Total synthesis of verucopeptin, an inhibitor of hypoxia-inducible factor 1 (HIF-1)

Article abstract of DOI:10.1039/c9cc06169j

Verucopeptin is an inhibitor of hypoxia-inducible factor 1 (HIF-1), which is a promising target for cancer chemotherapy. Here, we report the first total synthesis of verucopeptin via condensation of the depsipeptide core and the polyketide side chain unit including three branched methyl groups after the synthesis of each segment.

Full text of DOI:10.1039/c9cc06169j

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
Total synthesis of verucopeptin, an inhibitor
of hypoxia-inducible factor 1 (HIF-1)†
Cite this: Chem. Commun., 2019,
55, 11956
Nobuaki Takahashi, Hideaki Hayashi, Viktors Poznaks and Hideaki Kakeya
*
Received 9th August 2019,
Accepted 6th September 2019
DOI: 10.1039/c9cc06169j
rsc.li/chemcomm
Verucopeptin is an inhibitor of hypoxia-inducible factor 1 (HIF-1), To investigate its chemical and biological properties, we carried
which is a promising target for cancer chemotherapy. Here, we out the total synthesis of verucopeptin (1) and report it herein.
report the first total synthesis of verucopeptin via condensation of
The summary of the total synthesis of 1 is shown in
the depsipeptide core and the polyketide side chain unit including Scheme 1. Condensation of the depsipeptide core 2 and the
three branched methyl groups after the synthesis of each segment. side chain 3 followed by removal of the protective group for
construction of the THP ring was carried out in the final stage.
Tumor cells are usually exposed to hypoxic conditions or a Carboxylic acid 3 was obtained by coupling alkyne 7 and
starvation state. For survival and cell growth of tumor cells in aldehyde 6 following structural conversions. Aldehyde 6 was
these severe environments, hypoxia-inducible factor 1 (HIF-1)
plays an important role as a transcriptional factor that regulates
the expression of a number of genes involved in angiogenesis,
gluconeogenesis, and metastasis.1,2 Therefore, HIF-1 is a promising
target for cancer chemotherapy, and studies on HIF-1 by using
a chemical and biological approach have been carried out in our
group.3
For the purpose of identifying new HIF-1 inhibitors, we
screened natural resources using a hypoxia-responsive luciferase
reporter gene assay and we re-discovered verucopeptin (1) as a
potent inhibitor of HIF-1 from a culture broth of Streptomyces
sp. KUSC_A08.3b,4 Verucopeptin (1) consists of a cyclic depsi-
peptide core and a polyketide side chain including three branched
methyl groups. Unlike the other derivatives such as azinothricin5a
and dentigerumycin5b 1 has a unique feature in that it exists
in equilibrium between an open form and a closed form via its
tetrahydropyran (THP) ring.
Recently, we determined the stereochemistry of 1 and
revealed that 1 inhibits HIF-1 via the mTORC1 pathway.3b
For elucidation of the mode of action of 1 in more detail,
investigation of various derivatives of 1 is necessary. Although
the synthesis of the peptide core has been reported by the
Hale group,6 the total synthesis of 1 has not been achieved.
Department of System Chemotherapy and Molecular Sciences, Division of
Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical
Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
E-mail: scseigyo-hisyo@pharm.kyoto-u.ac.jp
† Electronic supplementary information (ESI) available: Supporting figures,
procedures for the syntheses of verucopeptin (1) and copies of 1H- and 13C-NMR
Scheme 1 Retrosynthesis of verucopeptin (1).
spectra. See DOI: 10.1039/c9cc06169j
11956 | Chem. Commun., 2019, 55, 11956--11959
This journal is ©The Royal Society of Chemistry 2019

View Article Online
Communication
ChemComm
transformed from known alcohol 8. Alkyne 7 was obtained from
alcohol 9, which was prepared from known monoacetate 10.7
Depsipeptide 2 was transformed from the linear peptide 4 by
the same method as reported in Hale’s work.6 Compound 4 was
prepared by Fmoc-based solid phase peptide synthesis (SPPS)
from Fmoc glycine loaded resin 5.
