Synthesis and biological evaluation of quinaldopeptin

Article abstract of DOI:10.1021/jo500039d

The second-generation total synthesis of quinaldopeptin (1) was established via a Staudinger/aza-Wittig/diastereoselective Ugi three-component reaction sequence and a racemization-free [5 + 5] coupling and macrolactamization. A single-crystal X-ray structure of the chromophore analogue 26 confirmed the structural and stereochemical assignments of the macrocycle. Synthetic 1 successfully unwound supercoiled DNA to form a relaxed DNA in a dose-dependent manner, the binding affinity of 1 to four dsODNs was within a similar range (K_b = 1.45-2.53 × 10⁷ M^-1), and the sequence selectivity was subtle. It was suggested that 1 possesses biological behaviors similar to those of sandramycin (2) in terms of cytotoxic activity against human cancer cell lines (IC₅₀ = 3.2-12 nM) and HIF-1 inhibitory activity.

Full text of DOI:10.1021/jo500039d

Article
pubs.acs.org/joc
Synthesis and Biological Evaluation of Quinaldopeptin
Katsushi Katayama,† Takuya Okamura,† Takuya Sunadome,† Koji Nakagawa,† Hiroshi Takeda,†
Motoo Shiro,§ Akira Matsuda,† and Satoshi Ichikawa†,‡,*
†Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
‡Center for Research and Education on Drug Discovery, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
§Rigaku Corporation, 3-9-12 Matsubara, Akishima, Tokyo 196-0003, Japan
S
* Supporting Information
ABSTRACT: The second-generation total synthesis of
quinaldopeptin (1) was established via a Staudinger/aza-
Wittig/diastereoselective Ugi three-component reaction se-
quence and a racemization-free [5 + 5] coupling and
macrolactamization. A single-crystal X-ray structure of the
chromophore analogue 26 confirmed the structural and
stereochemical assignments of the macrocycle. Synthetic 1
successfully unwound supercoiled DNA to form a relaxed
DNA in a dose-dependent manner, the binding affinity of 1 to
four dsODNs was within a similar range (Kb = 1.45−2.53 ×
107 M−1), and the sequence selectivity was subtle. It was
suggested that 1 possesses biological behaviors similar to those of sandramycin (2) in terms of cytotoxic activity against human
cancer cell lines (IC50 = 3.2−12 nM) and HIF-1 inhibitory activity.
symmetric cyclic octadecadepsipeptide, which similarly acts as a
double-strand DNA bisintercalator. It inhibits the binding of
hypoxia-inducible factor (HIF) proteins to a hypoxia-responsive
element (HRE) sequence. Considering the similarities of the
structures and the modes of binding to double-stranded DNA,
the ability of 1 and 2 to inhibit the HIF pathway was also
evaluated.
INTRODUCTION
■
Quinaldopeptin (1) was first isolated from the culture broth of
Streptoverticillium album (actinomycetes strain Q132-6) in 19901
and constitutes one of the members in a C2-symmetric cyclic
decapeptide class that includes sandramycin (2),2 luzopeptins,3
and quinoxapeptins (Figure 1).4 This class of natural products
possesses high-affinity double-strand DNA binding through a
phenomenon called bisintercalation.5 Quinaldopeptin (1) has a
strong activity against B16 (murine melanoma) and Moser
(human colorectal carcinoma) cells in vitro with IC50 values of
0.6 and 32 nM, respectively. In vivo antitumor activity was also
tested against lymphocytic leukemia P388 in mice, and 1 exhibits
greater potency than mitomycin C, one of the classical cancer
chemotherapeutic agents. Accordingly, 1 or 2 could be a
potential candidate for therapeutic use in cancer chemotherapy.
Although extensive efforts have been devoted to synthesize and
elucidate the DNA binding properties of 2,6,7 luzopeptins,8,9 and
quinoxapeptins,10 there has been no similar effort for 1. Recently,
we have accomplished the first total synthesis of 1 by solid-phase
peptide synthesis.11 Our first-generation synthesis featuring a
macrocyclization site was conducted at the less sterically
hindered glycine (Gly) residue as the N-terminus and L-pipecolic
aicd (L-Pip) residue as the C-terminus in the linear decapeptide 3
(Scheme 1). Unfortunately, the cyclization did not proceed
smoothly and severe racemization at the L-Pip residue occurred.
As a result, the undesired epimer was the major product
(desired/epimer = 1/1.5). In this study, we describe a
substantially improved second-generation total synthesis of 1.
DNA binding properties and cytotoxic activity against a range of
human cancer cell lines are also reported. Echinomycin is a C2-
RESULTS AND DISCUSSION
■
We have recently established the total synthesis of 2 as well as
analogues of the macrocyclic moiety.12 Our approach to the
synthesis includes a preparation of a pentadepsipeptide by a
Staudinger/aza-Wittig/diastereoselective Ugi three-component
reaction (U3CR) sequence and a racemization-free [5 + 5]
coupling and macrolactamization, which can be applicable to the
total synthesis of 1. Our revised retrosynthetic analysis of 1 is
illustrated in Scheme 2. The quinaldine chromophores 5 were
installed on macrocycle 4 in the late stages of the synthesis. The
macrocycle 4 was disconnected at the amide moiety linking
sarcosine (Sar) and L-pipecolic acid (L-Pip) residues. Since the
Sar residue has no substituent at the α position, the peptide
coupling is free from racemization during both [5 + 5] coupling
and macrolactamization. The sequential Staudinger/aza-Wittig/
diastereoselective U3CR13,14 of the azidoaldehyde 10 with the
isonitrile 7 and the carboxylic acid 8 to provide the pentapeptide
6 forms a nonproteinogenic amino acid, L-Pip residue, with
simultaneous linking to the two dipeptides 7 and 8 at the C- and
Received: January 8, 2014
Published: February 20, 2014
© 2014 American Chemical Society
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Scheme 2. Retrosynthetic Analysis of Quinaldopeptin
positions of six-membered cyclic imines.15,16 On the other hand,
the introduction of one substituent gave unsatisfactory
diastereoselectivity.14 In our previous study of the total synthesis
of 2,12 a bulky silyloxy substituent introduced at the 3-position of
9 was effective in controlling the diastereoselectivity up to S/R =
85/15; therefore, this strategy was used in the present study.
The dipeptide 8, the carboxylic component of the U3CR, was
prepared as shown in Scheme 3. The Cbz group of the known
Figure 1. Structures of quinaldopeptin, sandramycin, and luzopeptin A.
Scheme 1. Previous Total Synthesis of Quinaldopeptin
Scheme 3. Preparation of Dipeptide 8
alcohol 1127 was removed, and the liberated amine was
reprotected with a Boc group to give 12. The hydroxyl group
of 12 was mesylated, and SN2 displacement with azide ion
provided the azide 13 in 79% over two steps. Reduction of the
azide group of 13 by catalytic hydrogenation was followed by
protection of the resulting amine with a Cbz group to provide the
fully protected (2R,3R)-2,3-diaminobutanoic acid (Dab) de-
rivative 14. The Boc and the tert-butyl groups were both removed
with TFA in CH2Cl2, and the resulting amino acid was coupled
with Boc-L-Pip pentafluorophenyl ester (15) to afford the
dipeptide 8 in 78% yield over two steps.
N-termini. It is a key issue to control the stereoselectivity of the
newly formed stereogenic center in the U3CR using a cyclic
imine, and several approaches were investigated by introducing a
substituent to the cyclic imine.15−26 Most of the successful
examples of achieving good diastereoselectivity involve the
introduction of two substituents in a cis relationship at the 2,3-
positions of five-membered cyclic imines and at the 3,4- or 2,5-
With the dipeptide carboxylic acid component 8 in hand, the
total synthesis of 1 was investigated as shown in Scheme 4. The
azido alcohol 1612 was oxidized by SO3·pyridine and Et3N in
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Scheme 4. Second-Generation Total Synthesis of Quinaldopeptin
DMSO to afford the aldehyde 10 (Scheme 2), which was directly
used for the following Staudinger/aza-Wittig/U3CR sequence.
