Streamlined Total Synthesis of Shishijimicin A and Its Application to the Design, Synthesis, and Biological Evaluation of Analogues thereof and Practical Syntheses of PhthNSSMe and Related Sulfenylating Reagents

Article abstract of DOI:10.1021/jacs.8b06955

Shishijimicin A is a scarce marine natural product with highly potent cytotoxicities, making it a potential payload or a lead compound for designed antibody-drug conjugates. Herein, we describe an improved total synthesis of shishijimicin A and the design, synthesis, and biological evaluation of a series of analogues. Equipped with appropriate functionalities for linker attachment, a number of these analogues exhibited extremely potent cytotoxicities for the intended purposes. The synthetic strategies and tactics developed and employed in these studies included improved preparation of previously known and new sulfenylating reagents such as PhthNSSMe and related compounds.

Full text of DOI:10.1021/jacs.8b06955

Article
pubs.acs.org/JACS
Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Streamlined Total Synthesis of Shishijimicin A and Its Application to
the Design, Synthesis, and Biological Evaluation of Analogues
thereof and Practical Syntheses of PhthNSSMe and Related
Sulfenylating Reagents
K. C. Nicolaou,*,† Ruofan Li,†,# Zhaoyong Lu,†,# Emmanuel N. Pitsinos,†,§ Lawrence B. Alemany,†,‡
Monette Aujay,∥ Christina Lee,∥ Joseph Sandoval,∥ and Julia Gavrilyuk∥
‡
†Department of Chemistry, BioScience Research Collaborative and Shared Equipment Authority, Rice University, 6100 Main
Street, Houston, Texas 77005, United States
§Laboratory of Natural Products Synthesis & Bioorganic Chemistry, Institute of Nanoscience and Nanotechnology, National Centre
for Scientific Research “Demokritos”, 153 10 Agia Paraskevi, Greece
∥AbbVie Stemcentrx, LLC, 450 East Jamie Court, South San Francisco, California 94080, United States
S
* Supporting Information
ABSTRACT: Shishijimicin A is a scarce marine natural
product with highly potent cytotoxicities, making it a potential
payload or a lead compound for designed antibody−drug
conjugates. Herein, we describe an improved total synthesis of
shishijimicin A and the design, synthesis, and biological
evaluation of a series of analogues. Equipped with appropriate
functionalities for linker attachment, a number of these
analogues exhibited extremely potent cytotoxicities for the
intended purposes. The synthetic strategies and tactics
developed and employed in these studies included improved
preparation of previously known and new sulfenylating
reagents such as PhthNSSMe and related compounds.
reported, in a preliminary communication, the first total
synthesis of shishijimicin A.7c In this article we describe (a) an
improved version of this synthesis, (b) its application to the
synthesis of a series of designed shishijimicin A analogues (7−
16, Figure 2), (c) a number of methodological advances
regarding the preparation of the sulfenylating reagent
PhthNSSMe11 and a number of related sulfenylating reagents,
(d) biological evaluation of the synthesized compounds, and
(e) identification of a number of structurally simpler analogues,
equally or even more potent than the natural product.
1. INTRODUCTION
Antibody−drug conjugates (ADCs) have become a highly
sought after paradigm for targeted, personalized cancer
therapies.1 The clinically successful Mylotarg,2 Adcetris,3 and
Kadcyla4 gave momentum to this approach of chemotherapy
that currently accounts for tens of clinical candidates in
development.5 An essential part of ADCs is the payload, a
highly potent cytotoxic agent attached onto the antibody (the
delivery system) of the conjugate through a chemical linker.6
Natural products endowed with highly potent antitumor
properties or their analogues provide a useful pool of
compounds from which suitable payloads could be selected,
as demonstrated with the three clinically used ADCs
mentioned above and the several others currently in clinical
trials. Shishijimicin A7 (1, Figure 1) is the most potent
2. RESULTS AND DISCUSSION
2.1. Optimization of the Original Synthetic Strategy
for the Total Synthesis of Shishijimicin A. In order to
improve the efficiency and practicality of our original synthesis
of shishijimicin A and its application to analogue construction,
we undertook studies directed toward improvement of a
number of steps and modification of certain intermediates and
key building blocks along the way. Our first task became the
improvement of the synthesis of the enediyne fragment of
shishijimicin A (1), a domain common to a number of other
enediyne antitumor antibiotic discovered thus far (e.g., IC50
=
0.48 pM against P388 leukemia cells). Shishijimicins B (2)7a
and C (3),7a namenamicin (4),8 calicheamicin γI1 (5),9 and
esperamicin A1 (6)10 (Figure 1) are its close relatives. By virtue
of these properties and its rarity,7a shishijimicin A became an
attractive target for total synthesis. The latter would not only
serve to render the natural product available for further
biological investigations but also provide an entry to designed
analogues of the molecule for the same purposes. In 2015, we
Received: July 2, 2018
© XXXX American Chemical Society
A
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Table 1. Optimization of Intramolecular [3 + 2]
Cycloaddition
Figure 1. Representative 10-membered ring enediyne natural
products: shishijimicins A−C (1−3), namenamicin (4), calicheamicin
γI1 (5), and esperamicin A1 (6).
a
b
Original conditions as reported in ref 13. A 6.15 wt% aqueous
c
prominent enediyne antitumor antibiotics, including name-
namicin (4),8 calicheamicin γ1I (5),9 and esperamicin A1 (6).10
As shown in Table 1, we started with the intramolecular [3 +
2] dipolar cycloaddition of nitrile oxide 18, derived from
substrate 17,7c,9b aiming at the optimization of the yield and
selectivity for the desired product 19. The yield of this reaction
for 19 stood, at that time, at 51% with the desired product
accompanied by its diastereoisomer 20 (14% yield) and
fragmentation side-product 23 (20% yield) (Table 1, entry
1).12 Changing the solvent from CH2Cl2 to CHCl3 did not
significantly alter the outcome of this reaction (Table 1, entry
2). Our speculative mechanism, depicted in Table 1 (18 → 21
solution of NaClO (2.0 equiv) was used. The reaction was performed
by adding dropwise a benzene solution of 17 to t-BuOCl in benzene.
→ 22 → 23),12 proved inspirational and crucial in guiding us
to define better reaction conditions that improved the outcome
of this reaction to 91% yield for desired product 19,
contaminated with only small amounts of undesired stereo-
isomer 20 (4% yield) and side-product 23 (<1% yield) (Table
1, entry 7). Thus, reasoning that the fragmentation of initially
formed nitrile oxide intermediate 18 to ONC− and oxonium
species 21 is reversible, we hypothesized that formation of
Figure 2. Synthesized shishijimicin A analogues 7−16.
B
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side-product 23 via species 22 could be suppressed or
eliminated by changing the oxidant from NaClO (which
requires aqueous media) to t-BuOCl (which does not require
aqueous media) thereby eliminating H2O, the culprit for the
fragmentation reaction. Entries 3−7 (Table 1) show the results
of this change under a variety of conditions, with entry 7
depicting the optimal protocol. The final yield improvement of
this reaction was achieved through slow addition of the
substrate (17) to a benzene solution of the oxidant (t-BuOCl)
at the lowest possible temperature given the melting point of
the solvent (5 °C).
Having significantly improved the [3 + 2] cycloaddition
reaction, we then turned our attention to the installment of the
enediyne moiety into the growing molecule, a process that we
felt could benefit from improvement of its original
version.7c,12,13 Our initial aim was to convert both isoxazoline
diastereomers 19 and 20 (Table 1) into useful advanced
intermediates for further elaboration. Scheme 1A summarizes
addition of the acetylide unit occurred with exclusive
diastereoselectivity. The one-step introduction of the enediyne
system into the emerging molecule (as compared to the
stepwise original approach)12,13 represented a further signifi-
cant improvement in the synthesis of the targeted enediyne
domain. In an attempt to explore the possibility of trans-
forming the other diastereoisomer obtained from the [3 + 2]
cycloaddition reaction (i.e., 20 in Scheme 1B), we exposed the
latter to the same sequence of reactions as shown in Scheme
1A,B. The results included an even higher overall yield for the
final product (28 via 27, 93% overall yield from 20, Scheme
1B), which in contrast to the original approach,12 was formed
exclusively. Unfortunately, however, the configuration of the
newly generated stereogenic center [see red arrows on
structures 26 and 28 (Scheme 1A,B, respectively)] was proven
to be of the opposite configuration to that obtained from
isomer 19. This assertion was based on a NOESY experiment
(see the Supporting Information). Note that these inter-
mediates (i.e., 26 and 28) lose their other two stereocenters
downstream in the pending sequence, leaving only the
enediyne bearing center as the important one with regard to
these intermediates (i.e., 26a and 28a, respectively, as shown in
Scheme 1C). In that sense, while precursor intermediate 26 is
destined for the target molecule, its isomeric precursor 28 is
not. It could, however, serve as a useful precursor for the
antipodal molecule of the natural product, if one wishes to
synthesize enantiomeric shishijimicin A. This task would, of
course, require inversion of all the other eight stereogenic
centers of the aryl disaccharide fragment. However, the
corresponding (1R) diastereomer of shishijimicin A could be
derived from 28 simply by employing the same building blocks
as those used to construct the natural product, provided all
pending reactions and procedures proceed as those destined
for shishijimicin A. An explanation of the exclusive
diastereospecificities of these two enediyne addition reactions
is provided in Scheme 1D which shows the preferred
conformations of the two intermediates (24 and 27) based
on steric considerations and the allowable trajectories of attack
on the carbonyl moieties by the enediyne nucleophile. Manual
molecular models of intermediates 24 and 27 indicate that the
H atom attached to the angular C atom and one of the OCH2
structural motifs of the ketal should be in axial positions,
thereby blocking the enediyne approach from the same side
due to 1,3-diaxial interaction. The isoxazoline moieties lie in
equatorial positions exerting minimal steric bias, and thus
allowing the enediyne to approach from the less hindered face
of the carbonyl group (see Scheme 1D).
