Design, Synthesis, and Conformation-Activity Study of Unnatural Bridged Bicyclic Depsipeptides as Highly Potent Hypoxia Inducible Factor-1 Inhibitors and Antitumor Agents

Article abstract of DOI:10.1021/acs.jmedchem.9b02039

By carrying out structural modifications based on the bicyclic peptide structure of echinomycin, we successfully synthesized various powerful antitumor derivatives. The ring conformation in the obtained compounds was restricted by cross-linking with an unnatural bond. The prepared derivatives were demonstrated to strongly suppress the hypoxia inducible factor (HIF)-1 transcriptional activation and hypoxia induction of HIF-1 protein expression. Particularly, alkene-bridged derivative 12 exhibited remarkably potent cytotoxicity (IC₅₀ = 0.22 nM on the MCF-7 cell line) and HIF-1 inhibition (IC₅₀ = 0.09 nM), which considerably exceeded those of echinomycin. Conformational analyses and molecular modeling studies revealed that the biological activities were enhanced following restriction of the conformation by cross-linking through a metabolically stable and rigid bridge bond. In addition, we proposed a new globular conformation stabilized by intramolecular πstacking that can contribute to the biological effects of bicyclic depsipeptides. The developments presented in the current study serve as a useful guide to expand the chemical space of peptides in drug discovery.

Full text of DOI:10.1021/acs.jmedchem.9b02039

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Article
Design, Synthesis, and Conformation–Activity Study of Unnatural Bridged
Bicyclic Depsipeptides as Highly Potent HIF-1 Inhibitors and Antitumor Agents
Kota Koike, Masanobu Nagano, Masahiro Ebihara, Tasuku
Hirayama, Mieko Tsuji, Hiroaki Suga, and Hideko Nagasawa
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b02039 • Publication Date (Web): 23 Mar 2020
Downloaded from pubs.acs.org on March 27, 2020
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Design, Synthesis, and Conformation–Activity
Study of Unnatural Bridged Bicyclic Depsipeptides
as Highly Potent HIF-1 Inhibitors and Antitumor
Agents
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†
‡
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Kota Koike , Masanobu Nagano , Masahiro Ebihara , Tasuku Hirayama , Mieko Tsuji ,
‡
*,†
Hiroaki Suga and Hideko Nagasawa
†Laboratory of Pharmaceutical and Medicinal Chemistry, Gifu Pharmaceutical
University, Gifu-city, Gifu 501-1196, Japan
‡Department of Chemistry, The University of Tokyo, Bunkyoku, Tokyo 113-0033, Japan.
§Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu
University, Gifu-city, Gifu 501-1193, Japan
KEYWORDS. Bicyclic peptide, HIF-1 inhibitor, antitumor agent, molecular modeling.
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ABSTRACT
By carrying out structural modifications based on the bicyclic peptide structure of Echinomycin,
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we successfully synthesized various powerful antitumor derivatives. The ring
conformation in the obtained compounds was restricted by cross-linking with an unnatural
bond. The prepared derivatives were demonstrated to strongly suppress the hypoxia
inducible factor (HIF)-1 transcriptional activation and hypoxia induction of HIF-1 protein
expression. Particularly, alkene-bridged derivative 12 exhibited remarkably potent
cytotoxicity (IC = 0.22 nM on the MCF-7 cell line) and HIF-1 inhibition (IC = 0.09 nM),
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which considerably exceeded those of echinomycin. Conformational analyses and
molecular modeling studies revealed that the biological activities were enhanced following
restriction of the conformation by cross-linking through a metabolically stable and rigid
bridge bond. In addition, we proposed a new globular conformation stabilized by
intramolecular π stacking that can contribute to the biological effects of bicyclic
depsipeptides. The developments presented in the current study serve as a useful guide
to expand the chemical space of peptides in drug discovery.
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INTRODUCTION
With the growing interest in expanding the chemical space for exploring new therapies,
synthetic macrocycles and constrained peptides have recently attracted significant
attention.1, 2 Such molecules are characterized by medium size and cyclic structures.
They are also thought to combine the best properties of biologics and small molecules.
