Synthesis and anticancer activity of all known (-)-agelastatin alkaloids

Article abstract of DOI:10.1021/jo4020112

The full details of our enantioselective total syntheses of (-)-agelastatins A-F (1-6), the evolution of a new methodology for synthesis of substituted azaheterocycles, and the first side-by-side evaluation of all known (-)-agelastatin alkaloids against nine human cancer cell lines are described. Our concise synthesis of these alkaloids exploits the intrinsic chemistry of plausible biosynthetic precursors and capitalizes on a late-stage synthesis of the C-ring. The critical copper-mediated cross-coupling reaction was expanded to include guanidine-based systems, offering a versatile preparation of substituted imidazoles. The direct comparison of the anticancer activity of all naturally occurring (-)-agelastatins in addition to eight advanced synthetic intermediates enabled a systematic analysis of the structure-activity relationship within the natural series. Significantly, (-)-agelastatin A (1) is highly potent against six blood cancer cell lines (20-190 nM) without affecting normal red blood cells (>333 μM). (-)-Agelastatin A (1) and (-)-agelastatin D (4), the two most potent members of this family, induce dose-dependent apoptosis and arrest cells in the G2/M-phase of the cell cycle; however, using confocal microscopy, we have determined that neither alkaloid affects tubulin dynamics within cells.

Full text of DOI:10.1021/jo4020112

Article
pubs.acs.org/joc
Synthesis and Anticancer Activity of All Known (−)-Agelastatin
Alkaloids
Sunkyu Han,† Dustin S. Siegel,† Karen C. Morrison,‡ Paul J. Hergenrother,‡
and Mohammad Movassaghi*,†
†Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
‡Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States
S
* Supporting Information
ABSTRACT: The full details of our enantioselective total syntheses of
(−)-agelastatins A−F (1−6), the evolution of a new methodology for synthesis
of substituted azaheterocycles, and the first side-by-side evaluation of all known
(−)-agelastatin alkaloids against nine human cancer cell lines are described.
Our concise synthesis of these alkaloids exploits the intrinsic chemistry of
plausible biosynthetic precursors and capitalizes on a late-stage synthesis of the
C-ring. The critical copper-mediated cross-coupling reaction was expanded to
include guanidine-based systems, offering a versatile preparation of substituted
imidazoles. The direct comparison of the anticancer activity of all naturally
occurring (−)-agelastatins in addition to eight advanced synthetic
intermediates enabled a systematic analysis of the structure−activity relation-
ship within the natural series. Significantly, (−)-agelastatin A (1) is highly
potent against six blood cancer cell lines (20−190 nM) without affecting
normal red blood cells (>333 μM). (−)-Agelastatin A (1) and (−)-agelastatin D (4), the two most potent members of this
family, induce dose-dependent apoptosis and arrest cells in the G2/M-phase of the cell cycle; however, using confocal
microscopy, we have determined that neither alkaloid affects tubulin dynamics within cells.
(−)-Agelastatin A (1) was found to exhibit modest antineo-
plastic activity against multiple tumor cell lines.1,2,5,6 Specifi-
cally, (−)-1 inhibits osteopontin-mediated neoplastic trans-
formation and metastasis. In addition, (1)-1 causes cells to
arrest in the G2/M-phase of the cell cycle; further studies
suggested that the arrest could be specifically in the G2-phase.7
Additionally, recent in vivo pharmacokinetic studies in mice
demonstrated that (−)-agelastatin A (1) is able to penetrate
into CNS compartments.6,8 (−)-Agelastatin A (1) also exhibits
toxicity toward anthropods3 and moderately inhibits the
glycogen synthase kinase-3β.5,9
INTRODUCTION
■
The agelastatins are a family of cytotoxic pyrrole-imidazole
alkaloids exhibiting a unique tetracyclic framework with four
contiguous stereogenic centers on the carbocyclic C-ring
(Figure 1). In 1993, Pietra and co-workers first isolated and
studied (−)-agelastatins A (1) and B (2) from the Coral Sea
sponge Agelas dendromorpha.1,2 (−)-Agelastatins C (3) and D
(4) were isolated by Molinski and co-workers in 1998 from
Cymbastela sp. native to the Indian Ocean.3 In 2010, Al-
Mourabit’s group isolated (−)-agelastatins E (5) and F (6)
from the New Caledonian sponge A. dendromorpha.4
The agelastatins are the only isolated pyrrole-imidazole
alkaloids with C4−C8 and C7−N12 bonds (Figure 1) and are
likely derived from linear biogenetic precursors such as
clathrodin,10 hymenidin,11 or oroidin.12,13 Kerr and co-workers
showed that histidine and ornithine (or proline) are the amino
acid precursors for related pyrrole−imidazole alkaloids.14,15
Prior to our synthetic report of agelastatin alkaloids,16 there
were two hypotheses for the biogenesis of agelastatin A (1)
from a putative linear precursor.1a,17
Its potent biological activities, in conjunction with its
intriguing molecular structure, have prompted considerable
efforts toward the total synthesis of agelastatin A (1).18
Preceding our report on the enantioselective total synthesis of
Received: September 10, 2013
Figure 1. Structures of (−)-agelastatins A−F (1−6).
© XXXX American Chemical Society
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(−)-agelastatins A−F (1−6),16 10 different research groups had
reported inventive solutions to the total synthesis of alkaloid
1.19 Interestingly, each of these synthetic strategies were
contingent on an early introduction of the central C-ring
followed by further derivatization to the natural product 1.
Agelastatin A (1) has continued to serve as a source of
inspiration for development of new and innovative synthetic
solutions as demonstrated by four additional recent syntheses.20
Distinct from these approaches, our biosynthetically inspired
synthetic strategy involved a late-stage C-ring formation and
enabled the total synthesis of all known (−)-agelastatins (1−
6).16 Herein, we provide a detailed account of our
enantioselective total synthesis of these alkaloids and the
development of a key methodology for the synthesis of
imidazol-2-one and 2-aminoimidazoles. We also describe the
first comparative anticancer activity study of all known
(−)-agelastatins (1−6), with alkaloids (−)-1 and (−)-4 being
most active, demonstrating potent and selective activity against
human blood cancer cell lines by inducing apoptotic cell death.
Scheme 2. Early Approach toward (−)-Agelastatin A (1)
biosynthetic precursor,14,22 to introduce the C7 stereochemistry
of intermediate 12 was of additional interest.
This early synthetic approach to 1 commenced with a
Mitsunobu reaction involving L-proline derivative 14 and allylic
alcohol 15 followed by Dess−Martin periodinane (DMP) C5-
oxidation to give enone 16 in 73% yield over two steps
(Scheme 3). Exposure of carbamate 16 to trifluoroacetic acid
Scheme 3. Synthesis of Key Bicycle 18 and Attempted
a
Oxidation−Cyclization
RESULTS AND DISCUSSION
■
Biosynthetic Hypothesis of (−)-Agelastatin A. Our
retrobiosynthetic analysis of (−)-agelastatin A (1) is shown in
Scheme 1.16 The key transformation in our planning involved a
Scheme 1. Retrobiosynthetic Analysis of (−)-Agelastatin A
(1)
a
Reagents and conditions: (a) DEAD, PPh3, THF, 0 → 23 °C, 79%;
(b) DMP, CH2Cl2, 23 °C, 93%; (c) TFA, CH2Cl2, 0 °C, 85%; (d)
DMP, CH2Cl2, 23 °C, 42%; (e) PhSH, KOH, MeCN, 0 °C, 84%; (f)
N-tert-butylbenzenesulfinimidoyl chloride, DBU, CH2Cl2, −78 → 23
°C, 20%. DEAD = diethyl azodicarboxylate, and DBU = 1,8-
diazabicycloundec-7-ene.
5-exo-trig cyclization, forming the central carbocyclic C-ring
and introducing the desired C4-, C5-, and C8-stereogenic
centers in a single step. We anticipated that the key
intermediate, pre-agelastatin A (9), would be accessed from
the tricycle 10 through a late-stage C8-oxidation. Our planning
was partly motivated by the structural similarity of tricycle 10 to
the natural alkaloid cyclooroidin (11) and the potential
formation of the imidazolone D-ring of 10 through methylation
and hydrolysis of a related aminoimidazole.21
Initial Synthetic Studies toward Agelastatin A. Prior to
our discovery and the first use of the nucleophilic D-ring
imidazolone in synthesis of the C-ring of these alkaloids, we
had planned on the use of ketotriazones for the formation of
the C4−C8 bond (Scheme 2). We had expected to enable C-
ring generation via nucleophilic attack of a C4-enol to a C8
electrophilic derivative of ketone 12 after C8-oxidation. In this
early approach, we planned to introduce the AB-bicyclic
structure of ketone 12 via an intramolecular conjugate addition
of N1219a in enone 13 followed by an oxidative aromatization.
