Macrocyclic Immunoproteasome Inhibitors as a Potential Therapy for Alzheimer's Disease

Article abstract of DOI:10.1021/acs.jmedchem.1c00291

Previously, we reported that immunoproteasome (iP)-targeting linear peptide epoxyketones improve cognitive function in mouse models of Alzheimer's disease (AD) in a manner independent of amyloid β. However, these compounds' clinical prospect for AD is limited due to potential issues, such as poor brain penetration and metabolic instability. Here, we report the development of iP-selective macrocyclic peptide epoxyketones prepared by a ring-closing metathesis reaction between two terminal alkenes attached at the P2 and P3/P4 positions of linear counterparts. We show that a lead macrocyclic compound DB-60 (20) effectively inhibits the catalytic activity of iP in ABCB1-overexpressing cells (IC50: 105 nM) and has metabolic stability superior to its linear counterpart. DB-60 (20) also lowered the serum levels of IL-1α and ameliorated cognitive deficits in Tg2576 mice. The results collectively suggest that macrocyclic peptide epoxyketones have improved CNS drug properties than their linear counterparts and offer promising potential as an AD drug candidate.

Full text of DOI:10.1021/acs.jmedchem.1c00291

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Article
Macrocyclic Immunoproteasome Inhibitors as a Potential Therapy
for Alzheimer’s Disease
Min Jae Lee, Deepak Bhattarai, Hyeryung Jang, Ahreum Baek, In Jun Yeo, Seongsoo Lee, Zachary Miller,
Sukyeong Lee, Jin Tae Hong,* Dong-Eun Kim,* Wooin Lee,* and Kyung Bo Kim*
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ABSTRACT: Previously, we reported that immunoproteasome
(iP)-targeting linear peptide epoxyketones improve cognitive
function in mouse models of Alzheimer’s disease (AD) in a
manner independent of amyloid β. However, these compounds’
clinical prospect for AD is limited due to potential issues, such as
poor brain penetration and metabolic instability. Here, we report
the development of iP-selective macrocyclic peptide epoxyketones
prepared by a ring-closing metathesis reaction between two
terminal alkenes attached at the P2 and P3/P4 positions of linear
counterparts. We show that a lead macrocyclic compound DB-60
(20) effectively inhibits the catalytic activity of iP in ABCB1-overexpressing cells (IC50: 105 nM) and has metabolic stability superior
to its linear counterpart. DB-60 (20) also lowered the serum levels of IL-1α and ameliorated cognitive deficits in Tg2576 mice. The
results collectively suggest that macrocyclic peptide epoxyketones have improved CNS drug properties than their linear counterparts
and offer promising potential as an AD drug candidate.
commonly referred to as Y, Z, and X, respectively, are replaced
by three immunosubunits β1i, β2i, and β5i, commonly referred
to as LMP2 (low-molecular-mass polypeptide-2), MECL-1
(multicatalytic endopeptidase complex subunit-1), and LMP7
(low-molecular-mass polypeptide-7), respectively, to form the
iP. Functionally, the iP has been shown to modulate
inflammatory responses.13−15 As such, iP-targeting inhibitors
have been investigated in preclinical and clinical trials as
potential anti-inflammatory agents. For example, KZR-616, an
iP-selective linear peptide epoxyketone (targeting both LMP7
and LMP2, Figure 1a), is currently being evaluated in clinical
trials for systemic lupus erythematosus.16 Studies also revealed
that the iP is upregulated in the brain of AD patients compared
with healthy controls17−19 and that iP expression is closely
linked to the activated microglia, which are widely considered
as a primary culprit behind AD progression,17,20,21 suggesting a
potential connection between the iP and AD.
INTRODUCTION
■
In the past decades, almost all drugs developed based on their
ability to intervene with the amyloid β (Aβ) pathway have
failed in clinical trials for Alzheimer’s disease (AD).1,2 As a
result, much attention has been paid to developing anti-tau
drugs, but no approved anti-tau therapies are available so far.3,4
Strategies of targeting neuroinflammation (regarded as a
significant contributing factor to the progression of AD)
have also drawn considerable attention.5,6 Such strategies are
further corroborated by recent findings that inflammatory
cytokines released by activated microglia can induce astrocytes-
mediated7 or tau-mediated neurotoxicity.8 Nevertheless, multi-
ple clinical trials of FDA-approved anti-inflammatory agents
targeting cyclooxygenases (COXs) or tumor necrosis factor
(TNF)-α signaling have yielded no effective AD therapies.9−12
Thus, identifying new therapeutic agents not reliant on
conventional AD targets has become increasingly crucial in
AD drug discovery efforts. However, the lack of promising
novel drug targets is a major obstacle in making real progress
toward developing effective AD drugs.
In mammalian cells, there exist two main types of 20S
proteasomes: the constitutive proteasome (cP) and immuno-
proteasome (iP), which are constitutively expressed in immune
cells. The multiprotease complex 20S cP has three catalytic
subunits (β1, β2, β5) on inner two β-rings displaying three
unique substrate preferences: referred to as caspase-like (C-L),
trypsin-like (T-L), and chymotrypsin-like (CT-L) activities,
respectively. The cP subunits β1, β2, and β5, which are also
Recently, we reported that iP-targeting linear peptide
epoxyketones (e.g., DB-310, Figure 1b) have a previously
unrecognized effect of alleviating cognitive deficits in mouse
models of AD in a manner independent of Aβ and tau
Received: February 15, 2021
https://doi.org/10.1021/acs.jmedchem.1c00291
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Figure 1. Previously reported linear peptide epoxyketones targeting the immunoproteasome (iP) catalytic subunits LMP7 (a) and LMP2 (b).
Figure 2. Structure-aided design strategy for macrocyclic peptide epoxyketones. (a) Predicted docking model of DB-310 bound to LMP2 (PDB ID:
3UNF) from the mouse 20S immunoproteasome. (b) Proposed macrocyclization strategy between the P2 and P3/P4 residues.
aggregation.22,23 We also showed that the iP inhibitors lower
the levels of serum interleukin-1α (IL-1α) and protect the
retinal pigment epithelium (RPE) layer from the structural
destruction caused by Aβ-triggered inflammation in Tg2576
mice, potentially representing a new class of AD drugs that aim
at a previously untapped pathway in AD drug discovery efforts.
Despite promising efficacy, however, these linear peptide
epoxyketones’ clinical prospect seems limited at this time due
to issues commonly associated with linear peptides, such as
poor brain penetration and metabolic instability (mainly
attributable to transporter-mediated efflux and enzymatic
metabolism).23,26−30 Yet, the peptide epoxyketone family
(“short peptides with C-terminal α′,β′-epoxyketone warhead”)
provides an attractive drug development platform for related
diseases due to pharmacological advantages conferred by their
proven target specificity for the proteasomes and long-term
safety in the clinic.31
Macrocycles, prepared via constraining the freely rotatable
peptide bond, are often introduced to improve linear peptides’
physicochemical (drug-like) properties.32,33 This strategy has
been used to boost the potency and chemical stability of linear
peptides and improve metabolic stability, penetration across
the blood−brain barrier (BBB), and membrane permeabil-
ity.34−37 Typically, for short linear peptides like the iP inhibitor
DB-310 (Figure 1b), a macrocycle can be introduced by a ring-
closing reaction between the (n) and (n + 2) residues,38
forming a β-turn mimic macrocyclic peptide.
Here, we report the development of iP-targeting macrocyclic
peptide epoxyketones prepared via a ring-closing metathesis
reaction between two terminal alkenes attached at the P2 and
P3/P4 residues of their linear counterparts in the presence of a
second-generation Grubbs catalyst. We show that a lead
macrocyclic peptide epoxyketone DB-60 (20) potently inhibits
the catalytic activity of the iP catalytic subunit LMP2 with an
IC50 of 105 nM in cells overexpressing ABCB1 (a major efflux
transporter at the BBB), suggesting minimal interactions
between DB-60 (20) and ABCB1. DB-60 (20) also showed
no interaction with ABCG2, another major efflux transporter
that can limit the BBB penetration. Furthermore, DB-60 (20)
displayed metabolic stability superior to its linear counterpart.
When injected into Tg2576 mice, DB-60 (20) lowered the
serum levels of IL-1α and improved cognitive function while
showing no gross adverse effects. The results suggest that iP-
targeting macrocyclic peptide epoxyketones may offer a new
AD drug class that warrants further investigation for clinical
potential.
RESULTS AND DISCUSSION
■
Structure-Guided Design of Macrocyclic Peptide
Epoxyketones. To assess the feasibility of macrocycle
formation, we first built an LMP2 (PDB ID: 3UNF) molecular
model complexed with DB-310, previously developed by us as
an LMP2-selective linear peptide epoxyketone.23 As shown in
Figure 2, both P2-phenylalanine (blue circle) and P4-glycine/
B
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a
Scheme 1. Synthetic Strategy for Macrocyclic Analogues of DB-310
a
Reagents and conditions: a: (a) HN(CH3)OCH3, HOBt, EDCI·HCl, DIPEA, DCM, rt, 12 h; (b) isopropenylmagnesium bromide, THF, −78 °C,
12 h; (c) (i) benzonitrile, H2O2, DIPEA, methanol, 0 °C to rt, 2 h; (ii) TFA, DCM, rt, 1 h, then evaporated and vacuum-dried. b: (a) HBTU,
HOBt, DIPEA, DCM, rt, 18 h; (b) potassium carbonate, 4-bromo-1-butane, DMF, rt, 6 h; (c) allyl methyl carbonate, Pd(PPh3)4, THF, 60 °C, 3 h;
(d) (i) TFA, DCM, rt, 1 h, then evaporated and dried; (ii) N-Boc-glycine (for compound 13) or alkenyl carboxylic acid, HBTU, HOBt, DIPEA,
DCM, rt, 18 h; (e) (i) Grubb’s second-generation catalyst, toluene, 90 °C, 1 h, purified by flash column chromatography; (ii) H2, Pd/C, methanol,
rt, 1 h; (f) amine-deprotected epoxyketone (4a, 4b or 4c), HBTU, HOBt, DIPEA, DCM, rt, 18 h.
a
Scheme 2. Synthesis of Macrocyclic Analogues of DB-310 with P3-Proline Substitutions
a
Reagents and conditions: (a) allyl methyl carbonate, Pd(PPh3)4, THF, 60 °C, 3 h; (b) TFA, DCM, rt, 1 h, then evaporated and dried, N-Boc-
proline derivative, HBTU, HOBt, DIPEA, DCM, rt, 18 h; (c) (1) TFA, DCM, rt, 1 h, then evaporated and dried; (2) alkenyl carboxylic acid,
HBTU, HOBt, DIPEA, DCM, rt, 18 h; (d) (1) Grubb’s second-generation catalyst, toluene, 90 °C, 1 h, purified by flash column chromatography;
(2) H2, Pd/C, methanol, rt, 1 h; (e) amine-deprotected epoxyketone (4a), HBTU, HOBt, DIPEA, DCM, rt, 18 h.
N-cap-pyrazine (pink circle) residues of DB-310 are predicted
to extend to the surface of the LMP2 catalytic cavity,
potentially providing structural flexibility necessary to form a
macrocyclic ring between the P2 and P3/P4 residues without
C
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a
Table 1. In Vitro Proteasome Inhibition Profiles by Macrocyclic Peptide Epoxyketones
IC50 (nM)
LMP2
Y
LMP7
>10 000
>10 000
>10 000
>10 000
X
YU102
KZR-504
DB-310
16
17
18
19
20
21
22
23
24
25
26
27
32
33
34
105.2 6.2
157.9 8.5
70.8 1.7
206.7 5.5
4763 54
589.9 4.7
ND
>10 000
>10 000
>10 000
>10 000
422.8 68.0
129.3 21.8
414.5 30.1
167.7 39.4
184.1 19.0
412.8 151.1
563.4 87.0
158.1 30.4
522.6 67.5
251.1 35.8
69.4 12.8
65.6 14.5
2,131 383
237.7 24.4
4,583 601
261.5 53.2
1245 200
715.1 88.0
414.5 62.7
ND
8385 847
1503 398
ND
1516 337
341.8 50.9
2686 489
399.3 27.6
>10 000
4766 549
4998 704
4998 704
4640 781
1081 223
4640 781
>10 000
>10 000
>10 000
ND
ND
ND
5773 1007
>10 000
>10 000
>10 000
ND
>10 000
>10 000
ND
>10 000
>10 000
>10 000
ND
>10 000
>10 000
>10 000
>10 000
ND
>10 000
>10 000
35
36
ND
>10 000
>10 000
ND
>10 000
>10 000
a
The activity of individual proteasome subunits was measured using purified human 20S proteasomes and the respective fluorogenic substrates, Ac-
PAL-AMC (for LMP2), Ac-nLPnLD-AMC (for Y), Ac-ANW-AMC (for LMP7), and Ac-WLA-AMC (for X). Data were obtained based on the
results of three replicates per compound. N.D. denotes “not determined.”.
disrupting its interactions with LMP2. Based on the molecular
analysis, the P3-proline is expected to play a crucial role as an
anchor residue by allowing P2 and P3/P4 residues to be
positioned for macrocyclization (Figure 2b).
serine or tyrosine benzyl ester (5) using conventional amide
coupling reactions. O-alkylation of 6a in the presence of
potassium carbonate furnished the terminal alkene intermedi-
ate 7 in good yield. On the other hand, O-alkylated terminal
alkene 10 was prepared from 6b using allyl methyl carbonate
and a palladium catalyst, whereas 13 was prepared by adding
Boc-Gly to the N-terminus of 10. Di-ene intermediates (8, 11,
and 14) were prepared from their respective monoalkene
precursors (7, 10, and 13, respectively) via conventional amide
coupling reactions.
Chemistry. Our synthetic approach relied on a convergent
strategy that separately synthesizes α′,β′-epoxy amino acid
right-hand fragments and macrocyclic peptide left-hand
fragments before coupling via a conventional amide bond
formation reaction. The right-hand epoxyketone fragments of
macrocyclic compounds were prepared from Boc-protected
respective amino acids (1) using a previously reported
procedure to yield Boc-deprotected 4a−4c (Scheme 1a).23
For the macrocyclic left-hand fragments (Scheme 1b),
dipeptide intermediates 6a and 6b were first prepared from
A ring-closing metathesis reaction in the presence of Grubb’s
second-generation catalyst provided alkene-bridged macro-
cyclic analogues. Reduction of the cycloalkenes and depro-
tection of the benzyl ester were simultaneously performed via
D
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hydrogenation to afford the macrocyclic left-hand fragments
(9, 12, and 15), which were then coupled to the right-hand
epoxyketone fragments (4a−4c) to provide the final products
(16−27) in moderate to good yields.
