-bromobenzaldehyde (85.1 g, 0.46 mol) in MeOH (1 L) p-toluenesulfonyl
hydrazide (98,6 g, 0.53 mol) was added at room temperature (RT) (Figure 3). The reaction was stirred
for 30 min, then cooled to 0 °C. The precipitate was collected by filtration and washed with cooled
MeOH (50 mL × 2). The solid was dried in an oven to afford N’-(4-bromobenzylidene)-4-
methylbenzenesulfonohydrazide (II) (150.3 g, yield 92%). LC-MS: 354.1 (M+l).
(
1R,2R and 1S,2S)-methyl-2-(4-bromophenyl)-1-(tert-
butyloxycarbonylamino)cyclopropanecarboxylate (IIIa and IIIb).
A solution of N’-(4-bromobenzylidene)-4-methylbenzenesulfonohydrazide (56.4 g, 0.16 mol)
in THF (1 L) was kept at -78 °C. To the solution dropwise n-Butyllithium (64 mL, 0.16 mol) was
added. The reaction was stirred for 1 h at -78 °C, and the solvent was removed under vacuum. To the
residue was added a mixture of methyl 2-(tert-butyloxycarbonylamino)acrylate (I) (32.2 g, 0.16 mol),
benzyltriethylammonium chloride (0.91 g, 4 mmol), and catalyst ClFeTPP (0.56 g, 0.4 mmol) in
toluene (1 L). The reaction was stirred for 4 days at 50 °C under N protection. The solvent was
2
removed under vacuum and the residue was partitioned between dichloromethane (500 mL) and water
(
500 mL). The organic layer was collected, and the water layer was extracted with CH Cl (500 mL ×
2 2
2
). The organic phases were combined, dried over anhydrous Na SO , and concentrated. The crude
2 4
product was purified by HPFC (PE:EA=10:1) to afford a mixture of (1R,2R)- (IIIa) and (1S,2S)-
methyl-2-(4-bromophenyl)-1-(tert-butoxycarbonyl- amino)cyclopropanecarboxylate (IIIb) (29.1 g,
yield 49%). LC-MS: 371.2 (M+l).
1
-Methyl-4-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylsulfonyl]piperazine (2).
To a mixture of 1-(4-bromophenyl sulfonyl)-4-methylpiperazine (1) (20 g, 62.6 mmol),
pinacolatodiboron (11 g, 62.6 mmol), and potassium carbonate (17.8 g, 0.13 mol) in toluene Pd(PPh3)4
(
3 g) was added (1 L) under nitrogen protection. The mixture was heated under reflux overnight. After
cooling to RT, the solvent was removed under vacuum. To the residue were added water (500 mL) and
EA (500 mL). The organic phase was collected, and the water phase was extracted with EA (500 mL ×
7

2
). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated to
afford 2 (15.1 g, yield 65%). LC-MS: 367.2 (M+l).
1R,2R) and (1S,2S)-methyl 1-(tert-butyloxycarbonylamino)-2-[4’-(4-methyl-piperazin-1-yl-
(
sulfonyl)biphenyl-4-yl]cyclopropanecarboxylate (3a and 3b).
To a mixture of 2 (15 g, 40.9 mol), IIIa and IIIb (15.1 g, 40.9 mmol) and sodium carbonate
aqueous solution (8.67 g, 2 M) in 1,4-dioxane (1 L) PdCl (dppf) (2 g) was added under nitrogen
2
2
protection. The mixture was refluxed overnight. After cooling to RT, the solvent was removed under
vacuum. To the residue were added water (500 mL) and EA (500 mL). The organic phase was
collected, and the water phase was extracted with EA (500 mL × 2). The organic phases were
combined, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by
HPFC (AE /PE as eluent) to afford the 3a and 3b mixture (12.9 g, yield 60%). LC- MS: 530.2 (M +l).
(
1R,2R) and (1S,2S)-methyl-1-amino-2-[4’-(4-methylpiperazin-1-yl-sulfonyl)-biphenyl-4-yl-
cyclopropanecarboxylate (4).
To a solution of 3a and 3b (2.5 g, 4.7 mmol) in THF (30 mL) 100 mL of aqueous 6 N HCl
was added. The mixture was stirred for 4 h at 40 °C, then adjusted to pH 8 with sat. NaHCO . The
3
mixture was extracted with EA (200 mL × 3). The organic layers were separated, dried over anhydrous
sodium sulfate, and concentrated to afford 4 quantitatively.
(
1R,2R)-methyl-(S)-2-(tert-butyloxycarbonylamino)butanamido)-2-(4’-(4-methylpiperazin-1-
ylsulfonyl)blphenyl-4-yl)cyclopropanecarboxylate (5a) (1S,2S)-methyl-((S)-2-(tert-
butyloxycarbonylamino)butanamido)-2-(4’-(4-methyl-piperazin-1-ylsulfonyl)biphenyl-4-
yl)cyclopropanecarboxylate (5b).
To a solution of Boc-Abu-OH (2.35 g, 9.4 mmol) and NMM (1.77 g, 18.8 mmol) in CH Cl
2
2
(
50 mL) at 0 °C ethyl chloroformate (1.03 g, 9.4 mmol) was added. The reaction was stirred for 1 h at
0
°C, and a solution of 4 (2.03 g, 4.7 mmol) in CH Cl (10 mL) was added. The reaction was allowed
2 2
8

to warm to RT and stirred overnight. It was poured into 100 mL of water and extracted with
dichloromethane (100 mL). The CH Cl layer was separated, and the aqueous phase was extracted
2
2
with dichloromethane (100 mL × 2) and EA (100 mL × 2). The combined organic extract was dried
over anhydrous Na SO and concentrated under vacuum. The residue was purified by HPFC (ethyl
2
4
acetate petroleum ether as eluent) to yield a mixture of stereoisomers, which was separated by chiral-
HPLC (ChiralPak AD 20 µm, 5×50 cm column temperature 25 °C, detection wavelength 210 nm and
254 nm Flow Rate 70 mL/min Mobile Phase A:Hex 0.1% HOAc ,Mobile Phase B: IPrOH 0.1%
HOAc, analysis time 40 min) to afford 5a (t 6.9 min 700 mg) and 5b (t 14.1 min, 650 mg). LC-MS:
R
R
615.3 (M+l).
Tert-butyloxycarbonylamino-(S)-1-((1R,2R)-1-carbamoyl-2-(4’-(4-methylplperazin-1-yl-
sulfonyl)biphenyl-4-yl)cyclopropylamino)-1-oxobutan-2-ylcarbamate (6a) and Tert-
butyloxycarbonylamino-(S)-1-((1S,2S)-1-carbamoyl-2-(4’-(4-methylpiperazin-1-yl-sulfonyl)biphenyl-
4
-yl)cyclopropylamino)-1-oxobutan-2-ylcarbamate (6b).
To a solution of 5a (0.7 g, 1.1 mmol) in THF (20 mL) and water (20 mL) LiOH×H O (0.25 g,
2
5.5 mmol) was added The reaction was stirred for 3 h at RT. It was adjusted to pH 3 with aqueous 1 N
HCl and extracted with EA (100 mL × 3). The combined extract was dried over anhydrous
Na SO and concentrated under vacuum. The residue was dissolved in CH Cl (50 mL), and NMM
2
4
2
2
(
0.33 g, 3.3 mmol) was added. After cooling the solution to 0 °C, ethyl chloroformate (0.24 g, 2.2
mmol) was injected into the reaction mixture. The reaction was stirred for l h at 0 °C, and the mixture
was poured into a flask with 200 mL of ammonia. The reaction was stirred overnight at RT. The
mixture was extracted with CH Cl (200 mL × 3) and EA (200 mL × 3). The organic phases were
2
2
collected, dried over anhydrous Na SO , and concentrated to afford 6a (550 mg, 81%). LC-MS: 600.2
2
4
(
M+H).
To a solution of 5b (0.65 g, 1.0 mmol) in THF (20 mL) and water (20 mL) LiOH×H O (0.25
2
g, 5.5 mmol) was added. The reaction was stirred for 3 h at RT. It was adjusted to pH 3 with aqueous 1
N HCl and extracted with EA (100 mL × 3). The combined extract was dried over anhydrous
9

