Toward tubulysin: Gram-scale synthesis of tubuvaline-tubuphenylalanine fragment

Article abstract of DOI:10.1021/jo9015503

(Chemical Equation Presented) A practical and stereoselective synthesis of the tubuvaline-tubuphenylalanine (Tuv-Tup) fragment of tubulysin is achieved involving the opening of aziridine, crucial MacMillan α-hydroxylation on both fragments, and an epoxide

Full text of DOI:10.1021/jo9015503

pubs.acs.org/joc
Toward Tubulysin: Gram-Scale Synthesis of
Tubuvaline-Tubuphenylalanine Fragment
Srivari Chandrasekhar,* Bodugam Mahipal, and
Mitta Kavitha
Organic Division-I, Indian Institute of Chemical Technology,
Hyderabad 500 607, India
srivaric@iict.res.in
Received July 17, 2009
FIGURE 1. Structures of tubulysins.
While few efforts have resulted in meaningful total synth-
eses,5 others have led to new strategies for partial syntheses.6
More recently, simpler analogues of tubulysin have been
reported to retain a potent level of cytotoxicity.7 The potent
analogues in this report were devoid of the Mep part of the
molecule, indicating that the requirement of this part is “not
so critical”. Our continued interest in synthesis of peptide
natural products and unusual amino acids8 prompted us to
take up the synthesis of tubulysin D, which culminated in the
synthesis of the essential core Tuv-Tup portion, the results
being disclosed herein. Our objective has been to achieve
gram quantities of the core Tuv-Tup of the natural product
so that new analogues could be designed. The classical
retrosynthesis of this tetra peptide produced two fragments
Mep-Ile-OMe 2, N-boc-Tuv-Tup-OMe 3 (Scheme 1).
The synthesis of tubuphenylalanine (Tup) commenced
from epoxysilyl ether 4 obtained from (-)-citronellol in six
steps as reported by us recently9 (Scheme 2). This epoxide 4
was treated with the Grignard reagent derived from bromo-
benzene to provide the single regioisomer 7 in over 94%
yield. The secondary alcohol was silylated using TBSCl and
imidazole to disilyl ether 8 followed by mono-desilylation of
TBDPS resulted in 8a. This was converted in a two-step
process to olefinic compound 9 via iodo derivative followed
by 1,2-elimination.
A practical and stereoselective synthesis of the tubuva-
line-tubuphenylalanine (Tuv-Tup) fragment of tubulysin
is achieved involving the opening of aziridine, crucial
MacMillan R-hydroxylation on both fragments, and an
epoxide-opening reaction.
Tubulysin is a peptide natural product in clinical devel-
opment with potent anticancer activity against the P-glyco
protein-expressing KBV1 cell line.1 This scarce natural pro-
duct with very limited supplies from fermentation of Angio-
coccus disciformis An d48 (with an yield of 0.25-1 mg/L)
binds to the peptide-binding site located near the vinca
alkaloid binding site of β-tubulin (Figure 1).1-3 The aryl-
hydroxylated tubulysin A has shown interesting antiangio-
genic effects.4 This exotic profile triggered synthetic and
medicinal chemists to initiate program toward its synthesis.
The desilylation in 9 was achieved using TBAF to realize
alcohol 10 which was converted to the azido compound 11
via Mitsunobu type inversion process (TPP, DIAD, and
DPPA).10
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Chem., Int. Ed. 2007, 46, 3526–3529. (c) Balasubramanian, R.; Raghavan, B.;
Begaye, A.; Sackett, D. L.; Fecik, R. A. J. Med. Chem. 2009, 52, 238–240. (d)
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DOI: 10.1021/jo9015503
r
Published on Web 11/23/2009
J. Org. Chem. 2009, 74, 9531–9534 9531
2009 American Chemical Society

