Antineoplastic agents. 599. Total synthesis of dolastatin 16

Article abstract of DOI:10.1021/np500925y

The first 23-step total synthesis of the cyclodepsipeptide dolastatin 16 (1) has been achieved. Synthesis of the dolaphenvaline and dolamethylleuine amino acid units using simplified methods improved the overall efficiency. The formation of the 25-membere

Full text of DOI:10.1021/np500925y

Article
pubs.acs.org/jnp
Antineoplastic Agents. 599. Total Synthesis of Dolastatin 16
George R. Pettit,* Thomas H. Smith, Pablo M. Arce, Erik J. Flahive, Collin R. Anderson,
Jean-Charles Chapuis, Jun-Ping Xu, Thomas L. Groy, Paul E. Belcher, and Christian B. Macdonald
Department of Chemistry and Biochemistry, Arizona State University, P.O. Box 871604, Tempe, Arizona 85287-1604, United States
S
* Supporting Information
ABSTRACT: The first 23-step total synthesis of the cyclodepsipeptide dolastatin 16 (1) has been
achieved. Synthesis of the dolaphenvaline and dolamethylleuine amino acid units using simplified
methods improved the overall efficiency. The formation of the 25-membered macrocycle
employing lactonization with 2-methyl-6-nitrobenzoic anhydride completed a key step in the
synthesis. Regrettably, the synthetic dolastatin 16 (1), while otherwise identical (by X-ray crystal
structure and spectral analyses) with the natural product, did not reproduce the powerful
(nanomolar) cancer cell growth inhibition displayed by the natural isolate. Presumably this result
can be attributed to conformation(s) of the synthetic dolastatin 16 (1) or to a chemically
undetected component isolated with the natural product.
he isolation of dolastatin 16 (1) from Dolabella auricularia
RESULTS AND DISCUSSION
■
50 10−3−10−4
Tand its impressive activity as an inhibitor (GI
Inspection of the dolastatin 16 macrocycle revealed it to be
composed of six known α-amino or α-hydroxy acids, which
include three proline residues, one N-methylated valine, lactic
acid, and 2-hydroxyisovaleric acid, and two novel amino acids,
the β-amino acid residue dolamethylleuine (2) and the α-amino
acid residue dolaphenvaline (3). A retrosynthetic analysis of our
approach to dolastatin 16 is presented in Figure 2. In this
approach we sought to generate 1 via macrolactonization of an
acyclic precursor (4), which was to be prepared by a fragment
condensation approach as illustrated in Figure 2.
Intermediate 7 was synthesized in five steps (Scheme 1).
First, N-Cbz-N(Me)-D-valine was coupled to L-proline tert-butyl
ester in the presence of the peptide-coupling reagent
bromotripyrrolidinophosphonium hexafluorophosphate (Py-
BroP) to obtain compound 11 in 89% yield. Deprotection of
compound 11 using palladium-on-carbon in the presence of
hydrogen afforded compound 12 in a 92% yield. Compound 13
was prepared in 78% yield by treating S-2-hydroxy-3-
methylbutanoic acid (Hiv) with tert-butyldimethylsilyl chloride
(TBDMS-Cl) in the presence of imidazole. Compound 13 was
activated with oxalyl chloride and then treated with compound
12 in the presence of N,O-bis(trimethylsily)acetamide (BSA)
and triethylamine (TEA) to obtain compound 14 as a mixture
of rotamers in 82% yield. Next, compound 14 was deprotected
using tetrabutylammonium fluoride (TBAF) to obtain com-
pound 7 as a mixture of rotamers in 73% yield.
μg/mL) of cancer cell growth were reported in 1997.1 The
exceptional activity shown in cancer cell line biological assays
made 1 an obvious candidate for further preclinical develop-
ment, but this initiative was delayed by the need for
unequivocal configurational and conformational assignments
leading to a practical total synthesis. In 2011, we reported the
X-ray crystal structure of dolastatin 16 (1) as well as the
syntheses of synthons suitable for incorporation of the novel
amino acid units dolamethylleuine (Dml) (2) and dolaphenva-
line (Dpv) (3).2 We now are pleased to report a successful and
efficient total synthesis of dolastatin 16 (1).
Intermediate 5 was also synthesized in five steps using
intermediate 7 (Scheme 2). First, compound 16 was prepared
from methyl L-lactate according to the procedure of Qi and
McIntosh.3 Methyl L-lactate was treated with benzyl bromide in
Special Issue: Special Issue in Honor of William Fenical
Received: November 25, 2014
Figure 1. Structure of dolastatin 16 (1), dolamethylleuine (2), and
dolaphenvaline (3).
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Figure 2. Retrosynthetic analysis for dolastatin 16 (1).
Scheme 1. Synthesis of Intermediate 7
the presence of silver(I) oxide in order to obtain compound 15
in 51% yield. Compound 15 was hydrolyzed in the presence of
potassium hydroxide to obtain acid 16 in 88% yield. Then,
compound 16 was treated with L-proline tert-butyl ester in the
presence of the peptide coupling reagent PyBroP and
diisopropylethylamine (DIPEA) to obtain compound 8 as a
mixture of rotamers in 82% yield. Compound 8 was
deprotected with trifluoroacetic acid and coupled to inter-
mediate 7 using 2-methyl-6-nitrobenzoic anhydride (MNBA)
in the presence of 4-dimethylaminopyridne (DMAP) and
triethylamine to afford compound 17 as a complex mixture of
rotamers in 81% yield. Finally, compound 17 was treated with
trifluoroacetic acid (TFA) to obtain intermediate 5 again as a
mixture of rotamers in quantitative yield.
The synthesis of the hydrochloric salt of dolaphenvaline was
accomplished in six steps following a strategy developed by Li
B
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Scheme 2. Synthesis of Intermediate 5
Scheme 3. Synthesis of Dolaphenvaline·HCl (9)
et al. using an Evan’s-type chiral auxiliary to control the
stereochemistry (Scheme 3).4,5 First, compound 18 was
obtained in 94% yield by treating crotonic acid with pivaloyl
chloride in the presence of triethylamine and then with a
solution containing the lithium salt of R-4-phenyl-2-oxazolidi-
none. Compound 18 was treated with benzylmagnesium
chloride in the presence of copper(I) bromide dimethyl sulfide
complex followed by N-bromosuccinimide to obtain compound
19 in 67% yield over two steps. Compound 19 was treated with
sodium azide in order to obtain compound 20, which was
treated as a crude with lithium hydroxide in the presence of
hydrogen peroxide to obtain compound 21. Lastly, crude
compound 21 was treated with hydrogen in the presence of
palladium-on-carbon and then with 6 N HCl to obtain
dolaphenvaline hydrochloric salt (9) in 78% yields over three
steps. The synthesis proposed here for dolaphenvaline is
stereoselective as compared to the synthesis performed by
Kimura et al.6 and more efficient as compared to the synthesis
reported earlier by us.2
Finally, dolastatin 16 (1) was synthesized in seven steps
using intermediates 5 and 9 (Scheme 4). Compound 22 was
obtained in 92% yield by treating S-homo-β-valine with thionyl
chloride in anhydrous methanol. Compound 22 was alpha-
methylated and N-protected by first treating it with LiHMDS
and then with methyl iodide7,8 followed by Cbz-Cl in the
presence of potassium carbonate to obtain Cbz-protected
dolamethylleuine methyl ester (23) in 58% yield. In this step
the 2S,3R-Dml diastereomer was also obtained in about 20%
yield. The strategy followed here for the synthesis of Dml is
more efficient as compared to the synthesis proposed
previously.2 Compound 23 was treated with hydrogen in the
presence of palladium-on-carbon to deprotect the amino group.
