Largazole and analogues with modified metal-binding motifs targeting histone deacetylases: Synthesis and biological evaluation

Article abstract of DOI:10.1021/jm200432a

The histone deacetylase inhibitor largazole 1 was synthesized by a convergent approach that involved several efficient and high yielding single pot multistep protocols. Initial attempts using tert-butyl as thiol protecting group proved problematic, and synthesis was accomplished by switching to the trityl protecting group. This synthetic protocol provides a convenient approach to many new largazole analogues. Three side chain analogues with multiple heteroatoms for chelation with Zn²⁺ were synthesized, and their biological activities were evaluated. They were less potent than largazole 1 in growth inhibition of HCT116 colon carcinoma cell line and in inducing increases in global H3 acetylation. Largazole 1 and the three side chain analogues had no effect on HDAC6, as indicated by the lack of increased acetylation of α-tubulin.

Full text of DOI:10.1021/jm200432a

ARTICLE
pubs.acs.org/jmc
Largazole and Analogues with Modified Metal-Binding Motifs
Targeting Histone Deacetylases: Synthesis and Biological Evaluation
Pravin Bhansali,†,§ Christin L. Hanigan,‡,§ Robert A. Casero, Jr.,‡ and L. M. Viranga Tillekeratne*,†
†Department of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo,
2801 W. Bancroft Street, Toledo, Ohio 43606, United States
‡The Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Bunting-Blaustein Cancer
Research Building 1, 1650 Orleans Street, Room 551, Baltimore, Maryland 21231, United States
S Supporting Information
b
ABSTRACT: The histone deacetylase inhibitor largazole 1 was
synthesized by a convergent approach that involved several
efficient and high yielding single pot multistep protocols. Initial
attempts using tert-butyl as thiol protecting group proved
problematic, and synthesis was accomplished by switching to
the trityl protecting group. This synthetic protocol provides a
convenient approach to many new largazole analogues. Three
side chain analogues with multiple heteroatoms for chelation
with Zn2+ were synthesized, and their biological activities were
evaluated. They were less potent than largazole 1 in growth inhibition of HCT116 colon carcinoma cell line and in inducing
increases in global H3 acetylation. Largazole 1 and the three side chain analogues had no effect on HDAC6, as indicated by the lack
of increased acetylation of α-tubulin.
’ INTRODUCTION
non-histone substrate proteins. The deacetylated histone, posi-
tively charged at physiological pH, interacts with the negatively
charged DNA phosphate backbone and can form transcription-
ally inactive condensed forms of chromatin. These epigenetic
changes can result in silencing of tumor suppressor genes
promoting cancer initiation and/or progression. In some cases,
inhibition of HDACs can be used to restore hyperacetylation to
derepress or activate these undesirably silenced genes.4 HDAC
proteins are a family of at least 18 enzymes and are classified into
four groups based on their size, cellular localization, active
catalytic site numbers, and homology to yeast HDAC proteins:5
class I, HDAC1, -2, -3, and -8; class IIa, HDAC4, -5, -7, and -9;
class IIb, HDAC6 and -10; class III, sirtuin proteins; class IV,
HDAC11. The HDAC proteins are associated with basic cellular
functions and disease states such as cancer, but the importance of
individual isoforms in cell function and cancer biology is not well
understood. However, class I isoforms are regarded as promis-
ing cancer targets.6 Two HDAC inhibitors (HDACis) suberoyl-
anilide hydroxamic acid (SAHA, vorinostat) and FK228
(romidepsin) have recently been approved by the USFDA for
the treatment of cutaneous T-cell lymphoma (CTCL). SAHA
with a hydroxamic acid moiety as the metal binding domain is
a pan-inhibitor of HDACs. FK228 has a thiol masked as a cyclic
disulfide bond with a cysteine residue of the depsipeptide
core. Many other HDACis are at different stages of clinical
A major challenge in cancer chemotherapy is the development
of drugs that selectively kill cancer cells without affecting normal
cells for increased potency and reduced toxic side effects.
Changes in gene expression due to mutation, loss, or rearrange-
ment are associated with cancer initiation and progression. With
few exceptions, these changes have been difficult to target
therapeutically. A new paradigm that is being extensively inves-
tigated as an anticancer drug development strategy is the target-
ing of epigenetic regulation of gene expression. Epigenetics refer
to heritable alterations in gene expression that are not a result of
changes in DNA sequences. In cancer, aberrant epigenetic
silencing of several genes including tumor suppressor genes is
a common occurrence.1 Aberrant epigenetic silencing occurs as a
result of aberrant promoter region CpG island DNA methylation
in concert with changes in covalent modification of histone
proteins.1g Enzymatic modifications such as acetylation, methy-
lation, and phosphorylation of the lysine and arginine residues of
the N-terminus of histones regulate the access to DNA by
transcriptional factors, thereby regulating gene expression.2
Histone acetylation modulated by two protein families, histone
acetyltransferases (HATs) and histone deacetylases (HDACs), is
the most extensively studied of these processes.3 Most impor-
tantly, epigenetic changes, unlike DNA sequence changes, are
reversible and thus there is interest in targeting epigenetic gene
regulation as an anticancer strategy.
HDACs are a class of metalloenzymes responsible for removal
of the acetyl group from lysine residues of histone and other
Received: April 11, 2011
Published: September 21, 2011
r
2011 American Chemical Society
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Figure 2. Largazole analogues targeted to Zn2+ binding motif.
focused mostly on alterations in the cap group and to a lesser
extent on the side chain. The four-atom linker between the
hydrophobic cap group and the Zn2+ coordinating group in
largazole 1 and other HDACis with a masked thiol as the Zn2+
binding domain such as FK228, spiruchostatins, and FR901375 2
appears to be the optimal distance for highest potency in
comparison to side chains with five to seven atoms which are
common among most HDACis.10d,11c Replacement of the zinc-
binding thiol of largazole 1 with a number of benzamide and
thioamide headgroups failed to produce more potent
analogues.10c The active sites of HDAC isoforms are highly
conserved. However, the presence of an internal cavity within the
active site has been exploited in designing isoform-selective
HDACis with substituted benzamide derivatives as the zinc-
binding group.12 Therefore, it is conceivable that structural
alterations in the zinc-binding domain of largazole 1 to modulate
metal binding affinity may take advantage of such minor struc-
tural differences within the active site to develop isoform- or
class-selective inhibitors. Isoform-selective inhibitors may help in
understanding the role of different isoforms in cellular processes
and disease states. Our approach to modifying the metal-binding
domain consisted of introducing a second heteroatom in the side
chain two to three atoms apart from sulfur and is thus capable of
interacting with Zn2+ via a five- or six-membered cyclic transition
state (Figure 2).
