LMP2 Inhibitors as a Potential Treatment for Alzheimer's Disease

Article abstract of DOI:10.1021/acs.jmedchem.0c00416

The immunoproteasome (iP), an inducible proteasome variant harboring three immunosubunits, low molecular mass polypeptide-2 (LMP2), multicatalytic endopeptidase complex subunit-1, and low molecular mass polypeptide-7 (LMP7), is involved in multiple facets of inflammatory responses. We recently reported that YU102, a dual inhibitor of the iP subunit LMP2 and the constitutive proteasome catalytic subunit β1, ameliorates cognitive impairments in mouse models of Alzheimer's disease (AD) independently of amyloid deposits. To investigate whether inhibition of LMP2 is sufficient to improve the cognitive functions of AD mice, here we prepared 37 YU102 analogues and identified a potent LMP2 inhibitor DB-310 (28) (IC₅₀: 80.6 nM) with improved selectivity and permeability in cells overexpressing ABCB1 transporters. We show that DB-310 induces suppression of IL-1α production in microglia cells and improves cognitive functions in the Tg2576 transgenic mouse model of AD. This study supports that inhibition of LMP2 is a promising therapeutic strategy for treatment of AD.

Full text of DOI:10.1021/acs.jmedchem.0c00416

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Article
LMP2 inhibitors as a potential treatment for Alzheimer’s disease
Deepak Bhattarai, Min Jae Lee, Ahruem Baek, In Jun Yeo, Zachary Miller, Yu
Mi Baek, Sukyeong Lee, Dong-Eun Kim, Jin Tae Hong, and Kyung Bo Kim
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00416 • Publication Date (Web): 19 Mar 2020
Downloaded from pubs.acs.org on March 19, 2020
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LMP2 inhibitors as a potential treatment for
Alzheimer’s disease
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#
,a
#,a
#,b
#,c
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Deepak Bhattarai, Min Jae Lee, Ahruem Baek, In Jun Yeo, Zachary Miller, Yu Mi Baek,
d
b
c
,a
Sukyeong Lee, Dong-Eun Kim, Jin Tae Hong, and Kyung Bo Kim*
aDepartment of Pharmaceutical Sciences, University of Kentucky, 789 South Limestone,
b
Lexington, KY 40536-0596, USA; Department of Bioscience and Biotechnology, Konkuk
c
University, Seoul 05029, Republic of Korea; College of Pharmacy, Chungbuk National
d
University, Cheongju, Chungbuk 28160, Republic of Korea; Verna and Marrs McLean
Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX
7
7030, USA
Corresponding Author
*
Phone: 1-859-257-5301. E-mail: kbkim2@uky.edu
KEYWORDS. Proteasome, Immunoproteasome, neuroinflammation, LMP2, epoxyketone peptide
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ABSTRACT
The immunoproteasome (iP), an inducible proteasome variant harboring three immuno-subunits,
molecular mass polypeptide-2 (LMP2), multicatalytic endopeptidase complex subunit-1 (MECL-
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) and molecular mass polypeptide-7 (LMP7), is involved in multiple facets of inflammatory
responses. We recently reported that YU102, a dual inhibitor of the iP subunit LMP2 and the
constitutive proteasome (cP) catalytic subunit β1, ameliorates cognitive impairments in mouse
models of Alzheimer’s disease (AD) independently of amyloid deposits. To investigate whether
inhibition of LMP2 is sufficient to improve the cognitive functions of AD mice, here we prepared
3
7 YU102 analogs and identified a potent LMP2 inhibitor DB-310 (28) (IC50: 80.6 nM) with
improved selectivity and permeability in cells overexpressing ABCB1 transporters. We show that
DB-310 induces suppression of IL-1α production in microglia cells and improves cognitive
functions in the Tg2576 transgenic mouse model of AD. This study supports that inhibition of
LMP2 is a promising therapeutic strategy for treatment of AD.
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INTRODUCTION
The immunoproteasome (iP) is a proteasome subtype harboring a distinct set of three inducible
catalytic subunits, molecular mass polypeptide-7 (LMP7), molecular mass polypeptide-
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(LMP2) and multicatalytic endopeptidase complex subunit-1 (MECL-1), which replace their
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constitutive proteasome (cP) counterparts β5, β1 and β2, respectively. Accumulating evidence
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suggests that the iP is closely linked to both adaptive and innate immune responses, drawing
considerable interest in the development of a new class of anti-inflammatory agents. In
particular, inhibitors of LMP7 have become a major focus in recent drug development efforts.3-5
For example, KZR-616 (Figure 1a), a peptide epoxyketone-based inhibitor of LMP7 and to a
lesser degree, LMP2, is currently being evaluated in clinical trials for systemic lupus
erythematosus (SLE) and lupus nephritis (LN).6-7 While co-inhibition of LMP7 and LMP2
appears to be more effective than LMP7 inhibition alone in mouse models of inflammatory
diseases,6, 8 interestingly, it has been also reported that LMP2 inhibition alone does not affect
inflammatory responses.9
Chronic activation of the innate immune system is firmly established as a contributing factor to
the pathogenesis of Alzheimer’s disease (AD), thereby considered important for both diagnostic
and therapeutic purposes.10-11 As such, several clinically available anti-inflammatory drugs
targeting cyclooxygenases or TNF-α have been examined for their efficacy in AD patients via
randomized controlled clinical trials, but have not yet yielded effective therapies.12-16 In recent
years, researchers have found a strong correlation between elevated expression and activity of
the iP and microglial activation in the brains of AD patients17-18 and in a mouse model of brain
injury.19-20 Studies also show that LMP7 knockout directly disrupts cytokine release profiles
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from microglia and improves cognitive function in mouse models of AD, indicating a potential
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role of the iP in CNS inflammation and AD progression. Similarly, moderate upregulation of
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LMP2 in the brains of AD patients compared to healthy controls was also reported. However,
despite such a potential link between the iP and AD, the impact of pharmacological iP inhibition
on AD has not yet been investigated until recently.
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We recently reported that YU102 (Figure 1b), a dual inhibitor of the iP catalytic subunit LMP2
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and the cP catalytic subunit β1, leads to cognitive improvements in mouse models of AD. We
also found that YU102 lowers the levels of microglial inflammatory cytokines in vitro and in
vivo, indicating a potential link between YU102-targeted proteasome subunits and microglia-
mediated inflammatory responses. While there are some suggestive data in the report, it remains
to be further verified as to whether LMP2 inhibition alone is sufficient to attenuate disease
progression in AD mice. Unfortunately, there are currently no LMP2-selective inhibitors
available to test this in mouse models of AD. Inhibitors previously reported as LMP2-selective
are either brain impermeable (KZR-504, LU-001i)9, 25 or lacking necessary LMP2 specificity
(
UK101, NC-001).25-26
Given this, in this report we aimed to prepare LMP2 inhibitors with improved selectivity and
pharmaceutical properties and evaluate their in vivo efficacy. To do this, we first built a library of
YU102 analogs via a structure-activity-relationship (SAR)-aided medicinal chemistry approach
and then performed a step-wise screening with sequential assessment of in vitro LMP2 inhibitory
potency/specificity and the ability to bypass the efflux transporter ABCB1 (P-glycoprotein, Pgp),
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known as an important gatekeeper for brain penetration. We show that compound 28 (DB-310)
,
identified as a lead LMP2 inhibitor, suppresses the release of inflammatory cytokines from
microglial cells and blocks epithelial mesenchymal transition (EMT) triggered by TNF-α-
induced inflammation. Intraperitoneal injection of DB-310 (28) led to improved cognitive
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function in an AD mouse model of Aβ deposits. The impact of DB-310 (28) on
neuroinflammation was further verified by the complete protection of the AD mice from retinal
pigment epithelium (RPE) degeneration which is caused by Aβ-triggered neuroinflammation.
