Development of Macrocyclic Peptides Containing Epoxyketone with Oral Availability as Proteasome Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.8b00819

Macrocyclization has been frequently utilized for optimizing peptide or peptidomimetic-based compounds. In an attempt to obtain potent, metabolically stable, and orally available proteasome inhibitors, 30 oprozomib-derived macrocyclic peptides with structural diversity in their N-terminus and linker were successively designed and synthesized for structure-activity relationship (SAR) studies. As a consequence, the macrocyclic peptides with N-methyl-pyrazole (24p, 24x), imidazole (24t), and pyrazole (24v) as their respective N-termini exhibited favorable in vitro activity and metabolic stability, which translated into their potent in vivo proteasome inhibitory activity after oral administration. In particular, compound 24v, as the most distinguished one among this series, displayed excellent chymotrypsin-like (ChT-L, β5) inhibitory potency (IC₅₀ = 16 nM), low nanomolar antiproliferative activity against all three of the tested cell lines, and superior metabolic stability in mouse liver microsome (MLM), as well as favorable inhibition against ChT-L compared to that of oprozomib in BABL/c mice following po administration at a comparatively low dose, thereby representing a promising candidate for further development.

Full text of DOI:10.1021/acs.jmedchem.8b00819

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Article
Development of Macrocyclic Peptides Containing
Epoxyketone with Oral Availability as Proteasome Inhibitors
Daqiang Li, Xiaotuan Zhang, Xiaodong Ma, Lei Xu, Jianjun Yu, Lixin Gao, Xiaobei
Hu, Jiankang Zhang, Xiaowu Dong, Jia Li, Tao Liu, Yubo Zhou, and Yongzhou Hu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00819 • Publication Date (Web): 28 Sep 2018
Downloaded from http://pubs.acs.org on September 28, 2018
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Development of Macrocyclic Peptides
Containing Epoxyketone with Oral
Availability as Proteasome Inhibitors
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†
,#
‡,¥,#
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‡,º,¥
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‡
Daqiang Li , Xiaotuan Zhang , Xiaodong Ma , Lei Xu , Jianjun Yu , Lixin Gao ,
‡
⊥
†
‡
*,†
*,‡
Xiaobei Hu , Jiankang Zhang , Xiaowu Dong , Jia Li , Tao Liu , Yubo Zhou ,
*,†
Yongzhou Hu
†
ZJU-ENS Joint Laboratory of Medicinal Chemistry, Zhejiang Province Key
Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences,
Zhejiang University, Hangzhou, 310058, People's Republic of China
‡
National Center for Drug Screening, State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai
2
01203, China
º
School of Life Science and Technology, ShanghaiTech University, Shanghai, 201203,
China
¥
University of Chinese Academy of Sciences, Graduate School, No. 19A Yuquan Road,
Beijing, China
§
Department of Medicinal Chemistry, School of Pharmacy, Anhui University of
Chinese Medicine, Hefei 230031, China
⊥
Zhejiang University City College, Hangzhou 310015, Zhejiang, China
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KEYWORDS
Macrocycle, peptide, epoxyketone, proteasome inhibitor, liver microsome stability,
oral availability.
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ABSTRACT
Macrocyclization has been frequently utilized for optimizing peptide or
peptidomimeticꢀbased compounds. In an attempt to obtain potent, metabolically stable,
and orally available proteasome inhibitors, 30 oprozomibꢀderived macrocyclic
peptides with structural diversity in their Nꢀterminus and linker were successively
designed and synthesized for structureꢀactivity relationship (SAR) studies. As a
consequence, the macrocyclic peptides with Nꢀmethylꢀpyrazole (24p, 24x), imidazole
(24t) and pyrazole (24v) as their respective Nꢀtermini exhibited favorable in vitro
activity and metabolic stability, which translated into their potent in vivo proteasome
inhibitory activity after oral administration. In particular, compound 24v, as the most
distinguished one among this series, displayed excellent chymotrypsinꢀlike (ChTꢀL,
β5) inhibitory potency (IC50 = 16 nM), low nanomolar antiꢀproliferative activity
against all three of the tested cell lines, and superior metabolic stability in mouse liver
microsome (MLM), as well as favorable inhibition against ChTꢀL compared to that of
oprozomib in BABL/c mice following po administration at a comparatively low dose,
thereby representing a promising candidate for further development.
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INTRODUCTION
As the vital component of the ubiquitinꢀproteasome system (UPS), the proteasome
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plays an essential role in the degradation of unneeded or damaged proteins, and is
responsible for maintaining cellular homeostasis. Additionally, the proteasome
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4ꢀ5
mediates a variety of cellular activities, including apoptosis, cell cycle regulation,
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DNA repair, immune and inflammatory responses. In contrast, its inhibition leads
to the accumulation of unneeded protein, thereby inducing the apoptosis of cells,
especially in malignant ones featuring aberrant division and an excess of unwanted
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intracellular proteins. Structurally, the proteasome is a cylindric complex bearing a
20S core particle (20S CP), that contains two outer α rings and two inner β rings.
Each β ring comprises three active enzymatic subunits crucial for proteolysis,
including caspaseꢀlike (CꢀL, β1), trypsinꢀlike (TꢀL, β2), and chymotrypsinꢀlike
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(ChTꢀL, β5) activities.
Based on their expression pattern and constitution, CPs in
vertebrates can be divided into three types, termed the constitutive proteasome (cCP),
the immunoproteasome (iCP), and the thymoproteasome (tCP). Among the
proteasomes, cCP, composed of β1c, β2c and β5c, is ubiquitously expressed. Despite
their identical composition (β1i, β2i and β5t), iCP is predominantly expressed in
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monocytes and lymphocytes, while tCP is exclusively distributed in thymic cortical
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epithelial cells. Due to the greater impact on general proteolysis resulting from the
inhibition of ChTꢀL than that of other subunits, massive research endeavors have been
made in the exploration of peptidyl proteasome inhibitors (PIs) that interact with
ChTꢀL in light of the binding modes of its endogenous substrates. Upon interaction
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with the ChTꢀL site, a majority of these inhibitors adopt an antiparallel βꢀsheet
conformation. At the Cꢀterminus, these peptide inhibitors are commonly equipped
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with an electrophilic warhead for covalently contacting with Thr1 of the ChTꢀL.
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So far, bortezomib (1), carfilzomib (2), and ixazomib (3), which belong to this class,
have been approved for use in humans, which verifies the medicinal potential of the
proteasome as an antiꢀcancer therapeutic target.
