Synthesis of macrocyclic α-ketoamide as a selective and reversible immunoproteasome inhibitor

Article abstract of DOI:10.1007/s00044-020-02678-2

In recent years, the human immunoproteasome has emerged as an attractive therapeutic target for various diseases, leading to a growing interest in the discovery of immunoproteasome inhibitors that selectively target specific subunits. Herein we report the design, synthesis, and evaluation of a new immunoproteasome inhibitor that feature a macrocyclic ring containing an internal α-ketoamide warhead. This compound is a selective and reversible inhibitor of immunoproteasome subunits β1i and β5i and shows essentially no inhibition of constitutive proteasome subunits. [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-020-02678-2

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research (2021) 30:410–420
https://doi.org/10.1007/s00044-020-02678-2
ORIGINAL RESEARCH
Synthesis of macrocyclic α-ketoamide as a selective and reversible
immunoproteasome inhibitor
1
Rui Ding1 Daniel J. Wilson1 Liqiang Chen
●
●
Received: 29 October 2020 / Accepted: 2 December 2020 / Published online: 11 January 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature 2021
Abstract
In recent years, the human immunoproteasome has emerged as an attractive therapeutic target for various diseases, leading to
a growing interest in the discovery of immunoproteasome inhibitors that selectively target specific subunits. Herein we
report the design, synthesis, and evaluation of a new immunoproteasome inhibitor that feature a macrocyclic ring containing
an internal α-ketoamide warhead. This compound is a selective and reversible inhibitor of immunoproteasome subunits β1i
and β5i and shows essentially no inhibition of constitutive proteasome subunits.
Graphical Abstract
●
●
●
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Keywords Immunoproteasome Immunoproteasome inhibitors Ketoamide Macrocyclic ketoamide Reversible covalent
inhibitors
Abbreviations
IIDQ
2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydro-
quin-oline;
nuclear overhauser effect;
N-ethyl-N′-(3-dimethylaminopropyl)
carbodiimide.
UPS
CP
ubiquitin-proteasome system;
core particle;
NOE
EDC
RP
regulatory particle;
LMP2
low-molecular mass protein-2;
MECL1 multicatalytic endopeptidase complex-like 1;
LMP7
DMP
HBTU
low-molecular mass protein-7;
Dess-Martin periodinane;
N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-
yl)uronium hexafluorophosphate;
Introduction
The 26S proteasome is a key player in the ubiquitin-
proteasome system (UPS) that maintains protein home-
ostasis [1, 2]. It consists of the proteolytic 20S core particle
(CP) and two 19S regulatory particles (RP). In the eukar-
yotic CP, only three of the seven β subunits, namely β1, β2,
and β5, are catalytically active because of the presence of an
N-terminal threonine. While the constitutive proteasomes
are present in all eukaryotic cells, the immunoproteasomes,
a special type of proteasomes, are normally expressed in
monocytes and lymphocytes [3]. Moreover, immunopro-
teasomes are highly expressed in immune cells and non-
immune cells under conditions of stress and inflammation [3].
The immunoproteasomes and constitutive proteasomes
This paper is dedicated to Professor Robert Vince on the occasion of
his 80th birthday.
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02678-2) contains supplementary
material, which is available to authorized users.
* Liqiang Chen
chenx462@umn.edu
1
Center for Drug Design, College of Pharmacy, University of
Minnesota, Minneapolis, MN 55455, USA

Medicinal Chemistry Research (2021) 30:410–420
411
Fig. 1 Design of reversible
immunoproteasome inhibitors
based on a macrocyclic α-
ketoamide warhead
share the same structural architecture; but in the immuno-
proteasomes, the three catalytically active subunits of con-
stitutive proteasomes, β1c, β2c and β5c, are replaced with
β1i (low-molecular mass protein-2, LMP2), β2i (multi-
catalytic endopeptidase complex-like 1, MECL1), and β5i
(low-molecular mass protein-7, LMP7), respectively [4].
The proteasome is a well-established anticancer target as
evidenced by the FDA approval of three proteasome inhi-
bitors: bortezomib, ixazomib, and carfilzomib for multiple
myeloma [5]. In recent years, the immunoproteasome has
emerged as a potential therapeutic target for various dis-
eases including cancer, autoimmune disorders, and obesity
[6, 7]. Therefore, there is a growing interest in the discovery
of immunoproteasome inhibitors that selectively target
specific subunits [8, 9]. Majority of the immunoproteasome
inhibitors reported are those that selectively target β5i,
including α′,β′-epoxyketones [10, 11], thiasyrbactins [12],
peptides with a reactive sidechain [13], N,C-capped
dipeptides [14], and nonpeptidic inhibitors [15, 16]. For β1i
inhibitors, UK-101 [17] with an expoxyketone warhead,
aldehyde-based IPSI-001 [18], and boronic acid-based
ML604440 [19] are among the first reported. Recent stu-
dies have revealed that structural modifications in the pep-
tide backbone of α′,β′-epoxyketones can be used to elicit
β1i selectivity [20, 21].
report the design, synthesis, and evaluation of a new
immunoproteasome inhibitor that feature a macrocyclic ring
containing an internal α-ketoamide warhead.
Materials and methods
Chemistry
General procedures
All commercial reagents were used as provided unless
otherwise indicated. An anhydrous solvent dispensing sys-
tem (J. C. Meyer) using two packed columns of neutral
alumina was used for drying THF, Et2O, and CH2Cl2,
whereas two packed columns of molecular sieves were used
to dry DMF. Solvents were dispensed under argon. Flash
chromatography was performed with RediSep Rf silica gel
columns on a Teledyne ISCO CombiFlash® Rf system using
the solvents as indicated. Nuclear magnetic resonance
spectra were recorded on a Varian 600 MHz with Me4Si or
signals from residual solvent as the internal standard for 1H.
Chemical shifts are reported in ppm, and signals are
described as s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), br s (broad singlet), and dd (double doublet).
Values given for coupling constants are first order. High
resolution mass spectra were recorded on an Agilent TOF II
TOF/MS instrument equipped with either an ESI or APCI
interface.
ONX-0914/PR-957 (1, Fig. 1) is the prototype of selec-
tive β5i inhibitors that feature an α′,β′-epoxyketone war-
head. Compound 1 is moderately selective against β5i over
β5c [10]. Its selectivity has been mainly attributed to a
bulky phenyl group at the P1 position that is accommodated
by the larger S1 pocket of β5i but not that of β5c [10].