Compound 21 (n = 1), chosen based on the initial
assessment of its inhibitory potency, selectivity toward
LMP2, and low molecular weight, was further modified to
provide additional macrocyclic peptide epoxyketones, in which
its P3-proline anchor was substituted by proline mimics having
different ring sizes, fluorinated or methylated proline (Scheme
2). Due to the common constrained macrocyclic structures, we
expected that these additional analogues would have similar
molecular properties to 21.
SAR Analysis of Macrocyclic Peptide Epoxyketones.
Seventeen macrocyclic analogues of DB-310 were successfully
synthesized and tested for their in vitro activity using purified
proteasomes and subunit-selective fluorogenic substrates
(Table 1). Most of the macrocyclic compounds inhibited the
iP (primarily LMP2) with high selectivity and good potency.
Notably, the tetrapeptide backbone-based macrocyclic com-
pounds (26 and 27, resulted from a macrocyclization between
P2-serine and P4-glycine) potently inhibited LMP2, with an
IC50 value comparable to the previously optimized LMP2-
selective linear peptide epoxyketone DB-310: 65.6-69.4 nM for
26 and 27 and 70.8 nM for DB-310. While the tripeptide-
based products with P2-tyrosine (16−20) were overall
effective against the iP (for LMP2, IC50 = 129−420 nM),
17−19 containing a longer linker or cyclohexyl residue at the
P1 exhibited additional inhibitory activity against the
constitutive proteasome (cP) subunits X and Y. The
tripeptide-based macrocyclic analogues with P2-serine (21−
25) also provided good IC50 values against LMP2 (158-563
nM) but with higher selectivity for the iP (specifically LMP2)
than the P2-tyrosine-containing ones. Similarly, P1-leucine
substitution with cyclohexyl or phenyl residues (17 vs 19 and
20; 21 vs 24 and 25) yielded only mild variations in the
proteasome inhibition profile. On the other hand, the P3-
proline substitution of 21 with 4-(cis)-fluoroproline (32), 2-
methylproline (34), or cyclohexane (36) resulted in a
substantial decrease in the LMP2 inhibitory potency, while
33 (with P3-4,4-difluoroproline) and 35 (with P3-azetidine)
displayed LMP2 inhibitory potency similar to 21. Collectively,
we chose the most potent LMP2 inhibitors (26 and 27), two
P2-tyrosine-derived macrocyclic compounds (17 and 20,
lacking X inhibition), and a P2-serine-derived one (21, having
the smallest MW) for further testing.
Figure 3. (a) LMP2 inhibitory activity of DB-60 (20) and other
compounds in ABCB1-overexpressing RPMI-8226 cells. ABCB1-
overexpressing RPMI-8226 cells or the parental RPMI-8226 cells were
incubated with varying concentrations of respective compounds (n =
3 replicates per concentration). After 72 h, cell lysates were prepared
to measure proteasome catalytic subunit activities (LMP2 and
LMP7). Curve fitting analysis was performed to calculate IC50 values
reported as the mean SD. *Due to carfilzomib’s cytotoxic effect (a
dual inhibitor of cP and iP), cells were incubated for 3 h, followed by
the measurement of chymotrypsin-like (CT-L) activity. (b) Lack of
interactions between DB-60 (20) and ABCG2. The inhibitory
interactions with ABCG2 were assessed by measuring the changes
in the cellular accumulation of pheophorbide A (PhA, a fluorescent
ABCG2 substrate) using MDCKII cells stably expressing ABCG2
(established in our previous study41) and parental cells. The known
ABCG2 inhibitor Ko143 was used as a positive control. After cells
were preincubated in the complete medium containing PhA (1 μM)
with Ko143 (0.2 μM) or DB-60 (20) (2 or 5 μM) at 37 °C for 30
min, cells were then washed with ice-cold medium and incubated
again with Ko143 or DB-60 (20) for 45 min at 37 °C. Subsequently,
the fluorescent signal was measured via flow cytometry using the
excitation and emission wavelengths of 635 and 670 nm, respectively.
Colored lines represent the following groups: gray, PhA only; orange,
PhA in the presence of 20 (2 μM); red, PhA in the presence of 20 (5
μM); purple, PhA in the presence of Ko143 (0.2 μM) (n = 2 per
group).
Macrocyclic Compounds Are More Resistant to the
ABCB1-Mediated Efflux than Linear Counterparts. To
assess the potential ability to cross the BBB, we evaluated the
interaction of the representative macrocyclic inhibitors (17, 20,
21, 26, and 27) with ABCB1 by comparing their LMP2
inhibitory potency in the cell line model overexpressing
ABCB1 (RPMI-8226/ABCB1, previously established by us30)
to that in the parental RPMI-8226 cells. Linear peptide
epoxyketones, including carfilzomib (a known substrate of
ABCB130,39,40), were used for comparison. As expected,
carfilzomib required higher concentrations to inhibit the
proteasome chymotrypsin-like (CT-L) activity in RPMI-
8226/ABCB1 cells than in parental RPMI-8226 cells (∼39-
fold difference in IC50 values, Figure 3a). The potency of two
linear LMP2 inhibitors was also affected by ABCB1 (6.9-fold
for YU102 and 7.3-fold KZR-504) but to a lesser extent than
carfilzomib. The fold differences in the LMP2 inhibitory
potency of the macrocyclic compounds were notably less than
the linear counterparts: 3.3-, 4.1-, and 4.7-fold for 20, 21, and
17, respectively. On the other hand, the tetrapeptide-based
macrocyclic compounds 26 and 27 displayed no LMP2
inhibition in either cell line (likely attributable in part to low
membrane permeability), despite their potent LMP2 inhibitory
activity in purified human 20S proteasomes (Table 1). The
LMP7 inhibition in RPMI-8226/ABCB1 and RPMI-8226
parental cells by compound 20 showed a similar pattern (about
twofold decrease, Supporting Information Table S1) as the
LMP2 inhibition by 20, confirming a moderate interaction
between 20 and ABCB1. Similar results were obtained when
20 was incubated with another ABCB1-overexpressing cell line
(NCI/ADR-RES) in the presence and absence of ABCB1
inhibitor, verapamil (Supporting Information Table S2). These
results cautiously support that the tripeptide-based macrocyclic
E
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Figure 4. (a) In vitro metabolic stability of DB-60 (20) in the whole blood or liver homogenates from mice. The whole blood and the liver were
collected from ICR mice and incubated with the test compounds for the indicated time. The remaining drug levels were quantified (details
provided in the Methods). P values were calculated using Student’s t-test (GraphPad Prism, v.8.4.1). (b) Effects of DB-60 (20) on LMP2 inhibition
and IL-1α release in microglial BV-2 cells stimulated with lipopolysaccharide (LPS) (1 μg/mL). Error bars indicate standard deviation derived from
three technical replicates.
peptide epoxyketones (17, 20, and 21) are affected by the
ABCB1-mediated efflux to a much lesser extent than linear
counterparts.
hydrolases and peptidases (major contributors to the
metabolism of linear peptide epoxyketones), was superior to
YU102. DB-60 (20) was also more effective in suppressing the
catalytic activity of LMP2 in inflammatory microglial cells than
the linear compounds (YU102, KZR-504) (left panel, Figure
4b). Besides, the treatment of DB-60 (20) decreased the
release of IL-1α by LPS-activated microglial BV-2 cells (right
panel, Figure 4b), further verifying that iP-targeting macro-
cyclic peptide epoxyketones retain the inflammation suppres-
sion activity of linear counterparts mediated by iP inhibition.23
Encouraged by these results, we investigated the brain
permeability of DB-60 (20) via single intravenous dosing
(10 mg/kg) to the ICR mice (Supporting Information Figure
S1). When we analyzed brain tissue homogenates 1 h
postdosing, the brain homogenates contained a detectable
but low level of compound DB-60 (20) (3.50 1.46 pmole/g
tissue). When the same brain homogenates were analyzed for
LMP2 activity (Supporting Information Figure S1), however,
the results showed no appreciable inhibition of the LMP2
activity (Supporting Information Figure S1).
DB-60 (20) Ameliorates Cognitive Impairment in
Tg2576 Mice. Despite modest brain permeability shown in
the single intravenous dosing study, we examined whether
macrocyclic iP inhibitors are efficacious in a mouse model of
AD. Specifically, DB-60 (20) (10 mg/kg) was injected to
Tg2576 mice (9-month old) twice weekly for 3 weeks,
followed by the assessment of cognitive function via the Morris
water maze test for 5 trial days, a single probe trial on day 6,
and passive avoidance test on days 7 and 8.22 Similar to the
results obtained from its linear counterparts,22,23 mice treated
with DB-60 (20) improved the cognitive function of Tg2576
mice. Specifically, Tg2576 mice treated with DB-60 (20) were
For compound 20 (labeled “DB-60”) (least affected by the
ABCB1-mediated efflux in cell line studies), we further
examined for its potential interaction with ABCG2, another
major efflux transporter that can limit the BBB penetration.42
We used the MDCKII cells stably expressing ABCG2 to test
whether DB-60 (20) can inhibit the efflux of the fluorescent
probe substrate pheophorbide A (PhA). The known ABCG2
inhibitor Ko143 (0.2 μM) markedly increased the cellular
accumulation of PhA as expected (purple distribution showing
a rightward shift, left panel, Figure 3b). Under the same
conditions, DB-60 (20) (2 or 5 μM) showed no inhibition of
the ABCG2-mediated efflux of PhA (leading to no red shift,
left panel, Figure 3b), suggesting a lack of interaction between
DB-60 (20) and ABCG2. The extent of LMP2 inhibition by
DB-60 (20) was similar between MDCKII/ABCG2 and
parental MDCKII cells (Supporting Information Table S3).
Based on these results, we then selected DB-60 (20) as a lead
macrocyclic LMP2 inhibitor for further study.
Metabolic Stability and Microglial LMP2 Inhibition
Efficiency of DB-60 (20). To assess whether macrocyclic
peptide epoxyketones are more resistant to enzymatic
metabolism than linear peptide epoxyketones, we examined
the metabolic stability of DB-60 (20) using whole blood and
liver homogenates harvested from ICR mice (male, 7 weeks
old). The linear counterpart YU102 (containing the same P3-
proline as DB-60) was included for comparison. As shown in
Figure 4a, DB-60 (20) displayed greater metabolic stability
than YU102. In particular, DB-60 (20) stability in liver
homogenates, rich in metabolizing enzymes, including epoxide
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Figure 5. (a) DB-60 (20) improves cognitive function in Tg2576 mice. The Morris water maze test was performed to measure cognitive function:
escape distance (upper) and escape latency time (lower). Statistical analyses were performed using two-way ANOVA. *Difference in days 4−5
between control and DB-60 (20)-treated mice was statistically significant (p-value < 0.05, n = 12 for control and n = 5 for DB-60 (20)-treated
mice). (b) Probe trial (on day 6, upper panel) and the passive avoidance test (on days 7 and 8, lower panel) were performed following the Morris
water maze test on days 1−5. Student’s t-test was used for statistical analyses of probe trials and passive avoidance. Differences in time spent in the
target quadrant or step-through latency between control and DB-60 (20)-treated mice were statistically significant (p-value < 0.05, n = 5). (c)
Serum samples collected from mice treated with vehicle or DB-60 (20) were used to quantify IL-1α levels via ELISA (p-value < 0.05, n = 3).
more efficient at locating the platform in a pool of water during
training days 4 and 5, thus escaping the water with lower
latency and shorter distance compared to vehicle-treated mice
(Figure 5a).
probe/passive avoidance tests. When immunostained using an
anti-β-catenin antibody, RPE’s orderly standard structure was
prominent in age-matched nontransgenic ICR mice (upper
panel, Figure 6a). In contrast, the orderly structure of RPE was
severely damaged in Tg2576 mice treated with vehicle only
(upper panel, Figure 6b), as reported previously by us and
others.22,23,43
On the other hand, RPE collected from Tg2576 mice treated
with DB-60 (20) displayed the orderly structure (lower panel,
Figure 6a), demonstrating the ability of DB-60 (20) to block
RPE destruction caused by Aβ-triggered inflammation. RPE’s
The probe trial results showed that the percentages of time
spent in the target quadrant (the quadrant with the hidden
platform) for DB-60 (20)-treated Tg2576 mice were higher
than those for control mice (upper panel, Figure 5b),
demonstrating improved spatial learning and memory in
drug-treated Tg2576 mice. Similarly, mice treated with DB-
60 (20) performed better in a fear-aggravated test (passive
avoidance) than control mice (lower panel, Figure 5b). The
serum levels of IL-1α were lower in Tg2576 mice receiving
DB-60 (20) treatment than the control mice receiving the
vehicle only (Figure 5c), demonstrating the impact of DB-60
(20)-mediated iP inhibition in inflammatory cytokine
production. Taken together, we observed that the multiple
dosing of DB-60 (20) improved cognitive function in AD
mice. As no appreciable brain LMP2 inhibition was observed
in diseased mice that received a single dose of DB-60 (20)
(Supporting Information Figure S1), we hypothesize that the
efficacy of DB-60 (20) in mouse AD models may be due to the
gradual accumulation of LMP2:DB-60 (20) covalent adduct in
the brain resulting from repeated administration.
DB-60 (20) Protects Mouse Retinal Pigment Epithelial
(RPE) from Inflammation-Triggered Structural Destruc-
tion. As visual evidence for the potential involvement of the iP
in neuroinflammatory responses, we previously reported that
iP-targeting linear peptide epoxyketones inhibit RPE (retinal
pigment epithelium) degeneration caused by Aβ-triggered
inflammation in Tg2576 mice.22,23 We examined whether the
macrocyclic DB-60 (20) can also suppress inflammation-
triggered RPE degeneration by assessing the RPE samples from
Tg2576 mice that completed the Morris water maze and
Figure 6. Protection of Tg2576 mice from retinal pigment epithelium
(RPE) destruction by DB-60 (20). Upon completing behavior tests
for Tg2576 mice treated with DB-60 (20), RPE samples were
collected and immunostained using an anti-β-catenin antibody for
visualization. A parallel experiment was also performed using age-
matched nontransgenic ICR mice. The images are representative of
three independent replicates.
G
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structural integrity in nontransgenic ICR mice was not affected
by DB-60 (20) treatment (lower panel, Figure 6a). No sign of
overt toxicity was observed in Tg2576 or nontransgenic ICR
mice treated with DB-60 (20) during the test period, which is
consistent with our previous results obtained from iP-targeting
linear peptide epoxyketones.22,23
EXPERIMENTAL SECTION
■
Chemistry. All of the reagents and solvents used in experiments
were purchased commercially and used without additional purifica-
tion. A precoated TLC plate was used to monitor the reaction
progress. The different spots were detected by UV or/and staining
solutions such as phosphomolybdic acid hydrate (PMA), iodine, and
potassium permanganate. All of the reactions were performed under
inert conditions unless otherwise mentioned. Purification of
intermediates and final compounds was performed by flash column
chromatography on 60 Å silica gel, 230−400 mesh (Merck) unless
otherwise reported. A 400 MHz Varian spectrometer was used to
CONCLUSIONS
■
With the failure of nearly all clinical trials for Aβ or tau-
targeting AD drugs in the pipeline to date, identifying a new
class of drug candidates has become imperative to bring about
effective AD therapies. A major obstacle has been a lack of
promising new drug targets unrelated to the events leading to
the accumulation of the Aβ and tau protein. We previously
reported that linear peptide epoxyketones inhibiting the iP, a
previously untapped target in AD drug discovery efforts, have
promising anti-AD efficacy in mouse models, potentially
offering a new class of AD drugs.22,23 However, inherent
issues associated with the linear peptides prohibit further
clinical development. Therefore, in the current study, we aimed
to improve the iP-targeting peptide epoxyketones’ CNS
druggability to be further investigated as potential AD drug
candidates in preclinical and clinical studies.
