Design, synthesis, and in vitro evaluation of aza-peptide aldehydes and ketones as novel and selective protease inhibitors

Journal of Enzyme Inhibition and Medicinal Chemistry

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20

Design, synthesis, and in vitro evaluation of aza-

peptide aldehydes and ketones as novel and

selective protease inhibitors

Thomas S. Corrigan , Leilani M. Lotti Diaz , Sarah E. Border , Steven C.

Ratigan , Kayla Q. Kasper , Daniel Sojka , Pavla Fajtova , Conor R. Caffrey ,

Guy S. Salvesen , Craig A. McElroy , Christopher M. Hadad & Özlem Doğan

Ekici

To cite this article: Thomas S. Corrigan , Leilani M. Lotti Diaz , Sarah E. Border , Steven C.

Ratigan , Kayla Q. Kasper , Daniel Sojka , Pavla Fajtova , Conor R. Caffrey , Guy S. Salvesen ,

Craig A. McElroy , Christopher M. Hadad & Özlem Doğan Ekici (2020) Design, synthesis,

and in vitro evaluation of aza-peptide aldehydes and ketones as novel and selective protease

inhibitors, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1, 1387-1402, DOI:

10.1080/14756366.2020.1781107

To link to this article: https://doi.org/10.1080/14756366.2020.1781107

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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

2020, VOL. 35, NO. 1, 1387–1402

https://doi.org/10.1080/14756366.2020.1781107

SHORT COMMUNICATION

Design, synthesis, and in vitro evaluation of aza-peptide aldehydes and ketones as

novel and selective protease inhibitors

Thomas S. Corrigan^a, Leilani M. Lotti Diaz^a, Sarah E. Border^a, Steven C. Ratigan^b, Kayla Q. Kasper^a, Daniel Sojka^c,

Pavla Fajtova^d, Conor R. Caffrey^d, Guy S. Salvesen^e, Craig A. McElroy^b, Christopher M. Hadad^aand

a,f

€

ꢀ

Ozlem Dogan Ekici

b

^aDepartment of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, USA; Division of Medicinal Chemistry and

c

Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH, USA; Institute of Parasitology, Biology Centre of the Czech

d

Academy of Sciences, Ceske Budejovice, Czech Republic; Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy

e

and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA; Sanford Burnham Prebys Medical Discovery Institute, La

f

Jolla, CA, USA; Department of Chemistry and Biochemistry, The Ohio State University at Newark, Newark, OH, USA

ABSTRACT

ARTICLE HISTORY

Received 13 February 2020

Accepted 8 May 2020

Aza-peptide aldehydes and ketones are a new class of reversible protease inhibitors that are specific for

the proteasome and clan CD cysteine proteases. We designed and synthesised aza-Leu derivatives that

were specific for the chymotrypsin-like active site of the proteasome, aza-Asp derivatives that were effect-

ive inhibitors of caspases-3 and -6, and aza-Asn derivatives that inhibited S. mansoni and I. ricinus legu-

mains. The crystal structure of caspase-3 in complex with our caspase-specific aza-peptide methyl ketone

inhibitor with an aza-Asp residue at P1 revealed a covalent linkage between the inhibitor carbonyl carbon

and the active site cysteinyl sulphur. Aza-peptide aldehydes and ketones showed no cross-reactivity

towards cathepsin B or chymotrypsin. The initial in vitro selectivity of these inhibitors makes them suitable

candidates for further development into therapeutic agents to potentially treat multiple myeloma, neuro-

degenerative diseases, and parasitic infections.

KEYWORDS

Proteasome inhibitor;

caspase and legumain

inhibitors; aza-peptide

carbonyls; anticancer;

antiparasitic

Introduction

other classes of aza-peptidyl inhibitors have been reported as potent

and selective inhibitors of other proteases. Aza-peptide epoxides and

aza-peptide Michael acceptors have been shown to potently and

selectively inhibit clan CD proteases such as caspases, legumains, gin-

gipains, and clostripain by Powers and co-workers^4–7. Aza-peptide

nitriles were developed as effective and stable inhibitors for clan CA

Aza-peptides are peptidomimetics where the a-carbon centre of

one or more of the natural amino acids in a peptide chain is

replaced with a nitrogen atom (Figure 1). This modification results

in the loss of chirality of the amino acid and a bend in the pep-

tide chain. Previously, it has been shown that the aza-substitution

in biologically active peptide analogs has led to enhanced meta-

bolic stability and selectivity for the biological targets¹. Hence, in

medicinal chemistry, aza-peptides have been employed in many

roles including enzyme inhibitors, activity-based probes, ligands

for receptors, imaging agents, pro-drugs, and drugs. While studies

on retro hydrazino-aza-peptoids have been attempted with the

proteasome before², aza-peptide aldehydes and ketones emerge

as a new class of electrophilic protease inhibitors for multiple pro-

teases. Here we report the design, synthesis, and kinetic evalu-

ation of aza-peptide aldehydes and ketones that are specific for

the proteasome, caspases, and legumain.

proteases such as cathepsins L, S, and K by Guetschow^8,9

.

In our design, the a-carbon of the P1 amino acid is replaced

with a nitrogen, making it an aza-P₁residue (Figure 1). A carbonyl

group is attached to the P₁aza-amino acid replacing the scissile

bond of the natural substrate and creating the novel aza-peptide

aldehyde or ketone warhead.

The proteasome is a threonine protease and plays a central role

in the ubiquitin-mediated degradation of unwanted proteins in the

cell¹⁰. The proteasome is 750 kDa in molecular mass and has three

active sites within its 20S catalytic core: chymotrypsin-like (CT-L),

trypsin-like (T-L), and caspase-like (C-L)¹¹. Inhibition of the prote-

asome, which has been shown to be overexpressed in tumour

cells¹², leads to cellular apoptosis of the affected cells. Therefore,

this inhibition strategy has been validated as an effective treatment

for multiple myeloma patients¹³. Inhibitors, such as the aza-peptide

aldehydes and ketones described here, are designed to cease prote-

olysis of mis-folded and unwanted proteins through the selective

inhibition of the CT-L active site. Development of the proteasome

Incorporation of the aza-peptide motif in protease inhibition was

first introduced by Dolle and co-workers³. Several aza-peptidyl inhibi-

tors, including aza-peptide halomethyl ketones and aza-peptide

ketones, were designed and synthesised to target the clan CA cyst-

eine proteases cathepsin B and calpain, as well as the serine pro-

teases chymotrypsin and elastase. All of them were found to be

ineffective inhibitors of these enzymes. However, to date, several inhibitor blockbuster drugs bortezomib (FDA-approved in 2003),

€

ꢀ

CONTACT Ozlem Dogan Ekici

dogan-ekici.1@osu.edu

Department of Chemistry and Biochemistry, The Ohio State University at Newark, Newark, OH

43055, USA

Supplemental data for this article can be accessed here.

ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

1388

T. S. CORRIGAN ET AL.

Figure 1. Aza-peptide aldehyde and ketone design.

carfilzomib^14,15(FDA-approved in 2012) and ixazomib (FDA- at the P₁position in order to cleave peptide substrates³⁰. Legumain

approved in 2015) revolutionised the treatment of multiple mye-

loma, significantly prolonging the lives of patients¹⁶. However, bor-

tezomib shows severe side effects, such as peripheral neuropathy, in

up to 64% of newly diagnosed multiple myeloma patients,^17,18

largely due to off-target inhibition, by bortezomib’s boronic acid

warhead¹⁹, of HtrA2/Omi, a serine protease involved in neuronal sur-

vival. Carfilzomib has been shown to result in less peripheral neur-

opathy, but lately it has been associated with adverse cardiovascular

effects in up to 30% of the treated patients²⁰. Ixazomib is an oral

formulation and also possesses a boronic acid warhead; thus, per-

ipheral neuropathy is still a major side effect. Therefore, careful dos-

ing or discontinuation is also essential with ixazomib, as drug-

induced peripheral neuropathy can be irreversible²¹. Hence, the

search for new proteasome inhibitors is an ongoing challenge.

Caspases are a family of cysteine proteases that belong to a

small clan, the clan CD cysteine proteases. They have a strict

requirement for peptides containing an Asp residue at the P₁pos-

ition for substrate cleavage; hence, the name caspase evolved

from “cysteinyl aspartate specific protease.” To date, there are 14

caspases identified in humans. Most importantly, caspases are the

key-mediators of apoptosis (programmed cell death), and these

enzymes play essential roles in inflammation and neurodegenera-

tion^22,23. Hence, they have been recognised as novel therapeutic

targets for central nervous system diseases in which cell death

occurs mainly by an apoptotic mechanism²⁴. The roles of caspases

in the mechanism of apoptosis has been studied extensively since

the mid-1990s^25,26. Caspase-3 is a key executioner caspase respon-

sible either partially or totally for proteolytic cleavage of key apop-

totic proteins²⁷. It functions to decrease or destroy essential

homeostatic pathways during the effector phase of apoptosis.

Caspase-3 cleaves or activates nuclear enzymes, such as poly(ADP-

ribose) polymerase (PARP), the 70 kDa subunit of the U1 small

ribonucleoprotein, the catalytic subunit of DNA-dependent protein

kinase, and protein kinase Cd²⁸. Compared to the rest of the cas-

pases, caspase-3 is found to be inherently high in abundance in

biological environments. In addition, caspase-3 has higher abso-

lute k_cat/K_mvalues in general⁵. Therefore, most caspase-specific

inhibitors will show anti-apoptotic activity via inhibition of cas-

pase-3, also due to high cross-reactivity among caspases them-

selves. A good caspase inhibitor has to possess a P1 Asp as it is a

strict requirement for the caspase family. It is with this motivation,

we designed inhibitors specific for caspase-3 with the DEVD

sequence. Lately, caspase-6 has been shown to cleave many

proteins important in neurodegeneration such as tau, amyloid pre-

cursor protein and Huntingtin protein²⁹. Hence, an effective cas-

pase-6 inhibitor may offer treatments for neurodegenerative

diseases, including Alzheimer’s Disease and Huntington’s Disease.

Legumains are also clan CD cysteine proteases, sharing a com-

mon fold with caspases that is unique to this clan. Legumains are

is abundant in human solid tumours and promotes cell migration,

tissue invasion and metastasis³¹. The overexpression of legumain in

tumour tissues³²makes it an attractive target for cancer therapy.

Legumain has been described in plants; mammals^33,34; the human

blood fluke, Schistosoma mansoni³⁵; the protozoan parasite,

Trichomonas vaginalis⁷; and the ticks, Ixodes ricinus³⁶and

Haemaphysalis longicornis³¹. In the intestine of S. mansoni and I. rici-

nus, legumains contribute to digestion of the blood meal pro-

teins³⁷, including haemoglobin, and trans-activate other protease

zymogens involved in protein digestion³⁸. Therefore, in addition to

cancer therapy, the selective inhibition of legumains also offers a

therapeutic opportunity to effectively treat the diseases caused or

transmitted by these parasites³⁹which affect over 200 million peo-

ple worldwide, with a great majority being located in Africa⁴⁰.