The crude product was then treated with Cbz-L-proline in the
presence of N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)-
uronium hexafluorophosphate (HBTU) and triethylamine to
obtain compound 10 in 73% yield over two steps. Compound
10 was subjected to the same series of reactions as for 23 using
Boc-protected Dpv, which was prepared by treating 9 with
Boc2O in an aqueous solution of KOH, to obtain compound 24
C
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Scheme 4. Synthesis of Dolastatin 16
in 67% yield. Compound 24 was then hydrolyzed using LiOH
followed by treatment with benzyl bromide in the presence of
triethylamine to obtain compound 6 in 56% yield. Then,
compound 6 was treated with TFA to deprotect the amino
group. The crude product was next treated with compound 5 in
the presence of HBTU and triethylamine to obtain compound
4 as a mixture of rotamers in 87% yield over two steps. For the
last step, compound 4 was subjected to hydrogenolysis to
remove the two benzyl groups and then treated with MNBA in
the presence of DMAP and triethylamine under high dilution to
afford dolastatin 16 (1) in 22% yield.
The synthetic dolastatin 16 was found to be identical to the
natural product as compared by HPLC, NMR (400 MHz),
optical rotation, HRMS, and X-ray crystallography data (Figure
3). The X-ray crystal structure observed for the synthetic
dolastatin 16 showed the same stereochemistry as the natural
dolastatin 16; however, there are small differences in bond
angles due to the solvent used for crystallization.
Biological evaluation of the synthetic dolastatin 16 against a
small panel of cancer cell lines showed a surprising lack of
cancer cell growth inhibition (GI50 > 10 μg/mL) as compared
to the natural counterpart, which consistently led to GI50
0.0012−0.000 96 μg/mL cancer cell growth inhibition against
a minipanel of human cancer cell lines.1 The results of this
analysis suggest a conformational change in the synthetic
specimen or presence of a chemically undetected compound in
the sample that was isolated from the natural source in 1997.
Previously, it was observed that certain cyclic depsipeptides
could carry traces of compounds too small to be detected by
NMR or chromatographic techniques responsible for the
biological activity.9
In 2011, dolastatin 16 was also isolated from the
cyanobacterium Symploca cf. hydnoides by Luesch and
colleagues.10 The activity of dolastatin 16 isolated from this
particular organism was greatly lower (IC50’s of 69 and 51 μg/
mL for the HT-29 and HeLa cell lines, respectively) as
compared to the specimen isolated from D. auricularia.
Recently, it was shown that the activity of phakellistatin 2, a
cyclic peptide also containing proline residues in its sequence,
exhibited different cancer cell growth inhibition depending on if
methanol or dimethyl sulfoxide was used for the bioassay.11
These findings suggest that conformational changes, due to the
solvent used, can have a big impact on the biological activity of
certain macrocycles containing proline residues and possibly N-
alkylated amino acids. In fact, NMR data (not shown) were also
collected in deuterated solvents such as methanol and dimethyl
sulfoxide, and the presence of two or more conformers was
observed. Following the logic in the findings mentioned above,
the synthetic dolastatin 16 was also evaluated in methanol;
unfortunately, no activity was observed (Table 1). The results
shown for the natural sample of dolastatin 16 require some
background. Table 1 data (current, 2014) for the natural
dolastatin 16 were on a very small sample from the remaining
∼100 μg from the original isolation (3.1 mg, 10−7 % yield) from
1000 kg (wet weight) of the sea hare D. auricularia that we
collected in Papua New Guinea in 1983 and reported following
14 years of research by 1997.1 Before we used this sample, it
was repurified by reversed-phase HPLC using the same
conditions as for the purification of the synthetic dolastatin
D
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Figure 3. (A) Crystal structure of natural dolastatin 16 (1) and synthetic dolastatin 16 (2). Crystal structures were created using the Chem3D
program from CIF files. (B) HPLC trace comparison of synthetic (blue) and natural (red) dolastatin 16. (C) NMR (400 MHz) comparison of
synthetic (red) and natural (black) dolastatin 16.
Table 1. Human Cancer Cell Growth Evaluation of Natural and Synthetic Dolastatin 16, GI50 (μg/mL)
a
cell line
compound
solvent
BXPC-3
MCF-7
SF-268
NCI-H460
KM20L2
DU-145
dolastatin 16 (natural)
dolastatin 16 (synthetic)
DMSO
DMSO
MeOH
0.050
>10
0.027
>10
0.016
>10
0.270
>10
0.013
>10
0.009
>10
>10
>10
>10
>10
>10
>10
a
Cancer cell lines in order: pancreas (BXPC-3); breast (MCF-7); CNS (SF-268); lung (NCI-H460); colon (KM20L2); prostate (DU-145).
16. After purification, a reduced activity was observed in some
cancer cell lines as growth inhibitor, except for DU-145
(prostate), as compared to the previous results obtained for the
original dolastain 16.
Although the utility of the synthetic dolastatin 16 for cancer
cell growth inhibition is disappointing, we will begin an
evaluation against other medical indications and the potential of
SAR modifications especially involving the proline unit.12
In recent years other proline-rich cyclodepsipeptides,
analogues of dolastatin 16, have been isolated from different
organisms. Kulokekahilide-16 was isolated from the cepha-
laspidean mollusk Philinopsis speciosa collected from Shark’s
cove, Pupukea, O’ahu, in 2002. Homodolastatin 1613 and
pitiprolamide14 were later isolated by different research groups
(in 2003 and 2011, respectively) from the marine cyanobacte-
rium Lyngbya majuscula collected from different parts of the
E
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J = 6.8 Hz), and 0.96 (3H, d, J = 6.8 Hz); 13C NMR (CDCl3, 101
MHz) δ 173.7, 171.3, 80.9, 67.1, 59.7, 46.9, 34.8, 31.1, 28.9, 27.8, 24.6,
19.6, and 18.3.
world. These analogues of dolastatin 16 showed low activity as
cancer cell growth inhibitors, with GI50’s ranging from 2 to 29
μg/mL against different cancer cell lines.
tert-Butyldimethylsilyl 2-(S)-tert-Butyldimethylsilyloxy-3-
methylbutyrate (13). To a stirred solution of (S)-2-hydroxy-3-
methylbutanoic acid (3.00 g, 25.4 mmol) and TBDMSCl (9.96 g, 66.0
mmol) in anhydrous DMF (15 mL) was added imidazole (8.99 g, 132
mmol). The reaction mixture was stirred at 40 °C for 20 h and then
partitioned between 150 mL of EtOAc and 50 mL of H2O. The
organic phase was separated and washed with 80 mL of 10% citric acid,
80 mL of 6% NaHCO3, and 80 mL of brine. The organic solution was
dried over MgSO4 and concentrated under diminished pressure. The
residue was purified by chromatography on a silica gel column. Elution
with 19:1 hexanes−ethyl acetate gave the product as a colorless oil:
Taking into account the reduced activity observed for the
repurified sample of the natural dolastatin 16 isolated from D.
auricularia, a likely explanation for the superior activity as
cancer cell growth inhibitor of the natural dolastatin 16 as
compared to our synthetic dolastatin 16 and to the one isolated
from the cyanobacterium Symploca cf. hydnoides by Luesch and
colleagues resides in the presence of a highly active untraceable
impurity in the natural sample.14a
EXPERIMENTAL SECTION
yield 6.48 g (78%); [α]24 −31.5 (c 1.2, CHCl3); TLC Rf 0.63 (19:1
■
D
General Experimental Procedures. Reagents and anhydrous
solvents were purchased from Sigma-Aldrich Chemical Co. and Alfa-
Aesar Inc. and were used as received. The reactions were carried out
under an atmosphere of nitrogen unless specified. Column
chromatography was conducted using silica gel (E. Merck 60 Å,
230−400 mesh), applying a low-pressure stream of nitrogen.