Generally, cyclic peptides are considered to exhibit enhanced binding affinity, selectivity,
and metabolic stability compared with their linear counterparts. Among them, bicyclic
peptides display significantly enhanced conformational rigidity compared with monocyclic
peptides, which is crucial for increasing the affinity of the target compound. Accordingly,
as a result of greater conformational rigidity, metabolic stability, and higher target
selectivity, bridged cyclic peptides are considered potential drug scaffolds for the next-
generation therapeutics.3-5
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Although recent advances in biotechnologies involving the preparation and screening
of de novo designed bicyclic peptide libraries have made it possible to routinely discover
a variety of biologically active peptides in a high-throughput platform,6, 7 challenges in
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optimizing the pharmacological properties such as cell permeability and oral
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bioavailability still exist. Thus, lead optimization through the modification of biologically
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active natural membrane-penetrating peptides is still considered a promising strategy for
drug development. Echinomycin (Ec, 1) and related bisintercalating natural products
characterized by unusual thioacetal bridged N-methylated cyclic depsipeptide scaffold
with two pendant planar chromophores constitute a representative family of antitumor and
anti-viral bicyclic peptides.8, 9 Ec is known to exhibit highly potent antitumor activity
through binding DNA in a sequence-specific fashion. The molecule intercalates into the
consensus 5′-ACGT-3′ sequence of the hypoxia inducible factor (HIF)-1 recognition site,
namely, hypoxia response element (HRE).10-12 Consequently, Ec has been extensively
studied in the medicinal chemistry and synthetic biology fields with the aim to expand the
diversity of the structure and improve the bioactivity.13-16 Ec therefore appears to be a
promising candidate as the lead compound for structural modification studies of antitumor
bicyclic peptides. Hypoxia is recognized as a hallmark of solid tumors and plays a crucial
role in important physiological and pathological events including metabolism,
proliferation, survival, invasion, metastasis, and angiogenesis.17 Over the last two
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decades, HIF-1, a master transcription factor of hypoxic gene expression, has emerged
as a promising therapeutic target for drug development not only in the field of cancer
treatment but also for various other diseases related to ischemia and oxidative stress.18-
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Considerable efforts are unceasingly made to develop HIF-1 inhibitors for cancer
treatment.22-24 Moreover, Ec has been subjected to a variety of clinical trials due to its
highly potent tumor cytotoxicity; however, no significant effect was observed in the Phase
II studies.25, 26 Nevertheless, numerous preclinical studies focusing on the strong and
specific HIF-1 inhibitory effect of Ec have been conducted.27, 28 Recently, the importance
of Ec has been highlighted again with the effect of specifically inhibiting cancer stem
cells.29, 30 In addition, the development of an antibody-drug conjugate using Ec analogs
as payloads is also a promising approach.31
We have previously conducted structural development studies based on a unique
bicyclic depsipeptide scaffold of quinomycin antibiotics, focusing on their potent antitumor
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effects and the specific inhibitory effect of the transcriptional activation of HIF-1α. We
established a valuable and practical liquid-phase procedure for the total synthesis of
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Triostin A (TA, 2) and its derivatives. We also found that TA exhibits moderate antitumor
effects (IC : 2.0 μM) on the MCF-7 cells and a potent inhibiting effect of the HIF-1α
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transcriptional activity (IC : 26.7 nM) in a HRE-luciferase reporter gene assay (Figure 1).
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Intriguingly, it was found that quinomycin antibiotics such as Ec and TA containing a
common depsipeptide ring displayed strong inhibitory effect on the hypoxia-induced
protein expression of HIF-1α. This was a remarkable finding, because the protein
expression of HIF-1α is an event upstream of DNA binding, suggesting the existence of
a potential target molecule other than HRE.
Nonetheless, our previous structural modifications of the cyclic octadepsipeptide of TA
did not lead to improvement of the biological activities, which remained 2-3 orders of
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magnitude lower than Ec. Hence, to enhance the strong biological activities of Ec, we
decided to focus on the bridge scaffold, which is the only dissimilarity from the structure
of TA. TA contains a disulfide between cysteine residues, whereas Ec possesses an
unusual dithioacetal bond.33, 34 Overlaying the crystal structures of Echinomycin analog
(Ec-2QN), which is a result of a substitution of the bis-quinoxaline moiety with a bis-
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quinoline scaffold, and TA revealed that the two chromophore moieties are brought
significantly closer together in Ec-2QN than in TA (Figure 1).35, 36 As it can be seen, the
distance between the carbonyl groups of the aromatic moiety shown as “distance a” and
the distance between the two bridgehead carbons of Ec-2QN, i.e., “distance b” were
significantly shorter than those of TA. This observation implied that the cross-bridge bond
structure plays a critical role in the biological activities of quinomycin antibiotics and that
the more constrained ring structure results in a more potent antitumor activity and HIF-1
inhibitory effect.