1
record H and 13C NMR spectra. Chemical shifts were reported in
parts per million (ppm) and coupling constants (J) in Hz with
tetramethylsilane as a reference standard. Proton NMR multiplicities
were reported using different abbreviations such as singlet (s), doublet
(d), triplet (t), quartet (q), and multiplet (m). LC/MS data of final
compounds were recorded by an Agilent Technologies 6120
Quadrupole using a 0.1% formic acid solution of (A) H2O and (B)
acetonitrile (CH3CN) as an eluent. An Agilent Eclipse XBD-C18
column with 5 μm particle size, 4.6 mm diameter, and 150 mm length
was used in the system with solvent gradient B/A from 5:95 to 90:10
over 18 min and then 100:0 over 7 min. The flow rate of the eluent
was 0.4 mL/min. The same solvent gradient, column, and flow rate
were used to determine the purity of all final compounds in an Agilent
Technologies 1200 series equipped with a UV detector at 254 nm.
The purity of most of the final compounds was determined to be
≥95%. KZR-504 and YU102 used in experiments were synthesized
using the previously reported method.
General Procedure for the Boc Deprotection Reaction.
Excess of TFA was added to the stirred solution of the Boc-protected
intermediate in DCM at room temperature. The reaction mixture was
further stirred at the same temperature for 2 h. The reaction solution
was concentrated under reduced pressure, and the resulting residue
was dried under a strong vacuum. Dry TFA salt was used in the next
step without further purification.
General Procedure for the Synthesis of Macrocyclic
Carboxylic Acids. The second-generation Grubbs catalyst (10 mol
%) was added at room temperature to the stirred solution of diolefin
in toluene. The reaction temperature was increased gradually to 100
°C, and the reaction solution was stirred for 1 h at the same
temperature. After cooling, the solvent was removed under reduced
pressure, and the resulting residue was purified by flash chromatog-
raphy using 50% ethyl acetate in hexane. The above-purified product
(a mixture of geometric isomers) was dissolved in methanol, activated
palladium in charcoal (10 mol %) was added, and the reaction mixture
was stirred under a hydrogen gas environment at room temperature
for 2 h. The reaction solution was then filtered through Celite, and the
filtrate was concentrated to yield the corresponding macrocyclic
carboxylic acid, which was used in the next step without further
purification.
General Procedure for the Preparation of Weinreb Amide
2a−2c. (S)-tert-Butyl 1-(Methoxy(methyl)amino)-4-methyl-1-oxo-
pentan-2-ylcarbamate (2a). To the DCM solution of Boc-leucine (2
g, 8.64 mmol), EDCI·HCl (2.48 g, 12.96 mmol), HOBt (1.17 g, 8.64
mmol), N,O-dimethylhydroxylamine hydrochloride (0.84 g, 8.64
mmol), and DIPEA (3.77 mL, 21.6 mmol) were added. The reaction
solution was stirred at the same temperature overnight. The solvent
was removed under reduced pressure, and the residue was purified by
flash column chromatography using 20% ethyl acetate in hexane to
One common strategy to improve linear peptide-based drug
candidates’ CNS druggability is to form a macrocycle by
constraining the freely rotatable linear peptide backbone. To
this end, we were able to synthesize 17 macrocyclic peptides
retaining the epoxyketone pharmacophore. These macrocyclic
peptide epoxyketones are highly selective to iP, unlike the
previously reported macrocyclic analogues of the anti-cancer
agent oprozomib, which targets both cP and iP.44 Besides,
these compounds are structurally unique, having a proline or
proline mimic at the P3 position as a handle for P2−P3 or P2−
P4 macrocyclization. In particular, our lead macrocyclic iP
inhibitor, DB-60 (20), showed significantly improved meta-
bolic stability and decreased interaction with the two major
drug transporters in the BBB, ABCB1 and ABCG2. Further,
DB-60 (20) effectively reduced serum IL-1α and enhanced the
cognitive function of Tg2576 mice. Our results support that iP-
targeting macrocyclic peptide epoxyketones have promising
potential for further preclinical and clinical investigations as
AD drugs, not reliant on the strategies of targeting Aβ or tau
protein accumulation.
In addition to its role in cognitive deficits, accumulating
evidence support that Aβ-triggered inflammation plays a
crucial role in RPE degeneration in APP (human amyloid
precursor protein) transgenic mouse models including
Tg2576.24,25,45−47 The pathological role of Aβ in the eye has
been further supported by the detection of Aβ in RPE and
drusen (extracellular waste deposits that accumulate near the
RPE region) obtained from patients with age-related macular
degeneration (AMD).43,48−50 This has led researchers to
suggest a common neuroinflammation-associated pathogenic
mechanism between AMD and AD. Accordingly, the idea of
using the retina structure as a potential diagnostic tool in AD
has been proposed lately.51,52 In the current study, the iP-
selective inhibitor DB-60 (20) completely protected RPE from
an inflammation-triggered structural abnormality in Tg2576
mice, supporting that iP is potentially linked to RPE
degeneration and AD. Taken together, iP-targeting macrocyclic
peptide epoxyketones may also have the potential for AMD
treatment.
1
yield the pure product as a sticky colorless solid (2.1 g, 89%). H
NMR (400 MHz, CDCl3) 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-oxo-
propan-2-ylcarbamate (2b). 2b was prepared from 1b using the
same general procedure for the preparation of Weinreb amide as
1
colorless oil (90%). H NMR (400 MHz, CDCl3) δ 5.00 (d, J = 9.5
Hz, 1H), 4.71 (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, 12H), 1.17 (m, 3H), 1.01−0.78
(m, 2H).
H
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(S)-tert-Butyl 1-(Methoxy(methyl)amino)-1-oxo-3-phenylpro-
pan-2-ylcarbamate (2c). 2c was prepared from 1c using the same
general procedure for the preparation of Weinreb amide as colorless
4.63−4.52 (m, 1H), 3.42−3.22 (m, 2H), 3.08 (dd, J = 5.9, 14.0 Hz,
1H), 2.95 (dd, J = 6.4, 14.0 Hz, 1H), 2.02−1.95 (m, 2H), 1.76−1.70
(m, 2H), 1.41 (s, 9H).
1
thick oil (86%). H NMR (400 MHz, CDCl3) δ 7.25 (d, J = 8.2 Hz,
(S)-tert-Butyl 2-((S)-1-(Benzyloxy)-3-hydroxy-1-oxopropan-2-
ylcarbamoyl)pyrrolidine-1-carboxylate (6b). The general procedure
for the amide coupling reaction is as follows. To the stirred solution of
serine benzyl ester hydrochloride (1 g, 4.31 mmol) in DCM, Boc-
proline (0.93 g, 4.31 mmol), HATU (3.26 g, 8.62 mmol), HOBt (1.16
g, 8.62 mmol), and DIPEA (3.0 mL, 17.42 mmol) were added
respectively at room temperature. The reaction solution was stirred at
the same temperature overnight. The reaction mixture was then
washed with water, and the organic solution was collected. It was
dried over sodium sulfate, filtered, and concentrated. The crude
product was purified through flash column chromatography using
50% ethyl acetate in hexane to yield the pure product as colorless oil
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, 3H), 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).
General Procedure for the Preparation of Enone 3a−3c. (S)-
tert-Butyl 2,6-Dimethyl-3-oxohept-1-en-4-ylcarbamate (3a). A 0.5
M isopropenylmagnesium bromide solution in THF (58 mL, 29.15
mmol) was added dropwise to the stirred solution of 2a (2 g, 7.28
mmol) in THF at −78 °C, and the reaction mixture was stirred at the
same temperature overnight. The reaction solution was warmed
gradually to room temperature, and the reaction was quenched with a
saturated solution of ammonium chloride. The reaction product was
extracted with ethyl acetate three times. The combined organic layer
was dried in sodium sulfate, filtered, and concentrated. The crude
product was purified by flash column chromatography using 5% ethyl
acetate in hexane to yield the pure product as colorless oil (1.2 g,
1
(1.37 g, 81%). H NMR (400 MHz, CDCl3) 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, 2H), 1.83 (d, J = 5.7 Hz, 2H), 1.42 (s, 9H).
1
64%). H NMR (400 MHz, CDCl3) δ 6.06 (s, 1H), 5.86 (s, 1H),
(S)-tert-Butyl 2-((S)-1-(Benzyloxy)-3-(4-(but-3-enyloxy)phenyl)-1-
oxopropan-2-ylcarbamoyl)pyrrolidine-1-carboxylate (7). The DMF
solution of 6a (0.5 g, 1.06 mmol), potassium carbonate (0.44 g, 3.18
mmol), and 4-bromo-1-butene (0.22 mL, 2.13 mmol) was stirred at
room temperature for 6 h. Water was added to the reaction mixture,
and the product was extracted with ethyl acetate. The combined
solution of ethyl acetate was dried over sodium sulfate, filtered, and
concentrated under reduced pressure. The crude product was purified
through flash column chromatography using 50% ethyl acetate in
5.20−4.96 (m, 2H), 1.83 (s, 3H), 1.81−1.64 (m, 1H), 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-ylcarba-
mate (3b). 3b was prepared from 2b using the same general
1
procedure for the preparation of enone as a white solid (76%). H
NMR (400 MHz, CDCl3) 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, 2H).
(S)-tert-Butyl 4-Methyl-3-oxo-1-phenylpent-4-en-2-ylcarbamate
(3c). 3c was prepared from 2c using the same general procedure for
the preparation of enone as a white solid (79%). 1H NMR (400 MHz,
CDCl3) δ 7.30−7.12 (m, 3H), 7.05 (d, J = 7.2 Hz, 2H), 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), 2.97−2.80 (m, 1H), 1.84 (s, 3H), 1.39 (s, 9H).
1
hexane to yield the pure product as colorless oil (0.45 g, 81%). H
NMR (400 MHz, CDCl3) δ 7.42−7.18 (m, 5H), 6.96−6.81 (m, 2H),
6.71 (dq, J = 2.9, 8.9 Hz, 2H), 6.47 (s, 1H), 5.95−5.71 (m, 1H),
5.20−5.00 (m, 4H), 4.81 (s, 1H), 4.21 (d, J = 31.8 Hz, 1H), 3.93 (tq,
J = 2.9, 5.5 Hz, 2H), 3.48−3.19 (m, 2H), 3.07 (ddq, J = 2.9, 5.5, 14.4
Hz, 1H), 2.96 (ddq, J = 2.8, 6.0, 14.1 Hz, 1H), 2.58−2.43 (m, 2H),
1.92 (m, 4H), 1.50−1.25 (s, 9H).
General Procedure for the Preparation of Epoxide 4a−4c.
tert-Butyl (S)-4-Methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-
ylcarbamate (4a). DIPEA (6.83 mL, 39.14 mmol) was added
dropwise to 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 the
same temperature for 3 h. Water was added to the solution, and
methanol was removed under reduced pressure. The aqueous layer
was extracted with ethyl acetate three times. The collected organic
layer was dried with sodium sulfate, filtered, and concentrated. The
resulting crude product was purified by flash column chromatography
using 5% ethyl acetate in hexane to yield the pure product as a sticky
white solid (0.6 g, 57%).1H NMR (400 MHz, CDCl3) 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), 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-oxopro-
pan-2-ylcarbamate (4b). 4b was prepared from 3b using the same
general procedure for the preparation of epoxide as a sticky white
solid (41%). 1H NMR (400 MHz, CDCl3) 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, 2H).
(S)-Benzyl 3-(4-(But-3-enyloxy)phenyl)-2-((S)-1-pent-4-enoylpyr-
rolidine-2-carboxamido)propanoate (8a). Boc-deprotected 7 was
coupled with 4-pentenoic acid using the general procedure for the
amide coupling reaction to prepare 8a as colorless oil (63%). 1H
NMR (400 MHz, CDCl3) 7.47−7.21 (m, 5H), 6.91 (dd, J = 6.8, 8.8
Hz, 2H), 6.79−6.62 (m, 2H), 6.08−5.91 (m, 1H), 5.84−5.63 (m,
1H), 5.37 (dq, J = 1.6, 17.3 Hz, 1H), 5.25 (dq, J = 1.6, 10.5 Hz, 1H),
5.19−4.88 (m, 4H), 4.75 (td, J = 5.9, 7.2 Hz, 1H), 4.54 (dd, J = 1.9,
8.1 Hz, 1H), 4.45 (tt, J = 1.5, 5.3 Hz, 2H), 3.35 (ddt, J = 4.5, 9.2, 13.2
Hz, 2H), 3.07 (dd, J = 5.8, 14.0 Hz, 1H), 2.98−2.89 (m, 1H), 2.36−
2.26 (m, 1H), 2.20 (ddd, J = 6.8, 8.4, 10.8 Hz, 1H), 2.16−2.04 (m,
2H), 2.04−1.86 (m, 3H), 1.81−1.55 (m, 3H).
(S)-Benzyl 3-(4-(But-3-enyloxy)phenyl)-2-((S)-1-hex-5-enoylpyr-
rolidine-2-carboxamido)propanoate (8b). Boc-deprotected 7 was
coupled with 5-hexenoic acid using the general procedure for the
1
amide coupling reaction to prepare 8b as colorless oil (69%). H
NMR (400 MHz, CDCl3) δ 7.50−7.25 (m, 5H), 6.92 (d, J = 8.6 Hz,
2H), 6.70 (d, J = 8.6 Hz, 2H), 5.96−5.62 (m, 2H), 5.19−5.06 (m,
4H), 5.06−4.92 (m, 2H), 4.75 (ddd, J = 5.8, 6.9, 7.6 Hz, 1H), 4.54
(dd, J = 1.9, 8.1 Hz, 1H), 3.93 (t, J = 6.7 Hz, 2H), 3.44−3.27 (m,
2H), 3.07 (dd, J = 5.8, 14.0 Hz, 1H), 2.93 (dd, J = 6.9, 14.1 Hz, 1H),
2.50 (qt, J = 1.4, 6.7 Hz, 2H), 2.37−2.14 (m, 3H), 2.14−2.02 (m,
2H), 2.02−1.84 (m, 2H), 1.84−1.58 (m, 3H).
