Synthesis of virenamide B, a cytotoxic thiazole-containing peptide

Full text of DOI:10.1021/jo9908694

J . Org. Chem. 1999, 64, 8715-8717
8715
Syn th esis of Vir en a m id e B, a Cytotoxic
Th ia zole-Con ta in in g P ep tid e
Christopher J . Moody* and J ames C. A. Hunt
School of Chemistry, University of Exeter, Stocker Road,
Exeter EX4 4QD, U.K.
Received May 27, 1999
Marine organisms are a rich source of novel peptides,
both cyclic and acyclic, many of which show high levels
of biological activity.1-4 For example, the linear thiazole-
containing peptide dolastatin 10 is one of the most potent
antineoplastic agents known,5 and other thiazole-con-
taining cyclic peptides also exhibit pronounced levels of
cytotoxicity.2-4 In continuation of our interest in the
synthesis of biologically active thiazoles,6-8 we now report
the first synthesis of the cytotoxic linear peptide virena-
mide B (1).
The N-terminal amino acid derivative was obtained from
phenylalanine methyl ester hydrochloride by N-propar-
gylation using 3-chloro-3-methylbutyne under copper-
catalyzed conditions,14 followed by Lindlar reduction to
give the amino ester 6. The final coupling reaction was
Virenamide B is a member of a series of five cytotoxic
linear peptides isolated from the Australian ascidian
Diplosoma virens.9,10 The structure, which was assigned
on the basis of extensive NMR experiments, incorporates
a 1-(2-thiazolyl)ethylamine fragment and two phenyla-
lanine residues, one of which bears an inverted prenyl
group at the N-terminus. We have recently reported a
new asymmetric synthesis of 1-(2-thiazolyl)ethylamines
based on the diastereoselective addition of 2-lithiothia-
zoles to (R)- and (S)-O-(1-phenylbutyl) oxime ethers;11 the
starting point for the synthesis of virenamide B was the
(E)-(R)-oxime ether 2.12 Addition of 2-lithiothiazole gave
the hydroxylamine 3 in excellent yield (96%) and dia-
achieved as follows. Ester 6 was hydrolyzed using lithium
hydroxide in aqueous THF. Evaporation of the solvent
gave the solid lithium salt. Dipeptide 5 was deprotected
in methanolic HCl (from AcCl/MeOH) to give the amine
hydrochloride, which was used without purification.
Coupling of the lithium carboxylate with the amine
hydrochloride was carried out in THF-DMF in the
presence of DCC and pentafluorophenol and gave vire-
1
namide B (1) in 51% yield. The H and 13C NMR spectra
1
stereomeric excess (>95% as judged from the H NMR
spectrum of the product). Cleavage of the N-O bond
using the zinc/acetic acid/ultrasound protocol13 gave the
corresponding amine, which was converted into its N-Boc
derivative 4 for purification and characterization. Re-
moval of the Boc group (77%) and coupling with N-Boc-
phenylalanine gave the thiazole dipeptide 5 in good yield.
* Fax: (44) 1392 263434. Email: c.j.moody@ex.ac.uk..
(1) Faulkner, D. J . Nat. Prod. Rep. 1999, 16, 155.
(2) Davidson, B. S. Chem. Rev. 1993, 93, 1771.
(3) Fusetani, N.; Matsunaga, S. Chem. Rev. 1993, 93, 1793.
(4) Wipf, P. Chem. Rev. 1995, 95, 2115.
(5) Pettit, G. R.; Kamano, Y.; Herald, C. L.; Tuinman, A. A.;
Boettner, F. E.; Kizu, H.; Schmidt, J . M.; Baczynskyj, L.; Tomer, K.
B.; Bontems, R. J . J . Am. Chem. Soc. 1987, 109, 6883.
(6) Moody, C. J .; Swann, E. J . Chem. Soc., Perkin Trans. 1 1993,
2561.
(7) Moody, C. J .; Bagley, M. C. J . Chem. Soc., Perkin Trans. 1 1998,
601.
(8) Moody, C. J .; Bagley, M. C. Chem. Commun. 1998, 2049.
(9) Carroll, A. R.; Feng, Y. J .; Bowden, B. F.; Coll, J . C. J . Org. Chem.
1996, 61, 4059.
of the product closely match those described for the
natural product, although the measured optical rotation
{[R]2D5 -77 (c 0.1, CHCl3)} differs by a factor of 10 from
the extremely high value reported earlier {[R]2D5 -775 (c
0.1, CHCl3)}.9 Correspondence with the authors of the
original paper has uncovered an error;15 the correct
specific rotation of the natural material is [R]2D5 -77.5 (c
0.1, CHCl3), which closely matches that of our synthetic
material.