Herein, we show that aza-peptide aldehydes and ketones are

selective inhibitors with considerable activity against the prote-

asome, caspases-3 and -6, as well as the legumains derived from

S. Mansoni and I. ricinus.

Materials and methods

Chemistry

All described reactions were carried out in flame/oven-dried glass-

ware under a nitrogen or argon atmosphere, unless otherwise

noted. Starting chemical reagents were purchased from reputable

suppliers (Sigma Aldrich, St. Louis, MO, USA; Fisher Scientific,

Waltham, MA, USA; Matrix Scientific, Columbia, SC, USA; Acros

Organics, Fair Lawn, NJ, USA) and used without further purification

or purified in accordance with procedures described in Purification

of Common Laboratory Chemicals. Product purification by flash

chromatography was carried out using normal phase 40–64 mm

60 Å silica gel from Sigma Aldrich or using a Teledyne Isco

CombiFlash RF þ UV auto column system. Dichloromethane, tetra-

hydrofuran, N,N-dimethylformamide, diethyl ether, and pentane

solvents were obtained from a solvent purification system (with

activated alumina columns). Triethylamine and diisopropylethyl-

amine were freshly distilled over CaH₂prior to use and stored

under an argon atmosphere. NMR experiments were performed

on Bruker 400, 700 or 850 MHz spectrometers. Chemical shifts are

expressed in parts per million (d, ppm) while coupling constant

values (J) are given in Hertz (Hz). Residual solvent protons were

1

used as internal standards: for H NMR spectra CDCl₃¼ 7.26 ppm,

DMSO-d₆¼ 2.50 ppm and D₂O ¼ 4.79 ppm while for ¹³C NMR

spectra CDCl₃¼ 77.0 ppm and DMSO-d₆¼ 41.23 ppm; CDCl₃and

DMSO-d₆were purchased from Cambridge Isotope Laboratories.

High resolution mass spectra were recorded on a Bruker MicroTOF

II instrument with internal sodium formate calibrant under electro-

acidic lysosomal enzymes that strictly require an asparagine residue spray ionisation (ESI) conditions. Hydrogenations were carried out

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1389

using a Parr hydrogenation apparatus. Kinetic assays were per- 7.92 (d, J ¼ 7.8 Hz, 1H), 7.60 (d, J ¼ 8.3 Hz, 1H), 7.48–7.23 (m, 5H),

formed on a Molecular Devices SpectraMax i3.

5.04 (d, J ¼ 5.7 Hz, 2H), 4.42–4.30 (m, 1H), 4.00–4.19 (m, 2H), 3.62

(s, 3H), 2.65 (dd, J ¼ 15.75, 5.66 Hz, 1H), 2.43 (dd, J ¼ 16.1, 9.1 Hz,

1H), 2.22 (m, 2H), 2.04 (s, 1H), 1.94–1.81 (m, 1H), 1.79–1.67 (m, 1H),

1.40 (s, 10H, Boc and Val CH), 1.37 (s, 9H), 0.88 (t, J ¼ 7.4 Hz, 6H).

Abbreviations. The following abbreviations have been used:

AMC, 7-amino-4-methyl coumarin; AAla, aza-alanine residue; AAsp,

aza-aspartic acid residue; AAsn, aza-asparagine residue; AGly, aza-

glycine residue; ALeu, aza-leucine residue; Cbz, Ph-CH₂-OCO-; Pz,

pyrazinyl; DCM, dichloromethane; DMF, N,N-dimethylformamide;

DMSO, dimethyl sulfoxide; DTT, dithiothreitol; EtOAc, ethyl acetate;

iBCF, isobutyl chloroformate; MeOH, methanol; NMM, N-methyl-

morpholine; NTA, nitrilotriacetic acid; RT, room temperature; TEV,

tobacco etch virus; THF, tetrahydrofuran.

Peptide methyl esters

tert-Butyl

(S)-4-(((benzyloxy)carbonyl)amino)-5-(((S)-1-methoxy-3-

methyl-1-oxobutan-2-yl)amino)-5-oxopentanoate (Cbz-Glu(OtBu)-

Val-OMe). See procedure and workup of Cbz-Leu-Leu-OMe. The

product was obtained as a colourless oil in quantitative yield. ¹H

NMR (DMSO-d₆, 400 MHz): 0.88 (t, 6H), 1.39 (s, 9H), 1.72 (m, 1H),

1.85 (m, 1H), 2.05 (q, 1H), 2.25 (t, 2H), 3.63 (s, 3H), 4.16 (m, 2H),

5.02 (d, 2H), 7.35 (m, 5H), 7.42 (d, 1H), 8.10 (d, 1H).

Methyl ((benzyloxy)carbonyl)-L-leucyl-L-leucinate (Cbz-Leu-Leu-

OMe). Cbz-Leu-OH (5.3 g, 20 mmol) was dissolved in dry THF

(200 ml) and cooled to ꢀ20 ^ꢁC. NMM (2.18 ml, 20 mmol) and iBCF

(2.59ml, 20 mmol) were added dropwise and the mixture was

allowed to react for 30min. Separately, H-Leu-OMe (3.6 g, 20mmol)

was dissolved in dry THF (200ml) and cooled to ꢀ20^ꢁC and NMM

(2.18 ml, 20 mmol) was added dropwise. This mixture was allowed

to stir for 15min. The two mixtures were then combined and

allowed to stir for 1 h at ꢀ20^ꢁC. Following, the solution was

allowed to stir while gradually warming to RT for 18h. The solvent

was removed in vacuo, and the resulting residue was dissolved

with EtOAc, washed with 1 M HCl, H₂O, saturated NaHCO₃, and

saturated brine. The organic layer was dried over Na₂SO₄, and con-

centrated in vacuo to afford the product as a white solid (7.0 g,

90% yield). ¹H NMR (DMSO-d₆, 400 MHz): 8.19 (d, J ¼ 7.80 Hz, 1H),

7.39–7.28 (m, 5H), 5.00 (s, 2H), 4.33–4.25 (m, 1H), 4.12–4.01 (m, 1H),

3.59 (s, 3H), 1.71–1.36 (m, 6H), 0.93–0.81 (m, 12H).

tert-Butyl

(S)-4-(((benzyloxy)carbonyl)amino)-5-(((2S,3S)-3-(tert-

butoxy)-1-methoxy-1-oxobutan-2-yl)amino)-5-oxopentanoate (Cbz-

Glu(OtBu)-Thr(OtBu)-OMe). At ꢀ15 ^ꢁC a solution of Cbz-Glu(OtBu)-

OH (6.74g, 20mmol) in THF (200ml) was treated with NMM (2.97ml,

20 mmol) and allowed to stir for 10 min. The solution was then

treated with iBCF (2.59 ml, 20 mmol) and allowed to stir for 30 min at

ꢀ15 ^ꢁC. A second solution of H-Thr(tBu)-OMe (4.51 g, 20 mmol) in THF

(200 ml) at ꢀ15 ^ꢁC was treated with NMM (2.97 ml, 20 mmol) and

stirred for 15 min. The solutions were combined and stirred for 1 h at

ꢀ15 ^ꢁC, then allowed to stir while gradually warming to RT for 16 h.

The solvent was removed in vacuo and the resulting residue was dis-

solved in EtOAc, washed with 1 M HCl, H₂O, saturated Na₂HCO₃, and

saturated brine. The organic layer was dried over Na₂SO₄and

removed in vacuo to afford the product as a white solid (8.07 g, 79%

Methyl ((benzyloxy)carbonyl)-L-alanyl-L-alaninate (Cbz-Ala-Ala-

OMe). See procedure and workup of Cbz-Leu-Leu-OMe. A white

solid was obtained in 83% yield. ¹H NMR (DMSO-d₆, 400 MHz):

8.26 (d, J ¼ 7.0 Hz, 1H), 7.40 (d, J ¼ 7.6 Hz, 1H), 7.36 (m, 5H), 5.01

(d, J ¼ 3.26 Hz, 2H), 4.27 (p, J ¼ 7.16 Hz, 1H), 4.08 (p, J ¼ 7.35 Hz,

1H), 3.62 (s, 3H), 1.29 (d, J ¼ 7.3 Hz, 3H), 1.20 (d, J ¼ 7.1 Hz, 3H).

1

yield). H NMR (DMSO-d₆, 400 MHz) d: 7.67 (d, J¼ 8.8 Hz, 1H), 7.53 (d,

J¼ 8.2 Hz, 1H), 7.41–7.27 (m, 5H), 5.03 (s, 2H), 4.35 (dd, J¼ 8.8, 2.7 Hz,

1H), 4.24–4.12 (m, 2H), 3.61 (s, 3H), 2.35–2.21 (m, 2H), 1.97–1.83 (m,

1H), 1.78–1.66 (m, 1H), 1.38 (s, 9H), 1.07 (s, 12H); HRMS (ESI) calcd for

[C₂₆H₄₀N₂O₈þNa]^þ, 531.2685, found 531.2699 (M þ 23).

Methyl (5S,8S,11S)-5–(2-(tert-butoxy)-2-oxoethyl)-8–(3-(tert-butoxy)-

3-oxopropyl)-11-isopropyl-3,6,9-trioxo-1-phenyl-2-oxa-4,7, 10-triazado-

decan-12-oate (Cbz-Asp(OtBu)-Glu(OtBu)-Val-OMe). See procedure

and workup of Cbz-Leu-Leu-OMe. A white foam was obtained in

86% yield. ¹H NMR (DMSO-d₆, 400 MHz): 8.12 (d, J ¼ 7.8 Hz, 1H),

1390

T. S. CORRIGAN ET AL.

tert-Butyl (S)-4-amino-5-(((2S,3S)-3-(tert-butoxy)-1-methoxy-1-oxobu- (150 ml) and hydrazine was added dropwise (5.59 ml, 178 mmol).

tan-2-yl)amino)-5-oxopentanoate (H-Glu(OtBu)-Thr(OtBu)-OMe).

A

The solution was allowed to stir for 18 h at RT. The solvent was

removed in vacuo to afford Cbz-Leu-Leu-NHNH₂as white solid

(7.0 g, 99% yield). ¹H NMR (DMSO-d₆, 400 MHz): 9.18 (s, 1H), 7.86

(d, J ¼ 9.01 Hz, 1H), 7.44 (d, J ¼ 7.71 Hz, 1H), 7.44–7.27 (m, 5H), 5.03

(s, 2H), 4.32–4.23 (m, 1H), 4.10–4.00 (m, 1H), 1.68–1.51 (m, 2H),

1.50–1.28 (m, 4H), 0.82–0.88 (m, 12H).

solution of tert-butyl (S)-4-(((benzyloxy)carbonyl)amino)-5-(((2S,3S)-

3-(tert-butoxy)-1-methoxy-1-oxobutan-2-yl)amino)-5-oxopentanoate

(8.07 g, 15.87 mmol) in MeOH (300 ml) was treated with 10% Pd/C

and placed under H₂at 40 psi for 2 h. The solution was filtered

over Celite and concentrated in vacuo to afford the product as a

white solid (5.36 g, 90% yield). ¹H NMR (DMSO-d₆, 400 MHz) d:

8.03 (d, J ¼ 9.1 Hz, 1H), 4.30 (dd, J ¼ 9.0, 2.5 Hz, 1H), 4.16 (qd,

J ¼ 6.2, 2.6 Hz, 1H), 3.63 (s, 3H), 3.24 (dd, J ¼ 7.9, 4.9 Hz, 1H), 3.17

(s, 1H), 2.30 (dd, J ¼ 16.7, 8.5 Hz, 2H), 1.98–1.86 (m, 1H), 1.57 (dq,

J ¼ 13.7, 7.6 Hz, 1H), 1.39 (s, 9H), 1.08 (s, 12H); HRMS (ESI) calcd for

[C₁₈H₃4N₂O₆þH]^þ, 375.2495, found: 375.2486 (M þ 1).

Benzyl ((S)-4-methyl-1-(((S)-4-methyl-1–(2-((E)-2-methylpropyli-

dene)hydrazineyl)-1-oxopentan-2-yl)amino)-1-oxopentan-2-yl)car-

bamate (Cbz-Leu-Leu-NHN ¼ CHCH(CH₃)₂). Cbz-Leu-Leu-NHNH₂

(7.0 g, 17.8 mmol) was dissolved in dry THF (200 ml) followed by

the addition of isobutyraldehyde (1.79 ml, 19.6 mmol) and catalytic

acetic acid (1 drop). The solution was allowed to stir for 18 h at RT.