Scheme 1. Stereospecific Addition of Enediyne 25 to
a
Ketones 24 and 27 to Form Adducts 26 and 28
a
Reagents and conditions: (a) NaOMe (0.1 equiv), MeOH, 0 °C, 12
h, quant.; (b) Jones reagent (1.5 equiv), acetone, 0 °C, 2 h; (c) 25
(3.0 equiv), LiHMDS (2.8 equiv), LaCl3·2LiCl (5.0 equiv), THF,
−78 °C, 0.5 h; then 24, −78 °C, 0.5 h; then Ac2O (10.0 equiv), −78
to 25 °C, 2 h, 90% for the two steps; (d) NaOMe (0.2 equiv), MeOH,
0 °C, 12 h, quant.; (e) Jones reagent (1.5 equiv), acetone, 0 °C, 40
min; (f) 25 (4.0 equiv), LiHMDS (3.0 equiv), LaCl3·2LiCl (5.0
equiv), THF, −78 °C, 0.5 h; then 27, −78 °C, 0.5 h; then Ac2O (10.0
equiv), −78 to 25 °C, 1 h, 93% for the two steps.
Having successfully improved the installment of the
enediyne structural motif into the growing molecule, we
turned our attention to optimizing the intramolecular ring
closure of the acetylenic aldehyde as the next desired stage (see
Scheme 2 and Tables 2 and 3). Beginning with isoxazole 29
[synthesized from 26 through a high-yielding 6-step sequence
(77% overall yield, Scheme 2) as reported in 2015],7c we first
sought for a more practical procedure for the reduction of the
isoxazole structural motif within 29 (Scheme 2). Reductive
rupture of the isoxazole ring embedded within 29 could be
achieved more conveniently than before12,13 through the
addition of Fe powder in a solution of this substrate in a
mixture of EtOH and aqueous NH4Cl.7c,15,16 Although these
conditions delivered required amino aldehyde 30 in a single
step and high yield (83%) on a 100 mg scale, they proved
capricious and difficult to reproduce,17 especially on a larger
our studies toward this goal. Thus, debenzoylation of 19
(NaOMe, MeOH, quantitative yield) followed by Jones
oxidation of the resulting alcohol furnished ketone 24, whose
reaction with the organometallic species generated from
enediyne 25 and LaCl3·2LiCl,7c,14 followed by in situ
acetylation (Ac2O) of the resulting tertiary alcohol, led to
desired acetoxy enediyne 26, in 90% overall yield from 19.
Pleasantly, and as proven by NMR spectroscopic analysis, the
C
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a
Scheme 2. Synthesis of Cyclized Enediyne Lactone 33
Table 3. Optimization of Enediyne Cycloaddition Reaction
within Intermediate 34
a
b
Original conditions as reported in ref 13. Conditions as reported in
a
ref 7c.
Reagents and conditions: (a) Fe (25 equiv), NH4Cl (50 equiv),
EtOH/H2O (1:1, v/v), 60 °C, 8 h, 83%; (b) TMSOTf (1.5 equiv),
Et3N (2.0 equiv), Zn(OTf)2 (0.025 equiv), CH2Cl2, −20 °C, 10 min,
then 29, −20 to 25 °C, 12 h; (c) Mo(CO)6 (1.0 equiv), MeCN/H2O
(5:1, v/v), 80 °C, 1.5 h; (d) K2CO3 (1.0 equiv), MeOH/THF (2:1,
v/v), 0 °C, 2 h, 53% for the three steps; (e) ClCO2Me (10.0 equiv),
Et3N (20 equiv), CH2Cl2, 25 °C, 0.5 h, 53%; (f) LiHMDS (3.0
equiv), LaCl3·2LiCl (5.0 equiv), THF, −20 °C, 5 min, 88%; (g) MsCl
(5.0 equiv), pyridine (10.0 equiv), CH2Cl2, 0 °C, 15 min; (h) SiO2,
CH2Cl2, 25 °C, 2 h; (i) PhthCl (1.5 equiv), pyridine (4.0 equiv),
MeNO2, 0 °C, 0.5 h; (j) SiO2, CH2Cl2, 25 °C, 2 h; (k) Ac2O (excess),
25 °C, 1 h, 81% for the three steps; (l) LiHMDS (3.0 equiv), LaCl3·
2LiCl (2.0 equiv), THF, −78 °C, 1 h, 85%; (m) MeNHNH2 (10.0
equiv), PhH, 25 °C, 0.5 h; then triphosgene (3.0 equiv), pyridine (30
equiv), CH2Cl2, 0 °C, 1 h, MeOH, 0 °C, 1 h, 81% for the two steps.
MeOH), proved to be reliable and efficient, securing
multigram quantities of vinylogous formamide 30 in good
overall yield (53%) from isoxazole 29 as shown in Scheme 2.
We then focused our efforts on developing a more efficient
transformation of 30 to 33 (Scheme 2) which previously
required six steps and proceeded in 28% overall yield.13 Our
first attempt sought to circumvent the intermediacy of the
phthalimide intermediate and required direct conversion of
amino compound 30 to methyl carbamate 31, an operation
that proceeded in 53% yield, upon exposure of the former to
ClCO2Me and Et3N. Unfortunately, subsequent efforts to
convert intermediate 31 directly to the desired cyclization
product 33 under various basic conditions (see Table 2) were
met with failure, primarily due to the tendency of the initially
formed hydroxy intermediate 32 to undergo cyclization to the
6-membered ring carbamate 36 under the strong basic
conditions employed (Table 2, entries 1−3). In the presence
of a Lewis acid (e.g., LiHMDS, CeCl3 or LiHMDS, LaCl3·
2LiCl), however, β-hydroxy methyl ester 32 could be isolated
in 78% (Table 2, entry 4) or 88% (Table 2, entry 5) yield,
respectively. To our disappointment, this intermediate failed to
be converted to the targeted product 33 through a two-step
sequence [(i) MsCl, py.; (ii) SiO2] reported for the inversion
of the β-hydroxyl group on a similar system.12,13 Instead, a
rapid generation of cyclic carbamate 36 was observed under
the indicated reaction conditions, presumably due to the
basicity of the pyridine employed.
Table 2. Enediyne Aldehyde Cycloaddition Reaction within
Intermediate 31
Another innovation along the route from 34 to 33 via 35
and 35a (see Scheme 2) was discovered and optimized from a
separate study directed toward a one-step conversion of formyl
phthalimide 34 to intermediate lactone 35 as shown in Table
3. Thus, while the use of KHMDS or LiHMDS as a base for
the cyclization of acetylenic aldehyde 34 resulted in dominant
formation of β-hydroxy phthalimide 37 through, anionic
species 34b (Table 3, entries 1−3), the employment of a
Lewis acid (e.g., CeCl3 or LaCl3·2LiCl) as an additive favored
the formation of desired lactone 35 (Table 3, entries 4 and 5)
scale. Employment of Mo(CO)6 as the reducing agent18 on the
free terminal alkyne substrate 29 led to substantial side-
product formation, making this substrate unsuitable for these
conditions. Eventually, a sequence involving trimethylsilylation
[TMSOTf, Et3N, Zn(OTf)2] of offending terminal alkyne of
29,19 followed by reductive cleavage of the isoxazole N−O
bond [Mo(CO)6]12,13,18 and TMS group removal (K2CO3,
D
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with LiHMDS, LaCl3·2LiCl furnishing the highest yield (i.e.,
85%). Accomplishing direct conversion of 34 to targeted
intermediate 35 (see Scheme 2) via intermediate species 34a
and 34c (see Table 3), this new sequence shortens the route
from 30 to 33 (see Scheme 2) from eight to six steps and
improves its overall yield from 28 to 56%. Notably, the overall
number of steps starting from oxime 17 (Table 1) to advanced
intermediate 33 (see Scheme 2) has been shortened from 20
to 19 steps, while the overall yield was significantly improved
from 1.8%, from the original approach reported in 1992,12,13 to
16%.