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Figure 1. Comparison of conformations and biological activities of Ec (1) and TA (2). (A)
Crystal structure of Ec-2QN (CCDC no. 131833) and TA (2) (CCDC no. 131836) and
overlay of Ec-2QNꢀ(yellow) and TA (2) (green). Distance a: the distance between the
carbonyl groups of the quinoxaline moiety. Distance b: the distance between the two
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bridgehead position carbons. (B) Conformation parameters, and half maximal inhibition
concentration (IC ) values of Ec (1) and TA (2) for cytotoxicity against MCF-7 cells and
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of inhibition of hypoxia-induced HIF-1 activation. a. Measured at crystal structure of Ec-
2QN.
With this preliminary analysis of the conformation–activity relationship in hand, we
envisioned that stronger activity could be obtained by introducing unprecedented
appropriate bridge structure to increase the rigidity of the octadepsipeptide ring.
Consequently, we conducted structural modification of the bridged cyclic
octadepsipeptide scaffold focusing on the structure of cross-bridge moiety. Although Ec
can be biosynthesized from the precursor antibiotic TA by nonribosomal peptide
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synthase,36, 37 the total synthesis of Ec has not yet been achieved because of the difficulty
of constructing the unusual dithioacetal bridge by chemical synthesis. In addition, several
studies have been reported concerning the synthetic studies of the structure–activity
relationship (SAR) of natural or synthetic quinomycin antibiotics.13, 15, 38, 39 Nevertheless,
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only a few examples of oxidation or alkylation of the sulfur-containing moiety on the cross-
bridge scaffold have been described.32,
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Thus, establishment of the structural
requirements for the high potency of Ec by the 3-D SAR studies through the structural
modification of the cross-bridge on the bicyclic peptide scaffold is expected to provide
unique and valuable viewpoints for future peptide-based drug design.
Herein, we describe the design, synthesis, biological evaluation, and molecular
modeling studies on the conformationally constrained cyclic octadepsipeptide derivatives
with unnatural cross-bridge structures derived from quinoxaline antibiotics to elucidate
the structural requirements for highly potent antitumor and HIF-1 inhibiting effects.
RESULTS AND DISCUSSION
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Strategy for SAR study of bridged bicyclic octadepsipeptides.
As mentioned above, in our previous structural modification study of the disulfide-bridged
octadepsipeptide scaffold, tumor cytotoxicity and inhibiting effect of HIF-1 transcriptional
activation were significantly reduced for diastereoisomers containing one or two D-MeVal
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residues and a thiosulfonate group. We therefore subsequently planned to investigate
the relationship between conformation and activity by modifying the cross-bridge structure
on the cyclic octadepsipeptide scaffold, as shown in Figure 2. We designed thioether-
and selenoether-linked derivatives, since such bridge bonds were expected to constrain
the depsipeptide ring to the same extent as the unusual dithioacetal bridge of Ec. The
ring-closing metathesis (RCM), which is an effective orthogonal chemical cross-linking
reaction, allowed the synthesis of an olefin-bridged derivative with a greater ring strain.41
In addition to the constrained cyclic analogs, more flexible derivatives, including
compounds containing a cleaved disulfide bond and an elongated thioacetal moiety, were
also synthesized for comparison.
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Figure 2. Strategy for molecular modification and SAR study of quinomycin antibiotics.
Chemical synthesis
In the first instance, alkene (11) and alkane (13) type derivatives, in which a disulfide bond
was replaced with a C–C bond to improve the metabolic stability, were prepared (Scheme
1). We employed the RCM reaction, which enabled an efficient chemoselective and
regioselective synthesis of complex bicyclic peptides.41 The sequence started with the
synthesis of Cbz-D-Ser(Boc-MeVal)-OAll (3) according to an optimized procedure
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previously reported for the TA analogs. Compound 3 was subsequently condensed with
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N-methyl-allyl-glycine derivative 4 affording a mixture of diastereomers 5. The next step
involved conjugation of N-Boc-L-Ala-OH to 5 to give tetradepsipeptide 6 as a single
isomer in four steps and a total yield of 62%. Half of the obtained compound 6 was then
deallylated to give 7. Subsequently, N-Boc deprotection of 6 was followed by
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condensation with compound
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using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-
methylmorpholinium chloride (DMT-MM)42 to afford a linear octadepsipeptide 8.