The reaction was quenched with saturated NaHCO₃, extracted

with DCM. The organic layer was dried over Na₂SO₄and removed

in vacuo to afford Cbz-Leu-Leu-NHN ¼ CHCH(CH₃)₂as a white solid

(8 g, 99% yield). ¹H NMR (DMSO-d₆, 400 MHz): 10.81,11.01 (s, 1H),

7.93, 7.77 (d, J ¼ 8.3 Hz, 1H), 7.74 (d, 1H), 7.41 (d, 1H), 7.40–7.24

(m, 5H), 5.02 (s, 2H), 4.12–4.00 (m, 2H), 1.48–1.31 (m, 2H),

1.08–0.99 (m, 1H), 0.93–0.75 (m, 18H).

Methyl (5S,8S,11S)-8–(3-(tert-butoxy)-3-oxopropyl)-11-((S)-1-(tert-

butoxy)ethyl)-5-((R)-s-butyl)-3,6,9-trioxo-1-phenyl-2-oxa-4,7,10-tri-

azadodecan-12-oate tert-butyl (S)-4-(((benzyloxy)carbonyl)amino)-

5-(((2S,3S)-3-(tert-butoxy)-1-methoxy-1-oxobutan-2-yl)amino)-5-oxo-

pentanoate (Cbz-Ile-Glu(OtBu)-Thr(OtBu)-OMe). A solution of Cbz-

Ile-OH (3.80 g, 14.33 mmol) in THF (100 ml) was cooled to ꢀ15 ^ꢁC

followed by the addition NMM (1.58 ml, 14.33 mmol) and allowed

to stir for 10 min. The solution was then treated with iBCF

(1.86 ml, 14.33 mmol) and allowed to stir for 30 min at ꢀ15 ^ꢁC.

Separately, a solution of tert-butyl (S)-4-amino-5-(((2S,3S)-3-(tert-

butoxy)-1-methoxy-1-oxobutan-2-yl)amino)-5-oxopentanoate (5.37g,

14.33 mmol) in THF (100 ml) at ꢀ15 ^ꢁC was treated with NMM

(1.58 ml, 14.33 mmol) and stirred for 15 min. The solutions were

combined and allowed to stir for 1 h at ꢀ15 ^ꢁC. Following, the

solution was allowed to stir while gradually warming to RT for

16 h. The solvent was removed in vacuo, the resulting residue was

dissolved in EtOAc, washed with 1 M HCl, H₂O, saturated

Na₂HCO₃, and saturated brine. The organic layer was dried over

Na₂SO₄and concentrated in vacuo to afford the product as a

white solid (6.30 g, 10.13 mmol, 70% yield). ¹H NMR (DMSO-d₆,

400 MHz) d: 8.11 (d, J ¼ 8.0 Hz, 1H), 7.68 (d, J ¼ 8.8 Hz, 1H),

7.41–7.25 (m, 6H, aromatic þ NH), 5.03 (s, 2H), 4.52–4.42 (m, 1H),

4.36 (dd, J ¼ 8.99, 2.42 Hz, 1H), 4.18–4.11 (m, 1H), 3.93 (t,

J ¼ 8.51 Hz, 1H), 3.62 (s, 3H), 2.35–2.15 (m, 2H), 1.99–9.80 (m, 1H),

1.79–1.62 (m, 2H), 1.49–1.31 (m, 9H), 1.14–1.02 (m, 14H, CH₃þ Ile

CH₂), 0.93–0.73 (m, 6H); HRMS (ESI) calcd for [C₃₂H₅₁N₃O₉þNa]^þ,

644.3525, found: 644.3485 (M þ 23).

Benzyl

((S)-1-(((S)-1–(2-isobutylhydrazinyl)-4-methyl-1-oxopen-

(Cbz-Leu-

tan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate

Leu-NHNHCHCH(CH₃)₂)_.A solution of benzyl ((S)-4-methyl-1-(((S)-4-

methyl-1–(2-((E)-2-methylpropylidene)hydrazinyl)-1-oxopentan-2-yl)

amino)-1-oxopentan-2-yl)carbamate (1 g, 2.24 mmol) in MeOH

(25 ml) was treated with acetic acid (0.2 ml) followed by sodium

cyanoborohydride (170 mg, 2.69 mmol). The reaction was stirred at

RT for 16 h, then the pH was adjusted to 10 by addition of 1 M

NaOH. The solvent was removed in vacuo, and the residue was

dissolved in DCM. The organic layer was washed with water, satu-

rated brine, dried over Na₂SO₄and concentrated in vacuo to

afford the product as a white solid (874 mg, 1.95 mmol, 87% yield).

¹H NMR (DMSO-d₆, 400 MHz) d: 9.25 (d, J ¼ 4.2 Hz, 1H), 7.87 (d,

J ¼ 6.73 Hz, 1H), 7.30 (d, J ¼ 7.32, 1H), 7.28–7.39 (m, 5H), 6.47 (d,

1H), 5.10 (s, 2H), 4.33–4.42 (m, 1H), 4.10–4.19 (m, 1H), 2.60 (m, 1H),

1.42–1.82 (m, 9H), 0.8–1.05 (m, 18H). HRMS (ESI) calcd for

[C₂₄H₄₀NO_4þNa]^þ471.2927, Found: 471.2950 (M þ 23).

Proteasome inhibitors

Compound 3: Benzyl ((S)-1-(((S)-1–(2-isobutyl-2–(2-oxoacetyl)hy-

Benzyl

((S)-1-(((S)-1-hydrazineyl-4-methyl-1-oxopentan-2-yl)a-

mino)-4-methyl-1-oxopentan-2-yl)carbamate (Cbz-Leu-Leu-NHNH₂). drazinyl)-4-methyl-1-oxopentan-2-yl)amino)-4-methyl-1-oxopentan-

Cbz-Leu-Leu-OMe (7.0 g, 17.8 mmol) was dissolved in MeOH 2-yl)carbamate. (Cbz-Leu-Leu-ALeu-CHO). A 0 ^ꢁC solution of benzyl

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1391

((S)-1-(((S)-1–(2-isobutylhydrazinyl)-4-methyl-1-oxopentan-2-yl)a-

DCM (3 ml) and chilled to 0 ^ꢁC. To this chilled solution was added

mino)-4-methyl-1-oxopentan-2-yl)carbamate (50 mg, 0.11 mmol) in a solution of benzyl ((S)-1-(((S)-1–(2-isobutylhydrazinyl)-4-methyl-1-

dry DCM (2 ml) was treated with a solution of 2-oxoacetyl chloride oxopentan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate

(13 mg, 0.14 mmol) in DCM (0.5 ml) followed by DIPEA (0.02 ml, (168 mg, 0.37 mmol) in DCM (1 ml) followed by DIPEA (0.13 ml,

0.14 mmol). The reaction was warmed to RT and stirred for 2 h. 0.74 mmol). The reaction was warmed to RT and stirred 16 h. The

The reaction was quenched with H₂O and extracted with DCM. reaction mixture was diluted with H₂O and extracted with DCM.

The organic layer was washed with saturated NaHCO₃, dried over The organic layer was dried over Na_sSO₄and concentrated in

Na₂SO₄and concentrated in vacuo. The crude product was puri- vacuo. The crude product was purified by silica gel chromatog-

fied by silica gel chromatography (2% MeOH/DCM) to afford the raphy (5% MeOH/DCM) and recrystallised from EtOAc/Et₂O to

product as a white solid (18 mg, 32% yield). ¹H NMR (DMSO-d₆, afford the pure product as a white solid (38 mg, 17% yield). ¹H

400 MHz) d 9.08 (s, 1H), 7.96 (s, 1H), 7.49–7.24 (m, 5H), 5.01 (s, 2H), NMR (CDCl₃, 400 MHz) d: 8.92 (s, 1H), 7.27–7.41 (m, 10H), 7.15–7.20

4.37–4.20 (m, 1 H), 4.14–4.01 (m, 1H), 3.78–3.66 (m, 1H), 1.77–1.32 (d, J ¼ 7.07 Hz, 1H), 6.06 (d, J ¼ 9.07 Hz, 1H), 5.04–5.14 (m, 2H),

(m, 7 H), 0.94–0.71 (m, 18H); ¹³C NMR (213 MHz, DMSO-d₆) d (ppm)

4.31–4.92 (m, 1H), 3.92–4.20 (m, 3H, CH and Bn), 3.40–3.65 (s, 1H),

3.20–3.37 (m, 1H), 1.84–1.99 (m, 1H), 1.49–1.71 (m, 6H), 1.22–1.35

¼ 189.9, 173.9, 173.0, 157.6, 138.8, 130.2, 129.4, 129.3, 67.0, 54.6,

(m, 2H), 0.81–0.95 (m, 18H). ¹³C NMR (213 MHz, DMSO-d₆): d

54.3, 42.5, 42.1, 25.3, 24.2, 23.2, 22.0; HRMS (ESI) calcd for

[C₂₇H₄₄N₄O₇þNa]^þ559.3108, found 559.3098 (MeOH hemiacetal)

(ppm) ¼ 198.4, 173.9, 156.3, 137.6, 130.4, 130.1, 129.0, 128.9,

128.8, 128.7, 128.2, 128.1, 65.8, 53.9, 53.3, 42.2, 41.1, 24.6, 23.5,

23.3, 22.0, 21.9, 21.1, 20.3; HRMS (ESI) calcd for [C₃₃H₄₆N₄O₆þNa]^þ

617.3316, found: 617.3308.

Caspase-3 inhibitors

Compound 4: Benzyl ((S)-1-(((S)-1–(2-isobutyl-2–(2-oxopropa-

noyl)hydrazinyl)-4-methyl-1-oxopentan-2-yl)amino)-4-methyl-1-oxo-

pentan-2-yl)carbamate (Cbz-Leu-Leu-ALeu-COMe). A 0 ^ꢁC solution

of pyruvic acid (0.17 ml, 0.69 mmol) in DCM (10 ml) was treated

with oxalyl chloride (0.06 ml, 0.72 mmol) followed by DMF (1 drop).