Na SO and concentrated under vacuum. The residue was dissolved in CH Cl (50 mL) and NMM
2
4
2
2
(
0.33 g, 3.3 mmol) was added. After cooling the solution to 0 °C, ethyl chloroformate (0.24 g, 2.2
mmol) was injected into the reaction mixture. The reaction was stirred for 1 h at 0 °C, and the mixture
was poured into a flask with 200 mL of ammonia. The reaction was stirred overnight at RT. The
mixture was extracted with CH Cl (200 mL × 3) and EA (200 mL × 3). The organic phases were
2
2
collected, dried over anhydrous Na SO , and concentrated to afford 6b (520 mg, 82%). LC-MS: 600.3
2
4
(
M+H).
To a solution of 6a (550 mg, 0.92 mmol) in DMF (10 mL) cyanuric chloride (0.50 g, 2.75
mmol) was added. The reaction was stirred for 4 h at RT. Solvent was removed under vacuum, and the
residue was partitioned between water (30 mL) and EA (60 mL). The organic layer was dried over
anhydrous Na SO and concentrated under vacuum. The crude product was purified by HPFC (EA) to
2
4
afford (Boc-compound 8a) (150 mg). Boc-compound 8a (150 mg, 0.26 mmol) was treated with excess
HCOOH (10 mL) for 4 h at RT. The solution was slowly added into a mixture of NaHCO in water
3
and ice with stirring. After neutralization, the mixture was extracted with EA (200 mL × 3) and
CH Cl (200 mL × 3). The combined organic extract was dried over anhydrous Na SO , and
2
2
2
4
concentrated to yield compound 8a (60 mg). LC-MS: 482.1 (M+H)/calculated mass: 481.6.
1
H NMR (CDCI , 300 MHz) δ: 7.81 (d, J - 8.4 Hz, 2H, Ph), 7.68 (d, J - 8.4 Hz, 2H, Ph), 7.54
3
(
d+s, J = 8.1 Hz, 3H, Ph+CONH), 7.34 (d, J = 8.1 Hz, 2H, Ph), 3.19 (m, 1H, COCH), 3.07 (m, 5H,
NCH +PhCH), 2.51 (m, 4H, NCH , 2.28 (s, 3H, NCH ), 2.09 (m, 1H, cyclopropane-CHz), 1.84 (m,
2
2)
3
1
H, cyclopropane-CH ), 1.48 (m, 2H, CH ), 0.63 (t, J - 7.2 Hz, 3H, CH ).
2 2 3
To a solution of 6b (520 mg, 0.87 mmol) in DMF (10 mL) cyanuric chloride (0.50 g, 2.75
mmol) was added. The reaction was stirred for 4 h at RT. The solvent was removed under vacuum,
and the residue was partitioned between water (30 mL) and EA (60 mL). The organic layer was dried
over anhydrous Na SO and concentrated under vacuum. The crude product was purified by HPFC
2
4
(
EA) to afford Boc-compound 8b (120 mg). Boc-compound 8b (120 mg, 0.21 mmol) was treated with
excess HCOOH (10 ml) for 4 h at RT. The solution was slowly added into a mixture of NaHCO in
3
1
0

water and ice with stirring. After neutralization, the mixture was extracted with EA (200 ml × 3) and
dichloromethane (200 ml × 3). The combined organic extract was dried over anhydrous Na SO , and
2
4
concentrated to yield compound 8b (98 mg). LC-MS: 482.1 (M+H)/calculated mass: 481.6.
1
H NMR (DMSO-d6, 300 MHz) δ: 7.90 (d, J = 8.4 Hz, 2H, Ph), 7.78 (d, J = 8.4 Hz, 2H, Ph),
7
4
.66 (d, J = 8.1 Hz, 2H, Ph), 7.34 (d, J = 8.4 Hz, 2H, Ph), 3.10 (t, J = 9.0 Hz, 1H, COCH), 2.91 (m,
H, NCH ), 2.84 (m, 1H, PhCH), 2.36 (m, 4H, NCH ), 2.13 (s, 3H, NCH ), 1.99 (m, 2H,
2
2
3
cyclopropane-CH2), 1.09 (m, 1H, CH ), 0.97 (m, 1H, CH ), 0.37 (t, J = 8.0 Hz, 3H, CH ).
2
2
3
2
.1.2. Spectroscopic data of synthetized CatC inhibitors
Compound 1, (S)-N-((S)-2-([1,1’-biphenyl]-4-yl)-1-cyanoethyl)-2-aminobutanamide.
1
(
127 mg, 33% yield). LC-MS: 308.1(M+l)/calculated mass: 307.4. H NMR (CDCI , 300
3
MHz) δ: 7.26-7.62 (m, 9H, Ph), 8.52 (br, 2H, NH ), 8.18 (br, 1H, NH), 4.98 (m, 1H, CH), 3.27
2
(
m,1H,CH), 3.21-3.01 (m 2H CH ), 1.84 (br, 2H, CH ), 0.87 (br, 3H, CH ).
2 2 3
Compound 2, (S)-2-amino-N-((S)-1-cyano-2-(4’-fluoro-[1,1’-biphenyl]-4-yl)ethyl)butanamide.
1
(
61 mg, 63% yield). LC-MS: 326.1 (M+H)/calculated mass: 325.5. H NMR (CDCI , 300
3
MHz) δ: 7.13-7.65 (m, 8H, Ph), 8.76 (br, 2H, NH ), 8.20 (br, 1H, NH), 4.87 (m, 1H, CH), 3.23
2
(
m,1H,CH), 3.27-3.01 (m 2H CH ), 1.88 (br, 2H, CH ), 0.85 (br, 3H, CH ).
2 2 3
Compound 3, (S)-2-amino-N-((S)-1-cyano-2-(4’-(trifluoromethyl)-[1,1’-biphenyl]-4-
yl)ethyl)butanamide.
1
(
49 mg, 44 % yield). LC-MS: 376.1 (M+H)/calculated mass: 375.4. H NMR (CDCI , 300
3
MHz) δ: 7.28-7.74 (m, 8H, Ph), 8.81 (br, 2H, NH ), 8.10 (br, 1H, NH), 4.85 (m, 1H, CH), 3.21
2
(
m,1H,CH), 3.27-3.01 (m 2H CH ), 1.81 (br, 2H, CH ), 0.82 (br, 3H, CH ).
2 2 3
Compound 4a, (S)-2-amino-N-((1R,2R)-1-cyano-2-(4’-fluorobiphenyl-4-yl)cyclopropyl)butanamide.
1
1