With multigram quantities of key enediyne building block 33
readily available, we turned our attention to the preparation of
differently substituted enediyne fragments so as to gain
flexibility in the ensuing coupling reactions toward shishijimi-
cin A and other naturally occurring enediyne antitumor
antibiotics carrying the same enediyne “warhead” [e.g.,
namenamicin (4),8 calicheamicin γ1I (5),9 and esperamicin A1
(6),10 and their designed analogues]. As shown in Scheme 3,
Finally, precursor 40 was diverted to thioacetate fragment 41
through selective desilylation of the secondary TMS ether
(HF·py, 99% yield), and to methyl trisulfide fragment 42
through a sequence involving cleavage of the thioacetate
moiety (DIBAL-H; then MeOH), methyl trisulfide formation
(PhthNSSMe),11 and desilylation (HF·py) of the remaining
secondary and tertiary silyl ethers, in 89% overall yield as
summarized in Scheme 3. The availabilities of the last two
more advanced intermediates (i.e., 41 and 42) would allow us
to test new protocols for the final and challenging coupling of
the glycosyl donor and acceptor as we shall discuss below.
With practical and efficient processes for the synthesis of
glycosyl acceptors 39, 41, and 42 in hand, we turned our
attention to optimizing the construction of the β-carboline-
disaccharide fragment (i.e., 53, see Scheme 4) of shishijimicin
Scheme 4. Construction of β-Carboline Disaccharide 53 via
a
Pictet−Spengler Condensation
Scheme 3. Syntheses of Enediyne Glycosyl Acceptors 39, 41,
a
and 42
a
Reagents and conditions: (a) DIBAL-H (3.0 equiv), CH2Cl2, −78
°C, 0.5 h, 95%; (b) NaBH4 (excess), MeOH, 0 °C, 1 h, 88%; (c)
NaBH4 (2.0 equiv), CeCl3·7H2O (3.0 equiv), MeOH, 25 °C, 1 h,
97%; (d) BzCl (2.0 equiv), pyridine (3.0 equiv), CH2Cl2, −15 °C, 1
h, 84%; (e) TMSCN (excess), 25 °C, 0.5 h; then AcOH (5.0 equiv),
THF/H2O (5:1, v/v), 0 °C, 0.5 h; (f) PPh3 (5.0 equiv), DEAD (5.0
equiv), AcSH (5.0 equiv), THF, 0 °C, 5 min, 96% for the two steps;
(g) HF·py/THF (1:20, v/v), 0 °C, 0.5 h, 99%; (h) DIBAL-H (3.0
equiv), −78 °C, 0.5 h; then MeOH, −78 °C, 20 min; then
PhthNSSMe (4.0 equiv), −78 to 25 °C, 1 h; (i) HF·py/THF (1:20,
v/v), 0 to 25 °C, 1.5 h, 89% for the two steps.
a
Reagents and conditions: (a) 52 (3.0 equiv), t-BuLi (6.0 equiv),
THF, −78 °C, 0.5 h; then 51 (1.0 equiv), −78 to −35 °C, 40 min,
86% (ca. 1:1 dr); (b) NaOH (3.0 equiv), EtOH, 0 to 25 °C, 2.5 h; (c)
DMP (1.1 equiv), CHCl3, 0 to 35 °C, 10 min, 68% for the two steps;
(d) TMSCN (3.0 equiv), SnCl4 (1.5 equiv), CH2Cl2, −78 to 0 °C, 0.5
h; then TMSOTf (2.0 equiv), 2,6-lutidine (3.0 equiv), 0 to 25 °C, 2 h;
(e) DIBAL-H (3.0 equiv), CH2Cl2, −78 °C, 1.5 h, 76% for the two
steps; (f) HF·py/THF (1:20, v/v), 0 to 25 °C, 2 h; (g) DMP (1.5
equiv), CH2Cl2, 25 °C, 0.5 h, 63% for the two steps; (h) 57 (4.0
equiv), AcOH, 60 °C, 1.5 h; then O2, 1 h, 51% (i) TBSOTf (1.1
equiv), Et3N (2.0 equiv), CH2Cl2, 0 °C, 5 min, quant.
we first targeted previously synthesized allylic benzoate 39,9b
and new allylic thioacetate 41,7c and previously known allylic
methyl trisulfide 42.9c The previously reported two-step
reduction of lactone 33 (DIBAL-H; NaBH4, 84% overall
yield)13 was successfully replaced with the one-step Luche
reduction (NaBH4, CeCl3·7H2O) that proceeded in superior
yield (97%) to afford diol 38.7c,20 The latter compound served
as a common precursor to all three enediyne fragments shown
in Scheme 3 (i.e., 39, 41, and 42). Thus, selective benzoylation
of 38 (BzCl, py., 84%) yielded the previously synthesized
enediyne glycosyl acceptor 39,9b whereas sequential exposure
of 38 to TMSCN, AcOH, and then PPh3, DEAD, and AcSH
led to fully and orthogonally protected precursor 40, in 96%
overall yield for the three steps as shown in Scheme 3.7c
A (1). Our optimization studies began with reinvestigation of
the conversion of glycal 43 to glycoside 45b (β-anomer, Table
4), via epoxide 44 (Table 4), needed to build disaccharide 51
(see Table 5). As reported in our preliminary communica-
tion,7c the preparation of 45b from glycal 43 via epoxide 44
called upon the original Danishefsky conditions21 that
employed ZnCl2 as the Lewis acid to activate the epoxide
moiety (Table 4, entry 1).7c In that and the other experiments
reported herein and summarized in Table 4, glycal 43 was
exposed to in situ generated dimethyl dioxirane (DMDO)
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yield), 45a (11% yield)],7c and in order to make the reaction
more stereoselective and improve its yield, we focused on
varying the Lewis acid promoter, the solvent (Table 4), and in
one case the temperature (Table 4, entry 4). The improvement
of diastereoselectivity was important for carrying out
subsequent steps with homogeneous material, rather than
anomeric mixtures. We first examined Lewis acids with
reasonable solubilities in THF such as those shown in entries
2−5 (i.e., ZnBr2, BF3·Et2O, AlCl3, and InCl3, Table 4). Thus,
switching the promoter from ZnCl2 (Table 4, entry 1) to
Table 4. Lewis Acid Optimization of Glycosylation of 1,2-
Anhydro-6-deoxy-glucose 44
a
22
ZnBr2 (Table 4, entry 2) resulted in higher selectivity (β-
anomer: 70% yield, α-anomer: 5% yield) and better yield (75%
combined yield vs 65% combined yield for ZnCl2, Table 4,
entry 1). While the stronger Lewis acids BF3·Et2O23 (Table 4,
entry 3) and AlCl324 (Table 4, entry 4) gave lower selectivities
than ZnCl2 (Table 4, entry 1), they proved more efficient in
terms of combined yield, namely, 75 and 85%, respectively. We
then considered InCl3,25 expecting that indium’s larger than
zinc’s and aluminum’s ionic radius would influence the
anomeric selectivity of the glycosylation reaction with o-
NBOH. As shown in Table 4 (entry 5), the InCl3-facilitated
glycosylation of 44 with o-NBOH proceeded in high yield
(81%) and excellent anomeric selectivity for the β-anomer
(<1% of the α-anomer). The gold catalysts Ph3PAuOTf26 and
Ph3PAuNTf227 were also tested28 and interestingly were found
to perform well in terms of combined yields [entries 6 (74%)
and 7 (92%), respectively] but failed in terms of anomeric
selectivity, with Ph3PAuNTf2 reversing the anomeric ratio in
favor of the undesired α-anomer while outperforming all the
promoters and catalysts tested in terms of combined yield
(92%, Table 4, entry 7).
a
Reaction conditions: (a) oxone (5.0 equiv), acetone (8.0 equiv),
NaHCO3 (25 equiv), H2O/CH2Cl2 (3:4, v/v), 0 °C, 4 h, quant.; (b)
44 (0.5 mmol), o-nitrobenzyl alcohol (o-NBOH, 1.0 mmol), Lewis
acid (0.75 mmol), 4 Å MS (1.7 g), solvent (3.0 mL), unless otherwise
noted. Isolated yields of indicated anomers. Conditions as reported
in ref 7c. A 0.2 equiv amount of Lewis acid was added.
b
c
d
a
Table 5. Glycosylation of Alcohol 46 with Donors 47−49
With an improved and scalable synthesis of glycoside 45b
developed (see Table 4), we then proceeded to its conversion
to methylthio cyanide 46, a task accomplished through a high-
yielding six-step sequence as we previously described7c (40.3%
overall yield) as summarized in Table 5. The next challenge
was the glycosylation of this, rather complex, glycosyl acceptor
(i.e., 46) with its Alloc-protected aminosugar partner in the
form of glycosyl donors 47,7b 48,29 or 4930 (Table 5) with the
desired goal of selectively obtaining α-glycoside 50a31 (Table
5) as needed for constructing shishijimicin A. To this end,
glycosyl donors 47−49 (for preparations, see the Supporting
Information and refs 7b, 29, and 30) were reacted with 2-
hydroxy glycosyl acceptor 46 under a variety of conditions as
shown in Table 5. Thus, 46 and glycosyl fluoride 47 were
allowed to react in THF at −78 to 25 °C in the presence of
AgClO4 and SnCl2,32 furnishing desired α-glycoside 50a in
85% yield, together with only 9% of the β-glycoside (50b), the
two anomers being separated chromatographically (Table 5,
entry 1).7c The desired α-anomer was then converted to
disaccharide aldehyde 51 (DIBAL-H, 87% yield). As seen in
Table 5, adoption of glycosyl donors 48 and 49 and of
appropriate conditions for their coupling with glycosyl
acceptor 46, even though leading to good to excellent
combined yields of the α,β mixture of disaccharide anomers
(50a/b, Table 5, entries 2−5), failed to improve the α-
glycoside selectivity beyond that observed with glycosyl
fluoride 47 under the AgClO4−SnCl2 activation conditions
(Table 5, entry 1). Interestingly, replacing SnCl2 with
a
Glycosyl acceptor 46 (0.1 mmol, 1.0 equiv) was used as a limiting
b
c
reagent for each entry. Isolated yield based on 46. Conditions as
reported in ref 7c. DIBAL-H (3.0 equiv), CH2Cl2, −78 °C, 45 min,
87%.
d
(acetone, oxone, aqueous NaHCO3, CH2Cl2, 0 °C, quant., ca.