(S)-Benzyl 3-(4-(But-3-enyloxy)phenyl)-2-((S)-1-hept-6-enoylpyr-
rolidine-2-carboxamido)propanoate (8c). Boc-deprotected 7 was
coupled with 6-heptenoic acid using the general procedure for the
amide coupling reaction to prepare 8c as colorless oil (70%). 1H
NMR (400 MHz, CDCl3) δ 7.44 (dd, J = 2.3, 7.7 Hz, 1H), 7.37−7.14
(m, 5H), 6.89 (tt, J = 4.3, 8.2 Hz, 2H), 6.69 (dd, J = 2.5, 8.7 Hz, 2H),
5.81 (ddddd, J = 3.4, 6.6, 10.4, 14.1, 33.6 Hz, 2H), 5.21−5.01 (m,
4H), 5.01−4.86 (m, 2H), 4.74 (dd, J = 2.2, 7.2 Hz, 1H), 4.59−4.43
(m, 1H), 3.91 (td, J = 2.4, 6.7 Hz, 2H), 3.46−3.23 (m, 2H), 3.05
(ddd, J = 2.4, 5.9, 14.0 Hz, 1H), 2.92 (ddd, J = 2.4, 6.9, 14.1 Hz, 1H),
2.57−2.40 (m, 2H), 2.35−1.82 (m, 7H), 1.74 (dq, J = 3.5, 10.7 Hz,
1H), 1.66−1.49 (m, 2H), 1.41 (qd, J = 2.3, 7.8 Hz, 2H).
tert-Butyl (S)-1-((R)-2-Methyloxiran-2-yl)-1-oxo-3-phenylpropan-
2-ylcarbamate (4c). 4c was prepared from 3c using the same general
1
procedure for the preparation of epoxide as a white solid (32%), H
NMR (400 MHz, CDCl3) 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).
(S)-tert-Butyl 2-((S)-1-(Benzyloxy)-3-(4-hydroxyphenyl)-1-oxo-
propan-2-ylcarbamoyl)pyrrolidine-1-carboxylate (6a). The general
amide coupling reaction was used to prepare 6a from tyrosine benzyl
1
ester and Boc-proline as colorless oil (88%). H NMR (400 MHz,
CDCl3) δ 7.83 (s, 1H), 7.41−7.27 (m, 5H), 6.89 (d, J = 8.1 Hz, 2H),
6.76−6.67 (m, 2H), 5.12 (d, J = 5.9 Hz, 2H), 4.80−4.76 (s, 1H),
I
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(S)-tert-Butyl 2-((S)-3-(Allyloxy)-1-(benzyloxy)-1-oxopropan-2-
ylcarbamoyl)pyrrolidine-1-carboxylate (10). To the solution of 6b
(1 g, 2.54 mmol) in THF, Pd(PPh3)4 (0.15 g, 0.12 mmol) and allyl
methyl carbonate (0.43 mL, 3.82 mmol) were added under the
anhydrous condition at room temperature. The reaction mixture was
stirred at 60 °C for 2 h and then quenched with water. The crude
product was extracted with ethyl acetate three times. The combined
organic layer was dried under sodium sulfate, filtered, and
concentrated under reduced pressure. The crude product was purified
through flash column chromatography using 30% ethyl acetate in
4H), 5.06−4.90 (m, 2H), 4.71 (dt, J = 3.3, 8.2 Hz, 1H), 4.51 (dd, J =
3.0, 8.0 Hz, 1H), 4.10 (dd, J = 4.6, 17.8 Hz, 1H), 4.03−3.82 (m, 4H),
3.63 (dd, J = 3.3, 9.5 Hz, 1H), 3.55 (ddd, J = 3.4, 7.9, 10.6 Hz, 1H),
3.41 (dt, J = 7.6, 9.9 Hz, 1H), 2.31 (t, J = 7.5 Hz, 1H), 2.27−2.15 (m,
3H), 2.15−1.90 (m, 4H), 1.79−1.62 (m, 2H).
(12S,4S)-N-((S)-4-Methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopen-
tan-2-yl)-2,14-dioxo-7-oxa-3-aza-1(2,1)-pyrrolidina-6(1,4)-benze-
nacyclotetradecaphane-4-carboxamide (16). Macrocyclic carbox-
ylic acid intermediate 9a was prepared from 8a using the general
procedure for synthesis of macrocyclic carboxylic acid. 9a was coupled
with right-hand Boc-deprotected epoxyketone intermediate 4a using
the general procedure for the amide coupling reaction to prepare 16
1
hexane as yellowish oil (94%). H NMR (400 MHz, CDCl3) δ 7.30
(d, J = 3.5 Hz, 5H), 6.83 (s, 1H), 5.71 (d, J = 16.8 Hz, 1H), 5.31−
5.03 (m, 4H), 4.73 (s, 1H), 4.26 (d, J = 27.6 Hz, 1H), 3.98−3.73 (m,
3H), 3.61 (d, J = 9.1 Hz, 1H), 3.43 (d, J = 8.2 Hz, 2H), 2.10 (s, 2H),
1.84 (q, J = 6.3, 6.8 Hz, 2H), 1.42 (s, 9H).
1
as a white solid (41%). H NMR (400 MHz, CDCl3) δ 7.08 (d, J =
8.3 Hz, 1H), 7.01−6.89 (m, 2H), 6.86−6.76 (m, 2H), 5.85 (d, J = 9.3
Hz, 1H), 4.69−4.57 (m, 2H), 4.44 (td, J = 3.6, 9.2 Hz, 2H), 4.06 (td,
J = 2.4, 11.7 Hz, 1H), 3.55−3.39 (m, 3H), 3.38 (d, J = 5.0 Hz, 1H),
2.88−2.81 (m, 1H), 2.64 (dd, J = 6.3, 13.8 Hz, 1H), 2.22 (ddd, J =
3.4, 6.8, 13.7 Hz, 1H), 2.18−2.07 (m, 1H), 2.07−1.78 (m, 6H),
1.66−1.48 (m, 2H), 1.48−1.29 (m, 7H), 1.29−1.17 (m, 2H), 1.11 (d,
J = 3.3 Hz, 1H), 0.97 (d, J = 6.5 Hz, 3H), 0.88 (d, J = 6.6 Hz, 3H).13C
NMR (101 MHz, CDCl3) δ 207.42, 173.50, 171.00, 169.98, 156.20,
130.52, 127.24, 115.85, 66.97, 61.00, 59.19, 52.31, 52.29, 50.24, 47.59,
38.64, 35.41, 33.43, 28.92, 26.38, 24.91, 24.61, 23.63, 23.13, 21.28,
20.65, 16.75. LC−MS (ES+) m/z calcd for C30H44N3O6 [M + H]+
542.3, found: 542.3; purity: ≥96% and retention time is 19.07 min by
HPLC analysis.
(S)-Benzyl 3-(Allyloxy)-2-((S)-1-pent-4-enoylpyrrolidine-2-
carboxamido)propanoate (11a). Boc-deprotected 10 was coupled
with 4-pentenoic acid using the general procedure for the amide
1
coupling reaction to yield 11a as colorless oil (46%). H NMR (400
MHz, CDCl3) δ 7.42−7.25 (m, 5H), 5.94−5.64 (m, 2H), 5.28−5.07
(m, 4H), 5.07−4.87 (m, 2H), 4.68 (dt, J = 3.5, 7.5 Hz, 1H), 4.54 (dd,
J = 2.6, 8.1 Hz, 1H), 3.97−3.78 (m, 3H), 3.69−3.48 (m, 2H), 3.41
(td, J = 6.6, 9.3 Hz, 1H), 2.47−2.31 (m, 4H), 2.31−2.13 (m, 1H),
2.13−1.97 (m, 1H), 1.97−1.80 (m, 2H).
(S)-Benzyl 3-(Allyloxy)-2-((S)-1-hex-5-enoylpyrrolidine-2-
carboxamido)propanoate (11b). Boc-deprotected 10 was coupled
with 5-hexenoic acid using the general procedure for the amide
(12S,4S)-N-((S)-4-Methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopen-
tan-2-yl)-2,15-dioxo-7-oxa-3-aza-1(2,1)-pyrrolidina-6(1,4)-benze-
nacyclopentadecaphane-4-carboxamide (17). Macrocyclic carbox-
ylic acid intermediate 9b was prepared from 8b using the general
procedure for synthesis of macrocyclic carboxylic acid. 9b was
coupled with right-hand Boc-deprotected epoxyketone intermediate
4a using the general procedure for the amide coupling reaction to
1
coupling reaction to yield 11b as colorless oil (54%). H NMR (400
MHz, CDCl3) δ 7.43−7.22 (m, 5H), 5.83−5.63 (m, 2H), 5.27−5.05
(m, 4H), 5.05−4.85 (m, 2H), 4.67 (dt, J = 3.5, 8.1 Hz, 1H), 4.53 (dd,
J = 2.5, 8.3 Hz, 1H), 3.96−3.78 (m, 3H), 3.65−3.47 (m, 2H), 3.39
(td, J = 6.7, 9.3 Hz, 1H), 2.35−2.14 (m, 3H), 2.14−1.97 (m, 3H),
1.89 (dddd, J = 2.7, 5.6, 10.3, 11.8 Hz, 2H), 1.81−1.61 (m, 2H).
(S)-Benzyl 3-(Allyloxy)-2-((S)-1-hept-6-enoylpyrrolidine-2-
carboxamido)propanoate (11c). Boc-deprotected 10 was coupled
with 6-heptenoic acid using the general procedure for the amide
1
prepare 17 as a white solid (48%). H NMR (400 MHz, CDCl3) δ
7.01 (d, J = 8.3 Hz, 1H), 6.98−6.90 (m, 2H), 6.86−6.74 (m, 2H),
6.16 (d, J = 9.0 Hz, 1H), 4.73−4.54 (m, 2H), 4.46 (dd, J = 3.1, 8.5
Hz, 1H), 4.11 (t, J = 5.4 Hz, 2H), 3.56−3.38 (m, 3H), 3.37 (d, J = 5.2
Hz, 1H), 2.85 (d, J = 5.1 Hz, 1H), 2.67 (dd, J = 5.5, 13.9 Hz, 1H),
2.27 (ddd, J = 2.6, 5.6, 12.0 Hz, 1H), 2.15−1.90 (m, 5H), 1.71 (dt, J =
6.8, 10.3 Hz, 3H), 1.50−1.25 (m, 10H), 0.94 (d, J = 6.1 Hz, 3H), 0.86
(d, J = 6.2 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 207.66, 173.74,
170.95, 170.42, 158.22, 130.52, 130.33, 127.20, 114.82, 66.64, 61.02,
59.15, 52.79, 52.33, 50.16, 47.84, 38.96, 35.46, 34.61, 28.70, 27.83,
27.74, 26.28, 24.97, 24.57, 23.58, 23.24, 22.29, 20.87, 16.74. HRMS
m/z calcd for C31H46N3O6 [M + H]+ 556.3342, found: 556.3393.
LC−MS (ES+), found: 556.3; purity: ≥96% and retention time is
19.20 min by HPLC analysis.
1
coupling reaction to provide 11c as colorless oil (64%). H NMR
(400 MHz, CDCl3) δ 7.43−7.18 (m, 5H), 5.73 (dtd, J = 3.4, 7.0, 17.2
Hz, 2H), 5.28−5.02 (m, 4H), 5.02−4.81 (m, 2H), 4.65 (dd, J = 3.6,
7.8 Hz, 1H), 4.52 (dd, J = 4.3, 7.2 Hz, 1H), 3.96−3.74 (m, 3H),
3.68−3.44 (m, 2H), 3.38 (dt, J = 3.1, 6.3 Hz, 1H), 2.37−2.11 (m,
3H), 2.11−1.95 (m, 3H), 1.89 (dt, J = 5.6, 16.5 Hz, 2H), 1.61 (tt, J =
5.5, 10.8 Hz, 2H), 1.39 (td, J = 2.9, 7.7 Hz, 2H).
(S)-Benzyl 3-(Allyloxy)-2-((S)-1-(2-(tert-butoxycarbonylamino)-
acetyl)pyrrolidine-2-carboxamido)propanoate (13). Boc-depro-
tected 10 was coupled with Boc-glycine using the general procedure
for the amide coupling reaction to provide 13 as colorless oil (57%).
1H NMR (400 MHz, CDCl3) δ 7.41−7.27 (m, 5H), 7.15 (d, J = 8.2
(12S,4S)-N-((S)-4-Methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopen-
tan-2-yl)-2,16-dioxo-7-oxa-3-aza-1(2,1)-pyrrolidina-6(1,4)-benze-
nacyclohexadecaphane-4-carboxamide (18). Macrocyclic carbox-
ylic acid intermediate 9c was prepared from 8c using the general
procedure for synthesis of macrocyclic carboxylic acid. 9c was coupled
with right-hand Boc-deprotected epoxyketone intermediate 4a using
the general procedure for the amide coupling reaction to prepare 18
Hz, 1H), 5.84−5.66 (m, 1H), 5.41 (s, 1H), 5.29−5.04 (m, 4H), 4.69
(dt, J = 3.3, 8.1 Hz, 1H), 4.53 (dd, J = 2.7, 8.1 Hz, 1H), 4.03−3.78
(m, 5H), 3.63 (dd, J = 3.4, 9.6 Hz, 1H), 3.52 (ddd, J = 3.2, 8.1, 9.9
Hz, 1H), 3.38 (td, J = 6.7, 9.2 Hz, 1H), 2.31−2.17 (m, 1H), 2.17−
2.02 (m, 1H), 1.95 (tdd, J = 4.1, 7.4, 9.6 Hz, 2H), 1.42 (s, 9H).