Compound 9 obtained by deallylation of 8 was subjected to intramolecular cyclization
(RCM) using Grubbs second-generation catalyst,43 followed by intramolecular amide
bond formation to produce olefin-bridged bicyclic peptide 10 as a mixture of (E) and (Z)
isomers in three steps and 69% yield. Next, following the removal of the Cbz group by a
catalytic reduction, 10 was condensed with quinoxaline-2-carboxylic acid to obtain the
target alkene derivative 11 as a single isomer crystal in 55% yield (8.9% overall yield from
3
in 13 steps). Single crystal structure analysis of 11 revealed that the configuration of the
alkene moiety is Z (Figure 3). The Z isomer of 10 was isolated, deprotected, and then
44
condensed with 3-hydroxyquinoline-2-carboxylic acid to give 12. In addition, the alkane-
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type derivative 13 was also obtained by hydrogenation of 10 under a pressure of 3 atm,
followed by the introduction of quinoxaline-2-carboxylic acid.
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
Scheme 1. Synthesis of alkene derivatives 11, 12, and alkane 13.
a) HCl/AcOEt, (b) 4, DMT-MM, AcOEt, (c) TFA, (d) Boc-Ala-OH, DMT-MM, NMM, DMF,
(e) Pd (dba) , PPh , N-methylaniline, THF, (f) 7, DMT-MM, AcOEt (g) Grubbs 2nd
2
3
3
catalyst, CH Cl , (h) DMT-MM, NMM, DMF (1 mM), (i) Pd(OH) /C, H (1 atm), MeOH, (j)
2
2
2
2
quioxaline-2-carboxylic acid, DMT-MM, DMF, (k) thioanisole, TFA, (l) 3-hydroxyquinoline-
-carboxylic acid, EDCI·HCl, HOAt, NaHCO , DMF, (m) Pd(OH) /C, H (3 atm), MeOH.
2
3
2
2
The synthetic procedures for thioether-bridged analog as a conformational mimic of Ec
were subsequently investigated (Scheme 2 and 3). The linear octadepsipeptides 21 and
2
2 were synthesized by the condensation of half segments 18 and 1932 as shown in
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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
Scheme 2. Firstly, two different routes resulting in the cyclization of the linear
octadepsipeptide 21 or 22 were examined (Scheme 3). Route 1 involved an initial
thioether-bridged ring formation by intramolecular nucleophilic substitution or conjugate
addition; however, the reaction failed under various conditions. As shown in route 2, the
first cyclization of linear peptides 22 and 21 successfully yielded the intermediate cyclic
octadepsipeptides 25 and 26, respectively. Unfortunately, intramolecular thioether
formation was also not successful in this case, because the presence of N-MeSer at the
bridgehead position readily led to undesired β-elimination. Finally, we investigated route
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3, which involved a double cyclization of a symmetric H-shaped intermediate 27
containing two carboxyl and two amino end groups. However, cyclization of the H-shaped
intermediate was expected to form two isomers, 24 and 24a, possessing theta- and
45
manacle-shaped rings, respectively. It has previously been reported that both theta- and
manacle-shaped isomers of TA were obtained by a double cyclization of a H-shaped
46
intermediate in a solid phase procedure. In the study, double cyclization between N-Me-
Cys and N-Me-Val afforded an unnatural conformer of TA. On the other hand, based on
our earlier research, the synthetic strategy employed in the current study involved the
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selection of compound 27 as an H-shaped key intermediate for the double cyclization
sequence, because we previously determined that cyclization between D-Ser and Ala can
suppress racemization during the conjugation step.32
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
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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
Scheme 2. Synthesis of linear peptides 21 and 22.
(a) Boc O, 1,4-dioxane, NaOH aq., (b) imidazole, TBDMSCl, THF, (c) NaH, MeI, dry
2
THF, (d) allylbromide, K CO , DMF, (e) AcOH, THF, H O, (f) allyl ethyl carbonate,
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
2
Pd (dba) , PPh , THF, (g) Pd (dba) , PPh , N-methylaniline, THF, (h) HCl/AcOEt, (i) 16,
2
3
3
2
3
3
DMT-MM, AcOEt, (j) Boc-Ala-OH, DMT-MM, NMM, DMF, (k) 19, DMT-MM, AcOEt.