Analytical thin-layer chromatography separations were carried out on
glass plates coated with silica gel (Analtech, GHLF uniplates). The
TLC chromatograms were visualized using UV (short-wave) lamp
irradiation or by immersing the plates in 2.5% potassium
permanganate in water followed by heating with a heat gun. Melting
points are uncorrected and were determined with a Fisher-Johns
melting point apparatus. Optical rotations were measured by use of a
Perkin-Elmer 241 polarimeter, and the [α]D values are given in 10−1
deg cm2 g−1. 1H and 13 C NMR spectra were recorded on Varian Unity
hexanes−EtOAc); 1H NMR (CDCl3, 400 MHz) δ 3.91 (1H, d, J = 4.5
Hz), 2.03 (1H, m), 0.94−0.84 (24H, m), 0.28 (3H, s), 0.27 (3H, s),
0.06 (3H, s), and 0.03 (3H, s); 13C NMR (CDCl3, 101 MHz) δ 173.6,
32.7, 25.7, 25.5, 19.1, 18.2, 17.6, 16.8, −4.9, −5.0, and −5.5;
HRAPCIM m/z 347.2444 [M + H]+ (calcd for C17H39O3Si2,
347.2432); anal. C 55.55, H 10.85%, calcd for C17H38O3Si2·H2O, C
55.99, H 11.06%.
(TBDMSO)Hiv-D-N(Me)Val-Pro-OtBu (14). To a stirred solution
of compound 13 (5.00 g, 14.4 mmol) and DMF (1.07 mL, 1.02 g, 13.9
mmol) in anhydrous DCM (60 mL) at 0 °C was added a 2 M solution
of oxalyl chloride in DCM (9.45 mL, 18.9 mmol), dropwise (gas
evolution). The solution was stirred at 23 °C for 3 h and then
concentrated under diminished pressure, and the residue was dissolved
in anhydrous DCM (60 mL). The mixture was cooled to 0 °C, and a
solution of compound 12 (3.15 g, 11.1 mmol) and BSA (5.71 mL, 4.74
g, 23.3 mmol) in anhydrous DCM (60 mL) at 0 °C was added via
cannula. Next, triethylamine (5.23 mL, 3.85 g, 37.7 mmol) was added,
and the solution was stirred at 23 °C for 16 h. The mixture was
concentrated under diminished pressure, and the residue was dissolved
in ethyl acetate (150 mL). The organic solution was washed with 80
mL of 10% aqueous citric acid, 80 mL of 6% aqueous NaHCO3, and
80 mL of brine, dried over MgSO4, and concentrated under
diminished pressure. The residue was purified by chromatography
on a silica gel column. Elution with 4:1 hexanes−ethyl acetate gave the
1
INOVA 400 and 500 instruments with deuterated solvents. H NMR
chemical shifts were recorded relative to residual CHCl3 at 7.26 ppm,
MeOH at 3.31 ppm, DMSO at 2.50 ppm, or H2O at 4.87 ppm. 13C
NMR chemical shifts were reported relative to residual CHCl3 at 77.16
ppm, MeOH at 49.00 ppm, or DMSO at 39.52 ppm. High-resolution
mass spectra were obtained with a JEOL JMS-LCmate mass
spectrometer. Elemental analyses were determined by Galbraith
Laboratories, Inc. The X-ray crystal structure data were obtained on
a Bruker APEX2 CCD diffractometer using Mo Kα (0.710 73 Å)
radiation.
product as a colorless oil (two conformers ∼2:1): yield 4.55 g (82%);
Cbz-D-N(Me)Val-Pro-OtBu (11). To a stirred solution of Cbz-D-
N(Me)Val (3.85 g, 15.0 mmol), L-Pro-OtBu·HCl (3.43 g, 16.5 mmol),
and PyBrop (10.5 g, 22.5 mmol) in anhydrous DCM (60 mL) at 0 °C
was added DIPEA (4.01 mL, 2.97 g, 23.0 mmol). The solution was
then stirred at 23 °C for 3.5 h and then concentrated under diminished
pressure. The residue was dissolved in EtOAc (100 mL) and washed
with 80 mL of 10% aqueous citric acid, 80 mL of 6% NaHCO3, and 70
mL of brine. The organic solution was dried over MgSO4 and
concentrated under diminished pressure. The residue was purified by
chromatography on a silica gel column. Elution with 4:1 hexanes−
1
[α]24 +32 (c 1.14, CHCl3); TLC Rf 0.35 (4:1 hexanes−EtOAc); H
D
NMR (CDCl3, 400 MHz) major conformer δ 5.07 (1H, d, J = 10.8
Hz), 4.32 (1H, dd, J = 8.5 and 3.2 Hz), 4.21 (1H, d, J = 5.5 Hz), 3.78
(1H, m), 3.56 (1H, m), 3.03 (3H, s), 2.36 (1H, m), 2.14 (1H, m),
1.82−2.04 (4H, m), 1.44 (9H, s), 0.98 (6H, d, J = 6.7 Hz), 0.93 (9H,
s), 0.83 (6H, d, J = 7.6 Hz), and 0.07 (6H, s); 13C NMR (CDCl3, 101
MHz) major conformer δ 172.7, 171.1, 168.6, 80.8, 59.9, 59.6, 47.3,
31.7, 30.1, 29.1, 28.0, 26.5, 25.9, 25.8, 24.8, 19.9, 19.7, 18.3, 18.2, 17.5,
−3.5, −4.5, and −5.1; HRAPCIMS m/z 499.3558 [M + H]+ (calcd for
C26H51N2O5Si, 499.3567); anal. C 62.19, H 10.14, N 5.54%, calcd for
C26H50N2O5Si, C 62.61, H 10.10, N 5.62%.
EtOAc gave the product as a colorless oil: yield 5.56 g (89%); [α]24
D
+41.8 (c 1.0, CH3OH); TLC Rf 0.50 (1:1 hexanes−EtOAc); 1H NMR
(CDCl3, 400 MHz) δ 7.35 (5H, m), 5.14 (2H, m), 4.56 (1H, d, J = 8.7
Hz), 4.33 (1H, m), 3.60 (2H, t, J = 6.4 Hz), 2.88 (3H, s), 2.35 (1H,
m), 2.18 (1H, m), 1.90 (3H, m), 1.43 (9H, s), 0.93 (3H, d, J = 6.4
Hz), and 0.85 (3H, d, J = 6.8 Hz); 13C NMR (CDCl3, 101 MHz) δ
171.4, 168.3, 156.9, 136.7, 128.4, 127.8, 127.5, 80.9, 67.3, 62.1, 59.7,
46.9, 29.2, 28.9, 27.9, 26.6, 24.9, 19.8, and 17.9; HRFABMS m/z
419.2537 [M + H]+ (calcd for C23H35N2O5, 419.2546); anal. C 65.89,
H 8.28, N 6.78%, calcd for C23H34N2O5, C 66.00, H 8.19, N 6.69%.