16:1 dr), and the resulting epoxide (44) was used crude
without purification. Given that our original conditions with
ZnCl2 as a Lewis acid promoter (Table 4, entry 1) led to a
mixture of anomers of the glycosylated products [45b (54%
33
Cp2HfCl2 (Table 5, entry 2) as a partner to AgClO4 in
this glycosylation reaction not only resulted in a similarly
impressive combined yield of the α,β-disaccharide (mixture
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50a/50b) but also in reversal of the α,β-anomeric selectivity
(α/β ca. 34:62, Table 5, entry 2).
Scheme 5. Syntheses of Disaccharide Trichloroacetimidates
60, 63, 64, and 66 and o-Alkynylbenzoate 67 as Glycosyl
a
With an efficient synthesis of disaccharide aldehyde 51
developed, we proceeded with the installation of the β-
carboline moiety onto the growing molecule. Scheme 4
summarizes two approaches through which this objective was
achieved. The first access to targeted disaccharide-carboline
domain 53 proceeded through a three-step sequence involving
lithiation of 52 through iodide-lithium exchange (t-BuLi)
followed by sequential addition of aldehyde 51 and
saponification/decarboxylation (NaOH, EtOH) to afford the
corresponding secondary alcohol, whose oxidation with DMP
yielded ketone product 53, in 58.5% overall yield as previously
communicated7c and summarized in Scheme 4. At this point
we also opted to attempt a presumably biomimetic approach34
to targeted β-carboline fragment 53 starting from disaccharide
aldehyde 51, as shown in Scheme 4 (blue sequence). Thus,
exposure of aldehyde 51 to TMSCN in the presence of SnCl4
followed by addition of TMSOTf/2,6-lutidine furnished
corresponding cyanohydrin TMS-derivative (54) of the
initially formed cyanohydrin. The latter was reduced with
DIBAL-H to give aldehyde 55 (76% yield for the two steps,
mixture of inconsequential diastereoisomers), whose sequential
desilylation (HF·py) and DMP oxidation furnished dicarbonyl
compound 56, in 63% overall yield as shown in Scheme 4. The
latter served as a precursor to carboline disaccharide 59
through a cascade event involving sequential condensation
with serotonin hydrochloride (57, Pictet−Spengler reaction35)
in AcOH (60 °C), followed by spontaneous dehydrogenation/
aromatization, via tetrahydro-β-carboline intermediate 58,
through the action of O2 (51% overall yield).36 The so-
obtained 6″-hydroxy carboline (59) was then silylated
(TBSOTf, Et3N) to afford targeted carboline TBS-ether 53
in quantitative yield as shown in Scheme 4.
In order to explore the attachment of the carboline-
disaccharide domain 53 onto the enediyne core of shishijimicin
A, we synthesized a number of glycosyl donors (i.e., 60, 63, 64,
66, and 67 as depicted in Scheme 5A,B). Thus, photoinduced
(hν) cleavage of the NB group from 53 followed by installation
of the trichloroacetimidate moiety (Cl3CCN, DBU) led to
carbohydrate donor 60 in 79% overall yield as depicted in
Scheme 5A. Donors 63 and 64, in which the Nap group was
replaced with a TMS or an Ac group, were constructed from
53 via intermediates 61 and 62, respectively, as shown in
Scheme 5A. Thus, removal of the Nap protecting group from
53 by treatment with DDQ (86%) followed by silylation
(TMSCl, Et3N) or acetylation (Ac2O, py, DMAP) of the
resulting alcohol substrate furnished 61 (64% yield) or 62
(97% yield), respectively. The latter compounds were
converted to the corresponding trichloroacetimidates 63 and
64 in 77 and 94% yields, respectively, as shown in Scheme 5A.
Trichloroacetimidate 66 lacking the Nap protecting group was
also prepared from 53 as shown in Scheme 5B. Thus,
sequential removal of the NB (hν) and Nap (DDQ) protecting
groups followed by trichloroacetimidate formation (Cl3CCN,
NaH) produced carbohydrate donor 66 in 53% overall yield
for the three steps from 53. For the purposes of employing, in
addition to Schmidt glycosylations, gold catalysis37 in the final
coupling step, hydroxy glycosyl donor 67 was synthesized from
53 through a sequence involving NB removal (hν), Nap
deprotection (DDQ), and esterification with o-alkynylbenzoic
acid 65 (EDCI, i-Pr2NEt, DMAP) in 75% overall yield for the
three steps as shown in Scheme 5B.38 Both 66 and 67 are
Donors
a
Reagents and conditions: (a) hν, THF/H2O (10:1, v/v), 4.5 h; (b)
Cl3CCN/CH2Cl2 (1:10, v/v), DBU (1.0 equiv), 0 °C, 1.5 h, 79% for
the two steps; (c) DDQ (2.0 equiv), CH2Cl2/H2O (10:1, v/v), 25 °C,
4 h, 86%; (d) TMSCl (2.0 equiv), Et3N (3.0 equiv), CH2Cl2, 0 °C, 3
h; or Ac2O (2.0 equiv), pyridine (3.0 equiv), DMAP (0.1 equiv),
CH2Cl2, 25 °C, 7 h, 64% for 61, 97% for 62; (e) hν, THF/H2O (10:1,
v/v), 0 °C, 4.5 h; (f) Cl3CCN/CH2Cl2 (1:10, v/v), DBU (1.0 equiv),
0 °C, 1.5 h, 77% for 63 from 61, 94% for 64 from 62 over the two
steps; (g) hν, THF/H2O (10:1, v/v), 0 °C, 4.5 h; (h) DDQ (2.5
equiv), CH2Cl2/H2O (10:1, v/v), 30 °C, 1.5 h; (i) NaH (2.0 equiv),
Cl3CCN/CH2Cl2 (1:2, v/v), 25 °C, 5 min; or 65 (2.0 equiv), EDCI
(2.0 equiv), i-Pr2NEt (2.0 equiv), DMAP (1.0 equiv), CH2Cl2, 25 °C,
24 h, 53% for 66, 75% for 67 over the three steps.
lacking the Nap protecting group, for the latter bulky moiety
was shown to exert an inhibiting role in the pending
glycosylation reaction. Incidentally, as we will see below, it
was for the same reason that the Nap group was exchanged for
the TMS and Ac groups in carbohydrate donors 63 and 64,
respectively, as mentioned above.
With both the enediyne fragments (39, 41, and 42, Scheme
3) and carboline-disaccharide donors (60, 63, 64, 66, and 67,
Scheme 5) readily available, we were in a position to address
the glycosylation reaction that would join them together for
the final drive toward shishijimicin A (1). This objective
proved challenging, as we soon realized. Table 6 summarizes
some of our attempts to accomplish this goal.39 Reaction of
hydroxy enediyne fragment 39 with carbohydrate donor 60
under BF3·Et2O (3.5 equiv) conditions9b failed to produce any
of the desired coupling product (Table 6, entry 1). Reasoning
that the bulky Nap group was responsible for the intransigence
of this substrate, we prepared (as shown in Scheme 5A) and
employed trimethylsilyl (TMS) and acetyl (Ac) counterparts
of 60, namely, donors 63 and 64. Much to our disappointment,
and just like their precursor 60, these intermediates resisted
coupling under the same conditions as those used with 60,
with the carbohydrate acceptor (i.e., enediyne 39) being
recovered unchanged (Table 6, entries 2 and 3). We then
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Table 6. Glycosylation of Disaccharide Donors 60, 63, 64, 66, and 67 with Enediyne Acceptors 39, 41, and 43
a
b
c
Glycosyl donor (1.0 equiv) was used as a limiting reagent for each entry. In each reaction, 4 Å molecular sieves were used. Isolated yield of
d
e
indicated product. No desired product observed; glycosyl acceptor recovered. No desired product observed; glycosyl acceptor decomposed.
f
Conditions as reported in ref 7c.
decided to use glycosyl donor 66, which carries no protecting
group on its C3-hydroxyl group. As seen from entry 4 (Table
6), the desired product (68) was obtained, but only in low
yield (15%). Employing TMSOTf as a Lewis acid promoter40
in this glycosylation reaction (Table 6, entry 5) furnished
product 68 in an even lower yield than the previous
experiment with BF3·Et2O, due to a rapid formation of the
TMS-ether of carbohydrate acceptor 39,41 forcing us to switch
back to BF3·Et2O as the preferred promoter. This time, we
used thioacetate enediyne fragment 41 as the glycosyl acceptor
with hydroxy trichloroacetimidate donor 66 and obtained an
improved yield (26%, Table 6, entry 6).7c The employment of
methyltrisulfide glycosyl acceptor 429c with donor 66 under
the same conditions led to no product however, with the
glycosyl acceptor (42) decomposing under the conditions of
the reaction (Table 6, entry 7). Exploring the possibilities of
success under gold-promoted conditions using coupling
partners 67 and 41 and Ph3PAuOTf26 (Table 6, entry 8)
the glycosylation reaction of the glycosyl donor derived from
88 (Scheme 9) lacking the MeS group afforded desired β-
glycoside 89 in 39% yield. Similarly, β-glycoside 91 (Scheme
10) was obtained in 40% yield from the glycosyl donor
(lacking the axial MeS group) derived from 85. It is possible
that deactivation of the Lewis acid (BF3·Et2O) through
complexation with the MeS group may also contribute to the
failure of the trichloroacetimidate carbohydrate donors 60, 63,
64, and 66 (Table 6) to perform well in the respective
glycosylation reactions.
