Modular synthesis and biological activity of pyridyl-based analogs of the potent Class i Histone Deacetylase Inhibitor Largazole

Article abstract of DOI:10.1016/j.bmc.2015.03.063

The formation of a series of analogs containing a pyridine moiety in place of the natural thiazole heterocycle, based on the potent, naturally occurring HDAC inhibitor Largazole has been accomplished. The synthetic strategy was designed modularly to access multiple inhibitors with different aryl functionalities containing both the natural depsipeptide and peptide isostere variant of the macrocycle. The cytotoxicity and biochemical activity of the library of HDAC inhibitors is described herein.

Full text of DOI:10.1016/j.bmc.2015.03.063

Accepted Manuscript
Modular Synthesis and Biological Activity of Pyridyl-based Analogs of the Po-
tent Class I Histone Deacetylase Inhibitor Largazole
Dane J. Clausen, William B. Smith, Brandon E. Haines, Olaf Wiest, James E.
Bradner, Robert M. Williams
PII:
S0968-0896(15)00259-X
http://dx.doi.org/10.1016/j.bmc.2015.03.063
BMC 12189
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 January 2015
23 March 2015
24 March 2015
Please cite this article as: Clausen, D.J., Smith, W.B., Haines, B.E., Wiest, O., Bradner, J.E., Williams, R.M.,
Modular Synthesis and Biological Activity of Pyridyl-based Analogs of the Potent Class I Histone Deacetylase
Inhibitor Largazole, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.03.063
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Modular Synthesis and Biological Activity of Pyridyl-based Analogs of
the Potent Class I Histone Deacetylase Inhibitor Largazole
Dane J. Clausen,a William B. Smith,b Brandon E. Haines,c Olaf Wiest,c James E. Bradner,b and Robert
M. Williamsa,d,*
a Department of Chemistry, Colorado State University, Fort Collins, CO 80523, USA
b Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA
c Department of Chemistry & Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA
d University of Colorado Cancer Center, Aurora, CO 80045, USA
ABSTRACT
The formation of a series of analogs containing a pyridine moiety in place of the natural thiazole
heterocycle, based on the potent, naturally occurring HDAC inhibitor Largazole has been
accomplished. The synthetic strategy was designed modularly to access multiple inhibitors with
different aryl functionalities containing both the natural depsipeptide and peptide isostere variant of the
macrocycle. The cytotoxicity and biochemical activity of the library of HDAC inhibitors is described
herein.
Abbreviations
HDACi = Histone Deacetylase Inhibitor
Keywords
Largazole, Class I Histone Deacetylase Inhibitor, pyridine analogs, cytotoxic and biochemical assays
Corresponding author: e-mail: robert.williams@colostate.edu; phone: 970-491-6747; FAX: 970-491-
3944
1

1. INTRODUCTION
Largazole (1) has received a great deal of attention from the synthetic and medical communities
ever since its isolation from the marine cyanobacterium of the genus Symploca by the Leusch
laboratory.1,2 This depsipeptide natural product has been synthesized and modified by several research
groups due to its potent biochemical activity as a Class I Histone Deacetylase Inhibitor (HDACi) and
consequent anticancer properties.3-5 Largazole (1) is a pro-drug with the active form being the free thiol
species 2 (Figure 1) that is formed in vitro and in vivo by esterase or lipase-based cleavage of the
octanoyl residue.6 It has been suggested by us that the octanoyl tail present on Largazole allows for
better cell-permeability relative to the active, free thiol species.6 The nascent thiol group tightly
coordinates to the Zn2+-domain within the HDAC enzyme active site resulting in tight, non-covalent
binding of the drug and consequent enzymatic inhibition.
Figure 1. Structure of Largazole and Largazole thiol and their peptide isosteres.
There are currently eighteen known HDAC enzymes present in human cells that are separated
into four different classes based on their structural homology with yeast proteins.7 Of these, Class I
(HDACs 1, 2, 3, and 8), Class II (HDAC 4, 5, 6, 7, 9, and 10), and Class IV (HDAC 11) are Zn2+-
2

dependent while Class III (SirT1-7, known as the Sirtuins) are NAD+-dependent. A substantial body of
data has elucidated that the inhibition of the ubiquitous Class I HDACs is a viable anticancer
therapeutic target.8,9 Current research efforts in our laboratories have been to access inhibitors that are
selective for Class I HDACs over the tissue-dependent Class II HDACs. The goal of our efforts has
been to generate compounds based on the molecular scaffold of Largazole that exhibit superior
potency, selectivity and drug-like properties relative to that of the natural product.
We have been active in pursuing new analogs of Largazole (1) since the completion of our
concise total synthesis in 2008.6 The first key structural alteration we examined, was to replace the
depsipeptide oxygen atom present in the macrocycle with a lactam nitrogen atom providing the
corresponding more chemically robust peptide isostere 3 (Figure 1).10 We also examined other
functional group changes in the macrocycle, as well as the zinc-binding domain.11,12 The present work
described herein continues this exploration by investigating the exchange of the thiazole heterocycle in
the macrocyclic cap group for a pyridyl residue in both the natural depsipeptide and peptide isostere
variants of the macrocyclic core (Figure 2).
3