H-D-N(Me)Val-Pro-OtBu (12). To a stirred solution of Cbz-D-
N(Me)Val-L-Pro-OtBu (11) (5.19 g, 12.4 mmol) in methanol (60 mL)
was added 10% palladium-on-carbon (500 mg). Then, the mixture was
stirred under a hydrogen atmosphere (1 atm) at 23 °C for 3 h. The
reaction mixture was filtered through Celite, and the filtrate was
concentrated under diminished pressure to afford the product as a
colorless oil: yield 3.23 g (92%); TLC Rf 0.53 (95:5 DCM−MeOH);
1H NMR (CDCl3, 400 MHz) δ 4.40 (1H, dd, J = 12, 4 Hz), 3.74 (1H,
m), 3.56 (1H, m), 3.03 (1H, d, J = 6.6 Hz), 2.38 (3H, s), 2.04−2.22
(2H, m), 1.92−1.99 (2H, m), 1.81 (1H, m), 1.47 (9H, s), 0.99 (3H, d,
H-Hiv-D-N(Me)Val-Pro-OtBu (7). To a stirred solution of
compound 14 (1.47 g, 2.96 mmol) in THF (15 mL) at 0 °C was
added 80 μL of water followed by a 1 M solution of TBAF in THF
(7.70 mL, 7.70 mmol). The solution was stirred at 0 °C for 4 h. The
reaction was terminated with 100 mL of water and extracted with two
100 mL portions of EtOAc. The combined organic solution was
washed with 50 mL of brine, dried over MgSO4, and concentrated
under diminished pressure. The residue was purified by chromatog-
raphy on a silica gel column. Elution with 3:2 hexanes−EtOAc gave
the product as a colorless oil (two conformers ∼5:1): yield 834 mg
(73%); [α]24 +73 (c 0.86, CHCl3); TLC Rf 0.25 (3:2 hexanes−
D
1
EtOAc); H NMR (CDCl3, 400 MHz) major conformer δ 5.01 (1H,
d, J = 11.0 Hz), 4.34 (2H, m), 3.60 (2H, t, J = 6.2 Hz), 3.50 (1H, d, J =
7.8 Hz), 2.95 (3H, s), 2.38 (1H, m), 2.17 (1H, m), 1.90 (4H, m), 1.43
(9H, s), 1.11 (3H, d, J = 6.8 Hz), 0.95 (3H, d, J = 6.5 Hz), and 0.82
(6H, d, J = 6.7 Hz); 13C NMR (CDCl3, 101 MHz) δ 174.3, 171.2,
167.7, 81.1, 72.4, 60.6, 59.7, 47.2, 30.5, 29.9, 28.9, 27.9, 26.4, 24.9,
20.2, 19.8, 18.0, and 14.4; HRFABMS, m/z 385.2718 [M + H]+ (calcd
F
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Article
for C20H37N2O5, 385.2703); anal. C 61.66, H 9.69, N 7.16%, calcd for
C20H36N2O5·0.25 H2O, C 61.75, H 9.46, N 7.20%.
(OBn)Lac-Pro-O-Hiv-D-(NMe)Val-Pro-OH (5). To a stirred
solution of compound 17 (950 mg, 1.48 mmol) in anhydrous DCM
(10 mL) was added triethylsilane (3.54 mL, 2.58 g, 22.2 mmol)
followed by TFA (3.40 mL, 5.06 g, 44.4 mmol). The solution was
stirred at 23 °C for 4 h. The mixture was concentrated under
diminished pressure, and the residual TFA coevaorated several times
with toluene and finally with diethyl ether to afford the product as a
Methyl (S)-2-(Benzyloxy)propionate (15). To a stirred solution
of methyl L-lactate (1.00 mL, 1.09 g, 10.5 mmol) in DCM (40 mL)
was added benzyl bromide (1.37 mL, 1.97g, 12.6 mmol) followed by
Ag2O (2.92 g, 12.6 mmol). The reaction mixture was stirred in the
dark at 23 °C for 60 h followed by passage through Celite, and the
filtrate was concentrated under diminished pressure. The residue was
purified by chromatography on a silica gel column. Elution with 4:1
hexanes−ethyl acetate gave the product as a colorless oil: yield 1.03 g
colorless solid (mixture of conformers): yield 869 mg (100%); mp
1
48−55 °C; [α]24 +3.8 (c 0.31, EtOAc); H NMR (CD3OD, 400
D
MHz) and 13C NMR (CD3OD, 101 MHz) showed a complex mixture;
HRAPCIMS m/z 588.3278 [M + H]+ (calcd for C31H46N3O8,
588.3285).
1
(51%); TLC Rf 0.50 (4:1 hexanes−EtOAc); H NMR (CDCl3, 400
MHz) δ 7.36 (5H, m), 4.71 (1H, d, J = 11.6 Hz), 4.45 (1H, d, J = 11.6
Hz), 4.07 (1H, q, J = 6.9 Hz), 3.75 (3H, s), and 1.54 (3H, d, J = 6.8
Hz); 13C NMR (CDCl3, 101 MHz) δ 173.7, 137.6, 128.5, 128.0, 127.9,
74.0, 72.1, 51.9, and 18.8.
(R,E)-3-(But-2-enoyl)-4-phenyloxazolidin-2-one (18). To a
stirred solution of crotonic acid (2.00 g, 23.2 mmol) in anhydrous
THF (60 mL) at −78 °C was added TEA (3.22 mL, 2.34 g, 23.2
mmol) followed by trimethylacetyl chloride (2.86 mL, 2.80 g, 23.2
mmol). The reaction mixture was stirred at 0 °C for 60 min and then
cooled to −78 °C. Separately, to a stirred solution of R-(−)-4-phenyl-
2-oxazolidinone (3.78 g, 27.8 mmol) in anhydrous THF (80 mL) at
−78 °C was added n-BuLi (2.5 M in hexanes) (11.1 mL, 27.8 mmol).
The reaction mixture was stirred at −78 °C for 30 min and then
transferred via cannula to the mixture previously prepared (vide
supra). The reaction mixture was stirred at 23 °C for 16 h. The
mixture was then diluted with 250 mL of EtOAc and washed with 100
mL of 10% aqueous citric acid, 100 mL of saturated aqueous NaHCO3,
and 100 mL of brine. The organic solution was dried over MgSO4 and
concentrated under diminished pressure. The residue was purified by
chromatography on a silica gel column. Elution with 4:1 hexanes−
ethyl acetate gave the product as an off-white solid: yield 5.03 g (94%);
(S)-2-(Benzyloxy)propanoic Acid (16). To a stirred solution of
methyl (S)-2-(benzyloxy)propionate (15) (1.03 g, 5.30 mmol) in
EtOH (8 mL) at 0 °C was added a solution of KOH (327 mg, 5.83
mmol) in water (8 mL), and the mixture stirred at 0 °C for 60 min.
The mixture was diluted with 10 mL of water and washed with 20 mL
of ethyl acetate. The aqueous phase was set to pH ≤ 3 with 6 M HCl
and extracted with two 20 mL portions of ethyl acetate. The combined
organic solution was washed with 25 mL of brine, dried over MgSO4,
and concentrated under diminished pressure to afford the product as a
1
colorless oil: yield 833 mg (88%); H NMR (CDCl3, 400 MHz) δ
10.30 (1H, br s), 7.36 (5H, m), 4.70 (1H, d, J = 11.6 Hz), 4.56 (1H, d,
J = 11.6 Hz), 4.12 (1H, q, J = 6.9 Hz), and 1.50 (3H, d, J = 6.8 Hz);
13C NMR (CDCl3, 101 MHz) δ 178.9, 137.1, 128.5, 128.0, 128.0, 73.5,
72.1, and 18.4.