The potential stereoablative use of L-proline, a known
resulted in the unveiling of the N12 and spontaneous
intramolecular conjugate addition to afford pyrrolidine 17 in
85% yield as a 3.3:1 mixture of diastereomers at C7 (major
diastereomer shown). This modest level of diastereoselection
hinted at possible further refinement of this approach for
asymmetric synthesis of lactam 18 based on the C11-
stereochemistry of enone 16. With interest in addressing the
key C-ring cyclization, we proceeded with sequential treatment
of ketone 17 with DMP to give the corresponding pyrrole
derivative (42% yield) possibly through oxidation of the C11-
enol tautomer,23,24 followed by N9-desulfonylation to afford
the desired lactam 18 in 84% yield.25
While we successfully validated a range of conditions for
activation of ketone 18 as a C4-nucleophile,26 the oxidation of
C8 to generate the necessary electrophile 19 (Scheme 3)
proved difficult.27,28 In contrast to significant literature
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precedent for the oxidation of amines, the oxidation of amides
is rare.29 After the examination of a wide range of conditions,
we found that treatment of lactam 18 with N-tert-
butylbenzenesulfinimidoyl chloride30 in the presence of DBU
gave rise to the enamide 20 in 20% yield along with 20%
recovery of lactam 18. Indeed, when ketone 18 was dissolved in
methanol-d4 in the presence of DBU, complete deuterium
incorporation at C4 occurred in less than 1 min. The more
facile oxidation of the lactam 18 at positions other than C8 and
a favorable tautomerization of acylimine 19 to the highly stable
bicycle 20 (Scheme 3, arrow B) seemed to prevent the desired
formation of the C4−C8 bond (Scheme 3, arrow A). Thus, we
sought an alternate strategy for the introduction of the C8-
hemiaminal functional grouping of lactam 18.31
Our First-Generation Total Synthesis of ( )-Agelasta-
tin A. An orthogonal approach to the desired bicyclic
hemiaminal ether 21 was the partial reduction of the C8-
carbonyl of imide 23 (Scheme 4). Moreover, for the formation
of the key C4−C8 bond, we planned to use the imidazolone
heterocycle as a nucleophile in accord with our retrobiosyn-
thetic analysis (Scheme 1).
triazone intermediate,26 prepared by treatment of triazone 27
with sec-butyllithium, to methyl ester ( )-26a gave ketone
( )-28 in 70% yield.26 Exposure of triazone ( )-28 to
methanolic hydrogen chloride solution led to spontaneous
condensative cyclization35 to form the imidazolone D-ring in
66% yield (Scheme 5).26
Imidazolone ( )-29 was then treated with scandium
trifluoromethanesulfonate (Sc(OTf)3) to induce the acylimi-
nium ion formation and subsequent introduction of the C-ring;
however, the C8-hydroxy hemiaminal product ( )-30, isolated
in 36% yield, and pyrrolopyrazinone 31 (<5% yield) were the
only observed products (Scheme 6). When hemiaminal ( )-30
Scheme 6. Formation of Pyrrolopyrazinone 31 under
a
Various Conditions
Scheme 4. Two Strategies for the Introduction of the C8-
Hemiaminal Ether 21
a
Reagents and conditions: (a) Sc(OTf)3, H2O, MeCN, 23 °C
[( )-30, 36%; 31, <5%]; (b) EtSH, TFA, CH2Cl2, 23 °C [( )-32,
95%]; (c) CH2Cl2, TFA, 23 °C, 95%; (d) NaIO4, MeOH, H2O, 23 °C,
55%; (e) MeCN, 79 °C, 100%. TFA = trifluoroacetic acid.
was exposed to a dichloromethane and trifluoroacetic acid
(TFA) mixture, pyrrolopyrazinone 31 was obtained in
quantitative yield. These results suggested that the acyliminium
ion intermediate was transiently formed, but was not effectively
trapped by the C4 nucleophile. In an attempt to minimize
tautomerization of the acyliminium ion under milder, acid-free
reaction conditions, we planned to introduce a sulfoxide group
at the C8-position. Thus, imidazolone ( )-29 was treated with
a mixture of ethanethiol, trifluoroacetic acid, and dichloro-
methane (EtSH:TFA:CH2Cl2 = 1:2.5:10) at 23 °C to give
sulfide ( )-32 (95%), which was oxidized to sulfoxide ( )-33
in 55% yield upon treatment with sodium periodate (6:4 dr).
However, heating a solution of sulfoxide ( )-33 in acetonitrile
resulted in the exclusive formation of pyrrolopyrazinone 31
(Scheme 6).
Treatment of the known methyl ester 2432 with benzylamine
and methoxyamine resulted in the formation of imides ( )-25a
and ( )-25b in 86% and 48% yield, respectively (Scheme 5).32
a
Scheme 5. Synthesis of Imidazolone ( )-29
To minimize any potential adverse steric blocking imparted
by the benzyl group in the attempted C4−C8 bond formation
(Scheme 6), we turned our attention to ketotriazone ( )-34,
which was synthesized via the coupling of methyl ester ( )-26b
and α-lithiated triazone generated from tin−lithium exchange
of the organostannane derivative in 74% yield.26 The
ketotriazone ( )-34 was treated with samarium iodide (73%
yield) followed by solvolysis in an aqueous hydrochloric acid
and methanol mixture to afford imidazolone ( )-35 (67%
yield, Scheme 7). Unfortunately, when imidazolone ( )-35 was
treated with a TFA−water mixture in acetonitrile, the
elimination of methanol occurred to give pyrrolopyrazinone
36 in 95% yield (Scheme 7).36
We were cognizant that the formation of pyrrolopyrazinone
36 was a result of rapid C7-deprotonation of the C8-iminium
ion intermediate to generate the conjugated bicycle prior to the
trapping of the C8-electrophile with the C4-nucleophile
(Scheme 7). Therefore, we considered substrate modifications
that would lower the kinetic acidity of the proton at C7 and
a
Reagents and conditions: (a) H2NR, DMF, 23 °C (R = Bn, 86%; R =
OMe, 48%); (b) L-Selectride, −78 °C, THF (R = Bn, 78%; R = OMe,
58%); (c) TsOH·H2O, MeOH, 23 °C (R = Bn, 70%; R = OMe, 95%);
(d) 27, s-BuLi, TMEDA, THF, −78 °C, 70%; (e) HCl (aq), MeOH,
23 °C, 66%. TsOH = p-toluenesulfonic acid.
Each of these imides was obtained as a racemate, reflecting the
facile deprotonation of the acidic C7-proton.33 Imides ( )-25a
and ( )-25b were readily reduced with L-Selectride and
subsequent treatment with p-toluenesulfonic acid yielded
hemiaminal ethers ( )-26a and ( )-26b in 55% and 46%
yield (two steps), respectively. The addition of a lithiated34
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Scheme 7. Synthesis of Imidazolone Derivative ( )-35 and
Its Reactivity under Acidic Conditions
Scheme 8. Our First-Generation Total Synthesis of
( )-Agelastatin A (1)
a
a
a
Reagents and conditions: (a) SmI2, THF, MeOH, −78 °C, 73%; (b)
HCl(aq), MeOH, 23 °C, 67%; (c) TFA, H2O, MeCN, 23 °C, 95%.
provide greater opportunity for the formation of the desired
C4−C8 bond. We envisioned that the allylic strain between the
C13-bromide and the C6-methylene, present in all agelastatins,
might hinder this tautomerization event. Comparison of the
calculated minimum energy conformation of the acyliminium
ion derived from C8-ionization of 35 and brominated
acyliminium ion 8 (Scheme 1) revealed that the H7 of
intermediate 8 is ∼22° further away from the ideal
conformation for acidification as compared to that of the
nonbrominated acyliminiun ion.37 Thus, the C7-proton of C13-
brominated acyliminium ion 8 would be less susceptible to
deprotonation as the overlap of the C7−H7 σ-orbital with the
C8−N9 π*-orbital would be less than that of the non-
brominated acyliminium ion.38 We envisioned that the lowered
kinetic acidity of H7 in the C13-brominated acyliminium ion
intermediate 8 would provide greater prospect to trap the C8-
electrophilic center with the C4-nucleophile before the
undesired H7-deprotonation.
This analysis of the possible difference in the preferred H7−
C7−C8−H8 dihedral angles of the bromo versus desbromo
intermediates prompted us to evaluate the effect of C13-
bromide in the key C4−C8 bond-forming step. For the
introduction of the bromide at C13, ketotriazone 34 was
treated with N-bromosuccinimide (NBS) to give bromopyrrole
37 in 45% yield (Scheme 8). Samarium iodide-mediated
reduction of N-methoxylactam 37 (64% yield) followed by
treatment with aqueous hydrogen chloride in methanol
afforded ( )-O-methyl-pre-agelastatin A (39) in 65% yield.
Gratifyingly, when ( )-O-methyl-pre-agelastatin A (39) was
treated with TFA and water in acetonitrile, ( )-agelastatin A
(1) and ( )-di-epi-agelastatin A (40) were obtained as a 2:1
mixture in 47% combined yield.39 Careful monitoring of this
transformation revealed that ( )-4,5-di-epi-agelastatin A (40)
was the kinetic product, which equilibrated to the thermody-
namically favored ( )-agelastatin A (1). With confirmation of
the key step at hand, the synthesis of ( )-O-methyl-pre-
agelastatin A (39) was envisioned to be streamlined via a
fragment assembly that would obviate the need for the N-
methoxy substitution of the B-ring lactam.37
a
Reagents and conditions: (a) NBS, MeCN, 0 °C, 45%; (b) SmI2,
THF, −78 °C, 64%; (c) HCl(aq), MeOH, 23 °C, 65%; (d) H2O,
MeCN, TFA, 40 °C, 47% (2:1 ( )-1/( )-40). NBS = N-
bromosuccinimide.
Importantly, it took 1 h for the C7-methine to show 52%
deuterium incorporation and an additional 2 h to show
complete deuterium incorporation (Scheme 9), which we
reasoned could provide us with a small window of opportunity
to intercept the imide carbonyl C8 via a rapid reduction before
racemization.
Scheme 9. Cyclization and Deuterium Incorporation at C7
of Amide (+)-41
Thus, we exposed methyl ester (+)-41 to sodium
borohydride in methanol at 0 °C (Scheme 10), which led to
cyclization and reduction of the resulting imide to afford an α-
hydroxyamide intermediate. Subsequent addition of p-toluene-
sulfonic acid monohydrate to the reaction mixture enabled a
Our Second-Generation Total Synthesis of (−)-Age-
lastatin A. We rationalized that a facile and reversible
enolization at C7 caused the racemization of imide derivatives
25a and 25b (Scheme 5). Therefore, similar to our solution for
the successful key C4−C8 bond formation (Scheme 8), we
envisioned that the bromination at C13 of these imides would
lower the kinetic acidity of their H7 and potentially minimize
erosion of their enantiomeric excess.37 Amide (+)-41 could be
obtained in two steps from a known pyrrole derivative upon
bromination and acylation.16 When amide (+)-41 was dissolved
in methanol-d4, we observed facile B-ring cyclization.40
Scheme 10. Synthesis of Bicycle (+)-44
D
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a
Table 1. Copper-Mediated Cross-Coupling between Thioester (+)-45 and Stannyltriazone Derivatives
entry
triazone
catalyst (mol %)
additive (equiv)
temp (°C)
time (h)
yield of (+)-48 (%)
yield of 49 (%)
1
2
3
4
5
6
46
46
46
46
46
47
Pd2(dba)3 (5)
Pd2(dba)3 (10)
Pd(PPh3)4 (10)
Pd(PPh3)4 (9)
−
CuDPP (2.4), P(OEt)3 (0.4)
CuDPP (2.4), SPhos (0.8)
CuDPP (2.4)
23
23
50
50
50
50
24
24
2
0
0
0
0
50
60
63
50
40
36
0
CuTC (2.4)
2
CuTC (2.4)
1
b
−
CuTC (1.5)
0.5
96
a
Tol = C6H4-p-Me, c-Hx = cyclohexyl, Pd2(dba)3 = tris(dibenzylideneacetone)dipalladium(0), CuDPP = copper(I) diphenylphosphinate, SPhos =
b
2-dicyclohexylphosphino-2′,6′-dimethoxylbiphenyl, and CuTC = copper(I) thiophene-2-carboxylate. The reaction was performed on a >5 g scale.
methanolysis reaction to directly give bicycle (+)-44 in 90%
yield (>10 g scale) and 99% ee.