The azido aldehyde 10 was treated with PEt3 in THF, which
resulted in clean conversion to the corresponding cyclic imine
9.12 The imine 9 was subsequently reacted with the isonitrile 712
and the carboxylic acid 8 in toluene at 70 °C to afford the desired
pentapeptide 6 with 84/16 diastereoselectivity at the newly
formed stereogenic center of the Pip residue. The diastereomers
were easily separated by silica gel column chromatography, and
the major diastereomer 6 was obtained in 61% yield over three
steps from 16. The stereochemistry of the α position of the newly
constructed Pip residue of 6 was determined to be S by
conventional amino acid analysis, as described later. The impact
of the bulky silyloxy substituent introduced at the 3-position of 9
on the stereoselectivity was great, providing good control of the
diastereoselectivity up to 84/16, which was acceptable in
pursuing the total synthesis of 1 as well as its analogues.
Deprotection of the TIPS group of 6 by HF·Et3N in MeCN gave
17 in 84% yield. Then the deoxygenation of the resulting
hydroxyl group of 17 was examined. The resulting secondary
hydroxyl group was converted to the corresponding phenyl-
thionocarbonate (PhOC(S)Cl, Et3N, CH2Cl2). First, the
phenylthionocarbonate was heated under reflux in the presence
of 2,2′-azobis(isobutylonitrile) (AIBN) as a radical initiator.28
However, the desired deoxygenated product 18 was not obtained
at all, and β elimination was predominant under these conditions
to give the enamide 19 as a major product. The reaction at low
temperature by using Et3B or 2,2′-azobis(4-methoxy-2,4-
dimethylvaleronitrile) (V-70)29 as an initiator also gave 19, and
the desired 18 was not obtained at all. Extensive efforts to
circumvent the β elimination were conducted, and the use of
trifluoroethanol as a solvent was found to give a good result. That
is, the phenylthionocarbonate and AIBN were heated in
trifluoroethanol at 78 °C for 1 h to provide 18 in 55% yield
over two steps from 17. As described later, the α position of the
newly constructed Pip residue had the S configuration, so that the
acidic α-hydrogen and the phenoxylthiocarbonyloxy group had a
syn-periplanar relationship. Facile syn elimination initiated by
intramolecular deprotonation by the sulfur atom in the
phenoxylthiocarbonyloxy group results. The defeat of the syn
elimination by the choice of the solvent could be attributed to the
difference in conformation of the Pip residue. Trifluoroethanol
has a relatively acidic proton (pKa = 12.4), which can form a
hydrogen bond with the carboxamide oxygens of the peptide
frame.30 This in turn could cause a conformational change
including a distortion of the piperidine ring, which would
suppress the syn elimination, thereby favoring the desired radical
deoxygenation. In order to determine the absolute stereo-
chemistry at the newly formed stereogenic center of the Pip
residue, the deoxygenated pentapeptide 18 was heated under
reflux in 6 M aqueous HCl for 24 h, and the resulting mixture was
treated with Marfey’s reagent.31 The reaction mixture was
analyzed by reverse-phase HPLC (ODS, 10−60% MeCN/H2O
linear gradient containing 0.1% TFA). The peak corresponding
to the Pip derivative matched that of authentic 20 derived from L-
Pip (Supporting Information).
Deprotection of either the Boc group (HCl, dioxane) or the
allyl group (Pd(PPh3)4, morpholine, THF, 80%) of 18 gave the
amine 21 or the carboxylic acid 22, respectively. The [5 + 5]
assembly of 21 and 22 was conducted by the peptide coupling
under the conditions using 3-(diethoxyphosphoryloxy)-1,2,3-
benzotriazin-4(3H)-one (DEPBT)32 and NaHCO3 in CH2Cl2−
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Figure 2. X-ray crystal structure of analogue 26.
i
DMF to afford the decapeptide 23 in 62% yield. Deprotection of
the Boc and the allyl groups of 23 gave the free linear decapeptide
25, which was then cyclized by treatment with DEPBT in DMF
to afford the cyclic decapeptide 4 in 23% yield over three steps
from 23. The cyclic decapeptide 4 obtained in this study is
identical with material synthesized in our previous study.11 The
cyclization of 25 still gave a low chemical yield of 4 and required a
prolonged reaction time and higher reaction temperature in spite
of the alteration of the cyclization site from our previous
approach with 3 as a precursor (Scheme 1). It was observed that
quinaldopeptin (1) and its synthetic precursor 4 adopt multiple
nium hexafluorophosphate (HATU) and Pr2NEt in DMF to
afford 1 in 43% yield over two steps, establishing a second-
generation total synthesis of 1.
In a manner similar to the synthesis of 1, the chromophore
analogue 26 was also prepared, as shown in Figure 2. A single-
crystal X-ray structure of 26 was obtained and confirmed the
structural and stereochemical assignments of the macrocycle and
further revealed a rigid cyclic decapeptide conformation. The
overall shape of the crystal structure of 26 is rectangular with a 2-
fold axis of symmetry and is folded with six intramolecular
hydrogen bonds. The Gly amide carbonyl oxygen atom is
engaged in a transannular hydrogen bond (2.15 Å) to the β-NH
of the Dab residue across the ring, and the Dab amide carbonyl
oxygen atom has two hydrogen bond networks with both the
adjacent secondary amide NH of Gly (2.29 Å) and the α-NH of
the Dab (2.21 Å) residues across the ring. These conformational
features resulted in a kink at the L-Pip residue between the Gly
and the Dab residues, and two chromophores embedded inside
the kink, in which the interchromophore distance is 4−6.5 Å.
This structure is quite different from that found in the X-ray
structure of luzopeptin A (Figure 1).34 The macrocyclic
framework of luzopeptin A is rather flat and two chromophores
are oriented toward the perimeter of the macrocycle with an
interchromophore distance of 17−19.5 Å. It was reported that
natural 1 did not give clear signals in 1H and 13C NMR spectra.1
Those of 26 also exhibited broadened NMR spectra. This made it
difficult to elucidate their structures. The X-ray crystal structure
of 26 directly and unambiguously determined the relative and
absolute stereochemistry of the cyclic peptide framework of 1 as
well as 26, as shown in Schemes 1 and 2.
1
conformers in the solvents used to acquire their H NMR
spectra.11 This observation is in contrast to that of 2 and its cyclic
intermediate, which in solution adopts a single conformation.6,12
The cyclization of the corresponding linear decapeptide in the
synthesis of 2 proceeded smoothly. Presumably, the macrocyclic
framework of 4 would be inherently strained and, thus, it would
be difficult for the cyclization precursors such as 3 and 25 to
adopt conformations suitable for successful cyclization. How-
ever, in this strategy, no epimers were detected in the
macrocyclization of 25 or the [5 + 5] assemblage of 21 and 22.
This allowed us to subject the reaction mixture to harsher
reaction conditions and isolate the desired products easily.
Ciufolini et al. directly synthesized a maclocycle of luzopeptin E2
in 26% yield by a dimerization/cyclization strategy with the free
pentadepsipeptide.9 Accordingly, the dimerization/cyclization
route was also investigated in this study with the free
peptapeptide prepared by deprotection of both N- and C-
terminal protecting groups of the pentapeptide 18 in order to
directly obtain the macrocycle 4. However, none of these efforts
were successful. This was also true for our recent total synthesis
of 2. Finally, the Cbz groups of 4 were removed by
hydrogenolysis, and the liberated amines were coupled with
533 using 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluro-
The DNA binding properties of 1 were totally unknown.