(OBn)Lac-Pro-OtBu (8). To a stirred solution of (S)-2-
(benzyloxy)propanoic acid (16) (2.70 g, 15.0 mmol), L-Pro-OtBu·
HCl (3.43 g, 16.5 mmol), and PyBroP (10.5 g, 22.5 mmol) in
anhydrous DCM (60 mL) at 0 °C was added DIPEA (7.86 mL, 5.88 g,
45.0 mmol). The solution was stirred at 23 °C for 5 h and then
concentrated under diminished pressure. The residue was dissolved in
EtOAc (100 mL) and washed with 80 mL of 10% aqueous citric acid,
80 mL of 6% NaHCO3, and 70 mL of brine. The organic solution was
dried over MgSO4 and concentrated under diminished pressure. The
residue was purified by chromatography on a silica gel column. Elution
with 1:1 EtOAc−hexanes gave the product as a colorless oil (two
TLC Rf 0.35 (4:1 hexanes−EtOAc); mp 73−74 °C; [α]24 −123.2 (c
D
1
0.93, acetone); H NMR (CDCl3, 400 MHz) δ 7.44−7.27 (6H, m),
7.17−7.03 (1H, m), 5.48 (1H, dd, J = 8.7, 3.9 Hz), 4.70 (1H, t, J = 8.8
Hz), 4.27 (1H, dd, J = 8.8, 3.9 Hz), and 1.97−1.90 (3H, m); 13C NMR
(CDCl3, 101 MHz) δ 164.6, 153.9, 147.4, 139.2, 129.3, 128.8, 126.0,
121.9, 70.1, 57.9, and 18.7; HRFABMS m/z 232.09770 [M + H]+
(calcd for C13H14NO3, 232.09737).
(R)-3-[(2R,3R)-2-Bromo-3-methyl-4-phenylbutanoyl]-4-phe-
nyloxazolidin-2-one (19). To a stirred mixture of copper(I)
bromide dimethyl sulfide complex (4.93 g, 24.0 mmol) in anhydrous
THF (54 mL) at −78 °C was added 54 mL of anhydrous dimethyl
sulfide. The mixture was stirred for 5 min at −78 °C, and
benzylmagnesium chloride (2 M in THF) (24.0 mL, 48.0 mmol)
was added dropwise. The reaction mixture was stirred at −78 °C for
45 min, and a solution of compound 18 (5.03 g, 21.8 mmol) in
anhydrous THF (54 mL) at −78 °C was added via cannula. The
mixture was stirred at −78 °C for 90 min and then at 0 °C for 30 min
(formation of a gray-black precipitate). The mixture was cooled to
−78 °C, and a solution of NBS (12.8 g, 71.9 mmol) in anhydrous
THF (150 mL) at −78 °C was added via cannula. The reaction
mixture was stirred at −78 °C for 2 h. The reaction was terminated
with 300 mL of saturated aqueous NH4Cl and extracted with 300 mL
of EtOAc. The organic solution was washed with 300 mL of 10%
aqueous Na2S2O4 and 300 mL of brine, dried over MgSO4, and
concentrated under diminished pressure. The residue was purified by
chromatography on a silica gel column. Elution with 5:4:1 hexanes−
toluene−ethyl acetate gave the product as an off-white solid: yield 5.99
conformers ∼4:1): yield 4.11 g (82%); [α]24 −110 (c 2.04, CHCl3);
D
1
TLC Rf 0.48 (1:1 hexanes−EtOAc); H NMR (CDCl3, 400 MHz)
major conformer δ 7.36 (5H, m), 4.71 (1H, m), 4.43 (2H, m), 4.21
(1H, q, J = 6.7 Hz), 3.60 (2H, m), 2.17 (1H, m), 2.02 (1H, m), 1.90
(2H, m), 1.47 (9H, s), and 1.44 (3H, d, J = 6.8 Hz); 13C NMR
(CDCl3, 101 MHz) δ 171.2, 171.0, 137.7, 128.3, 127.9, 127.6, 81.7,
74.6, 71.0, 60.0, 46.5, 28.5, 27.9, 25.2, and 17.4; HRFABMS m/z
334.2028 [M + H]+ (calcd for C19H28NO4, 334.2018); anal. C 68.44,
H 8.36, N 4.44%, calcd for C19H27NO4, C 68.44, H 8.16, N 4.20%.
(OBn)Lac-Pro-O-Hiv-D-N(Me)Val-Pro-OtBu (17). To a stirred
solution of compound 8 (535 mg, 1.60 mmol) in anhydrous DCM (4
mL) was added 2 mL of TFA. The reaction mixture was then stirred at
23 °C for 4 h and then concentrated under diminished pressure. The
residual TFA was coevaporated with toluene. The residue was
dissolved in anhydrous DCM (13 mL), and compound 7 (576 mg,
1.50 mmol) was added followed by MNBA (661 mg, 1.92 mmol),
DMAP (78 mg, 0.64 mmol), and TEA (665 μL, 486 mg, 4.80 mmol).
The reaction mixture was stirred at 23 °C for 5 h. The mixture was
concentrated under diminished pressure. The residue was dissolved in
EtOAc (80 mL) and washed with 40 mL of 10% aqueous citric acid, 40
mL of 6% NaHCO3, and 40 mL of brine. The organic solution was
dried over MgSO4 and concentrated under diminished pressure. The
residue was purified by chromatography on a silica gel column. Elution
with 4:1 EtOAc−hexanes gave the product as a colorless solid
g (67%); TLC Rf 0.25 (5:4:1 hexanes−toluene−EtOAc); mp 112−113
1
°C; [α]24 −75.3 (c 0.43, acetone); H NMR (CDCl3, 400 MHz) δ
D
7.53−6.98 (10H, m), 5.64 (1H, d, J = 8.6 Hz), 5.41 (1H, dd, J = 8.8,
4.5 Hz), 4.67 (1H, t, J = 8.9 Hz), 4.24 (1H, dd, J = 8.9, 4.5 Hz), 3.26
(1H, d, J = 10.4 Hz), 2.52−2.31 (2H, m), and 0.91 (3H, d, J = 6.4 Hz);
13C NMR (CDCl3, 101 MHz) δ 167.9, 152.8, 139.5, 137.7, 129.3,
129.2, 128.9, 128.4, 126.3, 125.8, 69.9, 57.8, 50.3, 40.1, 37.7, and 16.8;
HRAPCIMS m/z 404.0683 [M + H]+ (calcd for C20H21NO381Br,
404.0684).
(mixture of conformers): yield 776 mg (81%); mp 53−57 °C; [α]24
D
−19 (c 1.00, CHCl3); TLC Rf 0.50 and 0.15 (4:1 EtOAc−hexanes);
1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 101 MHz)
show a complex mixture; HRAPCIMS m/z 644.3946 [M + H]+ (calcd
for C35H54N3O8, 644.3911); anal. C 64.55, H 8.34, N 6.02%, calcd for
C35H53N3O8·0.5 H2O, C 64.39, H 9.46, N 6.44%.