Figure 1. Natural HDAC inhibitors with anticancer properties.
investigation7 with a preference for isoform-selective or class-
selective inhibitors for higher therapeutic potential.5 X-ray
crystallography and SAR studies of HDAC inhibitors have shown
that three key structural elements are involved in the binding of
the inhibitor to the active site of the HDAC protein:8 (a) a metal
binding domain, which chelates with the Zn2+ cation present
at the active site; (b) a surface recognition cap group which
interacts with hydrophobic residues on the rim of the active site;
(c) a linker, which mimics the N-acetyllysine side chain of histone
and occupies a hydrophobic channel positioning the zinc binding
moiety and the cap group in binding orientation.
Recently, a new natural product largazole (1, Figure 1),
isolated from a marine cyanobacterium of the genus Symploca
was reported to possess remarkable selective activity against
highly invasive transformed human mammary epithelial cells
(MDA-MB-231) and transformed fibroblastic osteosarcoma
(U2OS) cells over the normal cell lines (NMuMG and NIH3T3,
respectively).9
Mechanistic studies showed that largazole 1 is an inhibitor of
HDACs.10 It is a prodrug that undergoes hydrolysis of the
thioester group by cellular esterases and/or lipases to release a
free thiol function that constitutes the domain that chelates Zn2+.
The depsipeptide ring system represents the surface recognition
cap group. The two domains are connected by a four-carbon
olefinic linker. A striking similarity of largazole 1 to other natural
HDAC inhibitors such as FK228,10b FR901375 2,10b and spir-
uchostatins (Figure 1) is that they all contain a masked 3-hydro-
xy-7-mercapto-4-heptenoic acid side chain. Upon activation, they
release the free thiol as the active species.
Because of its highly selective activity on specific cancer cell
lines and a strong and exceptional picomolar inhibitory bias for
class I HDACs (HDAC1, -2, and -3) over class II HDAC6,
largazole 1 has been subjected to extensive investigation since its
discovery in 2008.10b A number of synthetic approaches to
largazole 1 have appeared since then.10a,b,11 Its high potency
and selectivity, along with the presence of a metal-binding
domain and a surface recognition group interacting with differ-
ently conserved regions of the receptor, make largazole 1 a
fascinating lead molecule for further structural optimization in
pursuit of molecules of higher potency and/or selectivity. An
array of largazole analogues has been synthesized and their
biological properties evaluated to uncover some of the SAR
requirements of the molecule.10,11,11c,11gÀ11i These studies have
’ CHEMISTRY
The convergent synthetic approach adopted as shown in
retrosynthetic analysis (Scheme 1) involved macrolactamiza-
tion and side chain thioesterification of the intermediate 24
which could be derived by acyl transfer from fragment 20 to 22
followed by coupling with Fmoc-valine. Intermediate 22 can
be synthesized from the thiazole nitrile 7 and (R)-α-methyl-
cysteine hydrochloride 14. Synthesis of 14 would also gen-
erate its enantiomer useful for making the C-7 epimers of
largazole 1 and analogues. Fragment 20 is the product of
acetate aldol reaction of the aldehyde 18 using acetyl Nagao as
the chiral auxiliary.13 In our initial approach when tert-butyl
was used as the thiol protecting group, deprotection and
thioesterification to produce the final product became proble-
matic (vide infra).
The synthesis of thiazole derivative 7 is shown in Scheme 2.
Thiazole amide 6 was made in 60% overall yield from boc-
thioglycinamide 4 and bromopyruvic acid in a one-pot reaction
in which the initially formed carboxylic acid, after removal of
water, was activated and treated with ammonia. This one-pot
protocol was found to be more efficient and gave the product in
higher yields and in a much shorter period of time than the
previously reported methods.11a Amide 6 was converted to the
nitrile 7 using standard conditions in 99% yield.11a
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Scheme 1. Retrosynthetic Analysis of Largazole 1
Scheme 2. Synthesis of Fragment 7
The synthesis of (R)-α-methylcysteine HCl 14 and its (S)-
enantiomer 15 was achieved via the enzymatic resolution of
racemic precursor 11 (Scheme 3).14 Reaction of chloroacetone 8
and tert-butyl mercaptan under basic conditions gave 9, which
was subjected to BuchererÀBerg conditions to produce hydan-
toin 10. This one-pot protocol gave 90% yield of 10 after two
steps. Hydantoin 10 was converted to 11 in one pot by hydrolysis
with aqueous NaOH followed by KCNO treatment. Resolution
of 11 with D-hydantoinase afforded 12 and 13 which were
converted to 14 and 15, respectively, in quantitative yields.14
Turning our attention to the synthesis of 20 (Scheme 4),
conjugate addition of triphenylmethanethiol to acrolein 16 gave
17, which was used in a Wittig reaction to yield 18.15 This one-
pot protocol gave 65% overall yield for the two steps. Acetate
aldol condensation of the aldehyde 18 with acetyl Nagao auxiliary
was used to synthesize 20 in 84% diastereomeric excess.13 The S-
configuration of the newly created chiral center of molecule 20
was established by modified Mosher ester analysis.16
thiazole-thiazoline carboxylic acid 21.11a After simultaneous
removal of Boc group and esterification of the free carboxylic
acid under acidic conditions in MeOH, the acyl group was
transferred17 from 20 to 22 to obtain 23. The formation of 21
from nitrile 7 and the one pot conversion of 21 to 23 proved
very efficient with 78% yield for three steps. Yamaguchi
esterification10a,18 was used to couple Fmoc-valine to 23 to
afford the acyclic precursor 24. After saponification and
Fmoc group removal, macrocyclization with HOAt, HATU,
and Hunig’s base yielded the cyclized product 25 in 56%
overall yield over the three steps.10a Deprotection of trityl
group gave largazole thiol which was esterified with octanoyl
chloride using Hunig’s base to give largazole 1 in 79% yield
(based on recovered largazole thiol in esterification step)
over two steps.10b
Scheme 6 shows the general method used for the synthesis of
largazole analogues with modification in the zinc-binding motif.
These analogues are designed such that thiol and a second
heteroatom in the side chain are two to three atoms apart and
are thus capable of interacting with Zn2+ via five- or six-
membered cyclic transition states (Figure 2).
