Synthesis and X-ray crystal structure of the Dolabella auricularia peptide dolastatin 18

Article abstract of DOI:10.1021/jo030358o

A previously synthesized unit of dolastatin 10 (1), dolaphenine (Doe, 3), was converted in four steps to tripeptide 10. Subsequent condensation with carboxylic acid 11 (four steps from Meldrum's acid) provided a practical synthesis of the cancer cell growth inhibitor dolastatin 18 (2, Dhex-(S)-Leu-(R)-N-Me-Phe-Doe). The synthesis of dolastatin 18 (2) confirmed the R stereochemistry of the N-Me-Phe unit as originally assigned and unusual among amino acid components of the sea hare Dolabella auricularia. An X-ray crystal structure determination of dolastatin 18 was also completed.

Full text of DOI:10.1021/jo030358o

Syn th esis a n d X-r a y Cr ysta l Str u ctu r e of th e Dola bella
a u r icu la r ia P ep tid e Dola sta tin 18†,1
George R. Pettit,* Fiona Hogan, and Delbert L. Herald
Cancer Research Institute and Department of Chemistry and Biochemistry, Arizona State University,
P.O. Box 872404, Tempe, Arizona 85287-2404
bpettit@asu.edu
Received November 24, 2003
A previously synthesized unit of dolastatin 10 (1), dolaphenine (Doe, 3), was converted in four
steps to tripeptide 10. Subsequent condensation with carboxylic acid 11 (four steps from Meldrum’s
acid) provided a practical synthesis of the cancer cell growth inhibitor dolastatin 18 (2, Dhex-(S)-
Leu-(R)-N-Me-Phe-Doe). The synthesis of dolastatin 18 (2) confirmed the R stereochemistry of the
N-Me-Phe unit as originally assigned and unusual among amino acid components of the sea hare
Dolabella auricularia. An X-ray crystal structure determination of dolastatin 18 was also completed.
The marine shell-less mollusk Dolabella auricularia
contains a number of cancer cell line inhibitory and in
vivo antineoplastic peptides that we began to isolate in
1972. One of these, the exceptionally active anticancer
drug dolastatin 10 (1),2 an unusual thiazole-containing
pentapeptide that we isolated from a D. auricularia
collection made in the Indian Ocean, is presently under-
going human cancer clinical trials under the auspices of
the U.S. National Cancer Institute,3 and very encourag-
ing preclinical studies are also ongoing.4 More recently,
other research groups, especially that of Yamada,2b,5 have
considerably expanded the number and structural variety
of D. auricularia bioactive constituents. In addition, it
is now being realized that exogenous dietary sources
employed by D. auricularia, such as cyanobacteria, might
be responsible at least in part for the production of these
most important dolastatin peptides and probably other
classes.6a,b The isolation of symplostatins 16c and 3,6d
analogues of dolastatin 10, as well as peptide 16c itself
from Symploca marine cyanobacteria and of dolastatin
16 from Lyngbya majuscula6e provides ample support for
this viewpoint.
Resu lts a n d Discu ssion
In 1983, we extended our investigation of D. auricu-
laria anticancer constituents to specimens we collected
in Papua New Guinea, which led to the isolation of the
novel human cancer cell-growth-inhibitory dolastatins
16,7a 17,7b and 18.7c The composition and sequence
assignment of dolastatin 18 was determined by means
of carbon and proton NMR spectroscopy and analytical
HPLC to be Dhex-L-Leu-N-Me-D-Phe-Doe (2), where Dhex
(dolahexanoic acid) corresponds to the 2,2-dimethyl-3-
oxohexanoyl unit and Doe (3, dolaphenine) refers to the
C-terminal thiazole unit found in 1 and also in the
cyanobacterial metabolite barbamide.7d Dolastatin 18 (2)
was isolated in small quantity (1.51 mg), but a prelimi-
nary anticancer evaluation revealed significant inhibition
of growth of several human cancer cell lines (e.g., NCI-
H460, GI50 0.39 µg/mL).7c Synthesis of 2 became neces-
sary for confirmation of structure and further biological
evaluations. A convergent pathway was devised whereby
† Dedicated to Professor Carl Djerassi on the occasion of his 80th
birthday and in recognition of his world-class contributions to chem-
istry and medicine.
