Total synthesis of azumamide E and sugar amino acid-containing analogue

Article abstract of DOI:10.1021/jo8020264

(Chemical Equation Presented) An efficient and practical total synthesis of marine cyclic tetrapeptide, natural product azumamide E (1) is achieved via high-yielding reactions. The strategy also allowed us to synthesize the azumamide E-SAA (sugar amino ac

Full text of DOI:10.1021/jo8020264

SCHEME 1. Retrosynthetic Plan for Azumamide E
Total Synthesis of Azumamide E and Sugar
Amino Acid-Containing Analogue
Srivari Chandrasekhar,*,† Chennamaneni Lohitha Rao,†
Mallikanti Seenaiah,† Police Naresh,‡ Bharatam Jagadeesh,‡
Dharani Manjeera,§ Arpita Sarkar,§ and Manika Pal Bhadra§
Organic DiVision-I, Centre for Nuclear Magnetic Resonance,
and Centre for Chemical Biology, Indian Institute of
Chemical Technology, Hyderabad, India 500 607
sriVaric@iict.res.in
ReceiVed June 28, 2008
deacetylase) inhibitors3 incorporating four nonribosomal amino
acid residues; three of them are R-amino acids (D-series), while
the fourth one is a unique ꢀ-amino acid [(Z)-(2S,3R)-3-amino-
2-methyl-5-nonenedioic acid-9-amide (Amnaa)] in azumamide
A, B, and D and a free acid in C and E.
The azumamide E, in particular, has shown histone deacety-
lase (HDAC) inhibition at the lowest concentration among all
other congeners of azumamides.4,5 We have been fascinated both
by the structural elegance as it incorporates a ꢀ-amino acid
skeleton and by the novel biological profile. Recently, we have
synthesized novel ꢀ-amino acids as a part of our studies devoted
to the understanding of the folding patterns of short peptide
oligomers.6 We embarked on the total synthesis of azumamide
E (Scheme 1), while also being keen to incorporate one of our
own ꢀ-amino acids (ꢀ-SAA)6a into the structural motif of
azumamide, by replacing the D-alanine part. This novel ana-
logue, azumamide E-SAA hybrid, is expected to show a better
An efficient and practical total synthesis of marine cyclic
tetrapeptide, natural product azumamide E (1) is achieved
via high-yielding reactions. The strategy also allowed us to
synthesize the azumamide E-SAA (sugar amino acid)
analogue (2), whose solution-phase NMR and biological
activity studies were also carried out.
The major concern in cancer chemotherapy has been the drug
resistance. There have been several new chemical entities
brought into clinic with various structural diversities and
complexities to overcome this inherent drawback. More recently,
the marine invertebrates have been providing some of the most
interesting and complex secondary metabolites with diverse
affinities toward biological targets. Among these, the most
fascinating have been the cyclic peptides1 with impressive
antiproliferative properties. Fusetani et al. have isolated azuma-
mides A-E from the marine sponge Mycale izuensis.2 These
natural products belong to a rare class of HDAC (histone
(3) (a) Miller, T. A.; Witter, D. J.; Belvedere, S. J. Med. Chem. 2003, 46,
5097–5116. (b) Paris, M.; Porcelloni, M.; Binaschi, M.; Fattori, D. J. Med. Chem.
2008, 51, 1505–1529. (c) George, A. Y.; Cecil, A. R. L.; Mo, A. H. K.; Wen,
S.; Rogers, H.; Habens, F.; Maeda, S.; Yoshida, M.; Packham, G.; Ganesan, A.
J. Med. Chem. 2007, 50, 5720–5726. (d) Greshock, T. J.; Johns, D. M.; Noguchi,
Y.; Williams, R. M. Org. Lett. 2008, 10, 613–616. (e) George, A. Y.; Habens,
F.; Brimmel, M.; Packham, G.; Ganesan, A. J. Am. Chem. Soc. 2004, 126, 1030–
1031. (f) Takizawa, T.; Watanabe, K.; Narita, K.; Oguchi, T.; Abe, H.; Katoh,
T. Chem. Commun. 2008, 1677–1679.
(4) Nakao, Y.; Narazaki, G.; Hoshino, T.; Maeda, S.; Yoshida, M.; Maejima,
H.; Yamashita, J. K. Bioorg. Med. Chem. Lett. 2008, 18, 2982–2984.