Taken together, the results support that LMP2 plays a key role in microglia-mediated
inflammation and inhibition of LMP2 is a promising therapeutic strategy for AD treatment.
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a
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N-cap/P4
P1
KZR-616
KZR-504
P3
P2
YU102
Figure 1. a. Inhibitors of the immunoproteasome (iP), KZR-616 (primarily targets LMP7) and
KZR-504 (LMP2-selective). b. Predicted docking model of YU102 bound to LMP2 (PDB ID:
3
UNF) from the mammalian 20S iP.
RESULTS AND DISCUSSION
Structure-guided construction of a library of YU102 analogs. Prior to preparing a library of
YU102 analogs, we first investigated the structure-activity relationship (SAR) using a docking
structure of YU102 bound to the LMP2 subunit. This model was built based on the previously
reported X-ray crystal structure (PDB ID: 3UNF) of the mouse 20S iP in complex with an iP
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inhibitor (ONX 0914). In the docking model, the P2-phenylalanine residue (pink circle, Figure
b) is predicted to be exposed to solvent without engaging in interactions with the surrounding
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amino acid residues of the S2 subsite of LMP2, potentially providing a flexible chemical space
for further YU102 optimization. The P3-proline residue (blue circle, Figure 1b) is predicted to be
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positioned at the rim of the S3 pocket located between LMP2 and neighboring MECL-1,
providing YU102 a stabilizing effect via hydrophobic interactions with the residues His114 and
His116 from the neighboring MECL-1 subunit. This suggests a limited flexibility for P3
optimization in order to maintain the interaction between the P3-proline and the hydrophobic S3
pocket. The P1-leucine residue is predicted to make strong hydrophobic interactions with
surrounding residues in the well-defined S1 pocket located deep inside the LMP2 catalytic site,
indicating little room for extensive optimization for the position. The N-terminal acetyl (N-cap)
and P4-glycine are predicted to bind in a deep, narrow pocket formed at the boundary between
LMP2 and MECL-1, again indicating limited opportunity for optimization.
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Chemistry. Based on the SAR analysis, we prepared YU102 analogs having a wide range of
natural and non-natural amino acids at the P2 position following a similar synthetic route
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previously reported. For the P3 position, we only utilized proline or proline mimics to maintain
the β-turn-like conformation of YU102. The N-cap/P4 position were modified with chemical
groups that are reported to confer favorable oral bioavailability and aqueous solubility in the
context of other peptide epoxyketones.30
Scheme 1. Synthetic scheme for the right-hand fragments (epoxy ketones) of YU102 analogs.
Reagents and Conditions: (a) HN(CH
Isopropenylmagnesium bromide, THF, -78 °C, 12 h, 64-79%; (c) Benzonitrile, H
C to rt, 2 h, 32-64%.
3
)OCH
3
, HOBt, EDCI·HCl, DIPEA, DCM, rt, 12 h, 86-90%; (b)
O
2 2
, DIPEA, methanol, 0
°
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The right hand fragments of YU102 analogs were prepared as shown in Scheme 1. In brief,
respective Boc-protected amino acids (1a-1c) were treated with N,O-dimethylhydroxylamine
hydrochloride to prepare Weinreb amides (2a-2c) which were easily converted to keto-alkene
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(3a-3c) in the presence of isopropenylmagnesium bromide. Reactions of the conjugated alkenes
with hydrogen peroxide furnished the final products (4a-4c). The left hand peptide backbone of
each analog was prepared following conventional amide coupling reactions (Schemes 2-4).29
Scheme 2. Synthetic scheme for the left hand fragments of YU102 analogs.
Reagents and Conditions: (a) HBTU, HOBt, DIPEA, DCM, rt, 18 h, 54-96%; (b) i. TFA, DCM,
rt, 1 h. ii. N-Acetyl glycine or N-Boc glycine, HBTU, HOBt, DIPEA, DCM, rt, 18 h, 73-96%; (c)
i. TFA, DCM, rt, 1 h. ii. N-cap carboxylic acid, HBTU, HOBt, DIPEA, DCM, rt, 18 h, 56-80%;
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(
d) i. Piperidine, DMF, 30 min. ii. R -Carboxylic acid, HBTU, HOBt, DIPEA, or R -acyl
chloride, DIPEA DCM, rt, 18 h 45-94%; (e) i. H , Pd/C, MeOH, rt, 1h. ii. Boc-deprotected Leu-,
2
Phe- or cyclohexyl-epoxides, HBTU, HOBt, DIPEA, DCM, rt, 18 h, 12-65%.
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Scheme 3. Synthetic scheme for YU102 analogs with proline mimic groups at the P3.
Reagents and Conditions: (a) HBTU, HOBt, DIPEA, DCM, rt, 18 h, 56-97%; (b) i. TFA, DCM,
rt, 1 h. ii. N-Acetyl glycine, HBTU, HOBt, DIPEA, DCM, rt, 18 h, 85%; (c) i. H , Pd/C, MeOH,
2
rt, 1h. ii. Boc-deprotected Leu-epoxide, HBTU, HOBt, DIPEA, DCM, rt, 18 h, 18-49%.
Scheme 4. Synthetic scheme for N-cap/P4 analogs of YU102.
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Reagents and Conditions: (a) Respective carboxylic acid, HBTU, HOBt, DIPEA, DCM, rt, or
trifluoro acetyl chloride or sulfonyl chloride, DIPEA, DCM, rt, 18 h, 56-97%; (b) i. H , Pd/C,
2
MeOH, rt, 1h. ii. Boc-deprotected Leu-epoxide, HBTU, HOBt, DIPEA, DCM, rt, 18 h, 12-48%.
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SAR analysis. Altogether, we synthesized 37 YU102 analogs (Figure 2) and assessed their
proteasome inhibitory activity using two fluorogenic substrates (Suc-LLVY-AMC for
chymotrypsin-like (CT-L) activity (both LMP7 and β5 are responsible for the CT-L activity);
Ac-PAL-AMC for LMP2).
P1
P3
P2
N-cap/P4
YU102 (1)
Figure 2. Final YU102 analogs derivatized at the P1, P2, P3, or N-cap/P4 sites.
As shown in Table 1, the substitution of the leucyl residue at the P1 of YU102 with the bulkier
groups (phenyl for 2 and cyclohexyl for 3) resulted in the 2-3-fold loss of LMP2 inhibitory
potency (IC50 = 341 nM for 2 and 201 nM for 3 vs 102 nM for YU102). Replacement of the P2-
benzyl residue of YU102 by comparable or smaller substituents was well tolerated, mostly
retaining similar LMP2 inhibitory potency, as shown for compounds 4-7 and 16. On the
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contrary, compounds (8-10 & 13) with larger hydrophobic/aromatic groups at the P2 improved
their LMP2 inhibitory activity, which is in line with previously reported SAR.9, 25
Table 1. Proteasome inhibitory potencies (IC50 values) of YU102 analogs. Proteasome activity
assays were performed using RPMI 8226 cell lysates (for CT-L activity) or purified human 20S
immunoproteasomes (for LMP2 activity) and the respective fluorogenic substrates, Suc-LLVY-
AMC for CT-L activity and Ac-PAL-AMC for LMP2. Data were obtained based on n=3
replicates per compound per concentration.
