Expedient synthesis of N-methyl tubulysin analogues with high cytotoxicity

Article abstract of DOI:10.1021/jo800384x

(Chemical Equation Presented) An optimized and highly efficient synthesis of potent, bioactive N-methyl tubulysin analogues 2 and 4 has been achieved with > 40% overall yields. This synthesis represents a significant improvement over previously reported s

Full text of DOI:10.1021/jo800384x

Expedient Synthesis of N-Methyl Tubulysin Analogues with High
Cytotoxicity
Andrew W. Patterson, Hillary M. Peltier, and Jonathan A. Ellman*
Department of Chemistry, UniVersity of California, Berkeley, California 94720
jellman@berkeley.edu
ReceiVed February 15, 2008
An optimized and highly efficient synthesis of potent, bioactive N-methyl tubulysin analogues 2 and 4
has been achieved with > 40% overall yields. This synthesis represents a significant improvement over
previously reported syntheses of these and related tubulysin analogues. The stereoselective synthesis of
the unnatural amino acid tubuvaline is accomplished using tert-butanesulfinamide chemistry. N-Alkylation
to form N-methyl tubuvaline is performed without protection of the tubuvaline alcohol by implementing
a unique N-methylation strategy via formation and reduction of a 1,3-tetrahydrooxazine heterocycle.
Acylation of the hindered N-methyl tubuvaline amine utilizes a novel sequence of O-acylation followed
by an O- to N-acyl transfer to form the hindered amide bond between N-methyl tubuvaline and isoleucine.
This high-yielding synthesis should enable the production of large quantities of material for biological
studies.
Introduction
the Tuv-amide,2,3 while the less potent tubulysins (tubulysins
U-Z) lack this functionality.4,5
The tubulysins are exceptionally potent cell growth inhibitors
that act by inhibiting tubulin polymerization, thus inducing
apoptosis.1 With activity exceeding that of vinblastine, pacli-
taxel, and the epothilones,2 the tubulysins are exciting leads for
new anticancer therapeutics.
The tubulysin scaffold can be divided into four amino acid
segments as defined in Figure 1: D-Mep (D-N-methyl pipecolinic
acid), Ile (L-isoleucine), Tuv (tubuvaline), and Tup/Tut (tu-
buphenylalanine/tubutyrosine). The most potent tubulysins (tu-
bulysins A-I) incorporate a rare O-acyl N,O-acetal moiety on
The isolation of the tubulysins from myxobacterial cultures3
produces only small quantities of these natural products;
therefore, efficient synthesis routes to the natural tubulysins as
well as their analogues are needed to advance biological studies.
Indeed, much effort has been devoted to the synthesis of these
complex tetrapeptides. Partial syntheses of the unnatural amino
acid segments Tuv and Tup/Tut have been reported,6 as have
syntheses of unnatural tubulysin analogues7–10 and the less
potent tubulysins U and V.11 In 2006, our group reported the
(1) (a) Khalil, M. W.; Sasse, F.; Lu¨nsdorf, H.; Elnakady, Y. A.; Reichenbach,
H. ChemBioChem 2006, 7, 678–683. (b) Kaur, G.; Hollingshead, M.; Holbeck,
S.; Schauer-Vukasˇinovic´, V.; Camalier, R. F.; Do¨mling, A.; Agarwal, S. Biochem.
J. 2006, 396, 235–242.
(4) Do¨mling, A.; Beck, B.; Eichelberger, U.; Sakamuri, S.; Menon, S.; Chen,
Q.-Z.; Lu, Y.; Wessjohann, L. A. Angew. Chem., Int. Ed. 2006, 45, 7235–7239.
(5) Do¨mling, A.; Richter, W. Mol. DiVersity 2005, 9, 141–147.
(6) (a) Ho¨fle, G.; Glaser, N.; Leibold, T.; Karama, U.; Sasse, F.; Steinmetz,
H. Pure Appl. Chem. 2003, 75, 167–178. (b) Wipf, P.; Takada, T.; Rishel, M. J.
Org. Lett. 2004, 6, 4057–4060. (c) Friestad, G.; Marie´, J.-C.; Deveau, A. M.
Org. Lett. 2004, 6, 3249–3252.
(2) Steinmetz, H.; Glaser, N.; Herdtweck, E.; Sasse, F.; Reichenbach, H.;
Ho¨fle, G. Angew. Chem., Int. Ed. 2004, 43, 4888–4892, and references therein.
(3) Sasse, F.; Steinmetz, H.; Ho¨fle, G.; Reichenbach, H. J. Antibiot. 2000,
53, 879–885.
(7) Wipf, P.; Wang, Z. Org. Lett. 2007, 9, 1605–1607.
4362 J. Org. Chem. 2008, 73, 4362–4369
10.1021/jo800384x CCC: $40.75 2008 American Chemical Society
Published on Web 05/15/2008

Expedient Synthesis of N-Methyl Tubulysin Analogues
analogues that replaced the O-acyl N,O-acetal with a simplified
N-methyl amide.8 Upon biological evaluation of this focused
library, the SAR surprisingly revealed that compounds contain-
ing the Tuv N-methyl amide (Figure 2, 2 and 4) had activity
similar to that of the analogous compounds containing the
O-acyl N,O-acetal (Figure 2, 1 and 3). This finding is important
because exclusion of the labile O-acyl N,O-acetal should result
in compounds that are more biologically stable and easier to
prepare than the parent tubulysins. In parallel efforts, Peter Wipf
and co-workers have also synthesized and reported on the potent
cytotoxicity of 2.7,9
Our previously reported sequence for the preparation of
tubulysin D and analogues (Scheme 1)8,12 was predicated on a
synthetic strategy and protecting group scheme that was
compatible with the O-acyl N,O-acetal moiety present in
tubulysin D, which is labile under both acidic and basic
conditions. Herein, we report a new sequence for the preparation
of N-methyl tubulysin analogues that is considerably more
efficient than our previously reported syntheses of N-methyl
tubulysin derivatives 2 and 48 as well as the other reported
syntheses of 215 and related analogues. This sequence, which
provides N-methyl tubulysin analogues 2 and 4 in >40% overall
yields from commercial materials, should enable the straight-
forward production of sufficient quantities of N-methyl tubulysin
analogues for biochemical and pharmacological studies.
FIGURE 1. Structures of selected tubulysins.