Figure 1. Structures of bortezomib, carfilzomib, ixazomib, and oprozomib
Nonetheless, 1, the pioneering PI marketed for treating multiple myeloma (MM)
and mantle cell lymphoma (MCL), features a boronate moiety as the warhead, which
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is capable of interacting with HtrA2/Omi and thereby inducing peripheral
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neuropathy (PN).
Compound 2, introducing an α′, β′ꢀepoxyketone as a
replacement for the boronate warhead for irreversible interaction with Thr1, has some
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advantages over 1, including decreased side effects and the obviation of
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bortezomibꢀmediated drug resistance.
However, both 1 and 2 are routinely
intravenously administered due to poor oral bioavailability. To improve the patient
compliance, oprozomib (4), an analog of carfilzomib obtained by peptide truncation,
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was discovered and has been advanced into Phase 1/2 clinical trials in patients with
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hematological malignancy and solid tumors.
In addition, much efforts of
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medicinal chemistry on 2 delivered PRꢀ893 and PRꢀ957 with individual subunit
selectivity, respectively. Especially a dual β1i and β5i epoxyketone inhibitor,
KZRꢀ616, was successfully completed Phase I clinical trials for the treatment of
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systemic lupus erythematosus with and without nephritis. Aside from the boronate
and α´β´ꢀepoxyketone, other PIs equipped with aldehydes, vinyl sulfones,
vinylamides, αꢀketoaldehydes, and βꢀlactones were developed and reviewed in the
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literature. Among them, the most advanced one is marizomib, a derivative of
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omuralide, bearing γꢀlactamꢀβꢀlactone bicyclic core, which is now on Phase Ⅲ
clinical trials for the treatment of newly diagnosed glioblastoma due to the ability of
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penetrating the bloodꢀbrain barrier. Another βꢀlactone derivative, belactosin A and
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its derivatives,
with generally low cell permeability and limited bioavailability,
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were further optimized by Shuto and coworkers.
As a wellꢀestablished strategy for structural optimization, macrocyclization has
been utilized for constraining the conformation of peptide or peptidomimeticꢀbased
compounds to reduce the entropy loss correlated with ligandꢀreceptor interaction,
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enhancing the metabolic stability, and improving cellular penetration.
These
peptide macrocycles are commonly designed via cyclization between the P1 and P3 or
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the P2 and P4 sites on the basis of the βꢀstrand geometry of proteases. In our initial
efforts to explore macrocyclic oprozomib derivatives, cyclization between the P1 and
P3 was envisioned to be unsuitable because of the potential steric clash with Ala49.
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Recently, Götz and coworkers reported two macrocyclic peptide PIs bearing aldehyde
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as the warhead and an intramolecular linkage between the P2 and P4 side chains.
Despite the selectivity, the aldehyde functionality was relevant to undesired
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pharmacokinetics and low oral bioavailability. In addition, hydrophobic groups in
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each amino acid side chain of these were detrimental for gastrointestinal absorption.
For the macrocyclic protease inhibitors with intramolecular linkage between
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Nꢀterminal aromatic rings and the amino acid side chain,
synthetic accessibility
hindered the introduction of various aryl moieties for systematic structure activity
relationship (SAR) studies and their biological stability, especially oral availability,
remain to be investigated.
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Our group has always been committed to the research of 2 and its derivatives.
Considering the acyclic peptide backbone, 2 and 4, is also prone to hydrolysis by
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endogenous proteases and peptidases, which compromises its in vivo efficacy.
Hence, we hypothesized that the strategy of macrocyclization might be beneficial for
overcoming this shortcoming and provided a novel series of PIs with favorable
potency and oral bioavailability.
RESULTS AND DISCUSSION
To rationally design the macrocyclic peptides appropriate for oral administration,
we began by relying on insight from the Xꢀray coꢀcrystal structure of oprozomib
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bound to the human 20S proteasome (Figure 2a, PDB 5LEY). Accordingly, the
antiparallel βꢀsheet backbone of oprozomib was accommodated by the active sites of
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ChTꢀL and the β6 subunits with each amide from P1 to P3 engaged in an Hꢀbonding
interaction with them, and the α´β´ꢀepoxyketone warhead irreversibly interacted with
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Thr1, forming a sevenꢀmembered 1,4ꢀoxazepane. The phenylalanyl moiety at the P1
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site was projected into the hydrophobic S1 pocket, while the Oꢀmethylꢀseryl moieties
at P2 and P3 were located in the S2 and S3 pockets, respectively, without obvious
contacts. The thiazole, as the Nꢀterminal moiety, inserted into the lipophilic S4 pocket
and conferred hydrophobic interaction with residues Asp125, Pro126 and Ala127 in
the β6 subunit. In view of their close spatial proximity, we incorporated a wide
variety of chemical linkers between the P2 side chain and the Nꢀterminus for
constructing the macrocyclic PIs. To attempt to validate the design rationale, a
representative virtual molecule 24c was modeled onto the human 20S proteasome.
According to the overlay of 24c and oprozomib in the binding pocket (Figure 2b),
24c maintained the binding mode of oprozomib, and the linker of 24c was involved in
additional interaction with Ala22 and Gly23. Additionally, considering the synthetic
convenience, phenyl and other six or fiveꢀmembered aryl moieties, such as the
bioisosteres of thiazole, were introduced to replace this group at the Nꢀterminus.
(a)
(b)
Figure 2. (a) Crystal structure of human 20S proteasome in complex with oprozomib
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(
green) (PDB 5LEY). (b) Overlay of oprozomib (green) and 24c (cyan) bound to the
human 20S proteasome (van der Waals surfaces, PDB 5LEY). Activeꢀsite residues
participating in polar interactions with the ligand or structural water (red, H O) are
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shown as sticks and colored according to atom type (red, oxygen; blue, nitrogen; pink,
carbon). Hydrogen bonds are indicated by dashed yellow lines.
As illustrated in Table 1, compounds 24a-f, bearing 15 to 20ꢀmembered
phenylꢀfused macrocycles, exhibited moderate to potent ChTꢀL inhibitory activity.
Among these, compound 24a-d displayed comparable ChTꢀL inhibitory activity to
that of oprozomib, with IC values at the twoꢀdigit nanomolar level. Consistent with
50
this result, the four compounds demonstrated attractive cytotoxic activities against
both the MM.1S and MVꢀ4ꢀ11 cell lines with IC50 values at the twoꢀdigit nanomolar
level. According to the relatively weakened activity of their 19 and 20ꢀmembered
macrocyclic counterparts 24e and 24f, the macrocycle of excessive size was
detrimental for activity. Compounds 24a-c, with favorable ChTꢀL inhibitory activity,
were therefore selected for evaluating the blood proteasome inhibitory activity in
BABL/c mice following po administration. To our delight, 24c treatment at a dose of
1
00 mg/kg conferred an equivalent blood proteasome inhibition compared to that of
oprozomib treatment at a dose of 30 mg/kg at both 2 and 24 h postꢀadministration.
