Exploration of novel macrocyclic dipeptide N-benzyl amides as proteasome inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.12.072

As proteasome inhibitors, a series of novel macrocyclic dipeptide N-benzyl amides were designed, synthesized and evaluated. Most of them exhibited potent proteasome inhibition and excellent anti-proliferative activity against RPMI 8226, MM1S, and MV-4-11 cell lines. As the most distinguished one among this series, compound 23h displayed potent and selective proteasome inhibitory potency (IC₅₀: β5c = 29 nM, β5i = 35 nM, β_1c, β_2c β_1i β_2i > 10 μM), excellent anti-proliferative activity against RPMI 8226, MM1S, and MV-4-11 cell lines with IC₅₀ values of 18 nM, 15 nM, and 21 nM, respectively, as well as favorable metabolic stability in human liver microsomes (HLMs), highlighting that it is a promising lead compound for further development of proteasome inhibitors.

Full text of DOI:10.1016/j.ejmech.2018.12.072

European Journal of Medicinal Chemistry 164 (2019) 423e439
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Exploration of novel macrocyclic dipeptide N-benzyl amides as proteasome
inhibitors
,
e
,
,
c
,
,
, b,
Jianjun Yu a 1, Jieyu Liu b 1, Daqiang Li a 1, Lei Xu d c, Duidui Hong a, Shan Chang f,
Lei Xu f, Jia Li b c, Tao Liu a *, Yubo Zhou b
,
,
, c, **
a ZJU-ENS Joint Laboratory of Medicinal Chemistry, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences,
Zhejiang University, Hangzhou, Zhejiang, 310058, PR China
b National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai,
201203, PR China
c University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing, 100049, PR China
d School of Life Science and Technology, ShanghaiTech University, Shanghai, 201203, PR China
e School of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou, 510632, PR China
f Institute of Bioinformatics and Medical Engineering, School of Electrical and Information Engineering, Jiangsu University of Technology, Changzhou,
213001, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
As proteasome inhibitors, a series of novel macrocyclic dipeptide N-benzyl amides were designed,
synthesized and evaluated. Most of them exhibited potent proteasome inhibition and excellent anti-
proliferative activity against RPMI 8226, MM1S, and MV-4-11 cell lines. As the most distinguished one
Received 29 September 2018
Received in revised form
28 December 2018
Accepted 28 December 2018
Available online 31 December 2018
among this series, compound 23h displayed potent and selective proteasome inhibitory potency (IC50
:
b
5c ¼ 29 nM,
b
5i ¼ 35 nM, b1c
,
b2c, b1i, b2i > 10 M), excellent anti-proliferative activity against RPMI
m
8226, MM1S, and MV-4-11 cell lines with IC50 values of 18 nM, 15 nM, and 21 nM, respectively, as well as
favorable metabolic stability in human liver microsomes (HLMs), highlighting that it is a promising lead
compound for further development of proteasome inhibitors.
Keywords:
Proteasome inhibitors
Macrocyclic dipeptides
N-Benzyl amides
Metabolic stability
© 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction
There are two main proteasome subtypes: the constitutive pro-
teasome (cCP) which contains three catalytic subunits denoted as
The eukaryotic 26S proteasome is a large (1.6e2.4 MDa) ATP-
dependent proteolytic complex, composed of a cylindrical 20S
core particle (CP) capped by two 19S regulatory complexes [1]. The
20S proteasome is the proteolytically active key element of the
ubiquitin proteasome system (UPS) that directs the majority of
intracellular protein degradation in eukaryotic cells. It is composed
by four heptameric rings stacked in a a7b7b7a7 arrangement and
b
1c,
b
2c, and
b
5c and the immunoproteasome (iCP) which contains
1i, 2i, and 5i [3]. Inhibition of
three catalytic subunits denoted as
b
b
b
the 20S proteasome leads to the accumulation of substrate proteins
involved in signal transduction, antigen presentation, cell-cycle
progression and apoptosis, and is preferentially cytotoxic to can-
cer cells [4e8]. Indeed, proteasome inhibitors are recognized as
clinically effective anti-cancer agents, primarily for hematological
malignancies [9]. To date, three proteasome inhibitors Bortezomib,
Carfilzomib, and Ixazomib (Fig. 1) are FDA approved for the treat-
ment of multiple myeloma (MM) and mantle cell lymphoma
[10e12]. All three are covalent inhibitors with an electrophilic
warhead at the C-terminal end of a peptidyl backbone for covalent
attachment to the catalytic Thr1 residues of the proteasome [13].
However, the electrophilic warhead is often related to excessive
reactivity, lack of specificity and instability, which is believed to be
the major cause of side effects during therapy [12,14]. These in-
hibitors have also been unsuccessful in the treatment of solid
contains three proteolytic subunits, b1, b2, and b5 [2], respectively.
* Corresponding author. ZJU-ENS Joint Laboratory of Medicinal Chemistry, Zhe-
jiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharma-
ceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058, PR China.
** Corresponding author. National Center for Drug Screening, State Key Laboratory
of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sci-
ences, Shanghai, 201203, PR China.
E-mail addresses: Lt601@zju.edu.cn (T. Liu), ybzhou@simm.ac.cn (Y. Zhou).
Equal contribution.
1
https://doi.org/10.1016/j.ejmech.2018.12.072
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

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J. Yu et al. / European Journal of Medicinal Chemistry 164 (2019) 423e439
Fig. 1. FDA-approved proteasome inhibitor Bortezomib, Carfilzomib, and Ixazomib.
cancers [15,16], with the lack of efficacy most likely due to their
covalent binding to the proteasome, limiting their widespread tis-
sue distribution. In contrast, non-covalent proteasome inhibitors
do not possess an electrophilic warhead with less reactivity. Non-
covalent and reversible binding mode ensure them with rapid
binding and dissociation kinetics. These features may allow non-
covalent inhibitors to overcome drawbacks arising in therapeutics
of covalent ones.
Representative peptidyl non-covalent proteasome inhibitors
have been reported. TMC-95A (4, Fig. 2) is a macrocyclic natural
product isolated from Apiospora montagnei Sacc TC 1093, which
potently inhibits all the three proteasome catalytic activities with
However, linear peptides are always thought to be unstable with
unsatisfied pharmacokinetic profiles. In contrast, macrocyclic
peptides often show distinct and (in comparison to linear peptides)
superior pharmacokinetic and pharmacological properties [31,32].
Desirable properties introduced by the macrocycle include
increased metabolic stability, cellular penetration and selectivity
[33e35]. We have identified a series of oral and potent macrocyclic
dipeptide epoxyketones as covalent proteasome inhibitors in our
previous work [36]. Due to the disadvantages of the electrophilic
warhead discussed, in this manuscript, we explored novel protea-
some inhibitors based on the peptidyl macrocyclic skeleton of
macrocyclic dipeptide epoxyketones, replacing the electrophilic
warhead with substituted benzyl groups (Fig. 3). During further
optimization, a series of macrocyclic dipeptide N-benzyl amides
were designed and synthesized. Moreover, the anti-proliferative
activity against various cancer cells of selected compounds were
evaluated. In addition, we explored the differences in metabolic
stability between our macrocyclic dipeptides and the correspond-
ing linear analogues.
preference for
b
5c activity (IC50 ¼ 5.4 nM) [17,18]. However, the
structural complexity of TMC-95A maybe one of important obstacle
to its further development. Additionally, a series of 5-methoxy-1-
indanone di-peptide benzyl amides have been reported, including
CVT-659 (5, Fig. 2) which selectively inhibits
micromolar potency (IC50 ¼ 0.14 M), but this compound displayed
M) [19]. Researchers from Millen-
b5c site with sub-
m
poor cellular activity (IC50 ¼ 8
m
nium Pharmaceuticals, Inc. identified a series of di- and tripeptides
(e. g. 6 and 7, Fig. 2) with potency and selectivity for both consti-
2. Results and discussion
tutive proteasome and immunoproteasome b5 sites [20]. Moreover,
the X-ray structures of the inhibitors in complex with proteasome
suggest that the occupancy of S1 and S3 pockets was crucial to
inhibitor potency. Of note, the size of the hydrophobic benzyl group
was well suited to S1 pocket, which may be a reason for the wide
use of various substituted benzyl groups in peptidyl non-covalent
proteasome inhibitors. Meanwhile, the 2-(neopentylamino)-2-
oxoethyl group (as present in compound 6) has been reported to
provide a near-optimal fit for the S3 binding pocket [20e22]. Other
reported peptidyl non-covalent proteasome inhibitors include 2-
aminobenzylstatine derivatives [23,24] and linear TMC-95A ana-
logues [25,26]. Overall, linear peptide-based proteasome inhibitors
remain to be the mainstream of this field [13,21,27e30].
2.1. Chemistry
All the target compounds were synthesized by following routes
as described in Schemes 1e5. Schemes 1 and 2 show the synthetic
routes of the intermediates which would be used in Schemes 3e5.
Schemes 3 and 4 show the synthetic routs of macrocyclic di-
peptides 23a-23j and 31. Finally, Scheme 5 shows the synthetic rout
of linear analogue 33.
The synthetic routes of terminal alkene-containing carboxylic
intermediates 10a - 10e are displayed in Scheme 1. (a) Treatment of
8a with 5-bromopent-1-ene in the presence of potassium carbon-
ate, followed by removal of the methyl group yielded the carboxylic
intermediate 10a. (b) The preparation of 10b was initiated from
ethyl 5-bromo-1-methyl-1H-pyrazole-4-carboxylate 8b, through
nucleophilic displacement with pent-4-en-1-ol using NaHMDS to
yield alkenyl esters 9b, which was hydrolyzed to furnish 10b. (c)
The ethyl 5-bromo-1-methyl-1H-pyrazole-4-carboxylate 8b was
coupled with allyltributyltin to provide 9c, followed by hydrolysis
to obtain 10c. (d) Electrophilic substitution of ethyl 1H-imidazole-
2-carboxylate 8c with 4-bromobut-1-ene or 6-bromohex-1-ene,
afforded 9d or 9e, which were subsequently converted to 10d or
10e through hydrolysis.
The terminal alkene-containing amino acid intermediates 13a,
13b, and 17 were prepared through the following routes (Scheme
2). (a) Benzyl (tert-butoxycarbonyl)-
L
-serinate 11a or benzyl (tert-
butoxycarbonyl)- -threoninate coupled 11b with allylmethyl car-
L
bonate in the presence of a palladium catalyst to afford 12a or 12b.
Following deprotection in HCl-saturated ethyl acetate, 13a or 13b
was formed. (b) Boc- -serine 14 underwent nucleophilic substitu-
L
tion with 6-bromohex-1-ene, followed by benzylation and removal
of the Boc group to afford 17.
Fig. 2. Structures of representative peptidyl non-covalent proteasome inhibitors.
The synthetic routes for target compounds 23a-23j are outlined

J. Yu et al. / European Journal of Medicinal Chemistry 164 (2019) 423e439
425
Fig. 3. Design and optimization of the macrocyclic dipeptide N-benzyl amides.
Scheme 1. Synthetic routes for the carboxylic intermediates 10a e 10e. Reagents and conditions: (i) 5-bromopent-1-ene, K2CO3, DMF, 80 ꢀC, 2 h; (ii) 2M NaOH (aq), THF/MeOH, r.t.,
3 h; (iii) pent-4-en-1-ol, NaHMDS, THF, ꢁ8 ꢀC; (iv) allyltributyltin, Pd2(dba)3, PtBu3, CsF, THF, reflux, 5 h; (v) 4-bromobut-1-ene or 6-bromohex-1-ene, K2CO3, DMF, 80 ꢀC, 3 h.
Scheme 2. Synthetic routes for terminal alkene-containing amino acid intermediates 13a, 13b, 17. Reagents and conditions: (i) allylmethyl carbonate, Pd(PPh3)4, THF, 60 ꢀC, 4 h; (ii)
HCl-saturated ethyl acetate, 0ꢀС - r.t., 2 h; (iii) 6-bromohex-1-ene, NaH, DMF, rt, 18 h; (iv) BnBr, K2CO3, DMF, 0 ꢀC e r.t., 20 h.
in Scheme 3. Boc protected amino acids with different side chains
18a-18c, as the starting material, were firstly condensed with 13a,
13b or 17 to yield dipeptide intermediates 19a-19e, which
subsequently underwent deprotection of the Boc group and
condensation with the corresponding carboxylic intermediates
10a-10c or 10f to afford the diolefin derivatives 21a-21g. Following

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J. Yu et al. / European Journal of Medicinal Chemistry 164 (2019) 423e439
Scheme 3. Synthetic routes for target compounds 23a-23j. Reagents and conditions: (i) 13a, 13b or 17, HOBt, EDCI, DIPEA, DCM, 0 ꢀC - rt, 5 h; (ii) HCl-saturated ethyl acetate, 0ꢀС -
r.t., 2 h; (iii) 10a - 10c or 10f, HOBt, EDCI, DIPEA, DCM, 0 ꢀC - rt, 5 h; (iv) Grubbs second generation catalyst, toluene, 100 ꢀC; (v) 10% Pd/C, MeOH, rt, 2 h; (vi) phenylmethanamine, (2-
chlorophenyl)methanamine or p-tolylmethanamine, HOBt, EDCI, DIPEA, DCM, 0 ꢀC - rt, 5 h.
this, diolefin derivatives through ring-closing metathesis (RCM) in
the presence of Grubbs second generation catalyst, as well as
reduction of the CeC double bond and removal of the benzyl-
protecting group on one pot, gave the macrocyclic carboxylic de-
rivatives 22a-22g. Finally, 22a-22g was condensed with phenyl-
reduction produced target compound 31.
To explore the advantages of the designed macrocyclic dipeptide
N-benzyl amides, we synthesized linear analogue 33, correspond-
ing to 23h. As show in Scheme 5, the synthesis of 33 was initiated
from a diolefin intermediate 21e, which was converted into a car-
boxylic intermediate 32 through reduction and debenzylation in
the present of 10% Pd/C under a H2 atmosphere. Subsequently, 32
underwent condensation with p-tolylmethanamine to produce the
linear analogue 33.
methanamine,
tolylmethanamine to produce 23a-23j as the target compounds.
Target compound 31 was prepared through the following route
(Scheme 4). Firstly, commercially available Boc- -tryptophan 24,
(2-chlorophenyl)methanamine
or
p-
L
was used as the starting material, which underwent condensation
with p-tolylmethanamine to yield 25, which was converted to 26
through electrophilic substitution with 3-bromoprop-1-ene in the
presence of NaH in THF. Subsequent removal of the Boc-protecting
group and condensation with N2-(tert-butoxycarbonyl)-N4-neo-
2.2. Biological evaluation
All synthesized target compounds (23a-23j, 31) were evaluated
for their 20S proteasome
b5c inhibitory activity. Nine compounds
pentyl- -asparagine 18c provided dipeptide 28. This was then
L
with promising 5 inhibition were further tested for their anti-
b
converted into the hydrochloride 29 via unmasking the amine
group, followed by condensation with the carboxylic intermediate
10e to afford 30. Finally, diolefin intermediate via RCM and
proliferative activity against three tumor cell lines including RPMI
8226, MM1S, and MV-4-11 (all of the three are peripheral blood cell
lines). The results are summarized in Table 1.