Results and Discussion
The initial route to tubulysin D and analogues containing the
O-acyl N,O-acetal, which was also followed in the production
of N-methyl tubulysins, is shown in Scheme 1. ꢀ-Hydroxy
ketimine 7 was obtained by stereoselective addition of the
metalloenamine16 of N-tert-butanesulfinyl ketimine 517 to thia-
zole aldehyde 6, which was prepared in four steps according to
literature procedure.12,18 Reduction of imine 7 then efficiently
provided sulfinyl-protected tubuvaline methyl ester 8 with high
selectivity.16,19 Acidic cleavage of the sulfinyl group, followed
by acylation with R-azido isoleucine acid chloride (10), afforded
azide 11. The tubuvaline alcohol was then protected as a
triethylsilyl ether (TES), followed by alkylation of the tubuvaline
amide with either chloromethyl isobutyl carbonate (13) or
methyl iodide, to produce N-alkyl amides 14 and 15. Reduction
of the azide and in situ acylation with the pentafluorophenyl
ester of D-Mep (Mep-PFP) afforded Mep-coupled products 16
and 17. Use of the azide masking group was necessary for two
reasons: (1) typical carbamate protecting groups would be
competitively alkylated during the amide alkylation step, and
(2) unmasking the protected amine under mild conditions with
in situ acylation was essential given the highly labile nature of
the newly formed O-acyl N,O-acetal. Cleavage of the silyl
protecting group under mild conditions then provided 18 and
19. Selective hydrolysis of the thiazole methyl ester over the
more highly activated acyl group present in the O-acyl N,O-
acetal was next accomplished using Me3SnOH, which is
selective for sterically unhindered esters.20 The syntheses were
completed by coupling carboxylic acids 20 and 21 with either
FIGURE 2. Structure and cell-growth inhibitory activity of tubulysin
D (1) and selected analogues (2-4) against human colon adenocarci-
noma cell line SW-480 (ATCC CCL-228).8,14
first total synthesis of any naturally occurring tubulysin,
tubulysin D (Figure 2, 1),12 which is the most potent of the
tubulysin family.2 This synthesis represents the only route
capable of incorporating the O-acyl N,O-acetal, which is
extremely labile to both acid and base, thus presenting many
synthetic challenges.13
We also prepared a number of unnatural tubulysin analogues
that had modifications in the Mep and Tup regions, as well as
(8) Patterson, A. W.; Peltier, H. M.; Sasse, F.; Ellman, J. A. Chem. Eur. J.
2007, 13, 9534–9541.
(9) Wang, Z.; McPherson, P. A.; Raccor, B. S.; Balachandran, R.; Zhu, G.;
Day, B. W.; Vogt, A.; Wipf, P. Chem. Biol. Drug Des. 2007, 70, 75–86.
(10) Reference 4 originally reported the total synthesis of naturally occurring
tubulysins U and V, but was actually the synthesis of epimeric compounds.
Correction: Do¨mling, A.; Beck, B.; Eichelberger, U.; Sakamuri, S.; Menon, S.;
Chen, Q.-Z.; Lu, Y.; Wessjohann, L. A. Angew. Chem., Int. Ed. 2007, 46, 2337–
2348.
(11) Sani, M.; Fossati, G.; Huguenot, F.; Zanda, M. Angew. Chem., Int. Ed.
2007, 46, 3526–3529.
(15) For an alternative synthesis of 2, see ref 7.
(16) Kochi, T.; Tang, T. P.; Ellman, J. A. J. Am. Chem. Soc. 2003, 125,
11276–11282.
(12) Peltier, H. M.; McMahon, J. P.; Patterson, A. W.; Ellman, J. A. J. Am.
Chem. Soc. 2006, 128, 16018–16019.
(13) Iley, J.; Moreira, R.; Calheiros, T.; Mendes, E. Pharm. Res. 1997, 14,
1634–1639.
(17) Liu, G.; Cogan, D. A.; Owens, T. D.; Tang, T. P.; Ellman, J. A. J. Org.
Chem. 1999, 64, 1278–1284.
(14) Other cancer cell lines such as human cervix carcinoma KB-3-1 (DSMZ
ACC 158) and mouse fibroblast L929 (DSMZ ACC 2) also showed comparable
biological activities.
(18) Inami, K.; Shiba, T. Bull. Chem. Soc. Jpn. 1985, 58, 352–360.
(19) Borg, G.; Cogan, D. A.; Ellman, J. A. Tetrahedron Lett. 1999, 40, 6709–
6712.
J. Org. Chem. Vol. 73, No. 12, 2008 4363

Patterson et al.
SCHEME 1. Initial Synthesis of Tubulysin D (1) and Related Analogues 2-4
tubuphenylalanine hydrochloride (22)21 or methylamine to
provide tubulysin D (1) and analogues 2-4.
SCHEME 2. Synthesis of Thiazole Aldehyde 26
The high lability of the O-acyl N,O-acetal functionality was
an overarching design feature of our initial total synthesis of
tubulysin D, and as overviewed above, the same synthetic route
could also be employed to prepare N-methyl tubulysin ana-
logues. However, because these N-methyl analogues no longer
incorporate the labile O-acyl N,O-acetal, substantial synthetic
constraints were removed, providing the opportunity to modify
the initial route to achieve a considerably more efficient
synthesis. The use of R-azido acid chloride 10, which requires
two steps to synthesize using highly reactive triflyl azide,22 could
be eliminated by installing the N-methyl substituent at an earlier
stage. Additionally, it would be ideal if the tedious tubuvaline
alcohol silyl ether protection and deprotection steps could be
eliminated. Also, in the absence of the O-acyl N,O-acetal, a
less toxic and more practical reagent could be used in place of
Me3SnOH for hydrolysis of the late-stage thiazole ester inter-
mediate. More straightforward means for directly coupling the
amino acid subunits would also be preferred, for example, direct
coupling of N-methyl pipecolinic acid rather than coupling via
the pentafluorophenyl ester, which is readily epimerized and
cannot be stored.
enable selective late-stage hydrolysis of the thiazole methyl ester
in the presence of the labile O-acyl N,O-acetal. Addition of the
metalloenamine derived from N-tert-butanesulfinyl ketimine
512,16 to thiazole aldehyde 26 then provided ꢀ-hydroxy imine
27 (Scheme 3). Excess LDA (3.5 equiv) and ClTi(O-i-Pr)3 (3
equiv) were both necessary to achieve high conversion and
diastereoselectivity (93:7). Stereoselective reduction of imine
27wasaccomplishedwith92:8drusingNaBH4andTi(OEt)4.12,16,19
The reaction was performed at low temperatures to prevent
reduction of the ethyl ester and to increase selectivity. After
chromatography, N-tert-butanesulfinyl tubuvaline ethyl ester (28)
was isolated in diastereomerically pure form in 74% yield over
the two steps.
We next sought to install the N-methyl group without
protection of the tubuvaline alcohol. Not surprisingly, alkylation
of 28 with methyl iodide was not selective and provided a
mixture of products. We therefore envisioned an alternative
approach that proceeded through 1,3-tetrahydrooxazine 29 or
30, which could potentially be reduced to give N-methyl
tubuvaline ethyl ester 31 (Scheme 4).