Chandrasekhar et al.
JOCNote
SCHEME 1. Retrosynthetic Analysis of Tubulysin
SCHEME 3
SCHEME 2
the free amine. This on treatment with (Boc)2O in dichloro-
methane and TEA provided mono Boc derivative 14 which
on further reaction with BuLi and (Boc)2O produced diboc
derivative 14a in 93% yield. The oxidative cleavage of
olefin functionalily in 14a was realized with OsO4, 2,6
lutidine, and NaIO4 furnished aldehyde 15 amicable for
R-hydroxylation.
The aldehyde 15 was subjected to the crucial MacMillan
R-hydroxylation13 using nitrosobenzene and 40 mol % of
L-proline in DMSO, followed by rapid reduction with
sodium borohydride to furnish the unstable anilinoxy com-
pound which was further treated with 30 mol % of CuSO4 in
methanol at room temperature to cleave the O-N bond
providing the diol 16 with high enantio- and diastereoselec-
tivity (67% yield, 98% de). This resulting 1,2-diol 16 on
treatment with TBSCl, and imidazole was monosilylated
to afford 17 in 98% yield. On further reaction with
MOMCl and DIPEA in dichloromethane, this provided
The other key fragment Tuv is synthesized on gram scale
starting from L-valine and L-cysteine (Scheme 3). The
L-valine was converted to the aziridine 12 by a known
procedure.11 The aziridine 12 was opened regioselectively
with allylmagnesium bromide using CuCN as a catalyst to
furnish 13,12 which on exposure to Na-naphthalene realized
(11) (a) Kim, B. M.; So, S. M.; Choi, H. J. Org. Lett. 2002, 4, 949–952. (b)
Krauss, I. J.; Leighton, J. L. Org. Lett. 2003, 5, 3201–3203. (c) Ye, W.; Leow,
D.; Goh, S. L.; Tan, C. T.; Chian, C. H.; Tan, C. H. Tetrahedron Lett. 2006,
47, 1007–1010. (d) Argouarch, G.; Gibson, C. L.; Stones, G.; Sherrington, D.
C. Tetrahedron Lett. 2002, 43, 3795–3798. (e) Argouarch, G.; Stones, G.;
Gibson, C. L.; Kennedy, A. R.; Sherrington, D. C. Org. Biomol. Chem. 2003,
1, 4408–4417. (f) Pei, Y.; Brade, K.; Brule, E.; Hagberg, L.; Lake, F.;
Moberg, C. Eur. J. Org. Chem. 2005, 13, 2835–2840.
(13) (a) Mangion, I, K.; MacMillan, D. W. C. J. Am. Chem. Soc. 2005,
127, 3696–3697. (b) Varseev, G. N.; Maier, M. E. Org. Lett. 2007, 9, 1461–
1464. (c) Brown, S. P.; Brochu, M. P.; Sinz, C. J.; MacMillian, D. W. C. J.
Am. Chem. Soc. 2003, 125, 10808–10809. For other reports on proline-
catalyzed oxidation of aldehydes, see: (d) Zhong, G. Angew. Chem., Int. Ed.
2003, 42, 4247–4250. (e) Hayashi, Y.; Yamaguchi, J.; Hibino, K.; Shoji, M.
Tetrahedron Lett. 2003, 44, 8293–8296.
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2007, 48, 1907–1910.
9532 J. Org. Chem. Vol. 74, No. 24, 2009