1
(
62 mg, 68%). LC-MS: 338.1 (M+l)/calculated mass: 337.4. H NMR (DMSO-d6, 300 MHz)
δ: 7.25-7.68 (m, 8H, Ph), 3.08 (m, 1H, COCH), 3.05 (t, J = 6.4 Hz, 1H, PhCH), 1.97 (d, J = 9.0 Hz,
H, cyclopropane-CH ), 1.10 (m, 2H, CH ), 0.57 (t, J = 7.8 Hz, 3H, CH ).
2
2
2
3
Compound 4b, (S)-2-amino-N-((1S,2S)-1-cyano-2-(4’-fluorobiphenyl-4-yl)cyclopropyl)butanamide.
1
(
68 mg, 75%). LC-MS: 338.1 (M+l)/calculated mass: 337.4. H NMR (DMSO-d6, 300 MHz)
<
5: 7.25-7.68 (m, 8H, Ph), 3.80 (t, J = 9.1 Hz, 1H, COCH), 3.05 (t, J = 6.0 Hz, 1H, PhCH), 1.95 (m,
H, cyclopropane-CH ), 0.95-1.19 (m, 2H, CH ), 0.36 (m, J - 7.4 Hz, 3H, CH ).
2
2
2
3
Compound 5a, (S)-2-amino-N-((1R,2R)-1-cyano-2-(4’-(trifluoromethyl)biphenyl-4-
yl)cyclopropyl)butanamide.
1
(
70 mg, 74%). LC-MS: 388.1 (M+l)/calculated mass: 387.4. H NMR (DMSO-d6, 300 MHz)
δ-: 7.33-7.88 (m, 8H, Ph), 3.09 (t, J = 9.3 Hz, 1H, COCH), 2.92 (t, J = 6.5 Hz, 1H, PhCH), 1.97 (d,
J=9.0 Hz, 2H, cyclopropane-CH ), 1.10 (m, 2H, CH ), 0.47 (t, J=7.5 Hz, 3H, CH ).
2
2
3
Compound 5b, (S)-2-amino-N-((1S,2S)-1-cyano-2-(4’-(trifluoromethyl)biphenyl-4-
yl)cyclopropyl)butanamide.
1
(
72 mg, 75%). LC-MS: 388.1 (M+l)/calculated mass: 387.4. H NMR (DMSO-d , 300 MHz)
6
δ: 7.33-7.88 (m, 8H, Ph), 3.10 (t, J = 9.0 Hz, 1H, COCH), 2.86 (t, J = 6.3 Hz, 1H, PhCH), 1.98 (m, 2H,
cyclopropane-CH ), 0.93-1.18 (m, 2H, CH ), 0.38 (m, J = 7.6 Hz, 3H, CH ).
2
2
3
Compound 6a, (S)-2-amino-N-((1R,2R)-1-cyano-2-(4’-(4-methylpiperazin-1-yl)biphenyl-4-
yl)cyclopro-pyl)butanamide.
+
1
(
88 mg, 73% yield). LC-MS: 418.2 [M+H] /calculated mass: 417.5. H NMR (DMSO-i , 300
MHz): 6 7.49 (d, J = 8.1 Hz, 4H, Ph), 7.22 (d, J = 8.4 Hz, 2H, Ph), 7.00 (d, J = 8.7 Hz, 2H, Ph), 3.17
(
br, 4H, NCH ), 3.03 (m, 1H, COCH), 2.95 (m, 1H, PhCH), 2.46 (br, 4H, NCH ), 2.22 (s, 3H, NCH ),
2 2 3
1
.96 (m, 2H, cyclopropane-CH ), 1.19 (m, 2H, CH ), 0.62 (t, J - 7.1 Hz, 3H, CH ).
2 2 3
Compound 6b, (S)-2-amino-N-((1S,2S)-1-cyano-2-(4’-(4-methylpiperazin-1-yl)biphenyl-4-
yl)cyclopro-pyl)butanamide.
1
2

+
1
(
32 mg, 31% yield). LC-MS: 418.2 [M+H] /calculated mass: 417.5. H NM (DMSO-d , 300
6
MHz): δ 7.48 (d, J = 8.4 Hz, 4H, Ph), 7.22 (d, J = 8.1 Hz, 2H, Ph), 7.00 (d, J = 8.7 Hz, 2H, Ph), 3.17
(
br, 4H, NCHi), 3.08 (m, 1H, COCH), 2.93 (m, 1H, PhCH), 2.47 (br, 4H, NCH ), 2.22 (s, 3H, NCH ),
2 3
1
.99 (m, 1H, cyclopropane-CHa), 1.87 (m, 1H, cyclopropane-CH ), 1.15 (m, 2H, CH ), 0.36 (t, J = 7.2
2 2
Hz, 3H, CH3).
Compound 7a, (S)-2-amino-N-((1R,2R)-1-cyano-2-(4-(2-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[1,5-
a]pyridin-6-yl)phenyl)cyclopropyl)butanamide.
1
(
150 mg, 46%). LC-MS: 459.3 (M+l)/calculated mass: 458.6. H NMR (CDCl 300 MHz) 5:
3,
8.53 (s, 1H, NH), 7.62-7.46 (m, 5H, Ph+CH), 7.34 (d, J - 8.1 Hz, 2H, Ph), 3.70 (t, J = 4.8 Hz, 4H,
NCH ), 3.21 (m, 1H, CH), 3.10 (m, 1H, CH), 2.58 (t, J = 5.0 Hz, 4H, NCH ) 2.39 (s, 3H, NCH ), 2.10
2
2
3
(
m, 1H, cyclopropane-CH ), 1.84 (m, 1H, cyclopropane-CH , 1.27 (m, 2H, CH ), 0.70 (t, J = 7.6 HZ,
2 2 2
3
H CH ).
, 3
Compound 7b, (S)-2-amino-N-((1S,2S)-1-cyano-2-(4-(2-(4-methylpiperazin-1-yl)-[1,2,4]triazolo[1,5-
a]pyridin-6-yl)phenyl)cyclopropyl)butanamide.
1
1
(
80 mg, 35%). LC-MS: 459.3 (M+l)/calculated mass: 458.6. H NMR (CDCI , 300 MHz) δ: H
3
NMR (CDCl ,300 MHz) δ: 8.55 (s, 1H, NH), 7.63 (d, J = 9.0 Hz, 1H, CH), 7.55-7.47 (m, 4H,
3
Ph+CH), 7.31 (d, J - 8.1 Hz, 2H, Ph), 3.70 (t, J - 5.0 Hz, 4H, NCH ), 3.16 (m, 1H, CH), 3.05 (m, 1H,
2
CH), 2.59 (t, J - 4.8 Hz, 4H, NCH ) 2.39 (s, 3H, NCH ), 2.15 (m, 1H, cyclopropane-CH ), 1.85 (m,
2
3
2
1
H, cyclopropane-CH ), 1.75-1.38 (m, 2H, CH ), 0.84 (t, J = 7.5 Hz, 3H, CH ).
2 2 3
Compound 9a, 1-amino-N-((1R,2R)-1-cyano-2-(4’-((4-methylpiperazin-1-yl)sulfonyl)-[1,’-biphenyl]-
4
-yl)cyclopropyl)cyclohexane-1-carboxamide.
1
(
118 mg, 31%). H NMR (CDCl , 300 MHz) δ: 7.99 (s, 1H, NH), 7.82 (d, J = 8.4 Hz, 2H, Ph),
3
7
5
1
.70 (d, J = 8.4 Hz, 2H, Ph), 7.55 (d, J = 8.1 Hz, 2H, Ph), 7.33 (d, J = 8.1 Hz, 2H, Ph), 3.05-3.14 (m,
H, CH +NCH ), 2.60 (br, 4H, NCH ), 2.35 (s, 3H, NCH ), 2.09-2.12 (m, 1H, cyclopropane-CH ),
2
2
3
2
.82-1.89 (m, 1H, cyclopropane-CH ), 1.18-1.62 (m, 10H, cyclohexane).
2
1
3