With the necessary building blocks in hand, the assembly of
largazole 1 was undertaken as shown in Scheme 5. The nitrile 7
was condensed with (R)-α-methylcysteine HCl 14 to obtain the
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Scheme 3. Synthesis of (R)- and (S)-α-Methylcysteine
Scheme 4. Synthesis of the β-Hydroxycarboxamide 20
The analogues 27, 28, and 29 were synthesized following the
removal of trityl group by nucleophilic substitution of the
corresponding precursors by either method A (basic)19 or one-
pot method B (acidic)20 as shown in Scheme 6.
’ BIOLOGICAL EVALUATION
The antiproliferative activity of analogues 27, 28, and 29 were
evaluated in the HCT116 colon adenocarcinoma cell line by a
standard 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfophenyl)-2H-tetrazolium (MTS) reduction assay (Promega)
using largazole 1 as the control as previously reported.24 Figure 3
shows the results of the MTS assay after 96 h of treatment with 10
nM, 100 nM, 1 μM, and 5 μM concentrations of the compounds.
Largazole 1 inhibited the growth of HCT116 cells with a GI50 of
10 nM, whereas the three side chain analogues showed activity
only at higher concentrations. The pyridine analogue 27 demon-
strated the greatest effect on cell survival of the three with a GI50
of 1.0 μM.
In order to ascertain the inhibitory effects of these analogues,
in vitro fluorimetric assays on recombinant HDACs were per-
formed in the presence and absence of the analogues. Analogues
27 and 28 at 1 μM significantly inhibited HDAC1 activity by
almost 80% (Figure 4). Analogue 29 had a modest inhibition of
HDAC1, with only a ∼30% decrease of activity. Largazole 1
demonstrated a decrease of HDAC1 activity similar to analogues
27 and 28. Assays using HeLa nuclear extracts as a source for
class 1 and class 2 HDAC activity were nearly identical to what
In a previous attempt to synthesize largazole 1 by a similar
approach, we used the tert-butyl group as the protecting group for
thiol. The synthetic route to make the tert-butyl protected
alcohol 35 is depicted in Scheme 7. Synthesis of alcohol 35
started from glycerol formal 30 which was converted to dioxene
31 in two steps. Alkylation of 31 with 2-[(2-bromoethyl)-
sulfanyl]-2-methylpropane 32 via the lithium derivative gave 33.
Compound 33 underwent retro DielsÀAlder reaction to give the
aldehyde 3421 which was used in aldol reaction to furnish the
alcohol 35. It was used as described in Scheme 5 to make the
cyclic core 38. Attempts to remove the tert-butyl group as
illustrated in Scheme 8 (1 M BBr3,22 trifluoroacetic acid, anisole,
mercuric acetate23) and to esterify with octanoyl chloride to form
largazole 1 failed (see Supporting Information for details). With
BBr3, starting material was recovered. Treatment with TFA/
anisole/mercuric acetate indicated (TLC) the formation of a
polar product which was subjected to thioesterification, but no
largazole 1, largazole thiol, or starting material was recovered.
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Scheme 5. Synthesis of Largazole 1
Scheme 6. Synthesis of Analogues with Modified Metal-Binding Motif
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Scheme 7. Synthesis of tert-Butyl Protected β-Hydroxycarboxamide 35
Scheme 8. Attempted Synthesis of Largazole 1
Figure 3. Growth inhibitory effects of largazole 1 and analogues on
HCT116 colon carcinoma cells. Each point represents the mean of two
experiments; each with 4 replicates with the error bars indicating the SEM.
Figure 4. In vitro HDAC inhibitory activity À Recombinant HDAC1
and 6 activity was measured with and without the analogues. Reactions
were carried out for 1 h at a concentration of 1 μM for all analogues.
HDAC1 showed the greatest inhibition with analogues 27 and 28, while
HDAC6 inhibition was more modest and less specific to any of the
largazole based analogues.
was observed with HDAC1 (data not shown). In vitro evaluation
of the inhibition of recombinant HDAC6 enzyme showed ∼50%
decrease of activity with all of the analogues, including largazole
1, at a concentration of 1 μM (Figure 4).
In order to ascertain the effect of the analogues on HDAC6
activity, we studied the levels of α-tubulin acetylation after
exposure to 10 nM, 100 nM, and 1 μM. Again, we measured
the α-tubulin acetylation using Western blot analysis of whole
cell lysates from cells treated for 24 h. No changes in α-tubulin
acetylation were observed by exposure to any of the analogues,
including largazole 1 (Figure 5). This concurs with earlier
findings that HDAC6 is not a target for largazole 1.10b
To evaluate the effects of these compounds on HDAC activity
in a more physiologically relevant system, we treated a colorectal
cancer cell line HCT116 with these analogues at increasing
concentrations for 24 h. We examined the downstream effects
of HDAC inhibition on global histone H3 acetylation by Western
blot analysis after 24 h of cellular exposure to 10 nM, 100 nM, and
1 μM of each compound. No increase in global acetylation was
observed in treated cells exposed to any of the compounds at
10 nM (Figure 5). Largazole 1 showed an increase of global
acetylation at 100 nM. Analogues 27 and 28 showed a significant
increase in global acetylation at 1 μM, though much lower than
what was observed with largazole 1.
’ DISCUSSION
Histone deacetylase inhibitors that modulate post-transcrip-
tional modification of proteins and target reversible epigenetic
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replacement of dihydrothiazoline with dihydrooxazoline,10c and
the substitution of pyridine for thiazole10c to generate the most
potent HDACi known to date. Major SAR determinants of the
side chain are the preference for a trans-alkene vs a cis-alkene,11h
preference for thiol vs esters,11a ketones,11a benzamides, and α-
thioamides10c as the metal-binding moiety, and most importantly
the requirement of an optimal four-atom linker between the hydro-
phobic cap group and the Zn2+ coordination thiol group10d,11c in
comparison to five to seven atom side chains common among
most HDACis. We modified the metal-binding domain by
introducing a second heteroatom in the side chain two to three
atoms apart from sulfur to facilitate chelation with Zn2+ via a five- or
six-membered cyclic transition state.
We adopted a convergent approach to the synthesis of
largazole 1 and used it as a general method to synthesize side
chain analogues by nucleophilic substitution of the desired
precursors with largazole thiol. In our initial approach to
largazole 1 we used tert-butyl for thiol protection. The tert-butyl
protected 5-mercapto-2-pentenal 34 was conveniently obtained
by the retro DielsÀAlder reaction of the 6-substituted-1,3-
dioxene 33. After conversion to the tert-butyl protected 3-hydro-
xy-7-mercapto-4-heptenoic acid derivative 35, it was used in our
convergent approach to synthesize tert-butyl protected largazole
thio ether 38. Attempts to convert 38 to largazole 1 by removal of
the tert-butyl group by a number of standard protocols and
esterification with octanoyl chloride were unsuccessful. This was
overcome by switching to the trityl protecting group. Largazole 1
and the side chain analogues were synthesized via largazole thiol
following removal of trityl under acid conditions.