J. Yu et al. / European Journal of Medicinal Chemistry 164 (2019) 423e439
427
Scheme 4. Synthetic route for target compound 31. Reagents and conditions: (i) p-tolylmethanamine, HOBt, EDCI, DIPEA, DCM, 0 ꢀC - rt, 5 h; (ii) 3-bromoprop-1-ene, NaH, THF,
0ꢀС - r.t., 3 h; (iii) HCl-saturated ethyl acetate, 0ꢀС - r.t., 2 h; (iv) N2-(tert-butoxycarbonyl)-N4-neopentyl-L-asparagine, HOBt, EDCI, DIPEA, DCM, 0 ꢀC - rt, 5 h; (v) 10d, HOBt, EDCI,
DIPEA, DCM, 0 ꢀC - rt, 5 h; (vi) Grubbs second generation catalyst, toluene, 100 ꢀC, 3 h; (vii) 10% Pd/C, MeOH, rt, 2 h.
Scheme 5. Synthetic route for the corresponding linear analogue 33. Reagents and conditions: (i) 10% Pd/C, MeOH, rt, 2 h; (ii) p-tolylmethanamine, HOBt, EDCI, DIPEA, DCM, 0 ꢀC -
rt, 5 h.
Firstly, we found that R2 substituent groups significantly influ-
enced proteasome inhibitory activity. 2-(Neopentylamino)-2-
oxoethyl substituted compound 23e was more potent than
methoxymethyl and phenethyl substituted compounds (23a &
23b). Therefore, 2-(Neopentylamino)-2-oxoethyl was employed at
the R2 group for further optimization. Secondly, in order to ensure
suitable R1 substitutions, the H, 2-Cl, and 4-CH3 groups, which are
often used in linear non-covalent proteasome inhibitors (e. g.
compounds 6 and 7), were investigated with the 4-CH3 (as in 23f)
being 10-fold and 2-fold more potent than the H (as in 23d) and 2-
Cl (as in 23e), respectively. Furthermore, three different rings were
employed in R3 with the methylpyrazole and imidazole rings
improving inhibitory potency compared to the phenyl rings, as
exemplified by compounds 23g-23i, which were approximately 2-
fold more potent than 23f with IC50 values about 30 nM. Mean-
while, compounds 23g-23i exhibited excellent anti-proliferative
activity against the three cancer cell lines, particularly compound
23h and 23i. In addition, compared to 23g and 23h, we found that
the oxygen and carbon atoms were both suitable for the X atom.
Finally, different linkers (e. g. 23i, 23j, & 31) when introduced into
the skeleton displayed little impact on inhibitory potency, but the
anti-proliferative activity against MM1S and MV-4-11 of compound
23j significantly decreased compared to 23i or 31.
Compound 23h and compound 23i exhibited excellent anti-
proliferative activity against RPMI 8226, MM1S, and MV-4-11 cell
lines, and all the three cell lines are blood cancer cell lines. Several
studies [37,38] have reported that immunoproteasome expressed
more than constitutive proteasome in B-cell malignancies, sug-
gesting the importance of the immunoproteasome in hematologic
malignancies [39e41]. Therefore, we assessed compound 23h and
23i against different subunits of cCP and iCP (Table 2.). We found
our compounds displayed potent and selective inhibitory activity
against b5c and b5i. The co-inhibition against b5c and b5i of 23h and
23i may contribute to well anti-proliferative activity against the
three cancer cell lines.
To assess metabolic stability profiles of 23h and 23i, we tested
they metabolic stability in HLMs. As shown in Fig. 4, compound 23h
showed more than 66% remaining after 30 min incubation, while
23i showed less than 20% remaining. This result demonstrates that
23h was more stable than 23i.
In order to investigate the advantages of our marcocyclic di-
peptides compared to the linear dipeptides, we also synthesized
linear analogue 33 which correspond to 23h. The
b5c inhibitory
activity, anti-proliferative activity and metabolic stability profiles in

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J. Yu et al. / European Journal of Medicinal Chemistry 164 (2019) 423e439
Table 1
20S proteasome (
b5c) inhibitory activity and anti-proliferative activity of the target compounds (22a-22j, 31).
Compd.
23a
R1
R2
R3
X
O
O
Linker
b5c IC50 (nM)a/inhibition rateb
Anti-proliferative activity (IC50, nM)a
RPMI 8226
MM1S
MV-4-11
2-Cl
2-Cl
34.01%b
36.67%b
e
e
e
23b
e
e
e
e
e
e
e
e
e
23c
23d
4-CH3
O
O
38.39%b
H
660 30
23e
23f
23g
23h
23i
23j
31
2-Cl
O
O
O
C
C
C
C
127 36
301 20
343 11
718 58
4-CH3
4-CH3
4-CH3
4-CH3
4-CH3
4-CH3
67 12
175
58
18
15
27
59
6
37
23
15
6
2
3
76
55
21
5
2
9
30
29
34
50
26
4
3
6
7
7
9
2
1
2
4
1
0.5
12 0.3
208 41
220 32
17
1
52
1
3
Carfilzomib
Oprozomib
e
e
e
e
e
e
e
e
e
e
7
47
0.5
2
2
19
0.1
3
<1
e
9
1
28
a
IC50 values are shown as an average of three independent determinations.
Inhibition rates of b5c were determined at a concentration of 10 mM.
b

J. Yu et al. / European Journal of Medicinal Chemistry 164 (2019) 423e439
429
Table 2
The inhibition of b1c, b2c, b5c, b1i, b2i, and b5i of compound 23h, 23i, and Bortezomib.
Compd.
IC50(nM)a
b1c
b2c
b5c
b1i
b2i
b5i
23h
23i
>10000
>10000
>10000
>10000
29
34
3
6
1
>10000 >10000
>10000 >10000
35
19
4
5
2
Bortezomib 104 38 2119 819 11
3
1
517 10
1
a
The IC50 values are shown as an average of three independent determinations.
Fig. 5. Metabolic stability profiles of compound 23h and 33 in HLMs.
amides might possess improved metabolic stability profiles than
carfilzomib and their corresponding linear analogues.
2.3. Binding mode analysis
To explore the binding modes of these macrocyclic dipeptide N-
benzyl amides within the active site of proteasome, molecular
docking calculations were performed. First, the ligand in the crystal
structure of 3MG6 (PDB ID) was extracted and re-docked into the
binding pocket [43e46]. The root mean square deviation (RMSD)
between the docked pose and the original conformation of the
ligand was calculated, and a RMSD ¼ 0.3 Å suggests that the Glide
docking with the SP scoring can successfully recognize the near-
native conformations. Then, the binding mode of compound 23h
within the proteasome was predicted by the Glide docking and
shown in Fig. 6. The docking simulations clearly demonstrated the
Fig. 4. Metabolic stability profiles of compound 23h and 23i in HLMs.
HLMs of the linear analogue 33 were evaluated. The results are
summarized in Table 3.
As illustrated in Table 3, proteasome
b5c inhibition of linear
analogue 33 was better than that of the macrocyclic compound, but
its anti-proliferative activity against all of the three cancer cell lines
was lower. In comparison to 33 with 23h, ring breaking of 23h led
to a 5-fold, 5-fold, and 3-fold loss of anti-proliferative activity
against RPMI 8226, MM1S, and MV-4-11, respectively. These results
indicated that macrocylic dipeptide N-benzyl amides were likely to
display improved cellular penetration compared to linear ana-
logues. Furthermore, 23h demonstrated excellent metabolic sta-
bility in HLMs (Table 3. & Fig. 5), as compared to 33 and carfilzomib.
This result suggested that our macrocylic dipeptide N-benzyl
presence of 23h in the proximity of the
b5 site. As depicted in
Fig. 6A, the C-terminal 4-methylbenzyl group and the neo-
pentylamino group occupied the S1 and S3 pockets, respectively,
while the linker was exposed to the solvent. In addition, a similar
binding mode reported by Blackburn et al. [20] involving b-sheet-
type H-bonding interactions was also observed (Fig. 6B). Six critical
hydrogen bonds were formed between the carbonyl and amino
groups of the peptide skeleton and Gly47, Thr21, Ala49, Ala50 and
Table 3
Biological evaluation of compounds 23h & 33.
Compd.
Structure
b5c IC50 (nM)a
Anti-proliferative activity (IC50, nM)a
RPMI 8226 MM1S
18
HLMs Stabilityb
66%
MV-4-11
23h
29
3
2
15
1
21
9
33
9
0.5
97 26
64
8
73
3
<5%
Carfilzomib
e
7
0.5
2
0.1
<1
e
<5%c
a
IC50 values are shown as an average of three independent determinations.
% remaining was determined after 30 min of incubation in HLMs. The higher of % remaining, the more stable is the compound in HLMs. % remaining was calculated as
b
described in 4.2.3 section.
c
Data from reported reference [42].

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J. Yu et al. / European Journal of Medicinal Chemistry 164 (2019) 423e439
Fig. 6. Binding mode of compound 23h within the active site of proteasome. A: Occupancy of the S1 and S3 pockets of the 20S proteasome by the 4-methylbenzyl group and the
neopentylamino group of compound 23h displayed as Connolly surface. B: Predicted H-bonds between compound 23h and the protein represented as a ribbon model, H-bonds are
shown as dashed yellow lines. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Asp114 of the proteasome active site. Notably, 23h established H-
(200e300 mesh). All yields are unoptimized and generally
represent.
bonds with Ala50N and ASP114Og via a bridge water molecule. In
brief, specific binding to the S1 and S3 pockets and the b-sheet-type
H-bonding interactions of compound 23h contribute to its inhibi-
tory potency.
4.1. Chemistry
4.1.1. Methyl 2-(pent-4-en-1-yloxy)benzoate 9a
A mixture of methyl 2-hydroxybenzoate (5.00 g, 33.0 mmol),
potassium carbonate (9.11 g, 66.0 mmol) and 5-bromopent-1-ene
(6.81 g, 46.0 mmol) in 50 mL DMF was heated to 80 ꢀC for 2 h. TLC
analysis indicated that the reaction had ceased. After cooling to
room temperature, the solution was diluted with EtOAc (100 mL)
and washed with water (100 mL ꢂ 3), brine (50 mL ꢂ 3). The
organic layer was dried over Na2SO4, filtered, and concentrated
under reduced pressure. The residue was then purified by SiO2
chromatography (ethyl acetate: petroleum ether ¼ 1:15) to give
compound 9a as colorless oil (5.8 g, 80%). 1H NMR (500 MHz, CDCl3)
3. Conclusions
In this study, a series of novel macrocyclic dipeptide N-benzyl
amides were rationally designed, synthesized, and biologically
evaluated. Most of them exhibited potent proteasomal inhibition,
as well as excellent anti-proliferative activity against RPMI 8226,
MM1S, and MV-4-11 cell lines. Compound 23h and 23i displayed
potent and selective inhibition against b5c and b5i. Further studies
found that compound 23h was more potent and more stable than
its corresponding linear analogues, proving that the introduction of
a macrocycle might increase potency and metabolic stability.
Finally, docking studies were performed to analyze the binding
d
7.78 (dd, J ¼ 7.5, 1.5 Hz, 1H), 7.44 (ddd, J ¼ 8.0, 7.5, 1.5 Hz, 1H),
6.99e6.94 (m, 2H), 5.92e5.80 (m, 1H), 5.09e5.04 (m, 1H),
5.02e4.98 (m, 1H), 4.05 (t, J ¼ 6.5 Hz, 2H), 3.89 (s, 3H), 2.34e2.25
(m, 2H), 1.98e1.88 (m, 2H); ESI-MS: m/z ¼ 221 [MþH]þ.
mode of 23h within
b5 site of the proteasome. Overall, 23h
exhibited excellent metabolic stability with promising proteasome
inhibitory activity and vigorous cellular activity, which merits
further development as a potential anticancer agent.
4.1.2. Ethyl 5-allyl-1-methyl-1H-pyrazole-4-carboxylate 9c
Ethyl 5-bromo-1-methyl-1H-pyrazole-4-carboxylate 8b (1.23 g,
5.3 mmol), allyltributyltin (1.8 mL, 5.8 mmol), Pd2(dba)3 (240 mg,
5 mol%), PtBu3 (0.27 mL, 18 mol %), and CsF (1.61 g, 10.0 mmol) was
charged in a three-necked flask under N2. To this mixture was
added dry THF (25 mL), and the reaction mixture was heated to
reflux for 5 h. The reaction mixture was filtered off, the solvent
removed in a vacuum, and the crude product purified by SiO2
chromatography (ethyl acetate: petroleum ether ¼ 1:8) to give 9c
4. Experimental section
1H and 13C NMR spectra were recorded on Brüker 500 MHz
spectrometer (Brüker Bioscience, Billerica, MA, USA) with CDCl3 or
DMSO‑d6 as solvent. Chemical shifts (d) were reported in parts per
million (ppm) relative to internal TMS, and coupling constants (J)
were reported in Hertz (Hz). Splitting patterns were designated as
singlet (s), broad singlet (brs), doublet (d), double doublet (dd),
triplet (t), quartet (q) and multiplet (m). Melting points were
determined using a Buchi B-540 capillary melting point apparatus
and are uncorrected. Electrospray ionization mass spectroscopy
(ESI-MS) spectra were obtained with a Shimadzu LCMS-2020 mass
spectrometer with mobile phases as methanol and water contain-
ing 0.1% formic acid. HPLC analysis was performed using an Agilent
as colorless oil (0.82 g, 80%). 1H NMR (500 MHz, CDCl3)
d 7.85 (s,
1H), 5.94e5.80 (m, 1H), 5.16e5.06 (m, 1H), 5.02e4.90 (m, 1H), 4.28
(q, J ¼ 7.0 Hz, 2H), 3.85e3.73 (m, 5H),1.34 (t, J ¼ 7.0 Hz, 3H); ESI-MS:
m/z ¼ 195 [MþH]þ.
4.1.3. Ethyl 1-(but-3-en-1-yl)-1H-imidazole-2-carboxylate 9d
A
mixture of ethyl 1H-imidazole-2-carboxylate (700 mg,
1260 Series system with
a
COSMOSIL 5C18-MS-II (4.6 mm
5.0 mmol), potassium carbonate (1.38 g, 10.0 mmol) and 4-
bromobut-1-ene (800 mg, 6.0 mmol) in 5 mL DMF was heated to
80 ꢀC for 3 h. TLC analysis indicated that the reaction had ceased.
After cooling to room temperature, the solution was diluted with
EtOAc (15 mL) and washed with water (25 mL ꢂ 3), brine
(25 mL ꢂ 3). The organic layer was dried over Na2SO4, filtered, and
I.D. ꢂ 250 mm) column and detected at 254 nm wavelength. The
details of the HPLC method can be found in supplementary data.
Reagents and solvents were purchased from common commercial
suppliers and were used without further purification unless stated
otherwise. Column chromatography was performed using silica gel