(S)-Benzyl 3-(Allyloxy)-2-((S)-1-(2-pent-4-enamidoacetyl)-
pyrrolidine-2-carboxamido)propanoate (14a). Boc-deprotected 13
was coupled with 4-pentenoic acid using the general procedure for the
1
as a white solid (33%). H NMR (400 MHz, CDCl3) δ 7.01 (d, J =
1
8.2 Hz, 1H), 6.98−6.88 (m, 2H), 6.83−6.71 (m, 2H), 6.16 (d, J = 9.4
Hz, 1H), 4.65 (ddd, J = 3.3, 5.5, 9.1 Hz, 1H), 4.61−4.51 (m, 1H),
4.47 (dd, J = 3.2, 8.8 Hz, 1H), 4.02 (dt, J = 4.1, 8.5 Hz, 1H), 3.92 (td,
J = 2.8, 10.0 Hz, 1H), 3.52 (dd, J = 3.6, 6.9 Hz, 1H), 3.50−3.40 (m,
2H), 3.38 (d, J = 5.1 Hz, 1H), 2.84 (d, J = 5.1 Hz, 1H), 2.64 (dd, J =
5.6, 13.9 Hz, 1H), 2.29 (ddt, J = 3.7, 6.3, 12.7 Hz, 1H), 2.17−1.88 (m,
6H), 1.84−1.58 (m, 3H), 1.46 (d, J = 2.7 Hz, 3H), 1.43−1.13 (m,
11H), 0.93 (d, J = 5.8 Hz, 3H), 0.84 (d, J = 5.9 Hz, 3H). 13C NMR
(101 MHz, CDCl3) δ 207.56, 173.67, 170.82, 170.17, 158.54, 130.36,
127.04, 114.16, 65.39, 61.15, 59.18, 52.72, 52.33, 50.16, 47.76, 38.74,
35.49, 34.96, 28.79, 27.54, 27.44, 26.28, 24.90, 24.37, 23.76, 23.67,
22.39, 22.07, 20.91, 16.75. HRMS m/z calcd for C32H48N3O6 [M +
H]+ 570.3498, found: 570.3534. LC−MS (ES+), found: 570.3; purity:
≥96% and retention time is 21.22 min by HPLC analysis.
amide coupling reaction to prepare 14a as colorless oil (78%). H
NMR (400 MHz, CDCl3) δ 7.32 (m, 5H), 7.03 (d, J = 8.2 Hz, 1H),
6.47 (s, 1H), 5.77 (dddd, J = 4.9, 10.6, 16.1, 17.9 Hz, 2H), 5.33−5.18
(m, 2H), 5.17−4.91 (m, 4H), 4.71 (dt, J = 3.3, 8.1 Hz, 1H), 4.52 (dd,
J = 3.0, 8.1 Hz, 1H), 4.09 (dd, J = 4.7, 17.8 Hz, 1H), 4.02−3.83 (m,
4H), 3.64 (dd, J = 3.3, 9.5 Hz, 1H), 3.55 (ddd, J = 3.2, 7.9, 10.7 Hz,
1H), 3.42 (dd, J = 6.8, 9.3 Hz, 1H), 2.45−2.18 (m, 5H), 2.18−2.03
(m, 1H), 1.98 (dtd, J = 4.0, 7.3, 13.4 Hz, 2H).
(S)-Benzyl 3-(Allyloxy)-2-((S)-1-(2-hex-5-enamidoacetyl)-
pyrrolidine-2-carboxamido)propanoate (14b). Boc-deprotected 13
was coupled with 5-hexenoic acid using the general procedure for the
1
amide coupling reaction to prepare 14b as colorless oil (68%). H
NMR (400 MHz, CDCl3) δ 7.40−7.26 (m, 5H), 7.07 (d, J = 8.2 Hz,
1H), 6.53 (t, J = 4.2 Hz, 1H), 5.84−5.64 (m, 2H), 5.32−5.06 (m,
J
https://doi.org/10.1021/acs.jmedchem.1c00291
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(12S,4S)-N-((S)-3-Cyclohexyl-1-((R)-2-methyloxiran-2-yl)-1-oxo-
propan-2-yl)-2,15-dioxo-7-oxa-3-aza-1(2,1)-pyrrolidina-6(1,4)-ben-
zenacyclopentadecaphane-4-carboxamide (19). Macrocyclic car-
boxylic acid intermediate 9b was prepared from 8b using the general
procedure for synthesis of macrocyclic carboxylic acid. 9b was
coupled with right-hand Boc-deprotected epoxyketone intermediate
4b using the general procedure for the amide coupling reaction to
4a using the general procedure for the amide coupling reaction to
prepare 22 as a white solid (35%). H NMR (400 MHz, CDCl3) δ
1
7.47 (d, J = 8.3 Hz, 1H), 6.97 (d, J = 8.5 Hz, 1H), 4.63 (ddt, J = 3.7,
9.0, 22.3 Hz, 2H), 4.30 (dd, J = 3.3, 8.3 Hz, 1H), 3.87 (dd, J = 2.4, 9.7
Hz, 1H), 3.76−3.61 (m, 1H), 3.61−3.43 (m, 3H), 3.41−3.31 (m,
1H), 3.29 (d, J = 5.2 Hz, 1H), 2.82 (d, J = 5.1 Hz, 1H), 2.62−2.47
(m, 1H), 2.38−2.19 (m, 2H), 2.13 (dd, J = 7.8, 13.1 Hz, 1H), 2.05−
1.81 (m, 4H), 1.79−1.65 (m, 1H), 1.65−1.31 (m, 11H), 0.91 (t, J =
7.1 Hz, 6H).13C NMR (101 MHz, CDCl3) δ 207.41, 174.37, 170.61,
170.15, 72.15, 69.60, 60.93, 59.04, 53.67, 52.27, 50.27, 48.16, 39.54,
33.81, 28.42, 27.89, 27.64, 27.27, 25.01, 24.93, 24.69, 23.35, 21.10,
16.76. LC−MS (ES+) m/z calcd for C24H40N3O6 [M + H]+ 466.2,
found: 466.2; purity: ≥96% and retention time is 19.13 min by HPLC
analysis.
(3S,17aS)-N-((S)-4-Methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopen-
tan-2-yl)-1,13-dioxohexadecahydropyrrolo[2,1-f ][1]oxa[4,7]-
diazacyclopentadecine-3-carboxamide (23). Macrocyclic carboxylic
acid intermediate 12c was prepared from 11c using the procedure for
synthesis of macrocyclic carboxylic acid. 12c was coupled with right-
hand Boc-deprotected epoxyketone intermediate 4a using the general
procedure for amide coupling reaction to prepare 23 as a white solid
(20%). 1H NMR (400 MHz, CDCl3) δ 7.46 (d, J = 8.5 Hz, 1H), 6.89
(d, J = 9.0 Hz, 1H), 4.73−4.57 (m, 2H), 4.40 (ddd, J = 2.2, 3.2, 8.9
Hz, 1H), 3.86 (dd, J = 2.2, 9.1 Hz, 1H), 3.67 (ddd, J = 4.2, 7.2, 9.9
Hz, 1H), 3.57−3.42 (m, 3H), 3.42−3.34 (m, 1H), 3.33−3.24 (m,
1H), 2.83 (d, J = 5.0 Hz, 1H), 2.50−2.26 (m, 3H), 2.20−2.05 (m,
1H), 2.05−1.86 (m, 3H), 1.84−1.68 (m, 2H), 1.64−1.41 (m, 8H),
1.41−1.17 (m, 5H), 0.93 (dd, J = 4.7, 6.6 Hz, 6H).13C NMR (101
MHz, CDCl3) δ 207.63, 174.61, 170.89, 170.07, 72.34, 69.94, 61.32,
59.04, 54.10, 52.29, 50.13, 48.19, 39.66, 33.61, 29.16, 28.62, 28.43,
28.12, 28.08, 24.85, 24.83, 23.43, 23.10, 21.04, 16.76. LC−MS (ES+)
m/z calcd for C25H42N3O6 [M + H]+ 480.3, found: 480.3; purity:
≥96% and retention time is 14.82 min by HPLC analysis.
1
prepare 19 as a white solid (42%). H NMR (400 MHz, CDCl3) δ
7.00 (d, J = 8.2 Hz, 1H), 6.97−6.91 (m, 2H), 6.86−6.75 (m, 2H),
6.23 (d, J = 8.9 Hz, 1H), 4.69−4.59 (m, 2H), 4.46 (dd, J = 3.2, 8.4
Hz, 1H), 4.17−4.08 (m, 2H), 3.49−3.36 (m, 3H), 3.34 (d, J = 5.0 Hz,
1H), 2.83 (d, J = 5.0 Hz, 1H), 2.70 (dd, J = 5.7, 14.1 Hz, 1H), 2.34−
2.25 (m, 1H), 2.11−1.93 (m, 5H), 1.91−1.81 (m, 2H), 1.81−1.70
(m, 2H), 1.70−1.52 (m, 4H), 1.51−1.39 (m, 5H), 1.39−1.27 (m,
5H), 1.27−1.07 (m, 6H), 0.98−0.84 (m, 2H). 13C NMR (101 MHz,
cdcl3) δ 207.73, 173.64, 171.09, 170.53, 158.11, 130.35, 127.26,
114.83, 66.34, 60.83, 59.12, 52.75, 52.32, 49.46, 47.78, 37.70, 35.30,
34.57, 33.85, 33.71, 31.57, 28.50, 27.85, 27.59, 26.43, 26.14, 26.08,
25.95, 24.97, 22.91, 22.21, 16.78. HRMS m/z calcd for C34H50N3O6
[M + H]+ 596.3655, found: 596.3534. LC−MS (ES+) m/z calcd for
C34H50N3O6 [M + H]+ 596.3, found: 596.3; purity: ≥96% and
retention time is 21.49 min by HPLC analysis.
(12S,4S)-N-((S)-1-((R)-2-Methyloxiran-2-yl)-1-oxo-3-phenylpro-
pan-2-yl)-2,15-dioxo-7-oxa-3-aza-1(2,1)-pyrrolidina-6(1,4)-benze-
nacyclopentadecaphane-4-carboxamide (20). Macrocyclic carbox-
ylic acid intermediate 9b was prepared from 8b using the general
procedure for synthesis of macrocyclic carboxylic acid. 9b was
coupled with right-hand Boc-deprotected epoxyketone intermediate
4c using the general procedure for the amide coupling reaction to
1
prepare 20 as a white solid (38%). H NMR (400 MHz, CDCl3) δ
7.34−7.16 (m, 5H), 7.08 (d, J = 8.7 Hz, 1H), 6.90−6.81 (m, 2H),
6.74−6.64 (m, 2H), 6.48 (d, J = 8.0 Hz, 1H), 4.94 (ddd, J = 5.0, 7.4,
8.4 Hz, 1H), 4.59 (dt, J = 5.5, 8.4 Hz, 1H), 4.50−4.41 (m, 1H), 4.19−
4.02 (m, 2H), 3.37 (ddt, J = 3.5, 5.7, 7.8 Hz, 3H), 3.18 (dd, J = 5.8,
14.5 Hz, 1H), 3.11−3.00 (m, 1H), 2.84 (dd, J = 2.0, 5.0 Hz, 1H), 2.76
(ddd, J = 2.6, 8.0, 14.3 Hz, 2H), 2.31−2.23 (m, 1H), 2.08−1.88 (m,
5H), 1.88−1.70 (m, 2H), 1.64 (dt, J = 3.4, 6.9 Hz, 1H), 1.52−1.38
(m, 4H), 1.38−1.22 (m, 4H), 1.21−1.07 (m, 2H). 13C NMR (101
MHz, cdcl3) δ 207.37, 173.63, 171.19, 170.96, 157.80, 136.46, 130.10,
129.16, 128.38, 127.15, 126.72, 114.90, 66.35, 60.35, 59.19, 52.98,
52.29, 52.02, 47.65, 36.80, 35.26, 34.51, 28.04, 27.72, 27.62, 26.11,
24.99, 23.07, 22.32, 16.41. HRMS (FAB) m/z calcd for for
C34H44N3O6 [M + H]+ 590.3185, found: 590.3214. LC−MS (ES+),
found: 590.3; purity: ≥96% and retention time is 18.53 min by HPLC
analysis
(3S,15aS)-N-((S)-3-Cyclohexyl-1-((R)-2-methyloxiran-2-yl)-1-oxo-
propan-2-yl)-1,11-dioxotetradecahydropyrrolo[2,1-f ][1]oxa[4,7]-
diazacyclotridecine-3-carboxamide (24). Macrocyclic carboxylic
acid intermediate 12a was prepared from 11a using the general
procedure for synthesis of macrocyclic carboxylic acid. 12a was
coupled with right-hand Boc-deprotected epoxyketone intermediate
4b using the general procedure for the amide coupling reaction to
1
prepare 24 as a white solid (33%). H NMR (400 MHz, CDCl3) δ
7.66 (d, J = 7.9 Hz, 1H), 7.02 (d, J = 8.0 Hz, 1H), 4.82 (dd, J = 1.9,
8.2 Hz, 1H), 4.62−4.54 (m, 1H), 4.38 (ddd, J = 3.7, 5.9, 7.9 Hz, 1H),
3.75−3.70 (m, 1H), 3.59 (dd, J = 3.9, 10.1 Hz, 2H), 3.53−3.37 (m,
3H), 3.29 (d, J = 5.0 Hz, 1H), 2.83 (d, J = 5.0 Hz, 1H), 2.55−2.44
(m, 2H), 2.19 (ddd, J = 2.5, 8.2, 12.9 Hz, 1H), 2.01 (tdd, J = 2.8, 4.6,
8.4 Hz, 2H), 1.92−1.75 (m, 4H), 1.70−1.51 (m, 7H), 1.47 (s, 3H),
1.43−1.29 (m, 3H), 1.20−1.07 (m, 3H), 0.99−0.81 (m, 3H); 13C
NMR (101 MHz, cdcl3) δ 207.90, 174.93, 171.31, 169.75, 69.42,
68.07, 59.57, 59.05, 53.70, 52.35, 49.52, 47.90, 38.31, 34.34, 34.25,
33.87, 31.87, 27.60, 26.57, 26.38, 26.33, 26.04, 24.98, 24.01, 22.07,
16.73. LC−MS (ES+) m/z calcd for C26H42N3O6 [M + H]+ 492.3,
found: 492.3; purity: ≥96% and retention time is 17.81 min by HPLC
analysis.
(3S,15aS)-N-((S)-4-Methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopen-
tan-2-yl)-1,11-dioxotetradecahydropyrrolo[2,1-f ][1]oxa[4,7]-
diazacyclotridecine-3-carboxamide (21). Macrocyclic carboxylic
acid intermediate 12a was prepared from 11a using the general
procedure for synthesis of macrocyclic carboxylic acid. 12a was
coupled with right-hand Boc-deprotected epoxyketone intermediate
4a using the general procedure for the amide coupling reaction to
1
prepare 21 as a white solid (60%). H NMR (400 MHz, CDCl3) δ
7.68 (d, J = 7.9 Hz, 1H), 7.00 (d, J = 8.2 Hz, 1H), 4.82 (dd, J = 1.9,
8.1 Hz, 1H), 4.57 (ddd, J = 3.3, 8.2, 10.2 Hz, 1H), 4.39 (ddd, J = 3.7,
6.0, 7.9 Hz, 1H), 3.71 (dd, J = 6.0, 9.9 Hz, 1H), 3.58 (dt, J = 4.3, 10.9
Hz, 2H), 3.53−3.34 (m, 3H), 3.27 (d, J = 5.0 Hz, 1H), 2.83 (d, J =
5.0 Hz, 1H), 2.57−2.42 (m, 2H), 2.19 (ddd, J = 2.6, 8.2, 12.9 Hz,
1H), 2.08−1.94 (m, 2H), 1.94−1.73 (m, 3H), 1.73−1.61 (m, 2H),
1.61−1.43 (m, 6H), 1.43−1.28 (m, 2H), 0.91 (d, J = 6.6 Hz, 6H). 13C
NMR (101 MHz, CDCl3) δ 207.80, 174.95, 171.38, 169.69, 69.53,
67.96, 59.50, 58.99, 53.81, 52.33, 50.12, 47.88, 40.04, 34.21, 27.51,
26.50, 25.14, 24.97, 23.85, 23.24, 21.88, 21.38, 16.71. LC−MS (ES+)
m/z calcd for C23H38N3O6 [M + H]+ 452.2, found: 452.2; purity:
≥96% and retention time is 15.21 min by HPLC analysis.