(1) Antineoplastic Agents. 508. For part 507, see: Pettit, R. K.;
Pettit, G. R. Soil DNA Libraries for Anticancer Drug Discovery, in
preparation.
(2) (a) 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, 6883-6885. (b) Pettit,
G. R. In Progress in the Chemistry of Organic Natural Products; Herz,
W., Kirby, G. W., Moore, R. E., Steglich, W., Tamm, Ch., Eds; Springer-
Verlag: Vienna, 1997; Vol. 70, pp 1-79. (c) Pettit, G. R.; Singh, S. B.;
Hogan, F.; Lloyd-Williams, P.; Herald, D. L.; Burkett, D. D.; Clewlow,
P. J . J . Am. Chem. Soc. 1989, 111, 5463-5465. (d) Pettit, G. R.;
Srirangam, J . K.; Singh, S. B.; Williams, M. D.; Herald, D. L.; Barko´czy,
J . B.; Kantoci, D. K.; Hogan, F. J . Chem. Soc., Perkin Trans. 1 1996,
859-863. (e) Pettit, G. R.; Grealish, M. P. J . Org. Chem. 2001, 66,
8640-8642.
(6) (a) Gerwick, W. H.; Tan, L. T.; Sitachitta, N. In The Alkaloids;
Cordell, G. A., Ed.; Academic Press: San Diego, 2001; Vol. 57, pp 75-
184. (b) Luesch, H.; Harrigan, G. G.; Goetz, G.; Horgen, F. D. Curr.
Med. Chem. 2002, 9, 1791-1806. (c) Luesch, H.; Moore, R. E.; Paul,
V. J .; Mooberry, S. L.; Corbett, T. H. J . Nat. Prod. 2001, 64, 907-910.
(d) Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J .; Mooberry, S.
L.; Corbett, T. H. J . Nat. Prod. 2002, 65, 16-20. (e) Nogle, L. M.;
Gerwick, W. H. J . Nat. Prod. 2002, 65, 21-24.
(7) (a) Pettit, G. R., Xu, J .-P.; Hogan, F.; Williams, M. D.; Doubek,
D. L.; Schmidt, J . M.; Cerny, R. L.; Boyd, M. R. J . Nat. Prod. 1997,
60, 752-754. (b) Pettit, G. R.; Xu, J .-P.; Hogan, F.; Cerny, R. L.
Heterocycles 1998, 47, 491-496. (c) Pettit, G. R.; Xu, J .-P.; Williams,
M. D.; Hogan, F.; Schmidt, J . M. Bioorg. Med. Chem. Lett. 1997, 7,
827-832. (d) Orjala, J .; Gerwick, W. H. J . Nat. Prod. 1996, 59, 427-
430.
(3) (a) Margolin, K.; Longmate, J .; Synold, T. W.; Gandara, D. R.;
Weber, J .; Gonzalez, R.; J ohansen, M. J .; Newman, R.; Baratta, T.;
Doroshow, J . H. Invest. New Drugs 2001, 19, 335-340. (b) Pitot, H.
C.; McElroy, E. A., J r.; Reid, J . M.; Windebank, A. J .; Sloan, J . A.;
Erlichman, C.; Bagniewski, P. G.; Walker, D. L.; Rubin, J .; Goldberg,
R. M.; Adjei, A. A.; Ames, M. M. Clin. Cancer Res. 1945, 5, 525-531.
(4) (a) Dang, L. H.; Bettegowda, C.; Huso, D. L.; Kinzler, K. W.;
Vogelstein, B. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 15155-15160.
(b) Turner, T.; J ackson, W. H.; Pettit, G. R.; Wells, A.; Kraft, A. S.
Prostate 1998, 34, 175-181.
(5) Kigoshi, H.; Itoh, T.; Ogawa, T.; Ochi, K.; Okada, M.; Suenaga,
K.; Yamada, K. Tetrahedron Lett. 2001, 42, 7461-7464.
10.1021/jo030358o CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/07/2004
J . Org. Chem. 2004, 69, 4019-4022
4019

Pettit et al.
SCHEME 1
the C-terminal tripeptide and the dolahexanoic acid
moieties could be coupled in the last step (Scheme 1).