The reaction mixture was warmed to RT and stirred 2 h. The solv-

ent was removed in vacuo, and residue was dissolved in fresh

DCM (5 ml) and chilled to 0 ^ꢁC. A solution of benzyl ((S)-1-(((S)-

1–(2-isobutylhydrazinyl)-4-methyl-1-oxopentan-2-yl)amino)-4-

methyl-1-oxopentan-2-yl)carbamate (200 mg, 0.45 mmol) was

added, followed by the addition of DIPEA (0.15 ml, 0.9 mmol). The

reaction was stirred 1 h at 0 ^ꢁC, then warmed to RT and stirred for

an additional 16 h. The reaction mixture was diluted with H₂O and

extracted with EtOAc. Organic extracts were dried over Na₂SO₄

and concentrated in vacuo. The crude product was purified by sil-

ica gel chromatography (20–40% EtOAc/Hexanes) to afford the

product as a white solid (27 mg, 7.5% yield). ¹H NMR (DMSO-d₆,

400 MHz) d 9.12 (s, 1H), 7.28–7.47 (m, 5H), 6.38 (d, J ¼ 7.74 Hz, 1H),

5.24 (br s, 1H), 5.13 (d, J ¼ 3.64 Hz, 2H), 4.45 (br s, 1H), 4.05–4.22

(m, 1H), 3.21–3.56 (m, 2H), 2.44 (s, 1H), 1.87–1.98 (m, 1H),1.40–1.75

(m, 6H), 0.86–0.98 (m, 18H).¹³C NMR (213 MHz, DMSO-d₆): d (ppm)

¼ 200.3, 174.1, 157.6, 138.8, 130.0, 129.5, 129.3, 67.0, 55.1, 54.5,

51.1, 42.3, 41.7, 28.9, 25.8, 24.7, 24.3, 23.2, 21.5; HRMS (ESI) calcd

for [C₂₇H₄₂N₄O₆þNa]^þ541.2997, found: 541.2981.

tert-Butyl (S)-4-((S)-2-(((benzyloxy)carbonyl)amino)-4-(tert-butoxy)-4-

oxobutanamido)-5-(((S)-1-hydrazinyl-3-methyl-1-oxobutan-2-yl)a-

mino)-5-oxopentanoate (Cbz-Asp(OtBu)-Glu(OtBu)-Val-NHNH₂). A

solution of methyl (5S,8S,11S)-5–(2-(tert-butoxy)-2-oxoethyl)-8–(3-

(tert-butoxy)-3-oxopropyl)-11-isopropyl-3,6,9-trioxo-1-phenyl-2-oxa-

4,7,10-triazadodecan-12-oate (7.9 g, 12.7 mmol) in) MeOH (80 ml)

was treated with hydrazine (2.67 ml, 80 mmol). The reaction was

stirred at RT for 18 h. The solvent and excess hydrazine were

removed in vacuo to afford the product as a white solid. This was

used for further transformations without additional purification

(7.9 g, quantitative). ¹H NMR (DMSO-d₆, 400 MHz) d: 9.16 (s, 1H),

7.98 (d, J ¼ 8.1 Hz, 1H), 7.80 (d, J ¼ 8.8 Hz, 1H), 7.64 (d, J ¼ 8.3 Hz,

1H), 7.44–7.26 (m, 5H), 5.13–4.95 (m, 2H), 4.46–4.11 (m, 4H), 4.04

(t, J ¼ 6.83 Hz, 1H), 2.69–2.36 (m, 2H), 2.30–2.14 (m, 2H), 1.96–1.81

(m, 2H), 1.52–1.30 (m, 18H), 0.89–0.76 (m, 6H); HRMS (ESI) calcd

for [C₃₀H₄₈N₅O₉]^þ622.3447, found: 622.3415.

Compound 5: Benzyl ((S)-1-(((S)-1–(2-isobutyl-2–(2-oxo-3-phenyl- tert-Butyl

(5S,8S,11S)-5–(2-(tert-butoxy)-2-oxoethyl)-8–(3-(tert-

propanoyl)hydrazinyl)-4-methyl-1-oxopentan-2-yl)amino)-4-methyl-

butoxy)-3-oxopropyl)-11-isopropyl-3,6,9,12-tetraoxo-1-phenyl-2-

1-oxopentan-2-yl)carbamate (Cbz-Leu-Leu-ALeu-COBn). To a solu- oxa-4,7,10,13,14-pentaazahexadecan-16-oate (Cbz-Asp(OtBu)-Glu

tion of phenylpyruvic acid (73 mg, 0.44 mmol) in DCM (2 ml) oxalyl (OtBu)-Val-NHNHCH₂CO₂tBu). A ꢀ15 ^ꢁC solution of tert-butyl (S)-

chloride (0.04 ml, 0.44 mmol) was added, followed by DMF 4-((S)-2-(((benzyloxy)carbonyl)amino)-4-(tert-butoxy)-4-oxobutana-

(1 drop). The reaction mixture was stirred at RT for 2 h, then con- mido)-5-(((S)-1-hydrazinyl-3-methyl-1-oxobutan-2-yl)amino)-5-oxo-

centrated in vacuo. The resulting residue was dissolved in fresh pentanoate (1 g, 1.6 mmol) in DMF (6 ml) was treated with the

1392

T. S. CORRIGAN ET AL.

dropwise addition of t-butyl bromoacetate (0.26 ml, 1.76 mmol).

The reaction was stirred for 30 min, then warmed to RT and stirred

for 16 h. The solvent was removed in vacuo, and the residue was

purified by silica gel chromatography (4% MeOH/DCM) to afford

the title product as a white solid (115 mg, 10% yield); ¹H NMR

(DMSO-d₆, 400 MHz) d: 9.48 (d, J ¼ 6.16 Hz, 1H), 7.95 (d, J ¼ 7.44 Hz,

1H), 7.85 (d, J ¼ 8.76 Hz, 1H), 7.62 (d, J ¼ 8.76 Hz, 1H), 7.47–7.22 (m,

5 H), 5.18–5.11 (m, 1H), 5.10–4.98 (m, 2H), 4.51–4.19 (m, 2H),

4.11–3.99 (m, 1H), 3.41–3.38 (m, 2H), 2.27–2.16 (m, 2H), 1.96–1.69

(m, 5H), 1.46–1.34 (m, 27H), 0.86–0.77 (m, 6H).

Compound 6: (5S,8S,11S)-8–(2-Carboxyethyl)-5-(carboxymethyl)-

11-isopropyl-3,6,9,12-tetraoxo-14–(2-oxopropanoyl)-1-phenyl-2-oxa-

4,7,10,13,14-pentaazahexadecan-16-oic

acid

(Cbz-Asp-Glu-Val-

AAsp-COMe). A solution of tert-butyl (5S,8S,11S)-5–(2-(tert-butoxy)-

2-oxoethyl)-8–(3-(tert-butoxy)-3-oxopropyl)-11-isopropyl-3,6,9,12-

tetraoxo-14–(2-oxopropanoyl)-1-phenyl-2-oxa-4,7,10,13,14-

pentaazahexadecan-16-oate (36 mg, 0.045 mmol) in DCM

(0.5 ml) was treated with trifluoroacetic acid (0.5 ml). The reaction

was stirred 2 h, then concentrated to dryness in vacuo to afford

the product as a white solid (27 mg, 95% yield). ¹H NMR (DMSO-

d₆, 400 MHz) d: 11.02 (br s, 1H), 8.17–7.78 (m, 2H), 7.73–7.53 (d,

J ¼ 8.0 Hz, 1H), 7.42–7.29 (m, 5H), 5.04 (s, 2H), 4.42–4.29 (m, 2H),

4.13 (t, J ¼ 6.8 Hz, 1H), 2.70–2.57 (m, 2H), 2.48–2.43 (m, 1H),

2.29–2.18 (m, 5H), 1.96–1.82 (m, 2H), 0.82 (d, J ¼ 6.4 Hz, 6H). ¹³C

NMR (213 MHz, DMSO-d₆): d (ppm) ¼ 199.7, 175.7, 173.4, 173.2,

172.9, 172.8, 172.5, 170.4, 170.0, 157.5, 138.6, 130.1, 129.5, 129.4,

67.2, 57.8, 53.3, 53.0, 50.3, 37.9, 31.7, 31.6, 29.1, 20.5, 19.6; HRMS

(ESI)

calcd

for

[C₂₇H₃₅N₅O₁₃þNa]^þ

660.2124,

found

660.2116 (M þ 23).

tert-Butyl (5S,8S,11S)-5–(2-(tert-butoxy)-2-oxoethyl)-8–(3-(tert-butoxy)-

3-oxopropyl)-11-isopropyl-3,6,9,12-tetraoxo-14–(2-oxopropanoyl)-1-

phenyl-2-oxa-4,7,10,13,14-pentaazahexadecan-16-oate

(Cbz-Asp

(OtBu)-Glu(OtBu)-Val-AAsp(OtBu)-COMe). Prepared following the

general procedure for coupling of aza-peptide to pyruvic acid as

1

previously described; (36 mg, 29% yield); H NMR (CDCl₃, 400 MHz)

d: 9.02 (s, 1H), 7.90 (d, J ¼ 4.99 Hz, 1H), 7.46–7.33 (m, 5H),

6.11–5.99 (m, 1H), 5.22–5.10 (m, 2H), 4.55–4.45 (m, 1H), 4.37–4.25

(m, 2H), 4.24–4.16 (m, 1H), 3.01–2.74 (m, 2H), 2.57–2.39 (m 2H),

2.37 (s, 3H), 2.33–2.24 (m, 1H), 2.19–1.99 (m, 2H), 1.49–1.44 (m,

27H), 0.97–0.92 (m, 6H); HRMS (ESI) calcd for [C₃₉H₅₉N₅O₁₃þNa]:

828.4002, found: 828.3984.

Compound 7: (5S,8S,11S)-8–(2-Carboxyethyl)-5-(carboxymethyl)-

11-isopropyl-3,6,9,12-tetraoxo-14–(2-oxo-3-phenylpropanoyl)-1-phe-

nyl-2-oxa-4,7,10,13,14-pentaazahexadecan-16-oic acid (Cbz-Asp-Glu-

Val-AAsp-COBn). tert-Butyl (5S,8S,11S)-5–(2-(tert-butoxy)-2-oxoethyl)-

8–(3-(tert-butoxy)-3-oxopropyl)-11-isopropyl-3,6,9,12-tetraoxo-14–(2-

oxo-3-phenylpropanoyl)-1-phenyl-2-oxa-4,7,10,13,14-pentaazahexa-

decan-16-oate (18 mg, 0.02 mmol) was dissolved in a 1:1 DCM/tri-

fluoroacetic acid mixture (1.5 ml). The mixture was stirred for 2.5 h,

then solvent was removed, and product was dried in vacuo to

give the title compound as a white solid (13.6 g, 95%). ¹H NMR

(DMSO-d₆, 400 MHz) d: 11.06 (s, 1H), 8.02–7.91 (m, 2H), 7.61 (d,

J ¼ 8.0 Hz, 1H), 7.42–7.15 (m, 10H), 5.03 (s, 2H), 4.44–4.27 (m, 3H),

4.21–4.12 (m, 2H), 4.06–3.94 (m, 3H), 2.70–2.60 (m, 1H), 2.80–2.18

(m, 2H), 1.95–1.84 (m, 2H), 1.80–1.70 (m, 1H), 0.84–0.70 (m, 6H);

¹³C NMR (213 MHz, DMSO-d₆): d (ppm) ¼ 199.3, 173.4, 173.3,

173.0, 172.5, 170.4, 157.5, 138.6, 131.2, 131.7, 131.6, 130.0, 129.9,

129.5, 129.4, 128.6, 67.2, 57.8, 53.4, 53.0, 50.3, 46.8, 37.9, 31.7, 26.4,

20.7, 19.5; HRMS (ESI) calcd for [C₃₃H₃₉N₅O₁₃þNa]^þ736.2437,

found: 736.2415 (M þ 23).

tert-Butyl (5S,8S,11S)-5–(2-(tert-butoxy)-2-oxoethyl)-8–(3-(tert-butoxy)-

3-oxopropyl)-11-isopropyl-3,6,9,12-tetraoxo-14–(2-oxo-3-phenylpropa-

noyl)-1-phenyl-2-oxa-4,7,10,13,14-pentaazahexadecan-16-oate (Cbz-

Asp(OtBu)-Glu(OtBu)-Val-AAsp(OtBu)-COBn). This product was pre-

pared following the general procedure for coupling of an aza-pep-

tide to phenylpyruvic acid as previously described: (19 mg, 18%

yield); ¹H NMR (DMSO-d₆, 400 MHz) d: 11.07 (s, 1H), 8.00 (d,

J ¼ 8.3 Hz, 1H), 7.93 (d, J ¼ 7.7 Hz, 1H), 7.60 (d, J ¼ 8.3 Hz, 1H),

7.40–7.15 (m, 11H), 5.12–4.96 (m, 2H), 4.41–4.28 (m, 2H), 4.21–4.12

(m, 1H), 4.10–3.96 (m, 2H), 2.70–2.57 (m, 1H), 2.47–2.39 (m, 1H),

2.26–2.14 (m, 2H), 1.94–1.80 (m, 2H), 1.77–1.65 (m, 2H), 1.44 (s,

9 H), 1.37–1.32 (m, 18H), 0.81–0.75 (m, 6H); HRMS (ESI) calcd for

[C₄₅H₆₃N₅O₁₃þNa]: 904.4315, found: 904.4282.