Alcohol 9 with three branched methyl groups was prepared
from known monoacetate 107 in 6 steps (Scheme 2). After
oxidation of 10 with 2-iodoxybenzoic acid (IBX), the resulting
aldehyde was converted to propargyl alcohol 11 by Pd-catalyzed
propargylation8 using chiral (S)-mesylate 12.9 To determine the
absolute configuration, alcohol 11 was transformed to diol 13
whose enantiomer is a known compound10 and the spectral
data, including the sign of optical rotation, were compared.
After hydrogenation of the alkyne, the unnecessary hydroxy
group was removed by radical deoxygenation in two steps. Next,
hydrolysis of the acetyl group was conducted to afford alcohol 9
in 96% yield.
Scheme 3 Synthesis of diol 6; (a) TEMPO, Phl(OAc)2, CH2Cl2, r.t., 3 h,
88%; (b) 14, CH3CN, 80 1C, 12 h, 75%; (c) DIBAL-H, toluene, ꢀ78 1C, 4 h;
(d) DMP, CH2Cl2, r.t., 2 h, 75% (2 steps); and (e) 17, nBu2BOTf, NEt3,
CH2Cl2, ꢀ78 1C to 0 1C, 1 h, 96%; (f) LiCl, NaBH4, EtOH, THF, r.t., 2 h, 55%.
Next, we focused on the construction of the two adjacent
chiral centers of the methoxy group and allylic hydroxy group
(Scheme 3). After oxidation of 9 with 85% yield, the resulting
aldehyde was transformed to a,b-unsaturated ester 15 by the
Wittig reaction using 14. Conversion of 15 into a,b-unsaturated
aldehyde 16 was conducted by diisobutylaluminium hydride
(DIBAL-H) reduction and followed by oxidation with 2,2,6,6-
tetramethylpiperidine 1-oxyl (TEMPO) and PhI(OAc)2 with 75%
yield (2 steps). Next, the aldehyde 16 was subject to the Evans
aldol reaction with oxazolidinone 17. Under the typical conditions
with boron enolate, the diastereomerically pure aldol product 18
was obtained in 96% yield. Next, reductive removal of the chiral
auxiliary of 18 gave diol 1911 in 55% yield.
Scheme 4 Synthesis of alkyne 7; (a) TBSCI, imidazole, CH2Cl2, r.t., 2 h,
81%; (b) PhCOOH, DEAD, PPh3, THF, r.t., 15 h, 95%; (c) K2CO3, MeOH, r.t.,
12 h; (d) TBSOTf, 2,6-lutidine, CH2Cl2, ꢀ78 1C, 2 h, 98% (2 steps); (e) PPTS,
EtOH, r.t., 16 h, 80%; (f) DMP, CH2Cl2, r.t., 2 h; and (g) dimethyl(1-diazo-2-
oxopropyl)phosphonate, K2CO3, MeOH, r.t., 18 h, 58% (2 steps).
Next, the diol 19 was transformed to alkyne 7 for a coupling
reaction with aldehyde 6 (Scheme 4). After protection of the
primary alcohol with a tert-butyldimethylsilyl (TBS) group to selective deprotection of the primary alcohol, Dess–Martin
obtain the intermediate in 81% yield,12 the Mitsunobu reaction periodinane (DMP) oxidation, and alkyne formation using the
was conducted to obtain benzoate 20 whose stereochemistry Ohira–Bestmann reagent.13
was the same as that of verucopeptin (1). The benzoate group
Aldehyde 6 was prepared from known diol 814 as shown in
was removed by methanolysis with K2CO3 and the resulting Scheme 5. Protection of the diol by p-methoxybenzylidene
alcohol was protected by a TBS group to obtain a di-silylated acetal gave compound 22 in quantitative yields.15 After removal
compound 21. The compound 21 was converted to alkyne 7 by of the p-methoxybenzyl (PMB) ester group, TBS protection
of the hydroxy group gave O-silylated compound 23. Next,
reduction of the p-methoxybenzylidene acetal with DIBAL-H
was conducted to afford 24 whose tertiary alcohol was protected
by the PMB group. After oxidation of 24, the resulting aldehyde
was transformed to a dimethyl acetal to generate 25. Finally,
removal of the TBS group followed by oxidation gave the
aldehyde 6.