Treatment of N-benzyloxycarbonyl-(R)-phenylalanine with
iodomethane and sodium hydride according to the method
of Benoiton gave Z-N-Me-(R)-Phe (4) in good yield.8
Synthesis of dolaphenine (3) was carried out according
to earlier procedures.2b,9 Condensation of amino acid 4
and amine 3 by use of diethyl phosphorocyanidate10
(DEPC) in the presence of triethylamine gave the N-
protected dipeptide 5. Removal of the benzyloxycarbonyl
group was performed by treatment with hydrogen bro-
mide in acetic acid to give N-Me-(R)-Phe-Doe (6); the
usual method for Z-cleavage using palladium over carbon
with cyclohexene was precluded by the presence of the
thiazole. An attempt to couple dipeptide 6 to Fmoc-(S)-
Leu by treatment with BroP failed, but activation with
PyBroP,11 which has been shown to be successful in the
coupling of hindered N-methylated amino acids, in the
presence of diisopropylethylamine (DIEA) yielded Fmoc-
(S)-Leu-N-Me-(R)-Phe-Doe (7). An X-ray analysis of pep-
tide 7 confirmed its structure and stereochemistry.
Synthesis of tert-butyl 3-oxohexanoate (8) was ac-
complished cleanly according to the procedure of Yone-
mitsu and co-workers,12 via reaction between Meldrum’s
acid and butyryl chloride followed by lysis of the product
with tert-butyl alcohol. Methylation of ketone 8 with
iodomethane and sodium hydride afforded tert-butyl
dolahexanoate (9). Before the final coupling, the fluoren-
ylmethoxycarbonyl group was removed from N-protected
tripeptide 7 by treatment with trisaminoethylamine
followed by isolation using a phosphate buffer13 to yield
amine 10. The tert-butyl group was removed from ester
9 by treatment with trifluoroacetic acid14 to yield car-
boxylic acid 11. Amide formation between amine 10 and
acid 11 by use of DEPC10 in the presence of TEA gave
Dhex-(S)-Leu-N-Me-(R)-Phe-Doe (2), whose identity with
dolastatin 18 was demonstrated by TLC and NMR
spectroscopy. A high-field (500 MHz) 1H NMR spectrum
of an equimolar mixture of the natural and synthetic
specimens in CDCl3 showed exact coincidence of the
signals, except for an unidentified (impurity) singlet at
δ 1.27 in the spectrum of the natural isolate.15 Interest-
ingly, an X-ray analysis of synthetic dolastatin 18 (2)
showed that, in crystalline form, the peptide has a folded
orientation like that of dolastatin 10 (1).2b
In the next stage of cancer cell-line evaluations, the
activity of the synthetic compound was significant
(P388: ED50 7.11 µg/mL; human tumors: GI50 2.7-4.1
µg/mL) but less than that of the natural isolate by a factor
of nearly 10. Previously, we suggested that such a
discrepancy in the activities of natural and synthetic
marine invertebrate peptides may be due to an extremely
active (such as dolastatin 16) but chemically undetected
impurity that is present in the isolated material in a
quantity indicated only by biological methods.16 Further
biological studies of dolastatin 18 (2) are in progress.
(8) McDermott, J . R.; Benoiton, N. L. Can. J . Chem. 1973, 51, 1915-
1919.
Exp er im en ta l Section
(9) Irako, N.; Hamada, Y.; Shioiri, T. Tetrahedron 1992, 48, 7251-
N-Ben zyloxyca r bon yl-N-m eth yl-(R)-p h en yla la n in e (4).
A solution of N-benzyloxycarbonyl-(R)-phenylalanine (2.67 g;
8.9 mmol) in THF (60 mL) was stirred under argon. Iodo-
methane (5.1 mL; 81.1 mmol) was added, and the mixture was
cooled to 0 °C before the addition of sodium hydride (60%; 1.68
g from which the oil was removed by a hexane wash; 42 mmol).
7264.
(10) (a) Yamada, S.; Kasai, Y.; Shioiri, T. Tetrahedron Lett. 1973,
1595-1598. (b) Yamada, S.; Ikota, N.; Shioiri, T.; Tachibana, S. J . Am.
Chem. Soc., 1975, 97, 7174-7175.
