Total synthesis of largazole and analogues: HDAC inhibition, antiproliferative activity and metabolic stability

Article abstract of DOI:10.1016/j.bmc.2011.02.024

The total synthesis of largazole and four analogues is reported. All analogues were nanomolar HDAC inhibitors. The antiproliferative activity is driven by lipophilicity and cell permeability. In murine liver homogenates, largazole is rapidly metabolized (half-life ≤5 min) to the thiol which has a half-life of 51 min.

Full text of DOI:10.1016/j.bmc.2011.02.024

Bioorganic & Medicinal Chemistry 19 (2011) 3650–3658
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Total synthesis of largazole and analogues: HDAC inhibition,
antiproliferative activity and metabolic stability
Hanae Benelkebir a, Sabrina Marie a, Annette L. Hayden b, Jason Lyle c, Paul M. Loadman c,
Simon J. Crabb b, Graham Packham b, A. Ganesan a,
⇑
a School of Chemistry, University of Southampton, Southampton SO17 1BJ, United Kingdom
b Cancer Research UK Centre, University of Southampton, Faculty of Medicine, Southampton General Hospital, Tremona Road, Southampton SO16 6YD, United Kingdom
c Institute of Cancer Therapeutics, University of Bradford, Tumbling Hill Street, Bradford, West Yorkshire BD7 1DP, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
The total synthesis of largazole and four analogues is reported. All analogues were nanomolar HDAC
inhibitors. The antiproliferative activity is driven by lipophilicity and cell permeability. In murine liver
homogenates, largazole is rapidly metabolized (half-life 65 min) to the thiol which has a half-life of
51 min.
Received 31 August 2010
Revised 8 February 2011
Accepted 13 February 2011
Available online 17 February 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Epigenetics
Histone deacetylase
Enzyme inhibitors
Natural products
Depsipeptide
1. Introduction
and 10) and IV (HDAC 11) hydrolyze the amide bond using a cata-
lytic zinc ion at the active site while class III (sirtuins SIRT1-7)
The N-terminal tails of eukaryotic histone proteins are rich in
post-translational modifications such as acetylation, methylation,
phosphorylation, ADP-ribosylation, sumoylation and ubiquitina-
tion. These dynamic modifications represent a ‘histone code’ that
other proteins can read, write, erase, and modify with functional
consequences including chromatin remodeling, transcriptional
activation or silencing.1 Consequently, small molecule inhibitors of
histone modifying enzymes are important tools for chemical biology
and leads for drug discovery against diseases with mistimed gene
transcription or repression.2 Many of the inhibitors are natural prod-
ucts, a testament to the success of such compounds in possessing
unique biological activity and exploring chemical space that is
poorly populated by synthetic compounds.3
Among the histone modifying enzymes, histone deacetylases
(HDACs) catalyze the hydrolysis of acetyllysine residues to lysine.
The resulting change in shape, size and charge at physiological
pH is a powerful mechanism for cell signaling occurring not only
in histones but a large number of nuclear and cytoplasmic pro-
teins.4 There are 18 human HDACs subdivided into classes accord-
ing to sequence homology, cellular localization and enzyme
mechanism.5 Classes I (HDACs 1, 2, 3 and 8), II (HDACs 4, 6, 7, 9
HDACs are metal-free and employ NAD+ as cofactor.
Many synthetic hydroxamic acids inhibit the zinc-dependent
HDACs by reversibly binding the metal. Such compounds show
promising activity in both in vitro and in vivo disease models. Sev-
eral examples have reached clinical development with SAHA (mar-
keted by Merck as Zolinza) becoming the first HDAC inhibitor to
receive FDA approval for the treatment of cutaneous T-cell lym-
phoma.6 The second FDA approved HDAC inhibitor FK228
(marketed by Celgene as Istodax) is very different in structure
and comes from a much smaller class of bicyclic depispeptide bac-
terial natural products comprising FK228, the spiruchostatins and
FR901,375 isolated from fermentation extracts of Chromobacterium
violaceum and Pseudomonas sp. (Fig. 1). These natural products are
prodrugs that undergo intracellular disulfide reduction to release a
zinc-binding thiol at the HDAC active site. Compared to the
hydroxamic acid inhibitors, the depsipeptide macrocyclic scaffold
provides additional binding interactions to zinc chelation resulting
in higher potency and selectivity between HDAC isoforms. We7 and
others8 have extensively published on total syntheses of the bicy-
clic depsipeptides and unnatural analogues thereof.
In 2008, the depsipeptide largazole with high antiproliferative
activity was isolated from a marine cyanobacterium of the genus
Symploca.9 Although the original report claimed ‘the 3-hydroxy-
7-mercaptohept-4-enoic acid unit is unprecedented in natural
⇑
Corresponding author. Tel.: +44 02380 593897; fax: +44 02380 596805.
E-mail address: ganesan@soton.ac.uk (A. Ganesan).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.02.024

H. Benelkebir et al. / Bioorg. Med. Chem. 19 (2011) 3650–3658
3651
Largazole analogues with glycine replacements had not been
previously explored and we were interested in how analogue 1
(Fig. 2) with a phenylalanine side chain would compare in terms
of enzyme and cell growth inhibition.
Further analogues 2–4 had the goal of molecular simplification
and reduction of molecular weight of largazole. Analogue 2 re-
places the valine residue in largazole by the simpler glycine. We
had shown7d that FK228 analogues with an identical valine to gly-
cine substitution retained potent HDAC and cancer cell growth
inhibition. In analogue 3, the valine residue was replaced by b-
alanine, increasing the size of the macrocycle by one atom. Despite
the extensive studies with the bicyclic depsipeptides and largazole,
such ring expanded analogues were unknown and the effect of this
alteration on biological activity could not be predicted. Our next
analogue 4 probes the influence of the thiazoline ring in largazole.
Both epimers of the thiazoline are HDAC inhibitors as reported
by Williams and co-workers11i while FK228 contains an achiral
dehydrobutyrine instead. These observations suggest that this
region of the macrocycle is stereochemically tolerant. Instead of
the relatively complex thiazoline or dehydrobutyrine residues in
the natural products, analogue 4 contains a structurally simpler
achiral a-aminoisobutyric acid (Aib) residue.
Figure 1. Depsipeptide natural product HDAC inhibitors with thiol zinc-binding
groups.
2.1. Total synthesis of largazole and analogues
The total synthesis of depsipeptide HDAC inhibitors can be bro-
ken down to a synthesis of the zinc-binding 3-hydroxy-7-merca-
ptohept-4-enoic acid unit in enantiopure form, assembly of a
linear peptide or depsipeptide with appropriate protecting groups,
and macrocylization. We have routinely made the 3-hydroxy-7-
mercaptohept-4-enoic acid by an enantioselective aldol reaction
directed by the Fujita-Nagao auxiliary7a while our experiences7f
in FK228 total synthesis suggested macrolactamization rather than
macrolactonization for ring formation.
Table 1
Inhibition of total HDACs in HeLa nuclear cell extracts by
SAHA and depsipeptide natural products
Compound
HDAC inhibitiona
(nM)
SAHA
288 59
24.4 4.0
5.3 3.3
FK228 (dithiol)
spiruchostatin A
(dithiol)
largazole thiol
0.043 0.026
Our total synthesis of largazole (Scheme 1) began with the Han-
tzsch cyclization of Boc-protected glycine 5 (Scheme 1) and methyl
bromopyruvate. Conversion of the ester to the nitrile gave thiazole
13, an intermediate in previous largazole total syntheses.10,11 In
parallel, analogous chemistry starting with phenylalanine led to
thiazole 14. Condensation with (R)-methylcysteine13 hydrochlo-
ride salt under Cramer’s buffered conditions11e provided the thia-
zole–thiazoline fragments 15 and 16. These were coupled with
amine 17, an intermediate in our FK228 total synthesis.7f The linear
depsipeptides were then subjected to deprotection at the N- and
C-terminus followed by macrolactamization and thiol deprotection
to afford largazole thiol and analogue 1, respectively. Separately,
largazole thiol was acylated to complete a total synthesis of largaz-
ole. Overall, largazole was obtained in 0.24% yield in the longest
linear sequence.
a
Data obtained with the Flour-de-Lys assay in our
laboratory.
products’, this fragment is identical to the zinc-binding thiol in the
bicyclic depsipeptides. This suggests that largazole too is a HDAC
inhibitor. Indeed, our work on synthetic FK228 analogues had
demonstrated that the disulfide linkage can be replaced by an ester
which is cleavable intracellularly to release the active thiol.7d The
largazole hypothesis was soon confirmed and largazole thiol
shown to be a potent HDAC inhibitor.10 Since then, a steady stream
of largazole total and analogue syntheses has appeared.11 There are
two main reasons for this burst of activity in a natural product
reported only two years ago. Firstly, the pioneering efforts in the
synthesis of bicyclic depsipeptide HDAC inhibitors and thiazole/
thiazoline containing natural products enabled retrosynthetic
routes to be planned with high confidence and successfully exe-
cuted.12 Secondly, largazole thiol is an exciting lead as it is a more
potent HDAC inhibitor than the bicyclic depsipeptides (Table 1).