Caspase-6 inhibitor

tert-Butyl

(S)-4-((2S,3R)-2-(((benzyloxy)carbonyl)amino)-3-methyl-

pentanamido)-5-(((2S,3S)-3-(tert-butoxy)-1-hydrazineyl-1-oxobutan-

2-yl)amino)-5-oxopentanoate (Cbz-Ile-Glu(OtBu)-Thr(OtBu)-NHNH₂).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1393

A solution of methyl (5S,8S,11S)-8–(3-(tert-butoxy)-3-oxopropyl)-11- 8–(3-(tert-butoxy)-3-oxopropyl)-11-((S)-1-(tert-butoxy)ethyl)-5-((R)-s-

((S)-1-(tert-butoxy)ethyl)-5-((R)-s-butyl)-3,6,9-trioxo-1-phenyl-2-oxa-

4,7,10-triazadodecan-12-oate (2.08 g, 3.35 mmol) in MeOH (10 ml)

was treated with hydrazine (5.3 ml, 169.8 mmol). The reaction mix-

ture was stirred at RT for 16 h. Excess hydrazine and MeOH were

removed in vacuo to afford the product as a white solid (9.80 g,

98% yield). ¹H NMR (DMSO-d₆, 400 MHz) d: 8.88 (s, 1H), 8.12 (d,

1H), 7.46 (d, 1H), 7.46–7.35 (m, 6H), 5.02 (s, 2H), 4.38 (m, 1H), 4.25

(s, 2H) 4.15 (m, 1H), 3.40 (m, 2H), 2.30–2.12 (m, 2H), 1.93–1.79 (m,

1H), 1.76–1.63 (m, 2H), 1.13–1.06 (s, 9H), 1.10 (s, 11H), 0.99–0.93

(m, 3H), 0.77–0.82 (m, 6H); HRMS (ESI) calcd for

[C₃₁H₅₁N₅O₈þNa]^þ, 644.3638, found: 644.3624 (M þ 23).

butyl)-3,6,9,12-tetraoxo-1-phenyl-2-oxa-4,7,10,13,14-pentaazahexa-

decan-16-oate (100 mg, 0.136 mmol) in dry DCM (20 ml) and

DIPEA (0.05 ml, 0.272 mmol). The reaction was stirred for 16 h at

RT. The reaction was quenched with H₂O, and washed with satu-

rated brine. The organic layer was dried over Na₂SO₄and concen-

trated in vacuo The crude product was purified by silica gel

chromatography (10% MeOH/DCM) to afford product as a white

1

solid (22.8 mg, 21% yield). H NMR (DMSO-d₆, 400 MHz) d: 10.77 (s,

1H), 8.05 (d, J ¼ 7.9 Hz, 1H), 7.75 (d, J ¼ 7.8 Hz, 1H), 7.42–7.24 (m,

6H, aromatic CH þ NH), 5.02 (s, 2H), 4.54–4.44 (m, 1H), 4.27 (dd,

J ¼ 8.0, 4.5 Hz, 1H), 3.92 (t, J ¼ 8.1 Hz, 1H), 3.95–3.89 (m, 1H), 2.23

(s, 5H), 1.85 (m, 1H), 1.70 (m, 2H), 1.88–1.39 (m, 18H), 1.11 (s, 11H),

0.95 (m, 3H), 0.86–0.74 (m, 6H); HRMS (ESI) calcd for

[C₄₀H₆₃N₅O₁₂þNa]^þ828.4373, found 828.4360 (M þ 23).

tert-Butyl (5S,8S,11S)-8–(3-(tert-butoxy)-3-oxopropyl)-11-((S)-1-(tert-

butoxy)ethyl)-5-((R)- -butyl)-3,6,9,12-tetraoxo-1-phenyl-2-oxa-4,7,10,

13,14-pentaazahexadecan-16-oate

(Cbz-Ile-Glu(OtBu)-Thr(OtBu)-

NHNHCH₂CO₂tBu). A -15 ^ꢁC solution of tert-butyl (S)-4-((2S,3R)-2-

(((benzyloxy)carbonyl)amino)-3-methylpentanamido)-5-(((2S,3S)-

3-(tert-butoxy)-1-hydrazineyl-1-oxobutan-2-yl)amino)-5-oxopen-

tanoate (1.174 g, 1.89 mmol) in dry DMF (100 ml) was treated with

NMM (0.26 ml, 1.89 mmol) and left to stir 15 min. The solution was

then treated with t-butyl bromoacetate (0.42 ml, 2.835 mmol) and

allowed to stir for 30 min at -15 ^ꢁC. Following, the solution was

allowed to stir while gradually warming to RT for 16 h. The reac-

tion mixture was concentrated in vacuo and the crude product

was purified by silica gel chromatography (10% MeOH/DCM) to

afford the product as a white solid (0.4641 g, 0.6306 mmol, 33%

Compound 8: (5S,8S,11S)-5-((R)-s-butyl)-8–(2-carboxyethyl)-11-

((S)-1-hydroxyethyl)-3,6,9,12-tetraoxo-14–(2-oxopropanoyl)-1-phe-

nyl-2-oxa-4,7,10,13,14-pentaazahexadecan-16-oic acid (Cbz-Ile-Glu-

Thr-AAsp-COMe). tert-butyl (5S,8S,11S)-8–(3-(tert-butoxy)-3-oxo-

propyl)-11-((S)-1-(tert-butoxy)ethyl)-5-((R)-s-butyl)-3,6,9,12-tetraoxo-

14–(2-oxopropanoyl)-1-phenyl-2-oxa-4,7,10,13,14-pentaazahexade-

can-16-oate (22.8 mg, 0.028 mmol) was dissolved in 1:1 DCM/TFA

(20 ml) at 0 ^ꢁC and allowed to stir 30 min the warmed to RT and

stirred an additional 1 h. The solution was concentrated in vacuo

to afford the product as a white solid (15.8 mg, 85% yield). ¹H

NMR (DMSO-d₆, 400 MHz) d: 10.82 (s, 1H), 8.00 (d, J ¼ 7.99 Hz, 1H),

7.80 (d, J ¼ 7.96 Hz, 1H), 7.35 (m, 6H), 5.02 (s, 2H), 4.89 (m, 1H),

4.39 (m, 1H), 4.17 (m, 1H), 3.92 (m, 2H), 2.24 (s, 3H), 1.90 (m, 1H),

1.70 (m, 2H), 1.39 (m, 2H), 1.22 (s, 2H), 0.99 (m, 3H), 0.80 (m, 6H);

¹³C NMR (175 MHz, DMSO-d₆) d (ppm) ¼ 201.4, 178.6, 176.2, 172.2,

170.1, 167, 163, 149.1, 145.3, 135.9, 130.0, 129.9, 129.4, 67.1, 65.3,

62.3, 60.8, 57.4, 36.6, 31.6, 25.9, 21.2, 18.2, 12.7, 11.8. HRMS (ESI)

calcd for [C₂₈H₃₉N₅O₁₂þNa]^þ660.2487, found 660.2485 (M þ 23).

1

yield). H NMR (DMSO-d₆, 400 MHz) d: 9.14 (d, J ¼ 5.8 Hz, 1H), 8.11

(d, J ¼ 8.0 Hz, 1H), 7.55 (d, J ¼ 8.5 Hz, 1H), 7.36 (m, 6H, aromatic

CH þ NH), 5.12 (q, J ¼ 5.1 Hz, 1H), 5.02 (s, 2H), 4.42–4.31 (m, 1H),

4.18 (dd, J ¼ 8.6, 3.7 Hz, 1H), 3.92–3.88 (m, 2H, a CH þ CH Thr), 3.38

(s, 2H), 2.36–2.09 (m, 2H), 1.96–1.79 (m, 1H), 1.77–1.58 (m, 2H),

1.47–1.34 (m, 20H, Ile CH₂þ CH₃), 1.10 (s, 9H), 1.00–0.93 (m, 3H),

0.90–0.74 (m, 6H); HRMS (ESI) calcd for [C37H61N5O10 þNa]^þ

758.4318, found 758.4301 (M þ 23).

Legumain inhibitors

Benzyl ((S)-1-(((S)-1-hydrazinyl-1-oxopropan-2-yl)amino)-1-oxo-

propan-2-yl)carbamate (Cbz-Ala-Ala-NHNH₂). To

a solution of

methyl ((benzyloxy)carbonyl)-L-alanyl-L-alaninate (Cbz-Ala-Ala-

OMe) (2.0 g, 6.4 mmol) in MeOH (50 ml), hydrazine (2.12 ml,

64 mmol) was added dropwise. The reaction was allowed to stir at

RT for 18 h. MeOH and excess hydrazine were removed in vacuo

and the resulting white solid was washed with EtOAc to afford a

white solid (1.68 g, 5.4 mmol, 85% yield). ¹H NMR (DMSO-d₆,

400 MHz) d 9.05 (s, 1H), 7.90 (d, J ¼ 7.7 Hz, 1H), 7.42 (d, J ¼ 7.6 Hz,

1H), 7.41–7.26 (m, 5H), 4.97–5.07 (m, 2H), 4.20–4.26 (m, 2H),

tert-Butyl (5S,8S,11S)-8–(3-(tert-butoxy)-3-oxopropyl)-11-((S)-1-(tert-

butoxy)ethyl)-5-((R)-s-butyl)-3,6,9,12-tetraoxo-14–(2-oxopropanoyl)-

1-phenyl-2-oxa-4,7,10,13,14-pentaazahexadecan-16-oate (Cbz-Ile-

Glu(OtBu)-Thr(OtBu)-AAsp(OtBu)-COMe). A solution of pyruvic acid

(0.02 ml, 0.20 mmol) in dry DCM (50 ml) was treated with oxalyl

chloride (0.02 ml, 0.217 mmol) followed by 1 drop of DMF (cat).

The reaction was stirred at RT for 3 h, then concentrated in vacuo.

The resulting residue was dissolved in dry DCM (50 ml), chilled to

0 ^ꢁC and treated dropwise with a solution of tert-butyl (5S,8S,11S)- 4.04–4.11 (m, 2H), 1.19 (s, 3 H), 1.18 (s, 3H).

1394

T. S. CORRIGAN ET AL.

Ethyl ((S)-2-((S)-2-(((benzyloxy)carbonyl)amino)propanamido)pro-

panamido)glycinate (Cbz-Ala-Ala-NHNHCH₂CO₂Et). To solution of

benzyl ((S)-1-(((S)-1-hydrazinyl-1-oxopropan-2-yl)amino)-1-oxopro-

pan-2-yl)carbamate (1.68 g, 5.4 mmol) and NMM (0.65 ml,

5.9 mmol) in DMF (8 ml) at ꢀ10 ^ꢁC ethyl bromoacetate (0.65 ml,

5.9 mmol) was added dropwise. The reaction was stirred at ꢀ10 ^ꢁC

for 30 min, then warmed to RT and stirred for an additional 16 h.