4). Notably, this transformation could be carried out with equal
efficiency without the palladium catalyst (Table 1, entry 5).43
The undesired alkyl transfer from stannanes has been reported
during transmetalation involving an sp3-hybridized carbon in
the Stille cross-coupling reactions.44,45 In an attempt to
suppress this undesired alkyl transfer, we employed a
stannyltriazone substrate, 47, containing cyclohexyl groups as
auxiliary ligands to the tin.46 Importantly, when thioester
(+)-45 and triazone 47 were treated with CuTC (1.5 equiv) at
50 °C, the desired ketone (+)-48 was formed exclusively in
96% yield and 99% ee (>5 g scale, Table 1, entry 6).
Having established a reliable method to access ketone
(+)-48, we next focused on the optimization of the final steps
leading to (−)-agelastatin A (1). We found that ketotriazone
(+)-48 was most efficiently converted to (+)-O-methyl-pre-
agelastatin A (39) upon exposure to methanolic hydrogen
chloride at 65 °C (89% yield, 99% ee, Scheme 11).
Additionally, treatment of (+)-O-methyl-pre-agelastatin A
(39) with aqueous methanesulfonic acid at 100 °C, followed
by introduction of methanol to the resulting mixture of
(−)-agelastatin A (1) and (−)-di-epi-agelastatin A (40),
afforded (−)-agelastatin A (1) in 49% yield (99% ee, >1 g
scale) along with (−)-O-methyl-di-epi-agelastatin A (50; 22%
yield, Scheme 11). Not only could diastereomer 50 be readily
separated by flash column chromatography, but its resubmis-
sion to the above protocol provided another batch of
(−)-agelastatin A (1) in 66% yield along with recovered
(−)-50 in 30% yield.47 While treatment of an aqueous
tetrahydrofuran solution of (−)-agelastatin A (1) with NBS
and 2,6-di-tert-butyl-4-methylpyridine (DTBMP) efficiently
afforded (−)-agelastatin B (2) in 84% yield, the exposure of
a methanolic solution of (−)-1 to Amberlyst 15 resin provided
(−)-agelastatin E (5) in 96% yield.1c
While the C13-bromide served critical roles in both
suppressing C7-racemization (Scheme 10) and enabling the
key C-ring cyclization (Scheme 8), it was incompatible with the
addition of the lithiated triazone to methyl ester (+)-44 as
detailed in Scheme 5. When the same lithiated triazone was
exposed to methyl ester (+)-44, the reaction was plagued by
the undesired reactivity between the C13-bromide and the
organolithium species. The corresponding Grignard, organo-
cerium, organocuprate, and organozinc derivatives failed to add
to methyl ester (+)-44. However, we found that the
organocerium triazone derivative added smoothly to an
aldehyde variant of ester (+)-44 for the introduction of the
C4−C5 bond (64% yield), enabling our second-generation
total synthesis of (−)-agelastatins A (1) and B (2).37 Following
our successful access to enantiomerically enriched alkaloids
(−)-1 and (−)-2 via strategic introduction of C13-bromide at
an early stage of the synthesis, we pursued a more concise
strategy for the union of the desired triazone fragment and the
readily available bicyclic ester (+)-44.
Our Third-Generation Total Synthesis of (−)-Agelas-
tatins A−F. Inspired by the Liebeskind group’s reports on the
cross-coupling reaction between thioesters and organostan-
nanes,41 we set out to develop an efficient metal-mediated
cross-coupling reaction between thioester (+)-45, derived from
methyl ester (+)-44 in one step,16 and a stannyltriazone
derivative (Table 1).42 Under the previously reported reaction
conditions,41b thioester (+)-45 and triazone 46 in the presence
of Pd2(dba)3, copper(I) diphenylphosphinate (CuDPP), and
triethyl phosphite in THF at 23 °C failed to deliver the desired
product (Table 1, entry 1). Substitution of triethyl phosphite
with SPhos also showed no improvement (Table 1, entry 2).
Interestingly, upon treatment of thioester (+)-45 and triazone
46 with a catalytic amount of Pd(PPh3)4 and a stoichiometric
amount of CuDPP at 50 °C, the desired coupled product
(+)-48 was obtained in 50% yield. Under these conditions,
byproduct 49a, resulting from an undesired transfer of an n-
butyl group from the stannane 46, was also formed in 50% yield
(Table 1, entry 3). By switching the copper additive to
copper(I) thiophene-2-carboxylate (CuTC), we could favor the
formation of the desired coupled product (+)-48 (60% yield)
over the undesired byproduct 49a (40% yield, Table 1, entry
Concerning the synthesis of (−)-agelastatin C (3), a wide
range of oxidants tested were found to be ineffective for the
direct oxidation of the C4-methine of (−)-agelastatin A (1).
Thus, we devised a strategy based on the oxidation of
(−)-dehydroagelastatin A (51; Scheme 11). We were pleased
to find that (−)-O-methyl-di-epi-agelastatin A (50) could be
readily converted to (−)-dehydroagelastatin A (51) upon being
heated in pyridine at 115 °C in 95% yield (Scheme 11).48
Treatment of (−)-dehydroagelastatin A (51) with dimethyl-
dioxirane (DMDO) provided (−)-di-epi-agelastatin C (52) in
E
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Scheme 11. Enantioselective Synthesis of (−)-Agelastatins
Scheme 13. Formation of the C4−C13 Bond upon Acidic
Treatment of Enamide 57
a
A−C (1−3) and E (5)
propose that byproduct 56 is formed via protodebromination at
C13, followed by enamide formation and C4 to C13
cyclization. Since we were unable to confirm the intermediacy
of pyrrolopyrazinone 57 in the reaction mixture, we cannot
exclude a mechanism involving protodebromination after the
C4−C13 cyclization.
The formation of byproducts 55 and 56 is consistent with
the lower C4-nucleophilicity of (+)-O-methyl-pre-agelastatin D
(53) compared to that of (+)-O-methyl-pre-agelastatin A (39).
Indeed, monitoring the rates of deuterium incorporation at the
C4-position of (+)-O-methyl-pre-agelastatins A (39) and D
(53), respectively, revealed that deuterium incorporation at C4
occurred 10 times faster in (+)-39 as compared to (+)-53 (k1 =
3.094 × 10−5 s−1, k2 = 3.028 × 10−6 s−1),37 consistent with its
more efficient C4−C8 bond formation (Scheme 14). It is
a
Reagents and conditions: (a) HCl(aq), MeOH, 65 °C, 89%; (b)
MeSO3H, H2O, 100 °C; MeOH, 71% [2:1 (−)-1/(−)-50]; (c)
MeSO3H, H2O, 100 °C; MeOH [66% (−)-1 and 30% recovered
(−)-50]; (d) NBS, DTBMP, THF, H2O, 0 °C, 84% (1 → 2); (e)
Amberlyst 15, MeOH, 65 °C, 96% (1 → 5); (f) pyridine, 115 °C,
99%; (g) DMDO, acetone, H2O, 0 °C, 98%; (h) Amberlyst 15, H2O,
100 °C, 41%. DTBMP = 2,6-di-tert-butyl-4-methylpyridine, and
DMDO = dimethyldioxirane.
Scheme 14. Rate Constants for the Deuterium Incorporation
at C4 of (+)-39 and (+)-53
98% yield via oxidation on the convex face. Notably, heating an
aqueous solution of (−)-di-epi-agelastatin C (52) in the
presence of Amberlyst 15 resin afforded (−)-agelastatin C
(3) in 41% yield along with recovered (−)-di-epi-agelastatin C
(52; 42% yield).
Our copper-mediated cross-coupling reaction of thioester
and organostannane enabled an efficient synthesis of (+)-O-
methyl-pre-agelastatin D (53; Scheme 12) as described in the
interesting to note that the scarcity of natural (−)-agelastatin D
(4) compared to other N1-methyl agelastatin alkaloids is
consistent with this observation on the lower efficiency of the
desired cyclization with 53 as compared to 39.50
Scheme 12. Total Synthesis of (−)-Agelastatin D (4) and the
a
Formation of Byproducts 55 and 56
Copper-Mediated Cross-Coupling Reactions of Thio-
esters and Organostannanes: Expanded Scope and
Application. The successful copper-mediated cross-coupling
reactions between thioester 45 and organostannanes 46 and 47
(Table 1) prompted our examination of their scope and
potential broader application (Scheme 15). Of particular
Scheme 15. Versatile Synthesis of Azaheterocycle 63
a
Reagents and conditions: (a) MeSO3H, H2O, 100 °C; HCl, MeOH
[26% (−)-4, 9% (−)-54, 20% 55, 20% ( )-56]; (b) MeSO3H, H2O,
100 °C; HCl, MeOH, 23 °C, 68%; (c) NBS, DTBMP, THF, H2O, 0
°C, 86%.
following section (Table 2) and consequently the first synthetic
sample of (−)-agelastatin D (4). While we were pleased to
access alkaloid (−)-4, this cyclization was plagued by the
competing reaction pathways involving the C6−C7 bond
cleavage, resulting in byproduct 55 (20% yield) and C4−C13
cyclization, giving byproduct 56 (20% yield).