(3S,16aS)-N-((S)-4-Methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopen-
tan-2-yl)-1,12-dioxotetradecahydro-6H-pyrrolo[2,1-f ][1]oxa[4,7]-
diazacyclotetradecine-3-carboxamide (22). Macrocyclic carboxylic
acid intermediate 12b was prepared from 11b using the general
procedure for synthesis of macrocyclic carboxylic acid. 12b was
coupled with right-hand Boc-deprotected epoxyketone intermediate
(3S,15aS)-N-((S)-1-((R)-2-Methyloxiran-2-yl)-1-oxo-3-phenylpro-
pan-2-yl)-1,11-dioxotetradecahydropyrrolo[2,1-f ][1]oxa[4,7]-
diazacyclotridecine-3-carboxamide (25). Macrocyclic carboxylic
acid intermediate 12a was prepared from 11a using the general
procedure for synthesis of macrocyclic carboxylic acid. 12a was
coupled with right-hand Boc-deprotected epoxyketone intermediate
4c using the general procedure for the amide coupling reaction to
1
prepare 25 as a white solid (31%). H NMR (400 MHz, CDCl3) δ
7.77 (d, J = 7.7 Hz, 1H), 7.30−7.22 (m, 2H), 7.22−7.09 (m, 4H),
4.80 (td, J = 4.9, 8.2 Hz, 1H), 4.72 (dd, J = 1.7, 8.1 Hz, 1H), 4.38 (td,
J = 3.8, 7.3 Hz, 1H), 3.62 (dd, J = 3.8, 10.1 Hz, 1H), 3.59−3.49 (m,
2H), 3.49−3.42 (m, 1H), 3.35 (ddt, J = 4.0, 8.7, 11.7 Hz, 2H), 3.30
(d, J = 4.9 Hz, 1H), 3.11 (dd, J = 4.9, 14.0 Hz, 1H), 2.85 (d, J = 4.9
Hz, 1H), 2.82−2.73 (m, 1H), 2.53−2.41 (m, 2H), 2.20 (ddd, J = 2.7,
7.1, 12.9 Hz, 1H), 2.00 (ddt, J = 3.5, 8.4, 12.2 Hz, 2H), 1.86−1.75 (m,
2H), 1.55 (tt, J = 5.7, 13.1 Hz, 3H), 1.45 (s, 3H), 1.42−1.30 (m, 2H);
K
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13C NMR (101 MHz, cdcl3) δ 207.05, 175.03, 171.64, 169.73, 135.97,
coupling reaction to prepare 29b as colorless oil (87%). H NMR
(400 MHz, CDCl3) δ 7.47−7.23 (m, 5H), 6.91 (s, 1H), 5.72 (tt, J =
5.3, 10.8 Hz, 1H), 5.34−5.01 (m, 4H), 4.73 (d, J = 8.1 Hz, 1H), 4.49
(s, 1H), 4.04−3.75 (m, 4H), 3.65 (dt, J = 10.7, 22.5 Hz, 2H), 2.69 (d,
J = 60.0 Hz, 2H), 1.44 (s, 9H).
129.34, 128.35, 126.83, 69.05, 67.89, 59.20, 53.41, 52.39, 52.29, 47.85,
37.04, 34.08, 27.50, 26.13, 24.94, 23.87, 21.92, 16.48. LC−MS (ES+)
m/z calcd for C26H36N3O6 [M + H]+ 486.2, found: 486.2; purity:
≥96% and retention time is 15.50 min by HPLC analysis.
tert-Butyl (S)-2-(((S)-3-(Allyloxy)-1-(benzyloxy)-1-oxopropan-2-
yl)carbamoyl)-2-methylpyrrolidine-1-carboxylate (29c). Boc-depro-
tected 28 prepared from intermediate 28 using the general procedure
for the Boc deprotection reaction was coupled with N-Boc-α-
methylproline using the general procedure for the amide coupling
(3S,18aS)-N-((S)-4-Methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopen-
tan-2-yl)-1,11,14-trioxohexadecahydro-6H-pyrrolo[2,1-f ][1]oxa-
[4,7,10]triazacyclohexadecine-3-carboxamide (26). Macrocyclic
carboxylic acid intermediate 15a was prepared from 14a using the
general procedure for synthesis of macrocyclic carboxylic acid. 15a
was coupled with right-hand Boc-deprotected epoxyketone inter-
mediate 4a using the general procedure for the amide coupling
1
reaction to prepare 29c as colorless oil (65%). H NMR (400 MHz,
CDCl3) δ 7.31 (s, 5H), 5.72 (tt, J = 5.4, 10.7 Hz, 1H), 5.29−5.04 (m,
4H), 4.75−4.63 (m, 1H), 3.98−3.78 (m, 3H), 3.63 (dd, J = 3.3, 9.5
Hz, 1H), 3.53 (s, 2H), 1.76 (m, 4H), 1.57 (d, J = 16.0 Hz, 3H), 1.50−
1.32 (s, 9H).
1
reaction to prepare 26 as a white solid (47%). H NMR (400 MHz,
CDCl3) δ 7.62 (d, J = 7.3 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 6.32 (d, J
= 8.1 Hz, 1H), 4.76−4.66 (m, 2H), 4.62 (ddd, J = 3.3, 8.4, 9.9 Hz,
1H), 4.32 (dt, J = 3.5, 7.2 Hz, 1H), 3.72 (td, J = 3.6, 8.1, 8.9 Hz, 2H),
3.53 (dd, J = 3.1, 9.6 Hz, 1H), 3.49−3.31 (m, 4H), 3.27 (d, J = 4.9
Hz, 1H), 2.84 (d, J = 5.2 Hz, 1H), 2.56−2.43 (m, 1H), 2.43−2.32 (m,
1H), 2.12 (ddd, J = 2.9, 10.7, 13.7 Hz, 1H), 2.07−1.87 (m, 3H),
1.83−1.62 (m, 2H), 1.62−1.40 (m, 8H), 1.34 (ddd, J = 4.0, 9.3, 18.0
Hz, 2H), 0.92 (dd, J = 1.8, 6.7 Hz, 6H). 13C NMR (101 MHz,
CDCl3) δ 208.16, 173.95, 170.41, 169.99, 169.43, 70.54, 69.06, 60.60,
58.93, 54.70, 52.33, 50.00, 47.01, 41.46, 40.18, 36.47, 29.05, 27.17,
25.24, 25.03, 24.73, 23.39, 21.30, 16.67. LC−MS (ES+) m/z calcd for
C25H41N4O7 [M + H]+ 509.2, found: 509.3; purity: ≥96% and
retention time is 14.36 min by HPLC analysis.
tert-Butyl (S)-2-(((S)-3-(Allyloxy)-1-(benzyloxy)-1-oxopropan-2-
yl)carbamoyl)azetidine-1-carboxylate (29d). Boc-deprotected 28
prepared from intermediate 28 using the general procedure for the
Boc deprotection reaction was coupled with N-Boc-azetidine-2-
carboxylic acid using the general procedure for the amide coupling
1
reaction to prepare 29d as colorless oil (92%). H NMR (400 MHz,
CDCl3) δ 7.43−7.17 (m, 5H), 5.74 (ddt, J = 5.6, 10.5, 17.3 Hz, 1H),
5.28−5.03 (m, 4H), 4.75 (dt, J = 3.3, 8.3 Hz, 1H), 4.63 (t, J = 8.1 Hz,
1H), 3.95−3.83 (m, 4H), 3.77 (td, J = 5.8, 8.3 Hz, 1H), 3.65 (dd, J =
3.3, 9.6 Hz, 1H), 2.38 (d, J = 20.2 Hz, 2H), 1.42 (s, 9H).
tert-Butyl (S)-2-(((S)-3-(Allyloxy)-1-(benzyloxy)-1-oxopropan-2-
yl)carbamoyl)piperidine-1-carboxylate (29e). Boc-deprotected 28
prepared from intermediate 28 using the general procedure for the
Boc deprotection reaction was coupled with N-Boc-2-piperidinecar-
boxylic acid using the general procedure for the amide coupling
(3S,19aS)-N-((S)-4-Methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopen-
tan-2-yl)-1,12,15-trioxooctadecahydropyrrolo[2,1-f ][1]oxa[4,7,10]-
triazacycloheptadecine-3-carboxamide (27). Macrocyclic carboxylic
acid intermediate 15b was prepared from 14b using the general
procedure for synthesis of macrocyclic carboxylic acid. 15b was
coupled with right-hand Boc-deprotected epoxyketone intermediate
4a using the general procedure for the amide coupling reaction to
1
reaction to prepare 29e as colorless oil (81%). H NMR (400 MHz,
CDCl3) δ 7.46−7.23 (m, 5H), 6.80 (d, J = 26.1 Hz, 1H), 5.72 (ddt, J
= 5.6, 10.9, 17.3 Hz, 1H), 5.32−5.00 (m, 4H), 4.74 (d, J = 8.1 Hz,
2H), 3.98−3.79 (m, 4H), 3.70−3.59 (m, 2H), 2.93−2.65 (m, 1H),
2.24 (d, J = 12.1 Hz, 1H), 1.44 (m, 13H).
1
prepare 27 as a white solid (36%). H NMR (400 MHz, CDCl3) δ
7.35 (d, J = 8.4 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 6.47 (d, J = 7.1 Hz,
1H), 4.70 (d, J = 6.3 Hz, 1H), 4.67−4.55 (m, 2H), 4.38 (dd, J = 2.9,
8.5 Hz, 1H), 3.81 (dd, J = 2.8, 9.4 Hz, 1H), 3.70−3.57 (m, 2H),
3.47−3.37 (m, 2H), 3.37−3.31 (m, 2H), 3.28 (d, J = 5.0 Hz, 1H),
2.85 (d, J = 5.0 Hz, 1H), 2.47 (dd, J = 3.0, 5.9 Hz, 1H), 2.41−2.28
(m, 1H), 2.27−2.16 (m, 1H), 2.00 (dd, J = 4.6, 7.5 Hz, 3H), 1.87 (s,
2H), 1.72 (dt, J = 3.0, 8.7 Hz, 1H), 1.54−1.27 (m, 11H), 0.94 (t, J =
7.1 Hz, 6H).13C NMR (101 MHz, CDCl3) δ 208.21, 173.43, 170.30,
169.76, 169.43, 71.27, 69.95, 60.96, 58.95, 54.44, 52.33, 49.97, 46.82,
41.58, 40.13, 36.24, 30.21, 29.23, 27.49, 25.39, 25.00, 24.66, 24.63,
23.58, 21.31, 16.67. LC−MS (ES+) m/z calcd for C26H43N4O7 [M +
H]+ 523.3, found: 523.3; purity: ≥96% and retention time is 14.75
min by HPLC analysis.
Benzyl O-Allyl-N-(tert-butoxycarbonyl)-L-serinate (28). Inter-
mediate 28 was prepared from Boc-serine benzyl ester and allyl
methyl carbonate using the same procedure used for preparation of
intermediate 10 as colorless oil (78%). 1H NMR (400 MHz, CDCl3)
δ 7.32 (d, J = 3.1 Hz, 5H), 5.84−5.67 (m, 1H), 5.39 (d, J = 8.8 Hz,
1H), 5.31−5.19 (m, 2H), 5.13 (ddd, J = 3.6, 7.9, 12.4 Hz, 2H), 4.45
(dt, J = 3.2, 9.0 Hz, 1H), 3.96−3.80 (m, 3H), 3.63 (dd, J = 3.3, 9.4
Hz, 1H), 1.43 (s, 9H).
Benzyl O-Allyl-N-((2S,4S)-4-fluoro-1-(pent-4-enoyl)pyrrolidine-2-
carbonyl)-L-serinate (30a). Boc-deprotected 29a prepared from
intermediate 29a using the general procedure for the Boc
deprotection reaction was coupled with 4-pentenoic acid using the
general procedure for the amide coupling reaction to prepare 30a as
1
colorless oil (83%). H NMR (400 MHz, CDCl3) δ 7.32 (m, 5H),
6.93 (d, J = 8.8 Hz, 1H), 5.91−5.64 (m, 2H), 5.36−4.91 (m, 6H),
4.75 (m, 1H), 4.47 (d, J = 9.9 Hz, 1H), 3.97−3.64 (m, 4H), 3.61 (dd,
J = 3.4, 9.6 Hz, 1H), 3.51 (dd, J = 3.2, 9.5 Hz, 1H), 2.88−2.73 (m,
1H), 2.66 (t, J = 16.1 Hz, 1H), 2.57−2.07 (m, 5H).
Benzyl O-Allyl-N-((S)-4,4-difluoro-1-(pent-4-enoyl)pyrrolidine-2-
carbonyl)-L-serinate (30b). Boc-deprotected 29b prepared from
intermediate 29b using the general procedure for the Boc
deprotection reaction was coupled with 4-pentenoic acid using the
general procedure for the amide coupling reaction to prepare 30b as
1
colorless oil (97%). H NMR (400 MHz, CDCl3) δ 7.42−7.28 (m,
5H), 7.23 (d, J = 8.6 Hz, 1H), 5.91−5.67 (m, 2H), 5.32−4.93 (m,
6H), 4.81−4.66 (m, 2H), 3.99−3.74 (m, 5H), 3.63 (dd, J = 3.3, 9.6
Hz, 1H), 2.87−2.68 (m, 1H), 2.64−2.44 (m, 1H), 2.44−2.26 (m,
4H).