These were evaluated in comparison with 2, the DNA binding
properties of which were elucidated by Boger’s group.6−8,10
Treatment of negatively supercoiled ΦX174 DNA (form I,
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Figure 3, lane 6) with synthetic 1 resulted in successful
unwinding to form a relaxed DNA (form II) in a dose-dependent
Table 1. DNA Binding Properties
Figure 3. DNA unwinding experiment of 1 and 2. Lanes 1−5,
sandramycin-treated Φ×174 DNA; lane 6, untreated supercoiled
Φ×174 DNA, 95% supercoiled form and 5% relaxed DNA; lanes 7-
11, quinaldopeptin-treated Φ×174 DNA. The [agent]-to-[base pair]
ratios were 0.022 (lanes 1 and 7), 0.033 (lanes 2 and 8), 0.044 (lanes 3
and 9), 0.11 (lanes 4 and 10), 0.22 (lanes 5 and 11).
study, we synthesized a simple analogue of 1, desmethylqui-
naldopeptin (27, Figure 4a), with two methyl groups at the Dab
residues (indicated by the arrows) missing, by solid-phase
peptide synthesis. The cytotoxic activity of 27 was reduced
approximately by 2 orders of magnitude (171−457 nM; Table 2)
manner (lanes 7−10), and further treatment resulted in
rewinding (lane 11). This was in good accordance with the
results of 2 (lanes 1−5) and is indicative that 1 acts as a DNA
bisintercalator. The DNA binding affinity and sequence
selectivity of 1 were then evaluated by a fluorescence quenching
experiment, where a decrease of the fluorescence intensity of the
chromophores was measured during titrations with four self-
complementary hexamer double-stranded oligodeoxynucleoti-
des (dsODNs) (Figure 4b). The binding affinities to these
dsODNs were in a similar range (Kb = 1.45−2.53 × 107 M−1),
and 1 binds preferentially to a 5′-d(TA)-3′ sequence (Table 1).
The overall DNA binding ability of 1 was slightly weaker than
that of 2 (Kb = 8.0−23.0 × 107 M−1) but still of the same order of
magnitude under our experimental conditions. In the previous
a
Table 2. Cytotoxic Activity against Cancer Cell Lines
IC50 (nM)
HCT-118 RPMI8226 A431
RKO SU-DHL6 SU-DHL10
1
3.2
0.8
11
3.8
12
3.1
5
11
5.9
12
3.3
2
1.3
26
27
28
77
279
0.7
26
64
86
264
0.8
350
213
510
307
171
457
1.0
1.4
0.8
0.8
a
Definitions: HCT-118 and RKO, human colon cancer cells; A431,
human epidermal cancer cells; RPMI8226, human myeloma cells; SU-
DHL6 and SU-DHL10, human diffuse large B-cell lymphoma cells.
Figure 4. DNA binding properties of 1 and its desmethyl analogue 27: (a) structure of desmethylquinaldopeptin (27); (b) fluorescence quenching
experiment of 1 with four hexamer double-strand ODNs: (c) fluorescence quenching experiment of 27 with four hexamer double-strand ODNs.
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in comparison to synthetic 1 (3.2−12 nM). Presumably,
truncation of the methyl groups would cause a conformational
change of the macrocycle, which would not be preferred in DNA
binding. In order to support this hypothesis, the DNA binding
affinity of 27 was also evaluated (Figure 4c). Only ca. 30%
fluorescence quenching was observed even at higher concen-
trations of dsODNs in every experiment with four dsODNs, and
the DNA binding constants could not be measured. Thus, 27 is a
weak DNA binder in comparison to 1. The cytotoxic activities of
1, 2, and 26 against a range of human cancer cell lines were then
compared side by side (Table 2). The activity correlated well to
the DNA binding affinity, and 2 is more active than 1 against all
cell lines tested. The chromophore analogue 26 was 5−40-fold
less cytotoxic.
angiogenesis, migration, and invasion, all of which are important
for tumor progression and metastasis.38,39 HIF-1 consists of the
constitutively expressed subunit HIF-1β and the oxygen-
regulated subunit HIF-1α, HIF-2α, or HIF-3α. HIF-1α is
ubiquitous, and its paralog HIF-2α is more cell-specific. Both
1α and 2α subunits are highly conservative and bind to the same
hypoxia-response element (HRE), although their effect on the
expression of some genes may vary. Echinomycin is known to
inhibit the binding of transcription factor HIF-1α to HRE, which
contains 5′-ACGT-3′ in its core sequence.38 In this study, 28 also
exhibited a strong cytotoxic activity similar to that of 1 and 2
(Table 2). Considering the similarities of its structure and the
mode of action, the ability of 1 or 2 to inhibit the HIF-1 pathway
was the subject of a preliminary investigation by a reporter gene
assay in human embryonic kidney 293 (HEK293) cells (Figure
5b). First, the effect of 1, 2, and 27 or 28 as a positive control on a
HRE-dependent transcriptional activity was evaluated. That is,
HEK293 cells were transiently transfected with the pGL3-
5xHRE-Luc reporter plasmid and the internal control pGL4.75
(hRLuc/CMV) plasmid. After 48 h, the cells were incubated
under normoxia (21% O2) or hypoxia (1% O2) for 16 h in the
presence or absence of the 1, 2, and 27 or 28, and the luciferase
activities were measured. Echinomycin (28) completely
inhibited the HIF-dependent transcriptional activity in
HEK293 cells under hypoxia at 10 nM. In contrast to the case
for 28, 2 exhibited a very weak inhibitory activity of the
transcription activity at the same concentration12 and no
inhibitory activity was observed at all for 1 and 27. Next, the
selectivity between HIF-1α and HIF-2α inhibition at 10 nM of
agents was also investigated by transfecting HIF-1α or HIF-2α
expression vector together with the pGL3-5xHRE-Luc reporter
plasmid and the internal control pGL4.75 (hRLuc/CMV)
plasmid (Figure 5c). After transfection, the cells were exposed
to 10 nM of agents for 16 h under normoxic conditions, and the
luciferase activities were measured. A trend was observed similar
to that in the HRE-dependent transcriptional assay. That is, 2
showed a weak inhibition on both HIF-1α and HIF-2α
dependent transcriptional activities, which were strongly
inhibited by treatment with 28, and quinaldopeptin and its
analogue 27 did not affect the activities. In conjunction with the
fact that 1 and 2 exhibit cytotoxic activity with potency similar to
that of 28, these data suggest that the primary target of 1 and 2 is
not the binding of transcription factor HIF-1 to the HRE, and the
mode of action of 1 and 2 is different from that of 28.
Transcriptional pathways other than HIF could be a target of
cyclic decapeptides 1 and 2, and detailed studies will be necessary
to elucidate the mode of action.
Echinomycin35 (28, Figure 5a), which is another class of C2-
symmetric cyclic octadecadepsipeptide bisintercalator,36 exhibits
a strong cytotoxicity against human cancer cells, and phase I and
II clinical trials have been pursued.37 Hypoxia-inducible factor-1
(HIF-1) is a transcription factor that controls genes involved in
CONCLUSIONS
■
In summary, the second-generation total synthesis of 1 was
established via the diastereoselective U3CR. In conjunction with
our total synthesis of 2, this strategy is applicable to the synthesis
of this class of natural products as well as their analogues. A
single-crystal X-ray structure of the chromophore analogue 26
confirmed the structural and stereochemical assignments of the
macrocycle of 1. The DNA binding properties of 1 were
evaluated in comparison with those of 2. Synthetic 1 successfully
unwound supercoiled DNA to form a relaxed DNA in a dose-
dependent manner. The binding affinity of 1 to four dsODNs
was within a similar range, and the sequence selectivity was
subtle. The overall DNA binding ability of 1 was slightly weaker
than that of 2 but still the same order of magnitude. In addition, it
was suggested that 1 possesses biological behaviors similar to that
Figure 5. Effect of 1 and 2 on HIF-dependent transcriptional activity:
(a) structure of echinomycin (28); (b) HEK293 cells seeded transiently
transfected with the pGL3-5xHRE-Luc reporter plasmid; (c) HEK293
cells seeded in a 24-well plate transiently transfected with HIF-lα or
HIF-2β expression vector together with the pGL3-5xHRE-Luc reporter
plasmid. Echinomycin (28) was tested as a positive control.