Here, we report our efforts directed towards largazole and struc-
tural analogues and the first pharmacokinetic evaluation of such
compounds in terms of metabolic stability.
The synthesis of analogues 2–4 was achieved by the same route.
The union of glycine and b-alanine versions of amine 17 with
thiazole–thiazoline fragment 15 (Scheme 2) followed by macrocyc-
lization afforded analogues 2 and 3. The synthesis of analogue 4
proceeded via the coupling of thiazole 9 with
a-aminoisobutyric
acid to give 28, which was carried forward as in the earlier synthe-
ses (Scheme 3).
2.2. HDAC inhibition and antiproliferative activity
2. Results and discussion
We confirmed that our synthetic largazole inhibits HDAC activ-
ity in cells by monitoring histone H4K8(ac) accumulation in three
cancer cell lines (Fig. 3). As expected, largazole thiol was relatively
inactive due to its poor cell permeability.
Largazole and its free thiol were assayed for inhibition of HDAC
enzyme activity and growth of the MCF7 breast cancer cell line
(Table 2). Largazole thiol was a highly potent picomolar HDAC
Our initial objective was to complete a total synthesis of largaz-
ole to obtain a sample of the natural product and verify the validity
of the synthesis route. Once completed, this would then lead to the
more illuminating preparation of unnatural analogues. A compari-
son of the depsipeptides reveals a glycine residue in largazole that
corresponds to alanine or valine in FK228 and the spiruchostatins.

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H. Benelkebir et al. / Bioorg. Med. Chem. 19 (2011) 3650–3658
Figure 2. Structures of largazole thiol and synthetic analogues 1–4.
Scheme 1. Total synthesis of largazole and benzyl analogue 1.

H. Benelkebir et al. / Bioorg. Med. Chem. 19 (2011) 3650–3658
3653
Scheme 2. Synthesis of largazole analogues 2 and 3.
Scheme 3. Synthesis of largazole analogue 4.
Table 2
HDAC and MCF7 growth inhibition by largazole, largazole thiol and analogues 1–4
Compound
HDAC inhibition (nM)
MCF7 inhibition (nM)
Largazole
Largazole thiol
1
2
3
4
572 29
0.043 0.026
17.2 2.3
0.17 0.05
3.15 0.35
0.99 0.07
5
1
277 130
377 62
2458 1135
>10,000
5902 1698
The novel analogue 1 was a significantly weaker HDAC inhibitor
than largazole thiol. Nevertheless, in the growth inhibition assay
the two compounds had similar activity. This interesting result
shows that enzyme and cell activity are not always correlated in
the largazole series. It is tempting to suggest that the unexpectedly
high cell activity of 1 is due to the introduction of a lipophilic group
that aids passive membrane absorption.
Figure 3. Western blotting of H4K8(ac) in MCF7, MDA-MB-231 and SKBr3 cell lines
with DMSO control, largazole and largazole thiol at 100 nM.
Analogue 2 is a subnanomolar HDAC inhibitor indicating that a
valine to glycine substitution at this position is not detrimental,
matching the result we observed with FK228. Increasing the
macrocyle ring size in analogue 3 was also tolerated, leading to a
20-fold decrease in HDAC activity compared to 2 but still providing
a single digit nanomolar inhibitor. However, both 2 and 3 in partic-
ular are poor in growth inhibition compared to largazole. The Aib
containing analogue 4 shows the same pattern-nanomolar HDAC
inhibitor with an approximately 10,000 fold reduction in cellular
activity as first reported by Luesch and co-workers.9,10 Conversely,
the natural product largazole had potent antiproliferative activity
and some enzyme inhibition that we attribute to chemical hydro-
lysis back to largazole thiol under the assay conditions.

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H. Benelkebir et al. / Bioorg. Med. Chem. 19 (2011) 3650–3658
inhibition while much weaker in growth inhibition. Taken to-
gether, these results indicate that molecular simplification of the
largazole scaffold is possible without losing HDAC inhibitory activ-
ity. As analogues 2–4 have a reduced lipophilicity compared to
largazole thiol or 1, the decreased growth inhibitory activity may
reflect poorer cell uptake.
to a solution of 10 (1.14 g, 3.27 mmol, 1 equiv) in anhydrous
CH2Cl2 (70 mL). At 0 °C, ammonia solution (0.5 M in dioxane)
(65 mL, 32.74 mmol, 10 equiv) was added slowly. The solution
was stirred overnight under argon. The aqueous phase was ex-
tracted with CH2Cl2 (3 ꢀ 100 mL) and washed with brine
(2 ꢀ 100 mL). The combined organic layers were dried over MgSO4,
filtered and concentrated. Flash chromatography (CH2Cl2/MeOH
95:5) afforded 12 as a salmon solid (884 mg, 77%): mp 48–52 °C;
2.3. Metabolic stability
½
a 2D5 +12.0 (c 0.26, CHCl3); IR 3300, 1675, 1520, 1368, 1168 cmꢂ1
;
ꢁ
1H NMR (300 MHz, MeOD) d 7.30–7.18 (m, 5H), 4.29 (dd, J = 9.0,
5.7 1H), 3.11 (dd, J = 13.7, 5.3 Hz, 1H), 2.81 (dd, J = 13.7, 9.3 Hz,
1H), 1.36 (s, 9H); 13C NMR (75 MHz, MeOD) d 177.2, 157.6, 138.7,
130.4, 129.4, 127.7, 80.6, 57.1, 39.4, 28.7; ES+ MS m/z 370
([M+Na]+); HRMS (ESI) m/z calcd. for C17H21N3Na1O3S1 (M+Na)+
370.1196, found 370.1195.
The therapeutic potential of largazole and related analogues
will be determined not only by potency or selectivity but equally
by pharmacokinetic properties. Surprisingly, no discussion of the
latter is present in the extensive literature on largazole.9–11 We
have investigated the stability of largazole and our analogues in
the presence of mouse liver homogenate to determine their sus-
ceptibility to metabolism.
Largazole itself was highly unstable with a half-life 65 min. The
metabolism consists of thioester hydrolysis to largazole thiol and
reveals the potential disadvantage of ester prodrug thiol HDAC
inhibitors compared to the bicyclic disulfides such as the clinically
approved FK228. For in vivo applications, careful choice of the ester
group to slow down hydrolysis will be necessary. Once the ester is
cleaved, largazole thiol is relatively stable in murine liver homog-
enate with a half-life of 51 min at 37 °C. Analogue 2 was similar in
stability with a half-life of 32 min. In both cases, one major metab-
olite of unknown structure was observed. Analogues 3–4 had a
complex and rapid metabolism with a half-life 65 min.