Interestingly, heating an aqueous acidic solution of 13-
desbromoenamide 57 resulted in clean formation of tetracycle
56 (43%, Scheme 13).49 Consistent with this observation, we
interest was the possible general use of this methodology to
allow synthesis of versatile ketone 62 by formation of the C1−
C2 bond from thioester 60 and organostannane 61 (X = O or
NR). We envisioned that condensative cyclization of the urea
or guanidine function of intermediate 62 onto the C1-carbonyl
would provide an expeditious route to the corresponding
imidazol-2-one (X = O) or related azaheterocycle 63, common
substructures in various natural products.17
F
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Our cross-coupling reaction was effective for both alkyl and
aryl thioester coupling partners. Both cyclohexanecarbonyl
thioester 64 and benzoyl thioester 65 underwent efficient cross-
coupling with stannyltriazone 47 to give the corresponding
ketones 71 and 72 in 95% and 99% yield, respectively (Table 2,
entries 1 and 2). Consistent with our prior observation (Table
1), the use of organostannane 46, containing n-butyl auxiliary
ligands, as a coupling partner resulted in a lower yield (Table 2,
entry 3) due to competitive formation of the undesired n-butyl
ketone byproduct. Stannylguanidine 6737 proved to be highly
effective for the introduction of the guanidine functionality as
illustrated through its cross-coupling with thioesters 65 and 66,
affording the corresponding ketones 73 and 75, respectively
(Table 2, entries 4 and 6). Importantly, ketones 73 and 75
could be readily converted to the corresponding 2-amino-
imidazoles in quantitative yield upon treatment with TFA and
warming, highlighting the synthetic utility of our method as a
means to generate 2-aminoimidazoles (Table 2, entries 5 and
7). Stannyltriazone 68 was found to undergo smooth coupling
with adipic thioester 66 to provide diketone 77 in 82% yield
(Table 2, entry 8). Gratifyingly, under our standard reaction
conditions, the stannylurea 69 was found to afford the desired
cross-coupling with the complex thioester (+)-45 to give the
corresponding ketone, which after treatment with methanolic
hydrogen chloride at 65 °C provided (+)-O-methyl-pre-
agelastatin A (39; 58% yield and 99% ee, Table 2, entry 9).16
This direct coupling between thioester (+)-45 and stannylurea
69 enabled us to further streamline our synthesis of
(−)-agelastatin A (1) to seven steps from commercially
available material.16 Similarly, (+)-O-methyl-pre-agelastatin D
(53; Table 2, entry 10)16 could be prepared from thioester
(+)-45 and urea 70 in 62% yield (two steps), which enabled the
first synthetic access to (−)-agelastatin D (4; vide supra). The
efficient union of a variety of aminostannanes and thioester
fragments and subsequent direct conversion of the ketotriazone
and ketoguanidine intermediates to the corresponding
imidazolones and aminoimidazoles is promising for future
application of this chemistry to the synthesis of other oroidin-
based natural products such as cyclooroidin (11), nagelamides,
sceptrins, and many other derivatives.51
Table 2. Copper-Mediated Thioester−Aminostannane
Cross-Coupling and Application to Azaheterocycle
e
Synthesis
Anticancer Activity and Structure−Activity Relation-
ship Studies. The enantioselective total synthesis of all six
(−)-agelastatin alkaloids (1−6), as well as eight structurally
related alkaloids and advanced intermediates, provided an
opportunity to further probe the structure−activity relationship
for this class of natural products. Significantly, we set out to
provide the first comparative anticancer activity study involving
all natural members of this family of alkaloids in five human cell
lines. Previous reports have tested the activity of (−)-agelastatin
A (1) relative to other available derivatives in a single cell line,
showing that structural changes do affect the activity;1a,c,3,4
however, no comprehensive comparison of the entire class of
agelastatins against multiple cell lines has been performed.
Furthermore, although (−)-agelastatin A (1) has been
characterized for its antineoplastic activity in a range of cancer
types, including leukemia, prostate, breast, colon, uterine,
pancreatic, lung, and others,1a,c,2−7 the heterogeneity across
these studies makes it impossible to make a true assessment of
selectivity for any one type of cancer. We sought to refine the
structure−activity relationship of this class with our complete
panel of (−)-agelastatins A−F (1−6, respectively) and test it
more broadly against a range of cancer cell lines.
a
The ketone intermediate 73 was treated with TFA in toluene at 85
b
°C, yield over two steps. The ketone intermediate 75 was treated
with TFA in toluene at 70 °C, yield over two steps. The ketone
intermediate was filtered and treated with HCl in methanol at 65 °C,
one step. The cross-coupling intermediate was isolated and treated
with HCl in methanol at 45 °C, yield over two steps. Tol = C6H4-p-
Me, and c-Hx = cyclohexyl.
c
d
e
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Table 3. Assessment of the Anticancer Activity (IC50, μM) of (−)-Agelastatins A−F and Advanced Intermediates against Human
a
Cell Lines after a 48 h Exposure
compd
U-937
HeLa
A549
BT549
IMR90
(−)-1
(−)-2
(−)-3
0.067 0.003
0.708 0.090
4.8 1.2
>10
1.05 0.14
>10
0.278 0.076
4.8 1.1
>10
1.11 0.35
>10
1.06 0.16
>10
>10
>10
(−)-4
(−)-5
(−)-6
0.240 0.033
2.56 0.13
>10
1.00 0.20
8.60 0.81
>10
0.92 0.16
>10
0.631 0.082
6.9 2.5
>10
2.75 0.60
>10
>10
>10
(−)-50
(−)-51
(−)-52
(+)-39
(+)-44
(+)-45
(+)-48
(+)-53
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
a
Cell lines: U-937, lymphoma; HeLa, cervical carcinoma; A549, non-small-cell lung carcinoma; BT549, breast carcinoma; IMR90, immortalized lung
fibroblasts. Forty-eight hour IC50 values (μM) as determined by MTS (U-937) and SRB (HeLa, A549, BT549, and IMR90). MTS = 3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, and SRB = sulforhodamine B.
a
Table 4. Forty-Eight Hour Activity (μM) of (−)-Agelastatins A−F against Human Blood Cancer Cell Lines
compd
CEM
Jurkat
Daudi
HL-60
CA46
(−)-1
(−)-2
(−)-3
(−)-4
(−)-5
(−)-6
0.020 0.002
0.29 0.20
2.1 1.3
0.074 0.007
0.75 0.44
5.31 0.35
0.210 0.063
1.50 0.33
>10
0.020 0.003
0.46 0.28
7.2 2.9
0.138 0.066
2.4 1.0
>10
0.187 0.071
1.07 0.42
>10
0.074 0.025
0.83 0.37
>10
0.202 0.015
1.41 0.53
>10
0.54 0.12
4.6 3.6
>10
0.46 0.24
2.45 0.95
>10
a
Cell lines: CEM, acute lymphoblastic leukemia; Jurkat, acute T-cell leukemia; Daudi, Burkitt’s lymphoma; HL-60, acute promyelocytic leukemia;
CA46, Burkitt’s lymphoma. Forty-eight hour IC50 values (μM) as determined by MTS. MTS = 3-(4,5-dimethylthiazol-2-yl)-5-(3-
(carboxymethoxy)phenyl)-2-(4-sulfophenyl)-2H-tetrazolium.
While this total synthesis program generated more than four
dozen compounds for testing, after a preliminary examination
of HeLa and U-937 cells indicated many were completely
inactive, we decided to focus extensively on the six natural
(−)-agelastatins along with eight closely related derivatives that
either bear the tetracyclic framework or are immediate
precursors to the complex alkaloids (Table 3). The six
(−)-agelastatin alkaloids (1−6), (−)-O-methyl-di-epi-agelasta-
tin A (50), (−)-dehydroagelastatin A (51), (−)-di-epi-
agelastatin C (52), and (+)-O-methyl-pre-agelastatins A and
D (39 and 53, respectively), along with bicyclic pyrroles (+)-44
and (+)-45 and triazone (+)-48, were tested for their ability to
induce cell death in four human cancer cell lines (U-937,
lymphoma; HeLa, cervical carcinoma; A549, non-small-cell
lung carcinoma; BT549, breast carcinoma) and one immortal-
ized normal human cell line (IMR90, lung fibroblasts) after a
48 h exposure.37 As shown in Table 3, (−)-agelastatin A (1)
exhibited the highest potency in all cell lines tested, whereas
(−)-agelastatins C (3) and F (6) showed no activity at the
concentrations examined. Furthermore, we observed an
enhanced activity of (−)-agelastatin A (1) in U-937 (20×)
and BT549 (4×) cells relative to the other cell lines (Table 3).
(−)-Agelastatin D (4), which lacks a methyl group on the D-
ring, also showed reasonable activity against U-937 cells and
modest activity against BT549 cells. This activity was selective
relative to the other cell lines, and follows a trend in activity
similar to that of (−)-agelastatin A (1). (−)-Agelastatin B (2)
and (−)-agelastatin E (5) showed weak activity, albeit with the
same overall pattern as alkaloids (−)-1 and (−)-4.
Furthermore, none of the epi-agelastatin derivatives [(−)-50
or (−)-52], O-methyl-pre-agelastatins [(+)-39 or (+)-53],
dehydroagelastatin (−)-51, or any structurally simpler deriva-
tives [(+)-44, (+)-45, or (+)-48] showed any activity at the
concentrations tested against these cell lines (Table 3).
Together, this set of data allows the first direct comparison of
all naturally occurring agelastatin alkaloids and suggests that the
stereochemistry for the imidazolidinone ring is crucial
[compare (−)-5 vs (−)-50] while addition of the C14-bromide
substituent to the pyrrole is detrimental to the activity (5−20-
fold reduction) of the agelastatins [compare (−)-1 vs (−)-2
and (−)-4 vs (−)-6]. Demethylation of the imidazolidinone
ring gives more modest reductions (1−5-fold reductions) in
anticancer potency as seen by comparing (−)-agelastatin A (1)
and (−)-agelastatin D (4). Methylation of the C5-hydroxyl
reduces the activity by >10-fold as evident by comparing
(−)-agelastatin A (1) and (−)-agelastatin E (5). Finally, C4-
hydroxylation completely abolishes activity as demonstrated by
comparing (−)-agelastatin A (1) and (−)-agelastatin C (3).