The reaction was then concentrated in vacuo, and the resulting

residue was purified by silica gel chromatography (1:9 MeOH/

Compound 11: Benzyl ((S)-1-(((S)-1–(2–(2-amino-2-oxoethyl)-2-

(2-oxo-3-phenylpropanoyl)hydrazinyl)-1-oxopropan-2-yl)amino)-1-

oxopropan-2-yl)carbamate (Cbz-Ala-Ala-AAsn-COBn). A solution of

phenylpyruvic acid (172 mg, 1.05 mmol) in DCM (3 ml) was treated

with oxalyl chloride (0.09 ml, 1.05 mmol), followed by DMF

(1 drop), and the reaction was stirred for 1.5 h at RT, then concen-

trated in vacuo. The resulting residue was taken up in fresh DCM

(5 ml). To this solution was added a solution of benzyl ((S)-1-(((S)-

1–(2-(2-amino-2-oxoethyl)hydrazinyl)-1-oxopropan-2-yl)amino)-1-

oxopropan-2-yl)carbamate (80 mg, 0.21 mmol) in DCM (1.5 ml),

followed by DIPEA (0.07 ml, 0.21 mmol). The reaction was stirred at

RT for 16 h, then concentrated in vacuo and purified by silica gel

chromatography (2–10% MeOH/DCM) then recrystallised from

EtOAc/Et₂O to afford the product as a white solid (19 mg,

0.037 mmol, 18% yield). ¹H NMR (DMSO-d₆, 400 MHz) d: 8.13 (d,

J ¼ 7.89 Hz, 1H), 7.17–7.40 (m, 12H, ArH and NH2), 4.94–5.08 (m,

2H), 4.17–4.31 (m, 1H), 4.05–4.13 (m, 1H), 4.00 (s, 2H), 1.05–1.30

(m, 6H); ¹³C NMR (175 MHz, DMSO-d₆) d (ppm) ¼ 197.0, 168.4,

161.8, 157.4, 130.1, 130.0, 129.97, 129.94, 129.9, 129.5, 129.4, 67.0,

60.0, 59.5, 51.5, 45.9, 45.5, 19.8; HRMS (ESI) calcd for

[C₂₅H₂₉N₅O_7þNa]^þ534.1965, Found: 534.1975 (M þ 23).

1

DCM) to afford a white solid (375 mg, 20% yield). H NMR: (DMSO-

d₆, 400 MHz) d 9.34 (d, J ¼ 5.8 Hz, 1H), 7.92 (d, J ¼ 7.58 Hz, 1H),

7.41–7.47 (d, J ¼ 7.7 Hz, 1H), 7.28–7.41 (m, 5H), 5.18 (q, J ¼ 4.94 Hz,

1H), 5.02 (s, 2H), 4.18–4.26 (m, 1H), 4.01–4.14 (m, 2H), 3.48 (d,

J ¼ 5.24 Hz, 2H), 1.35–1.44 (m, 3H), 1.15–1.22 (m, 6H).

Benzyl

((S)-1-(((S)-1–(2-(2-amino-2-oxoethyl)hydrazinyl)-1-oxo-

(Cbz-Ala-Ala-

propan-2-yl)amino)-1-oxopropan-2-yl)carbamate

NHNHCH₂CONH₂). A 7 M solution of ammonia in MeOH (13.5 ml,

94.5 mmol) was added to a 0 ^ꢁC solution of ethyl ((S)-2-((S)-2-

(((benzyloxy)carbonyl)amino)propanamido)propanamido)glycinate

(375 mg, 0.95 mmol) in DMF (0.4 ml). NaCN (5 mg, 0.095 mmol) was

added, and the reaction vessel was sealed. The mixture was stirred

at 0 ^ꢁC for 2 h, then warmed to RT and stirred for 48 h. The reac-

tion was concentrated in vacuo and the product was precipitated

with 9:1 DCM/MeOH to afford a white solid (343 mg, 98% yield).

¹H NMR (DMSO-d₆, 400 MHz) d 9.33 (s, 1H), 7.98 (d, J ¼ 7.0 Hz, 1H),

7.30–7.47 (m, 5H), 7.12 (s, 1H), 5.22 (t, J ¼ 4.13 Hz, 1H), 4.97–5.08

(m, 2H), 4.15–4.24 (m, 1H), 4.01–4.11 (m, 1H), 3.20 (d, J ¼ 5.23 Hz,

2H), 1.20 (s, 3H), 1.18 (s, 3H).

Proteasome kinetic assay

Human 20S proteasome, 700 KDa, was purchased from Boston

Biochem (50 mg, 2 mM (1.4 mg/mL)) in 50 mM HEPES buffer (pH 7.6,

100 mM NaCl, 1 mM DTT). Assay buffer was prepared as follows:

20 mM HEPES buffer pH 7.8, 0.5 mM EDTA, 0.037% SDS.

Proteasome specific substrate Suc-LLVY-AMC was purchased from

Boston Biochem and used as a fluorogenic substrate; k_ex

¼

380 nm, k_em¼ 442 nm. 18 mL of 2 mM proteasome solution was

diluted with 425 mL H₂O to make a 0.06 mM stock enzyme solution

for kinetic assays. In a 96-well plate suitable for fluorometric

assays was added 86 mL assay buffer, 2 mL inhibitor, 2 mL enzyme

substrate, and 10 mL enzyme stock solution (0.06 nM enzyme con-

centration in well). Fluorescence at 442 nM was monitored for

10 min. The enzyme activity was measured in triplicate at varying

concentrations of inhibitor (0, 25, 50, 100 mM in DMSO) and sub-

strate (10, 20, 50, 100 mM in DMSO for K_imeasurements, and

100 mM for IC₅₀measurements) by converting the slope of the

plot of fluorescence intensity at 442 nm vs time for time points

between 4 and 8 min using Beer-Lambert’s law. IC₅₀values were

obtained from non-linear fitting of the data to a competitive

inhibition model using GraphPad Prism 7.0 software. The prote-

asome b5 chymotrypsin-like active site activity assay was per-

formed as previously described⁴¹.

Compound 10: Benzyl ((S)-1-(((S)-1–(2–(2-amino-2-oxoethyl)-2-

(2-oxopropanoyl)hydrazinyl)-1-oxopropan-2-yl)amino)-1-oxopro-

pan-2-yl)carbamate (Cbz-Ala-Ala-AAsn-COMe). A 0 ^ꢁC of pyruvic

acid (0.15 ml, 0.62 mmol) in DCM (10 ml) was treated with oxalyl

chloride (0.06 ml, 0.66 mmol), followed by one drop of DMF (cat).

The reaction was stirred at 0 ^ꢁC for 30 min, then warmed to RT

and stirred an additional 1.5 h. To this mixture was added DIPEA

(0.14 ml, 0.82 mmol) followed by a solution of benzyl ((S)-1-(((S)-

1–(2-(2-amino-2-oxoethyl)hydrazinyl)-1-oxopropan-2-yl)amino)-1-

oxopropan-2-yl)carbamate (150 mg, 0.41 mmol) in 1:2 DCM/DMF

(1 ml). The reaction was stirred for 16 h, then concentrated in

vacuo and purified by silica chromatography (1:9 MeOH/DCM) to

afford a white solid (24 mg, 13% yield). ¹H NMR (DMSO-d₆,

400 MHz) d: 10.41 (s, 1H), 9.05 (s, 1H), 8.11–8.00 (m, 1 H), 7.55–7.26

(m, 7H), 5.03 (s, 2H), 4.13–4.05 (m, 2H), 3.18 (d, J ¼ 5.24 Hz, 2H),

Caspase kinetic assay

Caspase-3 and caspase-6 were generous gifts from Prof. Guy

Salvesen’s laboratory and then used at Ohio State University.

Caspase activities were measured in 100 mM HEPES, 10% sucrose,

0.1% CHAPS, 10 mM DTT, pH 7.5 at 37 ^ꢁC. To 5,000 mL of this buf-

fer, 100 mL of freshly prepared 1 M DTT and 4,900 mL DI H₂O were

2.03–1.98 (m, 3H), 1.55–1.42 (m, 6 H); HRMS (ESI) calcd for added resulting in the caspase assay buffer. Studies were per-

[C₁₉H₂₅N₅O_7þNa]^þ458.1652, Found: 458.1634 (M þ 23).

formed at 37 ^ꢁC.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1395

Caspase-3

plate suitable for fluorometric assays was added 85 mL assay buf-

fer, 5 mL inhibitor, 5 mL enzyme substrate, and 5 mL enzyme stock

solution (0.145 mg/mL enzyme concentration in well). The enzyme

activity was measured in triplicate at varying concentrations of

Caspase-3 specific substrate Ac-DEVD-AMC was purchased from

Cayman Chemical Company and used as fluorogenic substrate; k_ex

¼ 353 nm, k_em¼ 442 nm. 2 mL of 33 mM caspase-3 solution was

diluted with 4998 mL caspase assay buffer to make a 13.2 nM inhibitor (0, 62.5, 125 mM in DMSO) and substrate (0.1 mM in

enzyme stock solution. In a 96-well plate suitable for fluorometric DMSO) by converting the slope of the plot of fluorescence inten-

assays, 80 mL assay buffer, 5 mL inhibitor, 5 mL enzyme substrate sity at 442 nm vs time for the time points between 4 and 8 min

(0.1 mM), and 10 mL enzyme stock solution were added. using Lambert-Beer’s law. IC₅₀values were obtained from non-lin-

Fluorescence at 442 nM was monitored for 10 min. The enzyme ear fitting of the data to a competitive inhibition model using

activity was measured in triplicate at varying concentrations of GraphPad Prism 7.0 software.

inhibitor (0, 25, 50, 100 mM in DMSO) and substrate (0.1 mM in

DMSO) by converting the slope of the plot of fluorescence inten-

a-Chymotrypsin kinetic assay

sity at 442 nm vs time for the time points between 4 and 8 min

using Beer-Lambert’s law. IC₅₀values were obtained from non-lin-

ear fitting of the data to a competitive inhibition model using

GraphPad Prism 7.0 software.

a-Chymotrypsin from bovine pancreas, type II lyophilised powder,

>40 units/mg protein activity was purchased from Sigma Aldrich

and was tested in 50 mM sodium phosphate, pH 7.5 buffer at

37 ^ꢁC. To prepare the enzyme stock solution, 1.6 mg a-chymotryp-

sin was dissolved in 1,600 mL assay buffer. The resulting stock

enzyme solution was further diluted 1:200 stock to assay buffer

for a final stock concentration of 5 mg/mL. a-Chymotrypsin specific

substrate Suc-LLVY-AMC was purchased from Boston Biochem and

used as a fluorogenic substrate; k_ex¼ 353 nm, k_em¼ 442 nm. In a

96-well plate suitable for fluorometric assays was added 80 mL

assay buffer, 5 mL inhibitor, 5 mL enzyme substrate, and 10 mL

enzyme stock solution (0.5 mg/mL enzyme concentration in well).

The enzyme activity was measured in triplicate at varying concen-

trations of inhibitor (0, 50, 125 mM in DMSO) and substrate

(0.1 mM in DMSO), by converting the slope of the plot of fluores-

cence intensity at 442 nm vs time for the time points between 4

and 8 min using Lambert-Beer’s law. IC₅₀values were obtained

from non-linear fitting of the data to a competitive inhibition

model using GraphPad Prism 7.0 software.

Caspase-6

Caspase-6 specific substrate Ac-VEID-AFC was purchased from

ChemCruz and used as a fluorogenic substrate; k_ex¼ 400 nm, k_em

¼ 505 nm. To 45 mL of assay buffer, 5 mL of the 19.7 mM enzyme

was added. This resulting enzyme solution was further diluted 1:5

with assay buffer resulting in a 0.32 mM solution. In a 96-well plate

suitable for fluorometric assays was added 80 mL assay buffer, 5 mL

inhibitor, 5 mL enzyme substrate (0.1 mM), and 10 mL enzyme stock

solution (32 nM enzyme concentration in well). The enzyme activ-

ity was measured in triplicate at varying concentrations of inhibi-

tor (0, 25, 50, 100 mM in DMSO) and substrate (0.1 mM in DMSO)

by converting the slope of the plot of fluorescence intensity at

442 nm vs time for the time points between 4 and 8 min using

Lambert-Beer’s law. IC₅₀values were obtained from non-linear fit-

ting of the data to a competitive inhibition model using GraphPad

Prism 7.0 software.