J. Yu et al. / European Journal of Medicinal Chemistry 164 (2019) 423e439
431
concentrated under reduced pressure. The residue was then puri-
fied by SiO2 chromatography (ethyl acetate: petroleum ether ¼ 1:1)
to give compound 9d as colorless oil (0.93 g, 96%).1H NMR
4.1.7. 5-Allyl-1-methyl-1H-pyrazole-4-carboxylic acid 10c
This compound was prepared from 9c (970 mg, 5.0 mmol),
10 mL MeOH/THF (VMeOH:VTHF ¼ 1:1) and 10 mL 2M NaOH (aq) in a
similar manner as described for compound 10a. The product was
obtained as colorless oil yellow solid (747 mg, 90%).1H NMR
(500 MHz, CDCl3)
d
7.14 (d, J ¼ 1.0 Hz, 1H), 7.05 (d, J ¼ 1.0 Hz, 1H),
5.80e5.70 (m, 1H), 5.08e5.01 (m, 2H), 4.47 (t, J ¼ 7.0 Hz, 2H), 4.41
(q, J ¼ 7.0 Hz, 2H), 2.59e2.50 (m, 2H),1.43 (t, J ¼ 7.0 Hz, 3H); ESI-MS:
m/z ¼ 195 [MþH]þ.
(500 MHz, DMSO‑d6) d 12.26 (s, 1H), 7.74 (s, 1H), 5.93e5.78 (m, 1H),
5.09e5.05 (m, 1H), 4.98e4.92 (m, 1H), 3.78e3.75 (m, 2H), 3.75 (s,
3H); ESI-MS: m/z ¼ 165 [M-H]-.
4.1.4. Ethyl 1-(hex-5-en-1-yl)-1H-imidazole-2-carboxylate 9e
This compound was prepared from 8c (700 mg, 5.0 mmol), 6-
bromohex-1-ene (972 mg, 6 mmol) and K2CO3 (1.38 g, 10.0 mmol)
in a similar manner as described for compound 9d. The product was
obtained as colorless oil (1.04 g, 94%). 1H NMR (500 MHz, CDCl3)
4.1.8. 1-(But-3-en-1-yl)-1H-imidazole-2-carboxylic acid 10d and 1-
(hex-5-en-1-yl)-1H-imidazole-2-carboxylic acid 10e
To a solution of compound 9d (0.93 g, 4.8 mmol) or 9e (1.11 g,
5.0 mmol) in 5 mL MeOH/THF (VMeOH:VTHF ¼ 1:1) at 0ꢀС was added
5 mL 2M NaOH (aq). The mixture was stirred at room temperature
for 3 h and MeOH and THF were evaporated in vacuo. The residual
water layer was washed with DCM (5 mL ꢂ 3), acidified to
pH ¼ 3e4 with 1 N HCl and extracted with N-butanol (5 mL ꢂ 3).
The combined organic layers were evaporated in vacuo at room
temperature to afford compound 10d or 10e, the crude product was
put into next step without further purification.
d
7.16 (s, 1H), 7.07 (s, 1H), 5.77 (ddt, J ¼ 17.0, 10.0, 7.0 Hz, 1H), 5.00
(dd, J ¼ 17.0, 1.0 Hz, 1H),4.96(d,J ¼ 10, 1H) 4.43e4.39 (m, 4H), 2.09
(dt, J ¼ 7.0, 7.0 Hz, 2H), 1.85e1.79 (m, 2H), 1.50e1.34 (m, 5H); ESI-
MS: m/z ¼ 223 [MþH]þ.
4.1.5. 2-(Pent-4-en-1-yloxy)benzoic acid 10a
To a solution of compound 9a (1.10 g, 5.0 mmol) in 10 mL MeOH/
THF (VMeOH:VTHF ¼ 1:1) at 0ꢀС was added 10 mL 2M NaOH (aq). The
mixture was stirred at room temperature for 3 h and MeOH and
THF were evaporated in vacuo. The residue was acidified to
pH ¼ 2e3 with 1 N HCl and extracted with ethyl acetate
(20 mL ꢂ 3). The combined organic layers were washed with brine
(20 mL ꢂ 3), dried over Na2SO4, filtered and concentrated under
reduced pressure to afford compound 10a as colorless oil (0.71 g,
4.1.9. Benzyl O-allyl-N-(tert-butoxycarbonyl)-L-serinate 12a
A three-necked flask was charged with 11a (4.42 g, 15.0 mmol)
and Pd(PPh3)4 (0.87 g, 0.75 mmol) under N2. The allylmethyl car-
bonate (2.3 mL, 21.0 mmol) in anhydrous THF (50 mL) was syringed
over 10 min. The reaction mixture was heated to 60 ꢀC for 4 h upon
which TLC indicated the loss of starting material. The solvent was
removed under reduced pressure and the residue was diluted with
EtOAc (40 mL) and washed with NaHCO3 (40 mL ꢂ 3) and brine
(40 mL ꢂ 3). The organic layer was dried over NaSO4, filtered, and
concentrated under reduced pressure. The residue was purified by
SiO2 chromatography (ethyl acetate: petroleum ether ¼ 1:2) to
afford compound 12a as colorless oil (4.11 g, 82%). 1H NMR
68%). 1H NMR (500 MHz, DMSO‑d6)
d
12.47 (s, 1H), 7.62 (dd, J ¼ 7.5,
2.0 Hz, 1H), 7.50e7.45 (m, 1H), 7.10 (d, J ¼ 8.5 Hz, 1H), 7.00e6.95 (m,
1H), 5.92e5.81 (m, 1H), 5.08e5.01 (m, 1H), 5.01e4.94 (m, 1H), 4.03
(t, J ¼ 6.0 Hz, 2H), 2.26e2.17 (m, 2H), 1.84e1.74 (m, 2H); ESI-MS: m/
z ¼ 205 [M-H]-.
(500 MHz, CDCl3)
d 7.41e7.30 (m, 5H), 5.86e5.68 (m, 1H), 5.41 (d,
J ¼ 8.5 Hz, 1H), 5.32e5.17 (m, 2H), 5.17e5.10 (m, 2H), 4.51e4.42 (m,
1H), 3.99e3.84 (m, 3H), 3.65 (dd, J ¼ 9.5, 3.0 Hz, 1H), 1.45 (s, 9H);
ESI-MS: m/z ¼ 336 [MþH]þ.
4.1.6. 1-Methyl-5-(pent-4-en-1-yloxy)-1H-pyrazole-4-carboxylic
acid 10b
A solution of pent-4-en-1-ol (1.45 mL, 14.0 mmol) in THF
(24.0 mL) was cooled to ꢁ8 ꢀC and treated with 1.0 M sodium
hexamethyldisilazane in THF (14.0 mL, 14.0 mmol). The reaction
mixture was stirred at the reduced temperature for 5 min before
the ice bath was removed. Stirring was continued for an additional
25 min before being treated with a solution of ethyl 5-bromo-1-
methyl-1H-pyrazole-4-carboxylate (1.1 g, 4.7 mmol) in THF
(24 mL). The reaction mixture was stirred for 1 hour at room
temperature before being quenched with 50 mL of sat. NH4Cl. The
crude product was extracted with DCM (60 mL x 3), dried with
Na2SO4 and concentrated under reduced pressure. This material
purified by SiO2 chromatography (ethyl acetate: petroleum
ether ¼ 1:5) to afford 0.65 g impure product 9b mixed with pent-4-
4.1.10. Benzyl O-allyl-N-(tert-butoxycarbonyl)-L-threoninate 12b
This compound was prepared from 11b (4.63 g, 15 mmol),
Pd(PPh3)4 (0.87 g, 0.75 mmol) and allylmethyl carbonate (2.3 mL,
21.0 mmol) in a similar manner as described for compound 12a. The
product was obtained as colorless oil (4.55 g, 87%).1H NMR
(500 MHz, CDCl3)
d
7.40e7.30 (m, 5H), 5.68 (ddt, J ¼ 16.5, 10.5,
5.5 Hz, 1H), 5.31e5.05 (m, 5H), 4.32 (d, J ¼ 9.5 Hz, 1H), 4.11e4.03 (m,
1H), 3.96e3.89 (m, 1H), 3.79e3.68 (m, 1H), 1.45 (s, 9H), 1.20 (d,
J ¼ 6.0 Hz, 3H); ESI-MS: m/z ¼ 350 [MþH]þ.
4.1.11. N-(tert-butoxycarbonyl)eO-(hex-5-en-1-yl)-L-serine 15
en-1-yl
1-methyl-5-(pent-4-en-1-yloxy)-1H-pyrazole-4-
(tert-Butoxycarbonyl)- -serine 14 (2.05 g, 10.0 mmol) was dis-
L
carboxylate (produced through transesterification reaction), the
mixed esters could both be the raw materials for next hydrolysis, so
the mixture was put into next step without further separation. To a
solution of the mixed esters in 5 mL MeOH/THF (VMeOH:VTHF ¼ 1:1)
at 0ꢀС was added 5 mL 2M NaOH (aq). The mixture was stirred at
room temperature for 3 h and MeOH and THF were evaporated in
vacuo. The residue was acidified to pH ¼ 2e3 with 1 N HCl and
extracted with ethyl acetate (10 mL ꢂ 3). The combined organic
layers were washed with brine, dried over Na2SO4, filtered and
concentrated under reduced pressure to afford compound 10b as
solved in DMF (25 mL). The solution was cooled to 0 ꢀC (ice bath)
before sodium hydride (60% dispersion in mineral oil, 0.60 g,
25.0 mmol) was added slowly under stirring. After gas evolution
had ceased 6-bromohex-1-ene (1.3 mL, 10.0 mmol) was added
dropwise and the reaction mixture stirred for 18 h at room tem-
perature. The reaction mixture was cooled to 0 ꢀC and diluted with
EtOAc (50 mL) and H2O (50 mL), the organic layer was separated
and washed with H2O (50 mL ꢂ 3), and brine (50 mL ꢂ 3). Dried
over Na2SO4, filtrated, and evaporated under reduced pressure to
yield compound 15 as colorless oil (1.61 g, 56%). 1H NMR (500 MHz,
yellow solid (0.45 g, 46%).1H NMR (500 MHz, CDCl3)
d
7.84 (s, 1H),
CDCl3)
d
5.84e5.72 (m, 1H), 5.42 (d, J ¼ 8.0 Hz, 1H), 5.06e4.88 (m,
5.89e5.77 (m, 1H), 5.10e5.00 (m, 2H), 4.46 (t, J ¼ 6.5 Hz, 2H), 3.70
(s, 4H), 2.28e2.19 (m, 2H), 1.93e1.85 (m, 2H); ESI-MS: m/z ¼ 209
[M-H]-.
2H), 4.47e4.37 (m, 1H), 3.87 (dd, J ¼ 11.0, 3.0 Hz, 2H), 3.65 (dd,
J ¼ 9.5, 3.5 Hz, 1H), 3.45 (t, J ¼ 6.5 Hz, 2H), 2.08e2.02 (m, 2H),
1.60e1.52 (m, 2H), 1.49e1.38 (m, 11H); ESI-MS: m/z ¼ 288 [MþH]þ.