Completion of the side chain unit is described in Scheme 6.
The addition of alkyne 7 to aldehyde 6 using lithium hexam-
ethyldisilazide (LHMDS) gave 26 in 95% yield as a mixture of
diastereomers. Oxidation of the resulting secondary alcohol
with DMP and the subsequent hydrogenation of the triple bond
were performed to obtain ketone 27. After deprotection of the
Scheme 2 Synthesis of alcohol 9; (a) IBX, DMSO, r.t., 3 h, 85%; (b) 12,
Pd(OAc)2, PPh3, ZnEt2, THF, ꢀ78 1C to ꢀ20 1C, 6 h, 70%; (c) Pd/C, H2,
dimethyl acetal in the presence of trimethylsilyl triflate
MeOH, r.t., 18 h, 85%; (d) phenyl chlorothionoformate, DMAP, pyridine,
(TMSOTf) and 2,4,6-collidine,16 the resulting aldehyde was
CH2CI2, r.t., 3.5 h, 74%; (e) nBu3SnH, AIBN, toluene, 100 1C, 16 h; (f) K2CO3,
MeOH, r.t., 2 h, 96% (2 steps); and (g) K2CO3, MeOH, r.t., 3 h, 85%.
oxidized to carboxylic acid 3 by the Pinnick oxidation.
This journal is ©The Royal Society of Chemistry 2019
Chem. Commun., 2019, 55, 11956--11959 | 11957

View Article Online
ChemComm
Communication
Scheme 7 Building blocks of the depsipeptide core 3; (a) FmocCl, sat.
NaHCO3 aq., 1,4-dioxane, r.t., 1 h, 83% and (b) TFA, CH2Cl2, r.t., 1 h, 90%.
Scheme 5 Synthesis of aldehyde 6; (a) p-methoxybenzaldehyde dimethyl
acetal, PPTS, CH2Cl2, r.t., 18 h, 98%; (b) NaOMe, MeOH, r.t., 18 h; (c) TBSCI,
imidazole, CH2Cl2, r.t., 16 h, 92% (2 steps); (d) DIBAL-H, CH2Cl2, 0 1C, 4 h,
36%; (e) SO3-pyridine, NEt3, DMSO, r.t., 2 h, 82%; (f) PPTS, trimethyl
orthoformate, CH2Cl2, r.t., 16 h; (g) TBAF, THF, r.t., 16 h, 91% (2 steps);
and (h) SO3-pyridine, NEt3, DMSO, r.t., 4 h, 69%.
Scheme 8 Synthesis of cyclic depsipeptide 2; (a) 20% piperidine/DMF, r.t.,
1 h; (b) 28, DIC, HOBt, DIEA, DMF, r.t., 2 h; (c) 20% piperidine/DMF, r.t., 1 h;
(d) 29, HATU, HOAt, DIEA, DMF, r.t., 1 h; (e) 20% piperidine/DMF, r.t., 1 h;
(f) 30, HATU, HOAt, DIEA, DMF, r.t., 12 h; (g) (COCl)2 benzene, r.t., 2.5 h; (h) 35,
AgCN, toluene, 60 1C, 20 min; (i) 98% TFA, r.t., 1 h, 36% (overall yield of Fmoc-
SPPS); (j) HATU, N-ethyl morpholine, CH2Cl2 (0.0004 M), 0 1C to r.t., 48 h, 54%;
(k) Zn, AcOH/H2O, r.t., 2 h; (l) CbzCl, 10% Na2CO3 aq., THF, r.t., 2 h, 79% (2 steps);
and (m) Pd/C (degussa type), H2, AcCl, MeOH, r.t., 24 h, 92%.
Scheme 6 Synthesis of carboxylic acid 3; (a) LHMDS, ꢀ78 1C to 0 1C, 1 h,
95%; (b) DMP, CH2Cl2, r.t., 1 h, 96%; (c) Pd/C, AcOEt, r.t., 3 h; (d) TMSOTf,
2,4,6-collidine, CH2Cl2, 0 1C, 5 h; and (e) NaClO2, NaH2PO4 monohydrate,
2-methyl-2-butene, H2O, tBuOH, r.t., 3 h, 69% (3 steps).