The observed necessity of alkylation of the imidazolidinone
ring is consistent with a previous report,7 which has also
indicated that chlorination rather than bromination does not
affect activity.
Anticancer Activity of (−)-Agelastatins A−F in Blood
Cancer Cell Lines. On the basis of the exceptional potency of
(−)-agelastatins A (1) and D (4) against U-937 cells relative to
the other cell lines tested, five additional blood cancer cell lines
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were tested for their sensitivity to the agelastatin alkaloids.
These cell lines spanned a variety of cancer types (CEM, acute
lymphoblastic leukemia; Jurkat, acute T-cell leukemia; Daudi,
Burkitt’s lymphoma; HL-60, acute promyelocytic leukemia;
CA46, Burkitt’s lymphoma). After a 48 h incubation, the ability
of (−)-agelastatins A−F (1−6) to induce cell death was
evaluated. As shown in Table 4, all five cell lines showed
remarkable sensitivity to (−)-agelastatin A (1). The same
trends in potency observed for the various agelastatins with the
general cell panel (Table 3) were also observed. These results,
the first indication of heightened potency of 1 versus a panel of
white blood cell cancer cell lines, highlight the outstanding
potential for the use of agelastatin A (1) against blood
cancers.52 This is especially encouraging given the difficulty of
treating blood cancers clinically.53
agelastatins A (1) and D (4) in these experiments were chosen
to provide a robust response after 24 h. Therefore, the
compounds were tested at 2×, 4×, and 6× concentration
relative to their IC50 determination experiments, which were
performed after 48 h.57 As shown in Figure 3, (−)-agelastatins
A (1) and D (4) induced dose-dependent activation of
procaspase-3 to active caspase-3 and cleavage of PARP-1,
consistent with apoptosis.
Hemolytic Activity of (−)-Agelastatins A−F and
Advanced Intermediates. To further explore the transla-
tional potential of the agelastatin alkaloids, all 14 compounds in
Table 3 were evaluated for their hemolytic activity (Figure 2).
Figure 3. (−)-Agelastatins A (1) and D (4) both induce caspase-
dependent apoptotic cell death. Western blot analysis of procaspase-3
and PARP-1 cleavage at 24 h in U-937 cells using β-actin as the
loading control. The compounds were tested at the indicated
concentrations (2, 4, and 6 times the 48 h IC50 value), and STS was
tested at 10 nM. C3 = caspase-3, PARP = poly(ADP-ribose)
polymerase 1, PC3 = procaspase-3, and STS = staurosporine.
We next examined whether (−)-agelastatins A (1) and D (4)
induced phosphatidylserine exposure prior to membrane
permeabilization. This was determined by evaluating the timing
of phosphatidylserine with FITC-labeled annexin-V relative to
the incorporation of propidium iodide, a DNA stain that can
only enter dead cells. The concentrations of agelastatins A (1)
and D (4) were chosen to provide a robust response after 21 h.
Therefore, the compounds were tested at 4× and 6×
concentration relative to their IC50 determination experiments,
which were performed after 48 h.57 As shown by the scatter
plots in Figure 4, the number of apoptotic cells (lower right
quadrant in Figure 4) increases with the dose of compound.
These results show, for the first time, that these compounds
induce apoptotic death of cancer cells.
Cell Cycle Arrest with (−)-Agelastatins A and D. After
apoptotic induction by (−)-agelastatins A (1) and D (4) was
established, experiments were conducted to probe the ability of
these compounds to induce cell cycle arrest in U-937 cells. As
shown in Figure 5, both compounds induced arrest in the G2/
M-phase, mirroring prior findings7 related to (−)-1 and now
demonstrating a direct relevance to the activity of (−)-agelas-
tatin D (4). Significantly, while arrest in the G2/M-phase is
commonly associated with disruption of microtubules within
the cell,58 using confocal microscopy, we have determined that
neither (−)-agelastatin A (1) nor (−)-agelastatin D (4) affects
tubulin dynamics within cells (Figure S4, Supporting
Information).59 Microtubule stabilization or destabilization
results in heightened or reduced levels of tubulin fluorescence,
respectively, as shown with the known tubulin binders
paclitaxel and colchicine. Treatment with (−)-agelastatins A
(1) and D (4) results in no change in tubulin fluorescence, an
experimental observation consistent with a prior hypothesis
regarding their mode of action.7
Figure 2. Hemolytic activity of (−)-agelastatins A−F and advanced
intermediates. Compounds were tested at 333 μM, and hemolysis was
evaluated after 2 h. DMSO and water served as the negative and
positive controls for hemolysis, respectively. Error bars represent the
standard deviation of the mean, n = 3.
Notably, none of the active agelastatin alkaloids [(−)-1, (−)-2,
(−)-4, or (−)-5] show any nonspecific hemolysis of red blood
cells. The triazone derivative (+)-48 showed some hemolytic
activity; however, this compound was completely inactive in all
cell lines tested (Table 3). In light of the exceptional potency of
(−)-agelastatin A (1) against a range of blood cancer cell lines,
this high degree of selectivity strongly suggests further
translational studies are warranted.
Apoptotic Activity of (−)-Agelastatins A and D. We
next sought to determine whether the most active (−)-agelas-
tatins A (1) and D (4) induce apoptotic cell death. In apoptosis
a cascade of events results in the activation of procaspase-3, a
low-activity zymogen, to caspase-3, a cysteine protease that
cleaves scores of cellular substrates, resulting in cell death.54
A
hallmark of cancer is a resistance to apoptosis;55 thus,
compounds that are able to induce apoptotic death in cancer
cells are interesting mechanistically and have potential as
chemotherapeutics. Experimentally, apoptosis is most readily
detected through (a) the observation of procaspase-3 (PC3)
maturation to caspase-3 (C3), (b) the cleavage of caspase-3
substrates, most notably the enzyme PARP-1, and (c) the
exposure of phosphatidylserine of the exterior cell membrane
before loss of membrane integrity.56 Procaspase-3 maturation
and PARP-1 cleavage can readily be detected via Western
blotting, and phosphatidylserine exposure is detected by
antibody staining and flow cytometry. Concentrations of
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imidazolones and related azaheterocycles, and (4) the
validation of our bioinspired use of the imidazolone for an
advanced-stage C-ring formation, C4−C8 bond formation, and
introduction of three stereogenic centers. The efficiency of our
synthetic sequence was highlighted by >1 g batch preparation
of (−)-agelastatin A (1). The generality of our synthesis
allowed for the first side-by-side testing of all known agelastatin
alkaloids for their ability to induce cell death in U-937
(lymphoma), HeLa (cervical carcinoma), A549 (non-small-cell
lung carcinoma), BT549 (breast carcinoma), and IMR90
(immortalized lung fibroblasts) human cell lines. (−)-Agelas-
tatin A (1) exhibited the highest potency in all cell lines tested,
while (−)-agelastatin D (4) retained antineoplastic activity,
albeit at a 4-fold lower potency than that of (−)-(1). Our
results show, for the first time, that both (−)-agelastatins A and
D induce apoptotic death of cancer cells and that neither affects
tubulin dynamics within cells. We also show that both of these
molecules arrested cell growth in the G2/M-phase. The
exceptional potency of (−)-agelastatins A (1) and D (4)
against U-937 cells led us to investigate all agelastatins (1−6) in
five additional human blood cancer cell lines (Table 4). From
these studies, we found that (−)-agelastatins A (1) is highly
potent against these blood cancer cell lines (20−190 nM)
without affecting normal red blood cells (>333 μM). These
compounds are particularly active against white blood cancer
cell lines, and the selectivity for these cells over normal cells
suggests considerable potential as anticancer agents.
Figure 4. (−)-Agelastatins A (1) and D (4) both induced dose-
dependent apoptosis. Analysis of phosphatidylserine exposure and
propidium iodide inclusion at 21 h in U-937 cells. The compounds
were tested at the indicated concentrations (4 and 6 times the 48 h
IC50 value), and STS was used as a positive control for apoptosis. The
samples were analyzed by flow cytometry for the relative timing of
phosphatidylserine exposure relative to PI inclusion. FITC =
fluorescein isothiocyanate, PI = propidium iodide, and STS =
staurosporine.
EXPERIMENTAL SECTION
■
Information for Key Compounds. For complete experimental
procedures and full characterization data for all (−)-agelastatin
alkaloids A−F (1−6, respectively) in addition to the eight advanced
derivatives (+)-39, (+)-44, (+)-45, (+)-48, (−)-50, (−)-51, (−)-52,
and (+)-53 examined in our anticancer activity assays, please see the
Supporting Information of our previous report of the total synthesis of
all (−)-agelastatins.16 For complete experimental procedures and full
characterization data for the key compounds (+)-41, 47, (−)-54, 55,
and ( )-56 discussed in our fully optimized route, please see the
Supporting Information of our previous report of the total synthesis of
all (−)-agelastatins.16 For complete experimental procedures and full
characterization data for all new substrates and products reported in
Table 3, please see below.