432
J. Yu et al. / European Journal of Medicinal Chemistry 164 (2019) 423e439
4.1.12. Benzyl N-(tert-butoxycarbonyl)eO-(hex-5-en-1-yl)-L-
serinate 16
as a white solid (5.09 g, 98%).1H NMR (500 MHz, CDCl3)
d 7.64 (d,
J ¼ 7.0 Hz,1H), 7.48e7.28 (m, 5H), 6.19 (d, J ¼ 5.0 Hz,1H), 5.99 (s,1H),
5.83e5.71 (m, 1H), 5.27e5.11 (m, 4H), 4.70 (dt, J ¼ 8.5, 3.5 Hz, 1H),
4.52 (d, J ¼ 3.5 Hz, 1H), 3.99e3.90 (m, 2H), 3.88 (dd, J ¼ 9.5, 3.5 Hz,
1H), 3.61 (dd, J ¼ 9.5, 3.5 Hz, 1H), 3.03 (d, J ¼ 6.0 Hz, 2H), 2.84 (dd,
J ¼ 14.0,2.0 Hz, 1H), 2.58 (dd, J ¼ 15.0, 6.0 Hz, 1H), 1.44 (s, 9H), 0.88
(s, 9H); ESI-MS: m/z ¼ 520 [MþH]þ.
Benzyl bromide (0.79 mL, 6.7 mmol) was slowly added to a
mixture of 15 (1.61 g, 5.6 mmol) and K2CO3 (0.92 g, 6.7 mmol) in
DMF (15 mL). The solution was stirred at room temperature for 20 h
and then diluted with EtOAc (30 mL) and H2O (30 mL). The organic
layer was separated and washed with saturated NaHCO3
(20 mL ꢂ 3) and brine (20 mL ꢂ 3), dried over Na2SO4. Filtrated, and
evaporated under vacuum to give compound 16 as colorless oil
4.1.17. Benzyl N-(N2-(tert-butoxycarbonyl)-N4-neopentyl-L-
asparaginyl)eO-(hex-5-en-1-yl)-L-serinate 19d
(1.10 g, 52%). 1H NMR (500 MHz, CDCl3)
d 7.41e7.29 (m, 5H),
5.85e5.71 (m, 1H), 5.38 (d, J ¼ 8.5 Hz, 1H), 5.28e5.09 (m, 2H),
5.05e4.89 (m, 2H), 4.52e4.39 (m, 1H), 3.84 (dd, J ¼ 9.5, 3.0 Hz, 1H),
3.63 (dd, J ¼ 9.5, 3.0 Hz, 1H), 3.45e3.27 (m, 2H), 2.07e1.99 (m, 2H),
1.55e1.33 (m, 13H); ESI-MS: m/z ¼ 378 [MþH]þ.
This compound was prepared from N2-(tert-butoxycarbonyl)-
N4-neopentyl-
L
-asparagine 18c (3.02 g, 10 mmol), HOBt (162 g,
12.0 mmol), EDCI (2.88 g, 15.0 mmol), compound 17 (2.91 g,
10.5 mmol) and diisopropylethylamine (6.61 mL) in similar
manner as described for compound 19a. The product was obtained
as a white solid (5.11 g, 91%). 1H NMR (500 MHz, CDCl3)
7.60 (d,
a
4.1.13. General procedure for compounds 13a, 13b, 17, 20a e 20e,
27 and 29
d
J ¼ 6.0 Hz, 1H), 7.41e7.29 (m, 5H), 6.16 (d,J ¼ 4.5 Hz,1H), 5.91 (s, 1H),
5.78 (ddt, J ¼ 17.0, 11.0, 6.5 Hz, 1H), 5.22 (d, J ¼ 12.5 Hz, 1H), 5.13 (d,
J ¼ 12.5 Hz, 1H), 4.99 (ddd, J ¼ 17.0, 3.5, 1.5 Hz, 1H), 4.97e4.92 (m,
1H), 4.68 (dt, J ¼ 8.0, 3.5 Hz, 1H), 4.52 (s, 1H), 3.86 (dd, J ¼ 9.5,
3.5 Hz, 1H), 3.59 (dd, J ¼ 9.5, 3.5 Hz, 1H), 3.45e3.29 (m, 2H),
3.07e2.97 (m, 2H), 2.84 (d, J ¼ 13.0 Hz,1H), 2.57 (dd, J ¼ 15.0, 6.0 Hz,
1H), 2.05e2.00 (m, 2H), 1.55e1.47 (m, 2H), 1.44 (s, 9H), 1.41e1.33
(m, 2H), 0.88 (s, 9H); ESI-MS: m/z ¼ 562 [MþH]þ.
To a solution of 12a, 12b, 16, 19a e 19e, 27 and 29 (3.0 mmol) in
6 mL ethyl acetate was added saturated hydrogen chloride in ethyl
acetate (12 mL) at 0ꢀС. After stirring at room temperature for 2 h,
the reaction mixture was concentrated. The residue was washed
with EtO2 (15 mL ꢂ 3) and evaporated under vacuum to give crude
products 13a, 13b, 17, 20a e 20e, 27 and 29, the crude products
were put into next step without further purification.
4.1.14. Benzyl O-allyl-N-(N-(tert-butoxycarbonyl)eO-methyl-L-
seryl)-L-serinate 19a
4.1.18. Benzyl O-allyl-N-(N2-(tert-butoxycarbonyl)-N4-neopentyl-L-
asparaginyl)-L-threoninate 19e
To a solution of N-(tert-butoxycarbonyl)eO-methyl-
L
-serine 18a
This compound was prepared from N2-(tert-butoxycarbonyl)-
(2.19 g, 10.0 mmol) in DCM (30 mL) were added HOBt (162 g,
12.0 mmol) and EDCI (2.88 g, 15.0 mmol) at 0ꢀС. The reaction
mixture was stirred for 30 min. Then compound 13a (2.47 mg,
10.5 mmol) and diisopropylethylamine (6.61 mL) were added. After
stirring at room temperature for another 5 h, the resulting mixture
was washed with aqueous NaHCO3 solution (30 mL ꢂ 3), brine
(30 mL ꢂ 3) and dried over Na2SO4. The organic layer was evapo-
rated in vacuo and the crude product was purified on silica gel to
afford the compound 19a as a white solid (3.61 mg, 82%).1H NMR
N4-neopentyl-
L
-asparagine 18c (3.02 g, 10 mmol), HOBt (162 g,
12.0 mmol), EDCI (2.88 g, 15.0 mmol), compound 13b (2.61 mg,
10.5 mmol) and diisopropylethylamine (6.61 mL) in similar
manner as described for compound 19a. The product was obtained
as a white solid (4.63 g, 87%).1H NMR (500 MHz, CDCl3)
7.48 (d,
a
d
J ¼ 9.5 Hz, 1H), 7.39e7.28 (m, 5H), 6.21 (s, 1H), 5.93 (s, 1H),
5.74e5.59 (m, 1H), 5.26e5.02 (m, 4H), 4.59 (dd, J ¼ 9.0, 2.5 Hz, 1H),
4.57e4.50 (m, 1H), 4.13e4.02 (m, 1H), 3.98e3.89 (m, 1H), 3.81e3.70
(m, 1H), 3.01 (d, J ¼ 6.0 Hz, 2H), 2.87 (d, J ¼ 13.5 Hz, 1H), 2.60 (dd,
J ¼ 15.0, 6.0 Hz, 1H), 1.44 (s, 9H), 1.13 (d, J ¼ 6.3 Hz, 3H), 0.88 (s, 9H);
ESI-MS: m/z ¼ 534 [MþH]þ.
(500 MHz, CDCl3)
d
7.39 (d, J ¼ 6.5 Hz, 1H), 7.36e7.29 (m, 5H),
5.80e5.70 (m, 1H), 5.43 (d, J ¼ 5.0 Hz, 1H), 5.26e5.21 (m, 1H),
5.21e5.16 (m, 1H), 5.16e5.11 (m, 2H), 4.74 (dt, J ¼ 8.0, 3.0 Hz, 1H),
4.31e4.24 (m, 1H), 3.94e3.87 (m, 3H), 3.77 (dd, J ¼ 9.0, 4.0 Hz, 1H),
3.65 (dd, J ¼ 9.5, 3.0 Hz, 1H), 3.43 (dd, J ¼ 9.0, 7.0 Hz, 1H), 3.33 (s,
3H), 1.44 (s, 9H); ESI-MS: m/z ¼ 437 [MþH]þ.
4.1.19. Benzyl O-allyl-N-(O-methyl-N-(2-(pent-4-en-1-yloxy)
benzoyl)-L-seryl)-L-serinate 21a
This compound was prepared from 10a (206 mg, 1.0 mmol),
HOBt (162 mg, 1.2 mmol), EDCI (288 mg, 1.5 mmol), 20a (390 mg,
4.1.15. Benzyl O-allyl-N-((S)-2-((tert-butoxycarbonyl)amino)-4-
phenylbutanoyl)-L-serinate 19b
This compound was prepared from (S)-2-((tert-butoxycarbonyl)
amino)-4-phenylbutanoic acid 18b (2.79 g, 10 mmol), HOBt (162 g,
12.0 mmol), EDCI (2.88 g, 15.0 mmol), compound 13a (2.47 mg,
1.05 mmol) and diisopropylethylamine (0.66 mL) in
manner as described for compound 19a. The product was obtained
as a white solid (445 mg, 85%). 1H NMR (500 MHz, CDCl3)
8.85 (d,
J ¼ 6.5 Hz, 1H), 8.21 (d, J ¼ 8.0 Hz, 1H), 7.48e7.42 (m, 1H), 7.40 (d,
J ¼ 8.0 Hz, 1H), 7.36e7.29 (m, 5H), 7.09e7.03 (m, 1H), 6.96 (d,
J ¼ 8.0 Hz, 1H), 5.89e5.79 (m, 1H), 5.79e5.69 (m, 1H), 5.27e4.98 (m,
6H), 4.88e4.82 (m, 1H), 4.81e4.77 (m, 1H), 4.16e4.09 (m, 2H),
3.97e3.87 (m, 4H), 3.70e3.65 (m, 1H), 3.55e3.49 (m, 1H), 3.38 (s,
3H), 2.30e2.22 (m, 2H), 2.11e1.97 (m, 2H); ESI-MS: m/z ¼ 525
[MþH]þ.
a similar
d
10.5 mmol) and diisopropylethylamine (6.61 mL) in
manner as described for compound 19a. The product was obtained
as a white solid (4.31 g, 87%).1H NMR (500 MHz, CDCl3)
7.36e7.30
a similar
d
(m, 5H), 7.29e7.24 (m, 2H), 7.21e7.15 (m, 3H), 6.71 (d, J ¼ 7.5 Hz,
1H), 5.82e5.69 (m, 1H), 5.29e5.06 (m, 5H), 4.81e4.71 (m, 1H),
4.23e4.11 (m, 1H), 3.98e3.82 (m, 3H), 3.63 (dd, J ¼ 9.5, 3.0 Hz, 1H),
2.70 (t, J ¼ 8.0 Hz, 2H), 2.19e2.08 (m, 1H), 1.99e1.88 (m, 1H), 1.44 (s,
9H); ESI-MS: m/z ¼ 497 [MþH]þ.
4.1.20. Benzyl O-allyl-N-((S)-2-(2-(pent-4-en-1-yloxy)benzamido)-
4-phenylbutanoyl)-L-serinate 21b
This compound was prepared from 10a (206 mg, 1.0 mmol),
HOBt (162 mg, 1.2 mmol), EDCI (288 mg, 1.5 mmol), 20b (454 mg,
4.1.16. Benzyl O-allyl-N-(N2-(tert-butoxycarbonyl)-N4-neopentyl-L-
asparaginyl)-L-serinate 19c
1.05 mmol) and diisopropylethylamine (0.66 mL) in
manner as described for compound 19a. The product was obtained
as a white solid (461 mg, 79%). 1H NMR (500 MHz, CDCl3)
8.63 (d,
a similar
This compound was prepared from N2-(tert-butoxycarbonyl)-
N4-neopentyl-
L
-asparagine 18c (3.02 g, 10 mmol), HOBt (162 g,
12.0 mmol), EDCI (2.88 g, 15.0 mmol), compound 13a (2.47 mg,
10.5 mmol) and diisopropylethylamine (6.61 mL) in similar
manner as described for compound 19a. The product was obtained
d
J ¼ 7.5 Hz, 1H), 8.21 (dd, J ¼ 8.0, 2.0 Hz, 1H), 7.47e7.41 (m, 1H),
7.35e7.30 (m, 5H), 7.28e7.23 (m, 2H), 7.21e7.15 (m, 3H), 7.09e7.04
(m, 1H), 6.97 (d, J ¼ 8.0 Hz, 1H), 6.85 (d, J ¼ 8.5 Hz, 1H), 5.88e5.78
a