However, the efficacy of 24c was significantly compromised when lowering the dose
to 30 mg/kg (Figure 3). To elucidate the underlying factors, a Cacoꢀ2 permeability
assay of 24c was performed and it exhibited a moderate membrane permeability
((BꢀA)/(AꢀB) ratio (4.4)) (Table 2). Additionally, in the following PK study, 24c
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demonstrated a lower bioavailability (2%) than that of oprozomib (6%, Table 3). In
particular, 24c displayed an extremely poor metabolic stability in MLM with more
than 95% degradation at 5 min postꢀincubation (Table 1). Taken together, the
moderate membrane permeability and undesired liver microsome stability of 24c
decreased its oral bioavailability, hence accounting for the lack of in vivo efficacy at
lower doses.
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blood proteasome activity (P.O)
150
2h
100
24h
50
0
CMC
Opr
24a
24b
24c
24a
24b
24c
3
0 mg/kg 30 mg/kg 30 mg/kg 30 mg/kg 100 mg/kg 100 mg/kg 100 mg/kg
Figure 3. Mouse oral PD data for oprozomib, 24a-c. CMC = 0.5%CMCꢀNa; Opr =
oprozomib.
Table 1. In vitro data of epoxyketone macrocycles.
ChTꢀL
Cytotoxicity IC50 (nM)
Microsome
Compd
4a
4b
4c
n
A
IC50 (nM) RPMI 8226 MM.1S MVꢀ4ꢀ11 Stability
2
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3
36 ± 6
21 ± 8
>100
74 ± 12 29 ± 3 13 ± 1
>100 58 ± 1 36 ± 7
65 ± 4
49
NT
NT
2
[
a]
2
54 ± 13
<5
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4d
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63 ± 15
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>100
NT
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
3
3
25 ± 1
4 ± 2
10 ± 2
5 ± 2
3 ± 0.1 9 ± 0.4
3 ± 0.01 6 ± 0.6
NT
[
a]
24h
<5
2
4i
4j
24k
4l
4m
4n
4o
24p
4q
4y
4z
Oprozomib
3
3
3
3
3
3
5
3
3
3
205 ± 35
253 ± 37
162 ± 12
100 ± 31
436 ± 59
248 ± 65
51 ± 16
>100
>100
>100
>100
NT
69 ± 6
43 ± 2
53 ± 4
NT
NT
NT
NT
NT
NT
NT
2
2
43 ± 1 26 ± 1
[a]
2
NT
NT
NT
NT
<5
2
>100
32 ± 1
20 ± 1
NT
37 ± 3
11 ± 1
NT
NT
2
[
b]
32 ± 0.5
232 ± 7
6 ± 0.5 4 ± 1
NT NT
63
2
NT
NT
NT
S
2
257 ± 6
128 ± 69 63 ± 7 131 ± 36
2
3
/
392 ± 10
47 ± 2
>100
65 ± 4 36 ± 2
9 ± 1 28 ± 3
[
b]
/
19 ± 3
60
[
a]
[b]
Mouse, % remaining at 5 min. Mouse, % remaining at 0.5 h. NT = not tested.
a
Table 2. Cacoꢀ2 permeability of 24c .
ꢀ
6
(BꢀA)/(AꢀB)
ratio
Cacoꢀ2 (×10 cm/s)
AꢀB
Compound
4c
BꢀA
2
6.00 ± 0.84
35.19 ± 1.89
0.41 ± 0.07
26.63 ± 0.47
25.94 ± 0.34
1.01 ± 0.08
15.11 ± 0.58
4.44
0.74
b
Metoprolol
b
Atenolol
2.45
b
c
Erythromycin
<0.19
>79.53
1
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a
b
Average values ± SD of two samples; See Supporting Information for experimental conditions. Low
c
to high permeable control, respectively; Papp values were expressed as “<” (less than) the values that
were calculated using the minimum concentration of the standards for receiver sides because the real
concentrations in the receivers were below the quantitation limit (BQL).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
a
a
a
Table 3. ICR mouse PK data of compounds oprozomib , 24c , 24t , and 24v .
Compound
oprozomib
5
24c
5
24t
NT
NT
NT
NT
24v
5
b
iv dose (mg/kg)
CL (mL/min/kg)
ss (L/kg)
164.30 ± 4.44
0.51 ± 0.02
2.4
67.20 ± 3.76
0.62 ± 0.08
9.6 ± 3.6
107 ± 11.8
0.48 ± 0.02
6.0 ± 3.6
V
T1/2 (min)
AUC0ꢀt (h × ng/mL)
506 ± 11
1238 ± 65
NT
784 ± 92
b
po dose (mg/kg)
25
25
519 ± 436
4.8
30
25
325 ± 82
4.8
C
max (ng/mL)
261 ± 35
13.2 ± 14.4
92 ± 60
Tmax (min)
AUC0ꢀt (h × ng/mL)
F (%)
13.2 ± 14.4
5
8 ± 32
142 ± 34
133 ± 57
141 ± 39
5.61 ± 1.35
2.15 ± 0.92
ND
3.72 ± 1.00
a
b
Average values ± SD of three rats (iv dose and po dose). Solution administration, iv formulation: 1%
DMSO, 10% Solutol, 10% EtOH, 75% saline; po formulation: 5% DMSO, 95% (0.5%MC). NT = not
tested. ND = data not determined.
To conquer the drawbacks of 24c, further structural optimization was demanded.
Due to the synthetic convenience, we were concerned about whether elaboration at
the phenyl moiety affected the biological profiles of compounds. As a result of
investigating the substitution pattern, a dramatic decrease in the ChTꢀL inhibitory
activity was observed when introducing replacements at the 4ꢀposition (24i-l) and the
5
ꢀposition (24m). This intolerance might be attributed to the steric clash between the
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5
5
6
substituents and residues Asp125, Pro126, and Ala127 in the β6 subunit. In contrast,
compounds 24g and 24h, with the 3ꢀposition substituted, displayed superior ChTꢀL
inhibitory activity to that of 24c. Notably, 24h, the methoxy counterpart, exerted
singleꢀdigit nanomolar IC50 values against ChTꢀL (IC50 = 4 nM) and all the three
tested cellꢀlines, including RPMI 8226 (IC50 = 5 nM), MM.1S (IC50 = 3 nM), and
MVꢀ4ꢀ11 (IC50 = 6 nM). Unfortunately, 24h was also metabolically unstable in MLM
0
1
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5
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Table 1). Afterwards, five or sixꢀmembered heteroaryl moieties were introduced as
surrogates for the phenyl group. Among the resultant counterparts, compound 24p,
with an Nꢀmethyl pyrazole embedded in the macrocycle, exhibited comparable ChTꢀL
inhibitory activity, as well as antiproliferative activity against RPMI 8226 and MM.1S
cell lines, to that of oprozomib. The cytotoxic activity of 24p against MVꢀ4ꢀ11
surpassed that of oprozomib, with an IC50 value at the singleꢀdigit nanomolar level.