Based on structural studies, it has been postulated that the α
′,β′-epoxyketone warhead adopts a bent conformation
before it reacts irreversibly with Thr1 to form a six- or
seven-membered morpholine ring [10, 22, 23]. Herein we
tert-Butyl (S)-(4-Cyano-3-oxo-1-phenyl-4-(triphenyl-λ5-
phosphanylidene)butan-2-yl)carbamate (5)
[24] To a solution of Boc-Phe-OH (4, 3.61 g, 13.6 mmol) in
CH2Cl2 (100 mL) was added EDC·HCl (2.61 g, 13.6 mmol)

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Medicinal Chemistry Research (2021) 30:410–420
and DMAP (41.6 mg, 0.340 mmol). After the resulting
mixture was stirred at rt for 1 h, (triphenylpho-
sphoranylidene)acetonitrile (4.10 g, 13.6 mmol) was added
and the solution was stirred overnight. The reaction was
quenched with sat. NaHCO3 and extracted with CH2Cl2
(×3). After the combined organic layer was dried over
Na2SO4 and filtered, the filtrate was concentrated in vacuo
and the residue was purified by flash column chromato-
graphy using EtOAc/hexanes (50%) to afford product 5 as a
2.75 mmol), and the solution was stirred at rt for 1 h. The
reaction was quenched with sat. NH4Cl and extracted with
CH2Cl2 (×3). After the combined organic layer was dried
over Na2SO4 and filtered, the filtrate was concentrated in
vacuo. To the solution of the residue in THF (8 mL) were
added triphosgene (64.1 mg, 0.216 mmol) and triethyla-
mine (131 mg, 1.29 mmol). After 5 min, 4-penten-1-amine
(110 mg, 1.29 mmol) was added and the solution was
stirred for 10 min. The reaction was then quenched with
sat. NaHCO3 and extracted with CH2Cl2 (×3). After the
combined organic layer was dried over Na2SO4 and fil-
tered, the filtrate was concentrated in vacuo and the resi-
due was purified by flash column chromatography using
EtOAc/hexanes (40%) to afford product 7a as a white
solid (178 mg, 76%). 1H NMR (600 MHz, CDCl3) δ
7.31–7.27 (m, 2H), 7.25–7.20 (m, 3H), 6.84 (br s, 1H),
5.82–5.74 (m, 1H), 5.55 (br s, 1H), 5.11 (br s, 1H), 5.02
(d, J = 17.2 Hz, 1H), 4.97 (d, J = 10.2 Hz, 1H), 4.08 (dd,
J1 = 6.4 Hz, J2 = 2.3 Hz, 1H), 3.97 (br s, 1H), 3.35–3.27
(m, 1H), 3.26–3.19 (m, 1H), 3.14–3.07 (m, 1H),
3.06–3.00 (m, 1H), 2.10–2.04 (m, 2H), 1.63–1.56 (m,
2H), 1.37 (s, 9H). 13C NMR (150 MHz, CDCl3) δ 172.5,
157.5, 138.1, 137.6, 129.3, 128.5, 126.6, 115.3, 80.4,
73.9, 55.6, 38.6, 36.4, 30.9, 28.7, 28.2. HRMS (ESI+)
1
white solid (5.50 g, 74%). H NMR (600 MHz, CDCl3) δ
7.68–7.60 (m, 3H), 7.60–7.46 (m, 12H), 7.30–7.16 (m, 5H),
5.19 (br s, 1H), 5.11 (br s, 1H), 3.38–3.27 (m, 1H),
3.06–3.97 (m, 1H), 1.38 (s, 9H). HRMS (ESI+) calcd for
25
D
C34H34N2O3P (M + H)+ 549.2302, found 549.2309. ½αŠ
À
35:6 (c = 1.0, CHCl3).
Methyl (2R,3S)-3-((tert-Butoxycarbonyl)amino)-2-hydroxy-
4-phenylbutanoate (6a) and Methyl (2S,3S)-3-((tert-
Butoxycarbonyl)amino)-2-hydroxy-4-phenylbutanoate (6b)
A solution of 5 (5.79 g, 10.5 mmol) in CH2Cl2 (100 mL)
was treated with O3 at –78 °C until the color of solution
remained yellow-blue. The excess O3 was removed by
purging with N2 at –78 °C until the blue color faded. To
this yellow solution was added slowly NaBH4 (600 mg,
15.9 mmol) and MeOH (20 mL). After the reaction mix-
ture being warmed up to rt, the solvents were removed in
vacuo and the residue was purified by flash chromato-
graphy over silica gel using EtOAc/hexanes (10%) to
afford products 6a (816 mg, 25%) and 6b (1.40 g, 43%) as
calcd for C20H31N2O4 (M + H)+ 363.2278, found
25
D
363.2273. ½αŠ À 25:2 (c = 1.0, CHCl3).