Figure 2. Pyridine containing library based on Largazole.
2. RESULTS AND DISCUSSION
2.1. Design. The macrocyclic cap group of Largazole not only contributes significantly to the overall
binding, but also offers unique opportunities for the design of even more potent and selective analogs
because it allows the precise positioning of functional groups on the surface of the protein by
appropriate functionalization of the macrocycle. The analysis of the position of Largazole in our model
of the HDAC1-Largazole thiol complex13 as well as the recently published crystal structure of the
HDAC8-Largazole thiol complex (pdb 3EQD)14 provide the structural basis for rational design that
exploits specific surface interactions. Based on the sequence alignment of the residues surrounding the
4

active site (Figure S1 in the Supporting Information), we envisioned the replacement of the thiazole ring
by a protonated pyridine to form a charge interaction with a nearby negatively charged residue (Asp),
as shown in Figure 3. In addition, the inclusion of a positive charge could modify the conformational
preferences of the macrocycle, which was shown previously to be important for the relative potency of
the depsipetide and its amide isostere,10 and control its overall shape, which is known to control isoform
selectivity.15 With these considerations in mind, two different isomers of the pyridyl-based analogs of
Largazole, compounds 6 and 8 their respective amide isosteres 10 and 12 were chosen for synthetic
studies and biochemical profiling.
Figure 3. Model of pyridyl Largazole thiol analogue bound to HDAC1
2.2. Chemistry. Our published total synthesis of Largazole (1) provided the technological framework
from which we have been able to access numerous new analogs.6 The retrosynthetic analysis
displayed in Scheme 1 outlines the modular nature of our approach. The key macrolactamization
reaction we have found is best performed on the least-hindered amide located at the bottom of
5

macrocycle 13. The target macrocycle has been bisected into two halves through the top amide
linkage. Bottom fragment 14 is constituted from L-Valine derivative 17 and both the requisite ꢀ-hydroxy
ester (16, X = OH, for the depsipeptides) and the corresponding ꢀ-amino ester (16, X = NH2, for the
peptide isosteres). The thiazoline/pyridyl-containing fragment 15 will be formed through a
cyclocondensation reaction between ꢁ-methyl-L-cysteine (18) and aryl nitrile 19.
Scheme 1. Retrosynthetic analysis of library.
The synthesis of fragment 23 for the depsipeptide version of the macrocycle is displayed in
Scheme 2. The formation of aldehyde 21 was achieved through a hetero-Michael addition of trityl
mercaptan into acrolein followed by a Wittig olefination. The stereocenter present in alcohol 22 was set
using a Crimmins’s type chiral auxiliary.16 This asymmetric aldol transformation occurred to produce
alcohol 22 in very high diastereomeric purity. This material was submitted to cleavage of the chiral
auxillary using 2-(trimethylsilyl)ethanol. Installation of the L-valine derived amino acid was mediated by
EDCI to access depsipeptide bottom fragment 23.
6

Scheme 2. Synthesis of fragment 23 for depsipeptide macrocycle. Reagents and conditions: (a) TrtSH,
Et3N, CH2Cl2; (b) (formylmethylene)triphenylphosphorane, PhH, 80 °C, 77% over 2 steps; (c) (R)-1-(4-
benzyl-2-thioxothiazolidin-3-yl)ethanone, TiCl4, DIPEA, CH2Cl2, –78 °C, 76%; (d) 2-
(trimethylsilyl)ethanol, imidazole, CH2Cl2, 83%; (e) N-Fmoc-Val-OH, EDCI, DIPEA, DMAP, CH2Cl2,
77%.
Peptide precursor 28 was furnished using the previously established protocol (Scheme 3).10
Commercially available amino acid derivative 24 was converted to the primary alcohol through
reduction of a mixed anhydride. Swern oxidation followed by Wittig olefination achieved formation of
terminal alkene 25. Scrambling of the stereogenic center was not apparent through an aqueous KHSO4
workup of the oxidation step and submission of the aldehyde directly to the olefination reaction. The
cross metathesis to construct alkene 26 proved to be troublesome in producing high yielding and
reproducible results.17 The primary alcohol was converted to the protected thiol through activation with
TsCl and displacement with trityl thiol anion. After methyl ester formation, the amine present on
substrate 27 was unmasked using TFA and coupled with an L-valine derived amino acid to yield peptide
precursor 28.
7