Figure 5. Western blot analysis of global acetylated H3 and acetylated
α-tubulin proteins following the treatment of HCT116 cells with
largazole 1 (L) and analogues at 10 nM, 100 nM and 1 μM concentra-
tions after 24 h of treatment. Increases of global acetylation were
observed with largazole 1 at 100 nM and 1 μM and modest increases
with analogues 27 and 28. α-tubulin levels remained unchanged.
Largazole 1 inhibited the growth of HCT116 cells with a GI50
of ∼10 nM. The three side chain analogues were less active, the
pyridine analogue 27 being the most active of the three with a
GI50 of ∼1.0 μM. The other two analogues were active at higher
concentrations. In vitro analysis of HDAC activity demonstrated
that all of these analogues have some inhibitory effects on
recombinant HDAC1 and HDAC6. The inhibition of HDAC6
observed with analogues 27 and 28 was modest in comparison to
their effects on HDAC1 or global class 1 and 2 HDACs. The
modest inhibition of HDAC1 by largazole 1 observed is some-
what in contrast to previous reports.10a,b However, the increased
global acetylation of H3 in treated cells indicates efficient in situ
inhibition of HDAC1. The effects on cellular proliferation are
consistent with significant increase of global H3 acetylation
observed with largazole 1 at 1 μM and modest induction of
global H3 acetylation observed for analogues 27 and 28. There
was no change in acetylated α-tubulin levels with any of the
compounds, indicating they have no effect on HDAC6. The
marked difference in the activity of largazole 1 in the cellular
assays compared to the in vitro HDAC activity assays can be
attributed to the hydrolysis of the prodrug largazole 1 to largazole
thiol by cellular esterases. Thioethers bind weakly to hard or
borderline metal ions like Mg2+, Mn2+, Cu2+, and Zn2+.27 These
largazole analogues were designed to see whether the introduc-
tion of a second heteroatom would lead to stronger binding to
the metal ion and also to isoform/class selectivity. However, it is
not possible to suggest if the diminished biological activity
observed is due to poor affinity of thioether group for Zn2+ ion
or incompatibility of the modified metal binding moiety with the
HDAC active site. Depsipeptides such as largazole 1 and FK228
constitute a distinct class of HDACis characterized by a masked
thiol function as the metal-binding domain and a side chain with
a characteristic four-atom spacer between the macrocycle and the
repression of gene expression are a promising source of selective
anticancer agents. Although the basic cellular functions of
individual HDAC isoforms are not fully characterized, the
aberrant expression of specific HDAC isoforms in some types
of cancer cells suggests that specific isoforms may be targeted to
develop selective anticancer agents. With two inhibitors SAHA
(vorinostat) and FK228 (romidepsin) already approved by the
USFDA for treatment of cutaneous T-cell lymphoma (CTCL)
and a number of other candidates in the drug pipelines, HDACis
are being extensively investigated as potential selective anticancer
agents. SAHA with a promiscuous hydroxamic acid group as the
metal-binding domain is a pan-inhibitor of HDACs, whereas
FK228 with a 3-hydroxy-7-mercapto-4-heptenoic acid side chain
masked as a disulfide bridge as the metal-binding domain is a
class I inhibitor with no effect on HDAC6. Although the thiol
group is not as strong a Zn2+ binding functionality as hydroxamic
acid,25 FK228 has a higher potency than SAHA due to additional
compensatory binding interaction of the cap group with the rim
region of the active site. The recently isolated depsipeptide
largazole 1 has a high degree of structural and functional
similarity to FK228 and, like FK228, is a prodrug of the active
thiol. A range of largazole analogues synthesized by a number of
groups has unraveled some SAR information related to potency
and selectivity. The cyclic cap of the molecule which interacts
with the less conserved rim region of the receptor has been the
major target of structural modifications. Notable among them are
the variable substitution of thiazoline methyl at C-7 or oxidation
of thiazoline to thiazole11i and replacement of L-valine with other
amino acids10d,11h without adverse effect on potency, reduction
in potency accompanying change of configuration at C210c,26
and C1710d and conversion of lactone to lactam,10e equipotent
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metal binding domain. The side chain occupies the hydrophobic
channel between the metal binding site and the hydrophobic rim
of the active site and positions the two end groups of the inhibitor
for binding interaction with the receptor. Despite the high
sequence similarity within the active site, the presence of discrete
binding cavities in the vicinity of the metal ion may be taken
advantage of to achieve isoform selectivity as exemplified by
HDACis with substituted benzamide metal-binding domains and
class I selectivity.12 The exploration of alternative metal-binding
domains is the way forward to the development of such isoform
and class-selective HDACis.
bromopyruvic acid (0.835 g, 5 mmol, 1 equiv) in dry THF (20 mL) was
stirred at 50 °C under nitrogen for 2 h. The reaction mixture was
concentrated to dryness, and the residue was dried by repeated
azeotropic removal of moisture with toluene. The crude carboxylic acid
thus obtained was dissolved in dry THF (50 mL), and triethylamine
(1.3 g, 12.94 mmol, 2.6 equiv) and ethyl chloroformate (0.681 g, 6.24 mmol,
1.25 equiv) were added at 0 °C and stirred for 30 min at the same
temperature. Ammonium hydroxide (∼ 1.1 g, 31.76 mmol, 5 equiv) was
added, and the reaction mixture was stirred at room temperature for 2 h.