(11) Coste, J .; Fre´rot, E.; J ouin, P. J . Org. Chem. 1994, 59, 2437-
2446.
(12) Oikawa, Y.; Sugano, K.; Yonemitsu, O. J . Org. Chem. 1978, 43,
2087-2088.
(13) Carpino, L. A.; Sadat-Aalaee, D.; Beyermann, M. J . Org. Chem.
1990, 55, 1673-1675.
(15) The published value (δ 5.11) for the H-9 shift in the 1H NMR
spectrum of the natural dolastatin 18 (see ref 7c) should be corrected
to δ 5.44.
(16) Pettit, G. R.; Lippert, J . W., III; Taylor, S. R.; Tan, R.; Williams,
M. D. J . Nat. Prod. 2001, 64, 883-891.
(14) (a) Bodanszky, M.; Bodanszky, A. The Practice of Peptide
Synthesis, 2nd ed.; Springer-Verlag: Berlin, 1994; pp 142-143. (b)
Kappeler, H.; Schwyzer, R. Helv. Chim. Acta 1960, 43, 1453-1459.
4020 J . Org. Chem., Vol. 69, No. 12, 2004

Synthesis and X-ray Crystal Structure of Dolastatin 18
The suspension was stirred under argon at 0 °C to room
temperature for 22 h. Ethyl acetate (50 mL) was then added,
followed by water, dropwise, until the mixture became a clear
solution. Removal of most of the solvent yielded a gummy
emulsion, which remained following the addition of ether (30
mL) and water (100 mL). Citric acid (10%; 100 mL) was added
to the slightly alkaline mixture, and the product was extracted
into ethyl acetate (100 mL) with loss of the emulsion. The
organic phase was washed successively with water (2 × 150
mL), sodium thiosulfate solution (5%; 100 mL), and water (50
mL) and was dried. Removal of solvent yielded a colorless gum
(2.73 g; 97.5%): 1H NMR (two conformers in a ratio of 2:1) δ
7.34-7.12 (m, 10 H), 5.11 and 5.03 (s, 2 H), 4.90 (m, 1 H),
3.35 (m, 1 H), 3.07 (m, 1 H), 2.86 and 2.79 (s, 3 H).
N-Ben zyloxyca r bon yl-N-m eth yl-(R)-P h e-Doe (5). To a
solution of Doe9 (3; 0.117 g; 0.57 mmol) in DCM (5 mL) was
added a solution of 4 (0.238 g; 0.76 mmol) in DCM (5 mL),
and a further 5 mL of solvent was used for transfer. Trieth-
ylamine (0.25 mL; 1.77 mmol) was added, and the mixture was
cooled to 0 °C before the addition of DEPC (1.0 mL; 6.13 mmol).
The mixture was stirred for 20 min, at which time reaction
was complete. After removal of solvent, the residue was
fractionated by flash chromatography (8:1 hexane-acetone)
to yield the product as a gum (0.280 g; 98%): 1H NMR δ 7.72
(bs, 1 H), 7.33-7.10 (m, 17 H), 5.58 (dd, 1 H, J ) 15 Hz, 7
Hz), 5.08 (brs, 2 H), 4.88 (m, 1 H), 3.26 (m, 3 H), 2.96 (m, 1
H), 2.78 (brs, 3 H); EIMS m/z (relative intensity) 499 (M+),
296, 224, 91 (100); HRMS (FAB) m/z (exact mass) 500.2035
[(MH)+, 100; calcd for C29H30N3O3S 500.2008].
N-Meth yl-(R)-P h e-Doe (6). Amide 5 (0.270 g; 0.54 mmol)
was treated with HBr in acetic acid (30%; 5 mL), and the
mixture was stirred at ambient temperature for 1 h in a flask
closed by a drying tube. Ether (35 mL) was added, causing
immediate precipitation of the hydrobromide salt as a gummy
solid, and the mixture was retained at 0 °C for 1.5 h. The
mother liquor was decanted, and the gum was washed with
ether (10 mL) that was then decanted. The salt was dissolved
in satd sodium bicarbonate solution (20 mL), and the free
amine was extracted with DCM (3 × 20 mL). The combined
organic phase was washed with water (10 mL) and dried.