4.1.2. (R)-tert-Butyl 1-(4-cyanothiazol-2-yl)-2-phenylethylcar
bamate (14)
Triethylamine (0.63 mL, 4.54 mmol, 2 equiv) and trifluoroacetic
anhydride (0.34 mL, 2.49 mmol, 1.1 equiv) were added to a solu-
tion of 12 (789 mg, 2.27 mmol, 1 equiv) in anhydrous CH2Cl2
(40 mL) at 0 °C under argon. The solution was stirred overnight
and the solvent removed. The residue was purified by flash chro-
matography (hexane/EtOAc 8:2) to give 14 as a white solid
(593 mg, 74%): mp 126–130 °C; ½a D25
ꢁ
+1.6 (c 0.24, CH3OH); IR
2979, 1705, 1508, 1365, 1255, 1164, 703 cmꢂ1
;
1H NMR
(300 MHz, MeOD) d 8.37 (s, 1H), 7.31–7.21 (m, 5H), 5.12 (dd,
J = 10.2, 4.9 Hz, 1H), 3.43 (dd, J = 13.9, 5.3 Hz, 1H), 3.04 (dd,
J = 13.6, 10.2 Hz, 1H), 1.35 (s, 9H); 13C NMR (75 MHz, CDCl3) d
174.4, 154.8, 135.6, 130.3, 129.2, 128.7, 127.1, 126.5, 113.8, 80.6,
53.7, 41.0, 28.1; ES+ MS m/z 352 ([M+Na]+), 681 ([2 M+Na]+); HRMS
(ESI) m/z calcd. for C17H19N3Na1O2S1 (M+Na)+ 352.1090, found
352.1094.
3. Conclusions
We report the total synthesis of largazole and four novel largaz-
ole analogues. Although the analogues are less active than largazole
thiol itself as HDAC inhibitors, they shed further light on the SAR of
this natural product. The extraordinary potency of largazole thiol in
enzyme assays is not matched in cell growth inhibitory activity,
whether as the free thiol or as the ester natural product. In cell as-
says the bicyclic depsipeptide natural products are superior despite
being significantly poorer in HDAC inhibition. These results imply
that largazole has some liabilities related to cell uptake, stability,
nonspecific binding or other aspects that lead to its reduced activity
in cell-based assays.
Our unnatural analogues 1–4 show that the glycine, valine and
thiazoline residues in largazole can all be replaced while retaining
nanomolar HDAC inhibition. Nevertheless, the analogues are less
potent compared to largazole thiol and indicate that the SAR of this
natural product is more subtle and less predictable than FK228.
Meanwhile, the cell growth inhibition appears to track with lipo-
philicity. Analogue 1, the weakest HDAC inhibitor in the series,
had the best cell growth inhibition and this is likely due to its
increased lipophilicity. Analogues 2–4, while simpler in structure
than largazole thiol, are more polar and this has a deleterious effect
on growth inhibition.
4.1.3. (R)-2-(2-((R)-1-(tert-Butoxycarbonylamino)-2-phenyl
ethyl) thiazol-4-yl)-4-methyl-4,5-dihydrothiazole-4-carboxylic
acid (16)
To a solution of 14 (329 mg, 1.04 mmol, 1 equiv) and (R)-meth-
ylcysteine hydrochloride (338 mg, 1.96 mmol, 1.9 equiv) in anhy-
drous methanol (20 mL), were added NaHCO3 (444 mg, 5.24 mmol,
5 equiv) and 6.46 mL of a solution of phosphate buffer (pH 6).
The reaction mixture was degassed by bubbling briefly argon
through it and stirred at 70 °C for 2 h. To the cold reaction mixture
were added water (20 mL) and saturated NaHCO3 solution (20 mL).
The solution was washed with EtOAc (3 ꢀ 20 mL). The aqueous
layer was acidified to pH 1–2 with a solution of saturated KHSO4
and extracted with EtOAc (3 ꢀ 20 mL). The combined organic
layers were washed with brine (4 ꢀ 50 mL), dried over MgSO4,
concentrated and purified by flash chromatography (CH2Cl2/MeOH/
AcOH 94:5:1) to give 16 as a white solid (399 mg, 85%): mp
60–70 °C. ½a 2D5
ꢂ13.8 (c 0.25, CH3OH); IR 2363, 2336, 1709, 1527,
ꢁ
673 cmꢂ1 1H NMR (300 MHz, CDCl3) d 7.98 (s, 1H), 7.31–7.12
;
Largazole was found to be rapidly degraded by mouse liver
homogenate to the thiol, which in turn had a half-life of 51 min at
37 °C. Our results highlight the importance of cell activity and AD-
METproperties suchasmetabolismratherthaninvitroenzymeinhi-
bition in the further development of largazole as a therapeutic lead.
(m, 5H), 5.31–5.24 (m, 2H), 3.90 (d, J = 11.7 Hz, 1H), 3.39 (d, J =
11.7 Hz, 1H), 3.32 (d, J = 6.2 Hz, 2H), 1.70 (s, 3H), 1.41 (s, 9H); 13C
NMR (100 MHz, CDCl3) d 175.6, 172.6, 165.0, 155.0, 147.9, 136.1,
129.4, 128.5, 126.9, 122.0, 84.2, 80.3, 53.7, 41.2, 40.9, 28.2, 24.1;
ES+ MS m/z 470 ([M+Na]+).
4. Experimental
4.1.4. (S,E)-2-(Trimethylsilyl)ethyl 3-((S)-2-((R)-2-(2-((R)-1-(tert-
butoxycarbonylamino)-2-phenylethyl)thiazol-4-yl)-4-methyl-
4,5-dihydrothiazole-4-carboxamido)-3-methylbutanoyl-oxy)-7-
(tritylthio)hept-4-enoate (19)
4.1. Compound synthesis
4.1.1. (R)-tert-Butyl 1-(4-carbamoylthiazol-2-yl)-2-phenylethyl-
carbamate (12)
HOBt (1.41 g, 9.82 mmol, 3 equiv), EDCI (1.89 g, 9.82 mmol,
3 equiv) and Hünig’s base (3 mL, 13.09 mmol, 4 equiv) were added
To a suspension of 16 (36 mg, 0.08 mmol, 1.1 equiv) and 17
(obtained by deprotection of 63 mg, 0.07 mmol, 1 equiv of the
Fmoc-carbamate) in anhydrous CH2Cl2 (5 mL) and anhydrous ace-
tonitrile (2 mL) was added PyBOP (44 mg, 0.08 mmol, 1.1 equiv) at

H. Benelkebir et al. / Bioorg. Med. Chem. 19 (2011) 3650–3658
3655
0 °C and Hünig’s base (32
was warmed to rt and stirred overnight. The solvent was then
l
L, 0.18 mmol, 2.5 equiv). The solution
4.1.7. (S,E)-2-(Trimethylsilyl)ethyl 3-(2-(((9H-fluoren-9-
yl)methoxy)carbonylamino)acetoxy)-7-(tritylthio)hept-4-
enoate (22)
removed and flash chromatography (hexane/EtOAc 8:2) gave 19
as an oil (55 mg, 65%): ½a D25
ꢁ
ꢂ24.4 (c 0.25, CHCl3); IR 2359, 2340,
The protected b-hydroxy ester (200 mg, 0.39 mmol, 1 equiv)
and N-Fmoc-glycine (126 mg, 0.42 mmol, 1.1 equiv) were dis-
solved in anhydrous CH2Cl2 (7 mL). DCC (96 mg, 0.46 mmol,
1.2 equiv) and DMAP (4.7 mg, 0.04 mmol, 0.1 equiv) were added
to the reaction mixture cooled to 0 °C. The reaction mixture was
stirred overnight. The precipitate was filtered and solvents were
evaporated. The crude was purified by flash chromatography
(EtOAc/hexane 5:95) to give 22 as a colorless oil (180 mg, 59%):
1731, 1504, 1168, 673 cmꢂ1
;
1H NMR (400 MHz, CDCl3) d 7.82 (s,
1H), 7.36–7.11 (m, 20H), 5.68–5.59 (m, 2H), 5.35 (dd, J = 15.4,
7.5 Hz, 1H), 5.26 (br s, 1H), 5.15 (br s, 1H), 4.46 (dd, J = 9.0,
4.6 Hz, 1H), 4.12 (m, 2H), 3.76 (dd, J = 11.4, 8.1 Hz), 3.31–3.28 (m,
3H), 2.66 (dd, J = 15.6, 7.7 Hz, 1H), 2.53 (dd, J = 15.6, 5.65 Hz, 1H),
2.16–2.10 (m, 3H), 2.03 (q, J = 6.7 Hz, 2H), 1.57 (s, 3H), 1.38 (s,
9H), 0.94 (m, 2H), 0.81 (d, J = 6.9 Hz, 3H), 0.72 (d, J = 6.8 Hz, 3H),
0.00 (s, 9H); 13C NMR (100 MHz, CDCl3) d 174.4, 170.3, 169.5,
163.4, 154.9, 148.8, 144.7, 136.1, 133.8, 133.8, 129.5, 128.5,
127.8, 126.9, 126.5, 120.6, 85.1, 80.2, 71.6, 66.5, 63.0, 56.7, 53.6,
41.3, 41.1, 39.6, 31.2, 31.1, 31.0, 28.2, 24.7, 19.0, 17.3, 17.2, ꢂ1.5;
ES+ MS m/z 1069 ([M+Na]+).