It was concentrated in vacuo and purified by flash column chromatog-
raphy on silica gel in ethyl acetate/hexanes, 33À100%, to yield 6 (0.768
g, 60% over two steps from boc-thioglycinamide): mp 153À154 °C
(lit.11c mp 153 °C). 1H NMR (400 MHz, CDCl3): δ 8.08 (s, 1H), 7.13
(br s, 1H), 5.94 (brs, 1H), 5.34 (br s, 1H), 4.59 (d, J = 5.6 Hz, 2H), 1.47
(s, 9H) ppm. 13C NMR (100 MHz, CDCl3): δ 169.7, 163.1, 155.8,
149.3, 124.8, 80.8, 42.5, 28.5 ppm.
tert-Butyl (4-Cyanothiazol-2-yl)methylcarbamate (7). To a
solution of amide 6 (0.204 g, 0.797 mmol, 1 equiv) in dichoromethane
(20 mL) at 0 °C was added triethylamine (0.174 g, 1.725 mmol, 2.16
equiv) followed by dropwise addition of trifluoroacetic anhydride (0.181
g, 0.863 mmol, 1.08 equiv). The reaction mixture was stirred at room
temperature for 1 h, concentrated in vacuo, and purified by flash
chromatography on silica gel in ethyl acetate/hexanes, 20À50%, to
obtain the nitrile 7 (0.189 g, 99%): mp 84À85 °C (lit.11c mp 84 °C). 1H
NMR (600 MHz, CDCl3): δ 7.95 (s, 1H), 5.3 (s, 1H), 4.62 (d, J = 6 Hz,
2H), 1.47 (s, 9H). 13C NMR (100 MHz, CDCl3): δ 171.7, 155.8, 131.1,
126.6, 114, 81, 42.5, 28.5.
(2E)-5-[(Triphenylmethyl)thio]-2-pentenal (18). To a solution
of triphenylmethanethiol (6.91 g, 25 mmol, 2.09 equiv) in dichloro-
methane (100 mL) was added acrolein 16 (1.965 g, 35 mmol, 2.9 equiv)
and triethylamine (3.56 g, 35 mmol, 2.9 equiv). The resulting mixture
was stirred for 1 h at room temperature and was concentrated to give the
aldehyde 17 as a white solid, which was used in the next step without
purification. A solution of the aldehyde 17 obtained above and (tri-
phenylphosphoranylidene)acetaldehyde (3.64 g, 11.96 mmol, 1 equiv)
in dry benzene (150 mL) was refluxed for 8 h. The reaction mixture was
concentrated and purified by flash chromatography on silica gel in
dichloromethane/hexanes, 20À25%, to afford aldehyde 18 (5.83 g, 65%
over the two steps): mp 140À141 °C. 1H NMR (400 MHz, CDCl3): δ
9.43 (d, J = 8.0 Hz, 1H), 7.42 (dd, J = 2.4, 7.6 Hz, 6H), 7.29 (dt, J = 2.0,
6.8 Hz, 6H), 7.22 (dt, J = 2.4, 7.2 Hz, 3H), 6.60À6.67 (m, 1H),
5.95À6.01 (dd, J = 8.0, 15.6 Hz, 1H), 2.29À2.37 (m, 4H). 13C NMR
(100 MHz, CDCl3): δ 194, 156, 144.7, 133.8, 129.7, 128.2, 127, 67.2,
31.9, 30.2.
’ CONCLUSION
Largazole 1 and its analogues were synthesized efficiently and
in high yields using several one-pot two-reaction protocols. The
stereochemistry of 20 was installed by acetate aldol reaction.
Macrolactamization with HATU, HOAt, and Hunig’s base gave
cyclic product 25. An initial attempt to synthesize largazole 1
using the tert-butyl group to protect thiol proved problematic
because of difficulty in removing the protecting group for
thioesterification. By use of the trityl group instead, deprotection
followed by thioesterification with octanoyl chloride provided
largazole 1. This method provides an efficient synthetic approach
to largazole analogues. Three side chain analogues with multiple
heteroatoms for metal binding synthesized using this approach
were found to be less potent than largazole 1 as measured by
growth inhibition in HCT116 colorectal carcinoma cell line and
induction of global H3 acetylation. However, the moderate
selectivity for HDAC1 over HDAC6 observed with analogues
27 and 28 in the in vitro HDAC inhibitory activity assay may
guide the structural modification of the metal-binding motif to
design selective HDAC inhibitors. Further studies in this direc-
tion are continuing.
’ EXPERIMENTAL SECTION
THF was refluxed with Na and benzophenone and freshly distilled
prior to use. NMR spectra were recorded on Varian INOVA 600 MHz
and Varian VXRS 400 MHz instruments and calibrated using residual
undeuterated solvent as internal reference (CDCl3, 1H NMR at δ 7.26,
13C NMR at δ 77.23; D2O, 1H NMR at δ 4.8; DMSO-d6, 1H NMR at δ
2.5, 13C NMR at δ 39.51). Optical rotations were recorded on an
AUTOPOL III 589/546 polarimeter. High-resolution mass spectra
(HRMS) were recorded on a Micromass LCT electrospray mass
spectrometer at the Central Instrument Facility, Wayne State University,
Detroit, MI, and on a Micromass Q-Tof II electrospray mass spectrom-
eter at the Mass Spectrometry and Proteomics Facility, The Ohio State
University, Columbus, OH. Crude products were purified by flash
column chromatography on silica gel (32À63 μm) purchased from
Dynamic Adsorbents Inc. and by preparative thin layer chromatography
on 1000 μm uniplates purchased from Analtech Inc. using commercial
solvents as specified. HPLC analyses were performed on a Waters 1525
binary pump HPLC system with Waters 2487 dual wavelength absor-
bance detector on a Symmetry C18 column (reverse phase, 5 μm, 4.6
mm  150 mm) using a linear gradient of 10À100% H2O/MeOH over
15À20 min; flow rate of 1 mL/min and UV detection at 254 nm.
Structural integrity and purity of the test compounds were determined
3S-Hydroxy-1-(4R-isopropyl-2-thioxothiazolidin-3-yl)-7-
tritylsulfanylhept-4E-en-1-one (20). To a solution of acetyl
Nagao chiral auxiliary (1.493 g, 7.355 mmol, 1 equiv) in dichloro-
methane (60 mL) at 0 °C was added TiCl4 (1.72 g, 9.05 mmol,
1.23equiv). After the reaction mixture was stirred for 5 min and cooled
to À78 °C, Hunig’s base (1.872 g, 9.2 mmol, 1.25 equiv) was added. The
mixture was stirred for 2 h at the same temperature, and to it was added
the aldehyde 18 (2.6 g, 7.263 mmol, 0.987 equiv) in dichloromethane
(8 mL) dropwise. The reaction mixture was stirred for 1 h at À78 °C. It
was removed from cooling bath, treated with water (15 mL), and diluted
with dichloromethane (50 mL). The aqueous portion was extracted with
dichloromethane; the organic layer was washed with saturated NaCl
(40 mL) and dried over anhydrous Na2SO4. It was concentrated in
vacuo and the residue was purified by flash chromatography on silica gel
in dichloromethane/hexanes, 25À90%, to obtain the major isomer 20 as
a thick yellow oil (1.963 g, 76.5% brsm), and the diastereomer 19 (0.193
g, 7.5%) with recovery of acetyl Nagao chiral auxiliary (0.513 g). Major
1
by the composite of H and 13C NMR, HRMS, and HPLC and were
found to be >95% pure. D-Hydantoinase, recombinant, immobilized
from E. coli was purchased from Fluka Chemie AG (catalog no. 53765,
CAS no. 9030-74-4).
tert-Butyl (4-Carbamoylthiazol-2-yl)methylcarbamate (6).