The remaining steps of the total synthesis of shishijimicin A
proceeded well as seen in Scheme 6. Thus, advanced
thioacetate derivative 69 was converted to its methyltrisulfide
counterpart 70 through a two-step, one-pot procedure
involving cleavage of the acetate group (NaSMe, MeOH)42
followed by addition of AcOH (neutralization), and reaction of
the resulting thiol with PhthNSSMe11 in 60−65% yield. Global
desilylation of the latter with HF·py led to bis-protected
precursor 71 in 80% yield. Finally, sequential removal of the
Alloc [Pd(PPh3)4 cat., morpholine] and ketal (p-TsOH)
protecting groups liberated shishijimicin A (1) in 75−80%
yield as shown in Scheme 6. Synthetic shishijimicin A exhibited
27
and Ph3PAuNTf2 (Table 6, entry 9) did not prove fruitful
either, with the latter catalyst furnishing <5% of the desired
product (69), while the former catalyst led to no product,
presumably due to complexation of Au(I) to the pyridine
moiety of the β-carboline structural motif, thereby, deactivating
the disaccharide donor and thus inhibiting the coupling
reaction. There is certainly room for improvement in this
glycosylation reaction, which is made so intransigent, no doubt
by the complexity of the partners involved and their unusual
structural motifs. In retrospect, we realized that the MeS group
on carbohydrate donors 60, 63, 64, 66, and 67 (Table 6)
resided most likely in an axial position hindering the formation
of the desired β-glycoside (formed in yields of ≤ 26%), a
speculation supported by subsequent experiments in our
syntheses of shishijimicin A analogues (section 2.2). Thus,
1
highly similar H and 13C NMR spectroscopic and optical
rotation data to those reported for the natural product, as
described in our previous communication.7c Additionally, the
two rather weak carbon signals (attributed to carbons 8 and 9,
see structure 1, Scheme 6, for numbering) that were barely
detectable in our previous work (see the Supporting
Information)7c,43 have now been unambiguously assigned by
HSQC and HMBC experiments (see Figure 3 and the
Supporting Information). Specifically, carbon 8 (C8) was
assigned to the now visible chemical shift at δ = 70.7 ppm
based on an HSQC experiment revealing a cross peak at (6.34,
H
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Scheme 6. Completion of Total Synthesis of Shishijimicin
a
A
a
Reagents and conditions: (a) NaSMe (15 equiv), MeOH, 0 °C, 1.5
h; then AcOH (15 equiv), 0 °C, 1 min; then PhthNSSMe (8.0 equiv),
0 to 25 °C, 0.5 h, 60−65%; (b) HF·py/THF (1:20, v/v), 25 °C, 12 h,
80%; (c) Pd(PPh3)4 (0.5 equiv), morpholine (10.0 equiv), THF, 0
°C, 2 h; (d) p-TsOH (5.0 equiv), THF/H2O/acetone (20:1:20, v/v/
v), 25 °C, 48 h, 75−80% for the two steps.
1
70.66 ppm) with respect to the JCH correlation between H8
and C8 (Figure 3A), and an HMBC experiment revealing a
3
cross peak at (4.95 and 70.66 ppm) with respect to the JCH
correlation between H1′ and C8 (Figure 3B). Carbon 9 (C9)
was unequivocally assigned the chemical shift at δ = 149.4 ppm
based on an HMBC experiment revealing a cross peak at (6.53
3
ppm, 149.35 ppm) with respect to the JCH correlation
between H14 and C9 (Figure 3C). With these NMR
assignments, all the NMR spectroscopic data of shishijimicin
1
A (1) and its H and 13C assignments are in good agreement
with those reported by the Fusetani group7a (see the
Supporting Information).
2.2. Design and Synthesis of Shishijimicin A
Analogues. The developed synthetic strategies and tech-
nologies were applied to the synthesis of designed shishijimicin
A analogues, aiming primarily to structure simplification and
potency sustainment or enhancement. Our first targets became
the thioacetate and methyldisulfide counterparts of shishijimi-
cin A, namely, analogues 7 and 8, respectively (see Scheme
7A). Thioacetate analogue 7 was inspired by calicheamicin θI1,
a synthetic analogue of calicheamicin γI1 that we prepared and
studied in the 1990s.44 Calicheamicin θ1I was proven to be
more potent than its parent, calicheamicin γI1, against certain
cancer cell lines,44,45 and more importantly, served as the
payload of one of the earliest antibody−drug conjugates
(ADCs) exhibiting effective suppression of growth and
dissemination of hepatic metastases of neuroblastoma in a
syngeneic mouse model.46 Shishijimicin A analogue 7 was
synthesized from advanced intermediate 69 (for preparation,
see Table 6, entry 6) as summarized in Scheme 7A. Thus,
cleavage of both silyl protecting groups from 69 (HF·py)
followed by sequential removal of the Alloc [Pd(PPh3)4 cat.,
Figure 3. (A) Assignment of 13C signal of C8 of the synthetic
shishijimicin A by HSQC NMR spectroscopy: δC(C8) = 70.67 ppm;
(B) assignment of 13C signal of C8 by HMBC NMR spectroscopy:
δC(C8) = 70.66 ppm; (C) assignment of 13C signal of C9 by HMBC
NMR spectroscopy: δC(C9) = 149.35 ppm.
morpholine] and ketal (p-TsOH) protecting groups furnished
7 in 56% overall yield.
Methyldisulfide shishijimicin A analogue 8 was inspired by
previous experimental47 and computational48 studies support-
ing the intermediacy of a calicheamicin γI1-glutathione disulfide
conjugate as a major precursor to the crucial dihydrothiophene
intermediate formed prior to the Bergman reaction,49 the latter
being responsible for the formation of DNA-cleaving
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Our attempt to directly form the N-acetyl shishijimicin A
(9) under the reported conditions for preparing N-acetyl
calicheamicin γ1I (3 vol% Ac2O in MeOH)51,52 was met with
failure as depicted in Scheme 7B, presumably due to the rather
hindered nature of the isopropyl amine structural motif and the
sensitivity of some of the various functionalities within
shishijimicin A. Coveted acetamide shishijimicin A analogue
9 was successfully prepared from N-acetyl disaccharide 53a
(generated from 53 by a two-step sequence) in seven steps by
following the described procedures for the conversion of
disaccharide 53 to shishijimicin A (1), in 1.1% overall yield
(unoptimized), as shown in Scheme 7B.
Scheme 7. Syntheses of Thioacetate Analogue 7, Disulfide
Analogue 8, and N-Acetyl Analogue 9
a
The truncated shishijimicin A analogue 10, which includes
in its structure a methyl group in the place of the aminosugar
(see Scheme 8), was designed in order to test the role of the
Scheme 8. Synthesis of Aminosugar Truncated Analogue
a
10
a
Reagents and conditions: (a) HF·py/THF (1:20, v/v), 25 °C, 12 h;
a
Reagents and conditions: (a) NaH (2.0 equiv), MeI (3.0 equiv),
(b) Pd(PPh3)4 (0.5 equiv), morpholine (10.0 equiv), THF, 0 °C, 2 h;
(c) p-TsOH (5.0 equiv), THF/H2O/acetone (20:1:20, v/v/v), 25 °C,
48 h, 56% for the three steps; (d) NaSMe (10.0 equiv), MeOH, 0 °C,
20 min; then AcOH (10.0 equiv), 0 °C, 5 min; then PhthNSMe (5.0
equiv), 0 to 25 °C, 0.5 h, 75%; (e) HF·py/THF (1:20, v/v), 25 °C, 12
h; (f) Pd(PPh3)4 (0.5 equiv), morpholine (10.0 equiv), THF, 0 °C, 2
h; (g) p-TsOH (5.0 equiv), THF/H2O/acetone (20:1:20, v/v/v), 25
°C, 48 h, 43% for the three steps; (h) Pd(PPh3)4 (0.5 equiv),
morpholine (10.0 equiv), THF, 60 °C, 40 min; (i) Ac2O (5.0 equiv),
pyridine (10.0 equiv), DMAP (1.0 equiv), CH2Cl2, 40 °C, 24 h, 76%
for the two steps; (j) Ac2O (3 vol% in MeOH, v/v), 25 °C, 7 d.