Intramolecular Cyclization [Route 1]
Cbz
Cbz
O
O
HN
O
HN
N
S
O
N
O
OH
N
O
OH
NH
H
N
O
HN
O
N
HN
O
N
O
H
H
N
O
N
O
N
O
O
O
N
O
N
O
O
Cbz
O
Cbz
O
OH
Cbz
O
S
SBam
O
Cbz
O
N
O
H
N
H
N
NH H
N
O
N
Boc
O
N
Boc
O
N
O
O
O
22
23
24
Intramolecular Cyclization [Route 2]
Cbz
O
O
O
HN
N
O
N
O
N
O
OH
NH
H
N
H
NH
O
N
HN
O
N
H
N
N
O
O
N
O
O
O
O
O
a, b
Cbz
O
Cbz
O
OH
Cbz
O
OAll
O
OAll
SBam
SBam
SBam
O
Cbz
O
Cbz
O
O
N
O
N
H
N
N
O
NH H
N
O
H
NH
N
O
N
Boc
O
N
O
N
O
21
25
26
26% over 2 steps
Cbz
O
N
O
N
O
HN
O
N
S
O
N
O
OH
NH
O
N
NH
H
N
HN
O
N
H
H
N
N
O
O
N
O
N
O
O
O
O
O
Cbz
O
Cbz
O
Cbz
OH
OH
a, b
SBam
SBam
O
Cbz
O
Cbz
O
N
O
O
N
O
H
N
N
O
NH H
NH H
N
O
N
Boc
O
N
O
O
O
22
26
ca. 38% over 2 steps
24
(moderate purity)
Intramolecular Cyclization [Route 3]
O
H
Cbz
O
N
O
HN
N
O HN
N
Cbz
Boc
N
O
O
O
NH
H
N
O
N
H
N
O
N
O
N
O
N
O O
O
N
O
OH
Cbz
O
S
S
+
O
N
S
O
O
Cbz
O O
N
O
N
OH
O
H
N
NH H
O
O
N
Boc
O
N
Cbz
NH
O
N
H
NH
O
O
O
Cbz
27
24
byproduct 24a
Scheme 3. Investigation of synthetic procedure for thioether bridged derivative 24.
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(
a) TFA, (b) DMT-MM, NMM, DMF (1 mM).
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
Scheme 4 demonstrates the synthesis of thioether- and selenoether-linked derivatives
39 and 40 via the corresponding H-type depsipeptide intermediate (route 3). For the
synthesis of the H-shaped intermediates, lanthionine derivatives 31 and 32 were first
prepared, followed by the extension of the two C-terminals, and then the addition of Boc-
Ala-OH to both of the N-terminals to obtain 27 and 37.
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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
R2
Boc
N
O
O
a
0
0
b
28
R1
N
N
S or Se
O
Boc
O
Boc
O
N
Boc O R2
0.55 equiv.
OH
OTs
15
28
1 2
2
9, R = S, R = O-allyl, 57% from 15
1
2
30, R = Se, R = O-allyl, 54% from 15
c
1
2
3
1, R = S, R = OH, 97%
1
2
32, R = Se, R = OH, 87%
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
O
O
R2
O
Cbz
Cbz
N
N
Boc
HN
H
H
O
O
O
O
H
N
O
O
Cbz
O
N
Boc
N
O
d, e
f, g
N
N
N
N
N
O
O
O
O
R1
O
Boc
R
N
Boc
N
O
O
O
O
3
NH
Boc
O
H
N
H
2.2 equiv.
N
Cbz
Cbz
O
O
R2
O
33, R = S
4, R = Se
1
2
3
5, R = S, R = O-allyl
3
1
2
3
6, R = Se, R = O-allyl
c
1
2
2
7, R = S, R = OH, 49% from 31
1
2
37, R = Se, R = OH, 39% from 32
O
O
N
O
N
N
R
O
NH
NH
H
N
O
H
N
O
O
Qx
N
O
N
O
f, h
Cbz
O
i, j
O
O
2
7 or 37
R
O
O
Cbz
O
O
O
N
O
N
Qx
N
NH H
NH H
O
N
O
N
O
O
2
3
4, R = S, 21%*
8, R = Se, 15%*
39, R = S, 37%* (total 1.1% from 15)
40, R = Se, 27%* (total 0.42% from 15)
Scheme 4. Synthesis of thioether and selenoether bridged derivatives, 39 and 40.