The optimized synthesis of N-methyl tubulysin analogues 2
and 4 began with the preparation of thiazole 26 in 78% yield
over three steps from commercially available diethoxyacetoni-
trile (23), according to literature methods (Scheme 2).18 This
pathway easily gives large quantities of thiazole precursor 26.
In contrast, the initial synthesis relied on thiazole methyl ester
6 (Scheme 1), which requires an additional step to prepare, to
(20) Nicolaou, K. C.; Estrada, A. A.; Zak, M.; Lee, S. H.; Safina, B. S. Angew.
Chem., Int. Ed. 2005, 44, 1378–1382.
(21) Prepared as described in ref 12 in three steps from phenylacetaldehyde.
(22) Lundquist, J. T., IV.; Pelletier, J. C. Org. Lett. 2001, 3, 781–783.
4364 J. Org. Chem. Vol. 73, No. 12, 2008

Expedient Synthesis of N-Methyl Tubulysin Analogues
SCHEME 3. Stereoselective Synthesis of
N-tert-Butanesulfinyl Tubuvaline Ethyl Ester (28)
the ratio of tetrahydrooxazines 29 and 30 to 88:12. An
examination of solvents using 0.01 equiv of TsOH established
that more polar solvents such as THF and dioxane gave
decomposition products (entries 11 and 12), while toluene gave
a slightly better ratio than benzene with similar conversions after
5 h (entries 8 and 13). Because even low concentrations of acid
catalyzed not only tetrahydrooxazine formation but also undes-
ired sulfinyl group cleavage, the condensation reaction with
paraformaldehyde was performed in the absence of any acid
(Table 1, entry 14). Gratifyingly, heating 28 with paraformal-
dehyde in toluene resulted in complete conversion to the desired
N-sulfinyl-protected tetrahydrooxazine 29.
When the reaction was performed on a preparative scale, 29
proved to be stable to silica gel chromatography and was
obtained in 87% yield (Scheme 5). Reduction of 2927 with
support-bound cyanoborohydride (MP-BH3CN) resin under
acidic conditions then readily provided 31 in 97% yield as the
free amine after silica gel chromatography in the presence of
NH4OH. Accordingly, N-methyl tubuvaline ethyl ester (31) was
prepared in 49% yield over seven steps and serves as a versatile
and scalable intermediate for the construction of N-methyl
tubulysin analogues with variation at the Mep, Ile, or Tup/Tut
positions.
Coupling of N-Boc-isoleucine (Boc-Ile-OH) with N-methyl
tubuvaline 31 to form the hindered tertiary amide linkage was
next attempted. However, using a variety of coupling reagents
and reaction parameters, ester 32 was surprisingly the predomi-
nant product (Table 2). Bisacylated product 33 was also
observed in some examples, but no more than 8% of the desired
amide product 34 was observed in any case. The predilection
for O-acylation with a secondary alcohol was quite unexpected,
although it is not without precedent.28,29 For amino alcohol 31,
selective esterification is possibly aided by intramolecular
general base catalysis provided by the proximal secondary
amine. EDC, which exists as an HCl salt, HOAt, and excess
base gave primarily ester 32 contaminated with significant
amounts of bisacylated product 33, but no amide product 34
was observed (entry 1). Decreasing the amount of base used in
the reaction decreased the amount of 33 (entry 2), as did using
the less active HOBt in place of HOAt (entry 3). EDC with
HOBt and 1.2 equiv of base provided only monoacylated
products with a 97:3 ratio of ester 32 to desired amide 34. Using
these conditions in DMF, however, failed to give a significantly
improved product ratio (entry 4). Similar base and HOBt/HOAt
dependence was observed when support-bound cyclohexylcar-
The direct alkylation of N-sulfinyl-1,3-amino alcohol 28 under
basic conditions was first attempted using dihalogenated me-
thylene reagents such as CH2Br2. This type of reactivity is
precedented for N-acyl- and N-alkyl-1,2-amino alcohol scaf-
folds,23 as well as for N-alkyl-1,3-amino alcohols,24 but to our
knowledge, not for N-acyl-1,3-amino alcohols. Exploration of
the alkylation of 28 with CH2Br2 using a variety of solvents
and bases as precedented for N-acyl-1,2-amino alcohol systems
(Table 1, entries 1-4) proved unsuccessful. We therefore
explored condensation of 28 with paraformaldehyde under acidic
conditions to yield tetrahydrooxazine 30 (Scheme 4). Formation
of tetrahydrooxazines and oxazolidines by reaction of formal-
dehyde with 1,3- and 1,2-amino alcohols with N-aryl- and
N-alkyl substituents is common. Reactions with N-acyl- and
N-sulfonyl-1,2-amino alcohols have also been reported in a few
cases,25 but there are no reports using N-acyl-1,3-amino alcohols.
In our initial attempt to condense 28 with paraformaldehyde, 1
equiv of TsOH in benzene with heating at 70 °C resulted in
complete conversion to sulfinyl-deprotected tetrahydrooxazine
30 in less than 2 h (Table 1, entry 5). Unfortunately, 30 was
prohibitively unstable and hydrolyzed upon purification via silica
gel or reverse-phase chromatography. Attempts to convert the
crude product mixture directly to the desired N-methyl tubu-
26
valine 31 via reduction with NaCNBH3 gave instead the
undesired dimethylated product due to contaminating amounts
of paraformaldehyde that remained after workup. Therefore, our
focus turned to the preparation of tetrahydrooxazine 29, which
should be more stable and isolable.
Incrementally decreasing the amount of TsOH from stoichio-
metric amounts to 0.001 equiv (Table 1, entries 6-9) increased
(26) For examples of borohydride reduction of 1,3-N-alkyl-tetrahydroox-
azines, see: (a) Kirst, H. A.; Wind, J. A.; Leeds, J. P.; Willard, K. E.; Debono,
M.; Bonjouklian, R.; Greene, J. M.; Sullivan, K. A.; Paschal, J. W.; Deeter,
J. B.; Jones, N. D.; Ott, J. L.; Felty-Duckworth, A. M.; Counter, F. T. J. Med.
Chem. 1990, 33, 3086–3094. (b) Mandville, G.; Bloch, R. Eur. J. Org. Chem.
1999, 9, 2303–2307. (c) Bandarage, U. K.; Chen, L.; Fang, X.; Garvey, D. S.;
Glavin, A.; Janero, D. R.; Letts, L. G.; Mercer, G. J.; Saha, J. K.; Schroeder,
J. D.; Shumway, M. J.; Tam, S. W. J. Med. Chem. 2000, 43, 4005–4016. (d)
Charmantray, F.; Demeunynck, M.; Carrez, D.; Croisy, A.; Lansiaux, A.; Bailly,
C.; Colson, P. J. Med. Chem. 2003, 46, 967–977. (e) Mueller, A.; Polborn, K.;
Wanner, K. T. J. Hetercycl. Chem. 2007, 44, 575–590.