J. Yu et al. / European Journal of Medicinal Chemistry 164 (2019) 423e439
433
(m, 1H), 5.79e5.67 (m, 1H), 5.27 (d, J ¼ 12.0 Hz, 1H), 5.21e5.13 (m,
2H), 5.13e5.09 (m, 1H), 5.09e5.04 (m, 1H), 5.03e4.99 (m, 1H),
4.83e4.72 (m, 2H), 4.18e4.10 (m, 2H), 3.96e3.85 (m, 3H), 3.62 (dd,
J ¼ 9.5, 3.0 Hz,1H), 2.80e2.74 (m, 2H), 2.34e2.25 (m, 3H), 2.13e2.01
(m, 3H); ESI-MS: m/z ¼ 607 [MþNa]þ.
3.57e3.52 (m, 1H), 3.05e3.00 (m, 2H), 2.80 (dd, J ¼ 15.0, 4.5 Hz, 1H),
2.62 (dd, J ¼ 15.0, 6.5 Hz, 1H), 2.06e1.95 (m, 2H), 1.78e1.68 (m, 2H),
1.21e1.17 (m, 2H), 0.80 (s, 9H); ESI-MS: m/z ¼ 596 [MþH]þ.
4.1.25. Benzyl O-allyl-N-(N2-(1-(hex-5-en-1-yl)-1H-imidazole-2-
carbonyl)-N4-neopentyl-L-asparaginyl)-L-threoninate 21g
This compound was prepared from 10e (194 mg, 1.0 mmol),
HOBt (162 mg, 1.2 mmol), EDCI (288 mg, 1.5 mmol), 20e (492 mg,
4.1.21. Benzyl O-allyl-N-(N4-neopentyl-N2-(2-(pent-4-en-1-yloxy)
benzoyl)-L-asparaginyl)-L-serinate 21c
This compound was prepared from 10a (206 mg, 1.0 mmol),
HOBt (162 mg, 1.2 mmol), EDCI (288 mg, 1.5 mmol), 20c (478 mg,
1.05 mmol) and diisopropylethylamine (0.66 mL) in
manner as described for compound 19a. The product was obtained
as a white solid (396 mg, 65%). 1H NMR (500 MHz, CDCl3)
8.78 (d,
a similar
1.05 mmol) and diisopropylethylamine (0.66 mL) in
manner as described for compound 19a. The product was obtained
as a white solid (461 mg, 76%). 1H NMR (500 MHz, CDCl3)
9.21 (d,
a
similar
d
J ¼ 8.0 Hz, 1H), 7.55 (d, J ¼ 9.0 Hz, 1H), 7.39e7.31 (m, 5H), 7.06 (d,
J ¼ 1.0 Hz, 1H), 7.01 (d, J ¼ 1.0 Hz, 1H), 5.99 (s, 1H), 5.83e5.73 (m,
1H), 5.71e5.64 (m, 1H), 5.23 (d, J ¼ 12.5 Hz, 1H), 5.17e5.10 (m, 2H),
5.06e4.94 (m, 4H), 4.64 (dd, J ¼ 9.0, 2.5 Hz, 1H), 4.51e4.39 (m, 2H),
4.09 (qd, J ¼ 6.5, 2.5 Hz, 1H), 3.95e3.91 (m, 1H), 3.76 (ddt, J ¼ 13.0,
5.5, 1.5 Hz, 1H), 3.12e3.02 (m, 2H), 2.91 (dd, J ¼ 15.0, 4.5 Hz, 1H),
2.72 (dd, J ¼ 15.0, 6.5 Hz, 1H), 2.12e2.06 (m, 2H), 1.87e1.77 (m, 2H),
1.47e1.38 (m, 2H), 1.14 (d, J ¼ 6.5 Hz, 3H), 0.89 (s, 9H); ESI-MS: m/
z ¼ 610 [MþH]þ.
d
J ¼ 6.5 Hz, 1H), 8.16 (dd, J ¼ 8.0, 1.5 Hz, 1H), 8.10e8.01 (m, 1H),
7.46e7.41 (m, 1H), 7.38e7.28 (m, 5H), 7.06e7.02 (m, 1H), 6.96 (d,
J ¼ 8.5 Hz, 1H), 6.11e6.02 (m, 1H), 5.90e5.80 (m, 1H), 5.79e5.69 (m,
1H), 5.24 (d, J ¼ 12.5 Hz, 1H), 5.21e5.15 (m, 1H), 5.13 (d, J ¼ 12.5 Hz,
1H), 5.10e4.96 (m, 4H), 4.78e4.70 (m, 1H), 4.15 (t, J ¼ 6.5 Hz, 2H),
3.97e3.85 (m, 3H), 3.64 (dd, J ¼ 9.5, 3.5 Hz, 1H), 3.05 (d, J ¼ 6.5 Hz,
2H), 2.92 (dd, J ¼ 15.0, 3.5 Hz, 1H), 2.67 (dd, J ¼ 15.0, 7.0 Hz, 1H),
2.32e2.17 (m, 2H), 2.16e1.98 (m, 2H), 0.89 (s, 9H); ESI-MS: m/
z ¼ 630 [MþNa]þ.
4.1.26. (10S,13S)-13-(Methoxymethyl)-12,15-dioxo-
2,3,4,5,6,7,10,11,12,13,14,15-dodecahydro-9H-benzo[i][1,11]dioxa
[4,7]diazacycloheptadecine-10-carboxylic acid 22a
4.1.22. Benzyl O-allyl-N-(N2-(1-methyl-5-(pent-4-en-1-yloxy)-1H-
pyrazole-4-carbonyl)-N4-neopentyl-L-asparaginyl)-L-serinate 21d
This compound was prepared from 10b (210 mg, 1.0 mmol),
HOBt (162 mg, 1.2 mmol), EDCI (288 mg, 1.5 mmol), 20c (478 mg,
A solution of 21a (105 mg, 0.20 mmol) in toluene (200 mL) was
degassed with dry nitrogen for 15 min. The mixture was stirred for
5 min at 100 ꢀC. after which, a degassed solution of grubbs second-
generation catalyst (17 mg, 0.02 mmol) in toluene (20 mL) was
injected with a syringe for 30 min. The reaction was stirred for half
an additional hour. After cooling to room temperature, the solvent
was evaporated under reduced pressure, and purified by flash
chromatography on silica gel. The product (a mixture of geometric
isomers) was redissolved in methanol (5 mL), stirred 3 h with 10%
Pd/C (10 mol %) under H2 atmosphere at room temperature. The
mixture was then filtered over Celite, concentrated to provide 22a
1.05 mmol) and diisopropylethylamine (0.66 mL) in
manner as described for compound 19a. The product was obtained
as
white solid (477 mg, 78%). 1H NMR (500 MHz, CDCl3)
7.99e7.85 (m, 2H), 7.74 (s, 1H), 7.39e7.29 (m, 5H), 6.03 (s, 1H),
a similar
a
d
5.88e5.70 (m, 2H), 5.27e4.99 (m, 6H), 4.92 (s, 1H), 4.75e4.67 (m,
1H), 4.34e4.20 (m, 2H), 3.97e3.85 (m, 3H), 3.70 (s, 3H), 3.63 (dd,
J ¼ 9.5, 3.0 Hz, 1H), 3.05 (d, J ¼ 6.5 Hz, 2H), 2.89 (dd, J ¼ 15.0,3.0 Hz,
1H), 2.61 (dd, J ¼ 15.0, 6.5 Hz, 1H), 2.24 (dd, J ¼ 14.0, 7.5 Hz, 2H),
2.02e1.90 (m, 2H), 0.89 (s, 9H); ESI-MS: m/z ¼ 612 [MþH]þ.
as white solid (62 mg, 76%). 1H NMR (500 MHz, CDCl3)
d 9.00 (d,
J ¼ 8.0 Hz, 1H), 8.18 (d, J ¼ 7.0 Hz, 1H), 7.47e7.38 (m, 1H), 7.23 (d,
J ¼ 6.5 Hz, 1H), 7.06e6.99 (m, 1H), 6.94 (d, J ¼ 8.5 Hz, 1H), 4.92e4.82
(m, 1H), 4.65e4.55 (m, 1H), 4.23e4.16 (m, 1H), 4.14e4.05 (m, 2H),
3.80e3.67 (m, 2H), 3.56e3.47 (m, 2H), 3.45e3.40 (m, 1H), 3.36 (s,
3H), 1.98e1.88 (m, 1H), 1.82e1.69 (m, 2H), 1.63e1.36 (m, 6H); ESI-
MS: m/z ¼ 407 [M-H]e.
4.1.23. Benzyl N-(N2-(5-allyl-1-methyl-1H-pyrazole-4-carbonyl)-
N4-neopentyl-L-asparaginyl)eO-(hex-5-en-1-yl)-L-serinate 21e
This compound was prepared from 10c (166 mg, 1.0 mmol),
HOBt (162 mg, 1.2 mmol), EDCI (288 mg, 1.5 mmol), 20d (522 mg,
1.05 mmol) and diisopropylethylamine (0.66 mL) in
manner as described for compound 19a. The product was obtained
as a white solid (438 mg, 72%). 1H NMR (500 MHz, CDCl3)
7.82 (d,
a similar
d
4.1.27. (10S,13S)-12,15-Dioxo-13-phenethyl-
J ¼ 8.0 Hz, 1H), 7.73 (d, J ¼ 5.5 Hz, 1H), 7.68 (s, 1H), 7.27e7.23 (m,
5H), 6.12 (s, 1H), 5.85e5.77 (m, 1H), 5.75e5.61 (m, 1H), 5.15 (d,
J ¼ 12.0 Hz, 1H), 5.06e5.02 (m, 2H), 4.94e4.85 (m, 4H), 4.62e4.60
(m, 1H), 3.79 (dd, J ¼ 9.5, 3.5 Hz, 1H), 3.77e3.64 (m, 5H), 3.53 (dd,
J ¼ 9.5, 3.5 Hz, 1H), 3.36e3.22 (m, 2H), 2.97 (d, J ¼ 6.5 Hz, 2H), 2.82
(dd, J ¼ 15.0, 3.5 Hz,1H), 2.53 (dd, J ¼ 15.0, 6.5 Hz,1H), 1.95e1.89 (m,
2H), 1.46e1.35 (m, 2H), 1.27e1.23 (m, 2H), 0.82 (s, 9H); ESI-MS: m/
z ¼ 610 [MþH]þ.
2,3,4,5,6,7,10,11,12,13,14,15-dodecahydro-9H-benzo[i][1,11]dioxa
[4,7]diazacycloheptadecine-10-carboxylic acid 22b
This compound was prepared from 21b (117 mg, 0.2 mmol) in a
similar manner as described for compound 21a. The product was
obtained as a white solid (64 mg, 69%). 1H NMR (500 MHz,
DMSO‑d6)
d
13.04 (brs, 1H), 8.46 (d, J ¼ 8.0 Hz, 1H), 8.12 (d,
J ¼ 8.5 Hz, 1H), 7.90 (d, J ¼ 8.0 Hz, 1H), 7.54e7.48 (m, 1H), 7.26 (t,
J ¼ 7.5 Hz, 2H), 7.21e7.13 (m, 4H), 7.07 (t, J ¼ 7.5 Hz, 1H), 4.68e4.58
(m, 2H), 4.25e4.17 (m, 1H), 4.16e4.05 (m, 1H), 3.74e3.60 (m, 2H),
3.47e3.36 (m, 2H), 2.71e2.55 (m, 2H), 2.17e2.05 (m, 1H), 1.96 (m,
1H), 1.87e1.68 (m, 2H), 1.69e1.50 (m, 2H), 1.50e1.30 (m, 4H); ESI-
MS: m/z ¼ 467 [M-H]e.
4.1.24. Benzyl O-allyl-N-(N2-(1-(hex-5-en-1-yl)-1H-imidazole-2-
carbonyl)-N4-neopentyl-L-asparaginyl)-L-serinate 21f
This compound was prepared from 10e (194 mg, 1.0 mmol),
HOBt (162 mg, 1.2 mmol), EDCI (288 mg, 1.5 mmol), 20c (478 mg,
1.05 mmol) and diisopropylethylamine (0.66 mL) in
manner as described for compound 19a. The product was obtained
as a white solid (410 mg, 69%). 1H NMR (500 MHz, CDCl3)
8.65 (d,
a
similar
4.1.28. (10S,13S)-13-(2-(Neopentylamino)-2-oxoethyl)-12,15-
dioxo-2,3,4,5,6,7,10,11,12,13,14,15-dodecahydro-9H-benzo[i][1,11]
dioxa[4,7]diazacycloheptadecine-10-carboxylic acid 22c
d
J ¼ 7.5 Hz, 1H), 7.57 (d, J ¼ 8.0 Hz, 1H), 7.26 (m, 5H),6.97 (d,
J ¼ 1.0 Hz, 1H), 6.92 (d, J ¼ 1.0 Hz, 1H), 5.91 (s, 1H), 5.76e5.61 (m,
2H), 5.30e7.21 (d, J ¼ 12.0 Hz, 1H), 5.12e5.06 (m, 1H), 5.05 (d,
J ¼ 12.0 Hz, 1H), 5.03e4.99 (m, 1H), 4.98e4.93 (m, 2H), 4.68e4.62
(m, 1H), 4.43e4.30 (m, 2H),4.09e4.00 (m, 1H), 3.86e3.78 (m, 3H),
This compound was prepared from 21c (121 mg, 0.2 mmol) in a
similar manner as described for compound 21a. The product was
obtained as a white solid (71 mg, 73%). 1H NMR (500 MHz,
DMSO‑d6)
d
12.93 (brs, 1H), 9.15 (d, J ¼ 8.5 Hz, 1H), 8.03 (dd, J ¼ 8.0,
2.0 Hz, 1H), 7.91 (t, J ¼ 6.0 Hz, 1H), 7.56e7.44 (m, 2H), 7.16 (d,

434
J. Yu et al. / European Journal of Medicinal Chemistry 164 (2019) 423e439
J ¼ 8.5 Hz, 1H), 7.06 (t, J ¼ 7.5 Hz, 1H), 4.99e4.90 (m, 1H), 4.52e4.42
(m, 1H), 4.22e4.07 (m, 2H), 3.68 (dd, J ¼ 10.0, 6.0 Hz, 1H), 3.57 (dd,
J ¼ 10.0, 3.0 Hz, 1H), 3.46e3.32 (m, 2H), 2.94 (dd, J ¼ 13.0, 7.0 Hz,
1H), 2.87 (dd, J ¼ 15.5, 5.5 Hz, 1H), 2.79 (dd, J ¼ 13.0, 6.0 Hz, 1H),
2.54 (dd, J ¼ 15.5, 4.5 Hz, 1H), 2.22e2.09 (m, 1H), 1.88e1.76 (m, 1H),
1.72e1.54 (m, 2H), 1.53e1.39 (m, 3H), 1.38e1.28 (m, 1H), 0.78 (s,
9H); ESI-MS: m/z ¼ 490 [M-H]e.
MS: m/z ¼ 492 [M-H]e.
4.1.33. (10S,13S)-N-(2-Chlorobenzyl)-13-(methoxymethyl)-12,15-
dioxo-2,3,4,5,6,7,10,11,12,13,14,15-dodecahydro-9H-benzo[i][1,11]
dioxa[4,7]diazacycloheptadecine-10-carboxamide 23a
This compound was prepared from 22a (40.8 mg, 0.1 mmol),
HOBt (16.2 mg, 0.12 mmol), EDCI (28.8 mg, 0.15 mmol), (2-
chlorophenyl)methanamine (14.8 mg, 0.105 mmol) and diisopro-
4.1.29. (6S,9S)-1-Methyl-6-(2-(neopentylamino)-2-oxoethyl)-4,7-
dioxo-1,4,5,6,7,8,9,10,12,13,14,15,16,17-tetradecahydropyrazolo[4,3-
i][1,11]dioxa[4,7]diazacycloheptadecine-9-carboxylic acid 22d
This compound was prepared from 21d (112 mg, 0.2 mmol) in a
similar manner as described for compound 21a. The product was
obtained as a white solid (60 mg, 61%). 1H NMR (500 MHz,
pylethylamine (66 mL) in a similar manner as described for com-
pound 19a. The product was obtained as a white solid (43.5 mg,
82%). Mp: 141.8e147.7 ꢀC. HPLC purity ¼ 100.00%, HPLC
tR ¼ 9.76 min; 1H NMR (500 MHz, CDCl3)
d
8.82 (d, J ¼ 6.0 Hz, 1H),
7.95 (dd, J ¼ 7.5, 2.0 Hz, 1H), 7.49e7.41 (m, 1H), 7.30e6.93 (m, 8H),
4.77e4.70 (m, 1H), 4.63e4.58 (m, 1H), 4.50 (d, J ¼ 6.0 Hz, 2H),
4.34e4.26 (m, 3H), 4.01 (dd, J ¼ 9.0, 2.0 Hz, 1H), 3.73e3.67 (m, 1H),
3.64 (dd, J ¼ 9.5, 3.5 Hz, 1H), 3.42 (s, 3H), 3.38 (dd, J ¼ 8.5, 3.5 Hz,
1H), 3.28e3.19 (m, 1H), 1.93e1.43 (m, 8H); 13C NMR (125 MHz,
DMSO‑d6)
d
12.87 (brs, 1H), 8.21 (d, J ¼ 7.5 Hz, 1H), 7.91 (d,
J ¼ 8.0 Hz, 1H), 7.75 (t, J ¼ 6.5 Hz, 1H), 7.69 (s, 1H), 4.73 (td, J ¼ 8.5,
5.0 Hz, 1H), 4.56e4.51 (m, 1H), 4.44e4.37 (m, 1H), 4.27e4.18 (m,
1H), 3.71e3.55 (m, 5H), 3.47e3.37 (m, 2H), 2.96 (dd, J ¼ 13.0, 6.5 Hz,
1H), 2.79 (dd, J ¼ 13.0, 5.5 Hz, 1H), 2.65 (dd, J ¼ 15.0, 9.0 Hz, 1H),
2.57 (dd, J ¼ 15.0, 5.0 Hz, 1H), 1.72 (m, 2H), 1.54e1.27 (m, 6H), 0.81
(s, 9H); ESI-MS: m/z ¼ 494 [M-H]e.
CDCl3) d 170.65,169.60,166.70,157.25,135.50,133.57,133.17,132.43,
129.17, 128.89, 128.19, 126.79, 121.14, 120.09, 111.79, 71.31, 70.68,
68.94, 68.70, 59.18, 55.59, 52.66, 41.30, 29.07, 28.40, 27.04, 26.88;
ESI-MS: m/z ¼ 554 [MþNa]þ.
4.1.30. (6S,9S)-1-Methyl-6-(2-(neopentylamino)-2-oxoethyl)-4,7-
dioxo-4,5,6,7,8,9,10,12,13,14,15,16,17,18-tetradecahydro-1H-
pyrazolo[4,3-i][1]oxa[4,7]diazacycloheptadecine-9-carboxylic acid
22e
This compound was prepared from 21e (121 mg, 0.2 mmol) in a
similar manner as described for compound 21a. The product was
obtained as a white solid (67 mg, 68%). 1H NMR (500 MHz,
4.1.34. (10S,13S)-N-(2-Chlorobenzyl)-12,15-dioxo-13-phenethyl-
2,3,4,5,6,7,10,11,12,13,14,15-dodecahydro-9H-benzo[i][1,11]dioxa
[4,7]diazacycloheptadecine-10-carboxamide 23b
This compound was prepared from 22b (46.8 mg, 0.1 mmol),
HOBt (16.2 mg, 0.12 mmol), EDCI (28.8 mg, 0.15 mmol), (2-
chlorophenyl)methanamine (14.8 mg, 0.105 mmol) and diisopro-
pylethylamine (66 mL) in a similar manner as described for com-
DMSO‑d6)
d
12.70 (s, 1H), 8.15 (d, J ¼ 8.0 Hz, 1H), 7.91 (d, J ¼ 8.0 Hz,
pound 19a. The product was obtained as a white solid (47 mg, 79%).
1H), 7.81 (s, 1H), 7.67 (t, J ¼ 6.0 Hz, 1H), 4.80e4.72 (m, 1H),
4.43e4.35 (m, 1H), 3.74 (s, 3H), 3.66 (dd, J ¼ 10.0, 5.0 Hz, 1H), 3.59
(dd, J ¼ 10.0, 3.5 Hz, 1H), 3.42e3.34 (m, 1H), 3.33e3.29 (m, 1H), 3.17
(d, J ¼ 3.5 Hz, 2H), 2.95 (dd, J ¼ 13.0, 6.5 Hz, 1H), 2.79 (dd, J ¼ 13.0,
5.5 Hz, 1H), 2.61 (d, J ¼ 7.5 Hz, 2H), 1.65e1.54 (m, 1H), 1.52e1.43 (m,
1H), 1.43e1.20 (m, 8H), 0.80 (s, 9H); ESI-MS: m/z ¼ 492 [M-H]e.
Mp: 154.3e158.4 ꢀC. HPLC purity ¼ 96.33%, HPLC tR ¼ 11.79 min; 1H
NMR (500 MHz, CDCl3)
d
8.26 (d, J ¼ 5.0 Hz, 1H), 7.97 (dd, J ¼ 7.5,
1.5 Hz, 1H), 7.51e7.44 (m, 1H), 7.32 (t, J ¼ 6.0 Hz, 1H), 7.25e7.22 (m,
4H), 7.20e7.14 (m, 3H), 7.14e7.06 (m, 2H), 7.03 (t, J ¼ 7.5 Hz, 1H),
6.96 (t, J ¼ 7.5 Hz, 2H), 4.72e4.67 (m, 1H), 4.55e4.47 (m, 3H),
4.32e4.25 (m, 1H), 4.21e4.13 (m, 1H), 3.95 (dd, J ¼ 9.5, 4.5 Hz, 1H),
3.60 (dd, J ¼ 9.5, 4.0 Hz, 1H), 3.54e3.47 (m, 1H), 3.43e3.36 (m, 1H),
2.86e2.73 (m, 2H), 2.44e2.34 (m, 1H), 2.11e2.00 (m, 1H), 1.97e1.78
4.1.31. (14S,17S)-17-(2-(Neopentylamino)-2-oxoethyl)-16,19-dioxo-
6,7,8,9,10,11,14,15,16,17,18,19-dodecahydro-5H,13H-imidazo[2,1-i]
[1]oxa[4,7,10]triazacycloheptadecine-14-carboxylic acid 22f
This compound was prepared from 21f (119 mg, 0.2 mmol) in a
similar manner as described for compound 21a. The product was
obtained as a white solid (52 mg, 55%). 1H NMR (500 MHz,
(m, 2H), 1.68e1.42 (m, 6H); 13C NMR (125 MHz, CDCl3)
d 171.59,
169.60, 166.94, 156.91, 140.39, 135.51, 133.60, 133.17, 132.44, 129.18,
128.94, 128.64, 128.37, 128.21, 126.80, 126.37, 121.32, 120.15, 112.01,
70.95, 69.09, 69.03, 55.62, 52.49, 41.33, 33.62, 32.69, 29.30, 28.62,
27.49, 27.28; ESI-MS: m/z ¼ 614 [MþNa]þ.
DMSO‑d6)
d
8.68 (d, J ¼ 8.0 Hz, 1H), 7.85 (t, J ¼ 6.5 Hz, 1H), 7.68 (d,
J ¼ 8.0 Hz, 1H), 7.39 (d, J ¼ 1.0 Hz, 1H), 6.99 (d, J ¼ 1.0 Hz, 1H),
4.82e4.72 (m, 2H), 4.39e4.32 (m, 1H), 4.07e3.98 (m, 1H), 3.66 (dd,
J ¼ 10.0, 5.0 Hz, 1H), 3.61 (dd, J ¼ 10.0, 3.5 Hz, 1H), 3.36e3.28 (m,
2H), 2.91 (dd, J ¼ 13.0, 6.5 Hz, 1H), 2.84 (dd, J ¼ 13.0, 6.0 Hz, 1H),
2.75 (dd, J ¼ 15.0, 8.5 Hz, 1H), 2.58 (dd, J ¼ 15.0, 4.5 Hz, 1H),
1.81e1.70 (m, 1H), 1.70e1.61 (m, 1H), 1.36e1.21 (m, 6H), 1.20e1.06
(m, 2H), 0.81 (s, 9H); ESI-MS: m/z ¼ 478 [M-H]e.
4.1.35. (10S,13S)-N-(4-Methylbenzyl)-12,15-dioxo-13-phenethyl-
2,3,4,5,6,7,10,11,12,13,14,15-dodecahydro-9H-benzo[i][1,11]dioxa
[4,7]diazacycloheptadecine-10-carboxamide 23c
This compound was prepared from 22b (46.8 mg, 0.1 mmol),
HOBt (16.2 mg, 0.12 mmol), EDCI (28.8 mg, 0.15 mmol), p-tolylme-
thanamine (12.7 mg, 0.105 mmol) and diisopropylethylamine
(66 mL) in a similar manner as described for compound 19a. The
product was obtained as
a white solid (48 mg, 85%). Mp:
4.1.32. (13R,14S,17S)-13-Methyl-17-(2-(neopentylamino)-2-
oxoethyl)-16,19-dioxo-6,7,8,9,10,11,14,15,16,17,18,19-dodecahydro-
5H,13H-imidazo[2,1-i][1]oxa[4,7,10]triazacycloheptadecine-14-
carboxylic acid 22g
This compound was prepared from 21g (121 mg, 0.2 mmol) in a
similar manner as described for compound 21a. The product was
obtained as a white solid (66 mg, 67%). 1H NMR (500 MHz,
208.1e208.7 ꢀC. HPLC purity ¼ 99.73%, HPLC tR ¼ 11.80 min; 1H
NMR (500 MHz, CDCl3)
d
8.26 (d, J ¼ 5.0 Hz, 1H), 7.96 (d, J ¼ 7.5 Hz,
1H), 7.48 (t, J ¼ 7.5 Hz, 1H), 7.24 (d, J ¼ 7.5 Hz, 2H), 7.21e7.14 (m, 4H),
7.10 (d, J ¼ 7.5 Hz, 2H), 7.06e6.94 (m, 4H), 6.90 (d, J ¼ 8.5 Hz, 1H),
4.71e4.65 (m, 1H), 4.54e4.48 (m, 1H), 4.41 (dd, J ¼ 15.0, 6.0 Hz, 1H),
4.35e4.25 (m, 2H), 4.19e4.10 (m, 1H), 3.96 (dd, J ¼ 9.5, 4.5 Hz, 1H),
3.58 (dd, J ¼ 9.5, 4.0 Hz, 1H), 3.54e3.47 (m, 1H), 3.42e3.35 (m, 1H),
2.85e2.71 (m, 2H), 2.43e2.32 (m, 1H), 2.28 (s, 3H), 2.09e1.98 (m,
1H), 1.95e1.77 (m, 2H), 1.68e1.41 (m, 6H); 13C NMR (125 MHz,
DMSO‑d6)
d
9.08 (d, J ¼ 8.0 Hz, 1H), 7.89 (t, J ¼ 6.0 Hz, 1H), 7.42 (s,
1H), 7.20 (d, J ¼ 9.0 Hz, 1H), 7.00 (s, 1H), 4.87e4.71 (m, 2H), 4.14 (dd,
J ¼ 9.0, 1.5 Hz, 1H), 4.08e3.97 (m, 1H), 3.98e3.87 (m, 1H), 3.43e3.33
(m, 1H), 3.19e3.10 (m, 1H), 2.91 (dd, J ¼ 13.0, 6.5 Hz, 1H), 2.88e2.75
(m, 2H), 2.60 (dd, J ¼ 15.5, 4.5 Hz, 1H), 1.86e1.73 (m, 1H), 1.73e1.57
(m, 1H), 1.34e1.06 (m, 8H), 1.00 (d, J ¼ 6.5 Hz, 3H), 0.79 (s, 9H); ESI-
CDCl3) d 171.47, 169.25, 166.84, 156.94, 140.42, 136.41, 135.17, 133.52,
132.47, 129.03, 128.63, 128.37, 127.51, 126.34, 121.27, 120.12, 111.98,
70.95, 69.19, 69.06, 55.52, 52.36, 43.23, 33.61, 32.68, 29.31, 28.68,
27.48, 27.32, 21.12; ESI-MS: m/z ¼ 594 [MþNa]þ.