½
a 2D3
ꢁ
ꢂ9.9 (c 0.54, CHCl3); IR 3368, 2964, 2918, 2843, 1735, 1520,
1448, 1247, 1172, 1051, 858, 839, 733, 692 cmꢂ1 1H NMR
;
(400 MHz, CDCl3) d 7.78 (d, J = 7.5 Hz, 2H), 7.60 (d, J = 7.0 Hz, 2H),
7.15–7.46 (m, 19H), 5.57–5.76 (m, 2H), 5.39 (dd, J = 15.6, 7.0 Hz,
1H), 5.24 (br s, 1H), 4.39 (d, J = 7.0 Hz, 2H), 4.24 (t, J = 7.0 Hz, 1H),
4.16 (t, J = 8.5 Hz, 2H), 3.96 (d, J = 5.5 Hz, 2H), 2.67 (dd, J = 15.8,
8.3 Hz, 1H), 2.55 (dd, J = 15.8, 5.3 Hz, 1H), 2.20 (t, J = 7.5 Hz, 2H),
2.08 (t, J = 7.0 Hz, 2H), 0.97 (t, J = 8.5 Hz, 2H), 0.04 (s, 9H); 13C
NMR (100 MHz, CDCl3) d 169.7, 168.9, 156.1, 144.8, 143.8, 141.3,
133.6, 129.6, 127.9, 127.7, 127.6, 127.1, 126.6, 125.1, 120.0, 71.9,
67.2, 66.6, 63.1, 47.1, 42.8, 39.6, 31.3, 31.1, 17.3, ꢂ1.5; ES+ MS
m/z 820 ([M+Na]+); HRMS (ESI) m/z calcd. for C48H51NNaO6SSi
(M+Na)+ 820.3099, found 820.3090.
4.1.5. (5R,8S,11S)-8-Isopropyl-5-methyl-11(2-4-tritylsulfanyl-
but-1-enyl)-10-oxa-3,17-dithia-7,14,19,20-tetraaza-tricyclo
[14.2.1.1ꢃ2,5ꢃ]icosa-1(18),2(20),16(19)-triene-6,9,13-trione (21)
Acyclic precursor 19 (58 mg, 0.05 mmol, 1 equiv) was dissolved
in CH2Cl2 (2.5 mL), cooled to 0 °C and treated with TFA (0.11 mL).
The reaction mixture was warmed to rt, stirred overnight, the sol-
vents evaporated and the crude amino acid coevaporated with tol-
uene to remove residual TFA. The crude amino acid was taken up in
CH2Cl2 (2.5 mL) and added dropwise to a stirred solution of iPr2NEt
(0.05 mL, 0.30 mmol, 6 equiv) in dry acetonitrile (50 mL). The mix-
ture was stirred for 10 min and a solution of HATU (40 mg,
0.10 mmol, 2 equiv) and HOBt (12 mg, 0.10 mmol, 2 equiv) in ace-
tonitrile (2.5 mL) was added dropwise. The solution was stirred
overnight. The solvent was then removed and flash chromatogra-
4.1.8. (S,E)-2-(Trimethylsilyl)ethyl 3-(2-((R)-2-(2-((tert-butoxy
carbonylamino)methyl)thiazol-4-yl)-4-methyl-4,5-dihydro
thiazole-4-carboxamido)acetoxy)-7-(tritylthio)hept-4-enoate (24)
Carbamate 22 (170 mg, 0.21 mmol, 1 equiv) was deprotected by
stirring for 2 h in anhydrous acetonitrile (5 mL) and diethylamine
(110 ll, 1.07 mmol, 5 equiv) followed by concentration in vacuo.
To a solution of 15 (84 mg, 0.24 mmol, 1.1 equiv) in anhydrous
CH2Cl2 (5 mL) cooled at 0 °C, PyBOP (151 mg, 0.26 mmol, 1.2 equiv)
and Hünig’s base (112 lL, 0.64 mmol, 3 equiv) were added. After
phy (CH2Cl2/MeOH 99:1) gave 21 as an oil (14 mg, 34%): ½a D25
ꢁ
ꢂ31.3 (c 0.37, CHCl3); 1H NMR (400 MHz, CDCl3) of the major com-
pound d 7.80 (s, 1H), 7.42–7.18 (m, 20H), 7.10 (d, J = 9.1 Hz, 1H),
5.78–5.70 (m, 1H), 5.67–5.64 (m, 1H), 5.58–5.54 (m, 1H), 5.34
(dd, J = 15.5, 7.1 Hz, 1H), 4.57 (dd, J = 3.2, 9.2 Hz, 1H), 4.09 (d,
J = 11.5 Hz, 1H), 3.30 (d, J = 11.6 Hz, 1H), 3.23 (d, J = 7.3 Hz, 1H),
3.17 (d, J = 7.3 Hz, 1H), 2.67–2.54 (m, 2H), 2.18–2.16 (m, 1H),
2.09–2.05 (m, 4H), 1.89 (s, 3H), 0.63 (d, J = 6.8 Hz, 3H), 0.46 (d,
J = 6.9 Hz, 3H); 13C NMR (100 MHz, CDCl3) of the major compound
d 173.7, 171.6, 170.3, 169.3, 169.1, 157.0, 145.2, 135.8, 133.6,
130.0, 129.8, 129.2, 128.9, 128.2, 128.1, 127.9, 127.0, 84.9, 72.6,
67.0, 58.0, 52.9, 43.5, 41.9, 40.8, 34.5, 31.8, 31.5, 24.3, 19.2, 16.8;
ES+ MS m/z 851 ([M+Na]+); HRMS (ESI) m/z calcd. for
10 min, the previous deprotected amine (123 mg, 0.21 mmol,
1 equiv) in solution in anhydrous CH2Cl2 (5 mL) was added drop-
wise. The reaction mixture was stirred at rt overnight and concen-
trated in vacuo. The crude material was purified by flash
chromatography (EtOAc/hexane 3:7) to afford 24 as a colorless
oil (153 mg, 78%): ½a D24
ꢂ40.3 (c 0.27, CHCl3); IR 3342, 2971,
ꢁ
2952, 1727, 1671, 1599, 1512, 1444, 1365, 1244, 1168, 858, 835,
741, 695 cmꢂ1 1H NMR (400 MHz, CDCl3) d 7.91 (s, 1H), 7.47–
;
7.34 (m, 6H), 7.34–7.12 (m, 9H), 5.73–5.54 (m, 2H), 5.36 (dd,
J = 15.6, 7.0 Hz, 1H), 5.26 (br s, 1H), 4.64 (d, J = 6.0 Hz, 2H), 4.22–
4.09 (m, 2H), 4.02 (dd, J = 7.5, 5.5 Hz, 2H), 3.76 (d, J = 11.5 Hz,
1H), 3.34 (d, J = 11.5 Hz, 1H), 2.66 (dd, J = 15.8, 8.3 Hz, 1H), 2.53
(dd, J = 15.8, 5.3 Hz, 1H), 2.18 (t, J = 7.0 Hz, 2H), 2.12–1.96 (m,
2H), 1.60 (s, 3H), 1.48 (s, 9H), 1.07–0.88 (m, 2H), 0.04 (s, 9H); ES+
MS m/z 937 ([M+Na]+); HRMS (ESI) m/z calcd. for C47H58N4NaO7S3-
Si (M+Na)+ 937.3129, found 937.3136.