(23) (a) Vega-Perez, J. M.; Espartero, J. L.; Alcudia, F. J. Carbohydr. Chem.
1993, 12, 477–486. (b) Vega-Perez, J. M.; Espartero, J. L.; Alcudia, F. Carbohydr.
Res. 1992, 235, C5-C7. (c) Vega-Perez, J. M.; Candela, J. I.; Blanco, E.; Iglesias-
Guerra, F. Tetrahedron: Asymmetry 2002, 13, 2471–2483. (d) Vega-Perez, J. M.;
Vega, M.; Blanco, E.; Iglesias-Guerra, F. Tetrahedron: Asymmetry 2004, 15,
3617–3633. (e) Hajji, C.; Testa, M. L.; Salud-Bea, R. D. L.; Zaballos-Garcia,
E.; Server-Carrio, J.; Sepulveda-Arques, J. Tetrahedron 2000, 56, 8173–8177.
(24) Comins, D. L.; Sahn, J. J. Org. Lett. 2005, 7, 5227–5228.
(25) (a) Bertolini, G.; Aquino, M.; Ferrario, F.; Pavich, G.; Zaliani, A.; Sala,
A. J. Org. Chem. 1996, 61, 3358–3361. (b) Wagner, B.; Beugelmans, R.; Zhu,
J. Tetrahedron Lett. 1996, 37, 6557–6560. (c) Baeckvall, J. E.; Oshima, K.;
Palermo, R. E.; Sharpless, K. B J. Org. Chem. 1979, 44, 1953–1957. (d) Schrey,
A.; Osterkamp, F.; Straudi, A.; Rickert, C.; Wagner, H.; Koert, U.; Herrschaft,
B.; Harms, K. Eur. J. Org. Chem. 1999, 11, 2977–2990. (e) Aurelio, L.;
Brownlee, R. T. C.; Hughes, A. B.; Sleebs, B. E. Aust. J. Chem. 2000, 53, 425–
433. (f) Reddy, G. V.; Rao, G. V.; Sreevani, V.; Iyengar, D. S. Tetrahedron
Lett. 2000, 41, 949–951. (g) Aurelio, L.; Brownlee, R. T. C.; Hughes, A. B.
Org. Lett. 2002, 4, 3767–3769. (h) Ashley, E. R.; Cruz, E. G.; Stoltz, B. M.
J. Am. Chem. Soc. 2003, 125, 15000–15001. (i) Bennett, N. J.; Prodger, J. C.;
Pattenden, G. Tetrahedron 2007, 63, 6216–6231. (j) Dai, C.-F.; Cheng, F.; Xu,
H.-C.; Ruan, Y.-P.; Huang, P.-Q. J. Comb. Chem. 2007, 9, 386–394.
(27) To our knowledge no examples exist of reducing 1,3-N-acyl-, 1,3-N-
sulfonyl-, or 1,3-N-sulfinyl-tetrahydrooxazines to the corresponding 1,3-amino
alcohol. For examples with 1,2-N-acyl-oxazolidines, see refs 25f, i, and 25j, as
well as: (a) Reddy, G. V.; Rao, G. V.; Iyengar, D. S. Chem. Commun. 1999, 4,
317–318. (b) Reddy, G. V.; Rao, G. V.; Sreevani, V.; Iyengar, D. S. Tetrahedron
Lett. 2000, 41, 953–954.
(28) (a) Ksander, G. M.; Erion, M.; Yuan, A. M.; Diefenbacher, C. G.; El-
Chehabi, L.; Cote, D.; Levens, N. J. Med. Chem. 1994, 37, 1823–1832. (b)
Kilonda, A.; Compernolle, F.; Peeters, K.; Joly, G. J.; Toppet, S.; Hoornaert,
G. J. Tetrahedron 2000, 56, 1005–1012. (c) Cheng, Q.; Kiyota, H.; Yamaguchi,
M.; Horiguchi, T.; Oritani, T. Bioorg. Med. Chem. Lett. 2003, 13, 1075–1077.
(29) Welsh, L. H. J. Am. Chem. Soc. 1947, 69, 128–134.
J. Org. Chem. Vol. 73, No. 12, 2008 4365

Patterson et al.
TABLE 1. Conditions for Formation of 1,3-Tetrahydrooxazines 29 and 30
entry
solvent
temp (°C)
time (h)
reagent (equiv)
acid/base (equiv)
product ratioa (28:29:30)
1
2
3
4
5
6
7
8
THF
60
60
60
100
70
70
70
70
70
23
70
70
70
70
24
24
20
24
2
2
4
5
22
48
17
17
5
CH2Br2 (8)
CH2Br2 (8)
CH2Br2 (5)
CH2Br2
K2CO3 (2)
100:0:0
100:0:0
100:0:0
100:0:0
0:0:100
0:28:72
0:78:22
10:77:13c
0:88:12
42:47:11c
decomp.e
decomp.e
13:84:3c
0:100:0
THF/DMSO (9:1)
pyridine
CH2Br2
benzene
benzene
benzene
benzene
benzene
benzene
THF
K2CO3 (2), TBACb(0.2)
pyridine
i-Pr2EtN (10)
TsOH (1)
TsOH (0.2)
TsOH (0.05)
TsOH (0.01)
TsOH (0.001)
TsOH (0.2)
TsOH (0.01)
TsOH (0.01)
TsOH (0.01)
(CH2O)n (20)
(CH2O)n (20)
(CH2O)n (20)
(CH2O)n (20)
(CH2O)n (20)d
(CH2O)n (10)
(CH2O)n (20)d
(CH2O)n (20)d
(CH2O)n (20)
(CH2O)n (20)
9
10
11
12
13
14
dioxane
toluene
toluene
50
a Product ratio of crude material determined by NMR. b Tetrabutylammonium chloride (TBAC) used as a phase-transfer catalyst. c Reaction progress
halted prior to completion. d Two additions of 10 equiv. e Complex mixture of decomposition products observed.
SCHEME 4. Outline of 1,3-Tetrahydrooxazine Formation
and Reduction from N-Sulfinyl-1,3-amino alcohol 28
with only a single chromatographic purification (Scheme 6).
This is an improvement over the prior synthesis (Scheme 1),8,12
which required preparation of the pentafluorophenyl ester of
D-Mep, whose byproducts were nontrivial to remove, and the
use of Me3SnOH for selective saponification of the thiazole
ester. Acetylation of the free alcohol then afforded 35 in 97%
yield.