(10) Feng, Y. J .; Bowden, B. F. Aust. J . Chem. 1997, 50, 337.
(11) Moody, C. J .; Hunt, J . C. A. Synlett 1999, 984.
(12) Moody, C. J .; Hunt, J . C. A. Synlett 1998, 733.
(13) Enders, D.; Kempen, H. Synlett 1994, 969.
(14) Hennion, G. F.; Hanzel, R. S. J . Am. Chem. Soc. 1960, 82, 4908.
Wipf, P.; Venkatraman, S. J . Org. Chem. 1996, 61, 6517.
(15) We thank Dr. Bruce Bowden for this information.
10.1021/jo9908694 CCC: $18.00 © 1999 American Chemical Society
Published on Web 10/23/1999

8716 J . Org. Chem., Vol. 64, No. 23, 1999
Notes
(S)-(-)-1-(2-Th ia zolyl)-2-m eth yl-1-p r op yla m in e Hyd r o-
ch lor id e. Acetyl chloride was added dropwise to a stirred
solution of (S)-(-)-N-(tert-butoxycarbonyl)-1-(2-thiazolyl)-2-me-
thylpropylamine (200 mg, 0.78 mmol) in dry MeOH (9 mL). After
3 h the solvent was removed in vacuo to give the title compound
as a colorless solid (154 mg, 77%): mp 185-186 °C (from
i-propanol/ether); [R]2D5 -16.9 (c 1.18, MeOH); IR (KBr) 3438
Exp er im en ta l Section
For general experimental details, see reference 16.
(E)-(R)-(+)-O-(1-P h en ylbu tyl) Isobu tyr a ld oxim e (2). Ob-
tained in 54% yield from the cleavage of (R)-N-(1-phenylbutoxy)
phthalimide (2.13 g, 7.22 mmol) with hydrazine monohydrate
(0.49 mL, 10.10 mmol) in ethanol (21 mL) and subsequent
condensation of the hydroxylamine with isobutyraldehyde (2.62
mL, 28.88 mmol) as previously described;12 a colorless oil, [R]2D2
+9.9 (c 0.95, CH2Cl2); 1H NMR (300 MHz, CDCl3) δ 7.31 (6H,
m), 5.02 (1H, t, J ) 7.2 Hz), 2.44 (1H, m), 1.93 (1H, m), 1.71
(1H, m), 1.34 (2H, m), 1.05 (3H, d, J ) 6.8 Hz), 1.03 (3H, d, J )
6.8 Hz), 0.92 (3H, t, J ) 7.3 Hz); 13C NMR (75 MHz, CDCl3) δ
156.0, 142.6, 128.2, 127.2, 126.8, 84.5, 38.4, 29.3, 20.3, 20.2, 18.9,
14.1 (Me).
(R,S)-N-(1-P h en ylbu toxy)-1-(2-th ia zolyl)-2-m eth yl-1-p r o-
p yla m in e (3). A solution of 2-lithiothiazole was prepared as
follows. Thiazole (0.85 mL, 12.25 mmol) was dissolved in ether
(5.5 mL) under nitrogen and cooled to -78 °C. n-Butyllithium
(4.90 mL, 2.5 M, 12.25 mmol) was added dropwise to the solution
over 15 min. The mixture turned pale yellow and was stirred
for a further 30 min.
1
cm-1; H NMR (400 MHz, CD3OD) δ 7.89 (1 H, d, J ) 3.3 Hz),
7.70 (1 H, d, J ) 3.3 Hz), 4.60 (1 H, d, J ) 6.9 Hz), 2.36 (1 H,
m), 1.11 (3 H, d, J ) 6.9 Hz), 0.97 (3 H, d, J ) 6.9 Hz); 13C NMR
(100 MHz, CDCl3) δ 163.9, 142.5, 120.8, 57.3, 32.5, 17.3, 17.0;
MS (EI) m/z (relative intensity) 157 ([M - Cl]+, 3%), 113 (100);
HRMS calcd for C7H13N2S (M - Cl) 157.0799, found 157.0802.
Anal. calcd for C7H13ClN2S‚0.5H2O: C, 41.7; H, 7.0; N, 13.9.
Found: C, 41.95; H, 6.7; N, 13.9.