(
a) Ts-Cl, pyridine, DMAP, dry CH Cl (b) LiBHEt , dry THF, (c) Pd (dba) , PPh , N-
2
2,
3
2
3
3
methylaniline, THF, (d) HCl/AcOEt, (e) 31 or 32, DMT-MM, AcOEt, (f) TFA, (g) Boc-Ala-
OH, DMT-MM, NMM, DMF, (h) DMT-MM, NMM, DMF (1 mM), (i) thioanisole, TFA, (j)
quioxaline-2-carboxylic acid, DMT-MM, NMM, DMF. *The reaction yield was calculated
from HPLC analysis.
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As shown in Scheme 4, N-Boc-N-Me-L-Ser-OAll (15) was obtained from L-Ser in five
steps and 42% yield. Tosyl ester 28 derived from 15 was then converted to a thiol or
selenol in the presence of elemental sulfur or selenium and LiBHEt3.47, 48 Subsequently,
this intermediate was subjected to a nucleophilic substitution reaction with an equivalent
of 28, followed by deallylation to give N-Me-Lan derivative 31 or 32 as a diastereomeric
mixture. After removal of the Boc group from dipeptide 3, the resulting amine was
condensed with dicarboxylic acid 31 or 32 to give hexadepsipeptide 33 or 34. Compound
33 or 34 was subjected to a condensation with Boc-Ala-OH immediately after the removal
of the Boc group, followed by deallylation to afford H-shaped octadepsipeptide 27 or 37
as a diastereomeric mixture. Subsequently, following the removal of the Boc group, the
resulting crude product was treated with DMT-MM in DMF under highly diluted conditions
to give a mixture of theta-shaped bicyclic peptide 24 and several isomers (Figure S16).
These isomers were found to have the same molecular weight as 24 by liquid
chromatography-mass spectrometry (LC-MS) analysis; however, they were challenging
to separate and identify. Thus, without further purification, the mixture of isomers was
treated with trifluoroacetic acid and thioanisole to remove the Cbz groups, followed by
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
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1
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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
condensation with quinoxaline-2-carboxylic acid to yield the target compound 39 as a
single isomer by crystallization. Furthermore, the selenoether-bridged bicyclic peptide 40
was also successfully prepared from 37 using the same procedure. Pleasingly, the
constrained bridged bicyclic octadepsipeptides 11, 39, and 40 all gave single crystals and
could be subjected to X-ray structural analysis (Figure 3, Figure S1 and Table S1).
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
Figure 3. X-ray crystal structural analyses of constrained bridged bicyclic
octadepsipeptides. (A) ORTEP drawing (50% probability ellipsoids) of 11(CCDC no.
1885767), 39(CCDC no. 1885766), and 40(CCDC no. 1885765) with atom numbering
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scheme. Crystal solvents were omitted and only one molecule in the asymmetric unit was
shown. (B) Superposition of crystal structures of alkene (11) (structure in green) and TA
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) (structure in yellow, CCDC no. 131836).
Scheme 5 demonstrates the syntheses of flexible ring structure derivatives. The
reduced form of monocyclic TA-SH (41) was obtained by the reductive cleavage of TA
using tributylphosphine and was further converted to TA-SAc (42), TA-SMe (43), and TA-
thioacetal (44).
Scheme 5. Syntheses of flexible cyclic derivatives 41, 42, 43, and 44.
n
(
a) P( Bu) , H O, MeCN, (b) Ac O, NEt , CH Cl , (c) MeI, DIPEA, CH Cl , (d) BF ·OEt ,
3
2
2
3
2
2
2
2
3
2
CH Cl , acetone.