tert-Butyl ((2S,3S)-3-Hydroxy-4-oxo-4-(pent-4-en-1-
ylamino)-1-phenylbutan-2-yl)carbamate (7b)
1
white solids. Compound 6a, H NMR (600 MHz, CDCl3)
Compound 7b was prepared in a manner similar to 7a
(white solid, 82%). 1H NMR (600 MHz, CDCl3) δ
7.30–7.24 (m, 2H), 7.22–7.18 (m, 3H), 7.06 (br s, 1H),
5.84–5.75 (m, 1H), 5.57 (br s, 1H), 5.06 (br s, 1H), 5.04 (d,
J = 17.2 Hz, 1H), 4.99 (d, J = 10.2 Hz, 1H), 4.26 (br s, 1H),
4.05 (br s, 1H), 3.34–3.22 (m, 2H), 3.06–2.99 (m, 1H),
2.95–2.88 (m, 1H), 2.13–2.06 (m, 2H), 1.66–1.58 (m, 2H),
1.38 (s, 9H). 13C NMR (150 MHz, CDCl3) δ 171.7, 157.9,
138.0, 137.5, 129.3, 128.5, 126.5, 115.3, 80.5, 75.1, 57.1,
δ 7.32–7.26 (m, 4H), 7.24–7.20 (m, 1H), 4.85 (d, J =
9.3 Hz, 1H), 4.30–4.23 (m, 1H), 4.06 (d, J = 3.6 Hz, 1H),
3.74 (s, 3H), 3.24 (d, J = 4.7 Hz, 1H), 2.97–2.87 (m, 2H),
1.38 (s, 9H). 13C NMR (150 MHz, CDCl3) δ 174.4, 155.2,
137.5, 129.4, 128.5, 126.6, 79.6, 70.2, 54.2, 52.8, 38.2,
28.2. HRMS (ESI+) calcd for C16H24NO5 (M + H)+
23
332.1468, found 332.1461. ½αŠ À 61:2 (c = 1.0, CHCl3);
D
1
Compound 6b, H NMR (600 MHz, CDCl3) δ 7.29–7.26
(m, 2H), 7.22–7.18 (m, 3H), 4.89 (d, J = 7.1 Hz, 1H),
4.35–4.29 (m, 2H), 3.59 (s, 3H), 3.38 (d, J = 4.1 Hz, 1H),
2.85–2.75 (m, 2H), 1.38 (s, 9H). 13C NMR (150 MHz,
CDCl3) δ 173.1, 155.5, 137.0, 129.5, 128.3, 126.6, 79.8,
38.5, 35.4, 31.0, 28.7, 28.2. HRMS (ESI+) calcd for
25
D
C20H31N2O4 (M + H)+ 363.2278, found 363.2271. ½αŠ
À
58:8 (c = 1.0, CHCl3).
72.5, 54.3, 52.5, 35.7, 28.2. HRMS (ESI+) calcd for
(2R,3S)-3-Amino-2-hydroxy-N-(pent-4-en-1-yl)-4-
phenylbutanamide (8a)
23
D
C16H24NO5 (M + H)+ 332.1468, found 332.1468. ½αŠ
18:0 (c = 1.0, CHCl3).
À
A solution of 7a (192 mg, 0.530 mmol) in CH2Cl2 (5 mL)
was treated with TFA (2 mL), and the solution was stirred at
rt for 2 h. The reaction was quenched with sat. NaHCO3 and
extracted with CH2Cl2 (×3). After the combined organic
layer was dried over Na2SO4 and filtered, the filtrate was
concentrated in vacuo and the residue was purified by flash
tert-Butyl ((2S,3R)-3-Hydroxy-4-oxo-4-(pent-4-en-1-
ylamino)-1-phenylbutan-2-yl)carbamate (7a)
To a solution of 6a (170 mg, 0.550 mmol) in MeOH
(4 mL) and H2O (2 mL) was added LiOH (66.0 mg,

Medicinal Chemistry Research (2021) 30:410–420
413
column chromatography using MeOH/DCM (5%) to afford
product 8a as a white solid (116 mg, 83%). 1H NMR
(600 MHz, CD3OD) δ 7.31–7.27 (m, 2H), 7.26–7.23 (m,
2H), 7.22–7.18 (m, 1H), 5.86–5.78 (m, 1H), 5.03 (d, J =
17.1 Hz, 1H), 4.95 (d, J = 10.1 Hz, 1H), 3.88 (d, J =
2.4 Hz, 1H), 3.35–3.25 (m, 2H), 3.23–3.16 (m, 1H), 2.87
(dd, J1 = 13.4 Hz, J2 = 7.1 Hz, 1H), 2.68 (dd, J1 = 13.4 Hz,
J2 = 7.8 Hz, 1H), 2.12–2.05 (m, 2H), 1.65–1.58 (m, 2H).
13C NMR (150 MHz, CD3OD) δ 175.8, 140.2, 139.1, 130.4,
129.6, 127.4, 115.5, 73.7, 56.4, 41.2, 39.6, 32.2, 29.7.
HRMS (ESI+) calcd for C15H23N2O2 (M + H)+ 263.1754,
tert-Butyl ((S)-1-(((2S,3S)-3-Hydroxy-4-oxo-4-(pent-4-en-1-
ylamino)-1-phenylbutan-2-yl)amino)-1-oxopent-4-en-2-yl)
carbamate (9b)
Compound 9b was prepared in a manner similar to 9a
(white solid, 89%). 1H NMR (600 MHz, CDCl3) δ
7.30–7.25 (m, 2H), 7.23–7.18 (m, 3H), 6.95 (t, J = 5.5 Hz,
1H), 6.64 (d, J = 6.8 Hz, 1H), 5.84–5.75 (m, 1H),
5.54–5.43 (m, 1H), 5.38 (br s, 1H), 5.10–5.01 (m, 3H),
5.00–4.96 (m, 1H), 4.79 (d, J = 6.4 Hz, 1H), 4.33–4.28 (m,
1H), 4.23 (br s, 1H), 4.06 (br s, 1H), 3.33–3.20 (m, 2H),
3.07–2.94 (m, 2H), 2.41–2.31 (m, 2H), 2.13–2.06 (m, 2H),
1.66–1.58 (m, 2H), 1.41 (s, 9H). 13C NMR (150 MHz,
CDCl3) δ 173.6, 171.2, 155.5, 137.6 (2C), 132.5, 129.2,
128.6, 126.7, 119.4, 115.3, 80.7, 74.3, 56.7, 53.9, 38.7,
found 263.1752. ½αŠ25 20:0 (c = 1.0, MeOH).
D
(2S,3S)-3-Amino-2-hydroxy-N-(pent-4-en-1-yl)-4-
phenylbutanamide (8b)
36.3, 35.1, 31.1, 28.6, 28.3. HRMS (ESI+) calcd for
25
D
Compound 8b was prepared in a manner similar to 8a
(white solid, 81%). 1H NMR (600 MHz, CD3OD) δ
7.38–7.32 (m, 2H), 7.31–7.24 (m, 3H), 5.93–5.85 (m,
1H), 5.09 (d, J = 17.1 Hz, 1H), 5.02 (d, J = 10.1 Hz, 1H),
4.14 (d, J = 3.6 Hz, 1H), 3.40–3.35 (m, 1H), 3.34–3.24
(m, 2H), 2.87 (dd, J1 = 13.6 Hz, J2 = 3.8 Hz, 1H), 2.62
(dd, J1 = 13.6 Hz, J2 = 9.9 Hz, 1H), 2.19–2.13 (m, 2H),
1.72–1.65 (m, 2H). 13C NMR (150 MHz, CD3OD) δ
175.0, 140.2, 139.0, 130.4, 129.5, 127.4, 115.5, 75.6,
C25H38N3O5 (M + H)+ 460.2806, found 460.2807. ½αŠ
À
33:4 (c = 1.0, MeOH).