J. Yu et al. / European Journal of Medicinal Chemistry 164 (2019) 423e439
435
4.1.36. (10S,13S)-N-Benzyl-13-(2-(neopentylamino)-2-oxoethyl)-
12,15-dioxo-2,3,4,5,6,7,10,11,12,13,14,15-dodecahydro-9H-benzo[i]
[1,11]dioxa[4,7]diazacycloheptadecine-10-carboxamide 23d
36.77, 31.89, 28.70, 28.43, 27.38, 27.25, 27.21, 21.08; ESI-MS: m/
z ¼ 617 [MþNa]þ.
This compound was prepared from 22c (49.1 mg, 0.1 mmol),
HOBt (16.2 mg, 0.12 mmol), EDCI (28.8 mg, 0.15 mmol), phenyl-
methanamine (10.7 mg, 0.105 mmol) and diisopropylethylamine
4.1.39. (6S,9S)-1-Methyl-N-(4-methylbenzyl)-6-(2-
(neopentylamino)-2-oxoethyl)-4,7-dioxo-
1,4,5,6,7,8,9,10,12,13,14,15,16,17-tetradecahydropyrazolo[4,3-i][1,11]
dioxa[4,7]diazacycloheptadecine-9-carboxamide 23g
(66 mL) in a similar manner as described for compound 19a. The
product was obtained as
a
white solid (42 mg, 73%). Mp:
This compound was prepared from 22d (49.5 mg, 0.1 mmol),
HOBt (16.2 mg, 0.12 mmol), EDCI (28.8 mg, 0.15 mmol), p-tolylme-
thanamine (12.7 mg, 0.105 mmol) and diisopropylethylamine
171.9e173.0 ꢀC. HPLC purity ¼ 99.80%, HPLC tR ¼ 10.14 min; 1H NMR
(500 MHz, CDCl3)
d
9.50 (d, J ¼ 6.5 Hz, 1H), 7.98 (dd, J ¼ 8.0, 2.0 Hz,
1H), 7.49e7.39 (m, 1H), 7.31 (t, J ¼ 6.0 Hz, 1H), 7.24e7.14 (m, 5H),
7.09 (d, J ¼ 8.5 Hz, 1H), 7.02e6.94 (m, 2H), 5.94 (s, 1H), 4.87e4.82
(m, 1H), 4.69e4.62 (m, 1H), 4.47 (dd, J ¼ 15.0, 6.5 Hz, 1H), 4.43e4.35
(m, 2H), 4.27e4.21 (m, 1H), 3.89 (dd, J ¼ 9.5, 4.5 Hz, 1H), 3.59 (dd,
J ¼ 9.5, 4.0 Hz, 1H), 3.54e3.46 (m, 1H), 3.43e3.35 (m, 1H),
3.08e2.93 (m, 3H), 2.65 (dd, J ¼ 15.0, 5.0 Hz, 1H), 2.15e2.05 (m, 1H),
1.90e1.80 (m, 1H), 1.67e1.56 (m, 2H), 1.51e1.39 (m, 4H), 0.88 (s,
(66 mL) in a similar manner as described for compound 19a. The
product was obtained as
a white solid (46 mg, 77%). Mp:
211.6e213.6 ꢀC. HPLC purity ¼ 97.17%, HPLC tR ¼ 9.45 min; 1H NMR
(500 MHz, CDCl3)
d
8.16 (d, J ¼ 5.5 Hz, 1H), 7.73 (s, 1H), 7.69 (t,
J ¼ 6.0 Hz,1H), 7.40 (d, J ¼ 8.0 Hz,1H), 7.16 (d, J ¼ 8.0 Hz, 2H), 7.05 (d,
J ¼ 8.0 Hz, 2H), 6.93 (t, J ¼ 6.0 Hz, 1H), 4.72e4.62 (m, 1H), 4.54 (dd,
J ¼ 14.5, 6.5 Hz, 1H), 4.50e4.41 (m, 2H), 4.26 (dd, J ¼ 14.5, 5.0 Hz,
1H), 4.03e3.94 (m, 1H), 3.91 (dd, J ¼ 9.0, 2.0 Hz, 1H), 3.63 (s, 3H),
3.57 (dd, J ¼ 9.0, 3.0 Hz, 1H), 3.47e3.33 (m, 2H), 3.01 (dd, J ¼ 13.5,
6.5 Hz,1H), 2.88 (dd, J ¼ 13.5, 6.0 Hz,1H), 2.82e2.70 (m, 2H), 2.28 (s,
3H), 1.82e1.67 (m, 1H), 1.63e1.50 (m, 1H), 1.50e1.35 (m, 3H),
9H); 13C NMR (126 MHz, CDCl3)
d 171.33, 170.09, 169.38, 167.07,
157.16, 138.26, 133.35, 132.30, 128.33, 127.43, 126.93, 120.68, 120.40,
112.03, 71.06, 69.01, 68.96, 53.03, 52.79, 50.70, 43.39, 36.88, 31.86,
28.66, 28.40, 27.37, 27.24, 27.20; ESI-MS: m/z ¼ 603 [MþNa]þ
1.34e1.19 (m, 3H), 0.83 (s, 9H); 13C NMR (125 MHz, CDCl3)
d 171.22,
4.1.37. (10S,13S)-N-(2-Chlorobenzyl)-13-(2-(neopentylamino)-2-
oxoethyl)-12,15-dioxo-2,3,4,5,6,7,10,11,12,13,14,15-dodecahydro-9H-
benzo[i][1,11]dioxa[4,7]diazacycloheptadecine-10-carboxamide 23e
This compound was prepared from 22c (49.1 mg, 0.1 mmol),
HOBt (16.2 mg, 0.12 mmol), EDCI (28.8 mg, 0.15 mmol), (2-
chlorophenyl)methanamine (14.8 mg, 0.105 mmol) and diisopro-
170.82, 169.86, 163.45, 153.88, 138.64, 136.51, 135.54, 129.04, 127.68,
101.25, 74.04, 70.98, 69.31, 54.12, 51.92, 50.56, 43.21, 35.87, 34.29,
31.94, 28.08, 27.95, 27.17, 24.43, 23.66, 21.06; ESI-MS: m/z ¼ 599
[MþH]þ.
4.1.40. (6S,9S)-1-Methyl-N-(4-methylbenzyl)-6-(2-
pylethylamine (66
mL) in a similar manner as described for com-
(neopentylamino)-2-oxoethyl)-4,7-dioxo-
pound 19a. The product was obtained as a white solid (44 mg, 71%).
4,5,6,7,8,9,10,12,13,14,15,16,17,18-tetradecahydro-1H-pyrazolo[4,3-i]
[1]oxa[4,7]diazacycloheptadecine-9-carboxamide 23h
This compound was prepared from 22e (49.3 mg, 0.1 mmol),
HOBt (16.2 mg, 0.12 mmol), EDCI (28.8 mg, 0.15 mmol), p-tolylme-
thanamine (12.7 mg, 0.105 mmol) and diisopropylethylamine
Mp: 195.7e198.1 ꢀC. HPLC purity ¼ 99.52%, HPLC tR ¼ 10.63 min; 1H
NMR (500 MHz, CDCl3)
d
9.53 (d, J ¼ 6.0 Hz, 1H), 7.96 (dd, J ¼ 7.0,
1.5 Hz, 1H), 7.47e7.36 (m, 2H), 7.26e7.21 (m, 2H), 7.16 (d, J ¼ 8.0 Hz,
1H), 7.13e7.04 (m, 2H), 7.00e6.94 (m, 2H), 6.23e6.08 (m, 1H),
4.89e4.81 (m, 1H), 4.69e4.61 (m, 1H), 4.57e4.44 (m, 2H),
4.44e4.38 (m, 1H), 4.29e4.22 (m, 1H), 3.87 (dd, J ¼ 9.5, 4.5 Hz, 1H),
3.59 (dd, J ¼ 9.5, 3.5 Hz, 1H), 3.53e3.45 (m, 1H), 3.44e3.34 (m, 1H),
3.05 (dd, J ¼ 13.0, 6.5 Hz, 1H), 3.02e2.94 (m, 2H), 2.66 (dd, J ¼ 14.5,
5.0 Hz, 1H), 2.17e2.05 (m, 1H), 1.91e1.79 (m 1H), 1.69e1.57 (m, 2H),
(66 mL) in a similar manner as described for compound 19a. The
product was obtained as
a white solid (43 mg, 72%). Mp:
227.9e229.7 ꢀC. HPLC purity ¼ 97.22%, HPLC tR ¼ 9.78 min; 1H NMR
(500 MHz, CDCl3)
d
8.18 (d, J ¼ 5.0 Hz, 1H), 7.81 (s, 1H), 7.74 (s, 1H),
7.33 (d, J ¼ 7.5 Hz, 1H), 7.18 (d, J ¼ 8.0 Hz, 2H), 7.08e6.95 (m, 3H),
4.64e4.58 (m, 1H), 4.50 (dd, J ¼ 14.5, 6.0 Hz, 1H), 4.40 (d, J ¼ 7.5 Hz,
1H), 4.29 (dd, J ¼ 14.5, 5.0 Hz, 1H), 3.94e3.88 (m, 1H), 3.75 (s, 3H),
3.51 (dd, J ¼ 8.5, 3.0 Hz, 1H), 3.40e3.33 (m, 1H), 3.32e3.23 (m, 2H),
3.00 (dd, J ¼ 13.0, 6.5 Hz, 1H), 2.87 (dd, J ¼ 13.0, 6.0 Hz, 1H),
2.82e2.67 (m, 2H), 2.49e2.39 (m, 1H), 2.26 (s, 3H), 1.58e1.11 (m,
1.53e1.40 (m, 4H), 0.87 (s, 9H); 13C NMR (126 MHz, CDCl3)
d 171.46,
170.11, 169.64, 167.11, 157.14, 135.52, 133.36, 133.15, 132.30, 129.18,
128.84, 128.19, 126.74, 120.66, 120.41, 112.02, 71.07, 68.98, 68.90,
53.13, 52.81, 50.69, 41.31, 36.82, 31.87, 28.63, 28.40, 27.39, 27.24,
27.18; ESI-MS: m/z ¼ 637 [MþNa]þ.
10H), 0.81 (s, 9H); 13C NMR (125 MHz, CDCl3)
d 171.13, 170.92,
4.1.38. (10S,13S)-N-(4-Methylbenzyl)-13-(2-(neopentylamino)-2-
oxoethyl)-12,15-dioxo-2,3,4,5,6,7,10,11,12,13,14,15-dodecahydro-9H-
benzo[i][1,11]dioxa[4,7]diazacycloheptadecine-10-carboxamide 23f
This compound was prepared from 22c (49.1 mg, 0.1 mmol),
HOBt (16.2 mg, 0.12 mmol), EDCI (28.8 mg, 0.15 mmol), p-tolylme-
thanamine (12.7 mg, 0.105 mmol) and diisopropylethylamine
170.07, 164.72, 147.11, 138.17, 136.44, 135.52, 129.01, 127.78, 112.59,
70.97, 69.24, 54.32, 52.11, 50.58, 43.38, 36.47, 35.78, 31.97, 27.81,
27.17, 26.79, 26.45, 25.91, 24.29, 23.52, 21.08; ESI-MS: m/z ¼ 597
[MþH]þ.
4.1.41. (14S,17S)-N-(4-Methylbenzyl)-17-(2-(neopentylamino)-2-
oxoethyl)-16,19-dioxo-6,7,8,9,10,11,14,15,16,17,18,19-dodecahydro-
5H,13H-imidazo[2,1-i][1]oxa[4,7,10]triazacycloheptadecine-14-
carboxamide 23i
This compound was prepared from 22f (49.3 mg, 0.1 mmol),
HOBt (16.2 mg, 0.12 mmol), EDCI (28.8 mg, 0.15 mmol), p-tolylme-
thanamine (12.7 mg, 0.105 mmol) and diisopropylethylamine
(66 mL) in a similar manner as described for compound 19a. The
product was obtained as
a white solid (47 mg, 79%). Mp:
183.6e186.1 ꢀC. HPLC purity ¼ 97.15%, HPLC tR ¼ 10.65 min; 1H NMR
(500 MHz, CDCl3)
d
9.52 (d, J ¼ 6.0 Hz, 1H), 7.94 (d, J ¼ 7.5 Hz, 1H),
7.43 (t, J ¼ 7.5 Hz, 1H), 7.31 (t, J ¼ 5.5 Hz, 1H), 7.13 (d, J ¼ 8.5 Hz, 1H),
7.08 (d, J ¼ 8.0 Hz, 2H), 7.01e6.90 (m, 4H), 6.44e6.32 (m, 1H),
4.86e4.74 (m, 1H), 4.65e4.57 (m, 1H), 4.46e4.36 (m, 2H), 4.29 (dd,
J ¼ 15.0, 5.0 Hz, 1H), 4.25e4.17 (m, 1H), 3.88 (dd, J ¼ 9.0, 4.0 Hz, 1H),
3.56 (dd, J ¼ 9.0, 3.5 Hz, 1H), 3.53e3.46 (m, 1H), 3.42e3.34 (m, 1H),
3.06e2.91 (m, 3H), 2.65 (dd, J ¼ 15.0, 5.0 Hz, 1H), 2.26 (s, 3H),
2.17e2.06 (m, 1H), 1.88e1.78 (m, 1H), 1.67e1.51 (m, 2H), 1.53e1.39
(66 mL) in a similar manner as described for compound 19a. The
product was obtained as
a white solid (33 mg, 69%). Mp:
184.4e187.5 ꢀC. HPLC purity ¼ 99.27%, HPLC tR ¼ 9.89 min; 1H NMR
(500 MHz, CDCl3)
d
8.99 (d, J ¼ 6.5 Hz, 1H), 7.48 (t, J ¼ 5.5 Hz, 1H),
7.22 (d, J ¼ 8.0 Hz, 2H), 7.17 (d, J ¼ 7.5 Hz,1H), 7.09 (d, J ¼ 8.0 Hz, 2H),
7.05 (s, 1H), 6.95 (s, 1H), 5.93 (s, 1H), 4.94e4.82 (m, 1H), 4.71e4.65
(m, 1H), 4.57 (dd, J ¼ 14.5, 6.5 Hz, 1H), 4.49e4.43 (m, 1H), 4.32 (dd,
J ¼ 14.5, 5.5 Hz, 1H), 3.95 (dd, J ¼ 9.0, 2.0 Hz, 1H), 3.76e3.67 (m, 1H),
(m, 4H), 0.86 (s, 9H). 13C NMR (125 MHz, CDCl3)
d 171.40, 170.15,
169.29, 167.03, 157.19, 136.37, 135.23, 133.27, 132.28, 129.00, 127.43,
120.59,120.42,111.99, 71.09, 69.06, 69.03, 53.03, 52.85, 50.66, 43.15,