A mixture of Boc-thioglycinamide 4 (0.95 g, 5 mmol, 1 equiv) and
20
1
isomer: [α]D À149 (c 3.7, CHCl3). H NMR (400 MHz, CDCl3):
δ 7.41 (d, J = 7.2 Hz, 6H), 7.28 (t, J = 7.2 Hz, 6H), 7.21 (t, J = 7.2 Hz, 3H),
5.61À5.55 (m, 1H), 5.46 (dd, J = 6.0, 15.2 Hz, 1H), 5.12 (t, J = 6.8 Hz,
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Journal of Medicinal Chemistry
ARTICLE
1H), 4.57 (t, J = 5.8 Hz, 1H), 3.56 (dd, J = 2.8, 17.6 Hz, 1H), 3.47 (dd, J =
7.6, 11.6 Hz, 1H), 3.28 (dd, J = 8.8, 17.6 Hz, 1H), 2.99 (d, J = 11.6 Hz,
1H), 2.82 (s, 1H), 2.37À2.32 (m, 1H), 2.21 (t, J = 7.2 Hz, 2H), 2.09 (q, J
= 7.2 Hz, 2H), 1.05 (d, J = 6.8 Hz, 3H), 0.97 (d, J = 6.8 Hz, 3H). 13C
NMR (100 MHz, CDCl3): δ 203.1, 172.7, 145, 132, 130.2, 129.7, 128,
126.8, 71.6, 68.6, 66.7, 45.4, 31.6, 31.6, 31, 30.8, 19.3, 18.0.
(R)-2-(2-((tert-Butoxycarbonylamino)methyl)thiazol-4-yl)-4,
5-dihydrothiazole-4-carboxylic Acid (21). To a well stirred
mixture of the nitrile 7 (0.096 g, 0.4 mmol, 1 equiv) and NaHCO3
(0.232 g, 2.76 mmol, 5.6 equiv) in methanol (5 mL) was added (R)-α-
methylcysteine hydrochloride 14 (0.084 g, 0.491, 1.23 equiv) followed
by phosphate buffer, pH 5.95 (2.5 mL). The reaction mixture was
degassed with nitrogen before stirring it under nitrogen at 70 °C for 1 h.
It was acidified with 1 M HCl and extracted with ethyl acetate (15 mL)
three times. The combined organic extract was washed with saturated
NaCl solution, dried over anhydrous sodium sulfate, and concentrated
to obtain the carboxylic acid 21 (0.137 g) which was used in the next step
without further purification.
127.9, 127.7, 127.3, 126.8, 125.2, 122.3, 120.15, 84.7, 72.42, 67.2, 66.8, 59.6,
53.1, 47.3, 41.7, 41.6, 41.3, 31.5, 31.3, 31, 29.9, 24.1, 19.2, 18.1. HRMS-ESI
(m/z): [M + H]+ calcd for C56H57N4O7S3, 993.3397; found, 993.3389.
Cyclic Core 25. To a stirred solution of 24 (133 mg, 0.134 mmol, 1
equiv) in THF/H2O (4:1, 4 mL) at 0 °C was added 0.1 M LiOH
(1.4 mL, 0.14 mmol, 1.045 equiv) dropwise over a period of 15 min.
After the mixture was stirred at 0 °C for 1 h, it was acidified with 1 M HCl
solution and was extracted with EtOAc three times. The combined
organic layer was washed with brine, dried over anhydrous sodium
sulfate, and concentrated. The reaction mixture was purified by pre-
parative TLC in ethyl acetate to get the carboxylic acid. It was dissolved
in dichloromethane (13 mL) and treated with diethylamine (0.462 g,
6.32 mmol, 47.16 equiv). After the mixture was stirred at room
temperature for 3 h, it was concentrated to dryness to afford the free
amino derivative. After the sample was dried azeotropically with toluene,
it was treated with HATU (0.105 g, 0.276 mmol, 2.06 equiv), HOAt
(0.038 g, 0.279 mmol, 2.08 equiv), dichloromethane (130 mL, ∼ 1 mmol),
and Hunig’s base (0.074 g, 0.575 mmol, 4.29 equiv) and the mixture
was stirred for 30 h at room temperature. The reaction mixture was
concentrated to dryness and was purified by flash chromatography on
silica gel in ethyl acetate/hexanes, 10À60%, to yield the cyclic core 25
(0.056 g, 57% over three steps from 24). [α]D20 +2.5 (c 0.95, CHCl3).
1H NMR (400 MHz, CDCl3): δ 7.73 (s, 1H), 7.37 (d, J = 8.4 Hz, 6H),
7.26 (t, J = 8 Hz, 6H), 7.20À7.15 (m, 4H), 6.49 (dd, J = 2.8, 9.2 Hz, 1H),
5.68À5.71 (m, 1H), 5.60 (m, 1H), 5.38 (dd, J = 6.8, 15.6 Hz, 1H), 5.19
(dd, J = 8.8, 17.6 Hz, 1H), 4.55 (dd, J = 3.2, 9.6 Hz, 1H), 4.11 (dd, J = 3.2,
17.6 Hz, 1H), 4.01 (d, J = 11.2 Hz, 1H), 3.26 (d, J = 11.6 Hz, 1H), 2.77
(dd, J = 9.6, 16.4 Hz, 1H), 2.64 (dd, J = 3.2, 16 Hz, 1H), 2.15À2.19 (m,
2H), 1.98À2.06 (m, 2H), 1.82 (s, 3H), 0.67 (d, J = 6.8 Hz, 3H), 0.50 (d, J
= 6.8 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 173.7, 169.4, 168.9,
168.1, 164.6, 147.6, 144.9, 133.3, 129.7, 128.1, 126.8, 124.3, 84.6, 72.0,
66.8, 58.0, 43.5, 41.2, 40.8, 34.2, 31.5, 31.4, 29.9, 24.4, 19.1, 16.9.