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IC50 (nM)
IC50 (nM)
Compound
Compound
CT-L
LMP2
CT-L
>10,000
LMP2
KZR-504
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
162.10 ± 25.49
102.98 ± 19.82
341.86 ± 77.53
201.32 ± 63.71
120.25 ± 21.41
57.96 ± 14.05
134.23 ± 19.10
120.89 ± 15.62
33.55 ± 7.77
20
100.16 ± 19.93
123.45 ± 28.66
152.42 ± 36.46
130.53 ± 31.35
1,218.28 ± 206.11
1,044.35 ± 192.87
1,005.61 ± 169.03
105.77 ± 12.09
80.62 ± 13.50
YU102 (1)
21
1,313.6 ± 464.7
2
3
22
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
23
4
24
5
25
6
26
27
7
8
28 (DB-310)
9
>10,000
5,297.5 ± 892.0
10,000
29.72 ± 6.84
21.66 ± 4.33
29
30
31
32
>10,000
>10,000
>10,000
>10,000
143.49 ± 30.89
114.54 ± 18.24
111.49 ± 19.47
102.99 ± 18.32
10
11
121.66 ± 24.02
72.01 ± 17.19
12
5,081.9 ± 1,001.2
1
3
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
33.16 ± 6.08
90.87 ± 24.17
81.99 ± 20.25
120.34 ± 36.59
151.27 ± 43.74
71.84 ± 22.32
622.23 ± 160.85
33
34
35
36
37
38
>10,000
10,000
183.11 ± 41.98
114.78 ± 21.44
131.11 ± 25.67
90.11 ± 14.85
62.83 ± 8.19
14
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17
18
>10,000
5,756.6 ± 909.8
10,000
10,000
131.98 ± 27.91
19
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More hydrophilic residues at the P2 position, such as pyridine (11) or flurophenyl (15), did not
alter the LMP2 inhibitory potency of the parent compound YU102. Introduction of a wide range
of proline mimics (17-22) at the P3 position also did not affect their LMP2 inhibitory activity,
with the exception of compound 19 which has an oxoproline residue and is ~6 times less potent
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(
IC50 of 622 nM) than YU102. Given that the P3-proline residue is predicted to be placed in a
hydrophobic environment formed between LMP2 and MECL-1 in our docking studies (Figure
b), we believe that the oxo group of the P3-oxoproline of 19 may engaged in less favorable
1
interactions with the hydrophobic S3 subsite, lowering its potency against LMP2. Tripeptide
YU102 analogs (24-26) containing morpholine or pyridine derivative at the P4/N-Cap position
almost completely lost their LMP2 inhibitory activities, whereas tetrapeptide analogs (23 and 28-
3
1) with heterocyclic N-cap residues mostly retained the potency of LMP2 inhibitory activity.
Finally, the combined derivatizations (32-38) at both P2 and N-cap/P4 did not improve their
LMP2 inhibitory potencies, showing similar potencies to YU102.
Identification of compound 28 (DB-310) as a lead LMP2 inhibitor. We next assessed the
potential ability of YU102 analogs to cross the blood brain barrier (BBB). Among efflux
transporters expressed in the BBB, ABCB1 plays a key role in maintaining brain homeostasis
and is shown to efflux peptide epoxyketones such as carfilzomib and epoxomicin.31-32 In order to
evaluate YU102 analogs for their interactions with ABCB1, we utilized human colon
adenocarcinoma DLD-1 cells overexpressing ABCB1 (DLD-1/ABCB1) previously established
3
2
by our laboratory. DLD-1/ABCB1 cells on 96-well plates were treated with YU102 analogs (1
µM, 4 h) and lysed to measure the extent of LMP2 inhibition using the fluorogenic substrate Ac-
PAL-AMC. When the extent of LMP2 inhibition in cellulo was plotted against in vitro LMP2
inhibitory potency (Figure 3a), most compounds including YU102 and KZR-504, a previously
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reported LMP2 inhibitor with very poor brain permeability, were not effective at inhibiting
LMP2 in cellulo despite their IC50 values at nanomolar ranges against purified 20S
immunoproteasome (for full sets of data, see Suppl. Figure 1). Compared to the rest, DB-310
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(
28), 34 and 35 (shown as closed circles in Figure 3a) appeared to be more effective in inhibiting
LMP2 in cells overexpressing ABCB1. Ultimately, DB-310 (28) was chosen for further
biological characterization considering the following properties: LMP2 inhibitory potency
against purified 20S proteasome (IC50 values: DB-310 (28) (80.6 nM) < 34 (114.8 nM) < 35
(
131.1 nM)), selective LMP2 inhibition without affecting the CT-L activity (DB-310 (28) & 35:
IC50 (CT-L activity) >10 µM) and low molecular weight (DB-310 (28) (578) < 35 (592) < 34
(674)). We then assessed the in cellulo LMP2 activity in the presence and absence of the ABCB1
inhibitor reversin 121 (7.5 µM) using DLD-1/ABCB1 cells following the incubation of YU102,
DB-310 (28), or KZR-504 for 4 hours. As shown in Figure 3b, reversin 121 enhanced the LMP2
inhibitory activity of YU102 and KZR-504, but not that of DB-310 (28), indicating that DB-310
(
28) is not a substrate of ABCB1. In this regard, the poor brain LMP2 inhibitory activity of
9
KZR-504 reported by Johnson et al. may be partially due to its interaction with ABCB1. The
detailed profiling of subunit selectivity using purified proteasomes confirmed that DB-310 (28)
is much more selective to LMP2 compared to YU102 and does not inhibit LMP7 or β5 subunits
at relevant concentrations (Figure 3c and Suppl. Fig 2).
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a
c
b
KZR-504
*
YU102
*
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-
+
Reversin 121
Reversin 121
100
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DB-310
28)
50
(
34
2
5
30
1
0
YU102 DB-310 (28) KZR-504
0
100
300
1000
10000
In vitro LMP2 activity (IC , nM)
50
Subunit
LMP2
β1
LMP7
β5
Substrate
Ac-PAL-AMC
Ac-nLPnLD-AMC Ac-ANW-AMC
IC50 (nM)
Ac-WLA-AMC
ONX 0914
YU102
1,009.0 ± 70.1
105.2 ± 6.2
> 10,000
> 10,000
206.7 ± 5.5
> 10,000
37.9 ± 1.1
> 10,000
> 10,000
> 10,000
> 10,000
472.7 ± 12.6
> 10,000
> 10,000
> 10,000
> 10,000
YU102 epi
DB-310 (28)
KZR-504
70.0 ± 1.7
589.9 ± 4.7
4,762.9 ± 53.9
157.9 ± 8.5
Figure 3. a. A scatter plot that shows in vitro LMP2 inhibitory potency (x-axis) and in cellulo
LMP2 inhibition (y-axis). Arrows indicate YU102 analogs (closed circle) that are most potent in
both purified iP and DLD-1/ABCB1 cell line. Previously reported LMP2 inhibitors (YU102 and
KZR-504) are also shown as a closed circle. b. Remaining LMP2 activity in the presence or
absence of the ABCB1 inhibitor reversin 121 (7.5 µM) in DLD-1/ABCB1 cells following
incubation of LMP2 inhibitors (3 µM) for 4 h. Error bars represent standard deviation derived
from three technical replicates (p value < 0.05, n=3, Student’s t-test comparing the log
transformed IC50 values obtained from three independent runs). c. Proteasome subunit inhibition
by proteasome inhibitors. Data is shown as mean ± SD derived from a non-linear regression
based on n=3 replicates.
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DB-310 (28) suppresses inflammatory cytokine release from microglial cells and blocks
epithelial mesenchymal transition (EMT) in a human retinal pigment epithelium (RPE) cell
line. To determine the impact of our lead LMP2 inhibitor DB-310 (28) on neuroinflammatory
response, we first measured the levels of inflammatory cytokines released from BV-2 murine
microglial cells, commonly used as a substitute for primary microglia in many experimental
0
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settings. When added to lipopolysaccharide (LPS)-stimulated BV-2 cells, DB-310 (28) was
effective in suppressing the release of IL- 1α from BV-2 cells (Figure 4a), comparable to ONX-
914 which is known to inhibit the production of inflammatory cytokines.5
0
Vehicle
TNF-α
DB-310 (28) + TNF-α
a
b
ONX0914
YU102
KZR-504
DB-310 (28)
1
00
7
5
5
0
20 μm
20 μm
20 μm
*
25
0
0
.1
0.3
0.6
1
3
Concentration (M)
20 μm
20 μm
20 μm
1
00
75
50
25
0
0
.1
0.3
0.6
1
3
Concentration (M)
20 μm
20 μm
20 μm
Figure 4. a. Effect of iP inhibitors on cytokine release in LPS-stimulated BV-2 cells. BV-2 cells
were incubated with LPS (1 g/mL) and iP inhibitors for 24 h before the cytokine release assay.