We started the synthesis of the depsipeptide core as shown tetrapeptide 34 was examined. After deprotection of the Fmoc
below. The building blocks for SPPS are described in Scheme 7. group, the resulting free amine was condensed with piperazic
Although resin 5 and Fmoc sarcosine 28 were commercially acid 30 using O-(7-aza-1H-benzotriazol-1-yl)-N,N,N0,N0-tetramethyl-
available, N-hydroxy glycine 29, piperazic acid 30 and dipeptide uronium hexafluorophosphate (HATU), 1-hydroxy-7-azabenzo-
31 needed to be synthesized. Among them, 3017 and 316 were triazole (HOAt), and N,N-diisopropylethylamine (DIEA). Because
synthesized by previously reported methods. However, N-hydroxy the reactivity of the Na-amine in the piperazic acid of 34 was
glycine 29 was an unknown compound, and thus it needed to be remarkably low,18 the protection of the Na-amine in 30 was not
synthesized. From the tert-butyl ester 32, protection of the amino necessary under these conditions. In the final step of the SPPS,
group with the Fmoc group and subsequent removal of the tert- introduction of dipeptide 31 was investigated to generate
butyl group under acidic conditions gave the amino acid 29.
the hexapeptide 36. After conversion of 31 to the acid chloride
The SPPS was started from the Fmoc-glycine loaded Wang 35,6,18 a condensation reaction with 34 in toluene at 60 1C in
resin 5 (Scheme 8). Two cycles of removal of the Fmoc group the presence of AgCN gave 36. Next, removal from the resin
using 20% piperidine/DMF and introduction of the amino acid and deprotection of the tert-butoxycarbonyl (Boc) group was
(Fmoc-sarcosine 28 or hydroxy glycine 29) in the presence of a conducted. Treatment with 98% trifluoroacetic acid (TFA) and
condensing agent gave the tripeptide 33. Then, synthesis of the purification by HPLC gave a pure linear peptide 4 in 36% yield
11958 | Chem. Commun., 2019, 55, 11956--11959
This journal is ©The Royal Society of Chemistry 2019

View Article Online
Communication
ChemComm
Conflicts of interest
There are no conflicts to declare.
Notes and references
1 G. L. Semenza, Annu. Rev. Pathol.: Mech. Dis., 2014, 9, 47.
2 S. Koyasu, M. Kobayashi, Y. Goto, M. Hiraoka and H. Harada, Cancer
Sci., 2018, 109, 560.
3 (a) Y. Yasuda, T. Arakawa, Y. Nawata, S. Shimada, S. Oishi, N. Fujii,
S. Nishimura, A. Hattori and H. Kakeya, Bioorg. Med. Chem., 2015,
23, 1776; (b) A. Yoshimura, S. Nishimura, S. Otsuka, A. Hattori and
H. Kakeya, Org. Lett., 2015, 17, 5364; (c) H. Kakeya, Nat. Prod. Rep.,
2016, 33, 648.
4 A. Yoshimura, S. Kishimoto, S. Nishimura, S. Otsuka, Y. Sakai,
A. Hattori and H. Kakeya, J. Org. Chem., 2014, 79, 6858.
5 (a) H. Maehr, C. M. Liu, N. J. Palleroni, J. Smallheer, L. Todaro,
T. H. Williams and J. F. Blount, J. Antibiot., 1986, 39, 17; (b) D. C. Oh,
M. Poulsen, C. R. Currie and J. Clardy, Nat. Chem. Biol., 2009, 5,
391.
6 K. J. Hale, L. Lazarides and J. Cai, Org. Lett., 2001, 3, 2927.
7 J. C. Anderson, S. V. Ley and S. P. Marsden, Tetrahedron Lett., 1994,
35, 2087.