Chandrasekhar et al.
JOCNote
SCHEME 4
tubulysin and biological evaluation of the analogues is in
progress.
Experimental Section
(2S,4R)-6-(tert-Butyldiphenylsilyloxy)-4-methyl-1-phenylhex-
an-2-ol (7). Magnesium turnings (1.36 g, 56.6 mmol) were placed
in a 250 mL, two-neck, round-bottomed flask equipped with a
reflux condenser and suspended in 80 mL of dry THF
under nitrogen atmosphere. Bromobenzene (8.94 g, 6.0 mL,
56.6 mmol) was added slowly, and the suspension was vigo-
rously stirred at room temperature by maintaining the cooled
condenser for 1 h. The generated Grignard reagent was slowly
added to epoxide 4 (7.0 g, 19.0 mmol) dissolved in dry THF
(30 mL) at 0 °C using a cannula under N2 atmosphere. The
reaction mixture was stirred at the same temperature for 3 h.
After completion of the reaction (monitored by TLC), the
reaction was quenched with satd NH4Cl solution (25 mL).
The reaction mixture was diluted with EtOAc (100 mL), and
the organic layer was separated. The organic layer was washed
with water (75 mL) and brine (60 mL), dried over anhydrous
Na2SO4 (5.0 g), and evaporated in vacuo. The crude product
was chromatographed over silica gel (4% EtOAc/petroleum
ether) to afford pure sigle regioisomer 7 (7.97 g, 94%) as a
colorless gummy liquid: [R]20D=þ0.5 (c 0.5, CHCl3); 1H NMR
(300 MHz, CDCl3) δH (ppm) 7.70-7.64 (m, 4H), 7.46-7.17 (m,
11H), 3.94-3.84 (m, 1H), 3.71 (dt, J=2.2, 6.7 Hz, 2H), 2.77 (dd,
J=4.3, 13.5 Hz, 1H), 2.63 (dd, J=8.3, 13.5 Hz, 1H), 1.94-1.80
(m, 1H), 1.69-1.37 (m, 3H), 1.30-1.20 (m, 1H), 1.04 (s, 9H),
0.86 (d, J=6.6 Hz, 3H); 13C NMR (75 MHz, CDCl3) δC (ppm)
138.6, 134.0, 129.5, 129.4, 128.5, 127.5, 126.3, 70.3, 62.0, 44.6,
44.2, 40.2, 26.8, 26.2, 19.4, 19.1; IR (neat) νmax 3422, 3067, 2929,
2858, 1462, 1427, 1108, 823, 739, 701, 612, 504 cm-1; MS (APCI)
m/z 447 (100) [M þ H]þ; HRMS (ESI) [M þ H]þ C29H39O2Si
calcd 447.2714, found 447.2734.
the diprotected compound 17a which on desilylation resulted
the free alcohol 17b in over 84% yield. This free alcohol in a
one-pot reaction was oxidized to aldehyde (unstable) which
without isolation was condensed with the methyl ester of
L-cysteine salt 6 using Swern oxidation conditions to afford
the thiazolidine 18 in 88% yield .14,15
Di- tert-butyl ((3R,5R)-5,6-Dihydroxy-2-methylhexan-3-yl)-
carbamate (16). (a) To a 250 mL, two-neck, round-bottomed
flask equipped with a magnetic stir bar was charged with
L-proline (0.98 g, 8.5 mmol), and DMSO (15 mL) was added
at room temperature under nitrogen atmosphere. After the
suspension was stirred for 10 min, nitrosobenzene (2.29 g,
21.4 mmol) was added in one portion at which time the solution
became green. Aldehyde 15 (14.1 g, 42.8 mmol) in DMSO
(25 mL) was added in one portion to the above greenish
suspension and stirring continued at room temperature until
the reaction was determined to be complete (the change of color
of the green color solution to a yellow homogeneous solution
was observed by TLC). The reaction mixture was then trans-
ferred to a suspension of NaBH4 (2.43 g, 63.9 mmol) in ethanol
(50 mL) at 0 °C. After 20 min of stirring, the reaction mixture
was treated with saturated aqueous NaHCO3 (50 mL) and
extracted with dichloromethane (3 ꢀ 100 mL). The combined
organic layers were washed with brine (50 mL), dried over
Na2SO4, filtered, and concentrated in vacuo. The crude com-
pound was subjected to flash chromatography using silica gel
(15% EtOAc/petroleum ether) to afford an unstable anilinoxy
compound (7.4 g, 85%) which was used immediately for the next
reaction.
(b) To a solution of the above anilinoxy compound (7.4 g,
16.8 mmol) in methanol (60 mL), was added CuSO4 (1.26 g,
5.0 mmol). The reaction mixture was stirred at room tempera-
ture overnight and then quenched with a cold saturated NH4Cl
solution (10 mL). The mixture was filtered on a Celite pad and
washed thoroughly with ethyl acetate (60 mL), and the complete
solvent was removed under reduced pressure. The compound
was extracted with ethyl acetate (3 ꢀ 60 mL). The com-
bined organic layers were washed with brine (40 mL), dried
over anhydrous Na2SO4, filtered, and concentrated in vacuo.
The key fragment (Tuv) 20 was obtained in over 73% yield
for the two-step reaction from 18 via the oxidation with
MnO214 followed by the saponification using LiOH H2O.
3
The azido group in 11 was subjected to LiAlH4 reaction
followed by coupling with acid 20 using EDCI/HOBT
conditions to deliver the dipeptide olefinic compound 21
without any detectable loss of stereochemical purity and
with very satisfactory yields (Scheme 4). This olefinic
compound 21 was subjected to OsO4 and 2,6-lutidine con-
ditions in a single step to give the aldehyde, which without
purification on reaction with bis(acetoxy)iodobenze and
TEMPO provided the triprotected Tup-Tuv-OH 22 in over
80% yield. The acid functionality was converted to the
methyl ester of the triprotected Tup-Tuv 23 in 98% yield.
Finally, the reaction with TFA/CH2Cl2 followed by treat-
ment with TEA and (Boc)2O afforded the desired N-Boc-
Tuv-Tup-OMe 3 in 78% yield with desirable functionality
for further elaborations and analogues.
Coincidentally, this fragment is also the late-stage inter-
mediate reported by Zanda and co-workers5b which has
indistinguishable spectroscopic data. In summary, we have
developed an effective gram-scale synthesis of the essential
core tubuvaline-tubuphenylalanine (Tuv-Tup) fragment.
Further work toward N-terminal modified analogues of
(14) (a) Hughes, R. A.; Thompson, S. P.; Alcaraj, L.; Moody, C. J. J. Am.
Chem. Soc. 2005, 127, 15644–15651. (b) Sani, M.; Fossati, G.; Huguenot, F.;
Zanda, M. Angew. Chem., Int. Ed. 2007, 46, 3526–3529.
(15) (a) Bachand, B.; DiMaio, J.; Siddiqui, M. A. Bioorg. Med. Chem.
Lett. 1999, 9, 913–918. (b) Szilagyi, L.; Gyorgydeak, Z. J. Am. Chem. Soc.
1979, 101, 427–432.
J. Org. Chem. Vol. 74, No. 24, 2009 9533