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A mixture of 13 (3.0 g, 9.8 mmol) and Pd/C (590 mg, 20% w/w) in
MeOH (100 mL) was vigorously stirred under a H2 atmosphere at room
temperature for 2 h. The catalyst was filtered off through a Celite pad,
and the filtrate was concentrated in vacuo to give the amine. A mixture of
the amine and NaHCO3 (820 mg, 98 mmol) in THF (74 mL) and H2O
(24 mL) was treated with CbzCl (2.1 mL, 15 mmol) at 0 °C and stirred
at room temperature for 11 h. The whole mixture was partitioned
between AcOEt (400 mL) and 1 M aqueous HCl (150 mL), and the
organic phase was washed with saturated aqueous NaHCO3 (150 mL)
and saturated aqueous NaCl, dried (Na2SO4), filtered, and concentrated
in vacuo. The residue was purified by silica gel column chromatography
(ϕ 3.0 × 18 cm, 14% AcOEt−86% hexane) to afford 14 (3.7 g, 93%) as a
colorless oil: [α]20D = +1.05° (c 1.37, CHCl3); 1H NMR (CDCl3, 500
MHz) δ 7.37−7.27 (m, 5H, Ph), 5.64 (br s, 1H, Boc-NH), 5.12 (q, 2H,
CH2Ph, J = 12.0 Hz), 5.01 (br s, 1H, Cbz-NH), 4.44 (m, 1H, H-α), 4.11
(m, 1H, H-β), 1.46 (s, 9H, C(CH3)3), 1.44 (s, 9H, C(CH3)3), 1.09 (d,
3H, CH3, J = 6.9 Hz); 13C NMR (CDCl3, 125 MHz) δ 169.6, 156.6,
155.5, 136.3, 128.7, 128.3, 127.1, 83.0, 79.8, 67.3, 65.5, 58.3, 48.8, 28.5,
28.1, 16.4; ESIMS-LR m/z 431 [(M + Na)+]; ESIMS-HR calcd for
C21H32N2NaO6 431.2158, found 431.2148.
of 2 in terms of cytotoxic activity against human cancer cell lines
and HIF-1 inhibitory activity.
EXPERIMENTAL SECTION
■
General Experimental Methods. 1H and 13C NMR chemical shifts
are reported in parts per million (δ) relative to tetramethylsilane (0.00
ppm) as internal standard unless otherwise noted. Coupling constants
(J) are reported in hertz (Hz). Multiplicities are given as follows; s,
singlet,; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. Data are
presented as follows: chemical shift (multiplicity, integration, coupling
constant). Assignments are based on 1H−1H COSY, HMBC, and
HMQC NMR spectra. The mass analyzer type used for the HRMS
measurements was TOF. All reactions except those carried out under
aqueous conditions were performed under an atmosphere of argon,
unless otherwise noted.
(2S,3R)-tert-Butyl 2-Hydroxy-3-tert-butoxycarbonylamino-
butanoate (12).
(2R,3R)-3-(N-tert-Butoxycarbonyl-L-pipecolylamino)-2-ben-
zyloxycarbonylaminobutanoate (8).
A mixture of 11 (5.0 g, 16 mmol) and Pd/C (1.0 g, 20% w/w) in MeOH
(80 mL) was vigorously stirred under a H2 atmosphere (balloon
pressure) at room temperature for 3 h. The catalyst was filtered off
through a Celite pad, and the filtrate was concentrated in vacuo to give
the amine. A mixture of the amine and NaHCO3 (14 g, 160 mmol) in
THF (60 mL) and H2O (20 mL) was treated with Boc2O (5.6 mL, 24
mmol) at 0 °C and stirred at room temperature for 11 h. The whole
mixture was partitioned between AcOEt (300 mL) and 1 M aqueous
HCl (100 mL), and the organic phase was washed with saturated
aqueous NaHCO3 (100 mL) and saturated aqueous NaCl, dried
(Na2SO4), filtered, and concentrated in vacuo. The residue was purified
by silica gel column chromatography (ϕ 3.0 × 18 cm, 25% AcOEt−75%
hexane) to afford 12 (3.7 g, 83%) as a colorless oil: [α]20D = +10.6° (c
0.89, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 4.72 (d, 1H, NH, J = 10.3
Hz), 4.17 (d, 1H, H-α, J = 8.1 Hz), 3.97 (dd, 1H, H-β, J = 2.3, J = 5.5 Hz),
3.10 (m, 1H, OH), 1.48 (s, 9H, C(CH3)3), 1.40 (s, 9H, C(CH3)3), 1.23
(d, 3H, CH3, J = 5.5 Hz); 13C NMR (CDCl3, 125 MHz) δ 172.8, 155.0,
83.5, 79.3, 73.6, 48.5, 28.5, 28.0, 18.7; ESIMS-LR m/z 298 [(M + Na)+];
ESIMS-HR calcd for C13H25NNaO5 298.1630, found 298.1621.
(2R,3R)-tert-Butyl 2-Azido-3-tert-butoxycarbonylaminobuta-
noate (13).
A solution of 14 (160 mg, 0.40 mmol) in 80% TFA/20% CH2Cl2 (4 mL)
was stirred at 0 °C for 12 h. The mixture was concentrated in vacuo. A
mixture of the residue and NaHCO3 (130 mg, 1.6 mmol) in DMF (4
mL) was treated with Boc-L-Pip pentafluorophenyl ester 15 (160 mg, 0.4
mmol) at 0 °C, and the mixture was stirred at room temperature for 24 h.
The mixture was partitioned between AcOEt (50 mL) and 1 M aqueous
HCl (30 mL × 3), and the organic phase was washed with saturated
aqueous NaCl, dried (Na2SO4), filtered, and concentrated in vacuo. The
residue was purified by silica gel column chromatography (ϕ 1.6 × 15
cm, 5% MeOH−CHCl3) to afford 8 (150 mg, 78%) as a colorless
amorphous solid: [α]23D = −28.6° (c 1.03, MeOH); 1H NMR (CD3OD,
500 MHz, a mixture of several rotamers at 20 °C; selected data for the
major rotamer) δ 7.37−7.30 (m, 5H, Ph), 5.12 (s, 2H, CH2Ph), 4.64 (s,
1H, Dab-NH), 4.47 (m, 1H, Dab-α-CH), 4.36 (m, 1H, Pip-α-CH), 3.91
(m, 1H, Pip-ε-CH), 2.99 (dd, 1H, Pip-ε-CH, J = 10.3, J = 13.2 Hz), 2.18
(d, 1H, Pip-β-CH, J = 11.5 Hz), 1.58−1.31 (m, 5H, Pip-β-CH, Pip-γ-
CH2 and Pip-δ-CH2), 1.45 (s, 9H, C(CH3)3), 1.11 (d, 3H, Dab-γ-CH3, J
= 6.3 Hz); 13C NMR (CDCl3, 125 MHz) δ174.2, 171.7, 157.5, 155.7,
136.8, 128.2, 127.8, 127.7, 80.3, 66.5, 58.6, 42.3, 40.9, 27.4, 26.6, 24.5,
20.0, 14.4; ESIMS-LR m/z 486 [(M + Na)+]; ESIMS-HR calcd for
C23H33N3NaO7 486.2216, found 486.2208.