DMF, 0 °C, 0.5 h, 82%; (b) DIBAL-H (1.5 equiv), toluene, −78 °C,
10 min, 79%; (c) 52 (3.0 equiv), t-BuLi (6.0 equiv), THF, −78 °C,
0.5 h; then 74 (1.0 equiv), −78 °C, 10 min, 83% (ca. 1:1 dr); (d)
NaOH (3.0 equiv), EtOH, 0 to 25 °C, 2 h; (e) DMP (2.0 equiv),
CHCl3, 25 °C, 2 h, 63% for the two steps; (f) hν, THF/H2O (10:1, v/
v), 0 °C, 4.5 h; (g) DDQ (2.5 equiv), CH2Cl2/H2O (10:1, v/ v), 30
°C, 1.5 h; (h) NaH (2.0 equiv), Cl3CCN/CH2Cl2 (1:2, v/v), 25 °C, 5
min; (i) 41 (2.0 equiv), BF3·Et2O (3.5 equiv), 4 Å MS, CH2Cl2, −78
to −40 °C, 1 h, 5.6% for the four steps; (j) LiOH·H2O (100 equiv),
MeOH, −17 to −10 °C, 20 min; then AcOH (100 equiv), 5 min;
then PhthNSSMe (8.0 equiv), −10 to 25 °C, 0.5 h; (k) HF·py/THF
(1:20, v/v), 25 °C, 12 h; (l) p-TsOH (5.0 equiv), THF/H2O/acetone
(20:1:20, v/v/ v), 25 °C, 48 h, 42% for the three steps.
benzenoid diradical species. The synthesis of disulfide analogue
8 was accomplished from the same advanced intermediate
thioacetate 69 (see Scheme 7A) through a three-step, one-pot
cascade reaction sequence initiated by excess NaSMe (for
deacetylation), followed by addition, first of AcOH (for
neutralization) and then of PhthNSMe,50 to afford triprotected
methyl disulfide 72. A three-step deprotection sequence [(i)
HF·py; (ii) Pd(PPh3)4 cat., morpholine; (iii) p-TsOH] then
furnished coveted methyldisulfide shishijimicin A analogue 8 in
43% overall yield as shown in Scheme 7A.
latter structural motif on cytotoxic potency. This analogue was
constructed from carbohydrate intermediate 46 (Scheme 8, see
Table 5 and ref 7c for preparation), carboline derivative 52,
and enediyne fragment 41 as depicted in Scheme 8. Thus,
methylation of the hydroxyl group of nitrile 46 (MeI, NaH,
82% yield) afforded methyl ether 73, whose DIBAL-H
reduction led to aldehyde 74 (79% yield). Coupling of
carboline derivative 52 with aldehyde 74 was achieved by
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generation of the lithio derivative of the former with t-BuLi at
−78 °C, followed by addition of the latter at the same
temperature, leading to the corresponding secondary alcohol as
a mixture of diastereomers (75a/b, 83% yield, ca. 1:1 dr) as
shown in Scheme 8. The latter mixture was converted to
carboline ketone 76 by treatment with NaOH/EtOH
(decarboxylation), followed by oxidation with DMP, in 63%
overall yield. Sequential removal of the NB (hν) and Nap
(DDQ) protecting groups from 76 followed by trichloroace-
timidate formation (Cl3CCN, NaH) and coupling of the
resulting carbohydrate donor with enediyne fragment 417c
under the influence of BF3·Et2O furnished advanced
intermediate 77 in 5.6% overall yield for the four steps from
76 as shown in Scheme 8. Finally, sequential deprotection/
sulfenylation [(i) LiOH, then AcOH, then PhthNSSMe; (ii)
HF·py; (iii) p-TsOH] of the latter gave analogue 10 in 42%
overall yield for the three steps.
Scheme 9. Synthesis of Structurally Simplified Shishijimicin
Analogues 11 and 12
a
In a rather bold move, we then decided to simplify the
hydroxy methylthio carbohydrate unit of shishijimicin A
(carbohydrate unit A) by deleting both its hydroxyl and
methylthioether functionalities and replacing them with
hydrogen atoms as in analogues 11 and 12 (see Scheme 9).
Their synthesis began with readily available keto sugar 7853 as
summarized in Scheme 9. The first task was the conversion of
78 to glycosyl acceptor 84, the latter intended for coupling
with glycosyl donor 47 in a pending glycosylation reaction.
Thus, treatment of 78 with TMSSMe in the presence of
TMSOTf furnished thioketal 79 (94% yield), whose exposure
to TMSCN and SnCl4 gave methylthio nitrile 80 (95% yield).
Replacement of the remaining methylthio group with a
hydrogen residue was then carried out with n-Bu3SnH in the
presence of AIBN54 to afford a mixture of diastereomers (at
the nitrile-bearing carbon center, 99% yield, 1:1.4 dr) 81 with
the CN group at the axial position (minor, undesired) and 82
with the CN group at the equatorial position (major, desired),
which were chromatographically separated. Undesired axial
isomer 81 was equilibrated [(i) LiOH·H2O; (ii) PivCl,
DMAP] to a 81/82 ca. 3:2 mixture, from which further
quantities of desired isomer 82 were isolated (93% yield based
on 60% starting material recovery), the former being recyclable
for further enrichment of desired compound 82. α-Methyl
glycoside 82 was then converted to β-o-nitrobenzyl (NB)
glycoside 83 through a four-step sequence [(i) Ac2O, H2SO4;
(ii) NH3, MeOH; (iii) Cl3CCN, DBU; and (iv) o-NBOH, BF3·
Et2O] in 63% overall yield as shown in Scheme 9. Removal of
the Piv group (LiOH·H2O, 78% yield) from the latter followed
by coupling of resulting carbohydrate acceptor 84 with glycosyl
donor 477b (SnCl2, AgClO4) furnished α-glycoside 85 in 87%
yield. Reduction of 85 (DIBAL-H, 85% yield) gave aldehyde
86, whose coupling with the lithio derivative obtained from
iodo-carboline fragment 52 (t-BuLi) afforded hydroxyl carbo-
line disaccharide 87a,b as a diastereomeric mixture (ca. 1:1 dr,
inconsequential) in 61% combined yield. Exposure of the latter
to NaOH in EtOH, resulting in cleavage of the methyl
carbamate functionality, was followed by DMP oxidation of the
so-obtained product (free carboline NH upon methyl
carbamate hydrolysis and decarboxylation) furnishing carbo-
line disaccharide 88 (70% overall yield for the two steps).
From the latter intermediate, the NB group was removed (hν)
and the so-obtained product was converted to its trichlor-
oacetimidate derivative (Cl3CCN, NaH), and thence to
advanced intermediate 89 (β-glycoside) through coupling
with thioacetate enediyne carbohydrate acceptor 41 (24%
a
Reagents and conditions: (a) TMSSMe (2.2 equiv), TMSOTf (1.5
equiv), toluene, −20 °C, 20 min; then sat. aq. NaHCO3 (0.75 equiv),
0 °C, 5 min, 94%; (b) TMSCN (3.0 equiv), SnCl4 (1.5 equiv),
CH2Cl2, 0 °C, 0.5 h, 95%; (c) n-Bu3SnH (2.0 equiv), AIBN (0.1
equiv), PhH, 80 °C, 1.5 h, 99% (ca. 1:1.4 dr); (d) LiOH·H2O (4.5
equiv), 60 °C, 4 h; (e) PivCl (5.0 equiv), pyridine (10.0 equiv),
DMAP (1.0 equiv), CH2Cl2, 40 °C, 10 h, 40% of 81 was converted to
82 after one round (93% brsm); (f) H2SO4 (0.8 equiv), Ac2O, 0 °C,
40 min; (g) NH3 (10.0 equiv), MeOH, 0 to 25 °C, 2 h; (h) Cl3CCN/
CH2Cl2 (1:10, v/v), DBU (0.1 equiv), 0 °C, 0.5 h; (i) o-NBOH (3.0
equiv), BF3·Et2O (2.0 equiv), 4 Å MS, CH2Cl2, −78 to −40 °C, 0.5 h,
63% for the four steps; (j) LiOH·H2O (13 equiv), MeOH, 0 to 25 °C,
3 h, 78%; (k) 47 (2.0 equiv), AgClO4 (2.5 equiv), SnCl2 (2.5 equiv),
4 Å MS, THF, −78 to 25 °C, 12 h, 87%; (l) DIBAL-H (3.0 equiv),
CH2Cl2, −78 °C, 45 min, 85%; (m) 52 (3.0 equiv), t-BuLi (6.0
equiv), THF, −78 °C, 5 min; then 86 (1.0 equiv), −78 to −35 °C, 40
min, 61% (ca. 1:1 dr); (n) NaOH (3.0 equiv), EtOH, 0 to 25 °C, 2 h;
(o) DMP (1.1 equiv), CHCl3, 0 to 35 °C, 10 min, 70% for the two
steps; (p) hν, THF/H2O (10:1, v/v), 0 °C, 4.5 h; (q) NaH (2.0
equiv), Cl3CCN/CH2Cl2 (1:2, v/v), 25 °C, 5 min; (r) 41 (2.0 equiv),
BF3·Et2O (3.5 equiv), 4 Å MS, CH2Cl2, −78 to −40 °C, 1 h, 24% for
the three steps; (s) LiOH·H2O (120 equiv), MeOH, −17 to −10 °C,
20 min; then AcOH (120 equiv), 5 min; then PhthNSSMe (8.0
equiv), −10 to 25 °C, 0.5 h, 75%; (t) HF·py/THF (1:20, v/v), 25 °C,
K
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Scheme 9. continued
Scheme 11. Synthesis of the β-Anomeric Isomer of 11:
a
Analogue 14
12 h; (u) Pd(PPh3)4 (0.5 equiv), morpholine (10.0 equiv), THF, 0
°C, 2 h; (v) p-TsOH (5.0 equiv), THF/H2O/acetone (20:1:20, v/v/
v), 25 °C, 48 h, 54% for the three steps; (w) HF·py/THF (1:20, v/v),
25 °C, 12 h; (x) Pd(PPh3)4 (0.5 equiv), morpholine (10.0 equiv),
THF, 0 °C, 2 h; (y) p-TsOH (5.0 equiv), THF/H2O/acetone
(20:1:20, v/v/v), 25 °C, 48 h, 61% for the three steps. Piv = pivaloyl.