Error bars indicate standard deviation derived from three technical replicates. Difference in
suppression of IL-1α secretion between YU102-treated and DB-310 (28)-treated BV-2 cells was
statistically significant (p-value < 0.05, n=3). b. Inhibition of TNF-α-triggered epithelial
mesenchymal transition (EMT) by DB-310 (28) in the human RPE cell line ARPE-19.
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We next investigated whether DB-310 (28) has an effect on inflammation-triggered cellular
events using a cell line model. It has been well established that induction of epithelial
mesenchymal transition (EMT) in human RPE cells underlies dysregulated inflammation and
RPE degeneration.34-35 Thus, we examined whether DB-310 (28) can block EMT in the human
RPE cell line ARPE-19, which is also known to form a characteristic mosaic-like monolayer.36
TNF-α-triggered induction of EMT in ARPE-19 is known to be mediated via inflammation and
accompanied by morphological and molecular changes in ARPE-19 that can be readily detected
by the loss of E-cadherin expression in the cell-cell junction and upregulation of vimentin in the
cytoplasm. When TNF-α-stimulated ARPE-19 cells were incubated with DB-310 (28) for 24 h,
its impact on EMT was evident in the immunofluorescence images of cells, suppressing changes
in E-cadherin and vimentin expression pattern associated with EMT and thus indicating that DB-
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10 (28) can suppress inflammatory responses in the RPE (Figure 4b).
DB-310 (28) ameliorates cognitive impairment in a mouse model of AD. We next
investigated the efficacy of DB-310 (28) using the transgenic mouse model of AD, Tg2576,
2
4
following a procedure previously reported by us. Tg2576 mice received intraperitoneal
injections of either DB-310 (28) (10 mg/kg) or vehicle twice a week for three weeks. While mice
treated with DB-310 (28) performed significantly better in the Morris water maze test than mice
treated with vehicle, they also showed moderately improved test performance compared to those
treated with YU102 (Figure 5a).
Next, we determined whether serum levels of cytokines in Tg2576 mice are also affected by the
treatment of DB-310 (28) using an enzyme-linked immunosorbent assay (ELISA) designed to
detect IL-1α, which is known to play a critical role in AD progression.37 As shown in Figure 5b,
serum IL-1α levels in DB-310 (28)-treated Tg2576 mice were significantly lower than in mice
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treated with vehicle, consistent with the previously reported effect of YU102 on the serum levels
2
4
of IL-1α in Tg2576 mice. To further verify the in vivo impact of DB-310 (28) on
neuroinflammatory responses, we examined the structural integrity of RPE in DB-310 (28)-
treated Tg2576 mice, which express human amyloid precursor protein (APP) and are known to
display RPE degeneration caused by Aβ-triggered inflammation in eyes.38-39 When RPE samples
were isolated and examined, the orderly mosaic structure of normal RPE was severely disrupted
in untreated or vehicle-treated Tg2576 mice (Figure 5c). In contrast, mice treated with DB-310
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(28) displayed the mosaic structure of normal RPE, indicating a nearly complete blockade of
RPE degeneration.
a
b
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DB-310 (28)
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e
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icl
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Days of testing
2
(
Days of testing
h
e
0
1
V
3
-
B
D
Tg2576 mouse RPE
Vehicle
c
Untreated
DB-310 (28)
50 μm
50 μm
50 μm
Figure 5. a. DB-310 (28) ameliorates cognitive deficits in Tg2576 mice. Cognitive function in
Tg2576 mice was evaluated by the Morris water maze test: escape latency time (left) and escape
distance of the mice (right). Statistical analyses of escape latency and escape distance were
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performed via two-way ANOVA. *Difference in escape latency on days 3-5 or distance on days
5
between control and DB-310 (28)-treated mice was statistically significant (p-value < 0.05,
n=12 for control and n=8 for DB-310 (28)-treated mice). b. Quantification of serum IL-1α levels
5-fold diluted) in vehicle- or DB-310 (28)-treated Tg2576 mice via ELISA (p-value <0.05,
1
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(
n=3). c. Immuno-stained RPE isolated from Tg2576 mice treated with vehicle or DB-310 (28).
The representative images are shown from 3 independent replicates.
Lastly, while we observed no signs of overt toxicity during the treatment period, we further
investigated whether DB-310 (28) has any adverse effects on cell viability using various cell
lines: RPMI 8266 (human myeloma cells), BV-2 (murine microglial cells) and WI-38 (normal
human fetal lung fibroblasts). Similar to other LMP2 inhibitors (YU102 and KZR-504), DB-310
(28) showed no negative impact up to 10 µM on the viability of all three cell lines tested (Suppl.
Figure 3).
CONCLUSIONS
In this study, we aimed to develop YU102 analogs with improved selectivity and pharmaceutical
properties and verify that LMP2 inhibition alone is sufficient to have an impact on cognitive
function in AD mouse models. YU102 analogs generated in this effort kept a proline residue at
the P3 position of YU102. This was done by design since proline-containing small peptides such
as Xaa-Pro-Xaa peptides are reported to be less labile to enzymatic hydrolysis than their
4
0
counterparts lacking proline. Given that some of previously reported LMP2 inhibitors are based
on the linear peptide epoxyketone backbone (UK101 & KZR-504)9, 26 or non-peptide N-
4
1
benzylamide pyridine scaffold, it will be interesting to compare LMP2 binding modes of these
compounds and use this information to guide the design of next-generation LMP2 inhibitors.
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Before evaluating the in vivo effect of our lead LMP2 inhibitor DB-310 (28), we first used the
human RPE ARPE-19 cell line and observed the dramatic impact of DB-310 (28) on TNF-α-
triggered EMT which is mediated via inflammatory responses. Since the EMT pathway has
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42
received great interest in cancer therapy in recent years, it will be also interesting whether the
LMP2-targeting strategy can be applied for therapeutic intervention of relevant cancer types.
While the positive effects of DB-310 (28) on cognitive function as well as RPE degeneration in
the Tg2576 mouse model were observed, it should be cautioned not to directly translate to
humans without further assessment. Perhaps, a study with another animal model such as a
tauopathy model (PS19), in which tau-associated neuroinflammation plays a role in disease
progression, may provide further validation of the LMP2-targeting strategy for
neurodegenerative diseases.
In summary, in this report we have developed a LMP2 inhibitor DB-310 (28) with improved
specificity and reduced ABCB1 interactions and demonstrated that LMP2 inhibition alone may
be sufficient to improve cognitive function in the AD mouse model. Considering recent failures
of a string of clinical trials aimed at the amyloid hypothesis, our study may offer a new
therapeutic strategy for AD that is not reliant on Aβ clearance. In addition, given increasing
evidence that microglia-mediated neuroinflammation is also a major contributing factor to the
pathogenesis of age-related macular degeneration (AMD), this study may potentially be
translatable into the development of AMD drugs.