Protein expression and purification

The expression and purification of caspase-3 was performed as

previously described^43,44with several modifications. The cDNA

encoding the caspase-3 sequence with the addition of an N-ter-

minal 6xHis-tag followed by a TEV protease recognition sequence

was codon optimised for expression in E. coli (GenScript) and

placed in a pET15b vector (Invitrogen). The protein was expressed

in E. coli at 30 ^ꢁC for 24 h after 200 mM IPTG induction. Cells were

pelleted by centrifugation and lysed by sonication. The lysate was

cleared by centrifugation and purified by Ni-NTA (GE Healthcare)

and Q-Sepharose (GE Healthcare) chromatography. TEV protease

(1.25 mg) was added to the purified protein during overnight dia-

lysis at 4 ^ꢁC and the TEV protease and free His-tag were removed

by reverse Ni-NTA purification. Any remaining impurities were

removed and the buffer was exchanged by size-exclusion chroma-

tography on a Superdex 200 Increase column equilibrated with

20 mM Tris, 20 mM NaCl, 10 mM DTT, pH ¼ 7.5 yielding protein

with purity >95% by SDS-PAGE. The protein was concentrated to

5 mg/mL by ultrafiltration (10 kDa MWCO, EMD Millipore), flash fro-

zen, and stored at ꢀ80 ^ꢁC until ready to use.

Legumain kinetic assay

Schistosoma mansoni and Ixodes ricinus legumain zymogens were

expressed in the yeast, Pichia pastoris, as described^36,42. Legumain

kinetic assay details: zymogens were activated for 3 h (Ir) or over-

night (Sm) at room temperature in 0.1 M Na-Ac pH 4.0; 5 mM DTT.

Activities were measured in 0.1 M Na-Ac pH 5.5; 2.5 mM DTT, 0.1%

PEG 6000 buffer. The substrate used was Cbz-AAN-AMC with

50 mM final concentration in the assay. Inhibitors were prepared in

DMSO (1% DMSO in activity assay). Measurements were taken

with an incubation period of 10 min at room temperature, and the

enzyme activity was measured in triplicate. Asparaginyl

Endopeptidase (AE) activity assay was performed as previously

described⁷.

Cathepsin B kinetic assay

Human Liver Cathepsin B (purified) was purchased from Enzo Life

Sciences (in 50 mM sodium acetate, pH 5.0 containing 1 mM

EDTA). The Cathepsin B activity assay is performed in 0.1 M phos-

phate, 1.25 mM EDTA, 0.01% Brij, pH 6.0 buffer at 37 ^ꢁC. Cathepsin

B specific substrate Cbz-Arg-Arg-AMC was purchased from Sigma

Crystallisation and structure determination

Aldrich and used as fluorogenic substrate; k_ex¼ 380 nm, k_em

¼

The protein was inhibited by adding 20 mM Cbz-DEVaD-COMe

inhibitor in DMSO to a final concentration of 2.1 mM giving a final

442 nm. The assay buffer was prepared as follows 0.1 M K₃PO₄,

1.25 mM EDTA, 0.01% Brij 35 with a pH of 6 was made with 1 mM

DTT added immediately prior to assay. To 100 mL of assay buffer, concentration of 4 mg/ml caspase-3/inhibitor complex. Protein

5 mL of the received 0.427 mg/mL Cathepsin B solution was added. crystallisation was performed by hanging-drop vapour diffusion at

The resulting solution was further diluted 1:6 of Cathepsin B to 298 K with 1:1 ratio of mother liquor (50 mM sodium citrate,

assay buffer for a final concentration of 2.9 mg/mL. In a 96-well 15.33% PEG 6000, 7.5% glycerol, 10 mM DTT, pH ¼ 6.2) to the

1396

T. S. CORRIGAN ET AL.

protein/inhibitor complex. Crystals were observed within one day electrophilic carbonyl warheads (Figure 4). The aza-peptide alde-

and reached their maximum dimensions within 7 days. Crystals hyde inhibitor was prepared by conversion of glyoxylic acid to the

corresponding acid chloride by refluxing in thionyl chloride, and

then coupling this intermediate to the aza-peptide hydrazides in a

manner analogous to the synthesis of the ketone inhibitors

(Figure 4(A)).

were cryoprotected in a 1:4 solution of glycerol:mother liquor and

frozen on the instrument in the cryo-stream at 100 K. X-ray diffrac-

tion data were collected in-house on a Compact HomeLab X-ray

diffractometer (Rigaku) with MicroMax 003i generator, AFC-11 4-

axis goniometer, and Pilatus 200 K detector.

Diffraction images were indexed, integrated, and scaled with

HKL3000 (HKL Research, Inc.) in the P22₁2₁space group with a

maximum resolution of 2.73 Å. Phases were determined by

molecular replacement with 2H5I⁴³as a model using Phaser-MR in

the Phenix software package⁴⁵. Coordinates and restraints for the

sulphur-bound ligand were created using eLBOW⁴⁶and PRODRG

(GlycoBioChem Ltd.) and modified with REEL⁴⁷. Refinement was

performed using phenix.refine⁴⁸in the Phenix software package

and all coordinates and electron density maps were visualised

using winCOOT⁴⁹. Statistical information for the solved structure

can be viewed in Supplemental information.

Initially, we proposed a methyl and a benzyl ketone to explore

the tolerance of functional groups in the S₁’ pocket where the

ketone R’ group was anticipated to reside. We have chosen the

methyl and benzyl groups for two reasons. (a) The methyl group

is the smallest group possible for our proposed aza-peptide

ketone design, and (b) the phenyl group, if tolerated, can be deri-

vatised further to make more specific interactions at the prime

site. Coupling of the warheads was performed by conversion of

readily available pyruvic acid or phenylpyruvic acid to the corre-

sponding acid chloride intermediates via reaction with oxalyl

chloride, and then acyl substitution of the acid chloride with the

aza-peptide hydrazide intermediates, thereby yielding the final

aza-peptide ketones (Figure 4(B)). These two described methods

were used for the synthesis of the aza-peptide aldehyde 3 as well

as ketones 4 and 5, as shown in Figure 4(C).

Results and discussion

A series of caspase-3 aza-peptide ketone inhibitors as well as

one caspase-6 aza-peptide ketone bearing a P₁aza-aspartate resi-

due were synthesised and evaluated (Figure 5).

Chemical synthesis

The general preparation of aza-peptide aldehydes and ketones

involves the synthesis of the substituted hydrazide precursor⁵and

its coupling to oxoacetyl chloride (prepared in situ with thionyl

chloride) for aldehydes and to methyl or benzyl pyruvic acid chlor-

ides for ketones, respectively.

The tripeptidyl inhibitor synthesis begins with an isobutyl

chloroformate mediated coupling between the methyl ester of the

P₂amino acid and the carboxybenzyl protected P₃amino acid

(Figure 2).

From the P₃-P₂dipeptide, the P₁aza-amino acid is added by,

first, the addition of excess hydrazine to the methyl ester to fur-

nish the corresponding hydrazide 1 in quantitative yield (Figure

3), followed by reductive amination with an appropriate aldehyde

to install the P₁side chain. The aza-leucine derivative was synthes-

ised by a two-step reductive amination, first reacting the hydra-

The tri-peptide hydrazides Z-Asp(O^tBu)-Glu(O^tBu)-Val-NHNH₂

and Z-Ile-Glu(O^tBu)-Thr(O^tBu)-NHNH₂were synthesised using the

general peptide coupling and hydrazide formation conditions

described for the proteasome inhibitors above. The aza-aspartate

residue was synthesised according to Figure 6. Nucleophilic substi-

tution of the hydrazide with tert-butyl bromoacetate gave the tert-

butyl aspartate protected intermediates of the general structure 9.

Analogous warhead coupling methods to those described in

Figure 4 were then used, followed by a trifluoroacetic acid depro-

tection of the tert-butyl groups, affording the final inhibitors 6, 7,

and 8.

Inhibitors were also designed and synthesised to target the

legumain protease. The clan CD cysteine protease, legumain, has

a preference to cleave substrates after an asparagine amino acid

residue and has the optimal substrate sequence Cbz-Ala-Ala-

Asn⁵⁰. The aza-peptide ketone inhibitors 10 and 11 depicted in

Figure 7, targeting inhibition of legumain, were synthesised.

The synthesis of legumain inhibitors 10 and 11 was performed

by adaptation of the previous syntheses. The alanine-alanine di-

zide Cbz-Leu-Leu-NHNH₂and isobutyraldehyde to afford

a

hydrazide intermediate, which was subsequently reduced with

sodium cyanoborohydride to effectively install the isobutyl side

chain on the hydrazide to form the aza-leucine intermediate 2 in

good yield (Figure 3).

With the synthesis of the inhibitor backbones complete, the peptide hydrazide 12 intermediate (Figure 8) was synthesised

final step was the coupling of the aza-peptide intermediate to the using an isobutyl chloroformate peptide coupling method,

Figure 2. General peptide coupling approach.

Figure 3. Substituted hydrazide precursor synthesis.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1397

Figure 4. (A) Coupling procedure for aza-peptide aldehyde warhead. (B) Coupling procedure for aza-peptide ketone warhead. (C) Library of synthesised aza-peptide

aldehyde and ketone inhibitors for the proteasome.

Figure 5. Aza-peptide ketone inhibitors designed for caspase-3 and -6.

Figure 6. Synthesis of the aza-aspartate caspase inhibitors.

Figure 7. Aza-peptide ketone inhibitors designed for the clan CD legumain protease.

1398

T. S. CORRIGAN ET AL.

Figure 8. Synthesis of aza-asparagine legumain inhibitors.

followed by the reaction of the resulting methyl ester with hydra-

zine. The intermediate 12 was subjected to reaction with ethyl

bromoacetate to afford the alkylated hydrazide 13. Amidation of

the ethyl ester was carried out with ammonia in a sealed pressure

tube to afford 14, as previously reported⁵. These precursors were

subsequently coupled with the methyl or benzyl keto-warhead to

give the final inhibitors 10 and 11.

Table 1. Inhibition of b5 active site of the human 20S proteasome by aza-pep-

tide aldehydes and ketones

Compound

Structure

IC₅₀(lM)

3

4

5

Cbz-Leu-Leu-ALeu-CHO

Cbz-Leu-Leu-ALeu-COMe

Cbz-Leu-Leu-ALeu-COBn

Cbz-Leu-Leu-Leu-CHO

9.02 1.82

14.56 2.39

10.11 4.49

0.0142 0.003^a

MG132

^aThis is a K_ivalue.

In vitro inhibition

Table 2. Inhibition of human caspase-3 and caspase-6 by aza-peptide ketones

IC₅₀(lM)

We synthesised a total of eight final compounds. Each compound

was characterised by ¹H and ¹³C nuclear magnetic resonance

spectra and electrospray ionisation mass spectra (see the Materials

and Methods section). The results of the inhibition of the various

aza-peptide aldehydes and ketones are summarised in Tables 1–3.

Compounds 3, 4 and 5 are designed to target the b5, chymo-

trypsin-like (CT-L) active site of the human 20S proteasome, and

thus are based on the ideal tripeptidyl sequence Leu-Leu-Leu⁵¹.

To our knowledge, this is the first study where a chemical electro-

philic warhead bearing an aza-P1 residue was designed and tested

with the proteasome. Compounds 3, 4 and 5 showed inhibition in

the mid-mM range, suggesting that the proteasome active site can

actually tolerate the aza-modification at the P₁position (Table 1).