Scheme 3. Synthesis of fragment 28 for peptide macrocycle.
Reagents and conditions: (a) 4-methylmorpholine, isobutyl chloroformate, THF, –40 °C; NaBH4, MeOH, –20 °C,
66%; (b) oxalyl chloride, DMSO, DIPEA, CH2Cl2, –65 °C; (c) methyltriphenylphosphonium bromide, KHMDS, THF,
–78 °C, 80% over 2 steps; (d) 3-buten-1-ol, Grubbs catalyst, 2nd generation, CH2Cl2, 50 °C, 25%; (e) TsCl, Et3N,
DMAP, CH2Cl2, 82%; (f) TrtSH, KOtBu, THF, 87%; (g) LiOH, THF, MeOH, 50 °C, 96%; (h) MeOH, EDCI, DIPEA,
DMAP, CH2Cl2, 77%; (i) TFA, CH2Cl2; (j) N-Boc-Val-OH, PyBOP, DIPEA, CH2Cl2, 88% over 2 steps.
Aryl fragment 32 was constructed starting with dicarboxylic acid 29 (Scheme 4). Fischer
esterification and reduction of one of the ethyl esters provided the requisite primary alcohol
functionality. After using NH4OH to generate an amide from the corresponding carboxylic acid, POCl3
successfully dehydrated the amide to generate the requisite nitrile and chloride present in compound
30. Gabriel’s protocol was utilized to convert the chloride into an amine, which was readily protected to
provide cyclization partner 31. Cyclocondensation with ꢁ-methyl-L-cysteine18 proceeded in high yields
to generate the pyridyl fragment 32.
Scheme 4. Synthesis of the pyridyl “IN” fragment 32.
Reagents and Conditions: (a) PTSA, EtOH, 80 °C, 100%; (b) NaBH4, EtOH, 80 °C, 53%; (c) NH4OH, EtOH; (d)
POCl3, DMF, 0 °C; (e) potassium phthalimide, DMF, 69% over three steps; (f) H2NNH2-H2O, MeOH, THF; (g)
Boc2O, Et3N, CH2Cl2, 43% over 2 steps; (h) ꢁ-methyl-L-cysteine, NaHCO3, pH 6.0 buffer, MeOH, 70 °C, 84%.
8

The route for the pyridyl “OUT” aryl fragment 36 is displayed in Scheme 5. The synthetic
strategy follows the same method mentioned previously for the pyridine “IN” variant except dicarboxylic
acid 33 was used instead.
Scheme 5. Synthesis of pyridine “OUT” fragment.
Reagents and conditions: (a) PTSA, EtOH, 80 °C, 100%; (b) NaBH4, CaCl2, EtOH, 80 °C, 61%; (c) NH4OH, EtOH;
(d) POCl3, DMF, 0 °C; (e) potassium phthalimide, DMF, 50% over three steps; (f) H2NNH2-H2O, MeOH, THF; (g)
Boc2O, Et3N, CH2Cl2, 84% over 2 steps; (h) ꢁ-methyl-L-cysteine, NaHCO3, pH 6.0 buffer, MeOH, 70 °C, 85%.
The final steps to access the desired inhibitors were performed upon the successful assembly of
the four key fragments. The depsipeptide class construction is shown in Scheme 6. The protected
amine present in substrate 23 was unmasked using diethylamine. The successful union between the
nascent amine and acid 32 or 36 was accomplished cleanly using PyBOP. The macrolactamization was
performed using HATU and HOBt after exposing the requisite functionality through acid-mediated
deprotection. Accessing pure material from the ring closure transformation required rigorous column
chromatography. Trityl cleavage granted the formation of inhibitors 6 and 8. Unfortunately, adding the
octanoyl residue was only viable using standard acid chloride procedure on pyridine “OUT”
depsipeptide thiol 8. Pyridine “IN” thiol 6 gave rise to dioctanoylation when exposed to the same
conditions. The N-octanoyl-imidazole reagent was prepared and then was exposed to thiol 6. The
change in the reactivity of the carbonyl gave rise to only the thioester being formed.
9

Scheme 6. Synthesis of depsipeptide containing analogs.
Reagents and conditions: (a) Et2NH, CH3CN; (b) 32, PyBOP, DIPEA, 79% over 2 steps; (c) 36, PyBOP, DIPEA,
81% over 2 steps; (d) TFA, CH2Cl2; (e) HATU, HOBt, DIPEA, CH3CN, 20% over 2 steps; (f) HATU, HOBt, DIPEA,
i
i
CH3CN, 23% over 2 steps; (g) TFA, Pr3SiH, CH2Cl2, 82%; (h) TFA, Pr3SiH, CH2Cl2, 89%; (i) N-octanoyl–
imidazole, imidazole, DMAP, THF, 50 °C, 69%; (j) octanoyl chloride, Et3N, 49%.
The creation of the peptide class of pyridine-thiazoline inhibitors 10 and 12 followed the same
method discussed for their depsipeptide counterparts. The only difference in their route arose during
10

the deprotection steps. The same strategy was employed as previously discussed to add the octanoyl
fragment to arrive at inhibitors 9 and 11.
Scheme 7. Synthesis of peptide containing inhibitors.
Reagents and conditions: (a) TFA, CH2Cl2; (b) 32, PyBOP, DIPEA, 84% over 2 steps; (c) 36, PyBOP, DIPEA,
92% over 2 steps; (d) LiOH, THF, H2O; (e) TFA, CH2Cl2; (f) HATU, HOBt, DIPEA, CH3CN, 33% over 3 steps; (g)
HATU, HOBt, DIPEA, CH3CN, 32% over 3 steps; (h) TFA, iPr3SiH, CH2Cl2, 94%; (i) TFA, iPr3SiH, CH2Cl2, 89%; (j)
N-octanoyl–imidazole, imidazole, DMAP, THF, 50 °C, 66%; (k) octanoyl chloride, Et3N, 55%.
11