EXPERIMENTAL SECTION
General chemistry. All reagents and solvents used in synthesis of intermediates and final
compounds were purchased from commercial vendors and used without further purification
unless otherwise reported. Reaction progress was monitored using TLC on precoated plates and
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compound spots were detected by using UV and staining solution like phosphomolybdic acid
hydrate (PMA), potassium permanganate and iodine. Flash column chromatography was
performed on 60 Å silica gel, 230-400 mesh (Merck). Proton and carbon NMR spectra were
recorded on 400 and 500 MHz Varian spectrometer. Chemical shifts are reported in ppm units
and coupling constant (J) is reported in Hz whereas tetramethylsilane was used as a reference
standard. NMR multiplicities were reported using different abbreviations such as singlet (s),
doublet (d), triplet (t), quartet (q) and multiplet (m). An Agilent Technologies 6120 Quadrupole
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4
5
6
7
8
9
0
LC/MS was used to record mass spectra. The eluent used were A: H
CH CN) both of which containing 0.1% formic acid. The linear gradient used was B/A from
5:95 to 90:10 over 18 min and then 100:0 over 7 min in an Agilent Eclipse XBD-C18 column
(5-μm particle size), 4.6 mm dia.  150 mm with a flow rate of 0.4 mL/min]. The purity of all
2
O and B: acetonitrile
(
3
[
the final compounds were checked in an Agilent Technologies 1200 series equipped with a UV
detector set at 254 nm. The same solvent gradient, column and method of LC/MS was used to
determine the purity of all final compounds. The purity of all final compounds determined to be
≥
95%. KZR-504 and ONX0914 used in experiments were synthesized using previously reported
method.5, 9
Synthesis
(S)-tert-Butyl 1-(methoxy(methyl)amino)-4-methyl-1-oxopentan-2-ylcarbamate 2a (General
procedure for the preparation of Weinreb amides 2a-2c)
To the stirred solution of Boc-leucine (2 g, 8.64 mmol), EDCI·HCl (2.48 g, 12.96 mmol) and
HOBt (1.17 g, 8.64 mmol) in DCM, N,O-dimethylhydroxylamine hydrochloride (0.84 g, 8.64
mmol) and DIPEA (3.77 mL, 21.6 mmol) were added. The reaction mixture was stirred at same
temperature for 18 hours. Solvent was removed under reduced pressure and residue was purified
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
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5
5
5
5
5
5
5
5
5
6
by flash column chromatography using 20% ethyl acetate in hexane to yield pure product as
1
sticky colorless solid (2.1 g, 89%). H NMR (400 MHz, CDCl
3
) δ 5.05 (d, J = 9.5 Hz, 1H), 4.70
(s, 1H), 3.76 (s, 3H), 3.17 (s, 3H), 1.69 (dt, J = 6.7, 13.5 Hz, 1H), 1.46-1.37 (m, 11H), 0.92 (dd, J
6.6, 14.0 Hz, 6H).
S)-tert-Butyl 3-cyclohexyl-1-(methoxy(methyl)amino)-1-oxopropan-2-ylcarbamate (2b)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
=
(
2
b was prepared from 1b following the same general procedure used for the preparation of
1
Weinreb amide as colorless oil (90%). H NMR (400 MHz, CDCl
3
) δ 5.00 (d, J = 9.5 Hz, 1H),
4
1
.71 (s, 1H), 3.75 (s, 3H), 3.16 (s, 3H), 1.87 (d, J = 13.0 Hz, 1H), 1.72 – 1.53 (m, 4H), 1.40 (s,
2H), 1.17 (m, 3H), 1.01 – 0.78 (m, 2H).
(S)-tert-Butyl 1-(methoxy(methyl)amino)-1-oxo-3-phenylpropan-2-ylcarbamate (2c)
2
c was prepared from 1c following the same general procedure used for the preparation of
1
Weinreb amide as colorless thick oil (86%). H NMR (400 MHz, CDCl
3
) δ 7.25 (d, J = 8.2 Hz,
2H), 7.24 – 7.19 (m, 1H), 7.15 (d, J = 7.4 Hz, 2H), 5.13 (d, J = 9.0 Hz, 1H), 4.93 (s, 1H), 3.64 (s,
H), 3.15 (s, 3H), 3.03 (dd, J = 6.1, 13.6 Hz, 1H), 2.86 (t, J = 10.5 Hz, 1H), 1.37 (s, 9H).
S)-tert-Butyl 2,6-dimethyl-3-oxohept-1-en-4-ylcarbamate 3a (General procedure for the
3
(
preparation of enone 3a-3c)
To the stirred solution of 2a (2 g, 7.28 mmol) in dry THF, 0.5 M isopropenylmagnesium bromide
solution in THF (58 mL, 29.15 mmol) was added drop-wise at -78 °C. The reaction mass was
stirred at same temperature overnight. Reaction solution was warmed gradually to room
temperature and the reaction was quenched with saturated solution of ammonium chloride. The
product was extracted with ethyl acetate 3 times. Combined organic layer was dried in sodium
sulfate, filtered and concentrated. Crude product was purified by flash column chromatography
1
using 5% ethyl acetate in hexane to yield pure product as colorless oil (1.2 g, 64%). H NMR
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(
400 MHz, CDCl
3
) δ 6.06 (s, 1H), 5.86 (s, 1H), 5.20 – 4.96 (m, 2H), 1.83 (s, 3H), 1.81 – 1.64 (m,
1
H), 1.41 (s, 9H), 1.36 – 1.18 (m, 2H), 0.98 (d, J = 6.5 Hz, 3H), 0.89 (d, J = 6.7 Hz, 3H).
(S)-tert-Butyl 1-cyclohexyl-4-methyl-3-oxopent-4-en-2-ylcarbamate (3b)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
b was prepared from 2b following the same general procedure used for the preparation of enone
1
as white solid (76%). H NMR (400 MHz, CDCl
3
) δ 6.04 (s, 1H), 5.84 (s, 1H), 5.07 (d, J = 6.7
Hz, 2H), 1.87 (s, 3H), 1.75 – 1.46 (m, 6H), 1.41 (s, 10H), 1.32 – 1.05 (m, 4H), 1.00 – 0.78 (m,
H).
S)-tert-Butyl 4-methyl-3-oxo-1-phenylpent-4-en-2-ylcarbamate (3c)
2
(
3
c was prepared from 2c following the same general procedure used for the preparation of enone
1
as white solid (79%). H NMR (400 MHz, CDCl
3
) δ 7.30 – 7.12 (m, 3H), 7.05 (d, J = 7.2 Hz,
2
2
H), 5.99 (s, 1H), 5.83 (p, J = 1.5 Hz, 1H), 5.25 (d, J = 8.7 Hz, 2H), 3.19 – 3.01 (m, 1H), 2.97 –
.80 (m, 1H), 1.84 (s, 3H), 1.39 (s, 9H).
tert-Butyl (S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-ylcarbamate 4a
General procedure for the preparation of epoxide 4a-4c)
(
DIPEA (6.83 mL, 39.14 mmol) was added drop-wise in the stirred solution of 3a (1 g, 3.91 mmol),
hydrogen peroxide (6.64 mL, 97.75 mmol) and benzonitrile (4.0 mL, 39.14 mmol) in methanol at
0
°C. The reaction mixture was stirred at same temperature for 3 hours. Water was added to the
solution and methanol was removed under reduced pressure. The aqueous layer was extracted with
ethyl acetate 3 times. Collected organic layer was dried with sodium sulfate, filtered and
concentrated. Crude product was purified by flash column chromatography using 5% ethyl acetate
1
in hexane to yield pure product as sticky white solid (0.6 g, 57%). H NMR (400 MHz, CDCl
3
) δ
4
.82 (d, J = 8.9 Hz, 1H), 4.35 – 4.24 (m, 1H), 3.27 (d, J = 5.0 Hz, 1H), 2.87 (d, J = 5.0 Hz, 1H),
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
4
4
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5
5
5
5
5
5
5
5
6
1
.78 – 1.64 (m, 1H), 1.50 (s, 4H), 1.39 (s, 9H), 1.15 (ddd, J = 4.3, 10.5, 14.3 Hz, 1H), 0.93 (dd, J
=
6.6, 13.0 Hz, 6H).
tert-Butyl (S)-3-cyclohexyl-1-((R)-2-methyloxiran-2-yl)-1-oxopropan-2-ylcarbamate (4b)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
b was prepared from 3b following the same general procedure used for the preparation of
1
epoxide as sticky white solid (41%). H NMR (400 MHz, CDCl
3
) δ 4.79 (d, J = 8.8 Hz, 1H),
4
.32 (td, J = 3.0, 8.6, 9.8 Hz, 1H), 3.26 (d, J = 5.1 Hz, 1H), 2.86 (d, J = 5.0 Hz, 1H), 1.93 – 1.79
m, 1H), 1.74 – 1.51 (m, 5H), 1.49 (s, 3H), 1.39 (s, 9H), 1.28 – 1.05 (m, 5H), 1.01 – 0.84 (m,
H).