Importantly, this result demonstrates the comparable metabolic stability in MLM to
that of oprozomib, with more than 60% remaining at 0.5 h postꢀincubation (Table 1).
From the SAR investigation undertaken above, we concluded that phenyl or a
5ꢀmembered Nꢀcontaining heteroaryl moiety, exemplified by pyrazole, at the
Nꢀterminus was probably beneficial for activity and that latter might also contribute to
improved metabolic stability. Bearing this in mind, we further broadened the
structural diversity of these macrocycle peptides. On the one hand, under the premise
of maintaining the phenyl as the Nꢀterminus, various types of linkers between the
moiety and P2 were examined. On the other hand, structural alteration was undertaken
via replacement of the pyrazole with its bioisosteres, exemplified by pyrrole and
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imidazole, or by modifying its orientation under the premise of maintaining the linker
of compound 24x, a close analog of 24p, obtained via reducing one oxygen atom at
the linker (Table 4). Compounds 24za and 24zb, both featuring phenyl as the
Nꢀterminus and a linker containing a single oxygen atom, exhibited slightly better
ChTꢀL inhibitory activity than that of oprozomib. However, their cellular potency was
inferior to that of oprozomib, probably attributed to the poor membrane permeability.
Similarly, compounds 30a and 30b, containing 3 and 5 oxygen atoms in the linker,
respectively, also failed to display acceptable in vitro potency. To our delight, several
promising macrocyclic peptides were identified among those bearing 5ꢀmembered
Nꢀcontaining heteroaryl moieties at the Nꢀterminus. The imidazole counterpart 24t
demonstrated more potent ChTꢀL inhibitory activity (IC50 = 14 nM) and
antiꢀproliferative efficacy against all the three tested cell lines (RPMI 8226, IC50 = 3
nM; MM.1S, IC50 = 1 nM; MVꢀ4ꢀ11, IC50 = 3 nM) than those of oprozomib. In
addition, compound 24v, characterized by pyrazole that bears a nitrogen atom
covalently attached to the linker as the Nꢀterminus, exerted comparable ChTꢀL
inhibitory activity (IC50 = 16 nM) and superior antiꢀproliferative efficacy against all
of the three tested cell lines (RPMI 8226, IC50 = 5 nM; MM.1S, IC50 = 2 nM;
MVꢀ4ꢀ11, IC50 = 3 nM) compared to those of oprozomib. In particular, both 24t and
1
1
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1
1
1
1
1
1
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2
2
2
2
2
2
2
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3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
24v were metabolically stable in MLM, with 92% and 75% remaining at 0.5 h
postꢀincubation (Table 4), respectively. The favorable in vitro potency and metabolic
stability of 24t and 24v were consequently transformed into remarkable proteasome
inhibition in vivo at both 2 h and 24 h after an oral dose of 30 mg/kg (Figure 4).
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a)
blood proteasome activity (P.O)
150
b)
blood proteasome activity (P.O)
1
50
00
2
h
2h
100
24h
24h
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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9
0
1
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9
0
1
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9
0
1
2
3
4
5
6
7
8
9
0
50
5
0
20%
1
3%
12%
10%
0
0
CMC
Opr
24c
24t
Opr
24c
24t
CMC
24t
24v
3
0 mg/kg 30 mg/kg 30 mg/kg 60 mg/kg 60 mg/kg 60 mg/kg
30 mg/kg
30 mg/kg
Figure 4. Mouse oral PD data for oprozomib, 24c, 24t, and 24v. CMC =
.5%CMCꢀNa; Opr = oprozomib.
0
Encouraged by the in vitro and in vivo biological activity, we then evaluated the PK
profiles of 24t and 24v in ICR mice following oral administration. As a result, 24t
displayed a dramatically lower oral exposure (AUC = 58 ng/ml after 1 h, 30 mg/kg)
0
ꢀt
than that of oprozomib (AUC0ꢀt = 142 ng/mL after 1 h, 25 mg/kg). By comparison,
4v exhibited better oral exposure (AUC0ꢀt = 141 ng/mL after 1 h, 25 mg/kg), thereby
2
meriting further investigation.
Table 4. In vitro data of epoxyketone macrocycles.
Cytotoxicity IC50 (nM)
ChTꢀL
Microsome
Stability
Compd
Linker
A
IC50 (nM)
352 ± 104
14 ± 4
RPMI 8226
MM.1S
MVꢀ4ꢀ11
24r
350 ± 17
13 ± 0.8
172 ± 21
421 ± 109
11 ± 3
NT
[a]
2
4s
4 ± 0.4
<5
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[
b]
2
4t
14 ± 4
37 ± 11
16 ± 1
3 ± 0.3
5 ± 1
1 ± 0.03
2 ± 0.2
3 ± 0.5
5 ± 1
92
2
4u
NT
[b]
24v
2 ± 0.2
5 ± 2
3 ± 0.3
2 ± 0.5
2 ± 0.4
32 ± 5
137 ± 7
12 ± 1
43 ± 4
9 ± 1
2 ± 0.1
5 ± 1
75
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4w
25 ± 2
NT
[b]
2
4x
20 ± 4
3 ± 0.8
73 ± 1
120 ± 42
55 ± 11
82 ± 9
19 ± 3
3 ± 1
48
2
4za
14 ± 1
66 ± 3
72 ± 16
33 ± 2
130 ± 5
28 ± 3
NT
NT
NT
NT
24zb
35 ± 9
3
0a
0b
116 ± 25
370 ± 42
47 ± 2
3
[b]
Oprozomib
/
/
60
a
b
Mouse, % remaining at 5 min. Mouse, % remaining at 0.5 h. NT = not tested.