Dip ep tid e 5. (tert-Butoxycarbonyl)-phenylalanine (281 mg,
1.06 mmol) was dissolved in dry THF (12 mL) under nitrogen
and cooled to 0 °C. N-Methylmorpholine (0.222 mL, 2.12 mmol)
and iso-butyl chloroformate (0.137 mL, 1.06 mmol) were added
sequentially to the solution and stirred for 30 min. (S)-(-)-1-(2-
Thiazolyl)-2-methyl-1-propylamine hydrochloride (170 mg, 0.88
mmol) in dry DMF (3 mL) was added in one portion, and the
mixture was stirred for 1 h. Water, brine, and EtOAc were
added, and the layers were separated. The aqueous phase was
further extracted with EtOAc. The combined EtOAc extracts
were washed with water, dried (Na2SO4), filtered, and evapo-
rated. The residue was purified by column chromatography on
silica gel eluting with EtOAc/light petroleum (1:4 to 1:2) to give
the title compound as a colorless solid (363 mg, 85%): mp 164-
165 °C (from EtOAc/light petroleum); [R]2D0 -49.50 (c 1.01,
(R)-O-(1-Phenylbutyl) isobutyraldoxime (2) (0.897 g, 4.09
mmol) was dissolved in toluene (9 mL) under nitrogen. The
solution was cooled to -78 °C and stirred for 15 min. Boron
trifluoride diethyl etherate (1.55 mL, 12.25 mmol) was added,
and the mixture was stirred for an additional 15 min. 2-Lithio-
thiazole (12.25 mL, 12 mmol) was added dropwise to the mixture,
care being taken not to allow the temperature to rise above -70
°C. The mixture was stirred until complete consumption of the
starting material, whereupon saturated ammonium chloride
solution was added. The reaction mixture was allowed to warm
to room temperature and was then extracted with ether. The
combined organic extracts were dried (Na2SO4), filtered, and
evaporated. The residue was purified by column chromatography
on silica gel eluting with EtOAc/light petroleum to give the title
compound as a pale yellow oil (1.187 g, 96%, de >95%): [R]D20
MeOH); IR (KBr) 3405, 3309, 1680, 1652 cm-1 1H NMR (400
;
MHz, CDCl3) δ 7.66 (1 H, d, J ) 3.3 Hz), 7.26-7.12 (6 H, m),
6.80 (1 H, br d, J ) 8.6 Hz), 5.16 (1 H, d, J ) 6.2 Hz), 5.13 (1 H,
d, J ) 6.2 Hz), 4.38 (1 H, m), 3.06 (2 H, d, J ) 6.9 Hz), 2.31-
2.26 (1 H, m), 1.41 (9 H, br s), 0.88 (3 H, d, J ) 6.9 Hz), 0.86 (3
H, d, J ) 6.8 Hz); 13C NMR (100 MHz, CDCl3) δ 170.9, 169.8,
155.4, 142.5, 136.5, 136.5, 129.2, 128.6, 126.8, 118.3, 80.2, 56.1,
55.9, 38.0, 33.4, 28.2, 19.0, 17.8; MS (CI) m/z (relative intensity)
404 (MH+, 49%), 142 (100); HRMS calcd for C21H30N3O3S (MH)
404.2008, found 404.2005. Anal. calcd for C21H29N3O3S: C, 62.5;
H, 7.2; N, 10.4. Found: C, 62.45; H, 7.3; N, 10.3.
1
+45.2 (c 1.0, CHCl3); IR (film) 3369 cm-1; H NMR (300 MHz,
CDCl3) δ 7.73 (1 H, d, J ) 3.3 Hz), 7.37-7.23 (6 H, m), 5.67 (1
H, br s), 4.59 (1 H, dd, J ) 5.6, 8.3 Hz), 4.10 (1 H, d, J ) 7.0
Hz), 1.98 (1 H, m), 1.64 (1 H, m), 1.43 (1 H, m), 1.27-1.20 (2 H,
m), 0.89 (3 H, d, J ) 11.4 Hz), 0.78 (3 H, t, J ) 7.2 Hz), 0.76 (3
H, d, J ) 11.4 Hz); 13C NMR (75 MHz, CDCl3) δ 172.9, 142.9,
141.8, 128.4, 127.4, 126.7, 126.5, 118.5, 85.6, 69.1, 38.6, 31.7,
19.4, 19.2, 18.9, 14.0; MS (CI) m/z (relative intensity) 304 (M+,
2%), 172 (58), 133 (71), 91 (100), 77 (20); HRMS calcd for
C17H24N2OS (M) 304.1609, found 304.1618.