(
2
tert-Butyl (S)-1-((R)-2-methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-ylcarbamate (4c)
4c was prepared from 3c following the same general procedure used for the preparation of
1
epoxide as white solid (32%), H NMR (400 MHz, CDCl
3
) δ 7.33 – 7.19 (m, 3H), 7.19 – 7.09
(m, 2H), 4.98 – 4.86 (m, 1H), 4.62 – 4.49 (m, 1H), 3.27 (d, J = 5.0 Hz, 1H), 3.09 (dd, J = 5.0,
13.9 Hz, 1H), 2.88 (d, J = 5.0 Hz, 1H), 2.72 (dd, J = 7.8, 13.9 Hz, 1H), 1.48 (s, 3H), 1.35 (s, 9H).
General procedure for the amide coupling reaction: To the stirring solution of carboxylic acid
(1 equiv) in DCM, HBTU (2 equiv), HOBt (2 equiv), amine (1 equiv) and DIPEA (3 equiv) were
added respectively at room temperature and the reaction solution was stirred at same temperature
for overnight. Solvent was removed under reduced pressure and residue was purified via flash
column chromatography.
General procedure for Boc deprotection reaction: Boc protected intermediate was dissolved
in DCM and excess of TFA was added. The reaction mixture was stirred at room temperature for
2
hours. Solvent was removed under reduced pressure and residue was used in the next step
without further purification.
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General procedure for benzyl deprotection reaction: Benzyl protected intermediate was
dissolved in methanol. Activated palladium in charcoal (10 mol%) was added to the solution at
room temperature and the reaction mixture was stirred under hydrogen gas environment at same
temperature for 2 hours. Reaction solution was filtered through celite and the collected filtrate
was evaporated under reduced pressure. The carboxylic acid was used in the next step without
further purification.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
General procedure for Fmoc deprotection reaction: 4 Equivalent of piperidine was added to
the stirred solution of Fmoc protected amine in DMF at room temperature and the reaction
solution was stirred at same temperature for 1 hour. Reaction solution was washed with water
and required product was extracted with ethyl acetate. Combined organic solution was dried with
sodium sulfate, filtered and concentrated under reduced pressure. Impure residue was purified
via flash column chromatography.
General procedure for acylation or sulfonylation reaction: To the stirred solution of Boc
deprotected amine or free amine in DCM at room temperature, triethyl amine (3 equiv) and acyl
chloride or sulfonyl chloride (1.5 equiv) were added respectively and the reaction solution was
stirred at same temperature for 3 hours. Solvent was removed under reduced pressure and residue
was purified via flash column chromatography.
General procedure for the TBDPS protection: Starting material was dissolved in DCM and 3
equiv of imidazole was added to it at room temperature. TBDPSCl was added drop-wise to the
reaction mixture and it was further stirred at same temperature for 2 hours. Reaction was
quenched with the addition of water. The product was extracted with DCM and collected
organic layer was dried under sodium sulfate, filtered and concentrated under reduced pressure.
The crude product was purified via flash column chromatography.
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4
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5
5
6
General procedure for the deprotection of TBDPS: To the stirred solution of TBDPS
protected starting material, TBAF (2 equiv) was added dropwise and the reaction mass was
stirred at same temperature for 1 hour. Solvent was removed under reduced pressure and the
residue was purified via flash column chromatography.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
S)-tert-Butyl 2-((S)-1-(benzyloxy)-1-oxo-3-phenylpropan-2-ylcarbamoyl)pyrrolidine-1-
carboxylate (5a)
General amide coupling reaction procedure was used to prepare 5a as colorless oil (96%). H
NMR (400 MHz, CDCl ) δ 7.30 (dt, J = 9.9, 20.8 Hz, 5H), 7.18 (d, J = 4.2 Hz, 3H), 6.99 (t, J =
.4 Hz, 2H), 5.13 (s, 2H), 4.86 (s, 1H), 4.32 – 4.03 (m, 2H), 3.45 – 3.22 (m, 2H), 3.15 (ddd, J =
1.9, 5.9, 13.9 Hz, 1H), 3.06 – 2.93 (m, 1H), 2.07 – 1.63 (m, 4H), 1.27 (s, 9H).
1
3
4
(S)-tert-Butyl 2-((S)-1-(benzyloxy)-1-oxohexan-2-ylcarbamoyl)pyrrolidine-1-carboxylate
(5b)
1
General amide coupling reaction procedure was used to prepare 5b as colorless oil (83%). H NMR
(
400 MHz, CDCl
3
) δ 7.46 – 7.28 (m, 5H), 5.22 – 5.04 (m, 2H), 4.56 (s, 1H), 4.25 (d, J = 31.5 Hz,
H), 3.36 (d, J = 45.7 Hz, 2H), 1.99 – 1.71 (m, 4H), 1.71 – 1.56 (m, 2H), 1.46 (d, J = 23.7 Hz, 9H),
.34 – 1.11 (m, 4H), 0.81 (dd, J = 6.0, 8.1 Hz, 3H).
1
1
(S)-tert-Butyl 2-((S)-1-(benzyloxy)-1-oxo-4-phenylbutan-2-ylcarbamoyl)pyrrolidine-1-
carboxylate (5c)
General amide coupling reaction procedure was used to prepare 5c as colorless sticky solid
1
(54%). H NMR (400 MHz, CDCl
3
) δ 7.33 (d, J = 3.0 Hz, 5H), 7.28 – 7.20 (m, 2H), 7.16 (d, J =
7
4
.2 Hz, 1H), 7.10 – 7.00 (m, 2H), 5.21 – 5.06 (m, 2H), 4.63 (d, J = 30.1 Hz, 1H), 4.27 (d, J =
3.4 Hz, 1H), 3.54 – 3.27 (m, 2H), 2.55 (q, J = 9.1, 9.5 Hz, 2H), 2.21 – 2.06 (m, 2H), 2.06 – 1.81
(m, 4H), 1.26 (s, 9H).
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(
S)-tert-Butyl 2-((S)-1-(benzyloxy)-3-(4-tert-butylphenyl)-1-oxopropan-2-
ylcarbamoyl)pyrrolidine-1-carboxylate (5e)
General amide coupling reaction procedure was used to prepare 5e as colorless oil (96%). H
NMR (400 MHz, CDCl ) δ 7.31 (q, J = 5.2, 5.9 Hz, 5H), 7.21 (d, J = 7.9 Hz, 2H), 6.95 (d, J = 7.8
Hz, 2H), 5.12 (s, 2H), 4.86 (s, 1H), 4.21 (d, J = 38.6 Hz, 1H), 3.47 – 3.19 (m, 2H), 3.13 (dd, J =
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
5
1
.9, 13.9 Hz, 1H), 2.96 (dd, J = 7.0, 14.0 Hz, 1H), 2.25 – 1.84 (m, 2H), 1.71 (s, 2H), 1.39 (s, 9H),
.26 (s, 9H).