8 (a) J. A. Marshall and N. D. Adams, J. Org. Chem., 1998, 63, 3813;
(b) J. A. Marshall and N. D. Adams, J. Org. Chem., 1999, 64, 5201;
(c) Y. Tamaru, S. Goto, A. Tanaka, M. Shimizu and M. Kimura,
Angew. Chem., Int. Ed. Engl., 1996, 35, 878.
9 J. R. Frost, C. M. Pearson, T. N. Snaddon, R. A. Booth, R. M. Turner,
J. Gold, D. M. Shaw, M. J. Gaunt and S. V. Ley, Chem. – Eur. J., 2015,
21, 13261.
Scheme 9 Completion of the total synthesis of verucopeptin (1);
(a) PyBop, NEt3, CH2Cl2, ꢀ78 1C to r.t., 3 h and (b) 1 N HCl aq., THF, r.t.,
8 h, 23% (2 steps).
from resin 5. Synthesis of the peptide core 2 from 4 was carried
out by Hale’s method.6 Macrolactamization, replacement of the
2,2,2-trichloroethoxycarbonyl (Troc) group to the benzyloxy-
carbonyl (Cbz) group, and removal of a benzyl group and two
Cbz groups gave 2, whose spectral data were consistent with
those in Hale’s report.
The final stage of the synthesis is shown in Scheme 9. The
depsipeptide core 2 and the side chain unit 3 were combined
in the presence of 1H-benzotriazol-1-yloxy-tri(pyrrolidino)phos-
phonium hexafluorophosphate (PyBop) and trimethylamine
(NEt3) to afford compound 37. In the last step, the TBS group
of 37 was removed to generate verucopeptin (1) in 23% (2 steps).
The spectral data of synthetic 1 were consisted with those of the
natural compound.
´
10 M. Jimenez, W. Zhu, A. Vogt, B. W. Day and D. P. Curran, Beilstein
J. Org. Chem., 2011, 7, 1372.
In conclusion, verucopeptin (1) was successfully synthesized.
First, the side chain unit 3 was created via construction of six chiral
centers, whereas the Fmoc-SPPS unit was subjected to macro-
lactamization to obtain the depsipeptide core 2. In the final stage,
2 and 3 were coupled to complete the first total synthesis of 1.
11 To confirm whether we obtained the syn aldol product, diol 19
was converted to an acetonide-protected compound and an NOE
experiment was conducted (for details see the ESI†).
12 The absolute configuration of the allylic position generated by the
Evans aldol reaction was determined by a modified Mosher method
(for details see the ESI†).
Taking advantage of the convergent properties in our present 13 (a) S. Ohira, Synth. Commun., 1989, 19, 561; (b) S. Mu¨ller, B. Liepold,
G. J. Roth and H. J. Bestmann, Synlett, 1996, 13.
14 T. Nagamitsu, D. Takano, M. Seki, S. Arima, M. Ohtawa, K. Shiomi,
synthetic scheme, derivatization of the synthetic product by
changing the partial structure in the units is underway for a
Y. Harigaya and S. Omura, Tetrahedron, 2008, 64, 8117.
structure–activity relationship (SAR) study and elucidation of 15 The conversion of 8 to 22 was carried out using the same method
with the case of the enantiomer; J. Xie and D. A. Horne, Tetrahedron
its mode of action.
Lett., 2009, 50, 4485.
This work was supported in part by a Grant-in-Aid for Scientific
ˆ
16 A. Ahlers, T. de Haro, B. Gabor and A. Fırstner, Angew. Chem., Int. Ed.,
Research from the Ministry of Education, Culture, Sports, Science
and Technology (MEXT), Japan (17H06401 and 19H02840) and a
research grant (JP19fk0310112 and JP19am0101092) from the Japan
Agency of Medical Research and Development (AMED), Japan.
2016, 55, 1406.
17 Y. Chen, Y. Lu, Q. Zou, H. Chen and D. Ma, Org. Process Res. Dev.,
2013, 17, 1209.
18 A. J. Oelke, D. J. France, T. Hofmann, G. Wuitschik and S. V. Ley,
Nat. Prod. Rep., 2011, 28, 1445.
This journal is ©The Royal Society of Chemistry 2019
Chem. Commun., 2019, 55, 11956--11959 | 11959