Chemistry
The synthesis of the dipeptide intermediates 10a-d, 17a, 17b is depicted in scheme
. a) The commercially available BocꢀLꢀserine 5, first underwent benzylation to yield
a, which was coupled with allylmethyl carbonate to afford 7a. Subsequent removal
1
6
of the Bocꢀprotecting group and condensation with BocꢀOꢀmethylꢀserine provided the
dipeptide 9a. It was then converted into the hydrochloride 10a via unmasking of the
amine group. The commercially available methyl Lꢀserinate hydrochloride 11 was
2
protected with (Boc) O to generate 6b. It was converted into the dipeptide 10b after
being subjected to a similar chemical transformation as that for preparing 10a. In the
presence of sodium hydride, the hydroxyl group of 5 was alkylated to provide 12. The
following benzylation, removal of the Bocꢀprotecting group, condensation with
BocꢀOꢀmethylꢀserine and deprotection furnished the intermediate 10c. The hydroxyl
1
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2
2
2
2
2
2
3
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3
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3
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5
5
5
5
5
5
5
5
5
6
group of 6a was transformed into the iodine via nucleophilic substitution. The newly
formed 13 was coupled with allyl chloride to afford 7d. It was then subjected to
similar chemical transformations to those for preparing 10a and the dipeptide 10d was
obtained as the intermediate.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
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8
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
b) BocꢀLꢀserine
5
was directly alkylated by the corresponding
4ꢀmethylbenzenesulfonate to yield 14a or 14b. Subsequent oneꢀpot removal of the
Bocꢀprotecting group and esterification afforded 15a or 15b. After condensation with
BocꢀOꢀmethylꢀserine and catalytic hydrogenation, the dipeptide 17a or 17b was
obtained as the intermediate.
a
Scheme 1. Synthesis of dipeptide intermediates 10a-d, 17a, 17b
a
2 3
Reagents and conditions: (a) BnBr, K CO , DMF, 0 °C to rt, 20 h; (b) allylmethyl
carbonate, Pd(PPh ) , THF, 60 °C, 4 h; (c) 4N HClꢀDioxane, rt; (d)
3
4
BocꢀOꢀmethylꢀserine, HOBt, EDCI, DIPEA, DCM, 0 °C to rt, 5 h; (e) 4N
HClꢀDioxane, rt; (f) (Boc) O, TEA, MeOH, rt, 18 h, 90%; (g) NaH,
2
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6ꢀbromohexꢀ1ꢀene, DMF, 0°C to rt, 18 h, 56%; (h) PPh
3
, I
9%; (i) Zn, allyl chloride, CuBr, DMF, 60%; (j) NaH, 2ꢀ(2ꢀ(benzyloxy)ethoxy)ethyl
ꢀmethylbenzenesulfonate or 2ꢀ(2ꢀ(2ꢀ(benzyloxy)ethoxy)ethoxy)ethyl
ꢀmethylbenzenesulfonate, DMF, 0°C to rt, 12 h; (k) SOCl , MeOH, 0 °C to 60 °C, 2
2
, pyridine, THF, 0 °C to rt,
8
4
4
2
h; and (l) 10% Pd/C, MeOH, rt.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The synthesis of various phenyl, heteroaryl or heterocycloalkyl acids 20a-z bearing
alkene substructure of different length are displayed in scheme 2. The
hydroxylꢀsubstituted aryl carboxylates 18a-j, 18q and 18r were subjected to
alkylation and subsequent hydrolysis to generate the corresponding carboxylic
N
derivatives 20a-o, 20y and 20z. S Ar reaction of pentꢀ4ꢀenꢀol with bromoꢀsubstituted
heteroaryl carboxylates 18k and 18l provided 19p and 19q. They were then
hydrolyzed to form the carboxylic derivatives 20p and 20q. 20r was prepared via
condensation of methyl Lꢀprolinate hydrochloride 18m with pentꢀ4ꢀenoic acid and
hydrolysis. Pyrrole carboxylate 18n, imidazole carboxylate 18o, or pyrazole
N
carboxylate 18p was subjected to an S 2 reaction with corresponding alkenyl
bromides, and the newly formed alkylated products were hydrolyzed to the carboxylic
derivates 20s-w. Stille coupling of the bromineꢀsubstituted Nꢀmethyl pyrazole
carboxylate 18k with allyltributyltin and the following hydrolysis afforded the
carboxylic derivate 20x.
a
Scheme 2. Synthesis of acids 20a-z
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and conditions: (a) Different alkenyl bromides, K CO , DMF, 80 °C; (b) 2
2
3
M NaOH, THF/MeOH, rt ꢀ 50 °C, 12 h; (c) NaHMDS, pentꢀ4ꢀenꢀ1ꢀol, THF, ꢀ8 °C to
rt; (d) pentꢀ4ꢀenoic acid, HOBt, EDCI, DIPEA, DCM, 0 °C to rt, 6 h, 92%; (e) 1 M
LiOH, THF/MeOH, rt; and (f) allyltributyltin, Pd
0%.
2 3 3
(dba) , PtBu , CsF, THF, reflux, 5 h,
8
The synthesis of target compounds 24a-z, 24za and 24zb is described in scheme 3.
The dipeptide 10a, was condensed with the corresponding carboxylic intermediates
0a-w for introducing the Nꢀterminus, which provided the diolefins 21a-w.
2
Afterwards, ringꢀclosing metathesis (RCM) of diolefin in the presence of the Grubbs
secondꢀgeneration catalyst, as well as a oneꢀpot reduction of the CꢀC double bond and
removal of the benzylꢀprotecting group, gave the macrocyclic carboxylic derivatives
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2a-w. Finally, the warhead was installed via condensation with Pheꢀepoxyketone 23
3
9
(23 was synthesized following the literature previously reported from our group) to
provide 24a-w as the target compounds. Similarly, the dipeptide 10c was condensed
with the carboxylic intermediate 20x to afford the diolefin 21x, which then underwent
a sequence of RCM, reduction of the CꢀC double bond, removal of the
benzylꢀprotecting group, and condensation with 23 to furnish 24x as the target
compound. 10b, as the common intermediate, was condensed with the carboxylic
intermediates 20y-z, and the newly formed diolefins were subjected to RCM, catalytic
hydrogenation, and hydrolysis to provide the carboxylic intermediates 22y and 22z.
Ultimately, they were condensed with 23 to generate the target compounds 24y and
24z. 10d, was condensed with the carboxylic intermediates 20b and 20c, and the
resulting newly formed diolefins 21za and 21zb experienced similar chemical
transformations as those for preparing compounds 24a-x to yield 24za and 24zb as
the target compounds.
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9
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a
Scheme 3. Synthesis of target compounds 24a-z, 24za, and 24zb
1
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a
Reagents and conditions: (a) HOBt, EDCI, DIPEA, DCM, 0 °C to rt; (b) (Ⅲ ) Grubbs
secondꢀgeneration catalyst, toluene, 100 °C; (Ⅲ ) 10% Pd/C, MeOH, rt; (c) 23, HOBt,
HBTU, DIPEA, THF, 0 °C to rt, 3 h. and (d) 0.3 M LiOH, acetone, 0 °C to rt, 6 h.