2.3. Biochemical & Biological Evaluation. Compounds 1–12 were tested for inhibitory activity against
HDACs 1–9, using an optimized homogenous assay performed in a 384-well plate, as described
previously.6 The results of these studies, summarized in Table 1 and Figure S5, identify that the thiol
derivatives of Largazole including compounds 2, 4, 6, 8, 10, and 12, are among the most potent with
IC50 values below 25nM for HDACs 1-3. Of these thiols, the depsipeptide pyridine thiols 6, 8 show
similar potency to Largazole thiol (2) and Largazole peptide isostere thiol (4) with IC50 values in the
single digit nM range for HDACs 1-3. Conversely, the peptide pyridine thiols 10, 12 show a decrease in
potency with IC50s in the double-digit nM range for HDACS 1-3. Largazole (1) and derivatives 3, 5, 7, 9,
11 display IC50 values in the 200nM-1µM range. Of these compounds, Largazole (1) and peptide
pyridine “OUT” derivative 11 were similar in potency for HDACs 1-3 with IC50s in the high nM-µM range
while the depsipeptide pyridines and peptide pyridine “IN” derivative 5, 7, 11 exhibited an increased
potency with IC50s in the 200nM-350nM range for HDAC 1. These studies demonstrate that the free
thiol is important for increased inhibition, as previously reported, with the depsipeptide pyridine
derivatives displaying increased inhibition over the peptide isostere derivatives.
Compound
HDAC1
10.09
2.51
HDAC2
18.65
4.19
HDAC3
9.09
HDAC6
HDAC8
1
2
165.6
1068
2.78
28.11
228.4
3
544.1
1.95
825.2
3.38
1151
2.59
-
-
4
102
255.3
5
203.6
2.68
349.5
4.39
332.1
3.07
-
-
6
48.55
341.3
7
340
655.4
4.42
319.5
2.31
-
-
8
2.20
35.16
101.8
9
340.3
42
471.8
69.8
332.4
42.5
-
-
10
11
12
-
-
-
-
816.9
13.2
1240
20.77
846.5
14.59
2849
1491
Table 1. HDAC inhibitory activity of Largazole analogs (IC50 nM).
12

Next, we tested the activity of the novel analogs in the 797 and 10326 NUT midline carcinoma
cell line. As can be seen in Table 2 and Figure S6, depsipeptide pyridine “OUT” 7 showed the greatest
potency against both cell lines with an IC50 of 10nM. This was comparable to both Largazole and
depsipeptide pyridine “IN” 1, 5 with IC50s of 20nM against both 797 and 30nM and 40nM against 10326
respectively. Compound 10 was not evaluated due to it’s low biochemical activity.
797
10326
Compound
797
10326
LogIC50
LogIC50
-7.621
0.06565
-7.606
0.02845
1
2
0.024
0.1
0.025
0.08
-7.016
0.03453
-7.083
0.06683
-6.971
0.04341
-6.763
0.06589
3
4
5
6
0.11
1.74
0.03
0.22
0.17
11.94
0.038
0.29
-5.761
0.05449
-4.923
0.9900
-7.529
0.05220
-7.422
0.04499
-6.654
0.04604
-6.526
0.09239
-7.949
0.07215
-7.873
0.04062
7
8
0.01
0.09
0.01
0.12
-7.031
0.06228
-6.913
0.03566
-6.509
0.02559
-
-6.263
0.07115
-
9
0.31
-
0.55
-
10
-7.122
0.05525
-6.873
0.06520
11
12
0.07
4.25
0.13
8.64
-5.371
0.04826
-5.063
0.1964
Table 2. Activity of Largazole analogs in 797 and 10326 cell lines (IC50s µM)
Conversely, the peptide isosteres 9, 11 exhibited ~10-fold decreased inhibition in comparison to
the depsipeptide pyridine derivatives, demonstrating that the depsipeptide mimetic may be important for
increased inhibition. Compound 11 is more active in cell culture but it appears to have less
activity in the biochemical assay compared to the other prodrugs derivatives. While this is an
13

unexpected result in cell culture as the other prodrugs and their thiols derivatives show
stronger activity biochemically, it may be possible that the compound 11 is cleaved and
activated within cells to a higher extent than it’s prodrug counterparts yielding higher activity in
cell culture.
2.4. Computational Studies of Selectivity. The biochemical profiling provides a number of interesting
results that, when combined with the results from molecular dynamics simulations, probe the structural
basis for the observed selectivity. We studied Largazole thiol 2 and its analogues 6, 8, 10 and 12, which
are postulated to be the active form of the corresponding Largazole derivatives,6 in our homology
model of HDAC1,13 HDAC6,15 and the crystal structure of HDAC8 (pdb 3EQD).14 Here, we will discuss
the cases of the largest experimentally observed selectivity in detail, while the full results for all five
compounds in the three HDAC isoforms studied can be found in the Supporting Information.
We will start the discussion of the potency of Largazole thiol and its derivatives in HDAC1.
Experimentally, the most notable finding is the 16-fold decrease in activity in going from 6, which is
similar to Largazole thiol 2, to its amide isostere 10. Figure 4 shows snapshots from the MD simulations
of 6 (blue, with protein in red) and 10 (green) in HDAC1. For comparison purposes, the previously
analyzed10 position of 2 is shown in grey. As can be seen, the overall positions of 2 and 6 at the mouth
of the active site pocket are similar. In addition, 6 does indeed establish an interaction between the
protonated pyridyl ring and the key aspartate D99, although the NH-O distance fluctuates to up to >3.5
Å over the course of the simulation. However, the macrocyclic ring of 6 has to rotate relative to the
position of 2 to enable this interaction, decreasing the covered surface area. As a result, the overall IC50
of the two compounds is similar. In contrast, 10 does not establish over the course of the simulation,
presumably because it would require a ring conformation that is unfavorable for the more rigid amide
isostere. Instead, the pyridyl and thiazole ring establish hydrophobic interactions at the opposite side of
the binding pocket, predominately with F205 and leaving the protonated pyridyl ring solvent exposed.
The resulting weaker interactions are consistent with the 16-fold decrease in binding affinity for 10.
14