(S)-(+)-Met h yl 2-[(2-Met h ylb u t -3-en -2-yl)a m in o]3-p h e-
n ylp r op a n oa te (6). (S)-Phenylalanine methyl ester hydrochlo-
ride (2 g, 9.27 mmol), copper metal (58.9 mg, 0.927 mmol), and
copper(I) chloride (92 mg, 0.927 mmol) were suspended in THF
(14 mL) and cooled to 0 °C. Triethylamine (2.84 mL, 20.4 mmol)
followed by 3-chloro-3-methylbut-1-yne (1.20 mL, 10.7 mmol) in
THF (6 mL) were added to the solution, and the mixture was
stirred for 3 h. The mixture was diluted with ether and filtered
through a pad of Celite. The filtrate was evaporated, and the
residue was partly purified by column chromatography on silica
gel eluting with EtOAc/light petroleum (1:10) to give a pale
yellow oil, which was used without further purification.
(S)-(-)-N-(ter t-Bu t oxyca r b on yl)-1-(2-t h ia zolyl)-2-m et h -
ylp r op yla m in e (4). Zinc dust (8.37 g, 128 mmol) was added to
a mixture of (R,S)-N-(1-phenylbutoxy)-1-(2-thiazolyl)-2-methyl-
1-propylamine (3) (0.967 g, 3.2 mmol) in acetic acid/water (20
mL, 1:1). The mixture was placed in a sonic bath at 40 °C, and
the was reaction followed by TLC until completion. The zinc was
filtered and washed with ether. The filtrate was basified with
sodium hydroxide solution (3 M), and the aqueous layer was
exhaustively extracted with dichloromethane. The extracts were
combined, dried (Na2SO4), filtered, and evaporated. The residue
was dissolved in dichloromethane (7 mL), and di-tert-butyl
dicarbonate (4 mmol) and DMAP (cat.) were added. The mixture
was stirred at room temperature for 12 h. Saturated aqueous
sodium bicarbonate (10 mL) was added, and the mixture was
stirred for 10 min. The mixture was extracted with dichlo-
romethane, and the organic extracts were combined, dried (Na2-
SO4), filtered and evaporated. The residue was purified by flash
chromatography on silica gel, eluting with EtOAc/light petro-
leum to give the title compound as a colorless oil (60%, ee 92%
by HPLC): [R]2D0 -33.6 (c 1.1, CHCl3); IR (film) 3299, 1704
Quinoline (0.95 mL) and 10% palladium on charcoal (100 mg)
were added to the yellow oil dissolved in light petroleum. A
balloon filled with hydrogen gas was fitted, and the mixture was
stirred for 30 min. The mixture was filtered, the solvent was
removed in vacuo, and the residue was purified by column
chromatography on silica gel eluting with ether/light petroleum
(1:20) to give the title compound as a colorless oil (1.168 g,
51%): [R]2D2 +24.73 (c 1.86, CHCl3); IR (film) 3328, 2971, 1737
cm-1; 1H NMR (300 MHz, CDCl3) δ 7.30 (5 H, m), 5.51 (1 H, dd,
J ) 10.4, 17.6 Hz), 4.96-4.89 (2 H, m), 3.58 (3 H, s), 3.43 (1 H,
dd, J ) 6.3, 7.7 Hz), 2.87 (1 H, dd, J ) 6.6, 13.2 Hz), 2.79 (1 H,
J ) 7.7, 13.2 Hz), 1.07 (3 H, s), 1.04 (3 H, s); 13C NMR (75 MHz,
CDCl3) δ 176.9, 145.6, 137.5, 129.4, 128.2, 126.6, 112.3, 57.7,
54.6, 51.5, 41.6, 27.1, 26.7; MS (EI) m/z (relative intensity) 248
(MH+, 4%), 188 (87), 156 (97), 120 (99), 91 (100); HRMS calcd
for C15H21NO2 (MH) 248.1651, found 248.1650.
cm-1 1H NMR (300 MHz, CDCl3) δ 7.68 (1 H, d, J ) 3.3 Hz),
;
7.19 (1 H, d, J ) 3.3 Hz), 5.37 (1 H, br d, J ) 8.9 Hz), 4.86 (1 H,
dd, J ) 5.5, 8.9 Hz), 2.34-2.23 (1 H, m), 1.40 (9 H, br s), 0.91 (3
H, d, J ) 6.8 Hz), 0.86 (3 H, d, J ) 6.8 Hz); 13C NMR (75 MHz,
CDCl3) δ 171.2, 155.5, 142.5, 118.3, 79.7, 57.7, 33.6, 28.3, 19.2,
17.5; MS (EI) m/z (relative intensity) 257 (MH+, 76%), 183 (100);
HRMS calcd for C12H21N2O2S (MH) 257.1323, found 257.1322.