The synthesis of macrocycles 30a and 30b bearing three and four oxygen atoms in
the linker, respectively, is depicted in scheme 4. Etherification of the alcohols 17a and
1
7b utilizing a Mitsunobu condition to yield 26a and 26b. For 26a and 26b, removal
of the allyl group of through a palladium catalyst and deprotection of Boc group with
TFA, followed by intramolecular cyclization in a low concentrated solution, led to the
cyclic peptides 28a and 28b, which were hydrolyzed and coupled with 23 to give 30a
and 30b, respectively.
a
a
Scheme 4. Synthesis of target compounds 30a and 30b
2
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9
0
a
Reagents and conditions: (a) allyl 2ꢀhydroxybenzoate, PPh , DIAD, THF, 0 °C to rt,
3
3 4
overnight; (b) Pd(PPh ) , 1,3ꢀdimethylbarbituric acid, DCM, rt, overnight; (c) (Ⅲ )
TFA, DCM, rt, 2 h; (Ⅲ ) HOBt, HBTU, DIPEA, THF, 0 °C to rt, 6 h; (d) 0.3 M LiOH,
acetone, 0 °C to rt, 6 h; and (e) 23, HOBt, HBTU, DIPEA, THF, 0 °C to rt, 3 h.
CONCLUSION
Upon attaching the P2 side chain to the aryl functionality at the Nꢀterminus through
a chemical linker, 30 macrocyclic peptides were successively designed and
synthesized on the basis of oprozomib for the pursuit of potent, metabolically stable,
and orally available proteasome inhibitors. As a result of the SAR investigation
focusing on the Nꢀterminus and the chemical linker, four compounds, including 24p,
24x, 24t, and 24v, have been identified to be biologically potent in vitro,
metabolically stable and effective in vivo following oral administration. Among the
compounds, the macrocyclic peptide 24v merits further development by virtue of its
excellent ChTꢀL inhibitory potency, low nanomolar antiꢀproliferative activity against
all the three tested cell lines, and superior metabolic stability in MLM, as well as
favorable inhibition against ChTꢀL in BABL/c mice following po administration
compared to that of oprozomib.
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6
EXPERIMENTAL SECTION
Chemistry. All reagents and solvents were purchased from commercial suppliers
and used without further purification unless otherwise indicated. Reactions were
monitored by thinꢀlayer chromatography (TLC) carried out on Silica gel 60 F254
plates supplied by Qingdao Puke Separation Material Corporation, and UV light was
used as the visualizing agent. Flash column chromatography was performed using
0
1
2
3
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8
9
0
1
2
3
4
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6
7
8
9
0
1
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9
0
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9
0
1
2
3
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8
9
0
200ꢀ300 mesh silica gel supplied by Qingdao Marine Chemical Factory, Qingdao,
China. A mixture of petroleum ether (PE, b.p. 60ꢀ90 ºC) and ethyl acetate (EtOAc)
was used as the eluting solvent for both TLC analysis and column chromatography
1
isolation. H NMR spectra were obtained on a Bruker Fourier transform spectrometer
13
(500 MHz) at 25
°C. C NMR spectra were recorded on a Bruker Fourier transform
spectrometer (125 MHz) spectrometer. All NMR spectra were calibrated using
1
13
residual solvents as internal references (for CDCl
3
: H NMR = 7.26, C NMR =
1
13
7
7.16; DMSO: H NMR = 2.50, C NMR = 39.52). All chemical shifts were reported
in parts per million (ppm) and coupling constants (J) in Hz. The following
abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t =
triplet, q = quartet, m = multiplet. Electrospray ionization mass spectroscopy
(ESIꢀMS) spectra were obtained with a Shimadzu LCMSꢀ2020 mass spectrometer
with mobile phases as methanol and water containing 0.1% formic acid. High
resolution mass spectra (HRMS) were measured on an Agilent 6224 TOF LC/MS
spectrometer using ESIꢀTOF (electrospray ionizationꢀtime of flight). Optical rotations
were measured on an Autopol SGWꢀ533 polarimeter (Shanghai Yidian Physical
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Optical Instrument Co., Ltd, Shanghai, China). Melting points were determined using
a Buchi Bꢀ540 capillary melting point apparatus and are uncorrected. Purity of all
biologically tested compounds was determined by HPLC to be >95%. HPLC analysis
was performed on Agilent 1260 infinity equipped with Cosmosil 5C18ꢀARꢀⅢ column
1
1
1
1
1
1
1
1
1
1
2
2
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0
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0
1
2
3
4
5
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7
8
9
0
(4.6 mm × 250 mm), eluting at 0.8 mL/min using a listed gradient: (mobile phase A,
H
2
O; mobile phase B, MeCN) (Table 5).
Table 5. Gradient of HPLC
t/min
mobile phase A (%) mobile phase B (%)
0
5
60%
60%
40%
40%
10%
10%
60%
60%
40%
40%
60%
60%
90%
90%
40%
40%
10
1
1
8
9
25
2
2
6
8
General procedure A for the synthesis of various phenyl, and heteroaryl carboxylates
19a-o, 19t-w and 19y-z) bearing alkene substructure of different length. A mixture of
(
commercial ester (33 mmol, 1.0 equiv), potassium carbonate (66 mmol, 2.0 equiv)
and corresponding different length of alkenyl bromide (46 mmol, 1.2 equiv) in 50 mL
DMF was heated to 80 ºC 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 x 2), brine (50 mL x 2). The organic layer was dried over
2 4
Na SO , filtered, and concentrated under reduced pressure. The residue was then
purified by flash chromatography on silica gel to give corresponding alkenyl ester.
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6
General procedure B for the synthesis of diolefins 21a-q, 21s-z, 21za, and 21zb. The
alkenyl ester (6.0 mmol, 1.0 equiv) was dissolved in THF/MeOH (1/1, 8 mL) and
aqueous NaOH 2 M (18.0 mmol, 3.0 equiv) was added. The solution was stirred at
room temperature for 12 h. The reaction mixture was then acidified to pH 2 with 3 N
HCl and extracted with EtOAc (a mixture of EtOAc: acetone = 3:1 for preparing 20t
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
5
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7
8
9
0
2 4
and 20u), washed with brine, dried (Na SO ), filtered, and concentrated in vacuo to
get the crude acid 20a-z and used for further reaction without further purification. The
crude acid (1.0 mmol, 1.0 equiv) was dissolved in 5 mL DCM and cooled to 0 °C. It
was sequentially treated with EDCI (1.8 mmol, 1.8 equiv) and HOBt (1.2 mmol, 1.2
equiv). After stirring at room temperature for 30 min, corresponding dipeptide
intermediate (1.2 mmol, 1.2 equiv) and DIPEA (3.0 mmol, 3.0 equiv) were added to
the reaction mixture and stirred for 30 min, raising the temperature to room
temperature and stirred for 3 h. The solvent was evaporated and the residue was
diluted with 10 mL EtOAc, then washed with saturated NH Cl solution (10 mL x 2),
4
3 2 4
saturated NaHCO (10 mL x 2) and brine (10 mL x 2), dried over Na SO , filtered,
and concentrated in vacuo. Chromatographic purification of the residue provided
diolefin derivatives.