Removal of solvent yielded a pale yellow oil (0.16 g; 0.43 mmol)
that was used immediately in the next reaction: 1H NMR δ
7.90 (d, 1 H, J ) 8.1 Hz), 7.78 (d, 1 H, J ) 3.3 Hz), 7.31-7.11
(m, 11 H), 5.65 (dt, 1 H, J ) 8.4 Hz, 6.0 Hz), 3.43 (dd, 1 H,
J ) 13.8 Hz, 6.0 Hz), 3.25 (dd, 1 H, J ) 13.8 Hz, 8.4 Hz), 3.16
(m, 2 H), 2.63 (m, 1 H), 2.01 (s, 3H).
X-r a y Cr ysta l Str u ctu r e Deter m in a tion of N-F m oc-(S)-
Leu -N-Me-(R)-P h e-Doe (7). A thick, plate-shaped X-ray
sample (∼0.44 × 0.32 × 0.38 mm) grown from ethyl acetate
was mounted on the tip of a glass fiber. Data collection was
performed at 297 ( 1 K. Accurate cell dimensions were
determined by least-squares fitting of 25 carefully centered
reflections in the range of 35° < θ < 40° using Cu KR radiation.
Crystal data: C42H44N4O4S, fw ) 700.87, orthorhombic, P212121,
a ) 9.842(2) Å, b ) 19.695(4) Å, c ) 20.024(4) Å, V ) 3881.6
(13) Å3, Z ) 4, Fc ) 1.199 Mg/m3, µ(CuKR) ) 1.101 mm-1, λ )
1.541 78 Å. All reflections corresponding to a complete octant
(0 e h e 11, 0 e k e 23, 0 e l e 23) were collected over the
range of 0< 2θ < 130° by use of the ω/2θ scan technique.
Friedel reflections were also collected (whenever possible)
immediately after each reflection. Three intensity control
reflections were also measured for every 60 min of X-ray
exposure time and showed a max variation of -0.5% over the
course of the collection. A total of 6995 reflections were
collected. Subsequent statistical analysis of the complete
reflection data set using the XPREP17 program, verified that
the space group was P212121. After Lorentz and polarization
corrections, merging of equivalent reflections and rejection of
systematic absences, 6249 unique reflections (Rint ) 0.0153)
remained, of which 5871 were considered observed (Io > 2σ(Io))
and were used in the subsequent structure determination and
refinement. Linear and anisotropic decay corrections were
applied to the intensity data as well as an empirical absorption
correction (based on a series of psi-scans).18 Structure deter-
mination was readily accomplished with the direct-methods
program SHELXS.19 All non-hydrogen atom coordinates were
located in a routine run using default values in that program.
The remaining H atom coordinates were calcd at optimum
positions. All non-hydrogen atoms were refined anisotropically
in a full-matrix least-squares refinement using SHELXL.19 The
H atoms were included, their Uiso thermal parameters fixed
at 1.2 the Uiso of the atom to which they were attached and
forced to ride that atom. The final residual R1 value for 7 was
0.0527 for observed data and 0.0572 for all data. The goodness-
of-fit on F2 was 1.048. The corresponding Sheldrick R values
were wR2 of 0.1381 and 0.1430, respectively. A final difference
Fourier map showed minimal residual electron density; the
largest difference peak and hole being 0.437 and -0.233 e/Å3,
respectively. Final bond distances and angles were all within
expected and acceptable limits. The absolute structure of
peptide 7 could be assigned with certainty based upon the
value of the Flack absolute structure parameter20 (0.01, esd
2). Consequently, the chiral centers of peptide 7 can be
assigned as follows: 18S,21R,24S.
N-(9-F lu or en ylm et h oxyca r b on yl)-(S)-Leu -N-Met h yl-
(R)-P h e-Doe (7). To a solution of dipeptide 6 (0.16 g; 0.43
mmol) in DCM (5 mL) were added PyBroP (301.9 mg; 0.65
mmol) and N-(9-fluorenylmethoxycarbonyl)-L-leucine (0.224 g;
0.63 mmol). The mixture was stirred under argon and cooled
to 0 °C before the addition of DIEA (0.3 mL). Stirring was
continued for 4 h. Removal of solvent yielded a yellow gummy
solid (1.0215 g) that was dissolved in ethyl acetate (50 mL).