Figure 4. Snapshots from the MD simulations of 6 (blue, with protein in red) and 10 (green) in HDAC1.
Position of 2 is shown in grey for reference purposes
Compounds 2 and 6 are experimentally found to be 11 and 18 times less potent in HDAC6 than
in HDAC1 while 12 is >200 times less potent. The results for 2 and 6 can be understood in terms of the
previously discussed15 differences in the shape of the protein surface between HDAC6 and the class 1
HDACs such as HDAC1 at the mouth of the active site. Specifically, HDAC6 selective inhibitors such
as tubacin or WT161 have a Y-shaped cap group that is quite dissimilar to the macrocycle in the
compounds studied here. As a result of this shape mismatch, significant loop reorganization in this
region is necessary to establish binding contacts, as can be seen from the RMSD plots in Figure S3 in
the Supporting Information.
Compounds 2 and 12 occupy very similar positions in HDAC6, as shown in the snapshots from
the MD simulations of HDAC6 in Figure 5. However, the long timescale simulations of 12 bound to
HDAC6 indicate a reorientation of a helix (shown on the top left in Figure 5) that is only observed in this
system. It can therefore be hypothesized that it is energetically unfavorable despite the short NH-O
hydrogen bond of 1.96 Å and correlates with the low activity of 12.
15

Figure 5. Snapshots from the MD simulations of 2 (grey) and 12 (green) in HDAC6. Position of 2 is
shown in grey for reference purposes
All compounds studied are found to have a ~100:1 selectivity favoring the class I HDAC1-3 over
HDAC8, another member of the same isoform class. These results can be rationalized by the
computational finding that, in order to accommodate the large macrocyclic cap group, HDAC8 has to
adopt the “open” conformation where the two binding sites observed in the crystal structures of HDAC8
(e.g. pdb code 1T6419) are connected through the movement of F152 and Y306, leading to a large
groove resembling the one observed experimentally (pdb code 1VKG).19 This groove is stable over the
course of the simulation (see Figure S4 in the Supporting Information) and specific for HDAC8.20
Compound 2 and analogs can only establish contacts to one side of the binding pocket, leading to the
experimentally observed selectivity. In the representation shown in Figure 6, 2 only binds to the right
side of the pocket, while the interaction with D88 leads to an interaction of 12 with the left side of the
binding pocket.
16

Figure 6. Snapshots from the MD simulations of 2 (grey) and 12 (green) in HDAC8.
3. CONCLUSION
Potent HDACis have been synthesized by manipulating the macrocycle present on the natural
product Largazole. Inhibitors with excellent selectivity for only HDAC1, HDAC2, and HDAC3 were
formed through altering the depsipeptide framework to the peptide isostere variant and changing the
thiazole to a pyridine.29 The depsipeptide pyridyl variants proved to be equipotent (pyridyl “IN”) and
twice as potent (pyridyl “OUT”) when tested against two cell lines. These biological observations were
further rationalized through molecular modeling.
4. EXPERIMENTAL SECTION
General experimental method. Unless otherwise noted, all reactions were run under an argon
17

atmosphere in flame or oven dried glassware. Reactions were monitored using thin layer silica gel
chromatography (TLC) using 0.25 mm silica gel 60F plates with fluorescent indicator from Merck.
Plates were visualized by treatment with anisaldehyde stain with gentle heating. Products were purified
via column chromatography using the solvent system(s) indicated. Silica gel 60, 230-400 mesh, was
purchased from Sorbent Technologies. Tetrahydrofuran (THF), dichloromethane (CH2Cl2), acetonitrile
(CH3CN), triethylamine (Et3N), toluene, diethyl ether (Et2O), and N,N-dimethylformamide (DMF) were
passed through an alumina drying column (Solv-Tek Inc.) using argon pressure. All other reagents
were purchased from commercial sources and used as received without additional purification. 1H NMR
13
and C NMR spectra were recorded on Varian 300, 400, or 500 MHz NMR spectrometers. Chemical
shifts are reported in ppm relative to CHCl3 at ꢂ = 7.27 (1H NMR) and ꢂ = 77.23 (13C NMR) or
tetramethylsilane (TMS) ꢂ = 0.00, where noted. Mass spectra were obtained on Fisions VG Autospec.
Optical rotations were collected at 589 nm on a Rudolph Research Automatic Polarimeter Autopol III.
All compounds tested in the biological assays were shown to have a purity of >95% via NMR.
(S,E)-2-(Trimethylsilyl)ethyl
3-(((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-
methylbutanoyl)oxy)-7-(tritylthio)hept-4-enoate (23): To a solution of 2-(trimethylsilyl)ethyl (S,E)-3-
hydroxy-7-(tritylthio)hept-4-enoate6 (300 mg, 0.578 mmol) in CH2Cl2 (15.0 mL) at ambient temperature
was added N-Fmoc-L-valine (981 mg, 2.89 mmol), EDCI HCl (665 mg, 3.47 mmol), DMAP (7.1 mg,
0.058 mmol), and DIPEA (0.60 mL, 3.47 mmol). After stirring for 18 h, the reaction mixture was
concentrated. The crude residue was purified using flash chromatography (1% to 20% ethyl acetate in
1
hexanes) to provide ester (375 mg, 77%): H-NMR (CDCl3, 400 MHz) ꢂ 7.71-7.75 (m, 2H), 7.56-7.58
(m, 2H), 7.18-7.38 (m, 19H), 5.58-5.69 (m, 2H), 5.34 (dd, J = 15.6, 7.6 Hz, 1H), 5.25 (d, J = 9.2 Hz,
1H), 4.30-4.40 (m, 2H), 4.25 (dd, J = 8.8, 4.4 Hz, 1H), 4.20 (t, J = 6.8 Hz, 1H), 4.12 (t, J = 8.8 Hz, 2H),
2.65 (dd, J = 16.0, 8.0 Hz, 1H), 2.52 (dd, J = 15.6, 5.2 Hz, 1H), 2.10-2.17 (m, 3H), 2.02 (q, J =6.8 Hz,
2H), 0.91-0.99 (m, 2H), 0.89 (d, J = 6.8 Hz, 3H), 0.77 (d, J = 6.8 Hz, 3H), –0.01 (s, 9H); 13C-NMR
(CDCl3, 100 MHz) ꢂ 170.9, 169.6, 156.1, 144.8, 143.9, 143.8, 141.3, 134.0, 129.6, 127.9, 127.7, 127.0,
126.6, 125.1, 120.0, 71.8, 67.0, 66.6, 63.1, 58.7, 47.2, 39.7, 31.4, 31.3, 31.1, 19.0, 17.3, –1.5; IR (neat)
18