Chandrasekhar et al.
JOCNote
The residue was then purified by silica gel chromatography (5%
EtOAc/petroleum ether) to afford diol 16 (4.63 g, 79% yield,
98% de) as a brownish gummy liquid: [R]30D = -26.4 (c 1.0,
CHCl3); 1H NMR (300 MHz, CDCl3) δH (ppm) 3.80-3.44 (m,
4H), 2.28-2.17(m, 1H), 1.89-1.63 (m, 2H), 1.49 (s, 18H), 0.93
(d, J=6.6 Hz, 6H); 13C NMR (75 MHz, CDCl3) δC (ppm) 154.3,
82.6, 68.9, 66.6, 61.8, 33.6, 31.0, 27.9, 20.7, 20.2.; IR (KBr) νmax
3430, 2973, 2930, 2361, 2335, 1736, 1693, 1460, 1368, 1247, 1165,
1127, 942, 854, 769, 669, 579 cm-1; MS (ESI) m/z 348 (100) [M þ
H]þ; HRMS (ESI) [M þ Na]þ C17H33NNaO6 calcd 370.2200,
found 370.2204.
(2S,4R)-Methyl 4-(2-((1R,3R)-3-(tert-Butoxycarbonylamino)-
1-hydroxy-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-
phenylpentanoate (3). The stirred solution of dipeptide ester 23
(3.0 g, 4.3 mmol) in dry CH2Cl2 (10 mL) at 0 °C under nitrogen
atmosphere was treated with CF3COOH (15 mL). The reaction
mixture was warmed to room temperature and stirred for 16 h.
The solvent and TFA were removed under reduced pressure.
The resulted salt was dissolved in dichloromethane (30 mL),
cooled to 0 °C, and treated with triethylamine (1.25 mL, 8.6
mmol). After the mixture was stirred for 10 min, (Boc)2O (1.0 g,
4.7 mmol) was added dropwise. The reaction was maintained at
room temperature for 30 min. The solvent was evaporated on a
rotary evaporator, and the crude compound was chromato-
graphed over SiO2 (35% EtOAc/petroleum ether) to afford the
pure N-Boc-protected dipeptide compound 3 (1.85 g, 78%) as a
colorless gummy liquid: [R]23D=þ16.1 (c 0.7, CHCl3); 1H NMR
(300 MHz, CDCl3) δH (ppm) 8.02 (s, 1H), 7.30-7.14 (m, 5H),
7.08 (d, J=9.3 Hz, 1H), 5.20 (br s, 1H), 4.87 (br d, J=10.1 Hz,
1H), 4.59 (d, J=9.4 Hz, 1H), 4.46-4.33 (m, 1H), 3.80-3.68 (m,
1H), 3.63 (s, 3H), 2.98-2.83 (m, 2H), 2.65-2.54 (m, 1H),
2.09-1.90 (m, 2H), 1.87-1.73 (m, 2H), 1.66-1.53 (m, 1H),
1.46 (s, 9H), 1.15 (d, J=7.0 Hz, 3H), 1.01 (d, J=6.7 Hz, 3H), 1.00
(d, J=6.7 Hz, 3H); 13C NMR (150 MHz, CDCl3) δC (ppm)
176.6, 175.2, 160.7, 157.9, 149.6, 137.4, 129.6, 128.3, 126.4,
122.9, 80.5, 68.7, 52.2, 51.7, 48.0, 41.5, 41.0, 37.7, 36.4, 32.2,
28.3, 19.3, 18.3, 17.8; IR (KBr) νmax 3364, 2970, 2915, 1742,
1703, 1650, 1536, 1483, 1460, 1376, 1269, 1227, 1105, 1086,
947, 761, 685, 670 cm-1; MS (ESI) m/z 538 (100) [M þ H]þ;
HRMS (ESI) [M þ H]þ C30H40N3O4S calcd 538.2734, found
538.2749.
Acknowledgment. B.M. and M.K. thank the Council of
Scientific and Industrial Research (CSIR), New Delhi, for
research fellowships and the Director, Indian Institute of
Chemical Technology (IICT), for research facilities.
Supporting Information Available: Experimental proce-
dures, melting points, specific rotation, and spectral data (1H
NMR, 13C NMR, IR, MS, HRMS) and copies of 1H NMR and
13C NMR for all compounds described in the paper. This
material is available free of charge via the Internet at http://
pubs.acs.org.
9534 J. Org. Chem. Vol. 74, No. 24, 2009