Largazole (1). To a solution of 25 (0.033 g, 0.045 mmol, 1 equiv) in
dichloromethane (5 mL) at 0 °C was added triisopropylsilane (0.013 g,
0.083 mmol, 1.85 equiv) followed by trifluoroacetic acid (0.307 g, 2.69
mmol, 59.85 equiv). After being stirred for 3 h at room temperature, the
reaction mixture was concentrated in vacuo. The crude product was
purified by flash chromatography on silica gel in 20% ethyl acetate/
hexanes to first remove impurity followed by ethyl acetate to obtain the
largazole thiol (0.022 g). To a stirred solution of largazole thiol in
dichloromethane (7 mL) at 0 °C were added Hunig’s base (0.045 g, 0.35
mmol, 7.7 equiv) and octanoyl chloride (0.044 g, 0.27 mmol, 6 equiv).
Catalytic DMAP (1 mg) in dichloromethane (0.1 mL) was added to the
reaction mixture. After the mixture was stirred for 4 h at room
temperature, the reaction was quenched with methanol and the mixture
was concentrated in vacuo. Purification of the crude product by
preparative thin layer chromatography on silica gel with ethyl acetate
as solvent gave largazole 1 (0.010 g, 79%, based on recovered largazole
(R)-Methyl 2-(2-((3S-Hydroxy-7-(tritylthio)hept-4E-enam-
ido)methyl)thiazol-4-yl)-4-methyl-4,5-dihydrothiazole-4-
carboxylate (23). A solution of carboxylic acid 21 (0.136 g, 0.38
mmol, 1 equiv) in anhydrous methanol (5 mL) was bubbled with HCl
gas for 5 min. The reaction mixture was stirred overnight and concen-
trated in vacuo to give compound 22 which was azeotroped using
toluene before taking it to the next step. A mixture of above obtained
compound 22 and DMAP (0.121 g, 0.992 mmol, 2.61 equiv) in
dichloromethane (2 mL) was stirred for 5 min, and a solution of aldol
product 20 (0.214, 0.38 mmol, 1 equiv) in dichloromethane (1 mL) was
added. The reaction mixture was stirred for 1 h, concentrated in vacuo,
and purified by flash chromatography on silica gel in ethyl acetate/
hexanes, 20À100%, to afford the alcohol 23 (0.191 g, 78% over three
20
steps aÀc). [α]D À11 (c 3.55, CHCl3). 1H NMR (400 MHz, CDCl3):
δ 7.90 (s, 1H), 7.38 (d, J = 8.4 Hz, 6H), 7.26 (t, J = 7.6 Hz, 6H), 7.19
(t, J = 7.6 Hz, 3H), 7.07 (t, J = 6.0 Hz, 1H), 5.50À5.58 (m, 1H),
5.37À5.43 (dd, J = 6.0, 15.2 Hz, 1H), 4.63À4.74 (m, 2H), 4.43 (m, 1H),
3.86 (d, J = 11.6 Hz, 1H), 3.78 (s, 3H), 3.48 (s, 1H), 3.25 (d, J = 11.6 Hz,
1H), 2.34À2.46 (m, 2H), 2.18 (t, J = 7.2 Hz, 2H), 2.05 (q, J = 7.2, Hz,
2H), 1.63 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 173.8, 172.0, 168.1,
162.9, 148.4, 144.9, 132.4, 130.3, 129.7, 128, 126.8, 122.4, 84.6, 69.2,
66.7, 53.1, 42.9, 41.6, 40.9, 31.6, 31.4, 24.1. HRMS-ESI (m/z): [M + H]+
calcd for C36H38N3O4S3, 672.2019; found, 672.2024
(4R)-Methyl 2-(2-((8S)-1-(9H-Fluoren-9-yl)-5-isopropyl-3,6,
10-trioxo-8-((E)-4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-dia-
zadodecan-12-yl)thiazol-4-yl)-4-methyl-4,5-dihydrothia-
zole-4-carboxylate (24). To a solution of Fmoc-L-valine (0.09 g,
0.0266 mmol, 1 equiv) in THF (1 mL) at 0 °C was added Hunig’s base
(0.045 g, 0.345 mmol, 1.29 equiv) and 2,4,6-trichlorobenzoyl chloride
(0.078 g, 0.32 mmol, 1.2 equiv). The reaction mixture was stirred at 0 °C
for 1 h. When TLC indicated formation of the anhydride, alcohol 23 in
THF (1 mL) was added to the reaction mixture at 0 °C. It was stirred
overnight at room temperature. The reaction mixture was concentrated
in vacuo, and flash chromatography purification on silica gel in ethyl
acetate/hexanes, 20À100%, yielded the acyclic precursor 24 (0.125 g,
20
1
thiol 0.012 g). [α]D +19.5 (c 0.2, CHCl3). H NMR (600 MHz,
CDCl3): δ 7.77 (s, 1H), 7.15 (d, J = 9.6 Hz, 1H), 6.41 (dd, J = 2.4, 9.0 Hz,
1H), 5.79À5.84 (m, 1H), 5.66 (m, 1H), 5.50 (dd, J = 6.6, 15.6 Hz, 1H),
5.29 (dd, J = 9.6, 18.0 Hz, 1H), 4.61 (dd, J = 3.0, 9.0 Hz, 1H), 4.27 (dd,
J = 3.0, 17.4 Hz, 1H), 4.04 (d, J = 11.4 Hz, 1H), 3.28 (d, 11.4 Hz, 1H), 2.90
(t, J = 7.2 Hz, 2H), 2.85 (dd, J = 10.8, 16.2 Hz, 1H), 2.53 (t, J = 7.8 Hz,
2H), 2.31 (q, J = 7.2, Hz, 2H), 2.10À2.12 (m, 1H), 1.87 (s, 3H),
1.62À1.67 (m, 2H), 1.26À1.31 (m, 8H), 0.87 (t, J = 7.2 Hz, 3H), 0.68
(d, J = 7.2 Hz, 3H), 0.5 (d, 6.6 Hz, 3H). 13C NMR (100 MHz, CDCl3):
δ = 199.6, 174.7, 169.6, 169.1, 168.1, 164.7, 147.7, 133.0, 128.6, 124.4,
84.7, 72.3, 57.9, 44.4, 43.6, 41.3, 40.7, 34.4, 32.5, 31.8, 29.1, 28.1, 25.9,
24.4, 22.8, 19.1, 16.8, 14.3. HRMS-ESI (m/z): [M + Na]+ calcd for
C29H42N4O5S3Na, 645.2211; found, 645.2215
20
1
94%). [α]D À12 (c 6.83, CHCl3). H NMR (600 MHz, CDCl3):
δ 7.89 (s, 1H), 7.76 (d, J = 7.2 Hz, 2H), 7.57 (d, J = 7.2 Hz, 2H), 7.41À7.37
(m, 8 H), 7.30 (t, J = 7.2 Hz, 2H), 7.26À7.29 (m, 6 H), 7.20 (t, J = 7.2
Hz, 3H), 6.74 (t, J = 8.4 Hz, 1H), 5.69À5.65 (m, 1H), 5.61 (dd, J = 6.0,
13.2 Hz, 1H), 5.42 (dd, J = 7.8, 15.0 Hz, 1H), 5.21 (d, J = 7.8 Hz, 1H), 4.7
(d, J = 6.0 Hz, 2H), 4.38 (dd, J = 7.2, 10.8 Hz, 1H), 4.33 (dd, J = 6.6, 10.8
Hz, 1H), 4.19 (t, J = 6.6 Hz, 1H), 4.05 (dd, J = 6.0, 8.4, 1H), 3.85 (d,
J= 10.8 Hz, 1H), 3.78 (s, 3H), 3.24 (d, J= 10.4 Hz, 1H), 2.58 (d, J=6.Hz,2H),
2.2À2.12 (m, 2H), 2.07À2.01 (m, 2H), 1.62 (s, 3H), 0.9 (d, J = 7.2 Hz,
3H), 0.85 (d, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 173.8,