3059, 2979, 1731, 1509, 1447, 1249, 1182, 1033, 858, 741; HRMS (ESI) m/z calcd for
C51H57NNaO6SSi [M+Na]+ 862.3574, found 862.3583; [ꢁ]D = –15.0 (c 1.18, CHCl3).
(S,E)-Methyl
3-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-7-(tritylthio)hept-4-
enoate (28): To a solution of Boc protected amine 2710 (0.650 g, 1.22 mmol) in CH2Cl2 (37 mL) at 0 °C
was treated with TFA (0.37 mL). After 2 h, the reaction mixture was concentrated under vacuum, taken
back up in toluene (15 mL), and concentrated again. In another flask, Boc-L-valine (0.531 g, 2.44
mmol), PyBOP (1.27 g, 2.44 mmol), and DIPEA (0.65 mL, 3.66 mmol) were combined in CH2Cl2 (60
mL). The freshly deprotected amine was added via CH2Cl2 (10 mL) to the flask containing the activated
acid at ambient temperature. After 3 h, the resulting mixture was concentrated under reduced pressure.
The crude residue was purified using flash chromatography (5% to 50% ethyl acetate in hexanes) to
obtain the desired amide (0.680 g, 88%): 1H-NMR (CDCl3, 400 MHz) ꢂ 7.15-7.37 (m, 15H), 6.49 (d, J =
8.0 Hz, 1H), 5.45 (dtd, J = 15.2, 6.4, 0.8 Hz, 1H), 5.34 (dd, J = 15.6, 6.0 Hz, 1H), 4.97-5.02 (m, 1H),
4.67-4.74 (m, 1H), 3.82-3.86 (m, 1H), 3.59 (s, 3H), 2.53 (d, J = 5.2 Hz, 2H), 2.05-2.16 (m, 3H), 2.01 (q,
J = 6.8 Hz, 2H), 1.40 (s, 9H), 0.89 (d, J = 6.8 Hz, 3H), 0.83 (d, J = 6.8 Hz, 3H); 13C-NMR (CDCl3, 100
MHz) ꢂ 171.6, 170.5, 155.8, 144.8, 130.5, 129.5, 127.8, 126.5, 79.7, 66.5, 59.9, 51.7, 47.2, 38.7, 31.3,
31.0, 28.3, 19.3, 17.6; IR (neat) 3304, 2965, 1738, 1685, 1650, 1522, 1492, 1365, 1172, 751; HRMS
(ESI) m/z calcd for C37H46N2NaO5S [M+Na]+ 653.3025, found 653.3032; [ꢁ]D = –14.0 (c 1.21, CHCl3).
6-((((tert-Butoxy)carbonyl)amino)methyl)picolinonitrile (31): To a solution of commercially available
chloride 30 (9.66 g, 63.3 mmol) in DMF (400 mL) at ambient temperature was treated with potassium
phthalimide (11.7 g, 63.3 mmol). After stirring for 5 h, the mixture was concentrated under vacuum. The
remaining mixture was taken up in H2O (200 mL) and was filtered to collect the solid. The solid was
washed with H2O (100 mL) and THF (100 mL) to obtain the desired phthalimide derivitive (11.5 g, 69%)
and was moved forward without further purification. To a solution of the crude phthalimide derivative
(5.84 g, 22.2 mmol) in THF/MeOH (200 mL, 1:1, v/v) at ambient temperature was treated with
hydrazine monohydrate (1.18 mL, 24.4 mmol). After 2 h, 1.0 M HCl (24.5 mL) was added to the mixture
19