C
47H48N4Na1O4S3 (M+Na)+ 851.2730, found 851.2730.
4.1.6. (5R,8S,11S)-8-Isopropyl-11-(2-4-mercapto-but-1-enyl)-5-
methyl-10-oxa-3,17-dithia-7,14,19,20-tetraaza-tricyclo[14.2.1.
1ꢃ.2,5ꢃ]icosa-1(18),2(20),16(19)-triene-6,9,13-trione (1)
At 0 °C, under argon, trifluoroacetic acid (0.05 mL, 0.64 mmol,
64 equiv) and triisopropylsilane (7 lL, 0.03 mmol, 3 equiv) were
added to a solution of 21 (14 mg, 0.01 mmol, 1 equiv) in CH2Cl2
(1.5 mL). The solution was warmed to rt and stirred for 4 h. The
solvent was then removed and the compound was purified by flash
chromatography (hexane/EtOAc 7:3) to give 1 as a yellowish oil
4.1.9. (5R,11S)-5-Methyl-11-((E)-4-(tritylthio)but-1-en-1-yl)-10-
oxa-3,17-dithia-7,14,19,20-tetraazatricyclo[14.2.1.12,5]icosa-
1(18),2(20),16(19)-triene-6,9,13-trione (26)
Acyclic precursor 24 (138 mg, 0.15 mmol) was dissolved in
CH2Cl2 (7 mL), cooled to 0 °C and treated with TFA (1.5 mL). The
reaction mixture was warmed to rt and stirred overnight. The
reaction mixture was concentrated in vacuo and then coevaporated
with toluene to remove residual TFA. The crude amino acid (108 mg,
0.15 mmol, 1 equiv) was taken up in anhydrous CH2Cl2 (13 mL) and
added dropwise to a vigorously stirred solution of Hünig’s base
(3 mg, 30%): ½a 2D5
ꢁ
ꢂ31.6 (c 0.13, CHCl3); 1H NMR (400 MHz, CD3OD)
of the major compound d 8.14 (s, 1H), 7.33–7.23 (m, 5H), 5.82–5.75
(m, 1H), 5.62–5.54 (m, 3H), 4.50–4.47 (m, 1H), 3.94 (d, J = 11.6 Hz,
1H), 3.40 (d, J = 11.5 Hz, 1H), 3.20–3.17 (m, 1H), 3.04–2.97 (m, 1H),
2.55–2.52 (m, 4H), 2.36–2.31 (m, 2H), 2.09–2.03 (m, 1H), 1.82 (s,
3H), 0.67 (d, J = 6.9 Hz, 3H), 0.44 (d, J = 6.9 Hz, 3H); 13C NMR
(100 MHz, CDCl3) of the major compound d 173.1, 171.7, 169.4,
168.2, 167.8, 135.4, 132.8, 129.5, 128.9, 128.5, 127.6, 127.2, 73.3,
58.0, 52.6, 42.9, 41.6, 36.3, 33.8, 29.7, 23.8, 22.7, 18.6, 16.6; ES+
MS m/z 609 ([M+Na]+); HRMS (ESI) m/z calcd. for C28H34N4Na1O4S3
(M+Na)+ 609.1634, found 609.1624.
(158 lL, 0.90 mmol, 6 equiv), HOBt (41 mg, 0.30 mmol, 2 equiv)
and HATU (115 mg, 0.30 mmol, 2 equiv) in anhydrous acetonitrile
(110 mL). The reaction mixture was stirred overnight, and then
concentrated in vacuo. The crude material was purified by flash
chromatography (hexane/EtOAc 10:1 then 100% EtOAc) to afford

3656
H. Benelkebir et al. / Bioorg. Med. Chem. 19 (2011) 3650–3658
26 as a pale yellow solid (55.4 mg, 53%): mp 136–138 °C; ½a D24
ꢁ
ꢂ51.2
5 equiv) was added to the reaction mixture. After stirring for 2 h, the
reaction mixture was concentrated in vacuo.
To a solution of 15 (90 mg, 0.25 mmol, 1.1 equiv) in anhydrous
CH2Cl2 (5 mL) cooled at 0 °C, PyBOP (162 mg, 0.28 mmol, 1.2 equiv)
(c 0.22, CH3OH); IR 3628, 2923, 1734, 1670, 1541, 1507, 1262,
840 cmꢂ1
;
1H NMR (400 MHz, CD3OD) d 8.02 (s, 1H), 7.40–7.29
(m, 6H), 7.29–7.10 (m, 9H), 5.74–5.53 (m, 2H), 5.41 (dd, J = 15.6,
7.0 Hz, 1H), 4.97 (d, J = 17.6 Hz, 1H), 4.34 (d, J = 17.6 Hz, 1H), 4.06
(d, J = 18.1 Hz, 1H), 3.97 (d, J = 11.5 Hz, 1H), 3.73 (d, J = 18.1 Hz,
1H), 3.31 (d, J = 11.5 Hz, 1H), 2.97 (dd, J = 17.3, 11.3 Hz, 1H), 2.63
(dd, J = 17.1, 2.0 Hz, 1H), 2.17 (t, J = 6.0 Hz, 2H), 2.02 (q, J = 6.5 Hz,
2H), 1.74 (s, 3H); 13C NMR (100 MHz, CD3OD) d 176.2, 172.8,
169.7, 168.0, 166.8, 148.4, 146.5, 134.4, 130.9, 129.5, 129.0, 127.9,
126.7, 85.4, 73.8, 67.9, 44.6, 42.7, 42.3, 40.2, 32.5, 32.4, 25.5; ES+
MS m/z 719 ([M+Na]+); HRMS (ESI) m/z calcd. for C37H36N4NaO4S3
(M+Na)+ 719.1791, found 719.1795.
and Hünig’s base (120 lL, 0.69 mmol, 3 equiv) were added. After
15 min, the previous deprotected amine (135 mg, 0.23 mmol,
1 equiv) in anhydrous CH2Cl2 (5 mL) was added dropwise. The
reaction mixture was stirred at rt overnight and concentrated in
vacuo. The crude material was purified by flash chromatography
(EtOAc/hexane 3:7) to afford 25 as a colorless oil (175 mg, 82%):
½
a 2D5
ꢁ
ꢂ39.8° (c 0.61, CHCl3); IR 3361, 2967, 2941, 2926, 1724,
1659, 1520, 1440, 1368, 1259, 1168, 1032, 866, 835, 745, 699 cmꢂ1
;
1H NMR (300 MHz, CDCl3) d 7.97 (br s, 1H), 7.39 (d, J = 7.5 Hz,
6H), 7.33–7.12 (m, 9H), 5.57 (dt, J = 14.6, 7.5 Hz, 2H), 5.35 (dd,
J = 15.1, 7.0 Hz, 1H), 5.28 (br s, 1H), 4.63 (d, J = 5.5 Hz, 2H), 4.21–
4.06 (m, 2H), 3.76 (d, J = 11.5 Hz, 1H), 3.43–3.59 (m, 2H), 3.33 (d,
J = 11.5 Hz, 1H), 2.62 (dd, J = 15.8, 7.8 Hz, 1H), 2.51 (dt, J = 10.3,
5.5 Hz, 3H), 2.18 (t, J = 7.5 Hz, 2H), 2.08–1.95 (m, 2H), 1.57 (s,
3H), 1.48 (s, 9H), 1.04–0.87 (m, 2H), 0.03 (s, 9H); 13C NMR
(100 MHz, CDCl3) d 174.5, 170.8, 169.9, 165.5, 154.3, 144.8,
132.9, 129.6, 128.1, 127.9, 126.6, 122.1, 85.0, 80.5, 77.2, 70.8,
66.6, 63.0, 42.3, 41.3, 39.7, 34.9, 34.2, 31.3, 31.2, 28.3, 24.8, 22.6,
17.3, ꢂ1.5; ES+ MS m/z 951 ([M+Na]+); HRMS (ESI) m/z calcd. for
4.1.10. (5R,11S)-11-((E)-4-Mercaptobut-1-en-1-yl)-5-methyl-10-
oxa-3,17-dithia-7,14,19,20-tetra-azatricyclo[14.2.1.12,5]icosa-
1(18),2(20),16(19)-triene-6,9,13-trione (2)
S-Trityl macrocycle 26 (45.3 mg, 65
in CH2Cl2 (8 mL) and cooled to 0 °C and successively treated with Et3-
SiH (21 L, 130 mol, 2 equiv) and TFA (320 L, 6.93 mmol,
lmol, 1 equiv) was dissolved
l
l
l
64 equiv). The reaction mixture was warmed to rt and stirred for
2 h, before being concentrated and purified by flash chromatogra-
phy with EtOAc as eluent to provide 2 as a white solid (6 mg, 20%):
mp 102–104 °C; ½a D24
ꢁ
+9.7 (c 0.24, CH3OH); IR 3380, 2964, 2930,
C
48H60N4NaO7S3Si (M+Na)+ 951.3286, found 951.3287.