A solution of 12 (3.4 g, 13 mmol) in CH2Cl2 (130 mL) was treated with
Et3N (2.6 mL, 19 mmol) and MsCl (1.5 mL, 19 mmol) at 0 °C, and the
mixture was stirred at room temperature for 1 h. The whole mixture was
partitioned between AcOEt (500 mL) and H2O (200 mL), and the
organic phase was washed with saturated aqueous NaCl, dried
(Na2SO4), filtered, and concentrated in vacuo. A solution of the residue
in DMF (130 mL) was treated with NaN3 (1.2 g, 19 mmol) at room
temperature, and the mixture was stirred at 70 °C for 16 h. The mixture
was concentrated in vacuo, and the residue was partitioned between
AcOEt (300 mL) and H2O (100 mL × 3). The organic phase was
washed with saturated aqueous NaCl, dried (Na2SO4), filtered, and
concentrated in vacuo. The residue was purified by silica gel column
Pentapeptide 6.
chromatography (ϕ 3.0 × 18 cm, 20% AcOEt−80% hexane) to afford 13
1
(3.0 g, 79%) as a colorless oil: [α]20 = +27.5° (c 1.62, CHCl3); H
D
NMR (CDCl3, 500 MHz) δ 4.73 (d, 1H, NH, J = 10.3 Hz), 4.19 (s, 2H,
H-α, β), 1.50 (s, 9H, C(CH3)3), 1.44 (s, 9H, C(CH3)3), 1.08 (d, 3H,
CH3, J = 6.9 Hz); 13C NMR (CDCl3, 125 MHz) δ 167.5, 155.0, 83.6,
80.0, 65.8, 47.6, 28.4, 28.1, 15.8; ESIMS-LR m/z 323 [(M + Na)+];
ESIMS-HR calcd for C13H24N4NaO4 323.1695, found 323.1686.
(2R,3R)-tert-Butyl 2-Benzyloxycarbonylamino-3-tert-butoxy-
carbonylaminobutanoate (14).
A solution of 16 (2.4 g, 8.0 mmol) and Et3N (3.3 mL, 24.0 mmol) in
DMSO (53 mL) was treated with sulfur trioxide pyridine complex (3.9
g, 24 mmol) at room temperature for 30 min. The reaction mixture was
neutralized by 1 M aqueous HCl at 0 °C, and the mixture was extracted
with AcOEt (300 mL). The organic phase was washed with saturated
aqueous NaHCO3 (100 mL), H2O (100 mL), and saturated aqueous
NaCl, dried (Na2SO4), filtered, and concentrated in vacuo to afford an
aldehyde (2.4 g) as a yellow oil. A solution of the aldehyde in THF (80
mL) was treated with PEt3 (20% toluene solution, 6.4 mL, 9.6 mmol) at
0 °C, and the mixture was stirred at room temperature for 30 h. The
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resulting mixture was concentrated in vacuo to afford 9 as a yellow oil.
This material was used in the next reaction without further purification.
A solution of carboxylic acid 8 (230 mg, 0.5 mmol), imine 9 (390 mg, 1.5
mmol), and isonitrile 7 (330 mg, 1.5 mmol) in toluene (1 mL) was
stirred at 70 °C for 48 h. The mixture was partitioned between AcOEt
(50 mL) and 1 M aqueous HCl (30 mL), and the organic phase was
washed with saturated aqueous NaHCO3 (30 mL), H2O (30 mL), and
saturated aqueous NaCl, dried (Na2SO4), filtered, and concentrated in
vacuo. The residue was purified by silica gel column chromatography (ϕ
1.6 × 19 cm, 33% AcOEt−67% hexane) to afford 6 (280 mg, 61%) as a
Pentapeptide 18.
A solution of 17 (100 mg, 0.13 mmol), DMAP (24 mg, 0.20 mmol), and
Et3N (55 μL, 0.40 mmol) in MeCN (1.5 mL) was treated with phenyl
chlorothioformate (54 μL, 0.40 mmol) at room temperature for 12 h.
The resulting mixture was partitioned between AcOEt (50 mL) and 1 M
aqueous HCl (30 mL), and the organic phase was washed with saturated
aqueous NaHCO3 (30 mL), H2O (30 mL), and saturated aqueous
NaCl, dried (Na2SO4), filtered, and concentrated in vacuo. The residue
in trifluoroethanol (1.3 mL) was treated with Bu3SnH (160 μL, 0.53
mmol) and AIBN (11 mg, 0.066 mmol), and the mixture was stirred at
78 °C for 1 h. The resulting mixture was partitioned between AcOEt (30
mL) and saturated aqueous KF (10 mL × 2), and the organic phase was
washed with H2O (10 mL), dried (Na2SO4), filtered, and concentrated
in vacuo. The residue was purified by flash column chromatography (ϕ
1.2 × 17 cm, 90% AcOEt−10% hexane) to afford 18 (54 mg, 55%) as a
1
colorless amorphous solid: H NMR (CDCl3, 500 MHz, a mixture of
several rotamers at 20 °C; selected data for the major rotamer) δ 8.29 (br
s, 1H, Gly-NH), 7.35−7.26 (m, 5H, Ph), 5.87 (ddd, 1H, CH2
CHCH2O, J = 5.2, J = 6.9, J = 14.2 Hz), 5.85−5.83 (m, 1H, Dab-α-NH),
5.43 (d, 1H, Sar-α-CH, J = 4.6 Hz), 5.24 (ddd, 2H, CH2=CHCH2O, J =
1.2, J = 9.1, J = 17.2 Hz), 5.15 (m, 1H, SiOPip-α-CH, J = 5.7 Hz), 5.07
(m, 1H, Pip-α-CH, J = 8.6 Hz), 5.02 (s, 2H, CH2Ph), 4.60 (d, 2H,
CH2CHCH2O, J = 5.2 Hz), 4.36 (dd, 1H, Dab-α-CH, J = 5.7, J = 17.2
Hz), 4.32−4.23 (m, 4H, SiOPip-ε-CH, Pip-ε-CH), 4.22 (d, 2H, Gly-α-
CH, J = 17.2 Hz), 4.08 (d, 1H, Sar-α-CH, J = 15.8 Hz), 3.92 (dd, 1H,
Dab-β-CH, J = 4.0, J = 17.8 Hz), 3.10−3.03 (m, 1H, SiOPip-β-CH), 3.02
(s, 3H, Sar-NCH3), 2.29 (s, 1H, Pip-β-CH), 1.95−1.40 (m, 8H, Pip-
(CH2)2), 1.43 (s, 9H, C(CH3)3), 1.18−1.13 (m, 3H, SiCH), 1.09−0.96
(m, 21H, SiCH(CH3)2, Dab-γ-CH); 13C NMR (CDCl3, 125 MHz)
δ171.2, 169.5, 168.8, 168.5, 168.3, 168.0, 156.6, 136.4, 131.6, 128.6,
128.2, 128.1, 118.8, 80.5, 70.3, 67.0, 66.4, 65.9, 56.9, 53.4, 50.6, 49.5,
42.6, 41.3, 35.4, 30.6, 28.4, 25.8, 25.7, 24.7, 20.9, 20.5, 18.1, 14.1, 12.1;
ESIMS-LR m/z 937 [(M + Na)+]; ESIMS-HR calcd for
C46H74N6NaO11Si 937.5083, found 937.5073.