In our biological evaluation of tubulysin D analogues, we
not only demonstrated that the O-acyl N,O-acetal is unnecessary
for potent biological activity, but also that broad modifications
at the Tup position have little effect on cell-based activity
(Figure 2).8 Therefore, compound 35, which was prepared in
44% yield over 13 steps, is a versatile intermediate for the
preparation of N-methyl tubulysin analogues with variation at
the Tup/Tut position, as demonstrated by the preparation of
bioactive compounds 2 and 4.
Compound 4 was prepared from 35 via PS-CCD conditions
with methylamine hydrochloride in 98% yield (Scheme 7). For
the formation of 2, the carboxylic acid of 35 was first activated
as the PFP-ester followed by the addition of tubuphenylalanine
hydrochloride (22) to afford 2 in 92% yield (Scheme 7).32
Conclusion
In summary, we have developed the asymmetric total
synthesis of N-methyl tubulysin 2 which proceeds in 15 linear
steps and in 40% overall yield. This synthesis represents by far
the most efficient and scalable preparation of this important
tubulysin analogue.7,8 N-Methyl tubulysin 4 was also prepared
in 43% overall yield over 14 steps through a nearly identical
route. Additionally, the synthesis is highly modular, enabling
modification at numerous positions for the preparation of diverse
N-methyl tubulysin analogues. Biochemical studies of N-methyl
tubulysin analogues are currently underway and will be reported
in due course.
bodiimide (PS-CCD) was used (entries 5-7). Other coupling
conditions such as PyBOP with HOBt (entry 8), acid fluoride
(Boc-Ile-F,30 entry 9), or isobutyl mixed anhydride (Boc-Ile-
O-CO2-i-Bu, entry 10) also failed to give appreciable amounts
of amide 34.
Because 32 could be prepared very cleanly without any of
the bisacylated product 33 using PS-CCD or PyBOP, we next
sought to transform ester 32 to N-methyl amide 34 (Scheme
6).29,31 Ester 32 was cleanly obtained on a preparative scale
from 31 using PS-CCD and HOBt in the absence of base. After
purification of 32 only by filtration and aqueous extraction,
heating at 90 °C in toluene under anhydrous conditions provided
the desired amide product 34 in 93% yield over the two steps.
Acidic cleavage of the Boc-group of 34, followed by
condensation with D-Mep and saponification of the ethyl ester
with LiOH, gave tripeptide 21 in 99% yield over the three steps
Experimental Section
(+)-2-[(Ss,1R)-1-Hydroxy-4-methyl-3-(2-methylpropane-2-sulfi-
nylimino)pentyl]thiazole-4-carboxylic acid ethyl ester (27). A 0.9
M solution of i-Pr2NH (0.669 mL, 4.76 mmol) in Et2O (5.5 mL)
was cooled to 0 °C, and n-BuLi (1.69 mL, 4.17 mmol, 2.47 M
solution in hexanes) was added. The solution was stirred for 20
(30) Carpino, L. A.; Sadat-Aalaee, D.; Chao, H. G.; DeSelms, R. H. J. Am.
Chem. Soc. 1990, 112, 9651–9652.
(31) Facile O- to N-acyl transfer has been reported, most notably in the
N-methyl-1,2-amino alcohol pseudoephedrine series. See ref 29 and: (a) Myers,
A. G.; Gleason, J. L.; Yoon, T.; Kung, D. W J. Am. Chem. Soc. 1997, 119,
656–673.
(32) Due to the unprotected carboxylic acid present in 22, PFP-ester activation
was used in preference to carbodiimide coupling procedures that would cause
oligomerization.
4366 J. Org. Chem. Vol. 73, No. 12, 2008

Expedient Synthesis of N-Methyl Tubulysin Analogues
TABLE 2. Acylation of N-Methyl Tubuvaline Ethyl Ester (31)
entrya
solvent
coupling reagents (equiv)
i-Pr2EtN (equiv)
Boc-Ile-X (equiv)
product ratiob (32:33:34)
1
2
3
4
5
6
7
8
9
10
CH2Cl2
CH2Cl2
CH2Cl2
DMF
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
EDC (1.1), HOAt (1.1)
EDC (1.2), HOAt (1.1)
EDC (1.1), HOBt (1.0)
EDC (1.2), HOBt (1.1)
PS-CCD (1.2), HOBt (1.05)
PS-CCD (1.2), HOBt (1.05)
PS-CCD (1), HOAt (1)
PyBOP (1.1), HOBt (1.1)
3
Boc-Ile-OH (1.1)
Boc-Ile-OH (1.05)
Boc-Ile-OH (1.1)
Boc-Ile-OH (1.05)
Boc-Ile-OH (1.05)
Boc-Ile-OH (1.1)
Boc-Ile-OH (1.05)
Boc-Ile-OH (1.1)
Boc-Ile-F (1.1)
70:30:0
86:8:6
97:0:3
86:6:8
96:2:2
100:0:0
96:4:0
100:0:0
decomp.c
83:17:0d
1.2
1.2
1.2
1.2
0
0
1.2
1.1
0
Boc-Ile-O-CO2-i-Bu (1.2)
a All reactions were performed at room temperature, with reaction times ranging from 12 to 24 h. b Product ratio of crude material determined by
reverse-phase HPLC. c A complex product mixture was observed, with 32 still the major product. d A complex mixture of other acylation and
Boc-deprotected products was also observed.
SCHEME 5. Synthesis of N-Methyl Tubuvaline Ethyl Ester
(31)
solution of the product mixture containing ꢀ-hydroxy imine 27
(∼0.943 mmol) and imine 5 (∼0.178 mmol) in THF (3.50 mL)
was cooled to -78 °C. Ti(OEt)4 (0.469 mL, 2.24 mmol) was added,
followed by NaBH4 (0.0847 g, 2.24 mmol), and the solution was
stirred at -78 °C for 12 h. The solution was acidified using a 4:1
(v/v) solution of THF/AcOH (3.2 mL) followed by addition of
EtOH (2 mL) and H2O (10 mL). The solution was warmed to rt
and diluted with EtOAc. The mixture was washed once with brine,
and the aqueous fraction was extracted once with EtOAc. The
combined organic fractions were dried with Na2SO4, filtered, and
concentrated. NMR analysis on the unpurified material established
a 92:8 ratio of diastereomers. HPFC purification (100% EtOAc)
produced 0.332 g (74%, over two steps) of the pure major
diastereomer 28 as a colorless oil: [R]25 ) +103.2 (c ) 1.0,
D
1
CHCl3); IR (film) 1474, 1718, 2868, 2929, 2960, 3283 cm-1; H
min at 0 °C and then cooled to -78 °C. A solution of sulfinyl
ketimine 517 (0.339 g, 1.79 mmol) in Et2O (3.6 mL) was added,
and the reaction mixture was stirred for 30 min at -78 °C.