overall yield for the three steps). Precursor 89 was transformed
to coveted thioacetate shishijimicin A analogue 11 through the
standard three-step global deprotection sequence [(i) HF·py;
(ii) Pd(PPh3)4 cat., morpholine; (iii) p-TsOH] in 61% overall
yield as shown in Scheme 9. The same advanced intermediate
(89) was diverted, first toward methyltrisulfide precursor 90 by
treatment with LiOH·H2O in MeOH, then AcOH and finally
PhthNSSMe,11 in one pot and 75% overall yield. The latter was
subjected to the standard global deprotection sequence as
mentioned above (89 → 11) to yield methyltrisulfide analogue
12 in 54% overall yield as shown in Scheme 9.
Scheme 10 summarizes the construction of β-carboline
truncated and simplified shishijimicin A analogue 13 whose
Scheme 10. Synthesis of β-Carboline Truncated Analogue
13
a
a
Reagents and conditions: (a) 49 (1.1 equiv), Ph3PAuOTf (0.1
equiv), 4 Å MS, CH2Cl2, −78 °C, 1 h, 89% (α/β ca. 1.6:1); (b)
DIBAL-H (3.0 equiv), CH2Cl2, −78 °C, 0.5 h, 85%; (c) 52 (3.0
equiv), t-BuLi (6.0 equiv), THF, −78 °C, 5 min; then 93 (1.0 equiv),
−78 to −35 °C, 40 min; (d) NaOH (3.0 equiv), EtOH, 0 to 25 °C, 2
h; (e) DMP (1.1 equiv), CHCl3, 0 to 35 °C, 10 min, 65% for the
three steps; (f) hν, THF/H2O (10:1, v/v), 0 °C, 4.5 h; (g) NaH (2.0
equiv), Cl3CCN/CH2Cl2 (1:2, v/v), 25 °C, 5 min; (h) 41 (2.0 equiv),
BF3·Et2O (3.5 equiv), 4 Å MS, CH2Cl2, −78 to −40 °C, 1 h, 11% for
the three steps; (i) HF·py/THF (1:20, v/v), 25 °C, 12 h; (j)
Pd(PPh3)4 (0.5 equiv), morpholine (10.0 equiv), THF, 0 to 25 °C, 1
h; (k) p-TsOH (5.0 equiv), THF/H2O/acetone (20:1:20, v/v/v), 25
°C, 48 h, 60% for the three steps.
preparation, see Scheme 9) and glycosyl donor 4930 under the
influence of Ph3PAuOTf cat. (see Scheme 11), yielding the
corresponding disaccharide as a mixture of α- and β-anomers
(α/β ca. 1.6:1), from which the desired β-anomer (92) was
chromatographically separated. Reduction of the nitrile moiety
within 92 (DIBAL-H, 85% yield) afforded aldehyde 93, which
was processed through a similar pathway, and in similar yields,
to afford targeted analogue 14 (see Scheme 11) as described
above for the synthesis of its α-anomer counterpart (see 11,
Scheme 9).
a
Reagents and conditions: (a) hν, THF/H2O (10:1, v/v), 0 °C, 2 h;
(b) Cl3CCN/CH2Cl2 (1:10, v/v), DBU (0.1 equiv), 0 °C, 0.5 h; (c)
41 (1.0 equiv), BF3·Et2O (3.5 equiv), 4 Å MS, CH2Cl2, −78 to −30
°C, 1 h, 33% for the three steps; (d) HF·py/THF (1:20, v/v), 25 °C,
12 h; (e) Pd(PPh3)4 (0.5 equiv), morpholine (10.0 equiv), THF, 0 to
25 °C, 2 h; (f) p-TsOH (5.0 equiv), THF/H2O/acetone (20:1:20, v/
v/v), 25 °C, 48 h, 73% for the three steps.
design was meant to test the limits of structural simplification
with regards to cytotoxicity potencies. Thus, disaccharide NB
derivative 85 (for preparation, see Scheme 9) was subjected to
photolytic cleavage of the NB protecting group (hν), followed
by activation of the resulting lactol through trichloroacetimi-
date formation (Cl3CCN, DBU), and coupling with hydroxy
thioacetate enediyne fragment 41 to afford triprotected
precursor 91 (33% overall yield). Analogue 13 was then
generated from 91 through the standard three-step global
deprotection sequence, in 73% overall yield, as depicted in
Scheme 10.
Scheme 11 shows the synthesis of shishijimicin analogue 14
(the β-anomer of 11, with regards to the aminosugar glycosidic
bond), whose design was intended to test the role for the α-
anomeric feature of the aminosugar structural motif of the
molecule for bioactivity. In order to obtain desired β-glycoside
disaccharide fragment 92, the Yu gold-promoted glycosylation
protocol55 was employed to couple glycosyl acceptor 84 (for
Inspired by the iodide residue of calicheamicin γI1 and its
importance to the binding of the molecule to duplex DNA,56
we ventured to design and synthesize thioacetate shishijimicin
A analogue 15 (Scheme 12A). Thus, thioacetate advanced
intermediate 89 (for preparation, see Scheme 9) was subjected
to Alloc-Boc exchange [(i) Pd(PPh3)4 cat., morpholine; (ii)
Boc2O, then DMAP] and subsequent desilylation (TBAF) to
afford phenol derivative 98 (equipped with three Boc groups).
The latter compound was treated, without purification, with
morpholine-I2 complex (99)57 to give 5″-iodide 100,
exclusively, whose exposure to formic acid furnished desired
iodo analogue 15 through global deprotection (three Boc
groups and a ketal), in 50% overall yield for the five steps from
89, as shown in Scheme 12A. The exclusive regioselectivity of
the iodination of the phenolic carboline moiety of 98 can be
rationalized by considering resonance structures 98a, 98b and
L
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Scheme 12. Synthesis of C5″-Iodo-Shishijimicin Analogue
Scheme 13. Modified Preparation of N-(Methyldithio)-
a
15
phthalimide [PhthNSSMe] and Synthesized Sulfenylating
a
Reagents 104−106
a
Reagents and conditions: (a) 101 (1.0 equiv), thiol (1.0 equiv),
benzene, reflux, 1.5−22 h, 74−90%; (b) 102 (1.0 equiv), SCl2 (1.0
equiv), Et3N (1.0 equiv), CH2Cl2, 0 °C, 40 min; then MeSH (1.0
equiv), Et3N (1.0 equiv), 0 °C, 8 h; then 25 °C, 40 min, 19%; (c) 102
(1.0 equiv), S2Cl2 (0.5 equiv), Et3N (1.2 equiv), THF, 0 to 25 °C, 6
h; (d) SO2Cl2 (excess), 70 °C, 12 h, 98% for the two steps; (e)
TMSSMe (1.0 equiv), 103 (1.0 equiv), CH2Cl2, −78 °C, 0.5 h, quant.
a
Reagents and conditions: (a) Pd(PPh3)4 (0.5 equiv), morpholine
(10.0 equiv), THF, 0 °C, 2 h; (b) Boc2O (10.0 equiv), MeCN, 80 °C,
36 h; then DMAP (1.0 equiv), 25 °C, 4 h; (c) TBAF (5.0 equiv),
THF, 0 °C, 0.5 h; (d) morpholine−I2 (99, 2.0 equiv), CH2Cl2, 25 °C,
20 min; (e) HCO2H, 25 °C, 12 h, 50% for the five steps.
we developed a convenient and high-yielding three-step
procedure for the preparation of the methyl Harpp-type
reagent (i.e., PhthNSSMe) as shown in Scheme 13B. Thus,
treatment of phthalimide (102) with S2Cl2 in the presence of
Et3N, followed by exposure of the resulting bis(1-
phthalimidyl)disulfane to excess SO2Cl2 generated intermedi-
ate 103 (stable for months in a desiccator at ambient
temperature) in 98% overall yield.60 Reaction of the latter
with TMSSMe at −78 °C led to PhthNSSMe in quantitative
yield and in pure form after evaporation of the byproduct (i.e.,
TMSCl; no further purification needed), presumably via
intermediate 103a through the mechanism shown in Scheme
13B.