1
colorless amorphous solid: H NMR (CDCl3, 500 MHz, a mixture of
several rotamers at 20 °C; selected data for the major rotamer) δ 7.33−
7.26 (m, 5H, Ph), 6.89 (br s, 1H, Gly-NH), 6.07 (br s, 1H, Dab-α-NH),
5.89 (ddd, 1H, CH2CHCH2O, J = 5.7, J = 6.9, J = 10.3 Hz), 5.33−5.25
(m, 1H, Sar-α-CH), 5.27 (dd, 2H, CH2=CHCH2O, J = 10.3, J = 16.4
Hz), 5.11−5.08 (m, 1H, Pip-α-CH), 5.06 (s, 2H, CH2Ph), 4.94 (m, 1H,
Pip-α-CH), 4.60 (d, 2H, CH2CHCH2O, J = 6.3 Hz), 4.25−3.90 (m,
5H, Dab-α−CH, Gly-α-CH, Sar-α-CH, Dab-β-CH), 3.30 (t, 2H, Pip-ε-
CH, J = 12.6 Hz), 3.10−3.04 (m, 3H, Sar-NCH3), 2.31−2.23 (br s, 2H,
Pip-β-CH), 1.80−1.46 (m, 12H, Pip-(CH2)3), 1.42 (s, 9H, C(CH3)3),
1.10 (d, 3H, Dab-γ-CH, J = 6.9 Hz); 13C NMR (CDCl3, 125 MHz) δ
171.4, 170.2, 169.8, 168.9, 168.6, 168.1, 156.7, 136.4, 131.6, 131.2, 128.6,
128.2, 128.1, 119.1, 80.5, 67.2, 66.0, 53.8, 53.0, 44.1, 41.5, 41.1, 35.6,
35.4, 29.9, 28.5, 26.2, 25.8, 25.7, 25.0, 20.6, 20.5, 15.3; ESIMS-LR m/z
765 [(M + Na)+]; ESIMS-HR calcd for C37H54N6NaO10 765.3799,
found 765.3794.
Pentapeptide 17.
Pentapeptide Carboxylic Acid 22.
A solution of 6 (300 mg, 0.33 mmol) in MeCN (3 mL) was treated with
Et3N·3HF (540 μL, 3.3 mmol) at room temperature, and the mixture
was stirred at 50 °C for 24 h. The resulting mixture was partitioned
between AcOEt (30 mL) and saturated aqueous NaHCO3 (50 mL × 3),
and the organic phase was washed with H2O (50 mL) and saturated
aqueous NaCl, dried (Na2SO4), filtered, and concentrated in vacuo. The
residue was purified by silica gel column chromatography (ϕ 2.2 × 20
cm, 90% AcOEt−10% hexane) to afford 17 (210 mg, 84%) as a yellow
oil: 1H NMR (CDCl3, 500 MHz, a mixture of several rotamers at 20 °C;
selected data for the major rotamer) δ 7.30 (br s, 5H, Ph), 6.93 (br s, 1H,
Gly-NH), 6.11 (d, 1H, Dab-α-NH, J = 6.3 Hz), 5.85 (ddd, 1H, CH2
CHCH2O, J = 5.8, J = 6.3, J = 10.3 Hz), 5.40 (d, 1H, Sar-α-CH, J = 5.8
Hz), 5.26 (dd, 2H, CH2=CHCH2O, J = 10.3 Hz, J = 16.1 Hz), 5.02 (s,
2H, CH2Ph), 4.72−4.61 (m, 1H, Pip-α-CH), 4.57 (d, 2H, CH2
CHCH2O, J = 10.9 Hz), 4.23−3.90 (m, 5H, Dab-α-CH, Gly-α-CH, Sar-
α-CH, Dab-β-CH), 3.78 (s, 1H, Pip-β-CH), 3.30 (t, 2H, Pip-ε-CH, J =
12.6 Hz), 3.03−2.94 (m, 3H, Sar-NCH3), 2.26 (br s, 2H, Pip-β-CH)
1.98−1.43 (m, 12H, Pip-(CH2)3), 1.42 (s, 9H, C(CH3)3), 1.06 (d, 3H,
Dab-γ-CH, J = 6.3 Hz); 13C NMR (CDCl3, 125 MHz) δ 171.3, 169.9,
169.1, 168.8, 168.5, 168.0, 156.5, 136.3, 131.5, 128.5, 128.2, 128.1, 119.0,
68.6, 67.0, 66.5, 66.0, 56.1, 53.5, 49.6, 42.9, 41.3, 35.4, 35.2, 29.8, 28.4,
25.8, 25.7, 25.0, 24.4, 24.3, 20.4, 14.3; ESIMS-LR m/z 781 [(M + Na)+];
ESIMS-HR calcd for C37H54N6NaO11 781.3748, found 781.3741.
A solution of compound 18 (420 mg, 0.57 mmol) and morpholine (150
μL, 0.17 mmol) in THF (6 mL) was treated with Pd(PPh3)4 (200 mg,
0.17 mmol) at room temperature for 30 min. The mixture was
partitioned between AcOEt (150 mL × 3) and 1 M aqueous HCl (50
mL), and the organic phase was washed with saturated aqueous NaCl,
dried (Na2SO4), filtered, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (ϕ 2.2 × 17 cm, 7%
MeOH−93% CHCl3) to afford 22 (320 mg, 80%) as a white amorphous
solid: 1H NMR (CD3OD, 500 MHz, a mixture of several rotamers at 20
°C; selected data for the major rotamer) δ 7.40−7.23 (m, 5H, Ph), 5.21
(m, 1H, Sar-α-CH), 5.08−5.02 (m, 2H, CH2Ph, J = 8.6 Hz), 5.00 (s, 1H,
Pip-α-CH), 4.67 (s, 1H, Pip-α-CH), 4.30−3.82 (m, 5H, Dab-α-CH,
Gly-α-CH, Sar-α-CH, Dab-β-CH), 3.60 (d, 1H, Pip-ε-CH, J = 12.6 Hz),
3.06−2.91 (m, 4H, Pip-ε-CH, Sar-NCH3), 2.36 (m, 1H, Pip-β-CH),
2.19 (m, 1H, Pip-β-CH), 1.80−1.46 (m, 12H, Pip-(CH2)3), 1.45 (s, 9H,
C(CH3)3), 1.14 (s 3H, Dab-γ-CH); 13C NMR (CD3OD, 125 MHz) δ
176.4, 173.2, 172.7, 171.7, 170.9, 170.6, 158.4, 137.9, 129.4, 129.0, 81.4,
67.8, 58.2, 55.3, 54.7, 54.7, 53.8, 52.6, 48.1, 44.9, 43.4, 42.1, 36.2, 35.8,
28.6, 27.7, 27.4, 26.7, 26.5, 25.9, 25.7, 22.1, 21.4, 18.4, 15.3; ESIMS-LR
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m/z 701 [(M + Na)+]; ESIMS-HR calcd for C34H49N6O10 701.3510,
found 701.3532.