Chlorotitanium triisopropoxide (0.744 mL, 3.57 mmol) was then
added, and the reaction mixture was stirred for an additional 45
min at -78 °C. Aldehyde 2618 (0.220 g, 1.19 mmol) was added in
one portion, and the solution was stirred at -78 °C for 46 h. The
solution was neutralized using a 4:1 (v/v) solution of THF/AcOH
(2.5 mL) followed by addition of H2O (15 mL). The resulting
mixture was warmed to rt and filtered through Celite, washing the
filter cake thoroughly with EtOAc. The solution was washed once
with brine, dried over MgSO4, filtered, and concentrated. NMR
analysis on the unpurified material established a 93:7 ratio of
diastereomers. HPFC purification (98:2 to 80:20 CH2Cl2:MTBE)
produced a ∼5.3:1 mixture of the pure major diastereomer 27 and
starting sulfinyl ketimine 5 as a yellow oil. The mixture was further
reacted without additional purification.
NMR (500 MHz, CDCl3) δ 0.90 (d, 3H, J ) 6.8 Hz), 0.93 (d, 3H,
J ) 6.8 Hz), 1.29 (s, 9H), 1.40 (t, 3H, J ) 7.1 Hz), 1.70 (doublet
of septets, 1H, J ) 2.3, 6.8 Hz), 1.91 (ddd, 1H, J ) 3.6, 11.5, 14.7
Hz), 2.30 (ddd, 1H, J ) 3.0, 11.8, 14.7 Hz), 3.30 (d, 1H, J ) 8.5
Hz), 3.40-3.47 (m, 1H), 4.38-4.45 (m, 2H), 5.18 (ddd, 1H, J )
3.1, 6.7, 11.6 Hz), 5.48 (d, 1H, J ) 6.7 Hz), 8.11 (s, 1H); 13C
NMR (125 MHz, CDCl3) δ 14.4, 17.3, 19.7, 23.0, 33.9, 40.6, 56.3,
58.5, 61.3, 67.8, 127.4, 147.0, 161.6, 177.6; HRMS (FAB) calcd
for C16H28N2O4NaS2 (M + Na) 399.1388, found 399.1389.
(+)-2-[(Ss,1R,3R)-4-Isopropyl-3-(2-methylpropane-2-sulfinyl)-
[1,3]oxazinan-6-yl]thiazole-4-carboxylic acid ethyl ester (29). A 0.1
M solution of N-sulfinyl amino alcohol 28 (0.575 g, 1.53 mmol) in
toluene (15.3 mL) with paraformaldehyde (0.917 g, 30.5 mmol)
was heated in a sealed vessel at 70 °C with stirring for 50 h. After
cooling to rt, the mixture was filtered through Celite, washing the
filter cake thoroughly with toluene. The solution was concentrated
and purified via HPFC (88:12 to 30:70 CH2Cl2:EtOAc) to afford
0.518 g of tetrahydrooxazine 29 (87%) as a foamy yellow solid:
A small sample was further purified by HPFC (90:10 to 80:20
toluene:EtOAc) for characterization purposes and was isolated as
[R]25 ) +108.4 (c ) 1.0, CHCl3); IR (film) 1011, 1076, 1087,
a yellow oil: [R]25 ) +67.3 (c ) 1.0, CHCl3); IR (film) 1476,
D
D
1163, 1195, 1474, 1729, 2957, 2979 cm-1; H NMR (500 MHz,
1
1
1625, 1732, 2871, 2931, 2970, 3220 cm-1; H NMR (500 MHz,
CDCl3) δ 1.01 (d, 3H, J ) 6.8 Hz), 1.02 (d, 3H, J ) 6.8 Hz), 1.17
(s, 9H), 1.35 (t, 3H, J ) 7.1 Hz), 2.08-2.16 (m, 1H), 2.28-2.40
(m, 1H), 2.31 (d, 1H, J ) 14.3 Hz), 3.02 (dd, 1H, J ) 4.7, 10.1
Hz), 4.37 (q, 2H, J ) 7.1 Hz), 4.72 (d, 1H, J ) 11.6 Hz), 5.14 (d,
1H, J ) 11.6 Hz), 5.20 (dd, 1H, J ) 2.5, 11.5 Hz), 8.11 (s, 1H);
13C NMR (125 MHz, CDCl3) δ 14.3, 20.5, 20.7, 22.8, 27.5, 32.6,
59.2, 61.3, 62.7, 71.4, 71.6, 127.8, 146.5, 161.3, 173.3; HRMS
(FAB) calcd for C17H29N2O4S2 (M + H) 389.1569, found 389.1566.
CDCl3) δ 1.15 (app t, 6H, J ) 8.5 Hz), 1.25 (s, 9H), 1.34 (t, 3H,
J ) 7.0 Hz), 2.75-2.85 (m, 1H), 3.22-3.32 (m, 2H), 4.35 (q, 2H,
J ) 7.0 Hz), 5.08 (m, 1H), 6.56 (d, 1H, J ) 8.5 Hz), 8.06 (s, 1H);
13C NMR (125 MHz, CDCl3) δ 14.2, 20.0, 20.8, 22.3, 38.8, 43.7,
58.3, 61.2, 67.3, 127.6, 147.1, 161.3, 178.1, 187.4; HRMS (FAB)
calcd for C16H26N2O4NaS2 (M + Na) 397.1232, found 397.1229.
(+)-2-[(Ss,1R,3R)-1-Hydroxy-4-methyl-3-(2-methylpropane-2-
sulfinylamino)pentyl]thiazole-4-carboxylic acid ethyl ester (28). A
J. Org. Chem. Vol. 73, No. 12, 2008 4367

Patterson et al.
SCHEME 6. Synthesis of Carboxylic Acid Intermediate 35
SCHEME 7. Synthesis of N-Methyl Tubulysins 2 and 4
were dried with Na2SO4, filtered, and concentrated to afford
intermediate 32, which was taken on without further purification.
A small sample of ester 32 was further purified for characteriza-
tion purposes. The intermediate was first isolated as the formic acid
salt via reverse-phase HPFC (80:20 to 0:100 H2O:MeCN with 0.1%
formic acid). The product fractions were concentrated, diluted with
EtOAc, and washed once with saturated NaHCO3(aq) to provide
the free amine. The organic fraction was dried with Na2SO4 and
concentrated to afford pure ester 32 as an amorphous solid: 1H NMR
(400 MHz, CDCl3) δ 0.81 (d, 3H, J ) 6.8 Hz), 0.86-0.93 (m,
6H), 0.95 (d, 3H, J ) 6.8 Hz), 1.11-1.25 (m, 1H), 1.37 (t, 3H, J
) 7.1 Hz), 1.34-1.48 (m, 2H), 1.40 (s, 9H), 1.82-1.99 (m, 3H),
2.04-2.14 (m, 1H), 2.29-2.36 (m, 1H), 2.34 (s, 3H), 4.39 (q, 2H,
J ) 7.1 Hz), 4.30-4.36 (m, 1H), 5.07 (d, 1H, J ) 9.2 Hz), 6.37
(dd, 1H, J ) 3.1, 9.9 Hz), 8.11 (s, 1H); 13C NMR (100 MHz,
CDCl3) δ 11.5, 14.3, 15.5, 16.6, 19.1, 24.7, 28.2, 28.6, 33.6, 36.1,
37.9, 58.2, 60.4, 61.4, 72.0, 79.7, 127.6, 147.1, 155.4, 161.1, 170.9,
171.5; HRMS (FAB) calcd for C24H42N3O6S (M + H) 500.2794,
found 500.2787.