Benzyl O-Allyl-N-((S)-2-methyl-1-(pent-4-enoyl)pyrrolidine-2-
carbonyl)-L-serinate (30c). Boc-deprotected 29c prepared from
intermediate 29c using the general procedure for the Boc
deprotection reaction was coupled with 4-pentenoic acid using the
general procedure for the amide coupling reaction to prepare 30c as
tert-Butyl (2S,4S)-2-(((S)-3-(Allyloxy)-1-(benzyloxy)-1-oxopropan-
2-yl)carbamoyl)-4-fluoropyrrolidine-1-carboxylate (29a). Boc-de-
protected 28 prepared from intermediate 28 using the general
procedure for the Boc deprotection reaction was coupled with N-Boc-
cis-4-fluoroproline using the general procedure for the amide coupling
1
1
colorless oil (96%). H NMR (400 MHz, CDCl3) δ 7.51 (d, J = 7.7
reaction to prepare 29a as colorless oil (93%). H NMR (400 MHz,
Hz, 1H), 7.36−7.22 (m, 5H), 5.92−5.65 (m, 2H), 5.28−4.87 (m,
6H), 4.76−4.58 (m, 1H), 3.96−3.78 (m, 3H), 3.65 (ddd, J = 0.8, 3.5,
9.6 Hz, 1H), 3.61−3.44 (m, 2H), 2.53−2.40 (m, 1H), 2.34 (s, 4H),
1.96−1.78 (m, 2H), 1.69 (ddd, J = 6.6, 8.7, 12.5 Hz, 1H), 1.63 (d, J =
0.8 Hz, 3H).
Benzyl O-Allyl-N-((S)-1-(pent-4-enoyl)azetidine-2-carbonyl)-L-
serinate (30d). Boc-deprotected 29d prepared from intermediate
29d using the general procedure for the Boc deprotection reaction
was coupled with 4-pentenoic acid using the general procedure for the
CDCl3) δ 7.37−7.16 (m, 5H), 7.02 (s, 1H), 5.71 (ddt, J = 5.6, 10.4,
17.2 Hz, 1H), 5.34−4.97 (m, 4H), 4.75 (dd, J = 3.5, 7.5 Hz, 1H), 4.42
(s, 1H), 3.98−3.79 (m, 2H), 3.79−3.41 (m, 3H), 2.64 (s, 1H), 2.26
(d, J = 43.7 Hz, 1H), 1.43 (s, 9H).
tert-Butyl (S)-2-(((S)-3-(Allyloxy)-1-(benzyloxy)-1-oxopropan-2-
yl)carbamoyl)-4,4-difluoropyrrolidine-1-carboxylate (29b). Boc-de-
protected 28 prepared from intermediate 28 using the general
procedure for the Boc deprotection reaction was coupled with N-Boc-
4,4-difluoro-proline using the general procedure for the amide
L
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amide coupling reaction to prepare 30d as colorless oil (78%). H
NMR (400 MHz, CDCl3) δ 8.30 (d, J = 7.8 Hz, 1H), 7.39−7.24 (m,
5H), 5.78 (dtd, J = 7.0, 10.5, 16.0 Hz, 2H), 5.30−4.91 (m, 6H), 4.87
(dd, J = 6.2, 9.4 Hz, 1H), 4.71 (dt, J = 3.9, 7.8 Hz, 1H), 4.10−3.98
(m, 2H), 3.92 (ddt, J = 1.4, 2.4, 4.0 Hz, 2H), 3.84 (dd, J = 4.0, 9.7 Hz,
1H), 3.65 (dd, J = 3.7, 9.6 Hz, 1H), 2.61 (ddd, J = 2.1, 5.7, 12.0 Hz,
1H), 2.39 (dq, J = 6.9, 22.3 Hz, 3H), 2.18 (dd, J = 5.8, 8.0 Hz, 2H).
Benzyl O-Allyl-N-((S)-1-(pent-4-enoyl)piperidine-2-carbonyl)-L-
serinate (30e). Boc-deprotected 29e prepared from intermediate
29e using the general procedure for the Boc deprotection reaction
was coupled with 4-pentenoic acid using the general procedure for the
5.6, 8.8 Hz, 1H), 3.28 (d, J = 4.9 Hz, 1H), 2.83 (d, J = 4.9 Hz, 1H),
2.55 (ddd, J = 1.9, 9.1, 11.3 Hz, 1H), 2.49−2.42 (m, 1H), 2.21−2.11
(m, 1H), 1.98−1.83 (m, 4H), 1.80 (s, 4H), 1.71−1.62 (m, 2H), 1.53
(t, J = 5.4 Hz, 2H), 1.47 (d, J = 3.4 Hz, 5H), 1.39 (dd, J = 4.4, 14.2
Hz, 1H), 0.91 (dd, J = 6.6, 9.5 Hz, 6H); 13C NMR (101 MHz,
CDCl3) δ 207.36, 174.21, 173.20, 170.07, 69.42, 68.95, 68.31, 58.90,
53.98, 52.27, 49.82, 49.75, 40.20, 39.00, 35.35, 27.74, 24.98, 24.40,
23.30, 23.17, 22.52, 22.40, 21.31, 16.76. LC−MS (ES+) m/z calcd for
C24H40N3O6 [M + H]+ 466.2, found: 466.2; purity: ≥96% and
retention time is 16.98 min by HPLC analysis.
(10S,13S)-N-((S)-4-Methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopen-
tan-2-yl)-2,12-dioxo-8-oxa-1,11-diazabicyclo[11.2.0]pentadecane-
10-carboxamide (35). Macrocyclic carboxylic acid intermediate 31d
was prepared from 30d using the general procedure for synthesis of
macrocyclic carboxylic acid. 31d was coupled with right-hand Boc-
deprotected epoxyketone intermediate 4a using the general procedure
for the amide coupling reaction to prepare 35 as a white solid (39%).
1H NMR (400 MHz, CDCl3) δ 8.20 (d, J = 7.5 Hz, 1H), 6.98 (d, J =
8.1 Hz, 1H), 4.98 (dd, J = 5.4, 9.6 Hz, 1H), 4.55 (ddd, J = 3.3, 8.0,
10.2 Hz, 1H), 4.46 (td, J = 3.7, 7.9 Hz, 1H), 4.11 (td, J = 6.5, 8.6 Hz,
2H), 3.75 (dd, J = 3.7, 9.8 Hz, 1H), 3.57 (dd, J = 8.1, 9.8 Hz, 1H),
3.42 (ddd, J = 4.1, 9.0, 10.4 Hz, 2H), 3.27 (d, J = 5.0 Hz, 1H), 2.84
(d, J = 5.0 Hz, 1H), 2.68−2.47 (m, 2H), 2.14 (ddd, J = 2.4, 11.0, 13.2
Hz, 1H), 2.02 (ddd, J = 3.1, 6.6, 12.9 Hz, 1H), 1.78−1.68 (m, 2H),
1.66−1.56 (m, 2H), 1.56−1.45 (m, 5H), 1.34 (dddd, J = 3.4, 10.1,
14.1, 28.3 Hz, 3H), 0.94−0.83 (m, 6H). 13C NMR (101 MHz,
CDCl3) δ 207.98, 175.42, 172.10, 169.44, 69.56, 67.83, 61.28, 59.01,
53.94, 52.36, 50.09, 48.65, 40.08, 31.02, 27.76, 25.13, 23.26, 23.19,
21.37, 21.30, 18.24, 16.70. LC−MS (ES+) m/z calcd for C22H36N3O6
[M + H]+ 438.2, found: 438.2; purity: ≥96% and retention time is
14.55 min by HPLC analysis.
1
amide coupling reaction to prepare 30e as colorless oil (97%). H
NMR (400 MHz, CDCl3) δ 7.39−7.25 (m, 5H), 6.74 (d, J = 7.9 Hz,
1H), 5.91−5.66 (m, 2H), 5.30−4.88 (m, 7H), 4.70 (dt, J = 3.4, 7.5
Hz, 1H), 3.94−3.79 (m, 3H), 3.77−3.69 (m, 1H), 3.65 (dd, J = 3.4,
9.6 Hz, 1H), 3.19 (td, J = 2.6, 13.1 Hz, 1H), 2.50−2.31 (m, 4H), 2.19
(dt, J = 2.5, 12.7 Hz, 1H), 1.71−1.48 (m, 4H), 1.48−1.32 (m, 1H).
(3S,14S,15aS)-14-Fluoro-N-((S)-4-methyl-1-((R)-2-methyloxiran-
2-yl)-1-oxopentan-2-yl)-1,11-dioxotetradecahydropyrrolo[2,1-f ]-
[1]oxa[4,7]diazacyclotridecine-3-carboxamide (32). Macrocyclic
carboxylic acid intermediate 31b was prepared from 30b using the
general procedure for synthesis of macrocyclic carboxylic acid. 31b
was coupled with right-hand Boc-deprotected epoxyketone inter-
mediate 4a using the general procedure for the amide coupling
1
reaction to prepare 33 as a white solid (43%). H NMR (400 MHz,
CDCl3) δ 7.16 (d, J = 8.4 Hz, 1H), 7.04 (d, J = 8.0 Hz, 1H), 4.90 (d, J
= 10.0 Hz, 1H), 4.61 (ddd, J = 3.8, 8.5, 9.6 Hz, 1H), 4.27 (ddd, J =
2.6, 3.6, 8.0 Hz, 1H), 4.03 (dd, J = 2.6, 9.7 Hz, 1H), 3.90 (ddd, J =
1.9, 12.4, 22.9 Hz, 1H), 3.82−3.69 (m, 1H), 3.53 (dt, J = 4.6, 9.1 Hz,
1H), 3.38−3.31 (m, 2H), 3.28 (d, J = 5.0 Hz, 1H), 2.92−2.85 (m,
1H), 2.83 (d, J = 4.9 Hz, 1H), 2.55 (ddd, J = 2.0, 8.0, 12.8 Hz, 1H),
2.30−2.15 (m, 2H), 1.92−1.77 (m, 3H), 1.71−1.58 (m, 3H), 1.54−
1.38 (m, 7H), 0.91 (dd, J = 1.8, 6.6 Hz, 6H). 13C NMR (101 MHz,
CDCl3) δ 207.52, 174.59, 169.68, 169.63, 93.17, 91.40, 69.60, 68.25,
58.98, 58.94, 54.64, 54.40, 53.87, 52.30, 50.14, 40.05, 34.69, 34.52,
34.30, 27.52, 25.00, 24.09, 23.26, 22.09, 21.33, 16.73. LC−MS (ES+)
m/z calcd for C23H37FN3O6 [M + H]+ 470.2, found: 470.2; purity:
≥96% and retention time is 15.30 min by HPLC analysis.
(3S,16aS)-N-((S)-4-Methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopen-
tan-2-yl)-1,11-dioxotetradecahydro-2H-pyrido[2,1-f ][1]oxa[4,7]-
diazacyclotridecine-3-carboxamide (36). Macrocyclic carboxylic
acid intermediate 31e was prepared from 30e using the general
procedure for synthesis of macrocyclic carboxylic acid. 31e was
coupled with right-hand Boc-deprotected epoxyketone intermediate
4a using the general procedure for the amide coupling reaction to
1
prepare 36 as a white solid (28%). H NMR (400 MHz, CDCl3) δ
(3S,15aS)-14,14-Difluoro-N-((S)-4-methyl-1-((R)-2-methyloxiran-
2-yl)-1-oxopentan-2-yl)-1,11-dioxotetradecahydropyrrolo[2,1-f ]-
[1]oxa[4,7]diazacyclotridecine-3-carboxamide (33). Macrocyclic
carboxylic acid intermediate 31a was prepared from 30a using the
general procedure for synthesis of macrocyclic carboxylic acid. 31a
was coupled with right-hand Boc-deprotected epoxyketone inter-
mediate 4a using the general procedure for the amide coupling
6.83 (d, J = 8.4 Hz, 1H), 6.78 (d, J = 8.2 Hz, 1H), 5.42−5.35 (m,
1H), 4.54 (ddd, J = 3.3, 8.1, 10.1 Hz, 1H), 4.46 (dt, J = 3.8, 7.9 Hz,
1H), 3.97 (dd, J = 3.6, 9.3 Hz, 1H), 3.81 (d, J = 13.4 Hz, 1H), 3.55−
3.47 (m, 1H), 3.35−3.23 (m, 3H), 2.97−2.89 (m, 1H), 2.83 (d, J =
4.9 Hz, 1H), 2.77 (s, 1H), 2.34 (dt, J = 2.6, 14.7 Hz, 1H), 2.19 (ddd, J
= 2.2, 10.2, 12.7 Hz, 1H), 1.77−1.57 (m, 7H), 1.56−1.36 (m, 9H),
1.30 (ddd, J = 4.5, 10.2, 13.7 Hz, 1H), 0.90 (dd, J = 1.8, 6.6 Hz,
6H).13C NMR (101 MHz, CDCl3) δ 207.88, 173.51, 171.11, 169.52,
69.63, 67.76, 59.01, 53.07, 52.34, 51.43, 50.33, 44.73, 40.03, 38.56,
33.18, 27.11, 25.57, 25.22, 24.07, 23.22, 22.50, 22.12, 21.49, 20.36,
16.70. LC−MS (ES+) m/z calcd for C24H40N3O6 [M + H]+ 466.2,
found: 466.3; purity: ≥96% and retention time is 16.31 min by HPLC
analysis.
Biology. All animal study protocols were approved by the Animal
Care and Use Committee (IACUC) of Chungbuk National
University (Approval No. CBNUA-144-1001-01) and Seoul National
University (SNU-200217-2). In addition, all cell culture experiments
were performed following the guidelines and regulations of the
University of Kentucky Biosafety Committee (Approval No. B17-
3000-M).
1
reaction to prepare 32 as a white solid (42%). H NMR (400 MHz,
CDCl3) δ 7.40 (d, J = 8.1 Hz, 1H), 6.83 (d, J = 8.3 Hz, 1H), 5.07 (dd,
J = 3.7, 9.9 Hz, 1H), 4.58 (ddd, J = 3.2, 8.3, 10.2 Hz, 1H), 4.42 (ddd,
J = 3.6, 5.3, 8.5 Hz, 1H), 4.00−3.81 (m, 2H), 3.80−3.75 (m, 1H),
3.54 (dd, J = 3.7, 9.8 Hz, 1H), 3.46 (dt, J = 3.9, 6.4 Hz, 1H), 3.40−
3.34 (m, 1H), 3.26 (d, J = 5.0 Hz, 1H), 3.18−3.04 (m, 1H), 2.84 (d, J
= 5.0 Hz, 1H), 2.56−2.40 (m, 2H), 2.26 (ddd, J = 2.6, 8.9, 13.0 Hz,
1H), 1.85−1.76 (m, 1H), 1.69−1.59 (m, 2H), 1.58−1.49 (m, 4H),
1.48 (s, 3H), 1.34 (ddd, J = 4.3, 10.3, 13.9 Hz, 2H), 0.91 (dd, J = 2.5,
6.5 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 207.83, 174.62, 169.20,
168.90, 69.55, 67.97, 58.99, 53.95, 52.33, 50.22, 40.11, 34.75, 34.21,
27.31, 25.14, 23.67, 23.25, 21.98, 21.36, 16.70. LC−MS (ES+) m/z
calcd for C23H36F2N3O6 [M + H]+ 488.2, found: 488.2; purity: ≥96%
and retention time is 16.15 min by HPLC analysis.
Cell Culture. BV-2 murine microglial cell line was a kind gift from
Dr. Won-Gon Kim (Korea Research Institute of Bioscience &
Biotechnology), and RPMI-8226 human multiple myeloma cell line
was obtained from the ATCC (American Type Culture Collection).
BV-2 cells and RPMI-8226 human multiple myeloma cells were
cultured as reported previously.22 RPMI-8226 cells with acquired
resistance to carfilzomib (RPMI-8226/ABCB1) previously established
by us were used and maintained in 80 nM carfilzomib.30 To assess the
in cellulo activity of macrocyclic proteasome inhibitors, RPMI-8226/
ABCB1 cells were kept in a drug-free medium for two weeks after
drug withdrawal.
