From the enediyne class of antitumor antibiotics, uncialamycin is among the rarest and most potent, yet one of the structurally simpler, making it attractive for chemical synthesis and potential applications in biology and medicine. In this article we describe a streamlined and practical enantioselective total synthesis of uncialamycin that is amenable to the synthesis of novel analogues and renders the natural product readily available for biological and drug development studies. Starting from hydroxy- or methoxyisatin, the synthesis features a Noyori enantioselective reduction, a Yamaguchi acetylide-pyridinium coupling, a stereoselective acetylide-aldehyde cyclization, and a newly developed annulation reaction that allows efficient coupling of a cyanophthalide and a p-methoxy semiquinone aminal to forge the anthraquinone moiety of the molecule. Overall, the developed streamlined synthesis proceeds in 22 linear steps (14 chromatographic separations) and 11% overall yield. The developed synthetic strategies and technologies were applied to the synthesis of a series of designed uncialamycin analogues equipped with suitable functional groups for conjugation to antibodies and other delivery systems. Biological evaluation of a select number of these analogues led to the identification of compounds with low picomolar potencies against certain cancer cell lines. These compounds and others like them may serve as powerful payloads for the development of antibody drug conjugates (ADCs) intended for personalized targeted cancer therapy.
The successful synthesis of dolastatin 11, a depsipeptide originally isolated from the mollusk Dolabella auricularia, permitted us to study its effects on cells. The compound arrested cells at cytokinesis by causing a rapid and massive rearrangement of the cellular actin filament network. In a dose-and time-dependent manner, F-actin was rearranged into aggregates, and subsequently the cells displayed dramatic cytoplasmic retraction. The effects of dolastatin 11 were most similar to those of the sponge-derived depsipeptide jasplakinolide, but dolastatin 11 was about 3-fold more cytotoxic than jasplakinolide in the cells studied. Like jasplakinolide, dolastatin 11 induced the hyperassembly of purified actin into filaments of apparently normal morphology. Dolastatin 11 was qualitatively more active than jasplakinolide and, in a quantitative assay we developed, dolastatin 11 was twice as active as jasplakinolide and 4-fold more active than phalloidin. However, in contrast to jasplakinolide and phalloidin, dolastatin 11 did not inhibit the binding of a fluorescent phalloidin derivative to actin polymer nor was it able to displace the phalloidin derivative from polymer. Thus, despite its structural similarity to other agents that induce actin assembly (all are peptides or depsipeptides), dolastatin 11 may interact with actin polymers at a distinct drug binding site.
Synthetic methodology for preparing novel esterase-sensitive
cyclic prodrugs of peptides with
increased protease stability and cell membrane permeability compared to
linear peptides is
described. Cyclic prodrug 1 of the hexapeptide
H-Trp-Ala-Gly-Gly-Asp-Ala-OH linked by the
N-terminal amino group to the C-terminal carboxyl group via an
(acyloxy)alkoxy promoiety was
synthesized. A convergent synthetic approach involving
Boc[[(alaninyloxy)methyl]carbonyl]-N-tryptophan (2) and H-Ala-Gly-Gly-Asp(OBzl)-OTce
(3) was used. The key fragment 2 has
the
promoiety inserted between the Ala and the Trp residues. Fragment
3 was synthesized by a
solution-phase approach using standard Boc-amino acid chemistry.
These fragments were coupled
to produce the protected linear hexapeptide, which after deprotection
was cyclized using standard
high-dilution techniques to yield cyclic prodrug 1. In
pH 7.4 buffer (HBSS) at 37 °C, cyclic prodrug
1 was shown to degrade quantitatively to the hexapeptide
(t
1/2 = 206 ± 11 min). The rate
of
hydrolysis of cyclic prodrug 1 was significantly faster in
human blood (t
1/2 = 132 ± 4 min) than
in
HBSS. Paraoxon, a known inhibitor of esterases, slowed this
hydrolysis of cyclic prodrug 1 to a
value (t
1/2 = 198 ± 9 min) comparable to
the chemical stability. In human blood, cyclic prodrug
1
was shown to be 25-fold more stable than the linear
hexapeptide.
This paper describes a unique strategy for preparing cyclic
prodrugs of peptides that have increased
metabolic stability and increased cell membrane permeability when
compared to the linear peptides.
By taking advantage of a unique “trimethyl lock”-facilitated
lactonization system, an esterase-sensitive cyclic prodrug of a model hexapeptide
H-Trp-Ala-Gly-Gly-Asp-Ala-OH was synthesized
by linking the N-terminal amino group to the C-terminal carboxyl group.
The key intermediate
for both approaches was compound 9 with Boc-Ala attached to
the phenol hydroxyl group of the
“trimethyl lock” linker through an ester bond, which can then be
incorporated into the peptide
using a normal coupling reagent for peptide synthesis. The
synthesis of the linear peptides was
accomplished using both solution-phase and solid-phase approaches with
the solution-phase
approach having the advantage of using the key intermediate 9
most efficiently. Cyclization using
standard high-dilution techniques provided cyclic prodrug
13. In 90% human plasma, prodrug
13
released the original peptide, as designed, through an apparent
esterase-catalyzed hydrolysis of
the phenol ester bond.
Intravenous administration of N-(beta-alanyl-L-leucyl-L-alanyl-L-leucyl)doxorubicin (4) induces an acute toxic reaction, killing animals in a few minutes. This results from its positive charge at physiological pH combined with its propensity to form large aggregates in aqueous solutions. Negatively charged N-capped versions of 4 such as the succinyl derivative 5 can be administered by the iv route at more than 10 times the LD(50) of doxorubicin without inducing the acute toxic reaction, and they are active in vivo.
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