Decapeptide 23.
with saturated aqueous NaHCO3 (10 mL), H2O (10 mL), and saturated
aqueous NaCl, dried (Na2SO4), filtered, and concentrated in vacuo. The
residue was purified by flash silica gel column chromatography (ϕ 0.9 ×
18 cm, 3% MeOH−CHCl3) to afford 4 (12 mg, 23%) as a colorless
amorphous solid: [α]20D = −37.0° (c 0.31, MeOH); 1H NMR (CD3OD,
500 MHz, a mixture of several rotamers at 20 °C; selected data for the
major rotamer) δ 7.45−7.21 (m, 10H, Ph), 5.35−4.90 (m, 6H, CH2Ph,
Gly-α-CH), 4.84−3.99 (m, 6H, Gly-α-CH, Pip-α-CH, Dab-α-CH),
3.82−3.35 (m, 4H, Sar-α-CH), 3.19−3.03 (m, 6H, Sar-NCH3), 2.94−
2.89 (m, 4H, Pip-ε-CH), 2.26−2.10 (m, 2H, Dab-β-CH), 1.62−1.23 (m,
20H, Pip-(CH2)3), 1.30−1.11 (m, 6H, Dab-γ-CH); 13C NMR (CD3OD,
125 MHz) δ 173.4, 172.5, 172.3, 172.2, 171.5, 171.0, 170.7, 170.2, 158.5,
158.4, 138.2, 129.5, 129.1, 79.5, 71.6, 67.8, 67.6, 67.5, 54.6, 45.1, 42.0,
41.7, 36.2, 35.9, 33.1, 30.8, 30.5, 28.7, 28.5, 27.6, 26.8, 26.4, 26.1, 25.6,
23.7, 21.8, 21.5, 21.4, 18.5, 14.4, 13.2; ESIMS-LR m/z 1191 [(M +
Na)+]; ESIMS-HR calcd for C58H80N12NaO14 (M + Na)+ 1191.5814,
found 1191.5798. These data are identical with those obtained
previously.11
Compound 18 (340 mg, 0.46 mmol) was treated with 4 M HCl in
dioxane (4.6 mL) at room temperature for 30 min. The mixture was
concentrated in vacuo to afford the crude amine hydrochloride 21 (300
mg, quantitative) as a white solid. The amine was added to a mixture of
22 (320 mg, 0.46 mmol), NaHCO3 (150 mg, 1.8 mmol), and (3-
(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT; 550
mg, 1.8 mmol) in CH2Cl2 (4 mL) and DMF (0.5 mL) at 0 °C, and the
whole mixture was stirred at room temperature for 24 h. The resulting
mixture was partitioned between AcOEt (50 mL) and 1 M aqueous HCl
(10 mL), and the organic phase was washed with saturated aqueous
NaHCO3 (30 mL), H2O (30 mL), and saturated aqueous NaCl, dried
(Na2SO4), filtered, and concentrated in vacuo. The residue was purified
by silica gel column chromatography (ϕ 2.2 × 18 cm, 90% AcOEt−10%
hexane) to afford 23 (380 mg, 62%) as a colorless amorphous solid: 1H
NMR (CDCl3, 500 MHz, a mixture of several rotamers at 20 °C;
selected data for the major rotamer) δ 7.38−7.23 (m, 10H, Ph), 6.93 (br
s, Dab-α-NH), 5.91 (m, 1H, CH2CHCH2O), 5.25 (m, 2H,
CH2=CHCH2O), 5.23−5.15 (m, 2H, Pip-α-CH), 5.11−5.00 (m, 4H,
CH2Ph), 4.66 (m, 2H, Dab-α-CH), 4.65−4.62 (m, 2H, CH2
CHCH2O), 4.30−3.91 (m, 10H, Gly-α-CH, Dab-β-CH, Sar-α-CH),
3.75−3.60 (m, 4H, Pip-ε-CH), 3.33 (m, 4H, Pip-ε-CH), 3.07−2.92 (m,
6H, Sar-NCH3), 2.30−2.15 (m, 4H, Pip-β-CH), 1.84−1.30 (m, 20H,
Pip-(CH2)2, Pip-β-CH), 1.44 (s, 9H, C(CH3)3), 1.24 (m, 3H, Dab-γ-
CH), 1.09 (m, 3H, Val-γ-CH); 13C NMR (CDCl3, 125 MHz) δ 171.3,
171.0, 170.8, 170.3, 170.0, 169.7, 168.8, 168.7, 168.5, 168.4, 168.2, 156.6,
156.0, 136.4, 136.3, 131.5, 128.49, 128.45, 128.0, 127.9, 127.8, 127.3,
118.9, 80.3, 67.6, 67.0, 65.9, 53.7, 52.9, 49.5, 43.9, 41.3, 41.2, 40.9, 35.6,
35.3, 35.1, 28.4, 25.8, 25.6, 25.5, 24.9, 21.0, 20.5, 20.4, 14.2; ESIMS-LR
m/z 1349 [(M + Na)+]; ESIMS-HR calcd for C66H94N12NaO17
1349.6758, found 1349.6741.
Quinaldopeptin 1.
A mixture of 4 (5.8 mg, 5.0 μmol) in MeOH (1 mL) and 10% Pd(OH)2/
C (1 mg) was vigorously stirred under a H2 atmosphere at room
temperature for 1 h. The catalyst was filtered off through a Celite pad,
and the filtrate was concentrated in vacuo to give the diamine. The
residue in DMF (1 mL) was treated with HATU (7.6 mg, 20 μmol),
iPr2NEt (3.5 μL, 20 μmol), and chromophore 5 (3.8 mg, 20 μmol) at 0
°C, and the mixture was stirred at room temperature for 17 h. The
resulting mixture was partitioned between AcOEt (30 mL) and 1 M
aqueous HCl (10 mL), and the organic phase was washed with saturated
aqueous NaHCO3 (10 mL), H2O (10 mL), and saturated aqueous
NaCl, dried (Na2SO4), filtered, and concentrated in vacuo. The residue
was purified by flash silica gel column chromatography (3% MeOH−
97% CHCl3) to afford quinaldopeptin (1; 2.7 mg, 43%) as a yellow solid:
1H NMR (CD3OD, 500 MHz; selected peaks are given because of
multiple conformers) δ 7.90 (br d, 1H, J = 5.8 Hz), 7.81−7.26 (m, 4H),
5.86 (br s, 1H), 5.67 (br s, 1H), 5.30 (d, 1H, J = 4.1 Hz), 5.12 (d, 1H, J =
4.0 Hz), 4.70 (d, 1H, J = 18.3 Hz), 4.60 (d, 1H, J = 10.3 Hz), 4.47 (d, 1H,
J = 13.2 Hz), 4.30 (br s, 1H), 4.23 (m, 2H), 4.07 (d, 1H, J = 18.9 Hz),
3.95 (d, 1H, J = 17.2 Hz), 3.65 (m, 1H), 3.43 (m, 1H), 2.87 (m, 1H),
3.16 (br s, 1H), 3.22−2.99 (m), 2.96 (s, 3H), 2.89−2.81 (m), 2.30 (br d,
1H, J = 12.1 Hz), 2.20 (br d, 1H, J = 13.7 Hz), 1.87−1.37 (m), 1.21 (d,
3H, J = 7.5 Hz); 13C NMR (CD3OD, 125 MHz; selected peaks are given
because of multiple conformers) δ 172.7, 172.6, 172.4, 171.9, 170.1,
169.7, 154.4, 135.8, 133.2, 130.5, 129.9, 129.8, 129.5, 128.5, 128.2, 127.4,
121.0, 57.1, 54.5, 54.4, 53.7, 52.6, 45.2, 42.0, 41.9, 36.2, 33.1, 30.7, 30.5,
30.3, 28.1, 27.9, 27.0, 26.4, 25.2, 23.7, 21.6, 21.4, 21.3, 14.4, 13.2; ESIMS-
LR m/z 1265 [(M + Na)+]; ESIMS-HR calcd for C62H78N14NaO14
1265.5720, found 1265.5728. These data are identical with those
obtained previously.11
Cyclic Decapeptide 4.