Following the same procedure, two previously reported
disulfenylating reagents (i.e., 10461b and 10511a,61b) and novel
phenylselenosulfenylating reagent 106 were successfully
synthesized from their corresponding thio- and seleno-TMS
ethers (i.e., PhSTMS, t-BuSTMS and PhSeTMS62) in 99, 96,
and 91% yields, respectively, as depicted in Scheme 13C (see
the Supporting Information for further details).
98c, with 98b being more favorable than 98c, as the
aromaticity and extended conjugation of the system are mostly
conserved in this resonance structure (i.e., 98b, see Scheme
12B).
2.3. New Procedures for the Synthesis of Old and
New Sulfenylating Reagents and Synthesis of Cysteine
Trisulfide Shishijimicin A Analogue 16. During these
studies, we recognized a number of issues with the published
procedures for the preparation of the methyl Harpp-type
reagent (PhthNSSMe).11 The original procedure reported by
Harpp11a featured an efficient generation of an array of the
Harpp reagents from N,N′-thiobisphthalimide (101)58 as
shown in Scheme 13A. Notably, however, methanethiol
(MeSH) was not tested using this procedure at the time and
its feasibility to synthesize the methyl Harpp reagent
(PhthNSSMe) is still unknown. The second procedure by
Danishefsky11b involves the use of in situ generated
phthalimidosulfenyl chloride (PhthNSCl, 103), whose reaction
with MeSH results in low isolated yield (19%) of the product
(see Scheme 13A). Inspired by these precedents, and the
previous work by Harpp et al. describing a facile synthesis of
disulfides from sulfenyl chloride and TMS thioether partners,59
As an extension of our synthetic investigations and in order
to enrich the conjugation options of the enediyne family of
payloads, we also developed disulfide phthalimide reagent 108
(see Scheme 14A) and employed it for the synthesis of
cysteine trisulfide shishijimicin A analogue 16 as shown in
Scheme 14B. Thus, reaction of thiol 10763 with TMSCl in the
presence of Et3N gave silyl thioether 107a, whose reaction with
M
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Scheme 14. Synthesis of Disulfenylation Reagent 108 and
Table 7. Cytotoxicity Data against the Cell Lines MES SA,
MES SA DX, and HEK 293T for Shishijimicin A (1) and Its
Analogues 7−16
Its Application to the Synthesis of Cysteine Trisulfide
a
a b
,
Shishijimicin Analogue 16
a
MSE SA = uterine sarcoma cell line; MES SA DX = MES SA cell line
with marked multidrug resistance; HEK 293T = immortalized human
b
embryonic kidney cell line. IC50 is the 50% inhibitory concentration
of the compound against cell growth, reported in nM. Data obtained
at AbbVie Stemcentrx.
a
Reagents and conditions: (a) TMSCl (1.1 equiv), Et3N (1.1 equiv),
potency against the multi-drug-resistant cell line (IC50 = 1.2
nM against MES SA DX). The thioacetate counterpart
analogue of shishijimicin A, analogue 7, was the third most
active compound tested, being only slightly less potent against
the MES SA and HEK 293T cell lines (IC50 = 0.023 and 0.033
nM, respectively) but over 250-fold more potent against the
multi-drug-resistant cancer cells (IC50 = 3.8 nM against MES
SA DX) than the natural product. Interestingly, analogue 13,
lacking the carboline domain of the shishijimicin A molecule,
showed subnanomolar potencies against both the MES SA and
the HEK 293T cell lines (IC50 = 0.20 and 0.46 nM,
respectively) but no significant activity against the more-
difficult-to-kill MES SA DX cell line. The thioester counterpart
(11) of simplified shishijimicin A analogue 12 also exhibited
subnanomolar potencies against the MES SA and the HEK
293T cell lines, while it was found to be devoid of significant
activity against the multi-drug-resistant MES SA DX cell line.
Also, analogues 9, 10, and 15, while exhibiting low nanomolar
potencies against the MES SA and HEK 293T cell lines were
considerably less potent against the drug-resistant cell line
MES SA DX. The lack of potent cytotoxicities against all three
of the tested cell lines by analogue 14 (the anomeric
diastereoisomer of the rather potent simplified analogue 11
against two of the cell lines) was also of note, as was the
decrease of potency of the iodo counterpart of analogue 11,
analogue 15 (see Table 7), indicating that these structural
changes are not tolerated with regard to biological activity.
Interestingly, cysteine trisulfide shishijimicin A analogue 16
demonstrated potent cytotoxicities against all three cell lines
tested (IC50 = 0.04 nM against MES SA, 2.5 nM against MES
SA DX, and 0.02 nM against HEK 293T), possessing the
second highest potency against the drug-resistant MES SA DX
cell line from all synthesized compounds evaluated.
CH2Cl2, 0 °C, 2 h; then PhthNSCl (103, 1.0 equiv), −78 °C, 0.5 h,
76%; (b) LiOH·H2O (100 equiv), MeOH, −15 to −10 °C, 20 min;
AcOH (100 equiv), 5 min; then 108 (5.0 equiv), −10 to 25 °C, 1 h,
73%; (c) HF·py/THF (1:20, v/v), 25 °C, 12 h; (d) Pd(PPh3)4 (0.5
equiv), morpholine (10.0 equiv), THF, 0 °C, 2 h; (e) p-TsOH (5.0
equiv), THF/H2O/acetone (20:1:20, v/v/v), 25 °C, 48 h, 46% for the
three steps.
PhthNSCl (103, for preparation, see Scheme 13) in the same
pot afforded reagent 108 in 76% overall yield from 107. As
shown in Scheme 14B, advanced intermediate 89 (for
preparation, see Scheme 9) reacted sequentially, and in the
same pot, with LiOH·H2O, then AcOH, and then reagent 108
to afford, in 73% overall yield, fully protected precursor 109.
The latter was subjected to our three-step global deprotection
sequence to afford targeted shishijimicin analogue 16 in 46%
overall yield, as shown in Scheme 14B.
2.4. Biological Evaluation of Synthesized Compounds
and Structure−Activity Relationships. The synthesized
shishijimicin A (1) and its analogues (7−16) were evaluated
for their antitumor activities against MES SA (uterine sarcoma
cells), MES SA DX (multi-drug-resistant uterine sarcoma
cells), and HEK 293T64 (immortalized human embryonic
kidney cells) using in vitro assays and with N-acetyl
calicheamicin γI1 as a positive control. The results of these
investigations (IC50 values in nM) are summarized in Table 7.
As seen in Table 7, analogues 7, 8, and 12 exhibited
comparable or higher potencies than those of the synthetic
natural product (1) with 8 being the most potent of all
compounds tested, against the MES SA and HEK 293T cells,
demonstrating subpicomolar potencies (i.e., 0.00067 nM
against MES SA and 0.0015 nM against HEK 293T). The
shishijimicin A analogue 12 lacking the methylthio and
hydroxyl groups from the ring A sugar is also impressive for
its single-digit picomolar potencies against these cell lines (i.e.,
IC50 = 0.006 and 0.008 nM against the MES SA and HEK
293T cell lines, respectively) and single-digit nanomolar
From these data, we were able to derive a set of structure−
activity relationships (SARs) within the shishijimicin family of
compounds that could facilitate further optimization studies
and preclinical development as shown in Figure 4. Thus, it
became evident that the methyltrisulfide moiety (the triggering
N
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Article
AUTHOR INFORMATION
Corresponding Author
*kcn@rice.edu
■
ORCID
K. C. Nicolaou: 0000-0001-5332-2511
Zhaoyong Lu: 0000-0002-9218-6337
Author Contributions
#R.L. and Z.L. contributed equally to this work.
Notes
The authors declare no competing financial interest.
Figure 4. Structure−activity relationships of shishijimicin A.
ACKNOWLEDGMENTS
■
We thank Dr. Q. Kleerekoper (Rice University) for NMR
spectroscopic assistance, and Drs. C. Pennington (Rice
University) and I. Riddington (University of Texas at Austin)
for mass spectrometric assistance. This work was supported by
the Cancer Prevention Research Institute of Texas (CPRIT),
The Welch Foundation (grant C-1819), AbbVie Stemcentrx,
and Rice University.
device of the molecule initiating the Bergman cycloaromatiza-
tion reaction) could be substituted with the methyldisulfide or
thioacetate moieties without significant loss of, if not
enhancing, the biological activity as predicted, in line with
our expectations based on previous studies.44−48 The same is
true for the simplification of the shishijimicin A structure by
replacing the methylthio and hydroxyl groups of the A
carbohydrate ring with hydrogen residues. However, removal
of the amino sugar residue, or acetylation of its amino group is
not tolerated, suggesting the important role of the basic
nitrogen atom in this region of the molecule. This possible role
may be a dipolar interaction of this basic nitrogen (after
protonation) with the negatively charged phosphate group of a
DNA molecule. Similarly, the carboline domain seems to be
playing an important role for the biological activity of the
molecule as evidenced from the significant loss of activity upon
its removal (see analogue 13, Table 7). This conclusion is also
supported by considerable loss of potency upon substituting
this moiety with an iodine residue (see analogue 15, Table 7).
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.8b06955.
Experimental procedures and characterization data for
all compounds (PDF)
O
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