(5) Synthesis: (a) Izzo, I.; Maulucci, N.; Bifulco, G.; De Riccardis, F. Angew.
Chem., Int. Ed. 2006, 45, 7557–7560. (b) Wen, S.; Carey, K. L.; Nakao, Y.;
Fusetani, N.; Packham, G.; Ganesan, A. Org. Lett. 2007, 9, 1105–1108. (c)
Maulucci, N.; Chini, M. G.; Di Micco, S.; Izzo, I.; Cafaro, E.; Russo, A.;
Gallinari, P.; Paolini, C.; Nardi, M. C.; Casapullo, A.; Riccio, R.; Bifulco, G.;
De Riccardis, F. J. Am. Chem. Soc. 2007, 129, 3007–3012.
† Organic Divison-1.
‡ Centre for Nuclear Magnetic Resonance.
§ Centre for Chemical Biology.
(1) (a) Xie, W.; Zou, B.; Pei, D.; Ma, D. Org. Lett. 2005, 7, 2775–2777. (b)
Meinke, P. T.; Colletti, S. L.; Doss, G.; Myers, R. W.; Gurnett, A. M.; Dulski,
P. M.; Darkin-Rattray, S. J.; Allocco, J. J.; Galuska, S.; Schmatz, D. M.; Wyvratt,
M. J.; Fisher, M. H. J. Med. Chem. 2000, 43, 4919–4922. (c) Singh, S. B.; Zink,
D. L.; Liesch, J. M.; Mosley, R. T.; Dombrowski, A. W.; Bills, G. F.; Darkin-
Rattray, S. J.; Schmatz, D. M.; Goetz, M. A. J. Org. Chem. 2002, 67, 815–825.
(2) Nakao, Y.; Yoshida, S.; Matsunaga, S.; Shindoh, N.; Terada, Y.; Nagai,
K.; Yamashita, J. K.; Ganesan, A.; van Soest, R. W. M.; Fusetani, N.
Angew.Chem., Int. Ed. 2006, 45, 7553–7557.
(6) (a) Chandrasekhar, S.; Reddy, M. S.; Jagadeesh, B.; Prabhakar, A.; Rao,
M. H. V. R.; Jagannadh, B. J. Am. Chem. Soc. 2004, 126, 13586–13587. (b)
Chandrasekhar, S.; Reddy, M. S.; Babu, B. N.; Jagadeesh, B.; Prabhakar, A.;
Jagannadh, B. J. Am. Chem. Soc. 2005, 127, 9664–9665. (c) Jagannadh, B.;
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10.1021/jo8020264 CCC: $40.75
Published on Web 11/24/2008
2009 American Chemical Society
J. Org. Chem. 2009, 74, 401–404 401

SCHEME 2. Synthesis of ꢀ-Amino Acid Methyl Ester 4
SCHEME 3. Synthesis of Trimer Acid 5
quantitative yields. This, on further reaction with DPPA under
Mitsunobu conditions,12 generated the azido derivative 15 in
86% yield with inversion of configuration. The azido group in
15 was reduced under Staudinger reaction conditions13 (PPh3,
THF/H2O) to furnish the ꢀ-amino acid methyl ester 4. This is
a key intermediate in the total synthesis of both targets. The
synthetic route described so far is well-standardized, and
multigram materials are obtained rather routinely.
The two other building blocks were synthesized following
the standard peptide synthesis protocol. The N-Boc-D-alanine
16 was coupled with the methyl ester of phenyl alanine 17 using
EDCI/HOBt to get the dipeptide 18 (86% yield). This was
further coupled with N-Boc-D-valine, after removal of the Boc
group in 18, to generate the tripeptide 19 that, on LiOH
hydrolysis, gave acid 5 (Scheme 3). This last compound was
amidated with amine 4 in the presence of EDCI/HOBt and
CH2Cl2 to furnish 3. A further reaction with TBSOTf and 2,6-
lutidine as base furnished silyl carbamate 20. We then performed
a LiOH-mediated double hydrolysis of silyl carbamate and
methyl ester functionalities to provide free amino acid 21 that
was ready for the macrolactamization. Luckily, the first at-
tempted EDCI/HOBt method was successful to realize the
protected azumamide 22 in 79% yield. The cleavage of MPM
ether under oxidative conditions (DDQ, CH2Cl2/H2O) provided
the free alcohol 23 that, after oxidation with BAIB and
TEMPO,14 afforded efficiently the target compound 1 in 84%
yield (Scheme 4). Thus, we were able to demonstrate the total
synthesis of a very potent natural product azumamide E via a
high-yielding strategy (overall ∼11% yield).
profile on the basis of the extensive reports of Kessler et al.7
Herein, we report the total synthesis of azumamide E (1) and
azumamide E-SAA (sugar amino acid) analogue (2) in a
stereoselective manner.