A solution of 23 (62 mg, 0.047 mmol) and morpholine (12 μL, 0.14
mmol) in THF (1 mL) was treated with Pd(PPh3)4 (16 mg, 14 μmol) at
room temperature for 30 min. The mixture was partitioned between
AcOEt (30 mL × 3) and 1 M aqueous HCl (10 mL), and the organic
phase was washed with saturated aqueous NaCl, dried (Na2SO4),
filtered, and concentrated in vacuo. The residue was purified by silica gel
column chromatography (ϕ 1.2 × 14 cm, 10% MeOH−90% CHCl3) to
afford 24 (46 mg, 0.036 mmol) as a white solid. The carboxylic acid 24
(46 mg, 0.036 mmol) was treated with 4 M HCl in dioxane (1 mL) for
30 min. The mixture was concentrated in vacuo to afford the crude
amino acid 25 (43 mg, 0.036 mmol, theoretical quantitative) as a white
solid. A solution of the residue and NaHCO3 (12 mg, 0.14 mmol) in
DMF (7 mL) was treated with DEPBT (42 mg, 0.14 mmol) at 0 °C, and
the reaction mixture was stirred at room temperature for 2 days and 40
°C for 2 days. The resulting mixture was partitioned between AcOEt (30
mL) and 1 M aqueous HCl (10 mL), and the organic phase was washed
Analogue 26.
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Article
A solution of the 2-quinoxalinecarboxylic acid (13.9 mg, 0.08 mmol) and
HOBt (16.2 mg, 0.12 mmol) in CH2Cl2 (1 mL) was treated with EDCI
(15.3 mg, 0.08 mmol), and the reaction mixture was stirred at room
temperature for 30 min. The mixture was added to the diamine obtained
from 4 by hydrogenolysis (18.1 mg, 0.02 mmol), and the reaction
mixture was stirred at room temperature for 24 h. The reaction mixture
was poured onto 1 M aqueous HCl and extracted with AcOEt. The
combined organic layer was washed with saturated aqueous NaHCO3
and saturated aqueous NaCl, dried (Na2SO4), filtered, and concentrated
in vacuo. The residue was purified by silica gel column chromatography
(SiO2, 1% MeOH−99% CHCl3) to afford 26 (9.7 mg, 40%) as a
colorless glass. Part of the material was crystallized from MeOH to give a
colorless prism, which was used for the X-ray crystal structure analysis:
mp >300 °C; [α]20D = −409.6° (c 0.64, CHCl3); 1H NMR (CDCl3, 500
MHz, a mixture of rotamers; selected data for the major rotamer) δ
10.34 (br s, 2H, NH), 8.88 (br s, 2H, NH), 8.01 (br s, 2H), 7.93 (br s,
2H), 7.80 (m, 6H), 7.63 (br s, 2H), 7.01 (d, 2H, J = 9.2 Hz), 5.90 (br s,
2H), 5.21 (s, 2H), 5.04 (br s, 2H), 4.73 (br s, 2H), 4.48 (m, 2H), 3.99 (br
s, 2H), 3.61 (br s, 2H), 3.20 (br s, 2H), 2.66 (br s, 6H), 2.46 (d, 2H, J =
12.6 Hz), 2.20−2.10 (m, 6H), 1.82 (m, 4H), 1.70−1.61 (m, 16H), 1.43
(d, 6H, Dab-CH3, J = 6.9 Hz); 13C NMR (CD3OD, 125 MHz, a mixture
of rotamers; selected data for the major rotamer) δ 172.7, 172.5, 172.2,
172.0, 171.9, 165.1, 145.1, 144.2, 133.1, 132.6, 132.3, 131.0, 130.9, 130.4,
129.9, 129.6, 57.1, 54.5, 527, 42.2, 41.8, 41.5, 36.3. 28.0, 26.7, 25.2, 23.8,
21.6, 21.3, 13.1; ESIMS-LR m/z 1235 [(M + Na)+]; ESIMS-HR calcd
for C60H76N16NaO12 1235.57208, found 1235.57142.
DNA Binding Constant Measurements. The temperature was
maintained at 24 °C throughout the experimental work. A 300 μL quartz
cuvette was used in all experiments. Double-stranded oligodeoxynucleo-
tide (dsODN) was dissolved in 10 mM Tris-HCl buffer solution
containing 75 mM NaCl. The excitation and emission spectra were
recorded with a sample (200 μL) containing 10 mM Tris-HCl, 75 mM
NaCl buffer, and 2 μL of a DMSO stock solution of the agent. The final
concentration for the agents was 10 μM. For the determination of the
dsODN binding constant, a 200 μL sample containing 10 μM 1 or 2 was
titrated with 2 μL of dsODN (320 μM) solution. The quenching of
fluorescence was measured by spectrofluorimeter 5 min after each
addition of dsODN to allow binding equilibration with 360 nm
excitation and 530 nm fluorescence, and the results are graphically
represented in Figure 3b,d.
General Procedure for Agarose Gel Electrophoresis. All agents
were dissolved in DMSO as stock solutions, stored at −30 °C in the
dark, and diluted to the working concentrations in DMSO prior to
addition to the DNA solution. A buffered DNA solution containing 0.25
μg of supercoiled ΦX 174 RF I DNA (1.0 × 10−8) in 9 μL of 50 mM
Tris-HCl buffer solution was treated with 1 μL of agent in DMSO (the
control DNA was treated with 1 μL of DMSO). The [agent] to [DNA]
base pair ratios were as follows: 0.022 (lane 1), 0.033 (lane 2), 0.044
(lane 3), 0.11 (lane 4), 0.22 (lane 5) for 2; 0 (lane 6 control DNA), 0.022
(lane 7), 0.033 (lane 8), 0.044 (lane 9), 0.11 (lane 10), 0.22 (lane 11) for
1. The reaction mixtures were incubated at 37 °C for 1 h and quenched
with 5 μL of loading buffer. Electrophoresis was conducted on a 0.9%
agarose gel at 90 V for 1 h. The gel was stained with 0.1 μg/mL ethidium
bromide, visualized on a lumino imaging analyzer.
Cytotoxicity Assay. Antiproliferative activities of the compounds
against HCT-116, HT-29, and CCRF-CME cells were measured using
the CellTiter-Glo Luminescent Cell Viability Assay according to the
manufacturer’s protocol. Briefly, cells (1.5−3 × 103 cells/well) in a 96-
well plate were cultured in RPMI medium containing 10% fetal bovine
serum in the presence of test compounds at 37 °C for 72 h under a 5%
CO2 atmosphere. Then, CellTiter-Glo reagent was added, and plates
were shaken and the luminescence was monitored 30 min after reagent
addition with the plates held at room temperature.
Reporter Gene Assay. HEK293 cells seeded in 48-well plates were
transfected with pGL3-5xHRE-Luc and pGL4.75 hRluc-CMV (Prom-
ega), together with HIF-1α or HIF-2α expression vectors as indicated.
Twenty-four hours after transfection, the cells were exposed to
echinomycin, sandramycin, quinaldpeptin, or desmethylquinaldpeptin
for 16 h under normoxic (21% O2) or hypoxic (1% O2) conditions.
Luciferase activities were measured using Dual-Luciferase Reporter
Assay System (Promega) and a luminescencer.
ASSOCIATED CONTENT
* Supporting Information
■
S
Text, figures, tables, and a CIF file giving amino acid analysis of
18, NMR spectra of new compounds, and an X-ray crystal
structure analysis of 26. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*S.I.: tel, (+81) 11-706-3229; fax, (+81) 11-706-4980; e-mail,
ichikawa@pharm.hokudai.ac.jp.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by JSPS Grants-in-Aid for
Challenging Exploratory Research (SI, Grant No. 22659020),
Scientific Research on Innovative Areas “Chemical Biology of
Natural Products” (SI, Grant No. 24102502), and Scientific
Research (B) (SI, Grant No. 25293026). We thank Ms. S. Oka
and Ms. A. Tokumitsu (Center for Instrumental Analysis,
Hokkaido University) for measurement of the mass spectra.
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