Dolaphenvaline·HCl (9). To a stirred mixture of compound 19
(5.99 g, 14.9 mmol) in anhydrous DMF (70 mL) at 0 °C was added
sodium azide (2.91 g, 44.7 mmol). The reaction mixture was stirred at
0 °C for 3 h. The solution was diluted with 300 mL of EtOAc and
washed with 300 mL of water followed by three 300 mL portions of
G
DOI: 10.1021/np500925y
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Journal of Natural Products
Article
brine. The organic solution was dried over MgSO4 and concentrated
under diminished pressure. The residue was dissolved in 3:1 THF−
water (180 mL) and cooled to 0 °C, and 35% aqueous H2O2 (8.69
mL, 89.4 mmol) was added dropwise followed by 1 M aqueous LiOH
(29.8 mL, 29.8 mmol). The reaction mixture was stirred at 0 °C for 3
h. Then, the reaction was terminated with 129 mL of 1.3 M aqueous
Na2SO3 and stirred at 23 °C for 30 min. The reaction mixture was
concentrated under diminished pressure, and the aqueous residue was
washed with three 100 mL portions of DCM. The aqueous phase was
cooled to 0 °C, brought to pH ∼1.5 with 6 N HCl, and extracted with
three 100 mL portions of DCM. The combined organic solution was
dried over MgSO4 and concentrated under diminished pressure to give
hydrogen atmosphere (1 atm) at 23 °C for 1 h. The reaction mixture
was filtered through Celite, and the filtrate was concentrated under
diminished pressure. The residual methanol was coevaporated with
toluene twice, and the residue was dissolved in anhydrous DMF (15
mL). The mixture was cooled to 0 °C before adding Cbz-L-Pro-OH
(944 mg, 3.79 mmol) followed by TEA (1.31 mL, 959 mg, 9.48 mmol)
and HBTU (1.50 g, 3.95 mmol). The resulting reaction mixture was
stirred at 23 °C for 16 h, diluted with 100 mL of ethyl acetate, and
washed with 80 mL of 0.5 N HCl, 80 mL of saturated aqueous
NaHCO3, and 80 mL of brine. The organic solution was dried over
MgSO4 and concentrated under diminished pressure. The residue was
purified by chromatography on a silica gel column. Elution with 1:1
hexanes−ethyl acetate gave the product as a colorless oil (mixture of
the crude product 21 as a colorless oil: yield 2.56 g (78%); [α]24
D
−89.4 (c 0.45, acetone); 1H NMR (CDCl3, 400 MHz) δ 11.29 (1H, s),
7.37−7.10 (5H, m), 3.92 (1H, d, J = 3.6 Hz), 2.74−2.59 (2H, m),
2.49−2.38 (1H, m), and 0.98 (3H, d, J = 6.7 Hz); 13C NMR (CDCl3,
101 MHz) δ 176.46, 139.12, 129.31, 129.05, 128.64, 128.36, 126.52,
64.81, 39.90, 37.72, and 14.78.
conformers ∼1:1): yield 900 mg (73%); TLC Rf 0.12 (2:1 hexane−
1
ethyl acetate); [α]24 −33.7 (c 0.38, MeOH); H NMR (DMSO-d6,
D
400 MHz) one conformer δ 7.47−7.16 (6H, m), 5.04 (2H, s), 4.22
(1H, d, J = 8.9 Hz), 3.79−3.63 (1H, m), 3.50 (3H, s), 3.45−3.31 (2H,
m), 2.72−2.56 (1H, m), 2.22−2.00 (1H, m), 1.84−1.60 (4H, m), 1.01
(3H, d, J = 6.8 Hz), and 0.82−0.63 (6H, m); 13C NMR (DMSO-d6,
101 MHz) δ 174.6, 171.6, 154.1, 136.9, 128.2, 127.7, 127.3, 65.8, 59.8,
55.7, 51.3, 47.1, 40.8, 31.5, 29.4, 22.9, 19.8, 17.5, and 14.0;
HRAPCIMS m/z 391.2236 [M + H]+ (calcd for C21H31N2O5,
391.2233).
Without further purification, crude product 21 (2.50 g, 11.4 mmol)
was dissolved in 2:1 AcOH−water (150 mL), and 10% palladium-on-
carbon (250 mg) was added. The reaction mixture was purged of air
and charged with hydrogen (36 psi). The reaction mixture was shaken
at 23 °C for 24 h in a Parr hydrogenator. The reaction was filtered
through Celite, and the filtrate concentrated under diminished
pressure. The residue was dissolved in 20 mL of 6 N HCl and
concentrated under diminished pressure. The residual water was
coevaporated with toluene several times, and finally the residue was
triturated with ether. The precipitate was filtered and dried to afford
the product (compound 9) as an off-white solid: yield 2.60 g (100%);
Boc-Dpv-Pro-Dml-OMe (24). To a stirred solution of compound
10 (480 mg, 1.23 mmol) in methanol (10 mL) was added 10%
palladium over activated carbon (48 mg). Then, the mixture was
stirred under a hydrogen atmosphere (1 atm) at 23 °C for 2.5 h. The
reaction mixture was filtered through Celite, and the filtrate was
concentrated under diminished pressure. The residual methanol was
coevaporated with toluene (twice), and the residue was dissolved in
anhydrous DMF (10 mL). The mixture was cooled to 0 °C before
adding Boc-Dpv-OH (410 mg, 1.40 mmol) followed by TEA (597 μL,
436 mg, 4.31 mmol) and HBTU (536 mg, 2.15 mmol). The resulting
reaction mixture was stirred at 23 °C for 16 h, diluted with 80 mL of
ethyl acetate, and washed with 80 mL of 10% aqueous citric acid, 80
mL of saturated aqueous NaHCO3, and 80 mL of brine. The organic
solution was dried over MgSO4 and concentrated under diminished
pressure. The residue was purified by chromatography on a silica gel
column. Elution with 1:1 hexanes−ethyl acetate gave the product as a
colorless foam: yield 439 mg (67%); TLC Rf 0.25 (1:1 hexane−ethyl
acetate); [α]24D −6.9 (c 0.14, EtOAc); 1H NMR (CDCl3, 400 MHz) δ
7.28−7.08 (5H, m), 6.83 (1H, d, J = 10.2 Hz), 5.42 (1H, d, J = 9.3
Hz), 4.55−4.42 (2H, m), 3.64 (1H, tt, J = 17.8, 8.8 Hz), 3.54 (3H, s),
3.38−3.20 (2H, m), 2.82−2.67 (2H, m), 2.45 (1H, dt, J = 22.1, 11.0
Hz), 2.27−2.17 (1H, m), 2.15−1.74 (4H, m), 1.52−1.36 (10H, m),
1.12 (3H, d, J = 7.1 Hz), and 0.89−0.79 (9H, m); 13C NMR (CDCl3,
101 MHz) δ 176.5, 172.1, 171.6, 156.0, 140.4, 129.5, 128.3, 126.1,
79.6, 60.7, 57.1, 54.0, 51.7, 46.8, 40.5, 39.7, 38.3, 31.8, 29.1, 28.4, 24.9,
19.9, 19.4, 15.9, and 14.1; HRAPCIMS m/z 532.3383 [M + H]+ (calcd
for C29H46N3O6, 532.3387).
mp 249−250 °C (dec); [α]24 +35.7 (c 0.28, MeOH); 1H NMR
D
(D2O, 400 MHz) δ 7.35−7.11 (5H, m), 3.85 (1H, d, J = 2.7 Hz),
2.77−2.65 (1H, m), 2.55−2.37 (2H, m), and 0.81 (3H, d, J = 6.5 Hz);
13C NMR (D2O, 101 MHz) δ 171.9, 138.9, 129.0, 128.7, 126.7, 56.9,
38.1, 35.7, and 13.2; HRAPCIMS m/z 194.1183 [M + H]+ (calcd for
C11H16NO2, 194.1181).