Compound 9b, 1-amino-N-((1S,2S)-1-cyano-2-(4’-((4-methylpiperazin-1-yl)sulfonyl)-[1,’-biphenyl]-
4
-yl)cyclopropyl)cyclohexane-1-carboxamide.
1
H NMR (CDCl , 300 MHz) δ: 7.98 (s, 1H, NH), 7.82 (d, J = 8.4 Hz, 2H, Ph), 7.70 (d, J = 8.4
3
Hz, 2H, Ph), 7.55 (d, J = 8.1 Hz, 2H, Ph), 7.33 (d, J = 8.1 Hz, 2H, Ph), 3.07 (br, 5H, CH +NCH ), 2.53
2
(
br, 4H, NCH ), 2.30 (s, 3H, NCH ), 2.06-2.13 (m, 1H, cyclopropane-CH ), 1.82-1.87 (m, 1H,
2 3 2
cyclopropane-CH ), 1.18-1.59 (m, 10H, cyclohexane).
2
Compound 10a, (S)-2-amino-N-((1R,2R)-1-cyano-2-(4’-(4-methylpiperazin-1-ylsulfonyl)biphenyl-4-
yl)cyclopro-pyl)-3-cyclopropylpropanamide.
1
(
80 mg, 46%). H NMR (DMSO-c 6, 300 MHz) 5: 7.91 (d, J = 8.1 Hz, 2H, Ph), 7.79 (d, J =
8
1
.1 Hz, 2H, Ph), 7.67 (d, J = 8.1 Hz, 2H, Ph), 7.34 (d, J = 7.8 Hz, 2H, Ph), 3.11 (m, 1H, CH), 3.03 (m,
H, CH), 2.89 (br, 4H, NCH ), 2.37 (m, 4H, NCH ), 2.13 (s, 3H, NCH ), 2.05 (m, 2H, cyclopropane-
2
2
3
CH ), 1.19 (m, 2H, CH ), 0.99-0.14 (m, 5H, cyclopropyl).
2
2
Compound 10b, (S)-2-amino-N-((1S,2S)-1-cyano-2-(4’-(4-methylpiperazin-1-ylsulfonyl)biphenyl-4-
yl)cyclopro-pyl)-3-cyclopropylpropanamide.
1
(
163 mg, 58%). H NMR (DMSO-i 6, 300 MHz) fl: 7.92 (d, J - 8.4 Hz, 2H, Ph), 7.79 (d, J =
8
5
.4 Hz, 2H, Ph), 7.67 (d, J = 8.1 Hz, 2H, Ph), 7.33 (d, J = 8.4 Hz, 2H, Ph), 3.13 (m, 1H, CH), 2.92 (br,
H, CH+NCH ), 2.36 (m, 4H, NCH ), 2.13 (s, 3H, NCH ), 1.98 (m, 2H, cyclopropane-CH ), 1.27-0.04
2
2
3
2
(
m, 7H, CH +cyclopropyl).
2
Compound 11a, (S)-2-amino-N-((1R,2R)-1-cyano-2-(4’-((4-methylpiperazin-1-yl)sulfonyl)-[1,1’-
biphenyl]-4-yl)cyclopropyl)pent-4-ynamide.
1
(
120 mg, 47%). H NMR (CDCI , 300 MHz) δ 7.83 (d, J = 8.4 Hz, 2H, Ph), 7.71 (d, J = 8.4
3
Hz, 2H, Ph), 7.57 (d, J = 7.5 Hz, 2H, Ph), 7.35 (d, J = 8.1 Hz, 2H, Ph), 3.38 (m, 1H, CH), 3.12. (br,
H, CH+NCH ), 2.53 (br, 4H, NCH ), 2.30 (s, 3H, NCH ), 2.14 (m, 3H, CH+cyclopropane-CHa), 1.88
5
2
2
3
(
m, 2H, CH2).
1
4

Compound 11b, (S)-2-amino-N-((1S,2S)-1-cyano-2-(4’-((4-methylpiperazin-1-yl)sulfonyl)-[1,1’-
biphenyl]-4-yl)cyclopropyl)pent-4-ynamide.
1
(
240 mg, 70%). H NMR (CDCI , 300 MHz) δ 7.84 (d, J = 8.4 Hz, 2H, Ph), 7.73 (d, J = 8.4
3
Hz, 2H, Ph), 7.58 (d, J = 8.4 Hz, 2H, Ph), 7.34 (d, J = 8.1 Hz, 2H, Ph), 3.34 (m, 1H, CH), 3.06 (br, 5H,
CH+NCH ), 2.60 (br, 2H, CH ), 2.53 (br, 4H, NCH ), 2.30 (s, 3H, NCH ), 2.17 (m, 1H, CH ), 2.02 (m,
2
2
2
3
2
1H, CH), 1.86 (m, 1H, CH2).
Compound 12a, (S)-2-amino-N-((1R,2R)-1-cyano-2-(4’-(4-methylpiperazin-1-ylsulfonyl)biphenyl-4-
yl)cyclopro-pyl)-3-phenylpropanamide.
1
(
90 mg, 13%). LC-MS: 544.2 (M+H)/calculated mass: 543.7. H NMR (CDCI , 300 MHz) δ:
3
7.56 (d, J = 8.1 Hz, 2H, Ph), 7.68 (d, J = 8.1 Hz, 2H, Ph), 7.57 (d, J = 8.1 Hz, 2H, Ph), 7.49 (s, 1H,
NH), 7.33 (d, J = 8.1 Hz, 2H, Ph), 7.25 (m, 3H, Ph), 7.08 (m, 2H, Ph), 3.48 (m, 1H, CH), 3.10 (m, 6H,
CH+Bn+NCH ), 2.52(m, 4H NCH ), 2.29 (s, 3H, NH ). 2.17 (m, 2H, Br+cyclopropane-CH ) 1.87 (m,
2
2
3
2
1H, cyclopropane-CH2).
Compound 12b, (S)-2-amino-N-((1S,2S)-1-cyano-2-(4’-(4-methylpiperazin-1-ylsulfonyl)biphenyl-4-
yl)cyclopro-pyl)-3-phenylpropanamide.
1
(
0.51 g, 67%). LC-MS: 544.2 (M+H)/calculated mass: 543.7. H NMR (CDCI , 300 MHz) δ:
3
7
7
.85 (d, J = 8.1 Hz, 2H, Ph), 7.72 (d, J = 8.1 Hz, 2H, Ph), 7.46 (m, 3H, Ph+NH), 7.31 (m, 3H, Ph),
.11 (m, 4H, Ph), 3.51 (m, 1H, CH), 3.07 (m, 7H, CH+Bn+NCH ), 2.54 (m, 4H, NCH ), 2.30 (s, 3H,
2
2
NCH ), 2.14 (m, 1H, cyclopropane-CH ), 1.74 (m, 1H, cyclopropane-CH ).
3
2
2
Compound 13a, (S)-2-amino-N-((1R,2R)-1-cyano-2-(4’-(4-methylpiperazin-1-ylsulfonyl)biphenyl-4-
yl)cyclopro-pyl)-3-(thiophen-2-yl)propanamide.
1
H NMR (CDCI , 300 MHz) δ: 7.82 (d, J = 8.4 Hz, 2H, Ph), 7.57 (d, J = 8.4 Hz, 2H, Ph),
3
7
6
6
2
.48-7.53 (m, 3H, Ph+thiophene), 7.34 (d, J = 8.1 Hz, 2H, Ph), 7.14 (d, J = 5.1 Hz, 1H, thiophene),
.89 (dd, J = 3.6 Hz, 5.1 Hz, 1H, thiophene), 6.72 (br, 1H, NH), 3.45-3.50 (m, 1H, CH), 3.06-3.18 (m,
H, thiophene-CH +NCH ), 2.55-2.64 (m, 1H, CH), 2.53 (m, 4H, NCH ), 2.89 (s, 3H, NCH ), 2.06-
2
2
2
3
.17 (m, 1H, cyclopropane-CH ), 1.84-1.90 (m, 1H, cyclopropane-CH ).
2
2
1
5