and was stirred for another 3 h before concentrating the reaction mixture under vacuum. The remaining
residue was taken up in H2O (200 mL) and the unwanted solid was removed through filtration. The
filtrate was concentrated and placed under vacuum to remove the remaining H2O. The crude solid was
taken up in CH2Cl2 (175 mL) and triethylamine (9.28 mL, 66.6 mmol) and Boc2O (4.86 g, 24.4 mmol)
was added. After stirring for 12 h at room temperature, the reaction was quenched with a saturated
solution of NaHCO3 (200 mL), extracted with CH2Cl2 (3 x 150 mL), dried over MgSO4, and concentrated
under reduced pressure. The residue was purified using flash chromatography (10% to 45% ethyl
1
acetate in hexanes) to provided the aryl pyridine “IN” fragement (2.24 g, 43%): H-NMR (CDCl3, 400
MHz) ꢂ 7.78 (t, J = 7.6 Hz, 1H), 7.56 (d, J = 7.6 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 5.51 (s, 1H), 4.44 (d,
J = 5.6 Hz, 2H), 1.43 (s, 9H); 13C-NMR (CDCl3, 100 MHz) 160.1, 155.9, 137.6, 133.0, 127.0, 125.1,
117.1, 79.9, 45.5, 28.3; IR (neat) 3347, 2979, 2934, 2239, 1699, 1518, 1453, 1250, 1170, 862; HRMS
(ESI) m/z calcd for C12H15N3NaO3 [M+Na]+ 256.1062, found 256.1062.
(R)-2-(6-(((tert-Butoxycarbonyl)amino)methyl)pyridin-2-yl)-4-methyl-4,5-dihydrothiazole-4-
carboxylic acid (32): To a solution of nitrile 31 (1.50 g, 6.43 mmol) in MeOH/pH 6.0 buffer (105 mL,
3:2, v/v) was added ꢁ-methyl-L-cysteine (1.32 g, 7.72 mmol) and NaHCO3 (1.08 g, 12.9 mmol). The
reaction mixture was heated to 70 °C for 48 h. The mixture was cooled to ambient temperature and the
MeOH was removed under reduced pressure. The aqueous layer was extracted with diethyl ether (3 x
30 mL) before being acidified to a pH of ~2 using 1.0 M HCl. The aqueous layer was then extracted
with ethyl acetate (3 x 30 mL) and the ethyl acetate layer was dried over MgSO4. The solution was
concentrated to give the desired acid (1.89 g, 84%): 1H-NMR (CDCl3, 400 MHz) ꢂ 7.97 (d, J = 7.6 Hz,
1H), 7.74 (t, J = 8.0 Hz, 1H), 7.37 (d, J = 7.6 Hz, 1H), 5.45 (bs, 1H), 4.46 (d, J = 5.2 Hz, 2H), 3.77 (d, J
= 11.6 Hz, 1H), 3.29 (d, J = 11.6 Hz, 1H), 1.65 (s, 3H), 1.45 (s, 9H); 13C-NMR (CDCl3, 100 MHz) ꢂ
176.3, 171.5, 157.5, 156.1, 149.6, 137.3, 123.9, 120.5, 84.9, 79.7, 45.3, 40.3, 28.4, 24.1; IR (neat)
3415, 2977, 1688, 1515, 1455, 1366, 1278, 1160, 754; HRMS (ESI) m/z calcd for C16H22N3O4S [M+H]+
352.1331, found 352.1329; [ꢁ]D = –36.6 (c 0.98, CHCl3).
20

2-((((tert-Butoxy)carbonyl)amino)methyl)isonicotinonitrile (35): To a solution of commercially
available chloride 34 (5.84 g, 38.3 mmol) in DMF (250 mL) at ambient temperature was treated with
potassium phthalimide (7.09 g, 38.3 mmol). After stirring for 5 h, the mixture was concentrated under
vacuum. The remaining mixture was taken up in H2O (100 mL) and was filtered to collect the solid. The
solid was washed with H2O (50 mL) and THF (50 mL) to obtain the desired phthalimide derivative (5.04
g, 50%) and was moved forward without further purification. To a solution of the crude phthalimide
derivative (5.04 g, 19.1 mmol) in THF/MeOH (170 mL, 1:1, v/v) at ambient temperature was treated
with hydrazine monohydrate (1.02 mL, 21.1 mmol). After 2 h, 1.0 M HCl (21.4 mL) was added to the
mixture and was stirred for another 3 h before concentrating the reaction mixture under vacuum. The
remaining residue was taken up in H2O (200 mL) and the unwanted solid was removed through
filtration. The filtrate was concentrated and placed under vacuum to remove the remaining H2O. The
crude solid was taken up in CH2Cl2 (150 mL) and triethylamine (8.00 mL, 57.4 mmol) and Boc2O (4.59
g, 21.1 mmol) was added. After stirring for 12 h at room temperature, the reaction was quenched with a
saturated solution of NaHCO3 (200 mL), extracted with CH2Cl2 (3 x 150 mL), dried over MgSO4, and
concentrated under reduced pressure. The residue was purified using flash chromatography (15% to
1
50% ethyl acetate in hexanes) to provided the aryl pyridine “IN” fragement (3.74 g, 84%): H-NMR
(CDCl3, 400 MHz) ꢂ 8.69 (d, J = 5.2 Hz, 1H), 7.51 (s, 1H), 7.39 (dd, J = 5.2, 0.8 Hz, 1H), 5.45 (s, 1H),
13
4.47 (d, J = 5.6 Hz, 2H), 1.44 (s, 9H); C-NMR (CDCl3, 100 MHz) 159.9, 155.9, 150.0, 123.6, 123.1,
121.1, 116.4, 80.1, 45.6, 28.3; IR (neat) 3337, 2978, 2934, 2245, 1709, 1514, 1246, 1168, 949; HRMS
(ESI) m/z calcd for C12H16N3O3 [M+H]+ 234.1243, found 234.1237.
(R)-2-(2-(((tert-Butoxycarbonyl)amino)methyl)pyridin-4-yl)-4-methyl-4,5-dihydrothiazole-4-
carboxylic acid (36): To a solution of nitrile 35 (1.50 g, 6.43 mmol) in MeOH/pH 6.0 buffer (105 mL,
3:2, v/v) was added ꢁ-methyl-L-cysteine (1.32 g, 7.72 mmol) and NaHCO3 (1.08 g, 12.9 mmol). The
reaction mixture was heated to 70 °C for 48 h. The mixture was cooled to ambient temperature and the
MeOH was removed under reduced pressure. The aqueous layer was extracted with diethyl ether (3 x
30 mL) before being acidified to a pH of ~2 using 1.0 M HCl. The aqueous layer was then extracted
21