(S)-Homo-β-Val-OMe·HCl (22). To 2.29 mL of anhydrous
methanol at 0 °C was added very slowly thionyl chloride (498 μL,
817 mg, 6.87 mmol). The reaction mixture was stirred for 10 min
before adding (S)-homo-β-Val (300 mg, 2.29 mmol). The reaction
mixture was stirred at 23 °C for 16 h and diluted with 60 mL of diethyl
ether. The precipitate was collected and dried under diminished
pressure to afford the product as a colorless solid: yield 383 mg (92%);
mp 133−134 °C; [α]24D −41.1 (c 0.13, MeOH); 1H NMR (D2O, 400
MHz) δ 3.62 (3H, s), 3.44−3.34 (1H, m), 2.65 (2H, m), 1.88 (1H,
m), and 0.86 (6H, m); 13C NMR (D2O, 101 MHz) δ 173.2, 53.4, 52.5,
33.4, 29.8, 17.2, and 16.7; HRAPCIMS m/z 146.1178 [M + H]+ (calcd
for C7H16NO2, 146.1181).
Cbz-Dml-OMe (23). To a stirred solution of compound 22 (801
mg, 4.67 mmol) in anhydrous THF (14 mL) at −10 °C was added a 1
M solution of LiHMDS in toluene (10.3 mL, 10.3 mmol). The
reaction was stirred for 10 min at −10 °C before adding (dropwise)
methyl iodide (436 μL, 995 mg, 7.01 mmol). The reaction mixture was
stirred at 23 °C for 2 h prior to adding 15 mL of 10% aqueous K2CO3
followed by benzyl chloroformate (4.01 mL, 4.78 g, 28.0 mmol). The
resulting mixture was stirred at 23 °C for 2 h, diluted with 50 mL of
ethyl acetate, washed with 40 mL of 10% aqueous citric acid and 40
mL of brine, dried over MgSO4, and concentrated under diminished
pressure. The residue was purified by chromatography on a silica gel
column. Elution with 9:1 hexanes−acetone gave the product as a
Boc-Dpv-Pro-Dml-OBn (6). To a stirred solution of compound
24 (200 mg, 0.38 mmol) in 2:1:1 MeOH−THF−water (1.2 mL) was
added LiOH·H2O (159 mg, 3.80 mmol). The reaction mixture was
stirred at 23 °C for 18 h and then concentrated under diminished
pressure. The residue was dissolved in 5 mL of water, and the pH was
adjusted to ≤3. The mixture was then extracted with three 10 mL
portions of ethyl acetate. The combined organic solution was washed
with 10 mL of brine, dried over MgSO4, and concentrated under
diminished pressure. The residue was dissolved in anhydrous DMF (1
mL), and TEA (316 μL, 231 mg, 2.28 mmol) was added followed by
benzyl bromide (135 μL, 195 mg, 1.14 mmol). The reaction mixture
was stirred at 23 °C for 6 h and then diluted with 50 mL of ethyl
acetate. The organic mixture was washed with 10 mL of 10% aqueous
citric acid, 10 mL of saturated aqueous sodium bicarbonate, 10 mL of
brine, dried over MgSO4, and concentrated under diminished pressure.
The residue was purified by chromatography on a silica gel column.
Elution with 1:1 hexanes−ethyl acetate gave the product as a colorless
colorless oil: yield 788 mg (58%); TLC Rf 0.30 (9:1 hexane−acetone);
1
[α]24 +16.7 (c 1.90, MeOH); H NMR (CDCl3, 400 MHz) δ 7.42−
D
7.21 (5H, m), 5.54 (1H, d, J = 10.2 Hz), 5.11 (2H, s), 3.64 (3H, s),
3.56−3.40 (1H, m), 2.81 (1H, dd, J = 6.9, 4.5 Hz), 1.69 (1H, m), 1.21
(3H, d, J = 7.1 Hz), and 0.93 (6H, m); 13C NMR (CDCl3, 101 MHz)
δ 176.0, 156.9, 136.9, 128.5, 128.0, 127.9, 66.6, 59.4, 51.7, 40.4, 31.8,
19.9, 19.2, and 15.8; HRAPCIMS m/z 294.1701 [M + H]+ (calcd for
C16H24NO4, 294.1705).
Cbz-Pro-Dml-OMe (10). To a stirred solution of compound 23
(928 mg, 3.16 mmol) in methanol (15 mL) was added 10% palladium
over activated carbon (98 mg). Then, the mixture was stirred under a
foam: yield 129 mg (56%); TLC Rf 0.26 (1:1 hexane−ethyl acetate);
1
[α]24 −2.1 (c 0.19, EtOAc); H NMR (CDCl3, 400 MHz) δ 7.43−
D
7.22 (9H, m), 7.21−7.08 (1H, m), 6.84 (1H, d, J = 10.2 Hz), 5.38
H
DOI: 10.1021/np500925y
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Journal of Natural Products
Article
(1H, d, J = 9.2 Hz), 5.13−4.87 (2H, m), 4.58−4.45 (2H, m), 3.67
(1H, dt, J = 17.1, 10.0 Hz), 3.40−3.21 (2H, m), 2.91−2.71 (2H, m),
2.49 (1H, dd, J = 13.3, 7.0 Hz), 2.31−2.20 (1H, m), 2.12−1.82 (4H,
m), 1.55−1.42 (10H, m), 1.18 (3H, d, J = 7.1 Hz), and 0.92−0.82
(9H, m); 13C NMR (CDCl3, 101 MHz) δ 175.9, 172.0, 171.6, 156.0,
140.4, 135.7, 129.6, 128.6, 128.4, 128.3, 128.1, 126.1, 79.6, 66.3, 60.7,
57.1, 54.0, 46.8, 40.6, 39.8, 38.4, 31.9, 29.2, 28.4, 25.0, 19.9, 19.6, 16.0,
and 14.1; HRAPCIMS m/z 608.3694 [M + H]+ (calcd for
C35H50N3O6, 608.3700).
unit cell yielded a total of 45 655 reflections to a maximum θ angle of
25.48° (0.83 Å resolution), of which 10 048 were independent
(average redundancy 4.544, completeness 99.6%, Rint = 6.61%) and
8727 (86.85%) were greater than 2σ(F2). The final cell constants of a
= 10.3987(10) Å, b = 18.7734(18) Å, c = 27.849(3) Å, volume =
5436.7(9) Å3, are based upon the refinement of the XYZ-centroids of
9674 reflections above 20σ(I) with 4.339° < 2θ < 45.71°. Data were
corrected for absorption effects using the multiscan method
(SADABS). The ratio of minimum to maximum apparent transmission
was 0.708. The calculated minimum and maximum transmission
coefficients (based on crystal size) are 0.9030 and 0.9780.
(OBn)Lac-Pro-O-Hiv-D-N(Me)Val-Pro-Dpv-Pro-Dml-OBn (4).
To a stirred solution of compound 6 (140 mg, 0.23 mmol) in
anhydrous DCM (2 mL) was added 2 mL of TFA. The reaction
mixture was stirred at 23 °C for 4 h and concentrated under
diminished pressure. The residual TFA was coevaporated with toluene.