2843, 1750, 1678, 1591, 1512, 1262, 1187, 1043 cmꢂ1
;
1H NMR
(400 MHz, CDCl3) d 7.74 (s, 1H), 7.09 (br s, 1H), 6.41 (br s, 1H),
5.94–5.71 (m, 2H), 5.52 (dd, J = 15.5, 7.3 Hz, 1H), 5.23 (dd, J = 17.4,
8.9 Hz, 1H), 4.27 (dd, J = 17.3, 4.0 Hz, 1H), 4.21–4.07 (m, 2H), 3.88
(dd, J = 18.8, 3.1 Hz, 1H), 3.25 (d, J = 11.3 Hz, 1H), 2.92 (dd, J = 16.7,
11.0 Hz, 1H), 2.69 (d, J = 15.2 Hz, 1H), 2.58 (q, J = 7.4 Hz, 2H), 2.37
(q, J = 6.7 Hz, 2H), 1.84 (s, 3H), 1.39 (t, J = 7.8 Hz, 1H); 13C NMR
(100 MHz, CDCl3) d 173.8, 169.5, 167.7, 167.5, 166.2, 135.4, 133.1,
128.8, 124.6, 84.3, 72.5, 43.7, 42.2, 41.3, 40.2, 36.2, 25.3, 23.8; ES+
MS m/z 477 ([M+Na]+); HRMS (ESI) m/z calcd. for C18H22N4NaO4S3
(M+Na)+ 477.0695, found 477.0702.
4.1.13. (5R,12S)-5-Methyl-12-((E)-4-(tritylthio)but-1-en-1-yl)-
11-oxa-3,18-dithia-7,15,20,21-tetraazatricyclo[15.2.1.12,5]
henicosa-1(19),2(21),17(20)-triene-6,10,14-trione (27)
Acyclic precursor 25 (152 mg, 0.16 mmol) was dissolved in
CH2Cl2 (7.6 mL), cooled to 0 °C and treated with TFA (1.6 mL).
The reaction mixture was warmed to rt, stirred overnight, concen-
trated in vacuo and then coevaporated with toluene to remove
residual TFA. The crude amino acid (120 mg, 0.17 mmol, 1 equiv)
was then taken up in anhydrous CH2Cl2 (14 mL) and added drop-
wise to a vigorously stirred solution of Hünig’s base (172 lL,
0.99 mmol, 6 equiv), HOBt (45 mg, 0.33 mmol, 2 equiv) and HATU
(125 mg, 0.33 mmol, 2 equiv) in anhydrous acetonitrile (120 mL).
The reaction mixture was stirred overnight, concentrated in vacuo
and the crude material was purified by flash chromatography (hex-
ane/EtOAc 10:1 then 100% EtOAc) to afford 27 as a colourless oil
4.1.11. (S,E)-2-(Trimethylsilyl)ethyl 3-(3-(((9H-fluoren-9-yl)
methoxy) carbonylamino)propanoy-loxy)-7-(tritylthio)hept-4-
enoate (23)
The protected b-hydroxy ester (208 mg, 0.40 mmol, 1 equiv)
and N-Fmoc-b-alanine (137 mg, 0.44 mmol, 1.1 equiv) were dis-
solved in anhydrous CH2Cl2 (8 mL), cooled to 0 °C and DCC
(99 mg, 0.48 mmol, 1.2 equiv) and DMAP (4.9 mg, 0.04 mmol,
0.1 equiv) were then added. The reaction mixture was stirred over-
night. The precipitate was filtered and solvents were evaporated.
The crude was purified by flash chromatography (EtOAc/hexane
(20 mg, 17%): ½a 2D4
ꢁ
+37.5 (c 0.01, CH3OH). IR 2960, 2926, 2850,
1739, 1671, 1463, 1266, 1085, 1028, 805 cmꢂ1
;
1H NMR
(400 MHz, CDCl3) d 7.71 (s, 1H), 7.36–7.16 (m, 15H), 6.96 (br s,
1H), 6.09 (dd, J = 15.6, 7.5 Hz, 1H), 5.73–5.59 (m, 1H), 5.55 (td,
J = 7.2, 3.7 Hz, 1H), 4.99 (dd, J = 16.8, 7.8 Hz, 1H), 4.03 (d,
J = 11.5 Hz, 1H), 3.94 (dd, J = 17.1, 4.0 Hz, 1H), 3.49 (d, J = 4.0 Hz,
2H), 3.33 (d, J = 11.5 Hz, 1H), 2.78–2.61 (m, 2H), 2.56–2.42 (m,
2H), 2.39–2.22 (m, 2H), 2.18–1.95 (m, 2H), 1.68 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 174.1, 171.5, 169.7, 167.5, 162.0, 148.0,
144.7, 132.0, 129.9, 129.6, 127.9, 126.7, 124.1, 84.6, 73.0, 66.9,
42.7; 42.3, 40.9, 35.4, 33.8, 31.4, 31.3, 25.5; ES+ MS m/z 733
([M+Na]+); HRMS (ESI) m/z calcd. for C38H38N4NaO4S3 (M+Na)+
733.1947, found 733.1947.
5:95) to give 23 as a colorless oil (207 mg, 64%): ½a D23
ꢂ9.9 (c
ꢁ
0.56, CHCl3); IR 3376, 3058, 2949, 1724, 1504, 1455, 1244, 1164,
1081, 1066, 1036, 990, 975, 862, 835, 737, 703 cmꢂ1 1H NMR
;
(400 MHz, CDCl3) d 7.75 (d, J = 7.5 Hz, 2H), 7.59 (d, J = 7.0 Hz, 2H),
7.52–7.34 (m, 19H), 5.70–5.53 (m, 2H), 5.48 (br s, 1H), 5.38 (dd,
J = 15.6, 7.0 Hz, 1H), 4.34 (d, J = 7.0 Hz, 2H), 4.28–4.07 (m, 3H),
3.40–3.50 (m, 2H), 2.69–2.46 (m, 4H), 2.19 (t, J = 7.0 Hz, 2H), 2.07
(t, J = 6.5 Hz, 2H), 0.95 (t, J = 8.5 Hz, 2H), 0.01 (s, 9H); 13C NMR
(100 MHz, CDCl3) d 171.2, 170.1, 156.3, 144.8, 144.0, 141.3,
133.1, 129.6, 128.0, 127.9, 127.7, 127.0, 126.6, 125.1, 119.9, 70.9,
66.8, 66.6, 63.1, 47.2, 39.7, 36.7, 34.8, 31.3, 31.2, 17.3, ꢂ1.5; ES+
MS m/z 834 ([M+Na]+); HRMS (ESI) m/z calcd. for C49H53NNaO6SSi
(M+Na)+ 834.3255, found 834.3270.