Inhibition rates by the aldehyde 3 and ketones 4 and 5 seemed

fairly close with the aldehyde 3 being slightly more potent with

an IC₅₀value of 9.02 mM. The benzyl group of the ketone inhibitor

5 was well accommodated at the prime site for this warhead

motif with an IC₅₀value of 10.11 mM, allowing further derivatisa-

tion on the aromatic ring for more potency and specificity. The

overall performance of our compounds, particularly compound 3,

can be directly compared to the commercially available aldehyde

inhibitor MG132 (Cbz-Leu-Leu-Leu-CHO). MG132 is a very potent,

but non-selective, proteasome inhibitor. In our assay, we have

determined the K_ivalue of MG132 of the b5, chymotrypsin-like

(CT-L) active site of the 20S proteasome as 14.28 3.06 nM. This

value compares well with the previously reported K_ivalues of

MG132 as 2–4 nM⁵², “few nanomolar”⁵³, and of the close analog

MG115 (Cbz-Leu-Leu-Nle-CHO) as 21 nM⁵⁴using the same sub-

strate Suc-LLVY-AMC as in our assay.

Compound

Structure

Caspase-3

Caspase-6

6

7

8

Cbz-Asp-Glu-Val-AAsp-COMe

Cbz-Asp-Glu-Val-AAsp-COBn

Cbz-Ile-Glu-Thr-AAsp-COMe

7.74 1.88

13.36 4.61

122 83.31

51.93 10.64

64.23 45.40

9.08 3.02

Table 3. Inhibition of S. mansoni and I. ricinus legumains by aza-peptide

ketones

IC₅₀(lM)

Compound

Structure

S. mansoni

I. ricinus

10

11

Cbz-Ala-Ala- AAsn-COMe

Cbz-Ala-Ala- AAsn-COBn

>100

22.23 4.21

>100

17.82 11.17

caspase-3 (Table 2). As expected, the caspase-3 specific com-

pounds 6 and 7 show less activity against caspase-6; however,

due to the strict P₁Asp requirement for cleavage for all caspases,

it is a challenge to obtain highly selective inhibition among differ-

ent caspase family members. Likewise, compound 8 inhibits cas-

pase-6 with an IC₅₀value of 9.08 mM, 13 times more effectively

than it inhibits caspase-3, emphasising the importance of the pref-

erence in caspase-3 for the P4 Asp residue. Within caspase-3 and

-6, the benzyl compound 7 demonstrates the lowest inhibition for

both enzymes, with IC₅₀values of 13.36 and 64.23 mM,

respectively.

Compounds 10 and 11 were tested for their ability to inhibit

the legumain proteases S. mansoni and I. ricinus and displayed

IC₅₀values in the mid-mM range (Table 3). Compound 11 inhibited

the I. ricinus and S. mansoni legumains with IC₅₀values of

17.82 mM and 22.23 mM, respectively, suggesting a favourable aro-

matic interaction on the prime site for both enzymes. The aza-

Compounds 6 and 7 were designed as tetrapeptides to target

human caspase-3 with the ideal sequence Asp-Glu-Val-Asp and

compound 8 targets human caspase-6 with its optimal sequence

Ile-Glu-Thr-Asp as caspases require a minimum of four amino acids

in their recognition sequence²⁶. Compounds 6, 7, and 8 show

low-to-mid mM range inhibition with their target enzyme, where

the best inhibitor is ketone 6 with an IC₅₀value of 7.74 mM against asparagine methyl ketone 10 inhibitor did not show inhibition

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1399

towards either of the legumains at the highest concentration a-chymotrypsin (Table 4). Cathepsin B was chosen to represent

tested (100 mM).

the clan CA type of cysteine proteases, and a-chymotrypsin repre-

senting the general chymotrypsin fold of serine proteases. The lit-

erature comparison compound, MG132 potently inhibits calpains

and various lysosomal cathepsins with K_ivalues of 5–12 nM⁵⁴in

addition to the proteasome⁵². Our compounds, on the other

In addition to screening all of the compounds with their

respective target enzymes, we also tested them for possible cross-

reactivities with human liver cathepsin B and bovine pancreas

Table 4. Cross-reactivity of human cathepsin B and bovine pancreas a-chymo- hand, showed no inhibition of either cathepsin B or a-chymotryp-

trypsin with aza-peptide aldehydes and ketones

sin at concentrations 50 mM and higher, suggesting no in vitro

Inhibition (lM)

cross-reactivity and high selectivity among different families of

proteases (see the Materials and Methods section for more

details). While in vitro and in vivo selectivities do not always correl-

ate, initial observation of in vitro selectivity is valuable information,

and then, one can interpret subsequent in vivo behaviour in light

of in vitro data more precisely. The initial in vitro selectivity is a

desired and promising asset to these compounds, rendering their

further development into potential drug candidates both compel-

ling and exciting.

Compound

Structure

Cathepsin B

a-Chymotrypsin

3

4

5

6

7

8

10

11

Cbz-Leu-Leu-ALeu-CHO

Cbz-Leu-Leu-ALeu-COMe

Cbz-Leu-Leu-ALeu-COBn

Cbz-Asp-Glu-Val-AAsp-COMe

Cbz-Asp-Glu-Val-AAsp-COBn

Cbz-Ile-Glu-Thr-AAsp-COMe

Cbz-Ala-Ala-AAsn-COMe

Cbz-Ala-Ala-AAsn-COBn

NI^a

ND

NI^b

NI^c

ND

NI^a

ND

NI^b

NI^c

NI^b

ND

NI: no inhibition; ND: not determined; NI up to 62.5 lM, ^b50 lM, 125 lM.

a

c

Binding mode and mechanism of inhibition

We determined the X-ray crystal structure of caspase-3 in complex

with our aza-peptide methyl ketone inhibitor 6, Cbz-Asp-Glu-Val-

AAsp-COMe (Figure 9).

We observed a similar binding mode at the active site in

accordance with the previously determined X-ray structures of cas-

pase-3 with inhibitors such as Ac-Asp-Glu-Val-Asp-H⁵⁵and Ac-Asp-

Val-Ala-Asp-FMK⁵⁶. In all of these structures, key hydrogen-bond-

ing interactions between the P1 Asp side chain of the inhibitor

with two Arg side chains (Arg64 and Arg207 in our structure) on

the protein backbone is the determinant of the strict P1 Asp spe-

cificity. We also observed that the P3 Glu forms a hydrogen bond

with Arg207 as well. In addition, the P2 Val side chain is well

accommodated by the Phe256, Trp206 and Tyr204 aromatic rings.

Most importantly, we observed the nucleophilic addition of the

active site Cys163 of caspase-3 at the carbonyl carbon resulting in

a tetrahedral thiohemiacetal adduct (Figure 10).

Based on our observations from the X-ray crystal structure of

caspase-3 in complex with compound 6, we propose that the

inhibition mechanism by the proteasome proceeds via a nucleo-

philic attack of the deprotonated Thr-O^–at the carbonyl carbon of

our warhead, thereby forming a reversible tetrahedral hemiacetal

adduct 15 as shown in Figure 11. Similarly, we propose that inhib-

ition by the other clan CD cysteine proteases, such as the

Figure 9. Caspase-3 in complex with Cbz-Asp-Glu-Val-AAsp-COMe (Compound 6).

Compound 6 is observed residing in the active site of caspase-3 at a resolution

of 2.73 Å after thiohemiacetal covalent-bond formation to the methyl ketone war-

head of 6.

Arg₂₀₇

HN Arg₆₄

H₂

NH

N

H₂N

NH

O

H₂N

O

OH

O

H

N

O

H

N

S

Cys₁₆₃

N

H

N

H

Cbz-A s p-G lu-Val

N

O

N

H

N

O

Phe₂₅₀

OH

O

S

Cys₁₆₃

O

H

N

Phe₂₅₆

Tyr₂₀₄

Trp₂₀₆

Figure 10. Mechanism of inhibition of caspase-3 by the aza-peptide methyl ketone inhibitor Cbz-Asp-Glu-Val-AAsp-COMe (Compound 6). The inhibitor ketone carbonyl

carbon is the site of nucleophilic addition by the active-site Cys163 sulphur atom, resulting in covalent bond formation.

1400

T. S. CORRIGAN ET AL.

Figure 11. Proposed mechanism of inhibition by aza-peptide aldehydes and ketones reacting with (a) the proteasome and with (b) other clan CD cysteine proteases.

The carbonyl carbon is expected to be the site of attack by nucleophilic residues: a Thr-O with the proteasome and a Cys-S with clan CD cysteine proteases.

legumains, occurs by nucleophilic addition of the deprotonated alkyl group which will render the ketone carbonyl to be more

active site Cys-S^–, similarly forming the reversible tetrahedral thio-

electrophilic. Our future, second generation compounds will be

hemiacetal adduct 16.

designed and synthesised accordingly. Overall, the in vitro selectiv-

ity and reversible nature of these inhibitors makes them more

desirable drug candidates, as any possible cross reactivity would

be reversible, arguably resulting in less severe side effects.

Conclusions

In summary, we have developed a new class of peptidyl protease

inhibitors bearing an aza-P1 residue that are effective against the

proteasome, caspases and legumains. The inhibitors were

designed and synthesised based on their ideal substrate sequence.

The tripeptide Leu-Leu-Aza-Leu sequence was chosen to target

the b5, chymotrypsin-like active site of the proteasome. Likewise,

Asp-Glu-Val-Aza-Asp, Ile-Glu-Thr-Aza-Asp and Ala-Ala-Aza-Asn were

chosen as preferred substrate sequences for the caspases-3, and

-6 and legumains, respectively.

Acknowledgements

The authors acknowledge Dr. James H. McKerrow (UC-San Diego)

in facilitating the logistics regarding the legumain assays to be

performed.

Eight novel compounds were synthesised and evaluated for

their ability to inhibit their respective target proteases in vitro, and

were shown to be low-to-mid-mM range inhibitors. Each inhibitor

was also tested for cross-reactivity with cathepsin B and a-chymo-

trypsin, and resulted in no inhibition (>50 mM). X-ray crystallo-

graphic data with caspase-3 revealed a tetrahedral adduct with

compound 6, providing insight into the mechanism of inhibition.

The micromolar range of inhibitory potency for these aza-peptide

aldehydes and ketones could be attributed to the fact that the

point of the nucleophilic attack is one chemical bond away

towards the C-terminus from where the scissile bond normally

would reside (Figure 1). However, it is also advantageous that the

proteasome, caspases-3, and -6, and legumains still tolerate this

aza-P1 design, whereas the classical cysteine protease cathepsin B

and a-chymotrypsin do not. These differences provide an oppor-

tunity for tuneability as well as selectivity, and possibly less off-tar-

get reactivity as observed for bortezomib, carfilzomib and

ixazomib.

Disclosure statement

The authors report no conflict of interest.

Funding

O.D.E. and C.M.H. acknowledge funding from The Ohio State

University Technology Commercialisation Office Accelerator Award

[GRT00051721]. G.S.S. acknowledges grant support from NIGMS

[R01GM099040]. D.S. acknowledges funding from the Centre for

Research of Pathogenicity and Virulence of Parasites [no.

CZ.02.1.01/0.0/0.0/16_019/0000759], funded by the European

Regional Development Fund (ERDF) and Ministry of Education,

Youth, and Sport, Czech Republic (MEYS). Research at the CDIPD

was funded in part by the NIH-NIAID award [R21AI126296]

We hypothesise that the potency of aza-peptide ketones can

be improved by increasing the inductive effect on the prime site’s to C.R.C.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY

1401

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Article Doi

Design, synthesis, and in vitro evaluation of aza-peptide aldehydes and ketones as novel and selective protease inhibitors

DOI: 10.1080/14756366.2020.1781107

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