General procedure C for the synthesis of macrocyclic carboxylic derivatives 22a-x,
22za-zb. A solution of the diolefin (0.20 mmol, 1.0 equiv) in toluene (0.01 molar)
was degassed with dry nitrogen for 15 min. The mixture was stirred for 5 min at 100
ºC. after which, a degassed solution of grubbs secondꢀgeneration catalyst (0.10 equiv)
in toluene (0.01 molar) was injected with a syringe for 30 min. The reaction was
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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 resulting products (a mixture of geometric isomers) were redissolved in
methanol (0.05 molar), stirred overnight with 10% Pd/C (0.10 equiv). The mixture
was then filtered over Celite, concentrated to provide the corresponding macrocyclic
carboxylic derivatives.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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9
0
General procedure D for the synthesis of target macrocycles 24a-z, 24za, 24zb. To a
solution of macrocyclic carboxylic derivate (0.10 mmol, 1.0 equiv), HOBt (0.12
mmol, 1.2 equiv), and HBTU (0.12 mmol, 1.2 equiv) in 2 mL THF at 0 ºC was added
Pheꢀepoxyketone (23, 0.12 mmol, 1.2 equiv), and DIPEA (0.15 mmol, 1.5 equiv). The
reaction mixture was stirred at room temperature for 3 h. The solvent was evaporated
and the residue was diluted with 5 mL EtOAc and then washed with saturated
3 2 4
NaHCO (5 mL x 2) and brine (5 mL x 2), dried over Na SO , filtered, and
concentrated in vacuo. Flash chromatography on C18 (50% to 80% MeOH in H O)
2
with a flow rate of 25 mL/min yielded the final product as a viscous liquid, which was
redissolved in EtOAc and subsequently added cyclohexane or petroleum ether. After
removal of the solvent, the corresponding product was afforded.
Benzyl (tert-butoxycarbonyl)-L-serinate (6a). Benzyl bromide (5.6 mL, 46.0 mmol)
was slowly added to a mixture of NꢀBocꢀLꢀSerine (5, 8.0 g, 39.0 mmol) and K CO
2
3
(6.4 g, 46.0 mmol) in DMF (100 mL) at 0 ºC. The solution was stirred at room
temperature for 20 h and then diluted with EtOAc (200 mL) and H O (200 mL). the
(100 mL x 2) and
2
organic layer was separated and washed with saturated NaHCO
3
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2 4
brine (100 mL x 2), dried over Na SO . Filtrated, and evaporated under vacuum. The
residue was resolved in EtOAc and added PE. Filtrated and washed with additional
1
PE to give 6a (5.2 g, 90%) as a while solid. mp = 68ꢀ70 ºC; H NMR (500 MHz,
0
1
2
3
4
5
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0
CDCl ) δ 7.40 – 7.31 (m, 5H), 5.45 (s, 1H), 5.28 – 5.16 (m, 2H), 4.48 – 4.38 (m, 1H),
3
3.99 (dd, J = 11.0, 3.5 Hz, 1H), 3.92 (dd, J = 11.0, 3.0 Hz, 1H), 1.94 (br s, 1H), 1.45
+
(
s, 9H) ppm; ESIꢀMS: m/z = 296 [M+H] .
Methyl (tert-butoxycarbonyl)-
hydrochloride (3.6 g, 30.0 mmol) in MeOH (50 mL), diꢀtertꢀbutyl dicarbonate (7.9 g,
6 mmol, 1.2 equiv) and TEA (8.3 mL, 60.0 mmol) was added slowly. The mixture
was stirred at room temperature for 18 h. The solvent was evaporated under vacuum
prior to addition of EtOAc (100 mL) and H O (100 mL). The organic layer was
separated and washed with saturated NH Cl (80 mL x 2), brine (80 mL x 2). Dried
over Na SO4, filtered and evaporated under vacuum. The residue was purified by
L-serinate (6b). To a solution of methyl Lꢀserinate
3
2
4
2
chromatography on silica gel to give 5.9 g of the BocꢀLꢀserꢀOMe in 90% yield as a
1
light yellow oil. H NMR (500 MHz, CDCl
3
) δ 5.47 (s, 1H), 4.43 – 4.33 (m, 1H), 3.96
(dd, J = 11.0, 3.5 Hz, 1H), 3.90 (dd, J = 11.0, 3.5 Hz, 1H), 3.78 (s, 3H), 2.27 (br s,
+
1
H), 1.45 (s, 9H) ppm; ESIꢀMS: m/z = 220 [M+H] .
-allyl- -(tert-butoxycarbonyl)- -serinate (7a). A threeꢀnecked flask was
charged with 6a (4.4 g, 15.0 mmol) and Pd(PPh ) (0.87 g, 0.75 mmol) under N . The
Benzyl
O
N
L
3
4
2
allylmethyl carbonate (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 4h upon which TLC (5/1
PE/EtOAc) indicated the loss of starting material. The solvent was removed under
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reduced pressure and the residue was diluted with EtOAc (40 mL) and washed with
3 4
NaHCO (40 mL x 2) and brine (40 mL x 2). the organic layer was dried over NaSO ,
filtered, and concentrated under reduced pressure. The residue was purified by
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
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3
3
3
3
3
3
4
4
4
4
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4
4
4
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5
5
5
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5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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8
9
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1
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3
4
5
6
7
8
9
0
column chromatography (SiO ; 0% to 30% EtOAc in PE) to afford 4.1 g (82%) of the
2
1
product 7a as a clear oil. H NMR (500 MHz, CDCl
3
) δ 7.41 – 7.30 (m, 5H), 5.86 –
5.68 (m, 1H), 5.41 (d, J = 8.5 Hz, 1H), 5.32 – 5.17 (m, 2H), 5.17 – 5.10 (m, 2H), 4.51
–
4.42 (m, 1H), 3.99 – 3.84 (m, 3H), 3.65 (dd, J = 9.5, 3.0 Hz, 1H), 1.45 (s, 9H) ppm;
+
ESIꢀMS: m/z = 336 [M+H] .