tert-Butyl ((3R,4S,7S,E)-4-Benzyl-3-hydroxy-2,6-dioxo-1,5-
diazacyclotridec-9-en-7-yl)carbamate (10a)
A solution of 9a (125 mg, 0.272 mmol) and Nitro-Grela
catalyst (36.5 mg, 0.0545 mmol) in toluene (150 mL) was
stirred at rt for 24 h. After the solvent was removed in
vacuo, the residue was purified by flash chromatography
using EtOAc/hexanes (80%) to afford product 10a as a
56.6, 39.5, 38.8, 32.2, 29.7. HRMS (ESI+) calcd for
25
D
C15H23N2O2 (M + H)+ 263.1754, found 263.1755. ½αŠ
24:6 (c = 1.0, MeOH).
À
1
white solid (62 mg, 53%). H NMR (600 MHz, CDCl3) δ
7.30–7.25 (m, 2H), 7.23–7.16 (m, 3H), 5.66–5.60 (m, 1H),
5.36–5.27 (m, 1H), 4.37 (br s, 1H), 4.12–4.04 (m, 2H),
3.62–3.55 (m, 1H), 3.25–3.17 (m, 1H), 3.16–3.05 (m, 2H),
2.81–2.73 (m, 1H), 2.32–2.24 (m, 2H), 2.14–2.06 (m, 1H),
1.83–1.73 (m, 1H), 1.68–1.59 (m, 1H), 1.37 (s, 9H). 13C
NMR (150 MHz, CDCl3) δ 174.7, 172.7, 154.7, 138.0,
136.2, 129.3, 128.8, 126.8, 122.2, 80.7, 74.6, 57.5, 54.5,
tert-Butyl ((S)-1-(((2S,3R)-3-Hydroxy-4-oxo-4-(pent-4-en-1-
ylamino)-1-phenylbutan-2-yl)amino)-1-oxopent-4-en-2-yl)
carbamate (9a)
A solution of (S)-N-Boc-allylglycine (159 mg, 0.740 mmol),
8a (97 mg, 0.37 mmol) and IIDQ (224 mg, 0.740 mmol) in
THF (30 mL) was stirred at rt for 24 h. The reaction was
quenched with sat. NaHCO3 and extracted with CH2Cl2
(×3). After the combined organic layer was dried over
Na2SO4 and filtered, the filtrate was concentrated in vacuo
and the residue was purified by flash column chromato-
graphy using EtOAc/hexanes (40%) to afford product 9a as
a white solid (150 mg, 88%). 1H NMR (600 MHz, CDCl3) δ
7.29–7.25 (m, 2H), 7.24–7.17 (m, 4H), 6.97 (t, J = 5.6 Hz,
1H), 5.89 (br s, 1H), 5.82–5.74 (m, 1H), 5.54–5.45 (m, 1H),
5.09–5.00 (m, 3H), 4.99–4.93 (m, 2H), 4.29–4.23 (m, 1H),
4.11–4.04 (m, 2H), 3.32–3.25 (m, 1H), 3.25–3.18 (m, 1H),
3.17–3.09 (m, 1H), 2.97–2.90 (m, 1H), 2.37–2.27 (m, 2H),
2.11–2.04 (m, 2H), 1.64–1.57 (m, 2H), 1.42 (s, 9H). 13C
NMR (150 MHz, CDCl3) δ 173.6, 172.6, 155.3, 137.9,
137.7, 132.8, 129.4, 128.7, 126.8, 119.3, 115.5, 80.4, 73.3,
55.5, 54.0, 38.8, 36.9, 35.9, 31.2, 28.7, 28.4. HRMS (ESI+)
40.8, 40.6, 35.6, 34.3, 33.2, 28.4. HRMS (ESI+) calcd for
25
D
C23H34N3O5 (M + H)+ 432.2493, found 432.2486. ½αŠ
À
17:2 (c = 1.0, CHCl3).
tert-Butyl ((3S,4S,7S,E)-4-Benzyl-3-hydroxy-2,6-dioxo-1,5-
diazacyclotridec-9-en-7-yl)carbamate (10b)
Compound 10b was prepared in a manner similar to 10a
(white solid, 51%). 1H NMR (600 MHz, CDCl3) δ
7.32–7.27 (m, 4H), 7.24–7.18 (m, 1H), 6.82 (br s, 1H), 6.18
(br s, 1H), 5.81 (br s, 1H), 5.56–5.48 (m, 1H), 5.37–5.30
(m, 1H), 5.05 (d, J = 7.4 Hz, 1H), 4.43–4.34 (m, 1H),
4.10–3.99 (m, 2H), 3.71–3.62 (m, 1H), 3.44–3.36 (m, 1H),
3.17–3.09 (m, 1H), 3.08–3.00 (m, 1H), 2.49–2.41 (m, 1H),
2.34–2.23 (m, 2H), 2.18–2.08 (m, 1H), 1.89–1.80 (m, 1H),
1.63–1.55 (m, 1H), 1.39 (s, 9H). 13C NMR (150 MHz,
CDCl3) δ 174.7, 171.6, 154.8, 137.0, 134.9, 129.3, 128.8,
126.9, 123.9, 80.2, 74.5, 58.4, 54.1, 40.1, 38.2, 36.2, 32.8,
calcd for C25H38N3O5 (M + H)+ 460.2806, found 460.2806.
25
D
½αŠ À 30:6 (c = 1.6, CHCl3).

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Medicinal Chemistry Research (2021) 30:410–420
28.3, 27.6. HRMS (ESI+) calcd for C23H34N3O5 (M + H)+
After the combined organic layer was dried over Na2SO4 and
filtered, the filtrate was concentrated in vacuo and the residue
was purified by flash column chromatography using EtOAc/
hexanes (80%) to afford product 12a as a white solid (42 mg,
24
432.2493, found 432.2493. ½αŠ À 138:1 (c = 0.32,
D
CHCl3).