The organic phase was washed successively with citric acid
solution (10%; 20 mL), saturated sodium bicarbonate solution
(20 mL), and water (20 mL) and dried. Removal of solvent
yielded a pale yellow foam that was fractionated by flash
chromatography (5:1 hexane-acetone) to yield peptide 7 as a
colorless solid that crystallized from ethyl acetate (0.174 g;
ter t-Bu tyl 2,2-Dim eth yl-3-oxoh exa n oa te (9). To a stirred
suspension of sodium hydride (60%; 0.23 g; 5.69 mmol) in DMF
(10 mL under argon) at 0 °C was added, dropwise, a solution
of tert-butyl 3-oxohexanoate12 (8, 512.5 mg; 2.75 mmol) in DMF
(3 mL). The mixture was stirred at 0 °C until evolution of gas
ceased (10 min), and a solution of iodomethane (0.37 mL; 5.88
mmol) in DMF (3 mL) was added dropwise. The mixture was
stirred for 18 h before being distributed between water (100
mL) and ether (100 mL). The ethereal layer was washed
successively with sodium thiosulfate solution (10%; 40 mL) and
water (50 mL) and dried. Removal of solvent yielded ketone 9
as a volatile colorless oil (215.2 mg; 36.5%): 1H NMR δ 2.43
1
58%): mp 164-165 °C; [R]D +45.3 (c 0.38, CHCl3); H NMR
(17) XPREP-The automatic space group determination program in
the SHELXTL (see ref 19).
(18) North, A. C.;Phillips, D. C.; Mathews, F. S. Acta Crystallogr.
1968, A2, 351.
(19) SHELXTL-PC Version 5.1, an integrated software system for
the determination of crystal structures from diffraction data, Bruker
Analytical X-ray Systems, Inc., Madison, WI 53719, 1997. This package
includes, among others: XPREP, an automatic space group determi-
nation program; XS, the Bruker SHELXS module for the solution of
X-ray crystal structures from diffraction data; XL, the Bruker SHELXL
module for structure refinement; XP, the Bruker interactive graphics
display module for crystal structures.
(500 Mz, CDCl3) δ 7.78 (bs, 1 H), 7.75 (d, 2 H, J ) 7.0 Hz),
7.58-7.52 (m, 3 H), 7.40-7.37 (m, 2 H), 7.30-7.11 (m, 13 H),
5.65 (m, 1 H), 5.52 (dd, 1 H, J ) 11.0 Hz, 5.0 Hz), 5.31 (d, 1 H,
J ) 8.0 Hz), 4.46 (m, 1 H), 4.40 (dd, 1 H, J ) 10.5 Hz, 7.0 Hz),
4.27 (dd, 1 H, J ) 10.5 Hz, 7.0 Hz), 4.17 (t, 1 H, J ) 7.0 Hz),
3.47 (m, 1 H), 3.39 (dd, 1 H, J ) 15.0 Hz, 5.0 Hz), 3.30 (m, 1
H), 2.92 (m, 1 H), 2.90 (s, 3 H), 1.22 (m, 1 H), 1.10 (m, 1 H),
0.85 (m, 1 H), 0.78 (d, 3 H, J ) 6.0 Hz), 0.73 (d, 3 H, J ) 6.0
Hz); HRMS (FAB) m/z (exact mass) 701.3145 [(MH)+, 20; calcd
for C42H45N4O4S 701.3161]. Anal. Calcd for C42H44N4O4S: C,
71.97; H, 6.33; N, 7.99. Found: C, 71.37; H, 6.45; N, 7.83.
(20) Flack, H. D. Acta Crystallogr. 1983, 876-881.
J . Org. Chem, Vol. 69, No. 12, 2004 4021

Pettit et al.
(t, 2 H, J ) 7.2 Hz), 1.62 (m, 2 H), 1.45 (s, 9 H), 1.31 (s, 6 H),
0.91 (t, 3 H, J ) 7.5 Hz); HRMS (APCI) m/z (exact mass)
213.1377 [(M - H)+, 23; calcd for C12H21O3 213.1491].