Compound 13b, (S)-2-amino-N-((1S,2S)-1-cyano-2-(4’-(4-methylpiperazin-1-ylsulfonyl)biphenyl-4-
yl)cyclopro-pyl)-3-(thiophen-2-yl)propanamide.
1
(
180 mg, 35%). H NMR (CDCI , 300 MHz) δ 7.83 (d, J = 8.4 Hz, 2H, Ph), 7.70 (d, J = 8.4
3
Hz, 2H, Ph), 7.48 (d, J = 8.4 Hz, 2H, Ph), 7.24 (m, 1H, thiophene), 7.12 (d, J = 8.4 Hz, 1H, thiophene),
7
1
2
.02 (dd, J = 3.3 Hz, 5.1 Hz, 1H, thiophene), 6.84 (br, 1H, NH), 3.46-3.51 (m, 1H, CH), 3.27-3.35 (m,
H, CH), 2.99-3.14 (m, 6H, thiophene-CH +NCH ), 2.52-2.54 (m, 4H, NCH ), 2.30 (s, 3H, NCH ),
2
2
2
3
.12-2.18 (m, 1H, cyclopropane-CH ), 1.80 (t, J = 7.7 Hz, 1H, cyclopropane-CH ).
2
2
Compound 14a, (S)-2-amino-N-((1R,2R)-1-cyano-2-(4’-(4-methylpiperazin-1-ylsulfonyl)biphenyl-4-
yl)cyclopro-pyl)-3-(furan-2-yl)propanamide.
1
(
63 mg). LC-MS: 534.1/calculated mass: 533.1. H NMR (CDCI , 300 MHz) δ: 7.80 (d, J =
3
8
2
.4 Hz, 2H, Ph), 7.68 (d, J = 8.1 Hz, 2H, Ph), 7.55 (d+s, J - 8.1 Hz, 3H, Ph+Fura), 7.33 (d, J = 8.1 Hz,
H, Ph), 6.23 (m, 1H, Fura), 5.98 (d, J = 3 Hz, 1H, Fura), 3.51 (m, 1H, COCH), 3.08 (m, 5H,
NCH +PhCH+FuraCH ), 2.51 (m, 4H, NCH ), 2.38 (m, 1H, FuraCH ), 2.28 (s, 3H, NCH ), 2.13 (m,
2
2
2
2
3
1
H, cyclopropane-CH ), 1.86 (m, 1H, cyclopropane-CH ).
2
2
Compound 14b, (S)-2-amino-N-((1S,2S)-1-cyano-2-(4’-(4-methylpiperazin-1-ylsulfonyl)biphenyl-4-
yl)cyclopro-pyl)-3-(furan-2-yl)propanamide.
1
(
55 mg). LC-MS: 534.1/calculated mass: 533.1. H NMR (CDCI , 300 MHz) δ 7.82 (d, J = 8.4
3
Hz, 2H, Ph), 7.70 (d, J = 8.4 Hz, 2H, Ph), 7.58 (br, 1H, CONH), 7.52 (d, J = 8.1 Hz, 2H, Ph), 7.34 (S,
H, Fura), 7.20 (d, J = 8.1 Hz, 2H, Ph), 6.33 (s, 1H, Fura), 6.09 (d, J = 2.7 Hz, 1H, Fura), 3.48 (m, 1H,
1
COCH), 3.07 (m, 7H, PhCHNCH +FuraCH ), 2.53 (s, 4H, NCH , 2.29 (s, 3H, NCH ), 2.18 (m, 1H,
2
2
2
3
cyclopropane-CH ), 1.77 (m, 1H, cyclopropane-CH ).
2
2
NMR analysis on synthesized compounds were performed by PepTech.
2
.2. IC determination
50
2
3
Human recombinant CatC was produced in baculovirus-infected cells (Unizyme
1
6

Laboratories A/S, Hørsholm, Denmark) and activated at 4-6 ºC with 5 mM cysteamin for 5
minutes and stored at -20 ºC in a buffer containing 2.5 mM Na-phosphate, 150 mM NaCl, 2
mM cysteamine, 50% glycerol, pH 7.0 at a concentration of 1-2 mg/mL (5-10 μM).
The IC values of the nitrile inhibitors (compounds 1-14) for human CatC were
5
0
determined using Gly-Phe-pNA (para-nitroanilide) (Sigma, Denmark) as a substrate in 20
mM citric acid, 150 mM NaCl, 2 mM EDTA pH 4.5. The substrate was solubilized in DMF to
give a 0.2-0.5 M stock solution, which was then further diluted with stirring in assay buffer to
a final concentration of 1 mM. The concentration of the substrate used in the assay was 0.75
mM which is about 5 times below the Km identified in this study (Km= 3.9 mM).
A stock solution of human CatC was diluted 500-1000 times in assay buffer to a
concentration of 10 nM. The assay was performed in 96-well plates. Diluted enzyme (25 μL)
was added to the well, followed by 25 µL of test substance in varying concentrations, and the
solution was mixed. The plate was incubated at 37 ºC for 5 min, followed by addition of 150
μL of 1 mM substrate prewarmed to 37 ºC (corresponding to a substrate concentration of 750
μM and a CatC concentration of 1.25 nM in the assay). The absorption was measured at 405
nm at 37 ºC for every 90 sec for 12 min or every 20 sec for 4 min. Each measurement was
made in duplicate. IC was determined using a 4-parameter logistic equation in a non-linear
5
0
curve fitting routine.
2
.3. Downstream effect of CatC inhibition on NE/PR3 and CatG activation
The pro-monocytic cell line U937 was purchased from Health Protection Agency
Culture Collection (HPACC) (Porton Down, Salisbury, UK) and maintained in suspension in
RPMI 1640 containing 10% fetal bovine serum, 10 mM Hepes, 1 mM sodium pyruvate and
1
3
00 units/mL of each of penicillin and streptomycin in a humidified atmosphere of 5% CO at
2
7 °C.
1
7

6
U937 cells were seeded in 12-well plates at 0.2 to 0.4 x 10 cells/mL in volumes of 1.5
mL per well in the presence of no or increasing concentrations of CatC inhibitor (compounds
2
-14). Twelve points in duplicate in the range of 0.02 nM to 50 μM inhibitor were tested.
After 24 or 48 h cells were harvested, washed twice with PBS and lysed in 20 mM Tris-HCl,
pH 7.5, 100 mM NaCl, 0.2% Triton X- 100. Debris was removed by centrifugation and
supernatants were retained. The extracts were mixed with assay buffer (50 mM Tris, 0.1%
Triton X-100, 0.5 M NaCl, pH 8.0) supplemented with NE/PR3 substrate MeOSuc-AAPV-
pNA (Bachem, Germany) to a final concentration of 0.9 mM, The activity of NE/PR3 was
determined by measuring the enzymatic release of chromogenic p-nitroaniline (pNA) from the
substrate which leads to an increase in absorbance at 405 nm. Assays were carried out in 96-
well plates in a final volume of 200 μL at 37 °C, and absorbance was measured 8 times during
3
5 min using a plate reader. IC was determined using a 4-parameter logistic equation in a
50
non-linear curve fitting routine.
2
.4. Crystallization and structure determination of dipeptidyl cyclopropyl nitrile inhibitors in
complex with human CatC
CatC preparation: CatC was diluted 1:1 in dialysis buffer and dialyzed (Spectra/Por
RC membrane molecular weight cutoff 6-8 kDa) three times overnight against the following
dialysis buffer: 2 mM cysteamin, 50 mM NaCl and 2.5 mM NaPO pH 7.0. The next day the
4
protein solution was supplemented with 1.5 mM dithiothreitol (DTT) and concentrated to 11
mg/mL in an Amicon concentrator with molecular weight cutoff 10 kDa. The protein was
used immediately for crystallization trials.
Crystallization: Compound 5a was diluted in DMSO to obtain a 50 mM solution. 49
µL of CatC solution were mixed with 1 µL of compound 5a. CatC was incubated with
compound 5a on ice for 1.5 h before crystallization. The crystal was grown from the JCSG-
plus screen (Molecular Dimensions), condition B9, at 18 °C from a 100 + 100 nL hanging
1
8