The most logical disconnection of this scarce natural product
made us visualize 4 and 5 as key intermediates. The synthesis
of azumamide E-SAA 2 required 28 as a key intermediate, and
the ꢀ-amino ester 4 is a common precursor for the total synthesis
of both 1 and 2.
The unusual ꢀ-amino acid methyl ester 4 was synthesized as
shown in Scheme 2. The commercially available 4-pentyne-1-
ol 7 was benzylated with PMBCl to realize the PMB ether 8 in
over 95% yield. The chlorobutyne-1-ol 6 was coupled with 8
in the presence of CuI, NaI, and K2CO3 to generate the skipped
bis(acetylene) 9 in 86% yield. The selective reduction of the
propargylic alcohol functionality to (E)-allyl alcohol was
8
achieved in 74% yield by LiAlH4 as reducing agent. The
Katsuki-Sharpless asymmetric epoxidation9 with L-(+)-diethyl
tartrate, Ti(O-i-Pr)4, and TBHP was uneventful with 97% yield
and over >98% ee. The three-step oxidation-esterfication was
done in one stroke, without isolating the intermediates, to furnish
the epoxy ester 12 in 74% overall yield for three steps. The
higher-order methyl cuprate was generated following literature
procedure10 and was added to the epoxy ester to realize the
single regioisomer 13 in 78% yield. The hydrogenation of 13
using Lindlar’s catalyst11 gave the (Z)-homoallyl alcohol 14 in
Inspired by the publication of Kessler et al.,7 wherein the
somatostain analogues incorporating ꢀ-amino acid (derived from
furanose sugar) have induced apoptosis in multi-drug-resistant
cells, we embarked on the synthesis of a similar analogue, as
the sugar ꢀ-amino acid methyl ester 24 was readily available
in our repertoire of building blocks. The building block 24 was
amidated with N-Boc-D-valine under well-established coupling
conditions to produce 25 in 85% yield, which, on hydrolysis
with LiOH, gave acid 26 (Scheme 5). The tripeptide acid 28
was assembled by incorporating the methyl ester of D-phenyl
alanine into 26 via 27 in two steps. The unusual ꢀ-amino acid
methyl ester 4 was reacted with 28, and the series of reactions
(7) Gruner, S. A. W.; Keri, G.; Schwab, R.; Venetianer, A.; Kessler, H. Org.
Lett. 2001, 3, 3723–3725.
(8) Eiter, K.; Lieb, F.; Disselnkotter, H.; Oediger, H. Liebigs Ann. Chem.
1978, 658–671.
(9) (a) Katsuki, T.; Sharpless, K. B. J. Am. Chem. Soc. 1980, 102, 5974–
5976. (b) Gao, Y.; Hanson, R. M.; Klunder, J. M.; Ko, S. Y.; Masamune, H.;
Sharpless, K. B. J. Am. Chem. Soc. 1987, 109, 5765–5780.
(10) Chong, J. M. Tetrahedron 1989, 45, 623–628.
(12) Lal, B.; Pramanik, B. N.; Manhas, M. S.; Bose, A. K. Tetrahedron Lett.
1977, 18, 1977–1980.
(13) (a) Vaultier, M.; Knouzi, N.; Carrie, R. Tetrahedron Lett. 1983, 24,
763–764. (b) Staudinger, H.; Meyer, J. HelV. Chim. Acta 1919, 2, 635–646.
(14) Epp, J. B.; Widlanski, T. S. J. Org. Chem. 1999, 64, 293–295.
(11) Lindlar, H. HelV. Chim. Acta 1952, 35, 446–450.