(3S,15aS)-15a-Methyl-N-((S)-4-methyl-1-((R)-2-methyloxiran-2-
yl)-1-oxopentan-2-yl)-1,11-dioxotetradecahydropyrrolo[2,1-f ][1]-
oxa[4,7]diazacyclotridecine-3-carboxamide (34). Macrocyclic car-
boxylic acid intermediate 31c was prepared from 30c using the
general procedure for synthesis of macrocyclic carboxylic acid. 31c
was coupled with right-hand Boc-deprotected epoxyketone inter-
mediate 4a using the general procedure for the amide coupling
1
reaction to prepare 34 as a white solid (32%). H NMR (400 MHz,
CDCl3) δ 7.60 (d, J = 8.6 Hz, 1H), 7.30 (d, J = 7.8 Hz, 1H), 4.66 (td,
J = 6.6, 8.5 Hz, 1H), 4.23 (dt, J = 3.6, 7.7 Hz, 1H), 3.97 (dd, J = 3.1,
10.1 Hz, 1H), 3.74−3.61 (m, 2H), 3.53−3.45 (m, 2H), 3.36 (dt, J =
M
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Animals. Nine-month-old Tg2576 mice were purchased from the
Division of Laboratory Animal Resources (Korea FDA, Osong, South
Korea). They were quarantined and acclimatized for a month before
use. Animals were kept in groups of three per cage, allowed free access
to water and food, and maintained on a 12 h light/dark cycle.
Proteasome Activity Assay. The in vitro assay of 20S
proteasome activities was performed as previously described.22 Briefly,
for the proteasome activity assay using purified human 20S
proteasome (Boston Biochem: constitutive proteasome, Cat. No. E-
360-050; immunoproteasome, Cat. No. E-370-025), 50 ng of diluted
purified human 20S proteasomes in the 20S proteasome assay buffer
(20 mM Tris−HCl, 0.5 mM EDTA, 0.035% SDS, pH 8.0) was
transferred to a 96-well microtiter plate and incubated with eight
concentrations of inhibitors (3, 10, 30, 100, 300, 1000, 3000, 10 000
nM) and control (DMSO only control) for 1 h. For the in cellulo
proteasome activity assay using cells, cells were treated with
proteasome inhibitors for 4 h and subsequently lysed using the
passive lysis buffer (Promega). Cell lysates were centrifuged at
14 000g for 20 min at 4 °C. Cell lysates containing 5 μg of total
protein (in 20S proteasome assay buffer; 20 mM Tris−HCl, 0.5 mM
EDTA, 0.035% SDS, pH 8.0) were added to a 96-well microplate. To
measure the proteasome subunit activity for purified proteasome or
cell lysates, the fluorescence substrates were incubated, and
fluorescence (360 nm excitation, 460 nm emission) was monitored
on a SpectraMax M5 microplate reader every minute for 1 h. For
individual proteasome subunit activity, the fluorescence substrates
were used as follows: Suc-LLVY-AMC (CT-L activity, 100 μM), Ac-
PAL-AMC (LMP2 activity, 100 μM), Ac-nLPnLD-AMC (β1
activity,100 μM), Ac-ANW-AMC (LMP7 activity, 100 μM), and
Ac-WLA-AMC (β5 activity, 20 μM).
In Vitro Metabolic Stability Assays. The whole blood and the
liver were collected from ICR mice (male, 7-week-old; DBL
(Eumseong, Korea)). The whole blood was treated with 25 IU/mL
heparin, and the liver was rinsed and homogenized in ice-cold
phosphate-buffered saline (PBS, 2.5 mL/g tissue). Following
preincubation at 37 °C, aliquots of whole blood or liver homogenates
(396 μL) were mixed with the stock solutions of the test compounds
(4 μL) to make the final concentration of 1 μM. From the reaction
mixture, an aliquot (40 μL) was taken at the predetermined time
points: 0, 10, and 30 min (for whole blood) and 0, 10, and 20 min
(for liver homogenates). Samples were immediately mixed with
acetonitrile (300 μL) containing 100 nM carfilzomib as an internal
standard. After vortexing and centrifugation (13 000 rpm, 10 min, 4
°C), the supernatant’s drug levels were quantified via LC−MS/MS.
Differences between the groups were assessed using Student’s t-test
(GraphPad Prism, v.8.4.1), and P values less than 0.05 were
considered statistically significant.
Quantitation of Drug Levels via LC−MS/MS. YU102 and DB-
60 (20) were quantified by an HPLC 1290 infinity II (Agilent, Palo
Alto, CA) coupled to an Agilent 6460 triple quadrupole (Agilent, Palo
Alto, CA) equipped with a Jet Stream Technology ion source
interface operating in the positive ion mode. The chromatographic
separation was performed on a Phenomenex Luna C18 column (50
mm × 2.0 mm id, 3 μm) with a mobile phase that consisted of 0.1%
formic acid in water (A)−0.1% formic acid in acetonitrile (B) at a
flow rate of 0.3 mL/min. The gradient is set up as follows: 0.5 min
85% A, 15% B; 3.5 min 100% B; 5.5 min 100% B; 5.6 min 85% A,
15% B; and 7.5 min 85% A, 15% B. The optimized parameters for the
source-dependent mass spectrometry are as follows: gas temperature
325 °C; gas flow 11 L/min; nebulizing gas 35 psi; sheath gas flow 12
L/min; and capillary voltage 4500 V. The compound-dependent
parameters, fragment voltage, collision energy (qualifier/quantifier),
and cell accelerator voltage are set as follows: for YU102, 150, 25/20,
and 4 V; for DB-60 (20), 176, 40/48, and 4 V; and for carfilzomib,
192, 29/29, and 4 V. Quantification was carried out in the selected
reaction monitoring (SRM) mode using the following transition: for
YU102, m/z 515.3 > 217.1 (qualifier), m/z 515.3 > 197.1
(quantifier); for DB-60 (20), m/z 590.3 > 260.2 (qualifier), m/z
590.3 > 70.2 (quantifier); and for carfilzomib, m/z 720.4 > 402.3
(qualifier), m/z 720.4 > 289.2 (qualifier). Linearity was verified in the
following concentration ranges: for whole blood, 10−2500 nM; and
for liver homogenates, 1−2000 nM.
ABCG2 Interaction Assays. The inhibitory interactions with
ABCG2 were assessed by measuring the changes in the cellular
accumulation of PhA (a fluorescent ABCG2 substrate) using
MDCKII cells stably expressing ABCG2 (established in our previous
study41) and parental cells. The known ABCG2 inhibitor Ko143 was
used as a positive control. Cells were preincubated in the complete
medium containing pheophorbide A (1 μM) with Ko143 (0.2 μM) or
DB-60 (20) (2 or 5 μM) at 37 °C for 30 min on a rotary shaker. Cells
were then washed with an ice-cold medium and resuspended in the
medium containing only Ko143 or DB-60 (20). After incubation for
45 min at 37 °C on a rotary shaker, cells were washed with 1 mL of
ice-cold PBS containing 2% fetal bovine serum. After centrifugation,
cell pellets were kept on ice until the fluorescent signal measurement
via flow cytometry (FACSCalibur Flow Cytometry System, BD
Biosciences, San Jose, CA) using the excitation and emission
wavelengths of 635 and 670 nm, respectively.
Cytokine Enzyme-Linked Immunosorbent Assay (ELISA).
This experiment was performed following a procedure reported
previously by us.23 Briefly, standards were diluted by seven serial
twofold dilutions for the generation of a standard curve. Prepared
standards and supernatants from BV-2 cells were incubated on a
capture antibody-coated plate overnight at 4 °C. For IL-1α level
measurement in Tg2576 mice serum, mouse serum samples were
diluted 1:5 in the diluent before ELISA, and IL-1α standard and
diluted serum samples were incubated at room temperature for 2 h.
Then, the plates were washed five times with the washing solution.
Next, a detection antibody was added and the plate was then
incubated for 1 h, followed by the addition of avidin conjugated with
horseradish peroxidase for 30 min. The plates were then washed five
times and developed by adding substrate solution to each well, and
afterward, the reaction was stopped by the addition of stop solution
(2 N H2SO4). The absorbance at 450 nm wavelength was measured
using a microplate reader.
Morris Water Maze Test. The Morris water maze test was
performed following a procedure described previously.22 Briefly, the
water inside a circular plastic pool was warmed at 25 1 °C, and the
escape platform was hidden 1−1.5 cm under the water level. The mice
were released into the water with their head pointing to the pool wall
and trained three times a day with randomized starting points over 5
days to promote the acquisition of the location of an escape platform
in the pool (trial duration: 60 s). A camera was positioned directly
above the center of the pool, which recorded the behaviors of mice.
The video was analyzed, and the swimming speed, escape latency, and
escape distance on every trial were calculated by the SMART-LD
program (Panlab, Spain). A probe test was performed 24 h after the
water maze test (i.e., day 6) to assess spatial memory consolidation
based on the preference for the quadrant where the platform was
placed. The swimming patterns of each mouse for 60 s were tracked
and analyzed by the SMART-LD program.
Passive Avoidance. As previously described by our group,22 the
effects of proteasome inhibitors on learning and memory function in
mice were evaluated using a step-through passive avoidance test. The
passive avoidance test was performed 48 h after the probe test. A
“step-through” apparatus (Med Associates Inc., Vermont) consists of
two connected chambers (an illuminated chamber and a dark
chamber) by a small gate. On the first day (i.e., day 7 of the behavior
study), the mice were placed in the illuminated compartment facing
away from the dark chamber for the learning trial. As soon as the mice
entered the darkroom, they received a punishing electric shock (0.45
mA, 3 s duration). The testing trial was given 24 h after termination of
the learning trial (i.e., day 8) using the same paradigm but without the
electric shock. Each mouse was released in the lighted compartment,
and the entrance latency until the mice re-entered the dark chamber
was determined and defined as the step-through latency. The
maximum latency time for entering the dark compartment was 120 s.
RPE Flat Mounts. RPE flat mounts were performed following the
previously published protocols by our group.22 Briefly, surgically
isolated eyes of Tg2576 transgenic mice were first fixed in 4%
N
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Article
paraformaldehyde for 1 h at room temperature. Next, anterior eyecups
were dissected on ice-cold PBS, followed by the removal of the retina.
The dissected RPE sheets were permeabilized with 0.2% Triton X-100
for 15 min and then blocked with 3% BSA for 1 h at room
temperature. The sections were then incubated with β-catenin (1:100;
Abcam, ab19381) overnight at 4 °C. After being rinsed in PBS
containing 0.5% BSA, the tissues were incubated further with
secondary antibodies coupled to Alexa 555 (1:1000; Invitrogen,
A21422) for 2 h at room temperature and mounted. The RPE sheets
were imaged using a confocal microscope (Carl Zeiss, LSM 800).
Computational Docking. AutoDock Vina was used to docking
DB-310 into the atomic structure of mouse LMP2 (PDB ID:
3UNF).23 A 3D docking box of 30 Å width covering the ligand-
binding site was used (for the PDB validation report, see the
Supporting Information). The highest-scoring, predicted ligand
model, with the epoxyketone positioned near the N-terminal
threonine for possible covalent interaction, was chosen for our
investigations. The chosen model shows 95% calculated affinity
compared to the highest-scoring model, with no covalent bond
formation.
Seongsoo Lee − College of Pharmacy and Research Institute of
Pharmaceutical Sciences, Seoul National University, Seoul
08826, Republic of Korea
Zachary Miller − Department of Pharmaceutical Sciences,
University of Kentucky, Lexington, Kentucky 40536-0596,
United States
Sukyeong Lee − Verna and Marrs McLean Department of
Biochemistry and Molecular Biology, Baylor College of
Medicine, Houston, Texas 77030, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00291
Author Contributions
M.J.L. and D.B. contributed equally. K.B.K., D-E.K., S.L., W.L.,
and J.T.H. conceived the study, designed the experiments, and
wrote the paper; D.B. (Schemes 1 and 2), M.J.L. (Table 1,
Figures 3a, 4b, and 5c, and Supplementary Table 1), H.J.
(Figures 3b and 4a), A.B. (Figure 6), Z.M. (Table 1), I.J.Y.
(Figure 5a,b), and S.L. (Supplementary Tables 2 and 3)
conducted experiments and acquired and analyzed the data. All
authors have given approval to the final version of the
manuscript.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00291.
Notes
The authors declare no competing financial interest.
Proteasome catalytic subunit inhibition of DB-60 (20)
in different cell lines; 1H/13C NMR spectra of all
macrocyclic compounds and HPLC elution profile of the
lead compound DB-60 (20) (PDF)
Molecular formula strings (CSV)
Molecular docking models (PDB)
ACKNOWLEDGMENTS
■
The authors thank the National Institutes of Health (R01
CA188354 to K.B.K. and R01 GM111084 to S.L.), the
N a t i o n a l R e s e a r c h F o u n d a t i o n o f K o r e a
(2017R1E1A1A01074656 to D-E.K. and MRC2017R
A5A2015541 to J.T.H), and the Creative-Pioneering Re-
searchers Program through Seoul National University (to
W.L.) for financially supporting this work.
AUTHOR INFORMATION
Corresponding Authors
■
Jin Tae Hong − College of Pharmacy, Chungbuk National
University, Cheongju, Chungbuk 28160, Republic of Korea;
Email: jinthong@chungbuk.ac.kr
Dong-Eun Kim − Department of Bioscience and
Biotechnology, Konkuk University, Seoul 05029, Republic of
Korea; orcid.org/0000-0001-6545-8387;
Email: kimde@konkuk.ac.kr
Wooin Lee − College of Pharmacy and Research Institute of
Pharmaceutical Sciences, Seoul National University, Seoul
08826, Republic of Korea; orcid.org/0000-0001-7805-
869X; Email: wooin.lee@snu.ac.kr
Kyung Bo Kim − Department of Pharmaceutical Sciences,
University of Kentucky, Lexington, Kentucky 40536-0596,
United States; orcid.org/0000-0003-4744-2466;
Email: kbkim2@uky.edu
ABBREVIATIONS USED
■
AD, Alzheimer’s disease; Aβ, amyloid-β; COX, cyclooxyge-
nase; TNF-α, tumor necrosis factor-α; iP, immunoproteasome;
LMP2, low-molecular-mass polypeptide-2; MECL-1, multi-
catalytic endopeptidase complex subunit-1; LMP7, low-
molecular-mass polypeptide-7; RPE, retinal pigment epithe-
lium; CNS, central nervous system; BBB, blood−brain barrier;
CT-L, chymotrypsin-like; LPS, lipopolysaccharide; cP, con-
stitutive proteasome; AMD, age-related macular degeneration;
APP, amyloid precursor protein; PhA, pheophorbide A
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