(+)-(1R,3R)-2-(1-Hydroxy-4-methyl-3-methylaminopentyl)thiaz-
ole-4-carboxylic acid ethyl ester (31). To a 0.18 M solution of
tetrahydrooxazine 29 (0.200 g, 0.514 mmol) in 9:1 MeCN/EtOH
(2.9 mL) with MP-BH3CN (2.52 mmol/g, 0.204 g, 0.514 mmol)
was added dropwise HCl (4.0 M in 1,4-dioxane, 0.515 mL, 2.06
mmol) with stirring. Stirring was continued for 3 h at rt, and then
the solution was concentrated and purified by HPFC (95:5:1 to 90:
10:1 CH2Cl2:EtOH:NH4OH). The product fractions were concen-
trated, diluted with EtOAc, and washed once with saturated
NaHCO3(aq) to remove excess ammonia salts. The aqueous fraction
was back-extracted once with EtOAc. The combined organic
fractions were dried with Na2SO4 and concentrated to afford 0.143
g of N-methyl tubuvaline ethyl ester 31 (97%) as a foamy solid:
(-)-2-{(1R,3R)-3-[(2S,3S)-(2-tert-Butoxycarbonylamino-3-meth-
ylpentanoyl)methylamino]-1-hydroxy-4-methylpentyl}thiazole-4-
carboxylic acid ethyl ester (34). Crude ester 32 (∼1.06 mmol) in
toluene (10.6 mL, 0.1 M) was heated to 90 °C in a sealed vessel
with stirring for 30 h. HPFC purification (88:12 to 0:100 hexanes:
EtOAc) afforded 0.492 g of N-methyl carbamate 34 (93%, two
1
steps) as an amorphous solid. The H NMR spectrum indicated
that the product existed as a 7:1 mixture of rotamers, with the major
isomer reported: [R]25D ) -11.7 (c ) 1.0, CHCl3); IR (film) 1016,
1094, 1166, 1206, 1234, 1366, 1495, 1617, 1713, 2875, 2934, 2965,
1
3325 cm-1; H NMR (500 MHz, d4-MeOD) δ 0.84 (d, 3H, J )
mp 85-88 °C; [R]25 ) +70.5 (c ) 0.8, CHCl3); IR (film) 1087,
7.4 Hz), 0.91 (t, 3H, J ) 7.4 Hz), 0.94-0.99 (m, 6H), 1.39 (t, 3H,
J ) 7.1 Hz), 1.42 (s, 9H), 1.58-1.69 (m, 1H), 1.77-1.87 (m, 2H),
1.88-1.95 (m, 1H), 2.14-2.24 (m, 1H), 3.15 (s, 3H), 4.38 (q, 2H,
J ) 7.1 Hz), 4.41 (d, 1H, J ) 8.8 Hz), 4.44-4.57 (br s, 1H),
4.63-4.68 (m, 1H), 8.30 (s, 1H); 13C NMR (125 MHz, d4-MeOD)
δ 11.3, 14.7, 16.2, 20.3, 20.6, 25.7, 28.8, 31.1, 37.6, 38.7, 56.8,
58.1, 62.5, 69.9, 80.5, 129.1, 147.8, 158.0, 162.8, 176.5, 180.5;
HRMS (FAB) calcd for C24H41N3O6NaS (M + Na) 522.2614, found
522.2605.
D
1
1232, 1315, 1492, 1713, 2947, 3119 cm-1; H NMR (500 MHz,
d4-MeOD) δ 0.89 (d, 3H, J ) 6.8 Hz), 0.90 (d, 3H, J ) 6.8 Hz),
1.39 (t, 3H, J ) 7.0 Hz), 1.88-2.04 (m, 3H), 2.38 (s, 3H),
2.43-2.48 (m, 1H), 4.38 (q, 2H, J ) 7.0 Hz), 5.14-5.18 (m, 1H),
8.32 (s, 1H); 13C NMR (125 MHz, d3-MeCN) δ 14.6, 17.3, 20.1,
28.8, 33.1, 33.9, 61.9, 63.5, 72.1, 128.6, 148.2, 162.4, 180.8; HRMS
(FAB) calcd for C13H23N2O3S (M + H) 287.1429, found 287.1427.
2-{(1R,3R)-1-[(2S,3S)-2-tert-Butoxycarbonylamino-3-methylpen-
tanoyloxy]-4-methyl-3-methylaminopentyl}thiazole-4-carboxylic acid
ethyl ester (32). A 0.1 M solution of N-methyl tubuvaline ethyl
ester 31 (0.305 g, 1.06 mmol), 1-hydroxybenzotriazole (HOBt)
(0.146 g, 1.08 mmol), and Boc-Ile-OH (0.267 g, 1.11 mmol) in
CH2Cl2 (10.6 mL) was cooled in a salted ice-water bath. PS-CCD
(1.38 mmol/g, 0.920 g, 1.27 mmol) was added with stirring, and
the bath was warmed to rt. Stirring was continued for 14 h, the
solution was filtered, and the resin was washed with CH2Cl2. The
filtrate was then concentrated, diluted with EtOAc, and washed once
with saturated NaHCO3(aq). The aqueous fraction was back-
extracted twice with EtOAc, and the combined organic fractions
(-)-2-[(1R,3R)-1-Hydroxy-4-methyl-3-(methyl-{(2S,3S)-3-meth-
yl-2-[((1R)-1-methylpiperidine-2-carbonyl)amino]pentanoyl}ami-
no)pentyl]thiazole-4-carboxylic acid (21). To a 0.1 M solution of
carbamate 34 (0.445 g, 0.891 mmol) in CH2Cl2 (9.00 mL) was
added an equal volume of TFA (9.00 mL) with stirring for 1 h.