436
J. Yu et al. / European Journal of Medicinal Chemistry 164 (2019) 423e439
3.51 (dd, J ¼ 9.0, 3.5 Hz, 1H), 3.38e3.33 (m, 1H), 3.33e3.26 (m, 1H),
3.10 (dd, J ¼ 13.5, 7.0 Hz, 1H), 2.94e2.86 (m, 2H), 2.82 (dd, J ¼ 14.5,
6.5 Hz, 1H), 2.31 (s, 3H), 1.71e1.60 (m, 1H), 1.35e1.11 (m, 8H),
4.1.45. tert-Butyl ((S)-1-(((S)-3-(1-allyl-1H-indol-3-yl)-1-((4-
methylbenzyl)amino)-1-oxopropan-2-yl)amino)-4-
(neopentylamino)-1,4-dioxobutan-2-yl)carbamate 28
This compound was prepared from 18c (302 mg, 1.0 mmol),
HOBt (162 mg, 1.2 mmol), EDCI (288 mg, 1.5 mmol), compound 27
(364 mg, 1.1 mmol) and diisopropylethylamine (0.66 mL) in a
similar manner as described for compound 19a. The product was
obtained as a white solid (517 g, 82%). 1H NMR (500 MHz, CDCl3)
1.06e0.94 (m, 1H), 0.86 (s, 9H). 13C NMR (125 MHz, CDCl3)
d13C
NMR (126 MHz, CDCl3)
d 170.39, 170.15, 169.92, 160.10, 137.17,
136.49, 135.50, 129.04, 129.00, 128.23, 127.82, 125.77, 70.88, 69.20,
54.26, 51.69, 50.53, 48.81, 43.38, 35.92, 31.80, 28.60, 27.60, 27.13,
26.59, 23.59, 23.49, 21.09; ESI-MS: m/z ¼ 583 [MþH]þ.
d
7.66 (d, J ¼ 8 Hz, 1H), 7.28 (s, 1H), 7.24e7.08 (m, 3H), 7.03 (d,
J ¼ 7.5 Hz, 1H), 7.00 (d, J ¼ 7.5 Hz, 2H), 6.97e6.83 (m, 4H), 5.98e5.82
(m, 2H), 5.18e5.12 (m, 1H), 5.07e4.98 (m, 1H), 4.75e4.66 (m, 1H),
4.63e4.56 (m, 2H), 4.40e4.24 (m, 2H), 4.23e4.18 (m, 1H), 3.47 (dd,
J ¼ 10.0, 4.0 Hz, 1H), 3.14e3.06 (m, 1H), 3.01e2.89 (m, 2H), 2.72 (dd,
J ¼ 15.0, 5.0 Hz, 1H), 2.58 (dd, J ¼ 15.0, 6.0 Hz, 1H), 2.28 (s, 3H), 1.21
(s, 9H), 0.86 (s, 9H); ESI-MS: m/z ¼ 632 [MþH]þ.
4.1.42. (13R,14S,17S)-13-Methyl-N-(4-methylbenzyl)-17-(2-
(neopentylamino)-2-oxoethyl)-16,19-dioxo-
6,7,8,9,10,11,14,15,16,17,18,19-dodecahydro-5H,13H-imidazo[2,1-i]
[1]oxa[4,7,10]triazacycloheptadecine-14-carboxamide 23j
This compound was prepared from 22f (49.3 mg, 0.1 mmol),
HOBt (16.2 mg, 0.12 mmol), EDCI (28.8 mg, 0.15 mmol), p-tolylme-
thanamine (12.7 mg, 0.105 mmol) and diisopropylethylamine
4.1.46. (S)-N1-((S)-3-(1-Allyl-1H-indol-3-yl)-1-((4-methylbenzyl)
amino)-1-oxopropan-2-yl)-2-(1-(but-3-en-1-yl)-1H-imidazole-2-
carboxamido)-N4-neopentylsuccinamide 30
This compound was prepared from 10d (166 mg, 1.0 mmol),
HOBt (162 mg, 1.2 mmol), EDCI (288 mg, 1.5 mmol), 29 (531 mg,
(66 mL) in a similar manner as described for compound 19a. The
product was obtained as
a white solid (47 mg, 80%). Mp:
204.4e206.4 ꢀC. HPLC purity ¼ 99.37%, HPLC tR ¼ 10.33 min; 1H
NMR (500 MHz, CDCl3)
d
9.30 (d, J ¼ 5.5 Hz, 1H), 7.42 (t, J ¼ 5.5 Hz,
1.05 mmol) and diisopropylethylamine (0.66 mL) in
manner as described for compound 19a. The product was obtained
as a white solid (421 mg, 62%). 1H NMR (500 MHz, CDCl3)
8.65 (d,
a similar
1H), 7.19 (d, J ¼ 8.0 Hz, 2H), 7.11 (d, J ¼ 8.5 Hz,1H), 7.06 (d, J ¼ 8.0 Hz,
2H), 7.04 (s, 1H), 6.94 (s, 1H), 6.46 (s, 1H), 4.90e4.77 (m, 1H),
4.68e4.59 (m, 1H), 4.46e4.32 (m, 2H), 4.24 (d, J ¼ 8.5 Hz, 1H),
4.19e4.09 (m, 1H), 3.81e3.66 (m, 1H), 3.54e3.41 (m, 1H), 3.11e2.98
(m, 2H), 2.98e2.86 (m, 2H), 2.80 (dd, J ¼ 14.5, 5.5 Hz, 1H), 2.28 (s,
3H), 1.74e1.60 (m, 1H), 1.36e0.99 (m, 12H), 0.82 (s, 9H); 13C NMR
d
J ¼ 7.5 Hz, 1H), 7.67 (d, J ¼ 8.0 Hz, 1H), 7.22 (d, J ¼ 8.5 Hz, 1H),
7.18e7.13 (m, 1H), 7.10 (d, J ¼ 8.0 Hz, 1H), 7.07e7.00 (m, 4H), 6.99 (d,
J ¼ 1.0 Hz, 1H), 6.94 (m, 3H), 6.69 (t, J ¼ 5.5 Hz, 1H), 5.94e5.65 (m,
3H), 5.13 (m, 1H), 5.05e5.01 (m, 2H), 5.01e4.97 (m, 1H), 4.79e4.70
(m, 2H), 4.57e4.48 (m, 2H), 4.37e4.30 (m, 1H), 4.28 (dd, J ¼ 6.0,
2.5 Hz, 2H), 4.26e4.18 (m, 1H), 3.46 (dd, J ¼ 14.5, 5.0 Hz, 1H), 3.15
(dd, J ¼ 14.5, 7.0 Hz, 1H), 3.02 (dd, J ¼ 13.5, 7.0 Hz, 1H), 2.90 (dd,
J ¼ 13.5, 6.0 Hz, 1H), 2.82 (dd, J ¼ 15.5, 4.5 Hz, 1H), 2.73 (dd, J ¼ 15.0,
7.0 Hz, 1H), 2.44 (q, J ¼ 7.5 Hz, 2H), 2.31 (s, 3H), 0.87 (s, 9H); ESI-MS:
m/z ¼ 680 [MþH]þ.
(126 MHz, CDCl3)
d 170.85, 170.32, 170.16, 160.05, 137.02, 136.62,
135.29, 129.08, 127.97, 127.92, 125.76, 73.80, 68.39, 58.78, 52.17,
50.58, 48.89, 43.47, 35.73, 31.88, 28.87, 28.02, 27.18, 26.69, 23.82,
23.63, 21.09, 16.50; ESI-MS: m/z ¼ 597 [MþH]þ.
4.1.43. tert-Butyl (S)-(3-(1H-indol-3-yl)-1-((4-methylbenzyl)
amino)-1-oxopropan-2-yl)carbamate 25
4.1.47. (6S,9S)-N-(4-Methylbenzyl)-6-(2-(neopentylamino)-2-
oxoethyl)-4,7-dioxo-4,5,6,7,8,9,17,18,19,20-decahydro-11,16-
(metheno)benzo[q]imidazo[1,2-g][1,7,10,13]
This compound was prepared from (tert-butoxycarbonyl)- -
L
tryptophan 24 (3.04 g, 10.0 mmol), HOBt (1.62 g, 12.0 mmol), EDCI
(2.88 g, 15.0 mmol), p-tolylmethanamine (1.27 g, 10.5 mmol) and
diisopropylethylamine (6.61 mL) in a similar manner as described
for compound 19a. The product was obtained as a white solid
tetraazacyclooctadecine-9-carboxamide 31
This compound was prepared from 30 (135 mg, 0.2 mmol) in a
similar manner as described for compound 21a. The product was
obtained as a white solid (58 mg, 45%). Mp: 278.6e282.2 ꢀC. HPLC
purity ¼ 98.31%, HPLC tR ¼ 10.70 min; 1H NMR (500 MHz,
(3.62 g, 89%). 1H NMR (500 MHz, DMSO‑d6)
d 10.81 (s, 1H), 8.35 (t,
J ¼ 6.0 Hz, 1H), 7.60 (d, J ¼ 8.0 Hz, 1H), 7.33 (d, J ¼ 8.0 Hz, 1H),
7.15e7.00 (m, 6H), 6.97 (t, J ¼ 7.5 Hz, 1H), 6.81 (d, J ¼ 8.0 Hz, 1H),
4.28e4.17 (m, 3H), 3.08 (dd, J ¼ 14.5, 5.0 Hz, 1H), 2.92 (dd, J ¼ 14.5,
9.0 Hz, 1H), 2.26 (s, 3H), 1.32 (s, 9H); ESI-MS: m/z ¼ 408 [MþH]þ.
DMSO‑d6)
d
8.62 (d, J ¼ 8.0 Hz, 1H), 8.56 (t, J ¼ 6.0 Hz, 1H), 8.26 (d,
J ¼ 8.0 Hz, 1H), 8.02 (t, J ¼ 6.5 Hz, 1H), 7.50 (d, J ¼ 7.5 Hz, 1H), 7.44 (d,
J ¼ 1.0 Hz, 1H), 7.37 (d, J ¼ 8.0 Hz, 1H), 7.16e7.07 (m, 5H), 7.03e6.97
(m, 3H), 5.01e4.93 (m, 1H), 4.75e4.67 (m, 1H), 4.63e4.57 (m, 1H),
4.34 (dd, J ¼ 15.0, 6.5 Hz, 1H), 4.24e4.11 (m, 2H), 4.08e4.01 (m, 1H),
3.88e3.79 (m,1H), 3.23e3.17 (m,1H), 3.05 (dd, J ¼ 16.0,11.0 Hz,1H),
2.85 (dd, J ¼ 13.5, 6.5 Hz, 1H), 2.79 (dd, J ¼ 13.5, 6.0 Hz, 1H), 2.68 (d,
J ¼ 7.0 Hz, 2H), 2.28 (s, 3H), 1.84e1.72 (m, 2H), 1.71e1.63 (m, 1H),
1.63e1.53 (m, 1H), 1.05e0.93 (m, 2H), 0.79 (s, 9H); 13C NMR
4.1.44. tert-Butyl (S)-(3-(1-allyl-1H-indol-3-yl)-1-((4-
methylbenzyl)amino)-1-oxopropan-2-yl)carbamate 26
To a solution of 25 (500 mg, 1.2 mmol) in anhydrous THF (10 mL)
were added NaH (98 mg, 12.0 mmol) and 3-bromoprop-1-ene
(295 mg, 2.5 mmol) at 0ꢀС. After stirring at room temperature for
3 h, 10 mL water was added at 0ꢀС. Then, the reaction mixture was
concentrated and dissolved with ethyl acetate (15 mL), washed
with brine (10 mL ꢂ 3). The organic layer was dried over Na2SO4,
concentrated under vacuum and purified by SiO2 chromatography
(ethyl acetate: petroleum ether ¼ 1:2) to afford the compound 26
(125 MHz, DMSO)
d 171.77, 171.57, 170.15, 158.86, 138.51, 136.79,
136.15, 136.07, 129.22, 128.32, 127.61, 126.69, 125.19, 121.53, 118.83,
118.79, 110.46, 109.99, 53.38, 50.39, 50.19, 46.30, 45.88, 42.47, 37.75,
32.27, 30.66, 28.85, 27.64, 27.50, 22.92, 21.15; ESI-MS: m/z ¼ 654
[MþH]þ.
as a white solid (430 mg, 79%). 1H NMR (500 MHz, CDCl3)
d
7.66 (d,
4.1.48. O-Hexyl-N-(N2-(1-methyl-5-propyl-1H-pyrazole-4-
carbonyl)-N4-neopentyl-L-asparaginyl)-L-serine 32
Compound 21e (183 mg, 0.3 mmol) was dissolved in methanol
(5 mL) in the presence of 10% palladium on carbon (10 mol %), and
the reaction mixture was stirred under an atmosphere of H2 for 2 h.
The Pd/C was removed via filtration through celite, and the solvent
was evaporated to obtain compound 32 as a white solid (150 mg,
J ¼ 8.0 Hz, 1H), 7.29 (d, J ¼ 8.0 Hz, 1H), 7.24e7.19 (m, 1H), 7.14e7.11
(m, 1H), 7.03 (d, J ¼ 7.5 Hz, 2H), 6.93e6.85 (m, 3H), 6.00e5.84 (m,
2H), 5.23e5.09 (m, 2H), 5.03 (d, J ¼ 17.0 Hz, 1H), 4.61 (dd, J ¼ 5.5,
1.5 Hz, 2H), 4.49e4.38 (m,1H), 4.25 (s, 2H), 3.33 (dd, J ¼ 14.5, 5.0 Hz,
1H), 3.16 (dd, J ¼ 14.5, 7.5 Hz, 1H), 2.30 (s, 3H), 1.41 (s, 9H); ESI-MS:
m/z ¼ 448 [MþH]þ.