2,2-Dim eth yl-3-oxoh exa n oic Acid (11). To a solution of
ketone 9 (103.6 mg; 0.48 mmol) in DCM (4 mL) that was
stirred under argon and cooled to 0 ° C was added TFA (3 mL;
38.9 mmol). The mixture was stirred for 2.5 h and removal of
solvent yielded the product as a pale yellow oil (70.1 mg; 0.44
mol) that was used without further purification in the final
coupling reaction.
Dola sta tin 18 (2, Dh ex-L-Leu -N-Me-D-P h e-Doe). To a
stirred solution of N-protected tripeptide 7 (0.116 g; 0.165
mmol) in DCM (5 mL) at ambient temperature under argon
was added tris(2-aminoethyl)amine (1.3 mL; 8.34 mmol). The
mixture was stirred for 1.5 h, during which time the yellow
solution became cloudy. DCM (20 mL) was added, and the
mixture was washed successively with saturated NaCl solution
(2 × 10 mL) and phosphate buffer (10 mL; 5 mL). The aqueous
layers were extracted with DCM (20 mL), and the combined
organic solution (40 mL) was washed with water and dried.
Removal of solvent yielded cream-colored solid 10 (92 mg) that
was used without further purification as follows.
sity control reflections were also measured for every 60 min
of X-ray exposure time and showed a max variation of -7.0%
over the course of the collection. A total of 10615 reflections
were collected. Subsequent statistical analysis of the complete
reflection data set using the XPREP17
program verified that
the space group was P21. After Lorentz and polarization
corrections, merging of equivalent reflections and rejection of
systematic absences, 9708 unique reflections (Rint ) 0.0741)
remained, of which 6588 were considered observed (Io > 2σ(Io))
and were used in the subsequent structure determination and
refinement. Linear and anisotropic decay corrections were
applied to the intensity data as well as an empirical absorption
correction (based on a series of psi-scans).18 Structure deter-
mination was readily accomplished with the direct-methods
program SHELXS.19 All non-hydrogen atom coordinates were
located in a routine run using default values in that program.
The remaining H atom coordinates were calculated at optimum
positions. All non-hydrogen atoms were refined anisotropically
in a full-matrix least-squares refinement using SHELXL.19 The
H atoms were included, their Uiso thermal parameters fixed
at 1.2 the Uiso of the atom to which they were attached and
forced to ride that atom. The final standard residual R1 value
for 2 was 0.1385 for observed data and 0.3437 for all data.
The goodness-of-fit on F2 was 1.425. The poor R value and
goodness-of-fit observed for the structural model are presum-
ably due to the apparent disorder exhibited by a number of
atoms in the two independent peptide molecules present in
the asymmetric cell unit. Thus, splitting of the atomic positions
were suggested for the following atoms: C1 of one molecule
and C1, C3, O5, C20, C24, C25, C42, and C44 of the second
molecule. A final difference Fourier map showed some residual
electron density, the largest difference peak and hole being
0.518 and -0.646 e/Å3, respectively. However, the final bond
distances and angles were all within expected and acceptable
limits. The absolute structure of dolastatin 18 could be
assigned with certainty based upon the value of the Flack
absolute structure parameter20 (-0.01, esd 6). Consequently,
the chiral centers of dolastatin 18 can be assigned as follows:
12S,21R,32S.