1
(3R,4S,7S,E)-7-Amino-4-benzyl-3-hydroxy-1,5-
diazacyclotridec-9-ene-2,6-dione (11a)
95%). H NMR (600 MHz, CD3OD) δ 7.28–7.22 (m, 4H),
7.16 (t, J = 6.5 Hz, 1H), 5.64–5.57 (m, 1H), 5.39–5.32 (m,
1H), 4.60–4.55 (m, 1H), 4.42–4.36 (m, 1H), 4.07–3.99 (m,
1H), 3.92–3.89 (m, 1H), 3.49–3.42 (m, 1H), 3.02–2.96 (m,
1H), 2.94–2.83 (m, 2H), 2.47–2.34 (m, 2H), 2.21–2.11 (m,
2H), 1.79–1.72 (m, 1H), 1.71–1.64 (m, 1H), 1.43 (s, 9H), 1.27
(d, J = 7.1 Hz, 3H). 13C NMR (150 MHz, CD3OD) δ 175.1,
174.9, 171.5, 157.6, 139.4, 135.1, 130.4, 129.4, 127.5, 124.7,
80.7, 72.3, 54.3, 54.2, 51.5, 41.5, 39.7, 36.0, 32.5, 28.7, 27.9,
A solution of 10a (62 mg, 0.144 mmol) in CH2Cl2 (5 mL) was
treated with TFA (2 mL), and the solution was stirred at rt for
2 h. The reaction was quenched with sat. NaHCO3 and
extracted with CH2Cl2 (×3). After the combined organic layer
was dried over Na2SO4 and filtered, the filtrate was con-
centrated in vacuo and the residue was purified by flash col-
umn chromatography using MeOH/DCM (5%) to afford
product 11a as a white solid (31 mg, 65%). 1H NMR
(600 MHz, CDCl3) δ 7.87 (d, J = 5.7 Hz, 1H), 7.46 (d, J =
8.2 Hz, 1H), 7.30–7.25 (m, 2H), 7.23–7.18 (m, 3H), 6.91 (d, J
= 6.9 Hz, 1H), 5.59–5.53 (m, 1H), 5.38–5.32 (m, 1H),
4.15–4.07 (m, 2H), 3.73–3.65 (m, 1H), 3.52–3.47 (m, 1H),
3.38–3.32 (m, 1H), 3.15–3.10 (m, 1H), 3.07–3.01 (m, 1H),
2.77–2.70 (m, 1H), 2.33–2.26 (m, 1H), 2.21–2.15 (m, 1H),
2.12–2.03 (m, 1H), 1.86–1.79 (m, 1H), 1.65–1.56 (m, 1H),
1.22 (br s, 2H). 13C NMR (150 MHz, CDCl3) δ 177.8, 172.8,
138.4, 135.6, 129.1, 128.5, 126.7, 121.9, 75.5, 58.0, 54.3,
18.1. HRMS (ESI+) calcd for C26H39N4O6 (M + H)+
22
D
503.2864, found 503.2857. ½αŠ À 67:8 (c = 1.0, MeOH).
tert-Butyl ((S)-1-(((3S,4S,7S,E)-4-Benzyl-3-hydroxy-2,6-
dioxo-1,5-diazacyclotridec-9-en-7-yl)amino)-1-oxopropan-
2-yl)carbamate (12b)
Compound 12b was prepared in a manner similar to 12a
(white solid, 54%). 1H NMR (600 MHz, DMSO-d6) δ 7.80 (d,
J = 7.1 Hz, 1H), 7.65 (d, J = 7.1 Hz, 1H), 7.46–7.41 (m, 1H),
7.26–7.19 (m, 4H), 7.19–7.14 (m, 1H), 7.01 (d, J = 7.4 Hz,
1H), 5.60–5.53 (m, 2H), 5.35–5.28 (m, 1H), 4.33–4.24 (m,
1H), 4.15–4.09 (m, 1H), 3.99–3.89 (m, 1H), 3.68–3.62 (m,
1H), 3.22–3.16 (m, 1H), 3.12–3.03 (m, 2H), 2.83–2.77 (m,
1H), 2.27–2.19 (m, 1H), 2.19–2.13 (m, 1H), 2.12–2.02 (m,
2H), 1.66–1.56 (m, 2H), 1.37 (s, 9H), 1.13 (d, J = 6.6 Hz, 3H).
13C NMR (150 MHz, DMSO-d6) δ 171.8, 171.2, 171.0, 154.9,
138.2, 133.0, 129.5, 128.0, 126.0, 124.3, 78.1, 72.1, 54.4,
51.8, 49.7, 40.1, 36.3, 35.4, 32.1, 28.2, 26.9, 18.0. HRMS
40.6, 36.6, 35.0, 33.1, 28.1. HRMS (ESI+) calcd for
24
D
C18H26N3O3 (M + H)+ 332.1969, found 332.1970. ½αŠ
5:8 (c = 1.0, CHCl3).
À
(3S,4S,7S,E)-7-Amino-4-benzyl-3-hydroxy-1,5-
diazacyclotridec-9-ene-2,6-dione (11b)
Compound 11b was prepared in a manner similar to 11a
(white solid, 65%). 1H NMR (600 MHz, CD3OD) δ
7.36–7.28 (m, 4H), 7.21 (t, J = 7.1 Hz, 1H), 5.62–5.55 (m,
1H), 5.48–5.39 (m, 1H), 4.38–4.33 (m, 1H), 3.98–3.94 (m,
1H), 3.55–3.48 (m, 1H), 3.37–3.27 (m, 2H), 3.11–3.05 (m,
1H), 2.99–2.93 (m, 1H), 2.30–2.21 (m, 3H), 2.19–2.11 (m,
1H), 1.83–1.76 (m, 1H), 1.73–1.65 (m, 1H). 13C NMR
(150 MHz, CD3OD) δ 178.7, 173.9, 139.6, 134.8, 130.5,
129.5, 127.6, 126.6, 74.9, 58.6, 55.6, 41.5, 40.0, 38.4, 33.7,
(ESI+) calcd for C26H39N4O6 (M + H)+ 503.2864, found
22
D
503.2856. ½αŠ À 106:8 (c = 0.1, MeOH).