(S)-Lith iu m 2-[(2-Meth ylbu t-3-en -2-yl)a m in o]3-p h en yl-
p r op a n oa te. Lithium hydroxide monohydrate (34 mg, 0.81
mmol) was added to a solution of the above ester 6 (200 mg,
0.81 mmol) in THF/water (15 mL:2 mL), and the mixture was
heated under reflux for 24 h. The solution was allowed to cool
(16) Bagley, M. C.; Buck, R. T.; Hind, S. L.; Moody, C. J . J . Chem.
Soc., Perkin Trans. 1 1998, 591.

Notes
J . Org. Chem., Vol. 64, No. 23, 1999 8717
to room temperature, and the solvent was removed in vacuo to
give a colorless solid, which was used without further purifica-
tion.
Dep r otection of Dip ep tid e 5. Acetyl chloride (0.21 mL, 2.98
mmol) was added over 3 h to a solution of dipeptide 5 dissolved
in dry MeOH (5 mL) under nitrogen. The solvent was removed
in vacuo to give a colorless oil, which was used without further
purification.
3087, 2969, 1675, 1660 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.11
(1 H, d, J ) 8.0 Hz), 7.69 (1 H, d, J ) 3.4 Hz), 7.29-7.09 (11 H,
m), 6.95 (1 H, d, J ) 8.9 Hz), 5.21 (1 H, m), 5.16 (1 H, dd, J )
6.1, 8.9 Hz), 4.86-4.81 (2 H, m), 4.64 (1 H, dd, J ) 7.2, 8.2 Hz),
3.27 (1 H, dd, J ) 4.1, 9.1 Hz), 3.10 (1 H, dd, J ) 7.2, 14.0 Hz),
3.05 (1 H, dd, J ) 7.2, 14.0 Hz), 3.99 (1 H, dd, J ) 4.1, 13.6 Hz),
2.41 (1 H, dd, J ) 9.1, 13.6 Hz), 2.28 (1 H, m), 0.92 (3 H, s), 0.90
(3 H, s), 0.88 (3 H, d, J ) 2.6 Hz), 0.86 (3 H, d, J ) 2.6 Hz); 13
C
Vir en a m id e B (1). (S)-Lithium 2-phenyl-N-2-(2-methylbut-
3-ene)ethylamino carboxylate (71.2 mg, 0.298 mmol), DCC (61.4
mg, 0.298 mmol), and pentafluorophenol (54.8 mg, 0.298 mmol)
were dissolved in THF under nitrogen. The above hydrochloride,
dissolved in DMF (2 mL), was added dropwise to the solution.
The mixture was stirred overnight, whereupon a white precipi-
tate had formed. Ethyl acetate was added, and the solution was
filtered. Saturated sodium bicarbonate solution was added to
the filtrate, and the layers were separated. A further portion of
sodium bicarbonate was added, and the layers were separated.
The combined aqueous layers were extracted with EtOAc. The
combined organic phases were washed with water, dried (Na2-
SO4), filtered, and evaporated to give a green residue. The
residue was purified by column chromatography on silica gel
eluting with EtOAc/light petroleum (1:2 to 2:3) to give the title
compound as a colorless oil (65.5 mg, 51%): [R]2D5 -77 (c 0.1,
CHCl3) (lit.9 [R]D25 -775 (c 0.10, CHCl3); IR (CHCl3) 3419, 3330,
NMR (75 MHz, CDCl3) δ 175.7, 170.6, 170.5, 144.7, 142.6, 137.1,
136.6, 129.5, 129.4, 128.6, 128.5, 127.0, 126.8, 118.3, 112.7, 58.0,
56.2, 54.8, 53.9, 39.9, 37.9, 33.2, 28.0, 25.0, 19.2, 17.8; MS (CI)
m/z (relative intensity) 519 (MH+, 11%), 188 (33), 142 (100), 120
(32); HRMS calcd for
519.2798.
C30H39N4O2S (MH) 519.2793, found
Ack n ow led gm en t. We thank the EPSRC for their
support of this work and the EPSRC Mass Spectrometry
Service at Swansea for mass spectrometric measure-
ments.
Su p p or tin g In for m a tion Ava ila ble: 13C NMR spectra of
compounds 1-6. This material is available free of charge via
the Internet at http://pubs.acs.org.
J O9908694