(S)-tert-Butyl 2-((S)-1-(benzyloxy)-3-(naphthalen-2-yl)-1-oxopropan-2-
ylcarbamoyl)pyrrolidine-1-carboxylate (5f)
General amide coupling reaction procedure was used to prepare 5f as colorless sticky solid
1
(89%). H NMR (400 MHz, CDCl
3
) δ 7.82 – 7.74 (m, 1H), 7.68 (t, J = 8.3 Hz, 2H), 7.53 – 7.39
(m, 3H), 7.39 – 7.26 (m, 2H), 7.26 – 7.18 (m, 3H), 7.11 (dd, J = 1.6, 8.5 Hz, 1H), 6.84 (d, J = 7.9
Hz, 1H), 5.18 – 5.01 (m, 2H), 4.93 (q, J = 6.6 Hz, 1H), 4.68 (s, 1H), 3.44 – 3.20 (m, 4H), 2.38 –
.25 (m, 1H), 2.02 – 1.81 (m, 3H), 1.27 (s, 9H).
S)-Benzyl 2-((S)-1-(2-acetamidoacetyl)pyrrolidine-2-carboxamido)-3-(naphthalen-1-
yl)propanoate (5g)
General amide coupling reaction procedure was used to prepare 5g as colorless oil (92%). H
NMR (400 MHz, CDCl ) δ 7.85 – 7.55 (m, 3H), 7.50 – 7.36 (m, 3H), 7.33 – 7.12 (m, 5H), 7.12 –
.05 (m, 1H), 6.23 (s, 1H), 5.23 – 5.02 (m, 2H), 5.04 – 4.87 (m, 1H), 4.46 (td, J = 3.1, 5.8, 6.3
Hz, 1H), 3.87 (ddd, J = 4.7, 9.6, 17.9 Hz, 1H), 3.34 (dd, J = 6.1, 13.9 Hz, 1H), 3.17 (dtd, J = 4.2,
.3, 9.9, 14.0 Hz, 2H), 2.36 – 2.11 (m, 2H), 1.80 – 1.67 (m, 2H), 1.40 (s, 9H).
S)-tert-Butyl 2-((S)-1-(benzyloxy)-3-(biphenyl-4-yl)-1-oxopropan-2-
ylcarbamoyl)pyrrolidine-1-carboxylate (5h)
2
(
1
3
7
8
(
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2
2
2
2
2
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3
3
3
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3
3
3
3
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4
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
General amide coupling reaction procedure was used to prepare 5h as colorless oil (88%). H
NMR (400 MHz, CDCl ) δ 7.52 (dt, J = 1.1, 8.1 Hz, 2H), 7.46 – 7.35 (m, 3H), 7.35 – 7.18 (m,
7H), 7.07 (d, J = 7.7 Hz, 2H), 5.13 (t, J = 10.0 Hz, 2H), 4.90 (s, 1H), 4.18 (s, 1H), 3.29 (s, 2H),
.20 (dd, J = 6.0, 13.9 Hz, 1H), 3.05 (dd, J = 6.5, 13.9 Hz, 1H), 2.10 – 1.86 (m, 2H), 1.56 – 1.43
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
(m, 2H), 1.25 (s, 9H).
(
S)-tert-Butyl 2-((S)-1-(benzyloxy)-1-oxo-3-(pyridin-4-yl)propan-2-
ylcarbamoyl)pyrrolidine-1-carboxylate (5i)
General amide coupling reaction procedure was used to prepare 5i as colorless oil (57%). H
NMR (400 MHz, CDCl ) δ 8.39 (d, J = 4.9 Hz, 2H), 7.41 – 7.34 (m, 2H), 7.29 (dd, J = 2.8, 6.7
Hz, 2H), 7.24 (d, J = 1.6 Hz, 1H), 6.90 (d, J = 4.9 Hz, 2H), 5.22 – 5.02 (m, 2H), 4.88 (s, 1H),
.25 (s, 1H), 3.39 – 3.11 (m, 3H), 3.04 – 2.89 (m, 1H), 1.80 (s, 2H), 1.65 (s, 2H), 1.40 (s, 9H).
1
3
4
(S)-Benzyl 3-(4-(((9H-fluoren-9-yl)methoxy)carbonylamino)phenyl)-2-((S)-1-(2-
acetamidoacetyl)pyrrolidine-2-carboxamido)propanoate (5j)
General amide coupling reaction procedure was used to prepare 5j as colorless sticky solid
1
(
(
(
93%). H NMR (400 MHz, CDCl
3
) δ 7.83 – 7.70 (m, 2H), 7.60 (d, J = 7.4 Hz, 2H), 7.49 – 7.28
m, 6H), 7.17 (d, J = 8.0 Hz, 2H), 6.85 (d, J = 8.3 Hz, 2H), 6.70 (s, 1H), 6.45 (s, 1H), 5.20 – 5.03
m, 2H), 4.81 (t, J = 6.6 Hz, 1H), 4.29 (t, J = 6.7 Hz, 1H), 4.01 – 3.80 (m, 2H), 3.41 – 3.23 (m,
2
H), 3.15 (dd, J = 5.9, 14.0 Hz, 1H), 2.96 (dd, J = 6.4, 14.0 Hz, 1H), 2.32 (d, J = 12.2 Hz, 1H),
2.08 – 1.91 (m, 1H), 1.89 – 1.69 (m, 1H), 1.38 (s, 9H).
S)-tert-Butyl 2-((S)-1-(benzyloxy)-3-(4-fluorophenyl)-1-oxopropan-2-
ylcarbamoyl)pyrrolidine-1-carboxylate (5k)
(
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General amide coupling reaction procedure was used to prepare 5k as colorless oil (91%). H
NMR (400 MHz, CDCl ) δ 7.41 – 7.26 (m, 5H), 6.93 (dd, J = 5.4, 8.4 Hz, 2H), 6.85 (t, J = 8.5
3
Hz, 2H), 5.13 (q, J = 12.1 Hz, 2H), 4.83 (s, 1H), 4.22 (d, J = 26.3 Hz, 1H), 3.29 (s, 2H), 3.12 (dd,
J = 5.9, 14.0 Hz, 1H), 2.97 (dd, J = 6.3, 14.0 Hz, 1H), 1.83 (d, J = 23.8 Hz, 2H), 1.62 (s, 2H),
1
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0
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0
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0
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0
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0
1
.49 – 1.29 (m, 9H).
S)-tert-Butyl 2-((S)-1-(benzyloxy)-3-hydroxy-1-oxopropan-2-ylcarbamoyl)pyrrolidine-1-
carboxylate (5l)
General amide coupling reaction procedure was used to prepare 5l as colorless oil (91%). H
NMR (400 MHz, CDCl ) δ 7.33 (d, J = 2.6 Hz, 5H), 7.17 – 7.02 (m, 1H), 5.20 (s, 2H), 4.62 (s,
1H), 4.16 (s, 1H), 4.11 – 3.80 (m, 2H), 3.52 – 3.31 (m, 2H), 2.25 – 2.09 (m, 1H), 2.09 – 1.95 (m,
H), 1.83 (d, J = 5.7 Hz, 2H), 1.42 (s, 9H).
S)-Benzyl 2-((S)-1-(2-acetamidoacetyl)pyrrolidine-2-carboxamido)-3-phenylpropanoate
(6a)
a was prepared from Boc deprotected 5a and acetyl glycine using general procedure for amide
(
1
3
2
(
6
1
coupling reaction as colorless sticky solid (93%). H NMR (400 MHz, CDCl
3
) δ 7.40 – 7.26 (m,
4
4
1
–
H), 7.17 (q, J = 2.3, 3.1 Hz, 4H), 7.08 – 6.96 (m, 2H), 6.41 (s, 1H), 5.21 – 5.06 (m, 2H), 4.92 –
.79 (m, 1H), 4.50 (dd, J = 2.0, 8.1 Hz, 1H), 3.97 (dd, J = 4.4, 17.7 Hz, 1H), 3.81 (dd, J = 3.8,
7.7 Hz, 1H), 3.69 (p, J = 6.7 Hz, 1H), 3.23 – 3.10 (m, 2H), 3.00 (dd, J = 6.9, 14.0 Hz, 1H), 2.34
2.24 (m, 1H), 2.02 (s, 3H), 1.97 – 1.86 (m, 2H), 1.80 (dt, J = 8.6, 12.2 Hz, 1H).