402 J. Org. Chem. Vol. 74, No. 1, 2009

SCHEME 4. Coupling of Amine 4 and Acid 5 and
Completion of the Synthesis of Azumamide E
SCHEME 6. Coupling of Acid 28 and Amine 4 and
Completion of the Synthesis of Azumamide E-SAA Analogue
2
SCHEME 5. Synthesis of Trimer Acid 28
single conformation for 2 during the time scale of observation.
Assignment of side chain protons was difficult because of
1
resonance overlaps in the H spectrum, which have limited us
to use only 10 spatial restraints in MD. Despite the flexibility
in the side chain, the orientation is restricted over the peptide
cavity, which was evidenced in simulated annealing structures
and supported by the observed NOEs between ꢀ SAA-CδH
and methylene protons of Amnda. The temperature coefficient
studies ∆δ/∆T > 4 for all NHs excluded the possibility of
hydrogen bonding and, thus, any defined secondary structure.
This solution structure of the sugar analogue is different from
its natural counterpart in orientation of the side chain.5a
were performed similar to the ones described in Scheme 4 to
synthesize for the first time azumamide E-SAA 2 in ∼10.3%
overall yield (Scheme 6).
To get an insight into the solution-state conformation for
azumamide E-SAA 2 (Figure 1), the restrained MD calculations
were carried out following simulated annealing protocol.
Superposition of energy-minimized structures derived from
these MD studies showed good convergence,15 where at least
the backbone has been well-defined, suggesting a predominantly
FIGURE 1. NMR solution-state (CD3OH) conformation of azumamide
E-SAA 2 obtained by restrained molecular dynamics calculations.
(15) See Supporting Information.
J. Org. Chem. Vol. 74, No. 1, 2009 403

TABLE 1. Biological Activities of Compounds TSA, 1, and 2 on
HeLa NE
mg, 84%) as a white solid: mp 257-259 °C; [R]D ) +72.0 (c 0.1,
MeOH); [Lit. isolation2 [R]D ) +53.0 (c 0.06, MeOH); Lit.
synthesis5a [R]D ) +58.2 (c 0.06, MeOH); Lit. synthesis5b [R]D )
deacetylated lysine/
abs HDAC(µM)
% of HDAC
inhibitiona
1
+84.7 (c 0.525, MeOH)]; H NMR (600 MHz, CD3OH) δ 9.50
compounds
(br s, 1H), 9.33 (br s, 1H), 8.20 (d, J ) 7.9 Hz, 1H), 7.83 (br s,
1H), 7.31-7.16 (m, 5H), 5.61-5.54 (m, 1H), 5.36-5.29 (m, 1H),
4.52-4.45 (m, 1H), 4.35 (br t, J ) 7.4 Hz, 1H), 4.32-4.27 (m,
1H), 3.94 (t, J ) 9.5 Hz, 1H), 3.11-3.00 (m, 2H), 2.90-2.66 (m,
3H), 2.44-2.37 (m, 1H), 2.36-2.21 (m, 4H), 1.35 (d, J ) 6.3 Hz,
3H), 1.33 (d, J ) 6.6 Hz, 3H), 1.04 (d, J ) 6.6 Hz, 3H), 1.00 (d,
J ) 6.6 Hz, 3H); 13C NMR (100 MHz, CD3OH) δ 176.9 (2C),
175.0, 174.6, 174.0, 138.4, 133.1, 130.1 (2C), 129.2 (2C), 127.6,
126.8, 65.0, 58.5, 52.6, 52.5, 44.9, 38.0, 37.9, 30.0, 28.8, 25.3, 20.0,
19.3, 16.1, 11.8; HRMS (ESIMS) calcd for C27H38N4O6Na 537.2689
[M + Na]+, found 537.2678 [M + Na]+.