A solution of 2,2-dimethyl-3-oxohexanoic acid (11, 30 mg)
in DCM (3 mL) was added to a stirred solution of tripeptide
10 (92 mg) in DCM (2 mL), and the mixture was cooled to
0 °C under argon. Triethylamine (1 mL) and DEPC (0.05 mL)
were added, and the mixture was stirred for 2.5 h, warming
to room temperature. Removal of solvent yielded a creamy oil
that was fractionated by flash chromatography (5:1 hexane-
acetone) to give dolastatin 18 (2, 40.5 mg; 0.065 mmol; 40%
yield based on peptide 7). Further purification on Sephadex
LH-20 [hexane-toluene-methanol (3:1:1)] gave a colorless
solid that crystallized from acetone-hexane in a cluster of
needles: mp 108-109 °C; [R]D + 44 (c 0.05 CH2Cl2); IR (neat)
3340.27, 1683.48, 1665.98, 1634.85, 1533.23 cm-1 1H NMR
;
(500 MHz, CDCl3), δ 7.74 (d, 1 H, J ) 3.3 Hz), 7.29-7.12 (m,
12 H), 6.25 (d, 1 H, J ) 6.0 Hz), 5.60-5.56 (m, 1 H), 5.54 (dd,
1 H, J ) 11.5, 5.5 Hz), 4.58-4.55 (m, 1 H), 3.42 (dd, 1 H, J )
14.0, 5.5 Hz), 3.38 (dd, 1 H, J ) 15.0, 5.5 Hz), 3.24-3.20 (m,
1 H), 2.92 (s, 3 H), 2.94-2.90 (m, 1 H), 2.45-2.41 (m, 2 H),
1.56-1.51 (m, 2 H), 1.32 (s, 3 H), 1.30 (s, 3 H), 1.12-1.06 (m,
2 H), 0.86-0.83 (m, 1 H), 0.84 (t, 3 H, J ) 7.5 Hz), 0.74 (d, 3
H, J ) 6.0 Hz), 0.71 (d, 3 H, J ) 6.0 Hz); 13C NMR (400 MHz,
CDCl3) δ 210.57, 174.06, 172.80, 171.62, 169.40, 142.71, 136.90,
136.71, 129.39, 128.75, 128.50, 128.48, 126.90, 126.72, 118.79,
57.13, 55.43, 52.85, 48.32, 41.12, 41.04, 40.04, 33.44, 31.02,
24.35, 22.95, 22.49, 22.42, 22.00, 17.08, 13.59; EIMS m/z
(relative intensity) 618 (M+, 27), 415 ([M - Doe]+, 100); HRMS
(FAB) m/z (exact mass) 619.3363 [(MH)+, 100; calcd for
Ack n ow led gm en t. We are pleased to thank the
following for very necessary financial support: Out-
standing Investigator Grant CA44344-05-12 and Grant
CA-90441-01-03 awarded by the Division of Cancer
Treatment and Diagnosis, National Cancer Institute,
DHHS; the Arizona Disease Control Research Commis-
sion; Dr. Alec D. Keith; the J . W. Kieckhefer Foundation;
the Margaret T. Morris Foundation; the Robert B.
Dalton Endowment Fund; Gary L. and Diane Tooker;
Polly J . Trautman; Dr. J ohn C. Budzinski; and the
Eagles Art Ehrmann Cancer Fund. For other helpful
assistance, we thank Drs. J un-Ping Xu, J ean-Charles
Chapuis, J ohn C. Knight, Venugopal Mukku, and
Michael D. Williams (NSF Grant CHE-9808 678).
C
35H47N4O4S 619.3318].
X-r a y Cr ysta l Str u ctu r e Deter m in a tion of Dola sta tin
18 (2). A colorless, plate-shaped X-ray sample (∼0.40 ×
0.16 × 0.12 mm) grown from acetone-hexane was mounted
on the tip of a glass fiber. Data collection was performed at
297 ( 1 K. Accurate cell dimensions were determined by least-
squares fitting of 25 carefully centered reflections in the range
of 35° < θ < 40° using Cu KR radiation. Crystal data:
C
35H46N4O4S, fw ) 618.82, monoclinic, P21, a ) 10.182(2) Å,
Su p p or tin g In for m a tion Ava ila ble: X-ray crystallo-
graphic tables of atomic coordinates, bond lengths and angles,
and anisotropic thermal parameters for peptide (7) and dola-
b ) 19.978(4) Å, c ) 17.471(4) Å, â ) 99.47(3)°, V ) 3505.6
(12) Å3, Z ) 4, Fc ) 1.173 Mg/m3, µ(Cu KR) ) 1.147 mm-1, λ )
1.541 78 Å, F(000) ) 1328. All reflections corresponding to a
complete octant (0 e h e 11, 0 e k e 23, 20 e l e 20) were
collected over a range of 0 < 2θ < 130°, by use of the ω/2θ
scan technique. Friedel reflections were also collected (when-
ever possible) immediately after each reflection. Three inten-
1
statin 18 (2) and H NMR spectra of the natural and synthetic
dolastatin 18. This material is available free of charge via the
Internet at http://pubs.acs.org.
J O030358O
4022 J . Org. Chem., Vol. 69, No. 12, 2004