drop containing 20% (w/v) PEG 6 000, 0.1 M sodium citrate, pH 5.0. An intergrown crystal
appeared within a few days. Cryoprotectant solution with the following composition was
added to the drop: 16% (w/v) PEG 3.000, 0.08 M sodium citrate pH 5.0, 2.5 mM DTT, 20%
v/v glycerol. The crystal was cut in pieces with crystal manipulation tools. The largest
fragment containing a single crystal measuring approximately 0.1 x 0.1 x 0.1 mm was cryo-
cooled in the liquid N stream at the beamline.
2
Compound 9a was diluted in DMSO to obtain a 100 mM solution. 49.5 µL of CatC
solution were mixed with 0.5 µL of compound 9a. The protease was incubated with
compound 9a on ice for 2 h before crystallization set-ups. The crystal was grown from a 2 + 2
μL sitting drop in a microbridge (Molecular Dimensions) placed in a NeXtal plate (Qiagen) at
1
8 °C containing: 20% (w/v) PEG 3 000, 0.2 M ammonium citrate dibasic. DTT was added to
the reservoir to 2 mM immediately after drop setup. Crystals appeared overnight.
Cryoprotectant solution with the following composition was used: 20% (w/v) PEG 3.000, 0.2
M ammonium citrate dibasic, 2.5 mM DTT, 20% (v/v) glycerol, 0.5 mM of compound 9a. A
crystal measuring 0.25 x 0.25 x 0.1 mm was flash-cooled in liquid N2.
Compound 13a was crystallized similarly to compound 9a, with the following
differences: the reservoir solution was 20% (w/v) PEG 3 000, 0.1 M sodium citrate pH 5.6
and the cryoprotectant solution was: 20% (w/v) PEG 3 000, 0.1 M sodium citrate pH 5.6, 2.5
mM DTT, 20% (v/v) glycerol, 0.5 mM of compound 13a. The crystal used measured 0.3 x 0.3
x 0.1 mm.
Data collection: Data for compound 5a were collected at 100 K at station I911-2 of
the MAX-lab synchrotron, Lund, Sweden ( = 1.03796 Å), equipped with a 165mm marCCD
detector. The data were collected in a single pass, processed using XDS and reduced using the
CCP4 suite. Data for compound 9a and compound 13a were collected at 100 K on a Proteum
X8 laboratory X-ray system from Bruker consisting of a Microstar microfocus X-ray source
1
9

(λ = 1.5418 Å), Montel optics, kappa goniometer, Pt135 CCD detector and Cryoflex cryo
system. Complete data were collected to 2.15 Å at the radial edge of the square detector.
Diffraction was obtained into the corners of the detector, to 2.0 Å resolution, albeit with lower
completeness. Data were processed using the Proteum2 software from Bruker and reduced
using the CCP4 suite.
Structure determination and refinement: The structures of CatC in complex with
compound 5a, compound 9a and compound 13a were solved by difference Fourier methods
using another in-house CatC complex. The difference map showed clear electron density for
all ligands. Geometric restraints files for the ligands and their covalent bonds to Cys234 were
generated using energy-minimized coordinates from Schrödinger Suite 2009 (Schrödinger
4
1
42
Inc.) and the JLigand tool in the CCP4 program suite. The model was refined using
4
3
44
Refmac5 combined with manual rebuilding using Coot. Riding hydrogen atoms were
4
5
added during refinement. A separate TLS group was used for each protein chain. Data
collection and refinement statistics for inhibitor/CatC complexes are shown in Table 2.
The structure of compound 8a was not determined by X-ray crystallography but
was computationally modelled. The necessary information about interactions of each end of
the compound 8a was given by the other structures in which these moieties were present, so it
was considered unnecessary to determine the structure of compound 8a. The ethyl moiety of
compound 8a is found in the crystal structure of compound 5a and the outer piperazine-
sulfonyl moiety is in the crystal structures of compound 9a and compound 13a.
2
.5. Human and mouse plasma stability analysis of IcatCXPZ-01
Studies (service provided by Absorption Systems (Exton, PA, USA)) were carried out
in mixed-gender plasma collected on sodium heparin. Plasma was adjusted to pH 7.4 prior to
initiating the experiments. DMSO stocks were first prepared for the test articles. Aliquots of
2
0

the DMSO solutions were dosed into 1 mL of plasma, which had been pre-warmed to 37 °C,
at a final IcatCXPZ-01 concentration of 1 μM. The vials were kept in a benchtop
Thermomixer® for the duration of the experiment. Aliquots (100 μL) were taken at each time
point (0, 15, 30, 60, and 120 min) and added to 96-well plates which had been pre-filled with
3
00 μL of acetonitrile (ACN). Samples were stored at 4 °C until the end of the experiment.
After the final time pointwas sampled, the plate was mixed and then centrifuged at 3.000 rpm
for 10 min. Aliquots of the supernatant were removed, diluted 1:1 into distilled water, and
analyzed by LC-MS/MS. The peak area response ratio (PARR) to internal standard
metoprolol was compared to the PARR at time 0 to determine the percent remaining at each
time point. Half-lives were calculated using GraphPad software, fitting to a single-
phaseexponential decay equation.
2
.6. Microsomal half-life determination of IcatCXPZ-01
Microsomal half-life determination was performed by Arbsorption Systems (Exton,
PA, USA). IcatCXPZ-01, compound 7a and compound 12a were dissolved in 100% DMSO at a
concentration of 10 mM. The reaction mixture, consisted of mouse or human liver
microsomes (1.0 mg/mL), 1 mM NADPH, 100 mM KH PO , 10 mM MgCl , pH 7.4, and the
2
4
2
test compound at a concentration of 5 μM.
An aliquot of the reaction mixture (without cofactors) was incubated in a shaking
water bath at 37 °C for 3 min. Another aliquot of the reaction mixture was prepared as the
negative control. The test compound was added into both the reaction mixture and the
negative control at a final concentration of 5 μM.
The reaction was initiated by the addition of NADPH to 1 mM (not into the negative
controls) and then incubated in a shaking water bath at 37 °C. Aliquots (100 μL) were
withdrawn at 0, 10, 20, 30, and 60 min or at 0, 15, 30 and 60 min and combined with 900 μL
2
1