4.1.14. (5R,12S)-12-((E)-4-Mercaptobut-1-en-1-yl)-5-methyl-11-
oxa-3,18-dithia-7,15,20,21-tetra-azatricyclo[15.2.1.12,5]
henicosa-1(19),2(21),17(20)-triene-6,10,14-trione (3)
S-Trityl macrocycle 27 (16.1 mg, 23
solved in CH2Cl2 (5.5 mL) and cooled to 0 °C. The mixture was suc-
cessively treated with Et3SiH (10 L, 130 mol, 2.7 equiv) and TFA
(320 L, 6.93 mmol, 64 equiv). The reaction mixture was warmed
lmol, 1 equiv) was dis-
l
l
4.1.12. (S,E)-2-(Trimethylsilyl)ethyl 3-(3-((R)-2-(2-((tert-butoxy
carbonylamino)methyl)thiazol-4-yl)-4-methyl-4,5-dihydro
thiazole-4-carboxamido)propanoyloxy)-7-(tritylthio)hept-4-
enoate (25)
l
to rt and stirred for 2 h, before being concentrated and purified
by flash chromatography with EtOAc as eluent to provide 3 as a
white solid (9 mg, 85%): mp 80–82 °C; ½a D24
ꢁ
+1.6 (c 0.36, CH3OH);
Carbamate 23 (185 mg, 0.23 mmol, 1 equiv) was dissolved in
IR 3353, 2926, 2854, 1739, 1671, 1542, 1179, 1039 cmꢂ1 1H
;
anhydrous acetonitrile (5 mL) and diethylamine (118
l
l, 1.14 mmol,
NMR (300 MHz, CDCl3/CD3OD) d 7.78 (br s, 1H), 7.65 (s, 1H), 7.19

H. Benelkebir et al. / Bioorg. Med. Chem. 19 (2011) 3650–3658
3657
(br s, 1H), 5.65 (t, J = 5.0 Hz, 2H), 5.43 (m, 1H), 4.94 (td, J = 16.6,
3.8 Hz, 1H), 4.24 (d, J = 16.6 Hz, 1H), 3.92 (d, J = 11.5 Hz, 1H), 3.39
(q, J = 5.0 Hz, 2H), 3.24 (m, J = 11.5 Hz, 1H), 2.72 (dd, J = 15.8,
6.8 Hz, 1H), 2.58–2.34 (m, 5H), 2.34–2.15 (m, 2H), 1.65 (s, 3H);
13C NMR (300 MHz, CDCl3/CD3OD) d 174.4, 171.3, 170.8, 167.8,
163.3, 147.5, 131.2, 129.9, 124.1, 84.1, 72.6, 42.1, 41.9, 40.5, 35.9,
34.8, 33.1, 25.1, 23.4; ES+ MS m/z 491 ([M+Na]+); HRMS (ESI) m/z
calcd. for C19H24N4NaO4S3 (M+Na)+ 491.0852, found 491.0850.
7.81 (s, 1H), 7.54–7.40 (m, 6H), 7.40–7.22 (m, 9H), 7.19 (d,
J = 8.5 Hz, 1H), 5.82–5.57 (m, 2H), 5.41 (dd, J = 7.5, 15.6 Hz, 1H),
5.34 (br s, 1H), 4.66 (d, J = 5.5 Hz, 2H), 4.58 (dd, J = 8.5, 4.0 Hz,
1H), 4.30–4.09 (m, 2H), 2.72 (dd, J = 15.8, 7.8 Hz, 1H), 2.58 (dd,
J = 15.6, 6.0 Hz, 1H), 2.35–2.15 (m, 3H), 2.09 (q, J = 6.5 Hz, 2H),
1.73 (s, 3H), 1.76 (s, 3H), 1.55 (s, 9H), 1.09–0.79 (m, 8H), 0.10 (s,
9H); 13C NMR (100 MHz, CDCl3) d 173.9, 170.7, 169.6, 169.4,
160.7, 155.6, 149.8, 144.8, 133.7, 129.5, 127.8, 127.8, 126.6,
123.8, 80.5, 71.6, 66.6, 63.1, 57.6, 57.1, 42.3, 39.7, 31.3, 31.1,
28.3, 25.8, 25.0, 19.0, 17.4, 17.3, ꢂ1.5; ES+ MS m/z 965 ([M+Na]+
100%); HRMS (ESI) m/z calcd. for C50H66N4NaO8S2Si (M+Na)+
965.3984, found 965.3964.
4.1.15. Methyl 2-(2-((tert-butoxycarbonylamino)methyl)thia
zole-4-carboxamido)-2-methylpro-panoate (28)
To a solution of 9 (200 mg, 0.77 mmol, 1.1 equiv) in CH2Cl2
(10 mL) cooled at 0 °C, PyBOP (549 mg, 0.93 mmol, 1.1 equiv) and
Hünig’s base (405 lL, 2.33 mmol, 3 equiv) were added. After
4.1.18. (7S,10S,E)-7-Isopropyl-4,4-dimethyl-10-(4-(tritylthio)
but-1-enyl)-9-oxa-16-thia-3,6,13,18-tetraaza-bicyclo[13.2.1]
octadec-1(17)-ene-2,5,8,12-tetraone (31)
Acyclic precursor 30 (218 mg, 0.23 mmol) was dissolved in
CH2Cl2 (11 mL), cooled to 0 °C and treated with TFA (2.3 mL). The
reaction mixture was warmed to rt, stirred overnight, concentrated
in vacuo and then coevaporated with toluene to remove residual
TFA.The crude amino acid (172 mg, 0.23 mmol, 1 equiv) was then
taken up in CH2Cl2 (20 mL) and added dropwise to a vigorously
10 min, 2-aminoisobutyric acid methyl ester hydrochloride
(108 mg, 0.70 mmol, 1 equiv) in solution in CH2Cl2 (10 mL) was
added dropwise. The reaction mixture was stirred at rt overnight
and concentrated in vacuo. The crude material was purified by
flash chromatography (EtOAc/hexane 3:7) to afford 28 as a white
solid (244 mg, 97%): mp 91.7–93.6 °C; IR 3338, 2986, 2933, 1746,
1712, 1667, 1538, 1448, 1368, 1285, 1247, 1149 cmꢂ1 1H NMR
;
(300 MHz, CDCl3) d 8.00 (s, 1H), 7.74 (br s, 1H), 5.28 (br s, 1H),
4.60 (d, J = 5.9 Hz, 2H), 3.77 (s, 3H), 1.67 (s, 6H), 1.48 (s, 9H); 13C
NMR (75 MHz, CDCl3) d 174.7, 169.2, 160.2, 150.0, 149.8, 123.6,
80.5, 56.4, 52.7, 42.3, 28.3, 25.0; ES+ MS m/z 380 ([M+Na]+); HRMS
(ESI) m/z calcd. for C15H23N3NaO5S (M+Na)+ 380.1251, found
380.1249.
stirred solution of Hünig’s base (242 lL, 1.39 mmol, 6 equiv), HOBt
(63 mg, 0.46 mmol, 2 equiv) and HATU (176 mg, 0.46 mmol,
2 equiv) in acetonitrile (165 mL). The reaction mixture was stirred
overnight, and then concentrated in vacuo. The crude material was
purified by flash chromatography (hexane/EtOAc 10:1 to 100%
EtOAc) to afford 31 as a pale yellow solid (108 mg, 64%): mp
4.1.16. 2-(2-((tert-Butoxycarbonylamino)methyl)thiazole-4-
carboxamido)-2-methylpropanoic acid (29)
132–134 °C; ½a 2D5
ꢁ
+32.3 (c 0.34, CHCl3); IR 3364, 2967, 2926,
2854, 1739, 1678, 1542, 1493, 1444, 1255, 1179, 839, 748,
To a solution of 28 (242 mg, 0.68 mmol, 1 equiv) in 8 mL of THF/
water (3:1) at 0 °C was added LiOH (49 mg, 2.03 mmol, 3 equiv).