The solution was then concentrated, diluted with EtOAc, and
washed once with saturated NaHCO3(aq). The aqueous fraction
was back-extracted twice with EtOAc, and the combined organic
fractions were dried with Na2SO4, filtered, and concentrated to
afford the Boc-deprotected free amine of 34, which was taken
on to the next step without further purification. To this amine
4368 J. Org. Chem. Vol. 73, No. 12, 2008

Expedient Synthesis of N-Methyl Tubulysin Analogues
in CH2Cl2 (0.1 M, 9.00 mL) were added HOBt (0.122 g, 0.908
mmol) and D-Mep12 (0.134 g, 0.935 mmol). The mixture was
then cooled in a salted ice-water bath with stirring, and PS-
CCD (1.38 mol/g, 0.775 g, 1.07 mmol) was added. The bath
was warmed to rt, and stirring was continued for 14 h. The
mixture was then filtered, the resin was washed with CH2Cl2,
and the filtrate was concentrated. The crude mixture was then
diluted with EtOAc and washed once with saturated
NaHCO3(aq). The aqueous fraction was back-extracted twice
with EtOAc, and the combined organic fractions were dried with
Na2SO4, filtered, and concentrated to afford the Mep-coupled
intermediate, which was taken on without further purification.
To the crude intermediate diluted in 1,4-dioxane (0.1 M, 9.00
mL) was added a 0.4 M solution of LiOH (85.3 mg, 3.56 mmol)
in degassed H2O (9.00 mL). After stirring for 5 h, the solution
was concentrated and HPFC purified (90:10:1 to 70:30:1 CH2Cl2:
MeOH:NH4OH) to afford 0.438 g of carboxylic acid 21 (99%, three
steps) as an amorphous solid. Spectral data of 21 matched that
previously reported by us for the preparation of 21 via Scheme 1.8
(-)-2-[(1R,3R)-1-Acetoxy-4-methyl-3-(methyl-{(2S,3S)-3-methyl-
2-[((1R)-1-methylpiperidine-2-carbonyl)amino]pentanoyl}amino)-
pentyl]thiazole-4-carboxylic acid (35). A 0.1 M solution of alcohol
21 (23.0 mg, 0.0463 mmol) in pyridine (0.460 mL) was cooled in
an ice-water bath, and Ac2O (21.9 µL, 0.232 mmol) was added
with stirring. The bath was warmed to rt, and stirring was continued
for 24 h. The solution was then cooled in an ice-water bath, and
a 1:1 (v/v) solution of degassed H2O/dioxane (2 mL) was added.
The bath was warmed to rt, and stirring was continued for 22 h.
Concentration, followed by HPFC purification (80:20:1 CH2Cl2:
MeOH:NH4OH), afforded 24.3 mg of acetate 35 (97%) as an
amorphous solid: [R]25D ) -14.8 (c ) 0.8, MeOH); IR (film) 1222,
s), 67.7, 69.6, 126.8, 150.1, 162.9, 169.1, 169.7, 171.5, 172.7;
HRMS (FAB) calcd for C26H43N4O6S (M + H) 539.2903, found
539.2884.
N-Methyl Tubulysin 2. A solution of carboxylic acid 35 (29.0
mg, 53.8 µmol) and pentafluorophenol (14.9 mg, 80.7 µmol) in
540 µL of CH2Cl2 was cooled in an ice-water bath followed by
addition of PS-CCD (1.38 mmol/g, 24.1 mg, 33.3 µmol). The bath
was warmed to rt and stirred for 15 h. The mixture was then filtered,
the resin was washed with CH2Cl2, and the filtrate was concentrated.
The crude PFP-ester was used without further purification. DMF
(215 µL, 0.25 M) was added to the crude product followed by
addition of the hydrochloride salt of tubuphenylalanine (22)12 (39.2
mg, 161 µmol) and i-Pr2EtN (47.0 µL, 269 µmol). The reaction
mixture was stirred for 24 h and concentrated. HPFC purification
(80:20 to 20:80 MeCN:H2O) afforded 36.1 mg (92%) of 2 as an
amorphous solid. Spectral data of 2 matched that previously reported
by us for the preparation of 2 via Scheme 1.8
N-Methyl Tubulysin 4. To a 0.1 M solution of carboxylic acid
35 (12.8 mg, 23.8 µmol), i-Pr2EtN (25.0 µL, 143 µmol), HOBt
(3.9 mg, 29 µmol), and CH3NH3Cl (6.4 mg, 95 µmol) in CH2Cl2
(238 µL) was added PS-CCD (1.38 mmol/g, 24.1 mg, 33.3 µmol)
in a sealed vessel. After stirring for 10 h, 6.4 mg (95 µmol) of
CH3NH3Cl and 25.0 µL (143 µmol) of i-Pr2EtN were added and
stirring was continued for an additional 10 h. The mixture was then
filtered, the resin was washed with CH2Cl2, and the filtrate was
concentrated. HPFC purification (80:20 to 20:80 H2O:MeCN)
afforded 12.9 mg of N-methyl tubulysin 4 (98%) as an amorphous
solid. Spectral data of 4 matched that previously reported by us
for the preparation of 4 via Scheme 1.8
Acknowledgment. This work was supported by the NSF
(CHE-0742565). A.W.P. acknowledges an ACS Medicinal
Chemistry Fellowship sponsored by BMS. We thank Prof.
Florenz Sasse for our continuing collaboration on the biological
evaluation of tubulysin derivatives.
1
1369, 1470, 1593, 1639, 1747, 2874, 2961 cm-1; H NMR (500
MHz, d6-DMSO) δ 0.67 (d, 3H, J ) 6.6 Hz), 0.82 (t, 3H, J ) 7.4
Hz), 0.86 (d, 3H, J ) 6.7 Hz), 0.91 (d, 3H, J ) 6.5 Hz), 0.99-1.10
(m, 1H), 1.10-1.22 (m, 1H), 1.29-1.40 (m, 1H), 1.40-1.51 (m,
2H), 1.52-1.58 (m, 1H), 1.58-1.67 (m, 2H), 1.71-1.82 (m, 2H),
1.97-2.04 (m, 1H), 2.08 (s, 3H), 2.11 (s, 3H), 2.12-2.17 (m, 1H),
2.17-2.25 (m, 1H), 2.52-2.56 (m, 1H), 2.84-2.89 (m, 1H), 2.97
(s, 3H), 4.23-4.43 (br s, 1H), 4.60 (app t, 1H, J ) 8.8 Hz), 5.53
(dd, 1H, J ) 2.4, 11.0 Hz), 7.69 (d, 1H, J ) 9.2 Hz), 8.32 (s, 1H);
13C NMR (125 MHz, d6-DMSO) δ 10.6, 15.4, 19.4, 20.0, 20.6,
22.4, 23.9, 24.3, 28.9, 29.4, 33.9, 35.8, 43.4, 52.5, 54.5, 55.1 (br
Supporting Information Available: General experimental
procedures and copies of 1H and 13C NMR spectra. This material
is available free of charge via the Internet at http://pubs.acs.org.
JO800384X
J. Org. Chem. Vol. 73, No. 12, 2008 4369