of ice-cold 50/50 acetonitrile/dH O to terminate the reaction. A control (testosterone) was run
2
simultaneously with the test compound in a separate reaction. LC/MS/MS is used to
determine the peak area response ratio (peak area corresponding to the test compound or
control divided by that of metoprolol used as the analytical internal standard). The natural log
of the percent remaining was plotted versus time. A linear fit was used to determine the rate
constant. The fit was truncated if the percent remaining of test compound was less than 10%.
The elimination half-lives associated with the disappearance of the test and control
compounds were determined to compare their relative metabolic stability.
2
.7. Test for inhibition of CYP2C9, CYP2D6 and CYP3A4 enzymes (IC Determination)
50
Testing for CYP inhibition potential was a service provided by Arbsorption Systems
Exton, PA, USA). The test compounds, at eight concentration levels (0, 0.137, 0.412, 1.23,
.70, 11.1, 33.3 and 100 μM), were incubated with pooled human liver microsomes (0.25 mg
(
3
protein/mL) at 37 ºC in the presence of phosphate buffer (100 mM, pH 7.4), MgCl (5 mM),
2
NADPH (1 mM), and CYP-specific probe substrate at approximately Km (6 μM diclofenac, 7
μM bufuralol and 75 µM testosterone for CYP2C9, CYP2D6, and CYP3A4, respectively).
After a period of incubation, samples were treated by the addition of protein precipitation
solvent and centrifuged. CYP enzyme activities were measured by determining the formation
of the CYP probe metabolites by LC-MS/MS, and the IC (the concentration of an inhibitor
5
0
causing 50% inhibition) was estimated using GraphPad Prism® software by fitting the
experimental data (percent of control activity remaining at each concentration of test
compound) to a sigmoidal model and non-linear regression analysis.
The CYP enzyme activities in the human liver microsomes were verified in parallel by
determining the inhibition of positive inhibitors on the CYP enzyme activities (sulfaphenazole,
quinidine and ketoconazole for CYP2C9, CYP2D6, and CYP3A4, respectively). All CYP
2
2

enzymes showed expected inhibitions by positive inhibitors, indicating that the human liver
microsomes used in this study were metabolically active and responsive.
2
.8. Test for hERG Channel Inhibition (IC Determination)
50
The experiments were performed (as a service provided by Cyprotex Discovery Ltd.
TM
(
Cheshire, UK)) on an IonWorks HT instrument (Molecular Devices Corporation), which
automatically performs electrophysiology measurements in 48 single cells simultaneously in a
TM
specialized 384-well plate (PatchPlate ). All cell suspensions, buffers and test compound
solutions were at RT during the experiment.
The cells used were Chinese hamster ovary (CHO) cells stably transfected with hERG
(cell line obtained from Cytomyx, UK). A single cell suspension was prepared in extracellular
solution (Dulbecco’s phosphate buffered saline with calcium and magnesium pH 7-7.2) and
TM
aliquots added automatically to each well of a PatchPlate . The cells were then positioned
over a small hole at the bottom of each well by applying a vacuum beneath the plate to form
an electrical seal. The vacuum was applied through a single compartment common to all
wells which was filled with intracellular solution (buffered to pH 7.2 with HEPES). The
resistance of each seal was measured via a common ground electrode in the intracellular
compartment and individual electrodes placed into each of the upper wells.
Electrical access to the cell was then achieved by circulating a perforating agent,
TM
amphotericin, underneath the PatchPlate and then measuring the pre-compound hERG current.
An electrode was positioned in the extracellular compartment and a holding potential of 80 mV
applied for 15 sec. The hERG channels were then activated by applying a depolarising step to +40
mV for 5 sec and then clamped at 50 mV for 4 sec to elicit the hERG tail current, before returning to
8
0 mV for 0.3 sec.
IcatCXPZ-01 was then added automatically to the upper wells of the PatchPlate from
a 96-well microtitre plate containing a range of concentrations of each compound. Solutions
TM
2
3

were prepared by diluting 10 mM DMSO solutions of the test compound into extracellular
buffer such that the final concentrations tested were 0.008, 0.04, 0.2, 1, 5 and 25 µM (final
DMSO concentration 0.25%). IcatCXPZ-01 was left in contact with the cells for 300 sec before
recording currents using the same voltage step protocol as in the pre-compound scan.
Quinidine, an established hERG inhibitor, was included as a positive control and buffer
containing 0.25% DMSO was included as a negative control. The results for all compounds
on the plate were rejected and the experiment repeated if the IC value for quinidine or the
5
0
negative control results were outside quality control limits.
TM
Each concentration was tested in 4 replicate wells on the PatchPlate . However, only
cells with a seal resistance greater than 50 MOhm and a pre-compound current of at least 0.1
nA were used to evaluate hERG blockade.
Post compound currents were then expressed as a percentage of pre-compound currents and
plotted against concentration for each compound. Where concentration dependent inhibition
was observed, the data were fitted to the following equation and an IC value calculated:
5
0
y =
ymax-ymin + ymin
s
1
+(x/x )
50
Where y = (post compound current/pre compound current) x 100, x = concentration,
x = concentration required to inhibit current by 50% (IC ) and s = slope of the graph
5
0
50
2
.9. Animal experiments, observations and examinations
The evaluation of IcatCXPZ-01 in the collagen antibody induced mouse model of
rheumatoid arthritis was performed by Inflammations Discovery Services at MD Biosciences
Glasgow, UK). 46 young adult BALB/c male mice (6-8 weeks at study initiation) in good
(
health were acclimatized to laboratory conditions. During 10-day acclimation period and
following dosing, animals were housed within a limited access rodent facility and kept in
groups of 5. Mice were housed in sterilized individually ventilated polysulfone cages with
2
4

sterile wood shavings as bedding materials. Animals were provided ad libitum a commercial
rodent diet and free access to drinking water, supplied to each cage via sterilized polyethylene
bottles with stainless steel sipper tubes. All food and water were sterilized. Automatically
controlled environmental conditions were set to maintain temperature at 20-24 C with
relative humidity (RH) of 30-70%, a 12:12 h light:dark cycle and 90 air changes/h in
individually ventilated cages. Temperature, RH and light cycle were monitored daily by the
control computer. Animals were given a unique animal identification number. This number
also appeared on a cage card, visible on the front of each cage. The cage card also contained
the study number. Animals were randomly assigned to experimental treatment groups.
Treatment groups were distributed across experimental cages. At the end of the study
surviving animals were euthanized by CO asphyxiation followed by cervical dislocation.
2
This study was performed according to the UK Home Office regulations for use of animals in
scientific procedures - Animals (Scientific Procedures) Act 1986.
Arthritis induction: As shown in the schematic depiction (Figure 7), all animals were
subjected on day 0 of the study (study commencement) to a tail vein injection of
TM
ArthritoMAb monoclonal antibody (mAb) cocktail (2 mg/mouse). mAb stock solution (10
mg/mL), supplied by MD Biosciences, Division of Morwell Diagnostics GmbH, was used as
provided. Three days post the single mAb administration, all animals were subjected to LPS
(MD Biosciences) administration by a single intraperitoneal injection (100 µg/mouse). LPS
was diluted with PBS to achieve the appropriate concentration. Thorough vortexing was
required just prior to its injection.
Treatment administration: IcatCXPZ-01 (1.2 mg/kg or 4.8 mg/kg at 10 mL/kg) and
vehicle control (10 mL/kg) were administered twice daily with a 12 h separation between
doses, from day -14 to day 11 by subcutaneous injection. To ensure that neutrophils in
circulation have been exposed to IcatCXPZ-01 at the promyelocyte stage and beyond where
2
5