2
2
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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
O
O
S
N
O
S
N
N
N
N
S
O
O
N
N
N
S
O
O
HN
O
N
O
N
N
N
S
O
O
H
HN
O
N
O
HN
O
N
O
H
N
H
N
O
N
O
O
N
O
O
S
N
O
N
O
N
O
O
O
O
O
O
O
N
N
O
O
N
N
N
O
O
N
N
N
H
N
H
NH
H
NH
NH
O
O
O
Ile,Val (45)
Ile,Ile (46)
(+)-Triostin A (47)
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
O
O
N
O
N
O
HN
O
N
O
HN
O
N
H
N
H
Me
O
O
N
N
N
O
O
N
N
O
S
N
O
N
O
S
N
O
H
H
O
S
O
O
S
O
N
O
N
H
Me
O
N
H
N
NH
N
NH
O
O
O
O
O
Ac (48)
Qxc-Gly (49)
Figure 4. Structures of new TA derivatives.
As illustrated in Figure 4, TA derivatives TA-Ile, Val (45) and TA-Ile, Ile (46), in which
one or two N-Me-Val moieties in the TA depsipeptide core were replaced with N-Me-Ile
were also synthesized (Scheme 6). Moreover, the TA enantiomer, (+)-TA (47), TA-Ac (48)
without pendant quinoxaline (Qx) and TA-Qx-Gly (49), in which Gly was inserted between
Qx and the depsipeptide core, were prepared according to the procedure previously
reported by our group (Scheme 7).32
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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5
5
5
5
5
6
0
1
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9
0
1
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9
0
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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
Scheme 6. Syntheses of 45 and 46.
(a) EDCI·HCl, HOAt, NEt ,CH Cl , (b) HCl/AcOEt, (c) Boc-MeCys(Bam)-OH, DMT-MM,
3
2
2
AcOEt, (d) Boc-Ala-OH, DMT-MM, NMM, DMF, (e) Pd (dba) , PPh , N-methylaniline,
2
3
3
THF, (f) 19 or 55, DMT-MM, AcOEt, (g) I , CH CN (1 mM), (h) EDCI·HCl, HOAt, CH Cl
2
3
2
2
(1 mM) (i) thioanisole, TFA, (j) quioxaline-2-carboxylic acid, DMT-MM, NMM, DMF.
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9
1
1
1
1
1
1
1
1
1
1
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2
2
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2
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4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 7. Syntheses of (+)-TA (47), 48 and 49.
(a) EDCI·HCl, HOAt, NEt ,CH Cl , (b) HCl/AcOEt, (c) 65, DMT-MM, AcOEt, (d) Boc-D-
3
2
2
Ala-OH, DMT-MM, NMM, DMF, (e) Pd (dba) , PPh , N-methylaniline, THF, (f) 68, DMT-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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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
MM, AcOEt, (g) I , CH CN (1 mM), (h) EDCI·HCl, HOAt, CH Cl (1 mM), (i) thioanisole,
2
3
2
2
TFA, (j) quioxaline-2-carboxylic acid, DMT-MM, NMM, DMF, (k) AcOH, DMT-MM, NMM,
DMF, (l) Qxc-Gly-OH, DMT-MM, NMM, DMF.
Biological evaluations
The biological evaluation of the obtained compounds involved the examination of the in
vitro cytotoxicity using the MTT assay and the inhibitory effect of HIF-1 activity utilizing
the luciferase reporter assay. The latter was performed using the stable transformants of
the HEK293 cells previously established49 by transfection of the luciferase-expressing
5
0
reporter plasmid p2.1, an HRE reporter construct . After the cells were incubated with
various concentrations of the test compounds for 24 h under normoxic or hypoxic (1% O2)
conditions, luciferase activity was assessed according to the assay kit procedure.