(S)-Benzyl 2-((S)-1-(2-acetamidoacetyl)pyrrolidine-2-carboxamido)hexanoate (6b)
6
b was prepared from Boc deprotected 5b and acetyl glycine using general procedure for amide
1
coupling reaction as colorless oil (76%). H NMR (400 MHz, CDCl
3
) δ 7.41 – 7.26 (m, 5H),
7.02 (d, J = 7.7 Hz, 1H), 6.53 (t, J = 4.4 Hz, 1H), 5.21 – 5.04 (m, 2H), 4.56 – 4.44 (m, 2H), 4.01
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(
qd, J = 4.2, 17.6 Hz, 2H), 3.54 (ddd, J = 3.3, 8.2, 10.0 Hz, 1H), 3.40 (td, J = 7.0, 9.4 Hz, 1H),
2
.32 – 2.03 (m, 2H), 2.03 – 1.73 (m, 6H), 1.73 – 1.57 (m, 1H), 1.32 – 1.10 (m, 4H), 0.88 – 0.73
(m, 3H).
S)-Benzyl 2-((S)-1-(2-acetamidoacetyl)pyrrolidine-2-carboxamido)-4-phenylbutanoate (6c)
0
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(
6
c was prepared from Boc deprotected 5c and acetyl glycine using general procedure for amide
1
coupling reaction as white solid (88%). H NMR (400 MHz, CDCl
3
) δ 7.22 (dd, J = 6.4, 7.6 Hz,
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3
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H), 7.19 – 7.07 (m, 8H), 6.62 (s, 1H), 4.50 (qd, J = 4.0, 7.8 Hz, 2H), 4.16 – 4.05 (m, 2H), 4.05 –
.90 (m, 2H), 3.54 (ddd, J = 3.3, 8.1, 9.8 Hz, 1H), 3.39 (td, J = 6.8, 9.1 Hz, 1H), 2.59 (dt, J = 5.6,
.3 Hz, 2H), 2.29 – 2.02 (m, 3H), 2.02 – 1.81 (m, 6H).
(S)-Benzyl 2-((S)-1-(2-acetamidoacetyl)pyrrolidine-2-carboxamido)-3-(4-tert-
butylphenyl)propanoate (6e)
6
e was prepared from Boc deprotected 5e and acetyl glycine using general procedure for amide
1
coupling reaction as colorless oil (96%). H NMR (400 MHz, CDCl
3
) δ 7.39 – 7.22 (m, 4H),
7
.17 (ddd, J = 2.9, 8.4, 13.3 Hz, 3H), 7.05 – 6.88 (m, 2H), 6.61 (d, J = 3.8 Hz, 1H), 5.17 – 5.00
(m, 2H), 4.90 – 4.73 (m, 1H), 4.55 – 4.42 (m, 1H), 4.13 – 3.91 (m, 1H), 3.81 (dt, J = 3.5, 17.7
Hz, 1H), 3.39 – 3.21 (m, 2H), 3.10 (ddd, J = 2.9, 5.8, 13.9 Hz, 1H), 2.94 (ddd, J = 3.1, 7.2, 14.1
Hz, 1H), 2.21 (tt, J = 3.8, 8.4 Hz, 1H), 2.05 – 1.91 (m, 3H), 1.91 – 1.69 (m, 3H), 1.24 (d, J = 3.3
Hz, 9H).
(S)-Benzyl 2-((S)-1-(2-acetamidoacetyl)pyrrolidine-2-carboxamido)-3-(naphthalen-2-
yl)propanoate (6f)
6
f was prepared from Boc deprotected 5f and acetyl glycine using general procedure for amide
1
coupling reaction as colorless sticky solid (90%). H NMR (400 MHz, CDCl
3
) δ 8.07 – 7.95 (m,
1H), 7.86 – 7.70 (m, 1H), 7.70 – 7.59 (m, 2H), 7.50 – 7.34 (m, 3H), 7.34 – 7.15 (m, 5H), 7.11
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(
ddd, J = 1.6, 4.2, 8.5 Hz, 1H), 6.49 – 6.26 (m, 1H), 5.21 – 5.04 (m, 2H), 5.04 – 4.87 (m, 2H),
4
.46 (dq, J = 1.9, 2.7, 5.4 Hz, 1H), 3.93 – 3.80 (m, 1H), 3.67 – 3.38 (m, 3H), 3.33 (dd, J = 5.8,
13.8 Hz, 1H), 2.22 (qd, J = 2.4, 4.7, 5.5 Hz, 1H), 2.03 – 1.87 (m, 3H), 1.87 – 1.64 (m, 3H).
S)-Benzyl 2-((S)-1-(2-acetamidoacetyl)pyrrolidine-2-carboxamido)-3-(naphthalen-1-
yl)propanoate (6g)
1
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(
6
g was prepared from Boc deprotected 5g and acetyl glycine using general procedure for amide
1
coupling reaction as colorless sticky solid (92%). H NMR (400 MHz, CDCl
3
) δ 7.83 – 7.59 (m,
3
2
H), 7.53 – 7.36 (m, 3H), 7.36 – 7.15 (m, 5H), 7.15 – 7.05 (m, 1H), 6.25 (s, 1H), 5.23 – 5.04 (m,
H), 5.04 – 4.89 (m, 1H), 4.46 (td, J = 3.1, 5.8, 6.3 Hz, 1H), 3.87 (ddd, J = 4.7, 9.6, 17.9 Hz,
1H), 3.69 – 3.39 (m, 2H), 3.34 (dd, J = 6.1, 13.9 Hz, 1H), 3.17 (dtd, J = 4.2, 8.3, 9.9, 14.0 Hz,
H), 2.39 – 2.13 (m, 2H), 2.00 (s, 3H), 1.88 – 1.67 (m, 3H), 1.40 – 1.31 (m, 1H).
S)-Benzyl 2-((S)-1-(2-acetamidoacetyl)pyrrolidine-2-carboxamido)-3-(biphenyl-4-
yl)propanoate (6h)
2
(
6
h was prepared from Boc deprotected 5h and acetyl glycine using general procedure for amide
1
coupling reaction as colorless sticky solid (89%). H NMR (400 MHz, CDCl
3
) δ 7.60 – 7.48 (m,
2
H), 7.41 (dtt, J = 1.6, 4.0, 5.9 Hz, 4H), 7.33 (pt, J = 2.6, 5.1 Hz, 5H), 7.22 – 7.00 (m, 3H), 6.34
(s, 1H), 5.16 (tdd, J = 1.8, 11.1, 13.9 Hz, 2H), 4.96 – 4.86 (m, 1H), 4.53 (d, J = 8.0 Hz, 1H), 4.09
–
3.97 (m, 1H), 3.92 – 3.79 (m, 1H), 3.40 – 3.26 (m, 2H), 3.26 – 3.13 (m, 1H), 3.13 – 2.98 (m,
H), 2.32 (d, J = 12.5 Hz, 1H), 2.05 – 1.73 (m, 6H).
1
(S)-Benzyl 2-((S)-1-(2-acetamidoacetyl)pyrrolidine-2-carboxamido)-3-(pyridin-4-
yl)propanoate (6i)
6
i was prepared from Boc deprotected 5i and acetyl glycine using general procedure for amide
1
coupling reaction as colorless oil (77%). H NMR (400 MHz, CDCl
3
) δ 7.28 (dd, J = 1.7, 5.1 Hz,
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