169.2, 168.6, 163, 156.6, 148.5, 144.9, 143.6, 141.5, 134.2, 129.7, 128.03,
Cytoproliferation Assay. Cell proliferation was measured using a
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-
phenyl)-2H-tetrazolium (MTS) reduction assay with the CellTiter
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Journal of Medicinal Chemistry
ARTICLE
96 One solution MTS assay as described by the manufacturer (Promega,
Madison, WI). Briefly, cells (3 Â 104 cells/well) were seeded in
quadruplicate in 96-well plates and allowed to attach overnight. The
medium was replaced with 100 μL of fresh medium containing the
appropriate concentration of 27, 28, 29, or largazole 1. Following incuba-
tion at 37 °C, 5% CO2, 20 μL of CellTiter 96 One solution was added per
well and incubated 1.5 h at 37 °C and absorbance measured at 490 nm.
HDAC Activity Assays. HDAC activity was assayed using the Enzo
Life Sciences (Plymouth Meeting, PA) fluorimetric drug discovery kits
with specificity for HDAC1 and -6 or using cellular extracts kits.
Inhibition of global class 1 and 2 HDAC activity was ascertained using
the included HeLa nuclear extracts. Experiments were performed as
described by the protocol supplied by the manufacturer. Briefly, the
indicated inhibitor, enzyme, and Fluor de Lys substrate were incubated
for 1 h when the developing solution was added and incubated for the
designated time prior to reading. Experiments were repeated 3 times and
done in triplicate each time. Analogues incubated with the substrate and
developer was performed as a control and showed no influence on the
fluorescent signal. Data show a representative experiment with error bars
indicating standard deviation of the triplicates.
Evaluation of Global Histone Acetylation Levels. HCT116
cells were treated for 24 h with 10 nM, 100 nM, or 1 μM or DMSO. Cell
lysates were extracted using RIPA buffer, and equal amounts (30 μg/
lane) were loaded onto an SDSÀPAGE gel. Standard Western blotting
protocols were used using the Invitrogen NuPAGE Western blotting
system. Primary antibodies used were AcH3 (Millipore), α-tubulin
(Sigma), and β-actin (Sigma). Dye-conjugated secondary antibodies
from Li-Cor Biosciences were used for detection and scanned using the
Odyssey infrared detection system (LI-COR Biosciences, Lincoln, NE).
fibroblastic osteosarcoma; NMuMG, nontransformed mouse
mammary gland epithelial cell line; HCT116, human colorectal
carcinoma cell line; HATU, 2-(1H-azabenzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate; HDACis, histone
deacetylase inhibitors; NMR, nuclear magnetic resonance; USFDA,
United States Food and Drug Administration; SAR, structureÀ
activity relationship; TIPS, triisopropylsilane; TBAB, tetrabutylam-
monium bromide; TFAA, trifluoroacetic anhydride; HOAt, 1-hydro-
xy-7-azabenzotriazole; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; TLC,
thin layer chromatography; CTCL, cutaneous T-cell lymphoma
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’ ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures, ana-
b
lytical and spectral characterization data, and NMR spectra for
compounds 10À14, 31À35 and analogues 27À29; NMR spec-
tra for compounds 6, 7, 18, 20, 23À25, and largazole 1. This
material is available free of charge via the Internet at http://pubs.
acs.org.
’ AUTHOR INFORMATION
(4) Conley, B. A.; Wright, J. J.; Kummar, S. Targeting epigenetic
abnormalities with histone deacetylase inhibitors. Cancer 2006, 107 (4),
832–840.
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deacetylase inhibitors. Chem. Soc. Rev. 2008, 37 (7), 1402–1413.
(6) Butler, K. V.; Kozikowski, A. P. Chemical origins of isoform
selectivity in histone deacetylase inhibitors. Curr. Pharm. Des. 2008, 14
(6), 505–528.
Corresponding Author
*Phone: +1-419-530-1983. Fax: +1-419-530-7946. E-mail: ltillek@
utnet.utoledo.edu.
Author Contributions
§P.B. (synthesis) and C.L.H. (biological assay) contributed
equally to this work.
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technol. 2007, 25 (1), 17–19.
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’ ACKNOWLEDGMENT
(9) Taori, K.; Paul, V. J.; Luesch, H. Structure and activity of
largazole, a potent antiproliferative agent from the floridian marine
Cyanobacterium symploca sp. J. Am. Chem. Soc. 2008, 130 (6), 1806–
1807.
(R)-α-Methylcysteine HCl was initially synthesized in the lab
using D-hydantoinase enzyme. We thank Nagase Inc. for provid-
ing a sample of (R)-α-methylcysteine HCl at concessionary price
when D-hydantoinase became commercially unavailable. Support
for this work included NIH Grant CA51085 (to R.A.C.), Samuel
Waxman Cancer Research Foundation (to R.A.C.), and Susan G.
Komen for the Cure Foundation (to R.A.C.).
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Williams, R. M. Synthesis and histone deacetylase inhibitory activity of
largazole analogs: alteration of the zinc-binding domain and macrocyclic
’ ABBREVIATIONS USED
HDAC, histone deacetylase; HAT, histone acetyltransferase; SAHA,
suberoylanilide hydroxamic acid; MDA-MB-231, invasive trans-
formed human mammary epithelial cells; U2OS, transformed
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