(S)-N-((3R,4S,7S,E)-4-Benzyl-3-hydroxy-2,6-dioxo-1,5-
diazacyclotridec-9-en-7-yl)-2-(2-morpholinoacetamido)
propanamide (13a)
A solution of 12a (42 mg, 0.0837 mmol) in CH2Cl2 (4 mL)
was treated with TFA (2 mL), and the solution was stirred at
rt for 2 h. The reaction was quenched with sat. NaHCO3 and
extracted with CH2Cl2 (×3). The combined organic layer
was dried over Na2SO4 and filtered, and the filtrate was
concentrated in vacuo. After the residue was dissolved in
CH2Cl2 (5 mL), 4-morpholineacetic acid·TFA (23.8 mg,
0.0985 mmol), HBTU (37.3 mg, 0.0985 mmol) and TEA
(33.2 mg, 0.328 mmol) were added. After being stirred at rt
for 1 h, the reaction was quenched with sat. NaHCO3 and
extracted with CH2Cl2 (×3). After the combined organic
layer was dried over Na2SO4 and filtered, the filtrate was
concentrated in vacuo and the residue was purified by flash
column chromatography using MeOH/DCM (5%) to afford
28.4. HRMS (ESI+) calcd for C18H26N3O3 (M + H)+
24
D
332.1969, found 332.1975. ½αŠ À 21:1 (c = 1.0, MeOH).
tert-Butyl ((S)-1-(((3R,4S,7S,E)-4-Benzyl-3-hydroxy-2,6-
dioxo-1,5-diazacyclotridec-9-en-7-yl)amino)-1-oxopropan-
2-yl)carbamate (12a)
To a solution of Boc-Ala-OH (19.9 mg, 0.105 mmol) in
CH2Cl2 (3 mL) were added HBTU (39.8 mg, 0.105 mmol) and
TEA (17.7 mg, 0.175 mmol). After the resulting mixture was
stirred at rt for 1 h, 11a (29.0 mg, 0.0876 mmol) was added,
and the solution was stirred overnight. The reaction was
quenched with sat. NaHCO3 and extracted with CH2Cl2 (×3).

Medicinal Chemistry Research (2021) 30:410–420
415
product 13a as a white solid (25 mg, 56%). 1H NMR
(600 MHz, CDCl3) δ 7.67 (d, J = 7.7 Hz, 1H), 7.60 (d, J =
6.5 Hz, 1H), 7.28–7.16 (m, 5H), 7.13 (d, J = 7.1 Hz, 1H),
7.07 (br s, 1H), 6.13 (br s, 1H), 5.60–5.53 (m, 1H),
5.36–5.29 (m, 1H), 4.68–4.63 (m, 1H), 4.58–4.51 (m, 1H),
4.29 (s, 1H), 4.05 (d, J = 4.8 Hz, 1H), 3.74 (s, 4H),
3.43–3.34 (m, 1H), 3.32–3.24 (m, 1H), 3.17–3.11 (m, 1H),
3.10–3.03 (m, 1H), 3.01 (s, 2H), 2.60–2.47 (m, 5H),
2.36–2.29 (m, 1H), 2.25–2.17 (m, 1H), 2.13–2.05 (m, 1H),
1.71–1.60 (m, 2H), 1.29 (d, J = 6.4 Hz, 3H). 13C NMR
(150 MHz, CDCl3) δ 172.4 (2C), 171.7, 170.1, 137.7,
135.7, 129.2, 128.5, 126.6, 122.4, 72.9, 66.8, 61.7, 55.6,
53.7, 53.3, 48.4, 40.5, 36.6, 35.5, 32.3, 28.1, 18.4. HRMS
1H), 3.36–3.27 (m, 1H), 3.20–3.10 (m, 1H), 3.02 (s, 2H),
2.53 (s, 4H), 2.44–2.36 (m, 1H), 2.36–2.29 (m, 1H),
2.28–2.20 (m, 1H), 2.19–2.09 (m, 1H), 1.77–1.68 (m, 1H),
1.67–1.58 (m, 1H), 1.28 (d, J = 6.6 Hz, 3H). 13C NMR
(150 MHz, CDCl3) δ 195.4, 172.0 (2 C), 169.5, 162.5,
136.2, 135.6, 129.2, 128.8, 127.2, 122.7, 67.1, 62.0, 60.0,
53.9, 52.6, 48.3, 40.3, 37.6, 37.2, 32.7, 27.7, 19.4. HRMS
(ESI+) calcd for C27H38N5O6 (M + H)+ 528.2817, found
528.2814. ½αŠ25 171 (c = 0.58, CHCl3).
D
Biological activity
Biochemical assay
(ESI+) calcd for C27H40N5O6 (M + H)+ 530.2973, found
23
D
530.2975. ½αŠ À 76:6 (c = 1.0, MeOH).
Human S20 constitutive proteasome and immunoprotea-
some were purchased from BostonBiochem. Substrates
Suc-LLVY-AMC, Ac-RLR-AMC, and Z-LLE-AMC
(BostonBiochem) were used for constitutive proteasome
subunits β5c, β2c, and β1c, respectively. Substrates Ac-
ANW-AMC, Ac-RLR-AMC, and Ac-PAL-AMC (Bos-
tonBiochem) were used for immunoproteasome subunits
β5i, β2i, and β1i, respectively. Reactions were carried out in
a black 384 well non-binding surface microplate at the final
volume of 50 µL. Inhibitors (0.046–100 µM final con-
centrations) were mixed with either 4 nM constitutive pro-
teasome or 1 nM immunoproteasome in reaction buffer
(50 mM HEPES pH 7.5, 0.5 mM EDTA, and 0.01% SDS,
45 µL) in a 384-well plate and incubated for 40 min at
37 °C. A second set of reaction without enzyme was also
prepared as a control. After the preincubation, 100 µM of
the appropriate substrate was added to each well for a total
of 50 µL and 1.5% DMSO (1% from the inhibitor and 0.5%
from the substrate that was diluted from 20 mM in DMSO
to 1 mM in water prior to addition). The plate was mixed for
30 s at 1500 RPM and the reactions were monitored on an
i3 multimode plate reader (Molecular Devices) using an
excitation of 380 nm and an emission of 460 nm to detect
the release of byproduct 7-amino-4-methylcoumarin
(AMC). The reactions were monitored for 1 h at 25 °C
and the linear portion of the reaction was used to determine
the IC50 value. The initial velocities were fit using Prism
(GraphPad) to the log(inhibitor) vs. response–variable slope
(four parameter model) with the top and bottom fixed at 1
and 0, respectively.