Methyl
O
-allyl-
N
-(tert-butoxycarbonyl)-
L
-serinate (7b). A threeꢀnecked flask was
(0.53 g, 0.46 mmol) under N . The
charged with 6b (2.0 g, 9.1 mmol) and Pd(PPh
3
)
4
2
allylmethyl carbonate (1.5 mL, 12.7 mmol) in anhydrous THF (40 mL) was syringed
over 10 min. The reaction mixture was heated to 60 °C for 4h upon which TLC
indicated the loss of starting material. The solvent was removed under reduced
pressure and the residue was diluted with EtOAc (50 mL) and washed with NaHCO3
(
50 mL x 2) and brine (40 mL x 2). the organic layer was dried over NaSO
4
, filtered,
and concentrated under reduced pressure. The residue was purified by column
chromatography (SiO
2
; 0% to 20% EtOAc in PE) to afford 1.4 g (59%) of the product
) δ 5.87 – 5.78 (m, 1H), 5.38 (d, J = 8.0
1
7
b as a clear oil. H NMR (500 MHz, CDCl
3
Hz, 1H), 5.27 – 5.20 (m, 1H), 5.20 – 5.15 (m, 1H), 4.42 (dt, J = 7.0, 3.0 Hz, 1H), 4.01
– 3.92 (m, 2H), 3.85 (dd, J = 9.5, 3.0 Hz, 1H), 3.76 (s, 3H), 3.64 (dd, J = 9.5, 3.0 Hz,
+
1
H), 1.45 (s, 9H) ppm; ESIꢀMS: m/z = 260 [M+H] .
2
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Benzyl
(0.79 mL, 6.7 mmol) was slowly added to a mixture of 12 (1.6 g, 5.6 mmol) and
CO (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 H O (30 mL). The
N-(tert-butoxycarbonyl)-O-(hex-5-en-1-yl)-L-serinate (7c). Benzyl bromide
K
2
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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0
2
3
organic layer was separated and washed with saturated NaHCO (20 mL x 2) and
brine (20 mL x 2), dried over Na
2
4
SO . Filtrated, and evaporated under vacuum to give
1
7
c (1.1 g, 52%) as a viscous liquid. H NMR (500 MHz, CDCl
3
) δ 7.41 – 7.29 (m,
5H), 5.85 – 5.71 (m, 1H), 5.38 (d, J = 8.5 Hz, 1H), 5.28 – 5.09 (m, 2H), 5.05 – 4.89
(m, 2H), 4.52 – 4.39 (m, 1H), 3.84 (dd, J = 9.5, 3.0 Hz, 1H), 3.63 (dd, J = 9.5, 3.0 Hz,
1H), 3.45 – 3.27 (m, 2H), 2.07 – 1.99 (m, 2H), 1.55 – 1.33 (m, 13H) ppm; ESIꢀMS:
+
m/z = 378 [M+H] .
Benzyl (
activated zinc (0.33 g, 4.9 mmol) at 0 ºC under N
3 (0.50 g, 1.2 mmol) in DMF. The reaction mixture was removed from ice bath and
allowed to stir at room temperature for 3 h, upon with TLC (1/4 EtOAc in PE)
indicated the loss of starting material and formation of a lower R spot. At this point,
S)-2-((tert-butoxycarbonyl)amino)hex-5-enoate (7d). To a flask containing
2
was added dropwise a solution of
1
f
the reaction mixture as stopped to let the solid settle to the bottom. The supernatant
was then carefully transferred by syringe to a suspension of copper (I) bromide (36
mg, 0.25 mmol) in DMF (2 mL) at ꢀ15 ºC that also contained allyl chloride (0.22 g,
1.9 mmol). After complete addition, the cooling bath was removed and stirring was
continued overnight. At this point, EtOAc (10 mL) was added to the reaction mixture
2 2 2 3
and stirred for 15 min. washed with H O (10 mL x 2), 1N Na S O (20 mL x 2), and
2
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2 4
brine (20 mL x 1). Dried over Na SO , filtrated and concentrated in vacuo. The crude
residue was purified by flash chromatography (SiO
2
, 1/8 EtOAc in PE) to yield 0.23 g
) δ 7.41 – 7.31 (m, 5H),
1
(
60%) of 7d as a colorless oil. H NMR (500 MHz, CDCl
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5.81 – 5.70 (m, 1H), 5.24 – 5.10 (m, 2H), 5.10 – 4.93 (m, 3H), 4.42 – 4.32 (m, 1H),
2.15 – 2.02 (m, 2H), 1.97 – 1.82 (m, 1H), 1.78 – 1.67 (m, 1H), 1.44 (s, 9H) ppm;
+
ESIꢀMS: m/z = 320 [M+H] .
Benzyl
O-allyl-N-(N-(tert-butoxycarbonyl)-O-methyl-L-seryl)-L-serinate (9a). To
a solution of 7a (4.1 g, 12.0 mmol) in 4N HClꢀDioxane (24 mL) was stirred at room
temperature for 2 h. The volatiles were removed in vacuo. The residual HCl was
2
further removed by adding Et O (12 mL x 2) to the hydrochloride salt followed by its
removal in vacuo. This residue 8a and BocꢀOꢀmethylꢀLꢀSerine (2.2 g, 10.0 mmol)
were dissolved in THF (30 mL). The mixture was then treated sequentially at 0 ºC
with DIPEA (2.5 mL, 15.0 mmol), HOBt (1.6 g, 12.0 mmol), and EDCI (3.5 g, 18.0
mmol). The mixture was stirred for 5 h at room temperature. The solvent was
evaporated in vacuum and the residue was diluted by EtOAc (50 mL) and H
mL). The organic layer was separated and washed with saturated NaHCO (50 mL x
) and brine (30 mL x 1). Dried over Na SO , filtered and concentrated in vacuo.
2
O (50
3
2
2
4
Chromatography (10% EtOAcꢀHexanes) provided 9a (2.7 g, 61%) as a viscous liquid.
1
H NMR (500 MHz, CDCl ) δ 7.39 (d, J = 6.5 Hz, 1H), 7.36 – 7.29 (m, 5H), 5.80 –
3
5.70 (m, 1H), 5.43 (d, J = 5.0 Hz, 1H), 5.26 – 5.21 (m, 1H), 5.21 – 5.16 (m, 1H), 5.16
–
5.11 (m, 2H), 4.74 (dt, J = 8.0, 3.0 Hz, 1H), 4.31 – 4.24 (m, 1H), 3.94 – 3.87 (m,
3H), 3.77 (dd, J = 9.0, 4.0 Hz, 1H), 3.65 (dd, J = 9.5, 3.0 Hz, 1H), 3.43 (dd, J = 9.0,
2
9
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