S,S-Di-p-tolyl hexanebis(thioate) (66). To a flask charged with
adipic acid (4.0 g, 27 mmol, 1 equiv) was added thionyl chloride (5.0
mL, 68 mmol, 2.5 equiv), and the reaction flask was equipped with a
reflux condensor and heated to 85 °C (the exhaust gases were passed
through a 5 N aqueous potassium hydroxide solution). After 1.5 h, the
reaction mixture was allowed to cool to 23 °C and concentrated under
reduced pressure to afford an acyl chloride derivative as a viscous oil.60
The resulting crude material was dissolved in dichloromethane (10
mL), and the resulting mixture was transferred to a solution of 4-
methylbenzenethiol (7.3 g, 57 mmol, 2.1 equiv) in dichloromethane
(30 mL) via cannula at 0 °C. The reaction mixture was allowed to
gently warm to 23 °C. After 2 h, triethylamine (2.0 mL, 14 mmol, 0.52
equiv) was added to the reaction mixture. After 25 min, more
triethylamine (3.0 mL, 22 mmol, 0.79 equiv) was added to the reaction
mixture. After 16.5 h, the reaction mixture was diluted with
dichloromethane (210 mL) and saturated aqueous sodium bicarbonate
solution (250 mL), and the layers were separated. The aqueous layer
was extracted with dichloromethane (250 mL), and the combined
organic layers were dried over anhydrous sodium sulfate, were filtered,
and were concentrated under reduced pressure. The residue was
purified by flash column chromatography (silica gel, diameter 9.0 cm,
height 12 cm; eluent 11% ethyl acetate in hexanes) to afford thioester
Figure 5. (−)-Agelastatins A (1) and D (4) both exhibit dose-
dependent G2/M cell cycle arrest in synchronized U-937 cells after 16
h. The samples were fixed, treated with an RNase, stained with PI, and
analyzed by flow cytometry. Paclitaxel, a microtubule stabilizer, was
used as a positive control. Error bars represent the standard deviation
of the mean, n = 3. An asterisk indicates p < 0.01 based on a two-tailed
Student’s t-test. PI = propidium iodide.
CONCLUSIONS
■
We describe the development of a concise, stereocontrolled,
and biosynthetically inspired synthetic strategy toward the
agelastatin alkaloids, the development of a versatile new
synthetic methodology for azaheterocycle synthesis, and its
successful implementation in the synthesis of all known
(−)-agelastatins (1−6) and many derivatives. In addition, we
describe the first comparative study of the anticancer activity of
all known agelastatin alkaloids. Key features of our syntheses
include (1) the early introduction of C13-bromide to suppress
C7-enolization, (2) the development of a CuTC-mediated
cross-coupling reaction between thioester and organostannane,
(3) a new [4 + 1] annulation approach for the synthesis of
1
66 (4.6 g, 47%) as a white solid. Mp: 105−107 °C. H NMR (500
MHz, CDCl3, 21 °C): δ 7.27 (app d, J = 8.1 Hz, 4H), 7.20 (app d, J =
J
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7.9 Hz, 4H), 2.67−2.64 (m, 4H), 2.36 (s, 6H), 1.78−1.75 (m, 4H).
13C NMR (125.8 MHz, CDCl3, 21 °C): δ 197.7, 139.9, 134.6, 130.2,
ketotriazone 71 (16 mg, 95%) as a colorless oil. 1H NMR (500 MHz,
CDCl3, 21 °C): δ 7.08 (d, J =8.0 Hz, 2H), 6.94 (d, J =8.6 Hz, 2H),
4.69 (s, 2H), 4.66 (s, 2H), 4.09 (s, 2H), 2.88 (s, 3H), 2.32 (tt, J =11.4,
3.4 Hz, 1H), 2.27 (s, 3H), 1.80 (dd, J = 13.3, 2.2 Hz, 2H), 1.76−1.72
(m, 2H), 1.66−1.60 (m, 2H), 1.32 (ddd, J = 15.2, 12.4, 3.1 Hz, 2H),
1.25−1.15 (m, 2H). 13C NMR (125.8 MHz, CDCl3, 21 °C): δ 210.4,
156.1, 146.0, 132.6, 130.1, 119.8, 67.4, 67.4, 53.5, 48.2, 32.4, 28.4, 25.9,
25.7, 20.8. FTIR (neat, cm−1): 2929 (s), 2855 (m), 1720 (m), 1647
(s), 1514 (s), 1300 (m), 1198 (m), 829 (m). HRMS (ESI, TOF) (m/
z): calcd for C19H26N3O2 [M − H]− 328.2025, found 328.2030. TLC
(ethyl acetate): Rf = 0.40 (CAM).
124.3, 43.2, 25.0, 21.5. FTIR (neat, cm−1): 2929 (m), 1687 (s), 1493
(m), 1034 (m), 813 (s). HRMS (ESI, TOF) (m/z): calcd for
C20H22NaO2S2 [M + Na]+ 381.0959, found 381.0965. TLC (17%
ethyl acetate in hexanes): Rf = 0.53 (CAM, UV).
1-((Tricyclohexylstannyl)methyl)-N,N′-di-tert-butylcarba-
moylguanidine (67). (Tricyclohexylstannyl)methanamine (910 mg,
2.28 mmol, 1 equiv) was dissolved in acetonitrile (60 mL), and (E)-
tert-butyl(((tert-butoxycarbonyl)imino)(1H-pyrazol-1-yl)methyl)-
carbamate (1.09 g, 3.43 mmol, 1.50 equiv) was added sequentially at
23 °C. After 11 h, the resulting mixture was concentrated under
reduced pressure, and the crude residue, adsorbed onto silica gel, was
purified by flash column chromatography (silica gel, diameter 4.0 cm,
height 12 cm, eluent 3.3% ethyl acetate in hexanes) to afford
tricyclohexyltin reagent 67 (1.3 g, 91%) as a white amorphous residue.
1H NMR (500 MHz, CDCl3, 21 °C): δ 11.39 (s, 1H), 8.44 (t, J = 5.1
Hz, 1H), 3.08 (d, J = 5.6 Hz, 2H), 1.84 (t, J = 4.4 Hz, 6H), 1.62 (d, J =
9.6 Hz, 9H), 1.53 (t, J = 7.1 Hz, 9H), 1.48 (s, 9H), 1.45 (s, 9H), 1.28−
1.21 (m, 9H). 13C NMR (125.8 MHz, CDCl3, 21 °C): δ 163.9, 155.8,
153.6, 82.9, 78.9, 32.4, 29.5, 28.6, 28.2, 27.5, 27.4, 24.1. FTIR (neat,
cm−1): 3327 (s), 2917 (s), 2846 (s), 1717 (s), 1642 (s), 1575 (s),
1409 (s), 1337 (s), 1157 (s), 1052 (s), 909 (m), 735 (m). HRMS
(ESI, TOF) (m/z): calcd for C30H56N3O4Sn [M + H]+ 642.3293,
found 642.3290. TLC (10% ethyl acetate in hexanes): Rf = 0.56
(CAM).
5-Benzyl-1-methyl-3-((tricyclohexylstannyl)methyl)-1,3,5-
triazinan-2-one (68). To a solution of triazone 5-benzyl-1,3-
dimethyl-1,3,5-triazinan-2-one (1.5 g, 6.8 mmol, 2.0 equiv) in
tetrahydrofuran (40 mL) at −78 °C was added sec-butyllithium (1.4
M in cyclohexane, 4.9 mL, 6.8 mmol, 2.0 equiv) via syringe to result in
an orange homogeneous solution. After 5 min, a solution of
tricyclohexyltin chloride (1.42 g, 3.42 mmol, 1 equiv) in tetrahy-
drofuran (10 mL) at −78 °C was transferred to the resulting bright
orange mixture via cannula over a 3 min period. After 8 min, saturated
aqueous ammonium chloride solution (5 mL) was added via syringe.
The resulting mixture was partitioned between dichloromethane (250
mL) and water (200 mL). The layers were separated, the aqueous
layer was extracted with dichloromethane (250 mL), and the
combined organic layers were dried over anhydrous sodium sulfate,
were filtered, and were concentrated under reduced pressure. The
crude residue was purified by flash column chromatography (silica gel,
diameter 5 cm, height 10 cm, eluent hexanes and then 17% ethyl
acetate in hexanes) to afford stannyltriazone 68 (1.8 g, 90%) as a white
amorphous residue. 1H NMR (500 MHz, CDCl3, 21 °C): δ 7.34−7.24
(m, 5H), 4.10 (s, 2H), 4.03 (s, 2H), 3.90 (s, 2H), 2.81 (s, 3H), 2.75 (s,
2H), 1.84 (dd, J = 12.5, 2.0 Hz, 3H), 1.63 (d, J = 8.9 Hz, 12H), 1.55−
1.39 (m, 6H), 1.31−1.17 (m, 12H). 13C NMR (125.8 MHz, CDCl3,
21 °C): δ 156.1, 137.9, 129.1, 128.7, 127.7, 69.7, 67.6, 55.8, 32.9, 32.4,
29.5, 29.0, 27.9, 27.4. FTIR (neat, cm−1): 2917 (s), 2229 (m), 1634
(s), 1519 (s), 1445 (s), 1408 (m), 1297 (s), 1144 (m), 908 (s), 735
(s). HRMS (ESI, TOF) (m/z): calcd for C30H50N3OSn [M + H]+
588.2987, found 588.2976. TLC (17% ethyl acetate in hexanes): Rf =
0.32 (CAM, UV).
1-(2-Cyclohexyl-2-oxoethyl)-3-methyl-5-(p-tolyl)-1,3,5-tria-
zinan-2-one (71). Anhydrous tetrahydrofuran (1.0 mL, degassed
thoroughly by passage of a stream of argon) was added via syringe to a
flask charged with thioester 64 (12.0 mg, 51.2 μmol, 1 equiv), triazone
4716 (36.0 mg, 61.4 μmol, 1.20 equiv), and copper(I) thiophene-2-
carboxylate (CuTC; 15.3 mg, 76.8 μmol, 1.50 equiv) at 23 °C under
an argon atmosphere, and the reaction mixture was heated to 50 °C.
After 1 h, the reaction mixture was allowed to cool to 23 °C and was
filtered through a plug of Celite with ethyl acetate washings (3 × 1
mL). The resulting mixture was partitioned between ethyl acetate (20
mL) and saturated ammonium chloride aqueous solution (20 mL).