The reaction mixture was stirred at rt overnight. The solution
was diluted with water (3 mL) and acidified to pH 1–2 with a sat-
urated KHSO4 solution. This aqueous layer was extracted twice
with EtOAc (10 mL). The combined organic layers were washed
with brine, dried over MgSO4 and concentrated in vacuo to provide
29 as a yellow solid (225 mg, 96%): mp 171–173 °C; IR 3342, 2930,
2850, 2363, 2340, 1712, 1656, 1542, 1455, 1376, 1278, 1251,
695 cmꢂ1 1H NMR (400 MHz, CDCl3/CD3OD) d 8.32 (s, 1H), 8.05
;
(d, J = 9.0 Hz, 1H), 7.87 (d, J = 2.5 Hz, 1H), 7.64–7.37 (m, 15H),
6.00–5.82 (m, 2H), 5.82–5.68 (m, 1H), 5.26 (d, J = 17.6 Hz, 1H),
4.78 (dd, J = 9.3, 4.8 Hz, 1H), 4.47 (d, J = 17.1 Hz, 1H), 3.06 (dd,
J = 17.1, 9.5 Hz, 1H), 2.90 (d, J = 16.1 Hz, 1H), 2.60–2.40 (m, 3H),
2.40–2.17 (m, 2H), 2.12 (s, 3H), 1.86 (s, 3H), 1.14 (d, J = 7.0 Hz,
3H), 0.97 (d, J = 7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3/CD3OD) d
175.9, 171.8, 170.7, 167.9, 163.1, 149.9, 145.5, 133.7, 130.3,
128.9, 128.6, 127.4, 124.0, 72.7, 67.5, 60.0, 58.4, 41.1, 40.8, 32.0,
31.9, 31.8, 25.6, 25.3, 19.8, 17.5; ES+ MS m/z 747 ([M+Na]+); HRMS
(ESI) m/z calcd. for C40H44N4NaO5S2 (M+Na)+ 747.2645, found
747.2641.
1160 cmꢂ1 1H NMR (400 MHz, CD3OD) d 8.10 (s, 1H) 4.55 (s, 2H)
;
1.66 (s, 6H) 1.50 (s, 9H); 13C NMR (100 MHz, CD3OD) d 177.8,
173.0, 162.7, 158.5, 150.9, 124.9, 81.1, 57.7, 43.3, 28.8, 25.3; ES+
MS m/z 366 ([M+Na]+); HRMS (ESI) m/z calcd. for C14H21N3NaO5S
(M+Na)+ 366.1094, found 366.1094.
4.1.19. (7S,10S,E)-7-Isopropyl-10-(4-mercaptobut-1-enyl)-4,4-
dimethyl-9-oxa-16-thia-3,6,13,18-tetraaza-bicyclo[13.2.1]
octadec-1(17)-ene-2,5,8,12-tetraone (4)
S-Trityl macrocycle 31 (73.1 mg, 0.10 mmol, 1 equiv) was dis-
solved in CH2Cl2 (12 mL) and cooled to 0 °C. The mixture was suc-
4.1.17. (S,E)-2-(Trimethylsilyl)ethyl 3-((S)-2-(2-(2-((tert-butoxy
carbonylamino)methyl)thiazole-4-carboxamido)-2-methylprop
anamido)-3-methylbutanoyloxy)-7-(tritylthio)hept-4-enoate
(30)
cessively treated with Et3SiH (32
lL, 0.20 mmol, 2 equiv) and TFA
Carbamate 17 (371 mg, 0.44 mmol) was dissolved in anhydrous
(495 L, 6.93 mmol, 61 equiv). The reaction mixture was warmed
l
acetonitrile (4 mL) and diethylamine (229
ll, 2.21 mmol). After stir-
to rt and stirred for 2 h, before being concentrated and purified
ring for 2 h, the reaction mixturewas concentrated in vacuo. The free
amine was purified by flash chromatography (EtOAc/hexane 1:1).
To a solution of 29 (86.2 mg, 0.25 mmol, 1.1 equiv) in anhy-
drous CH2Cl2 (5 mL) cooled to 0 °C, PyBOP (162 mg, 0.27 mmol,
by flash chromatography with EtOAc as eluent to provide 4 as a
white solid (6 mg, 12%): mp 56–58 °C; ½a D25
ꢁ
+61.3 (c 0.24, CHCl3);
IR 3334, 2960, 2926, 2854, 1734, 1678, 1542, 1463, 1383, 1256,
974 cmꢂ1 1H NMR (400 MHz, CDCl3) d 8.03 (s, 1H), 7.65 (br s,
;
1.2 equiv) and Hünig’s base (119
l
L, 0.69 mmol, 3 equiv) were
1H), 6.60–6.39 (m, 2H), 5.84–5.70 (m, 3H), 5.16 (dd, J = 17.3,
7.8 Hz, 1H), 4.64 (dd, J = 9.5, 4.0 Hz, 1H), 4.37 (dd, J = 17.6, 4.0 Hz,
1H), 2.86–2.62 (m, 2H), 2.62–2.46 (m, 2H), 2.46–2.21 (m, 3H),
1.90 (s, 3H), 1.62 (s, 3H), 1.35 (t, J = 7.5 Hz, 1H), 0.91 (d,
J = 6.5 Hz, 3H), 0.72 (d, J = 7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3)
d 174.4, 170.0, 168.9, 166.9, 161.0, 149.5, 132.1, 129.0, 123.3,
71.8, 59.6, 57.4, 41.3, 40.8, 36.0, 31.3, 25.8, 24.8, 23.8, 19.1, 16.8;
ES+ MS m/z 505 ([M+Na]+); HRMS (ESI) m/z calcd. for
added. After 10 min, the previous deprotected 17 (141 mg,
0.23 mmol, 1 equiv) in solution in CH2Cl2 (5 mL) was added drop-
wise. The reaction mixture was left at rt overnight and concen-
trated in vacuo. The crude material was purified by flash
chromatography (EtOAc/hexane 3:7) to afford 30 as a white solid
(134 mg, 62%): mp 61–63 °C; ½a D25
ꢁ
+5.4 (c 0.26, CHCl3); IR 3338,
2960, 2926, 1731, 1682, 1535, 1497, 1444, 1391, 1365, 1251,
1172, 1028, 975 cmꢂ1
;
1H NMR (400 MHz, CDCl3) d 8.10 (s, 1H),
C
21H30N4NaO5S2 (M+Na)+ 505.1550, found 505.1551.

3658
H. Benelkebir et al. / Bioorg. Med. Chem. 19 (2011) 3650–3658
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4.2. HDAC enzyme assay and MCF7 growth inhibition assay
These were performed according to standard procedures as pre-
viously reported.7d The HDAC enzyme assay of largazole thiol and
analogues 1–4 was performed in the presence of DDT, by incubat-
ing 1 mM of compound with 10 mM DTT (10-fold excess) for 1 h
prior to the assay.
4.3. Metabolic stability studies
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Murine liver homogenates were prepared by adding 10 mM Tris
buffer (pH 7.4, 1:3 w:v) to the tissue and gently breaking it up with
a pestle and mortar. The assay commenced with the addition of
compound to preincubated homogenate at 37 °C to give a final
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drug concentration of 100
l
M. Samples (20
l
L) were taken at 0,
15, 30, 45, 60 and 120 min, diluted with methanol (60
lL) and
stored on ice prior to centrifugation at 10,000g for 5 min at 4 °C
to precipitate liver proteins. The supernatant was transferred to a
polypropylene vial and stored at ꢂ20 °C before analysis.
Samples (2 lL) were analyzed by UPLC/MS employing gradient
separation. Mobile phase A consisted of 10% MeOH/90% dH2O con-
taining 0.05% TFA, and mobile phase B of 90% MeOH/10% dH2O
containing 0.05% TFA. Analysis was performed on a Waters C18
10 cm Acquity column 1.7
l
M (10 cm ꢀ 2.1 mm, Milford) with a
flow rate of 0.3 mL/min. The gradient started with 25% B which in-
creased to 30% over the first 2 min. From 2 to 30 min %B increased
from 30% to 80%, from 30 to 32 min %B decreased from 80% to 25%
and remained at 25% until the end of the run. Peaks were detected
by ESI MS using Selected Ion Recording for the parent ions.
Acknowledgements
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We are grateful to Cancer Research UK and EPSRC for student-
ships to H.B. and S.M., respectively. We thank Cancer Research
UK, the Southampton Cancer Research UK Centre and Experimental
Cancer Medicine Centre and the COST Action TD0905 ‘Epigenetics:
Bench to Bedside’ for financial support.
Supplementary data
Supplementary data (Synthetic procedures for preparation of
known compounds and NMR spectra for novel compounds) associ-
ated with this article can be found, in the online version, at
doi:10.1016/j.bmc.2011.02.024.
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