Azumamide E-SAA (2). The oxidation procedure described
above to obtain 1 from 23 was applied to 33 (15 mg, 0.024 mmol)
with BAIB (23 mg, 0.073 mmol) and TEMPO (1.1 mg, 0.0073
mmol), followed by silica gel column chromatography
(MeOH-CH2Cl2, 11:89), yielding 2 (12 mg, 82% yield) as a white
solid: mp 142-146 °C; [R]D ) +93.0 (c 0.17, MeOH); 1H
NMR(500 MHz, CD3OH) δ 8.45 (d, J ) 7.7 Hz, 1H), 8.31 (br s,
1H), 7.93 (d, J ) 6.9 Hz, 1H), 7.48 (br s, 1H), 7.27-7.14 (m, 5H),
6.06 (d, J ) 3.8 Hz, 1H), 5.49-5.42 (m, 1H), 5.36-5.29 (m, 1H),
4.57-4.51 (m, 2H), 4.47 (d, J ) 3.1 Hz, 1H), 4.11-4.02 (m, 1H),
3.48 (t, J ) 8.5 Hz, 1H), 3.31-3.30 (m, 1H), 3.08 (dd, J ) 13.9,
4.6 Hz, 1H), 2.93 (dd, J ) 13.9, 10.1 Hz, 1H), 2.81-2.73 (m, 1H),
2.72-2.64 (m, 1H), 2.47-2.28 (m, 6H), 1.41 (s, 3H), 1.28 (s, 3H),
1.26 (d, J ) 7.3 Hz, 3H), 0.90 (d, J ) 6.9 Hz, 6H); 13C NMR (100
MHz, CD3OH) δ 179.9, 179.8, 177.0, 175.9, 172.2, 140.8, 134.1,
132.6 (2C), 132.0 (2C), 130.6, 130.3, 116.2, 109.2, 88.4, 82.9, 67.7,
60.2, 55.3, 46.3, 41.3, 37.5, 33.2, 32.6, 30.9, 29.4, 28.8, 26.5, 22.5
(2C), 15.6; ESI (MS) 651 [M + Na]+; HRMS (ESI) calcd for
C32H44N4O9Na 651.3000 [M + Na]+, found 651.3005 [M + Na]+.
TSA (10 µM)
TSA (20 µM)
1 (10 µM)
62.77 ( 2.71
25.53 ( 1.08
81.24 ( 2.19
29.61 ( 1.23
52.01 ( 1.59
4.3 ( 0.019
37
74
19
71
48
96
1 (20 µM)
2 (10 µM)
2 (20 µM)
a HeLa nuclear extract without any HDAC inhibitor, which shows 100
µM of deacetylated lysine and 0% HDAC inhibition.
To characterize whether the compounds are reliable and
potential histone deacetylase inhibitors, the effect of the
compounds on HDAC activity was determined by in vitro
HDAC enzyme assays. Trichostatin A (TSA), a known histone
deacetylase inhibitor, was used as positive control to compare
the activity of compounds 1 and 2. Parallel to its effect on cell
cycle arrest, compounds 1 and 2 were found to inhibit cellular
histone deacetylase activity derived from cell lysate directly.
The histone deacetylase activity was almost completely inhibited
by the 20 µM compound 2 (Table 1) in a cell-free system.
In summary, we have demonstrated a successful synthesis
of azumamide E 1 and azumamide E-SAA 2 involving a
solution-phase peptide synthesis strategy in good overall yields.
The preliminary biological data of azumamide E-SAA 2 was
found to be superior over the synthetic natural product 1.
Experimental Section
(Z)-6-((2R,5R,8R,11R,12S)-8-Benzyl-2-isopropyl-5,12-dimethyl-
3,6,9,13-tetraoxo-1,4,7,10-tetraazacyclotridecan-11-yl)hex-4-enoic
Acid (Azumamide E) (1). To a solution of 23 (30 mg, 0.06 mmol,
1 equiv) in a 1:1 mixture of CH3CN/H2O (0.4 mL) were added
TEMPO (3 mg, 0.02 mmol, 0.3 equiv) and BAIB (57 mg, 0.18
mmol, 3 equiv). The reaction mixture was stirred at ambient
temperature for 6 h and then quenched by the addition of an aqueous
Na2S2O3 solution (1.5 mL). The aqueous phase was then extracted
with CHCl3 (5 × 5 mL), and the combined organic extracts were
dried (Na2SO4) and concentrated in vacuo. Silica gel column
chromatography (MeOH-CH2Cl2, 10:90) of the residue gave 1 (26
Acknowledgment. C.L.R., M.S., and P.N. thank CSIR-New
Delhi for the award of research fellowships.
Supporting Information Available: General experimental
procedures and spectroscopic data for the compounds and copies
1
of H NMR and 13C NMR spectra are provided. This material
is available free of charge via the Internet at http://pubs.acs.org.
JO8020264
404 J. Org. Chem. Vol. 74, No. 1, 2009