The aqueous layer was extracted with ethyl acetate (2 × 20 mL), and
the combined organic layers were dried over anhydrous sodium sulfate
and were concentrated under reduced pressure. The crude residue was
purified by flash column chromatography (silica gel, diameter 2 cm,
height 10 cm, eluent 25% ethyl acetate in hexanes) to afford
1-Methyl-3-(2-oxo-2-phenylethyl)-5-(p-tolyl)-1,3,5-triazinan-
2-one (72). Anhydrous tetrahydrofuran (1.1 mL, degassed thoroughly
by passage of a stream of argon) was added via syringe to a flask
charged with thioester 65 (12.0 mg, 52.6 μmol, 1 equiv), triazone 4761
(37.0 mg, 63.1 μmol, 1.20 equiv), and CuTC (15.7 mg, 78.8 μmol,
1.50 equiv) at 23 °C under an argon atmosphere, and the reaction
mixture was heated to 50 °C. After 1 h, the reaction mixture was
allowed to cool to 23 °C and was filtered through a plug of Celite with
ethyl acetate washings (3 × 1 mL). The resulting mixture was
partitioned between ethyl acetate (20 mL) and saturated ammonium
chloride aqueous solution (20 mL). The aqueous layer was extracted
with ethyl acetate (2 × 20 mL), and the combined organic layers were
dried over anhydrous sodium sulfate, were filtered, and were
concentrated under reduced pressure. The crude residue was purified
by flash column chromatography (silica gel, diameter 2 cm, height 10
cm, eluent 25% ethyl acetate in hexanes) to afford ketotriazone 72 (17
1
mg, 99%) as a colorless oil. H NMR (500 MHz, CDCl3, 21 °C): δ
7.92 (dd, J = 8.4, 1.2 Hz, 2H), 7.55 (tt, J = 7.4, 1.2 Hz, 1H), 7.42 (app
t, J = 7.8 Hz, 2H), 7.07 (d, J = 8.0 Hz, 2H), 6.95 (app d, J = 8.5 Hz,
2H), 4.80 (s, 2H), 4.73 (s, 2H), 4.72 (s, 2H), 2.92 (s, 3H), 2.28 (s,
3H). 13C NMR (125.8 MHz, CDCl3, 21 °C): δ 196.1, 156.1, 145.9,
135.2, 133.8, 132.6, 130.1, 128.9, 128.3, 119.8, 67.4, 67.3, 52.5, 32.5,
20.3. FTIR (neat, cm−1): 2888 (m), 1694 (m), 1638 (s), 1513 (s),
1288 (m), 1219 (m), 750 (m). HRMS (ESI, TOF) (m/z): calcd for
C19H20N3O2 [M − H]− 322.1556, found 322.1562. TLC (ethyl
acetate): Rf = 0.46 (CAM, UV).
(E)-1,2-Bis(tert-butoxycarbonyl)-3-(2-oxo-2-phenylethyl)-
guanidine (73). Anhydrous tetrahydrofuran (6.0 mL, degassed
thoroughly by passage of a stream of argon) was added via syringe
to a flask charged with thioester 65 (36.6 mg, 0.160 mmol, 1 equiv),
stannylguanidine 67 (113 mg, 0.176 mmol, 1.10 equiv), and CuTC
(32.8 mg, 0.165 mmol, 1.03 equiv) at 23 °C under an argon
atmosphere, and the reaction mixture was heated to 50 °C. After 1 h,
the reaction mixture was allowed to cool to 23 °C, and 5% ammonium
hydroxide aqueous solution (15 mL) was added to the reaction
mixture. The resulting mixture was extracted with dichloromethane (2
× 15 mL), and the combined organic layers were dried over anhydrous
sodium sulfate, were filtered, and were concentrated under reduced
pressure. The crude residue was purified by flash column
chromatography (silica gel, diameter 2.5 cm, height 15 cm, eluent
11% ethyl acetate in hexanes) to afford ketoguanidine 73 (58 mg,
1
96%) as a white foam. Mp: 75−78 °C. H NMR (500 MHz, CDCl3,
21 °C): δ 11.5 (s, 1H, NH), 9.41 (s, 1H, NH), 7.98 (dd, J = 8.5, 1.2
Hz, 2H), 7.59 (tt, J = 7.4, 1.3 Hz, 1H), 7.46 (app t, J = 7.7 Hz, 1H),
4.92 (d, J = 4.1 Hz, 2H), 1.51 (s, 9H), 1.50 (s, 9H). 13C NMR (125.8
MHz, CDCl3, 21 °C): δ 193.4, 163.5, 156.1, 153.1, 134.4, 134.3, 129.1,
128.3, 83.5, 79.8, 48.4, 28.4, 28.3. FTIR (neat, cm−1): 3319 (m), 2980
(m), 1727 (s), 1697 (m), 1642 (s), 1618 (s), 1409 (m), 1308 (s),
1148 (s), 734 (m). HRMS (ESI, TOF) (m/z): calcd for C19H28N3O5
[M + H]+ 378.2029, found 378.2035. TLC (20% ethyl acetate in
hexanes): Rf = 0.44 (CAM, UV).
2-Amino-5-phenyl-1H-imidazol-3-ium 2,2,2-Trifluoroacetate
(74). To a flask charged with ketoguanidine 73 (57.9 mg, 0.153 mmol,
1 equiv) were added toluene (4 mL) and trifluoroacetic acid (120 μL,
1.53 mmol, 10.0 equiv) via syringe, and the reaction mixture was
heated to 85 °C. After 13.5 h, the reaction mixture was allowed to cool
to 23 °C and was concentrated under reduced pressure. Water (2 mL)
and trifluoroacetic acid (120 μL, 1.53 mmol, 10.0 equiv) were added
via syringe to the residue, and the resulting mixture was heated to 85
K
dx.doi.org/10.1021/jo4020112 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
°C. After 1.5 h, the reaction mixture was allowed to cool to 23 °C and
was concentrated under reduced pressure to give 2-aminoimidazole 74
(34 mg, 82%) as a pale yellow solid. Mp: 154−156 °C. 1H NMR (500
MHz, CD3OD, 21 °C): δ 7.56 (app d, J = 7.2 Hz, 2H), 7.43 (app t, J =
7.7 Hz, 2H), 7.34 (app tt, J = 7.4, 1.2 Hz, 1H), 7.13 (s, 1H). 13C NMR
(125.8 MHz, CD3OD, 21 °C): δ 163.1, 149.7, 130.3, 129.7, 129.2,
129.1, 125.6, 109.9, 101.4. FTIR (neat, cm−1): 3182 (br s), 1682 (br
s), 1204 (s), 1139 (s), 842 (m). HRMS (ESI, TOF) (m/z): calcd for
C9H10N3 [M + H]+ 160.0869, found 160.0867.
(E,E)-1,1′-(2,7-Dioxooctane-1,8-diyl)bis(2,3-bis(tert-
butoxycarbonyl)guanidine) (75). Anhydrous tetrahydrofuran (34
mL, degassed thoroughly by passage of a stream of argon) was added
via syringe to a flask charged with thioester 66 (300 mg, 0.836 mmol, 1
equiv), stannylguanidine 67 (1.34 g, 2.09 mmol, 2.50 equiv), and
CuTC (374 mg, 1.88 mmol, 2.25 equiv) at 23 °C under an argon
atmosphere, and the reaction mixture was heated to 50 °C. After 1 h,
the reaction mixture was allowed to cool to 23 °C. The resulting
mixture was partitioned between dichloromethane (145 mL) and 5%
ammonium hydroxide aqueous solution (145 mL). The aqueous layer
was extracted with dichloromethane (3 × 145 mL), and the combined
organic layers were dried over anhydrous sodium sulfate, were filtered,
and were concentrated under reduced pressure. The crude residue was
purified by flash column chromatography (silica gel, diameter 4 cm,
height 10 cm, eluent 11% ethyl acetate in hexanes) to afford
ketoguanidine 75 (549 mg, 100%) as a white foam. Mp: 133−136 °C.
1H NMR (500 MHz, CDCl3, 21 °C): δ 11.37 (s, 2H), 9.02 (s, 2H),
4.28 (d, J = 4.3 Hz, 4H), 2.42 (app s, 4H), 1.60 (t, J = 3.1 Hz, 4H),
1.47 (s, 9H), 1.46 (s, 9H). 13C NMR (125.8 MHz, CDCl3, 21 °C): δ
203.9, 163.4, 155.9, 153.0, 83.5, 79.7, 51.0, 39.9, 28.4, 28.2, 23.1. FTIR
(neat, cm−1): 3319 (br m), 2980 (m), 2253 (w), 1726 (s), 1643 (s),
1618 (s), 1408 (m), 1309 (s), 1151 (s), 1058 (m), 734 (m). HRMS
(ESI, TOF) (m/z): calcd for C30H53N6O10 [M + H]+ 657.3823, found
657.3801. TLC (50% ethyl acetate in hexanes): Rf = 0.44 (CAM).
4,4′-(Butane-1,4-diyl)bis(2-amino-1H-imidazol-3-ium) 2,2,2-
Trifluoroacetate (76). Toluene (40 mL) and trifluoroacetic acid
(650 μL, 8.36 mmol, 10.0 equiv) were added via syringe to a flask
charged with ketoguanidine 75 (549 mg, 0.836 mmol, 1 equiv), and
the resulting mixture was heated to 75 °C. After 16 h, the reaction
mixture was allowed to cool to 23 °C and was concentrated under
reduced pressure. Water (20 mL) and trifluoroacetic acid (650 μL,
8.36 mmol, 10.0 equiv) were added via syringe to the residue, and the
resulting mixture was heated to 100 °C. After 45 h, the reaction
mixture was allowed to cool to 23 °C and was concentrated under
reduced pressure to give 2-aminoimidazole 76 (375 mg, 100%) as a
brown solid. Mp: 200−203 °C. 1H NMR (500 MHz, CD3OD, 21 °C):
δ 6.49 (s, 2H), 2.53 (app t, J = 6.5 Hz, 4H), 1.65 (app t, J = 7.1 Hz,
4H). 13C NMR (125.8 MHz, CD3OD, 21 °C): δ 162.6, 148.8, 128.8,
109.8, 101.4, 28.7, 25.2. FTIR (neat, cm−1): 3179 (br s), 2361 (w),
1702 (s), 1442 (m), 1205 (s), 1134 (s), 845 (m), 723 (m). HRMS
(ESI, TOF) (m/z): calcd for C10H17N6 [M + H]+ 221.1509, found
221.1512.