The residue was dissolved in anhydrous DMF (3 mL), and compound
5 (135 mg, 0.23 mmol) was added followed by TEA (97 μL, 70 mg,
0.69 mmol) and HBTU (86 mg, 0.35 mmol). The mixture was stirred
at 23 °C for 16 h. The mixture was then diluted with 80 mL of ethyl
acetate and washed with two 40 mL portions of brine. The organic
solution was dried over MgSO4 and concentrated under diminished
pressure. The residue was purified by chromatography on a silica gel
column. Elution with 1:1 hexanes−acetone gave the product as a
The final anisotropic full-matrix least-squares refinement on F2 with
633 variables converged at R1 = 6.01% for the observed data and wR2
= 16.78% for all data. The goodness-of-fit was 1.085. The largest peak
in the final difference electron density synthesis was 1.501 e−/Å3, and
the largest hole was −0.518 e−/Å3 with an RMS deviation of 0.074 e−/
Å3. On the basis of the final model, the calculated density is 1.242 g/
cm3 and F(000) is 2168 e−.
Cancer Cell Line Procedures. Inhibition of human cancer cell
growth was assessed using the National Cancer Institute’s standard
sulforhodamine B assay as previously described.15 Briefly, cells in a 5%
fetal bovine serum/RPMI1640 medium were inoculated in 96-well
plates and incubated for 24 h. Serial dilutions of the compounds were
then added. After 48 h, the plates were fixed with trichloroacetic acid,
stained with sulforhodamine B, and read with an automated microplate
reader. A growth inhibition of 50% (GI50, or the drug concentration
causing a 50% reduction in the net protein increase) was calculated
from optical density data with Immunosoft software.
colorless foam (mixture of conformers): yield 216 mg (87%); TLC Rf
1
0.3 and 0.4 (1:1 hexane−acetone); [α]24 +5.0 (c 0.06, EtOAc); H
D
NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 101 MHz)
showed a complex mixture; HRAPCIMS m/z 1077.631 [M + H]+
(calcd for C61H85N6O11, 1077.628).
Dolastatin 16 (1). To a stirred solution of compound 4 (204 mg,
0.19 mmol) in ethanol (3 mL) was added 20% Pd(OH)2 on carbon
(41 mg). The mixture was stirred under a hydrogen atmosphere (1
atm) at 23 °C for 4 h. The reaction mixture was filtered through
Celite, and the filtrate was concentrated under diminished pressure.
The residue was dissolved in 5 mL of anhydrous toluene containing
TEA (26 μL, 19 mg, 0.19 mmol) and added (at 0.25 mL/h, using a
syringe pump) to a solution containing MNBA (327 mg, 0.95 mmol)
and DMAP (232 mg, 1.90 mmol) in anhydrous toluene (126 mL).
After the addition was complete the reaction mixture was stirred at 23
°C for 16 h. The reaction mixture was concentrated under diminished
pressure, and the residue was dissolved in ethyl acetate (100 mL). The
organic solution was washed with 50 mL of 1 N HCl, 50 mL of
saturated aqueous sodium bicarbonate, and 50 mL of brine, dried over
MgSO4, and concentrated under diminished pressure. The residue was
purified by chromatography on a silica gel column. Elution with 2:3
hexanes−acetone gave dolastatin 16 as an off-white solid: yield 36 mg
(22%); TLC Rf 0.55 (2:3 hexanes−acetone). An analytical sample was
purified by reversed-phase HPLC (Zorbax-SB C-18 column, 250 × 9.4
mm, flow rate 3.5 mL/min, elution gradient 40% ACN in water to 99%
ASSOCIATED CONTENT
■
S
* Supporting Information
X-ray crystallographic data for 1 as well as copies of the 1H and
13C NMR spectra of compounds 1, 4−19, and 22−24. This
material is available free of charge via the Internet at http://
pubs.acs.org. Crystallographic data have been deposited with
Cambridge Crystallographic Data Center as supplementary
publication no. CCDC 1035008. This can be obtained free of
charge on application to the Cambridge Crystallographic Data
Center, 2 Union Rd, Cambridge CBZ 1EZ, UK [fax: (+44)
1223-336 033; e-mail: deposit@ccdc.cam.ac.uk].
AUTHOR INFORMATION
■
Corresponding Author
*Tel: (480) 965-3351. Fax: (480) 965-2747. E-mail: bpettit@
asu.edu.
ACN in water in 20 min, retention time 16.4 min); mp 199−201 °C;
1
[α]24 +14.5 (c 0.20, MeOH); H NMR (CDCl3, 400 MHz) δ 7.73
D
(1H, d, J = 10.1 Hz), 7.35 (2H, d, J = 7.4 Hz), 7.29 (2H, d, J = 7.4
Hz), 7.22−7.13 (1H, m), 6.79 (1H, d, J = 8.8 Hz), 5.43 (1H, d, J = 2.9
Hz), 5.19 (2H, m), 4.96 (1H, d, J = 8.8 Hz), 4.65 (1H, dd, J = 8.7, 2.1
Hz), 4.57 (1H, d, J = 7.5 Hz), 4.47 (1H, d, J = 6.7 Hz), 3.96−3.88
(1H, m), 3.73−3.61 (2H, m), 3.47 (2H, m), 3.10 (3H, s), 2.90−2.81
(2H, m), 2.56−2.48 (2H, m), 2.46−1.69 (15H, m), 1.60−1.48 (2H,
m), 1.46 (3H, d, J = 6.8 Hz), 1.13−0.97 (9H, m), and 0.97−0.77
(15H, m); 13C NMR (CDCl3, 101 MHz) δ 174.8, 172.6, 171.4, 171.1,
171.0, 169.7, 169.3, 169.2, 140.6, 129.6, 128.4, 126.3, 76.5, 66.7, 61.3,
59.6, 58.9, 57.9, 56.5, 50.6, 47.6, 46.5, 46.0, 41.0, 40.9, 38.7, 32.4, 31.0,
30.8, 29.8, 28.3, 25.6, 25.5, 25.0, 24.8, 21.8, 20.3, 19.74, 19.73, 19.72,
17.9, 17.2, 16.1, 15.1, and 14.9; HRESIMS m/z 879.5225 [M + H]+
(calcd for C47H71N6O10, 879.5231).
X-ray Crystal Structure of Synthetic Dolastatin 16 (1). A
plate-like specimen of C47H70N6O10·1.62(CH2Cl2), approximate
dimensions 0.095 mm × 0.289 mm × 0.435 mm, was obtained from
dichloromethane−hexanes and used for X-ray crystallographic analysis.
The X-ray intensity data were measured. A total of 1092 frames were
collected. The total exposure time was 18.20 h. The frames were
integrated with the Bruker SAINT software package using a narrow-
frame algorithm. The integration of the data using an orthorhombic
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are pleased to acknowledge the financial support provided
by grants R01CA90441-01-05, 2R56CA090441-06A1, and 5-
RO1CA90441-07-08 from the Division of Cancer Treatment
and Diagnosis, NCI, DHHS; the Arizona Biomedical Research
Commission; J. W. Kieckhefer Foundation; Margaret T. Morris
Foundation; the Robert B. Dalton Endowment Fund; as well as
Dr. W. Crisp and Mrs. A. Crisp. We also wish to thank Dr. N.
Melody for her assistance.
DEDICATION
■
Dedicated to Dr. William Fenical of Scripps Institution of
Oceanography, University of California−San Diego, for his
pioneering work on bioactive natural products.
I
DOI: 10.1021/np500925y
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
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