elastase-like proteases are formed and stored, animals were treated with IcatCXPZ-01 for 14
days before arthritis induction. The vehicle 2-hydroxypropyl-β-cyclodextrin (HPβCD) (Sigma)
was dissolved in 50 mM citrate buffer (pH 5.0) (Sigma-Aldrich, Denmark) to make a 15%
HPβCD solution for injection. The positive control (Enbrel) was administered once daily from
day 0 to day 11 by intraperitoneal injection (6.25 mg/kg at 10 mL/kg). A stock solution of
Enbrel (Wyeth Pharmaceuticals) was formulated according to the manufacturer’s instructions
and diluted in PBS to the required concentration shortly before each daily administration.
On day -15 all the animals were assigned to treatment groups 1, 2, 3 and 4. The dose
administered was calculated based on the assumption that the animals weighed, on average,
2
0 g. A fixed volume of 200 µL was administered to each mouse. No analgesia was used
during the course of the study.
Bone marrow sample collection: Four mice from the vehicle (GROUP 1), IcatCXPZ-01
1
.2 mg/kg (GROUP 2) and IcatCXPZ-01 4.8 mg/kg (GROUP 3) treated groups were terminated
at 1 h (day 11) and 12 h (day 12) post the last dose. Four mice from the IcatCXPZ-01 1.2
mg/kg and IcatCXPZ-01 4.8 mg/kg treated groups and five mice from the vehicle treated group
were terminated at 24 h (day 12) after the last dose was administered. No samples were
obtained from mice treated with the positive control (Enbrel). At each time point, both rear
paws from all animals were removed and used to extract bone marrow. The bone marrow was
made into a single cell suspension, cell number was determined and the cells were then
pelleted and snap frozen. The pellets were dried using a pipette to ensure a minimal amount
of liquid remained. The cells were stored at -80 °C.
Bone marrow extract preparation: Pellets of bone marrow cells were resuspended in
4
00 µL buffer C (50 mM Tris, 0.5 M NaCl, pH 7.5 supplemented with Benzonase (Merck,
Cat.no.: 101654) to 250 u/mL and supplemented with Triton X-100 to a concentration of
.2%). Suspensions became clear within a few minutes and were then diluted with 300 µL
0
2
6

buffer B (50 mM Tris, 0.5 M NaCl, pH 7.5 supplemented with Benzonase). Extracts were
mixed and cell debris was removed by centrifugation 20.000 x g, 4 °C, 2 min. Concentrations
of protein in supernatants were determined using the Bradford method. All samples were
measured in duplicate and in two different dilutions.
Proteolytic activity measurement: 50 mM stock solution of NE/PR3 substrate
MeOSuc-Ala-Ala-Pro-Val-pNA (Bachem Cat. No.: L-1335) prepared in DMSO was stored at
RT and used for assays within 10 days after preparation. 1.09 mM working solution was
prepared from the 50 mM stock solution by dilution in 50 mM Tris, 0.5 M NaCl, pH 7.5 and
pre-warmed to 37 °C just prior to assay. 50 mM stock solution of CatG substrate Suc-Ala-
Ala-Pro-Phe-pNA (Bachem Cat. No.: L-1400) prepared in DMSO was stored at RT and used
for assays within 10 days after preparation. 2.5 mM working solution was prepared from the
5
3
0 mM stock solution by dilution in 50 mM Tris, 0.5 M NaCl, pH 7.5 and pre-warmed to
7 °C just prior to assay.
1
60 µL of NE/PR3 or CatG substrate (working solution) were mixed with 40 µL of
bone marrow supernatants (previously diluted 10-20 times in 50 mM Tris, 0.5 M NaCl, pH
.5) in 96-well plates and assayed in a Microplate Reader BioTek Elx808) NE/PR3 and CatG
7
substrates were used, respectively, at 0.875 and 2 mM final in assay. Absorbance (405 nm)
were measured every 2 min for 30 min at 37 °C.
Body weight measurement: Determination of individual body weights of animals were
made three times weekly from study day -14, shortly before arthritis induction on day 0 (study
commencement) and subsequently on study days 3-8, 10, 11 and 12.
Clinical signs examination: Careful clinical examinations were carried out three times
weekly from study day -14, shortly before arthritis induction on day 0 and subsequently on
study days 3-8, 10, 11 and 12. Observations included changes in skin, fur, eyes, mucous
membranes, occurrence of secretions and excretions (e.g. diarrhoea) and autonomic activity
2
7

(e.g. lacrimation, salivation, piloerection, pupil size, unusual respiratory pattern). Changes in
gait, posture and response to handling, as well as the presence of bizarre behaviour, tremors,
convulsions, sleep and coma were also noted.
Arthritis reactions measurements: All paws (front and rear) of each animal were
examined for signs of arthritogenic responses prior to Test Item or control item
administration on study day 0 (study commencement) and subsequently on study days 3-
8
, 10, 11 and 12. Arthritis reactions were scored and recorded according to a 0-4 scale in
ascending order of severity as shown below:
Arthritis Score
Grade
0
No reaction, normal
Mild, but definite redness and swelling of the ankle/wrist or apparent redness and
swelling limited to individual digits, regardless of the number of affected digits
Moderate to severe redness and swelling of the ankle/wrist
Redness and swelling of the entire paw including digits
Maximally inflamed limb with involvement of multiple joints
1
2
3
4
The thickness of both hind paws (left & right, just above the foot pad and below the
calcaneum) of each animal was measured in mm on study day 0 (study commencement) and
subsequently on study days 3-8, 10, 11 and 12. Paw thickness was measured using a dial
caliper (Kroeplin, Munich, Germany).
Evaluation was primarily based on the mean values for arthritis scoring and paw
thickness measurements. Where appropriate, analysis of the data by ANOVA with Tukey
post hoc analysis was applied to determine significance of treatment effects.
3
. Results
3
.1. Design and synthesis of novel potent cell-permeable dipeptidyl cyclopropyl nitrile
inhibitors of human CatC
2
8

The dipeptidyl nitrile compound 1 with a (S)-2-amino butyric acid at P2 position and a
biphenyl at P1 position has been previously identified as a potent and selective dipeptidyl
31
nitrile inhibitor of human CatC (half maximal inhibitory concentration, IC = 13±3 nM ) and
5
0
was used here as a template for the development of new nitrile inhibitors of CatC. The
addition of p-fluoro or p-trifluoromethyl on the biphenyl moiety of compound 1 yielding
compound 2 and compound 3 resulted in most potent CatC inhibitors with IC values of 5.5
50
nM and 1.7 nM, respectively (Table 3). However, all three inhibitors displayed relatively
poor reactivity in cellular assays using U937 cells due to the rapid hydrolysis of their amide
bond in the cell assay medium and in plasma (not shown).
We inserted a 1,1-cyclopropyl amino nitrile moiety at P1 in compound 2 and
compound 3 to tentatively prevent hydrolysis and increase the ex-vivo and in vivo stability of
the inhibitor. The resulting compounds were fractionated into S,R,R (compounds 4a and 5a)
and S,S,S (compounds 4b and 5b) isomers. S,R,R isomers were clearly more efficient than
S,S,S isomers at inhibiting CatC (IC = 18 nM for compound 4a vs 1060 nM for compound
5
0
4
b; IC = 15 nM for compound 5a vs 1390 nM for compound 5b). Further, compound 4a and
50
compound 5a resisted hydrolysis in the cell assay medium and in plasma though IC values
5
0
increased significantly using a U937 cell based inhibition assay (Table 3). We then solved the
crystal structure of the human CatC-compound 5a complex to direct the synthesis of
additional dipeptidyl cyclopropyl nitrile inhibitors from compound 5a with improved cell
permeability and cellular potency. Compound 5a is located in the active site area, with the P1
side chain 4’-(trifluoromethyl)biphenyl pointing toward the solvent and the trifluoromethyl
group facing proline 3 (measured distance ~3Å) in the exclusion domain whereas the nitrile
group clearly made a covalent bond with Cys234 of the CatC active site (Figure 4A). The
amino group of the inhibitor makes two potential H-bonds to the side chain of Asp1. Its
peptide carbonyl group makes an H-bond to the main chain amine group of Gly277 and the
2
9