1,8-Bis(5-benzyl-3-methyl-2-oxo-1,3,5-triazinan-1-yl)octane-
2,7-dione (77). Anhydrous tetrahydrofuran (8.0 mL, degassed
thoroughly by passage of a stream of argon) was added via syringe
to a flask charged with thioester 66 (70.9 mg, 0.198 mmol, 1 equiv),
triazone 68 (290 mg, 0.494 mmol, 2.50 equiv), and CuTC (88.4 mg,
0.448 mmol, 2.25 equiv) at 23 °C under an argon atmosphere, and the
reaction mixture was heated to 50 °C. After 1.5 h, the reaction mixture
was allowed to cool to 23 °C. The resulting mixture was partitioned
between dichloromethane (30 mL) and 5% ammonium hydroxide
aqueous solution (30 mL). The aqueous layer was extracted with
dichloromethane (30 mL), and the combined organic layers were
dried over anhydrous sodium sulfate, were filtered, and were
concentrated under reduced pressure. The crude residue was purified
by flash column chromatography (silica gel, diameter 3 cm, height 11
cm, eluent 24% chloroform, 5% methanol, and 0.6% ammonium
hydroxide in dichloromethane) to afford ketotriazone 77 (89 mg,
82%) as a colorless oil. 1H NMR (500 MHz, CDCl3, 21 °C): δ 7.33−
7.26 (m, 10H), 4.19 (s, 4H), 4.15 (s, 4H), 4.00 (s, 4H), 3.99 (s, 4H),
2.84 (s, 6H), 2.40 (t, J = 6.9 Hz, 4H), 1.57 (q, J = 3.3, 4H). 13C NMR
(100.6 MHz, CDCl3, 21 °C): δ 206.9, 155.7, 137.8, 129.3, 128.8,
127.8, 67.8, 67.5, 55.6, 54.6, 39.6, 32.6, 23.0. FTIR (neat, cm−1): 3442
(br s), 2929 (m), 2237 (w), 1717 (m), 1635 (m), 1506 (m), 1266 (s),
739 (s). HRMS (ESI, TOF) (m/z): calcd for C30H41N6O4 [M + H]+
549.3189, found 549.3183. TLC (24% chloroform, 5.4% methanol,
and 0.6% ammonium hydroxide in dichloromethane): Rf = 0.27
(CAM, UV).
Cell Culture Information. Cells were grown in media
supplemented with fetal bovine serum (FBS) and antibiotics (100
μg/mL penicillin and 100 U/mL streptomycin). Specifically, experi-
ments were performed using the following cell lines and media
compositions: U-937, HeLa, A549, BT549, CEM, Daudi, and Jurkat
(RPMI-1640 + 10% FBS), CA46 (DMEM + 10% FBS), HL-60
(IMDM + 10% FBS), and IMR90 (EMEM + 10% FBS). The cells
were incubated at 37 °C in a 5% CO2, 95% humidity atmosphere.
IC50 Value Determination for Adherent Cells using Sulfo-
rhodamine B (SRB). Adherent cells (HeLa, A549, BT549, and
IMR90) were added into 96-well plates (5000 cells/well for the HeLa
cell line, 2000 cells/well for the A549, BT549, and IMR90 cell lines) in
100 μL of medium and were allowed to adhere for 2−3 h. The
compounds were solubilized in DMSO as 100× stocks, added directly
to the cells (100 μL final volume), and tested over a range of
concentrations (1 nM to 10 μM) in triplicate (1% DMSO final) on a
half-log scale. DMSO and cell-free wells served as the live and dead
controls, respectively. After 48 h of continuous exposure, the plates
were evaluated using the SRB colorimetric assay as described
previously.62 Briefly, medium was removed from the plate, and the
cells were fixed by the addition of 100 μL of cold 10% trichloroacetic
acid in water. After incubation at 4 °C for 1 h, the plates were washed
in water and allowed to dry. Sulforhodamine B was added as a 0.057%
solution in 1% acetic acid (100 μL), and the plates were incubated at
room temperature for 30 min, washed in 1% acetic acid, and allowed
to dry. The dye was solubilized by adding 10 mM Tris base solution
(pH 10.5, 200 μL) and incubating at room temperature for 30 min.
The plates were read at λ = 510 nm. IC50 values were determined from
three or more independent experiments using TableCurve (San Jose,
CA).
IC50 Value Determination for Nonadherent Cells Using MTS.
In a 96-well plate, the compounds were preadded as DMSO stocks
(1% final) in triplicate to achieve final concentrations of 1 nM to 10
μM on a half-log scale. DMSO and cell-free wells served as the live and
dead controls, respectively. Suspension cells (U-937, CEM, CA46,
Daudi, HL-60, and Jurkat, 10 000 cells/well) were distributed in 100
μL of medium to the compound-containing plate. After 48 h, cell
viability was assessed by adding 20 μL of a PMS/MTS solution63 to
each well, allowing the dye to develop at 37 °C until the live control
had processed MTS, and reading the absorbance at λ = 490 nm. IC50
values were determined from three or more independent experiments.
Hemolysis Assay Using Human Erythrocytes. To prepare the
erythrocytes, 0.1 mL of human blood was centrifuged (10000g, 2 min).
The pellet was washed three times with saline (0.9% NaCl) via gentle
resuspension and centrifugation (10000g, 2 min). Following the final
wash, the erythrocytes were resuspended in 0.4 mL of red blood cell
(RBC) buffer (10 mM Na2HPO4, 150 mM NaCl, 1 mM MgCl2, pH
7.4).
DMSO stocks of the compounds were added to 0.5 mL tubes in
singlicate (1 μL, 3.3% DMSO final). The stocks were diluted with 19
μL of RBC buffer. Positive control tubes contained DMSO in water,
and negative control tubes contained DMSO in RBC buffer. A
suspension of washed erythrocytes (10 μL) was added to each tube,
and the samples were incubated at 37 °C for 2 h. The samples were
centrifuged (10000g, 2 min), and the supernatant was transferred to a
clear, sterile 384-well plate. The absorbance of the supernatants was
measured at λ = 540 nm, and the percent hemolysis was calculated
relative to the average absorbance values measured for the controls.
Apoptosis in U-937 Cells with Annexin V−FITC and
Propidium Iodide (AnnV/PI). DMSO stocks of the compounds
were added to a 24-well plate in singlicate (0.2% DMSO final). After
compound addition, 0.5 mL of a U-937 cell suspension (250 000 cells/
mL) was added and allowed to incubate for 21 h. Following treatment,
L
dx.doi.org/10.1021/jo4020112 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
the cell suspensions were transferred to flow cytometry tubes and
pelleted (500g, 3 min). The medium was removed by aspiration, and
the cells were resuspended in 200 μL of AnnV binding buffer (10 mM
HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) with 5 μg/mL PI and
a 1:90 dilution of AnnV. The samples were analyzed using flow
cytometry.
Cell Cycle Arrest in Thymidine-Synchronized U-937 Cells. U-
937 cells were split to 50% confluency (250 000 cells/mL) and treated
with 2 mM thymidine for 10 h. The cells were then pelleted (500g, 3
min) and washed with PBS before being resuspended in thymidine-
free medium and allowed to recover for 13 h. The cells were then
reblocked with 2 mM thymidine for 10 h, then washed with PBS as
before, and resuspended in medium (250 000 cells/mL).
DMSO stocks of the compounds were added to a 24-well plate in
triplicate (0.2% DMSO final), after which 1 mL of the prepared cell
suspension was added. Following a 16 h incubation, the cell
suspensions were transferred to 2 mL tubes and pelleted (600g, 3
min). The medium was removed by aspiration, and the cells were fixed
with 0.5 mL of ice cold 70% ethanol with vortexing. The samples were
fully fixed at −20 °C for 3 h. The samples were then pelleted (1000g, 5
min), and the supernatant was removed via aspiration. The cells were
incubated with 50 μL of 5 μg/mL RNaseA in PBS for 3 h at room
temperature. Prior to reading, the samples were taken up in 150 μL of
50 μg/mL propidium iodide in PBS and transferred to flow cytometry
tubes. The samples were analyzed on the basis of whole, single cells.
Tubulin Microscopy with HeLa Cells. Round, 1.5 mm coverslips
(no. 1.5) were sterilized with ultraviolet light and placed in a 12-well
plate. HeLa cells (100 000 cells/well) were added and allowed to
adhere for 8 h. The compounds were then added as DMSO stocks to
achieve a final DMSO concentration of 1%, and the plates were
returned to the incubator for 16 h.
Following incubation, the medium was removed, and the coverslips
were fixed with 0.5 mL of microtubulin-stabilizing buffer (MTSB; 80
mM PIPES, pH 6.8, 1 mM MgCl2, 5 mM EGTA, 0.5% TX-100) +
0.5% glutaraldehyde for 10 min at room temperature, after which the
fixative was removed and the sample was quenched with the addition
of 0.5 mL of freshly prepared 1 mg/mL NaBH4 in PBS. After a 5 min
incubation, the solution was removed by aspiration, and the coverslips
were washed with PBS.
ACKNOWLEDGMENTS
■
We acknowledge financial support by the National Institutes of
Health National Institute of General Medical Sciences (NIH-
NIGMS) (Grant GM074825) and the University of Illinois. We
thank Dr. Justin Kim and Dr. Peter Muller for assistance with
̈
X-ray crystallographic analysis. We thank Dr. Stephen Lathrop
for preparation of new samples for biological study. K.C.M. is a
National Science Foundation predoctoral fellow and a Robert
C. and Carolyn J. Springborn graduate fellow.
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ASSOCIATED CONTENT
■
S
* Supporting Information
General procedures, additional notes concerning our earlier
routes to agelastatins A (1) and B (2) and the rate of C4-
deuterium incorporation in (+)-O-methyl-preagelastatins A
(39) and D (53), tubulin microscopy images of HeLa Cells,
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Dong, G. Pure Appl. Chem. 2010, 82, 2231.
crystal structure of desbromomethyl ester S20, H and 13C
1
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NMR spectra, and CIF data. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: movassag@mit.edu.
Notes
The authors declare no competing financial interest.
M
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