Cytotoxic effects on various cancer cell lines were evaluated by the cell viability assay
using MTT reagent [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. For
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initial screening of the compounds synthesized here, half-maximal inhibitory
concentration (IC ) values of HIF-1 inhibition and cytotoxicity were determined from the
50
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
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3
4
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4
5
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5
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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
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8
9
0
1
2
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4
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8
9
0
1
2
3
4
5
6
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8
9
0
HRE-luciferase reporter assay and MTT assay against MCF-7 (Table 1). As shown in our
previous report, molecular modification of the depsipeptide core and the aromatic moiety
32
of TA did not improve biological activities. Altering the constituent amino acids of the
depsipeptide core decreased both the cytotoxic activity and the HIF-1 inhibitory activity
(Table 1a, runs 4-7). In addition, no activity was observed for the TA enantiomer, (+)-TA
(47) (run 3). A quinoxaline pendant with fixed rotation was also essential for activity (run
8, 9). Regrettably, none of the above structural changes based on the disulfide-bridged
bicyclic peptide scaffold resulted in an increase in the biological activities. These data
suggest that a symmetrical cyclic octadepsipeptide core containing two chromophores is
essential for the biological activity of quinomycin antibiotics. Subsequently, by introducing
an unnatural cross-bridge bond, we ultimately succeeded in achieving strong antitumor
and HIF-1 inhibitory activities at nM levels comparable with Ec (runs 10-14). When the
alkene-bridge bond was reduced, the activities marginally decreased (run 12). This
indicates that the rigidity of the macrocycle is a key factor for enhanced biological
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6
activities. Furthermore, the introduction of a hydroxyquinoline ring into the alkene-bridged
depsipeptide core afforded the most potent compound 12, which exhibited approximately
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
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9
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
10 times stronger cytotoxicity (IC50 220 pM) and four times higher inhibitory activity of
HIF-1 transcription activation (IC 90 pM) than Ec 1 (run 11). Based on the observation
50
of a rapid decrease in activity upon cleaving or extending the bridge bond, it is evident
that the constrained macrocyclic conformation plays an essential role in potent biological
activity (runs 15–18). Virtually analogous SAR trends were observed in cytotoxicity and
HIF-1 inhibitory activity evaluations. It is noteworthy however that derivative 44 displayed
relatively potent HIF-1 inhibitory activity, which was comparable with TA, whereas the
cytotoxicity of 44 decreased. This suggests that the cytotoxicity and the HIF-1 inhibitory
activity do not share the same target.
51
Rm values were shown for comparison of hydrophobicity of cyclic octadepsipeptides
(Table 1a). Furthermore, physicochemical parameters such as ALogP, polar surface area
(PSA)52 and solvent accessible surface area of the global minimum conformation (3D-
®
SASA) of Ec (1), TA (2), 11, 12, 39 and 40 were calculated using Discovery Studio 3.1 for
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the prediction of bioavailability based on the 3D structure53 (Table S18 in Supporting
Information). Although it is known that there are quite a few compounds with favorable
pharmacokinetic properties among cyclic peptides, these medium-sized molecules do not
meet common physicochemical guidelines for cell permeability and bioavailability of drug-
like small molecules due to their large and complex structures. Despite low water solubility
of Ec (1), it is considered to have passive membrane permeability because it exhibits
potent cytotoxicity at very low doses and acts as a DNA intercalator. Among the new
cyclic peptides, alkene bridged molecules 11 and 12 had smaller PSA and 3D-SASA than
those of Ec (1), especially 12 had the highest lipophilicity. These results suggest that
compound 12 may have better pharmacokinetic profile than Ec (1). On the other hand,
TA (2) was predicted to have a poor PK profile from all parameters.
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
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
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5
5
6
Table 1a. Cytotoxic activity against MCF-7 and HIF-1 inhibitory activity of quinomycin
derivatives.
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
IC50b
Rm
value
Compounda
HIF-1
run
1
cytotoxicity
for 24 h [nM] for 72 h [nM]
cytotoxicity
f
Inhibitory
c
d
e
activity [nM]
R2 = R3
=
S
S
TA (2) R1
=
N
N
26.9±1.3g
2000±500
5.5±0.2f
212±14
2.0±0.1
0.15
0.28
R4
=
2
Ec (1)
0.35±0.03g
Modification of depsipeptide core
3
47
ent-TA
>100000
-
-
>100000
0.15
0.24
4
73f
24300±2000
2000±800
5
6
7
74f
45
46
6100±1000
4100±900
4000±700
-
-
-
590±120
55.5±2.7
60.0±5.6
0.25
0.39
0.45
Modification of aromatic moiety
48
8
15500±1500
-
>100000
-0.3
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49
13600±1000
-
>100000
-0.05
Modification of bridge bond
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
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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
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8
9
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
0
1
11
12
13
39
40
41
42
43
13.4±1.5
2.9±0.3
1.0±0.2
0.09±0.01
6.3±1.2
2.4±0.3
2.0±0.1
32.3±4.3
235±28
222±56
0.25
0.52
0.32
0.2
OH
R1
=
R4
=
1
-
0.22±0.03
N
12
123±5
23.5±0.1
13
14
15
16
-
9.7±1.2
-
7.1±0.6
0.2
2700±300
8700±600
4300±10
-
-
-
0.28
0.23
0.34
17
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