(S)-N-((3S,4S,7S,E)-4-Benzyl-3-hydroxy-2,6-dioxo-1,5-
diazacyclotridec-9-en-7-yl)-2-(2-morpholinoacetamido)
propanamide (13b)
Compound 13b was prepared in a manner similar to 13a
(white solid, 71%). 1H NMR (600 MHz, DMSO-d6) δ 8.00 (d,
J = 7.4 Hz, 1H), 7.86 (br s, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.44
(t, J = 5.3 Hz, 1H), 7.25–7.21 (m, 4H), 7.20–7.15 (m, 1H),
5.60–5.53 (m, 2H), 5.36–5.29 (m, 1H), 4.37–4.28 (m, 2H),
4.14–4.08 (m, 1H), 3.68–3.64 (m, 1H), 3.60 (br s, 4H),
3.22–3.15 (m, 1H), 3.13–3.07 (m, 1H), 3.04 (dd, J1 = 13.5 Hz,
J2 = 5.4 Hz, 1H), 2.96 (d, J = 14.8 Hz, 1H), 2.89 (d, J =
14.8 Hz, 1H), 2.83 (dd, J1 = 13.5 Hz, J2 = 7.1 Hz, 1H), 2.43
(br s, 4H), 2.28–2.20 (m, 1H), 2.19–2.12 (m, 1H), 2.12–2.01
(m, 2H), 1.61 (br s, 2H), 1.17 (d, J = 6.8 Hz, 3H). 13C NMR
(150 MHz, DMSO-d6) δ 171.3 (2C), 171.2, 171.0, 138.3,
133.0, 129.6, 128.0, 126.0, 124.4, 72.2, 66.2, 61.3, 54.5, 53.2,
52.0, 47.4, 40.1, 36.4, 35.2, 32.1, 26.8, 18.7. HRMS (ESI+)
calcd for C27H40N5O6 (M + H)+ 530.2973, found 530.2965.
The optical rotation of 13b was not determined due to its low
solubility in common organic solvents.
(S)-N-((4S,7S,E)-4-Benzyl-2,3,6-trioxo-1,5-diazacyclotridec-9-
en-7-yl)-2-(2-morpholinoacetamido)propanamide (2)
To a solution of 13a (13 mg, 0.0245 mmol) in CH2Cl2
(3 mL) was added DMP (31.2 mg, 0.0736 mmol). After
10 min, the reaction was quenched with sat. NaHCO3 and
extracted with CH2Cl2 (×3). After the combined organic
layer was dried over Na2SO4 and filtered, the filtrate was
concentrated in vacuo and the residue was purified by flash
column chromatography using acetone/CH2Cl2 (50%) to
Determination of reversibility
Compound 2 was tested against the human S20 immuno-
proteasome using substrates Ac-ANW-AMC and Ac-PAL-
AMC for immunoproteasome subunits β5i and β1i,
respectively. Compound 2 was incubated with immuno-
proteasome (100 nM) in reaction buffer (50 mM HEPES pH
7.5, 0.5 mM EDTA, 0.01% SDS) at a final volume of 10 µL.
1
afford product 2 as a white solid (11.5 mg, 89%). H NMR
(600 MHz, CDCl3) δ 7.85 (br s, 1H), 7.73 (br s, 1H), 7.72
(br s, 1H), 7.31–7.24 (m, 2H), 7.24–7.16 (m, 3H), 6.96 (br
s, 1H), 5.43–5.31 (m, 2H), 4.57–4.49 (m, 2H), 4.41–4.35
(m, 1H), 3.75 (s, 4H), 3.68–3.59 (m, 1H), 3.44–3.37 (m,

416
Medicinal Chemistry Research (2021) 30:410–420
Scheme 1 Synthesis of
intermediates 6a and 6b
The concentrations of compound 2 were 10 µM and 3 µM
using the substrates Ac-ANW-AMC and Ac-PAL-AMC,
respectively. In addition, a positive control using 1% DMSO
instead of an inhibitor was included. The inhibitor-
immunoproteasome mixtures were incubated for 40 min at
37 °C. After preincubation, 0.5 µL of the reactions containing
DMSO, compound or water (negative control) were added to a
384 well black non-binding surface plate containing master
mix (49 µL) consisting of 100 µM substrate in reaction buffer
and an additional 0.5 µL DMSO. Reactions containing com-
pound 2 were also added to wells containing preincubation
concentrations of inhibitors instead of the additional DMSO
(undiluted inhibitor control). The plate was read for 3.5 h at rt
monitoring ex. 380 em. 460.
irreversible inhibition [35], we incorporated reversible α-
ketoamide as a warhead within the macrocyclic ring. Acyclic
ketoamides have been investigated as reversible proteasome
inhibitors [36–38]. Furthermore, ketoamide-based HCV pro-
tease inhibitors have been approved by the FDA, attesting
ketoamide’s clinical safety [39].
While acyclic α-ketoamides have been incorporated in
inhibitors of a wide range of targets including HDAC, HCV
NS3/4A protease and proteasome [40], to our best knowl-
edge there is no report on the synthesis and drug design
application of macrocyclic rings containing an internal α-
ketoamide even though they are present in several natural
products [40]. To prepare compound 2, we have developed
a synthetic strategy (Schemes 1–3) in which a ring-closing
metathesis was used to construct the requisite macrocyclic
ring. Furthermore, to avoid α-ketoamides being exposed to
various synthetic conditions, we used α-hydroxylamide as
an α-ketoamide precursor which was oxidized by mild
Dess-Martin periodinane (DMP) at the late stage of the
synthetic sequence.
All graphs were generated using Prism 5 (GraphPad) and
data fit lines were added for visualization purposes only.
Results and discussion
Design and chemical synthesis
To obtain the requisite α-hydroxylamides, Boc-Phe-OH
was first coupled with 2-(triphenyl-λ5-phosphanylidene)
acetonitrile to give ketone 5 (Scheme 1) [24]. Under ozo-
nolysis conditions at low temperature, compound 5 was
converted into a diketo nitrile intermediate, which could be
trapped as an α-ketomethyl ester and α-ketoamide in the
presence of methanol and an amine, respectively. Unfortu-
nately, we observed severe racemization under these con-
ditions. Nevertheless, ozonolysis at low temperature
followed by in situ reduction with NaBH4 in the presence of
methanol gave a mixture of α-hydroxylmethyl esters 6a and
6b, which were readily separated by flash column chro-
matography. The absolute stereochemistry of 6a and 6b was
established by comparison of their measured optical rotation
values and NMR spectra with those reported for 6a [41].