J. Yu et al. / European Journal of Medicinal Chemistry 164 (2019) 423e439
437
95%). 1H NMR (500 MHz, DMSO‑d6)
d
12.90 (brs, 1H), 8.12 (d,
4.2.2. Cancer cell proliferation assay
J ¼ 8.0 Hz, 1H), 7.94 (d, J ¼ 8.0 Hz, 1H), 7.86 (s, 1H), 7.74 (t, J ¼ 6.0 Hz,
1H), 4.84e4.75 (m, 1H), 4.38e4.30 (m, 1H), 3.75 (s, 3H), 3.69 (dd,
J ¼ 10.0, 5.0 Hz, 1H), 3.54 (dd, J ¼ 10.0, 4.0 Hz, 1H), 3.37e3.30 (m,
2H), 2.96e2.85 (m, 3H), 2.81 (dd, J ¼ 13.0, 6.0 Hz, 1H), 2.64 (dd,
J ¼ 14.5, 5.0 Hz, 1H), 2.56 (dd, J ¼ 14.5, 9.5 Hz, 1H), 1.55e1.46 (m,
2H), 1.44e1.36 (m, 2H), 1.26e1.15 (m, 6H), 0.88 (t, J ¼ 7.5 Hz, 3H),
0.84 (t, J ¼ 7.0 Hz, 3H), 0.79 (s, 9H); ESI-MS: m/z ¼ 524 [MþH]þ.
4.2.2.1. Cell culture. RPMI 8226, MM.1S and MV-4-11 cell lines were
obtained from the American Type Culture Collection (ATCC)
(Manassas, VA, USA) and maintained at 37 ꢀC in a humidified at-
mosphere containing 5% CO2. The human MM cell lines PRMI 8226
and MM.1S were cultured in RPMI-1640 media supplemented with
10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL
streptomycin from Invitrogen (Grand Island, NY, USA). The human
AML cell line MV-4-11 was cultured in Iscove's modified Dulbecco's
medium supplemented with 10% FBS, 100 U/mL penicillin, and
4.1.49. (S)-N1-((S)-3-(Hexyloxy)-1-((4-methylbenzyl)amino)-1-
oxopropan-2-yl)-2-(1-methyl-5-propyl-1H-pyrazole-4-
100 mg/mL streptomycin.
carboxamido)-N4-neopentylsuccinamide 33
4.2.2.2. Cell proliferation assays. A 90 mL aliquot of RPMI 8226
This compound was prepared from 32a (52.3 mg, 0.1 mmol),
HOBt (16.2 mg, 0.12 mmol), EDCI (28.8 mg, 0.15 mmol), p-tolylme-
thanamine (12.7 mg, 0.105 mmol) and diisopropylethylamine
(5 ꢂ 103 cells per well), MM-1S (3 ꢂ 104 cells per well), or MV-4-
11 cells (8 ꢂ 103 cells per well) was seeded into 96-well plates and
then treated with 10 mL of 0.2% DMSO or varying concentrations of
(66
product was obtained as a white solid (47 mg, 75%). 1H NMR
(500 MHz, CDCl3) 7.84e7.69 (m, 2H), 7.51 (s, 1H), 7.28 (s, 1H), 7.14
mL) in a similar manner as described for compound 19a. The
tested compounds for 72 h. Cell viability was measured using the
CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay
d
(MTS; Promega, Madison, WI). Briefly, 20 mL of the combined MTS/
(d, J ¼ 7.5 Hz, 2H), 7.06 (d, J ¼ 7.5 Hz, 2H), 6.29 (s, 1H), 4.74 (s, 1H),
4.56e4.42 (m, 2H), 4.39e4.26 (m, 1H), 3.98e3.88 (m, 1H), 3.78 (s,
3H), 3.47 (s, 1H), 3.42e3.30 (m, 2H), 3.06e2.66 (m, 6H), 2.29 (s, 3H),
1.62e1.48 (m, 2H), 1.46e1.33 (m, 2H), 1.28e1.10 (m, 6H), 0.91 (t,
J ¼ 7.0 Hz, 3H), 0.87e0.80 (m, 12H); 13C NMR (126 MHz, CDCl3)
PMS solution was pipetted into each of the 96-wells plate and then
incubated for 2e4 h at 37 ꢀC in a humidified, 5% CO2 atmosphere.
The optical density was determined at 490 nm (background sub-
traction at 690 nm) using a SpectraMax 340 microplate reader
(Molecular Devices, Sunnyvale, CA, USA). Growth inhibitory ratios
d
170.89, 169.77, 164.22, 146.36, 137.70, 136.54, 135.34, 129.08,
were calculated as follows: Growth inhibitory ratio ¼ (Acontrol
-
127.35, 116.79, 113.22, 71.63, 69.64, 53.57, 51.22, 50.66, 43.18, 37.11,
36.33, 31.88, 31.60, 29.39, 27.17, 26.35, 25.59, 22.49, 22.19, 21.08,
14.07, 13.82; ESI-MS: m/z ¼ 627 [MþH]þ.
Asample)/Acontrol. IC50 values were derived from a nonlinear regres-
sion model (curvefit) based on a sigmoidal dose response curve
(variable slope) and computed using GraphPad Prism version 5.02,
GraphPad Software.
4.2. Biological evaluation
4.2.3. Human liver microsomal stability assays
4.2.1. In vitro assays of chymotrypsin-like, trypsin-like, and
The metabolic stability profiles of 23h and 33 were assessed by
monitoring the loss of test compounds in the presence of human
caspase-like activities of the proteasome and immunoproteasome
The human constitutive proteasome was given by Dr. Jiang-ping
Wu (Notre-Dame Hospital, Montreal, QC, Canada), derived from
human hepatic cells; the immunoproteasome was purchased from
Boston Biochem, derived from human peripheral blood mono-
nuclear cell. We used several fluorogenic substrates, the fluorogenic
substrate Suc-LLE-AMC was used to measure the Capase-Like ac-
liver microsomes. A typical incubation mixture (100
mL total vol-
ume) for metabolic stability studies contained 1 M of test com-
m
pounds, 1.0 mg/mL microsomal protein (pooled Balb/c mouse liver
microsomes prepared in-house or BD UltraPool human liver mi-
crosomes), 0.1M phosphate buffered saline (pH 7.4), and 1 mM
NADPH. After preincubation at 37 ꢀC for 5 min, reactions were
started by the addition of test compounds and further incubated for
0, 10, 20, and 30 min. Reactions were terminated by the addition of
tivity of the proteasome,
the Trypsin-Like activity of the proteasome,
was used to measure the Chymotrypsin-Like activity of the pro-
teasome, 5c. The fluorogenic substrate Suc-PAL-AMC was used to
measure the Capase-Like activity of the immunoproteasome, 1i.
Suc-VGR-AMC was used to measure the Trypsin-Like activity of the
immunoproteasome, 2i. Suc-ANW-AMC was used to measure the
Chymotrypsin-Like activity of the immunoproteasome, 5i. All
b
1c. Suc-KQL-AMC was used to measure
b2c. Suc-WLA-AMC
400
lowed by centrifugation at 15000g for 10 min to obtain the super-
natant. Aliquots (100 L) of the supernatant were taken, which
mL ice-cold methanol containing internal standard (1 mM), fol-
b
b
m
were subsequently analyzed using a Shimadzu LCMS-2020 mass
spectrometer. The peak area response ratio to internal standard
(PARR) of the compounds at different time points were compared
to the PARR at 0 min to determine the percentage of test com-
pounds remaining.
b
b
compounds were solved in DMSO, then diluted with water to be
10% DMSO containing solution. The reaction under the following
buffer conditions: 100 mM Tris-HCl, pH7.5. Positive control: bor-
tezomib. Negative control: 2% DMSO. Blank: no enzyme, replaced
by reaction buffer. All the final concentration of the substrates were
4.3. Molecular modeling
100
pound (contain 10% DMSO, the fianl concentration of DMSO is 2%),
at different concentrations were added to 20 L human proteasome
(40 g/mL) or immunoproteasome (1 g/mL), after 15 min, the
flurogenic substrates were added into the reaction plate, the plates
were covered with a plate sealer and incubated at room tempera-
ture (25 ꢀC) for 60 min. The AMC of the probe was detected by
monitoring the increase in fluorescence with EnVision, at a 355 nm
excitation and 460 nm emission wavelength. The calculation for
m
M. All assays were carried out in a 50
m
L volume. 10
m
L com-
Docking calculations were performed using the Glide module in
Schrodinger (version 11.1) with the default option [47,48]. The X-ray
crystal structure of the proteasome (PDB entry: 3MG6) was used as
the docking template, and it was prepared by the protein prepa-
ration Wizard in Schrodinger by adding hydrogens and disulfide
bridges, removing crystallographic waters and ions, fixing bond
orders, assigning partial charges with the OPLS force field. The
ligand 23h was prepared using the Ligprep module in Schrodinger.
The binding box with the size of 10 Å ꢂ 10 Å ꢂ 10 Å centered on the
centroid of the ligand in the crystal structure of 3MG6. For the
docking calculations of 23h, the standard precision (SP) scoring
function of Glide was used. All graphical images were created using
Pymol.
m
m
m
Inhibition% as following formula: Inhibition%
¼
(ODDMSO e
ODCompound)/(ODDMSO e ODBlank) ꢂ 100%. IC50 data were calculated
using GraphPad Prism software, and the equation ‘sigmoidal
doseeresponse (variable slope)’ was chosen for curve fitting.

438
J. Yu et al. / European Journal of Medicinal Chemistry 164 (2019) 423e439
derivatives as proteasome inhibitors, Eur. J. Med. Chem. 126 (2017)
Acknowledgments
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
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