With these two isomers available, they were individually
subjected to the subsequent chemical transformations as
As our initial effort to discover selective and reversible
immunoproteasome inhibitors, we designed compound 2, in
which a phenyl group was placed at the P1 position to elicit
immunoproteasome selectivity like compound 1 [10]. We also
adopted the tail of compound 1, in which the small L-alanine
was optimal for β5i selectivity while the morpholine was
originally introduced to improve aqueous solubility [10]. To
enforce a bent conformation, we introduced a macrocyclic ring
similar to the one in natural product syringolin A (3, Fig. 1)
[25]. Syringolin A belongs to syrbactins, a group of protea-
some inhibitors that contain a macrocyclic, internal Michael
acceptor warhead [26]. Syringolin A and its analogues have
been actively sought for their anticancer applications through
total syntheses [27–30] and further structural manipulation
[12, 31–34]. To avoid potential side effects associated with

Medicinal Chemistry Research (2021) 30:410–420
417
Scheme 2 Synthesis of cyclic
intermediates 10a and 10b
Scheme 3 Synthesis of
compound 2
Table 1 Inhibition of constitutive proteasome and immunoproteasome
subunits
shown in Scheme 2. After methyl ester 6a underwent
hydrolysis, the resulting acid was coupled with pent-4-en-1-
amine in an amide formation reaction mediated by tri-
phosgene, a reagent that proved to be advantageous over
conventional coupling reagents such as N,N,N′,N′-tetra-
methyl-O-(1H-benzotriazol-1-yl)uronium hexafluoropho-
sphate (HBTU). The Boc protective group in β-amino acid
7a was then removed to give free amine 8a, which under-
went an 2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydroquin-
oline (IIDQ)-mediated amide formation with commercially
available (S)-N-Boc-allylglycine to afford di-olefin 9a in
high yields. IIDQ was adopted because it has been suc-
cessfully used to effect a coupling reaction between a
hydroxylamine and a protected vinyl glycine during a total
synthesis of syringolin A and B [28]. With di-olefin 9a
available, macrocyclic 10a was obtained in good yields
after a key ring-closing metathesis catalyzed by Nitro-Grela
catalyst, which has been used in an efficient synthesis of
HCV protease inhibitor BILN 2061 via ring-closing
metathesis macrocyclization [42]. As depicted in Scheme
2, methyl ester 6b was also converted into the corre-
sponding isomeric macrocyclic α-hydroxylamide 10b in an
identical synthetic sequence.
Compound IC50 (µM)
β1i
β2i
β5i
β1c
β2c
β5c
2
0.29 0.02 33.5 3.3 2.05 0.22 >100 >100 >100
13a
13b
1
>100
>100
76%a
>100
>100
9%a
>100
>100
91%a
>100 >100 >100
>100 >100 >100
5%a 28%a 52%a
aPercentage of inhibition at 1 µM.
coupled with protected alanine Boc-Ala-OH to give 12a,
which subsequently underwent deprotection and amide
formation with 2-morpholinoacetic acid to give macrocyclic
α-hydroxylamide 13a. Isomeric 13b was obtained in an
identical sequence. Preparation of compound 2 was finally
accomplished by brief oxidation of alcohol 13a with DMP
while alcohol 13b was not used for such purpose due to its
surprisingly low solubility in common organic solvents.
Biological activity
Deprotection of 10a led to free amine 11a, whose trans-
double bond was determined by nuclear overhauser effect
(NOE) (Scheme 3). Free amine 11b was also obtained after
deprotection. With a macrocyclic ring in place, 11a and 11b
were ready for introduction of a tail. Amine 11a was
Compounds 2 as well as intermediates 13a and 13b were
tested against human constitutive proteasome and immu-
noproteasome subunits and their IC50 values were listed in
Table 1. Compound 2 possessed IC50 values of ~300 nM
and 2 µM against immunoproteasome subunits β1i and β5i,

418
Medicinal Chemistry Research (2021) 30:410–420
Fig. 2 Determination of the reversibility of immunoproteasome inhi-
bitors 2. A Reversibility of inhibition of β5i by 2. Ac-ANW-AMC was
used at 100 µM and compound 2 was used at 0 (■), 10 µM diluted to
0.1 µM (▲), 10 µM (▼) and 0 without enzyme (●). B Reversibility
of inhibition of β1i by 2. Ac-PAL-AMC was used at 100 µM and
compound 2 was used at 0 (■), 3 µM diluted to 0.03 µM (▲), 3 µM
(▼) and 0 without enzyme (●). Unless noted, all reactions contained
50 mM HEPES (pH 7.5), 0.5 mM EDTA, 0.01% SDS, and a final
concentration of 1 nM of human S20 immunoproteasome (0 nM for
negative control)
respectively, indicative of a slight preference for β1i over
β5i. Furthermore, it showed high selectivity (>100-fold)
against β1i over β2i. More remarkably, compound 2
exhibited essentially no inhibition of constitutive protea-
some subunits β5c, β2c or β1c at 100 µM. This profile
compared favorably with compound 1 in terms of immuno-
selectivity (β1i vs. β1c and β5i vs. β5c) even though a
precise comparison was difficult due to different modes of
inhibition (reversible vs. irreversible). α-Hydroxylamides
13a and 13b displayed no activity against either constitutive
proteasome or immunoproteasome subunits, indicating that
the α-ketoamide warhead was responsible for inhibitory
activities.
selectivity in our future effort to develop new immuno-
proteasome inhibitors.
Acknowledgements This work was supported by the Center for Drug
Design at the University of Minnesota. We thank Prof Rodney John-
son on the use of the ozone generator in his laboratory.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
To determine the reversibility of compound 2, it was
tested under “jump dilution” conditions [43]. In this tech-
nique, an enzyme incubated with an inhibitor at a high
concentration (generally 10× IC50, expected to inhibit 91%
of enzyme activity) is promptly diluted 100-fold and the
reaction is monitored under diluted conditions that are
expected to inhibit 9% of activity. Slow recovery of the β1i
and β5i activity (Fig. 2) indicated that compound 2 was
indeed a reversible inhibitor.
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