with ethyl acetate (3 x 30 mL) and the ethyl acetate layer was dried over MgSO4. The solution was
concentrated to give the desired acid (1.89 g, 85%): 1H-NMR (CDCl3, 400 MHz) ꢂ 8.61 (d, J = 4.8 Hz,
1H), 7.68 (s, 1H), 7.58 (dd, J = 5.2, 1.6 Hz, 1H), 5.64 (bs, 1H), 4.48 (d, J = 4.8 Hz, 2H) 3.94 (d, J = 11.6
Hz, 1H), 3.38 (d, J = 11.6 Hz, 1H), 1.66 (s, 3H), 1.43 (s, 9H); 13C-NMR (CDCl3, 100 MHz) ꢂ 175.5,
166.5, 158.5, 156.1, 148.7, 141.4, 121.2, 120.9, 85.0, 79.8, 45.1, 41.8, 28.3, 23.8; IR (neat) 3355,
2979, 2359, 1707, 1517, 1284, 1167, 754; HRMS (ESI) m/z calcd for C16H22N3O4S [M+H]+ 352.1331,
found 352.1334; [ꢁ]D = –39.3 (c 1.08, CHCl3).
(S,E)-2-(Trimethylsilyl)ethyl 3-(((S)-2-((R)-2-(6-(((tert-butoxycarbonyl)amino)methyl)pyridin-2-yl)-
4-methyl-4,5-dihydrothiazole-4-carboxamido)-3-methylbutanoyl)oxy)-7-(tritylthio)hept-4-enoate
(37): To a solution of Fmoc protected amine 23 (2.39 g, 2.85 mmol) in acetonitrile (195 mL) at ambient
temperature was added diethylamine (15.0 mL). After 2 h, the reaction mixture was concentrated under
reduced vacuum, taken back up in ethyl acetate (50 mL), and concentrated again. In a separate flask,
acid 32 (1.00 g, 2.85 mmol), PyBOP (2.97 g, 5.70 mmol), and DIPEA (1.49 mL, 8.55 mmol) was
combined in CH2Cl2 (195 mL). The freshly deprotected amine was added via CH2Cl2 (10 mL) to the
flask containing the activated acid at ambient temperature. After 3 h, the resulting mixture was
concentrated under reduced pressure. The crude residue was purified using flash chromatography (5%
to 40% ethyl acetate in hexanes) to obtain the desired amide (2.13 g, 79%): 1H-NMR (CDCl3, 400 MHz)
ꢂ 8.04 (d, J = 8.0 Hz, 1H), 7.69 (t, J = 7.6 Hz, 1H), 7.15-7.38 (m, 16H), 5.67 (dt, J = 15.2, 6.8 Hz, 1H),
5.59-5.65 (m, 1H), 5,45-5.47 (m, 1H), 5.36 (dd, J = 15.6, 7.6 Hz, 1H), 4.45-4.48 (m, 3H), 4.14 (dd, J =
9.6, 7.6 Hz, 2H), 3.74 (d, J = 11.6 Hz, 1H), 3.28 (d, J = 12.0 Hz, 1H), 2.68 (dd, J = 15.6, 7.6 Hz, 1H),
2.54 (dd, J = 15.6, 5.6 Hz, 1H), 2.00-2.18 (m, 5H), 1.60 (s, 3H), 1.46 (s, 9H), 0.93-0.97 (m, 2H), 0.81 (d,
J = 6.8 Hz, 3H), 0.74 (d, J = 6.8 Hz, 3H), 0.01 (s, 9H); 13C-NMR (CDCl3, 100 MHz) ꢂ 174.4, 170.9,
170.4,169.6, 157.6, 156.0, 149.9, 144.8, 137.3, 133.9, 129.5, 127.8, 127.7, 126.6, 123.8, 120.0, 85.6,
79.6, 71.8, 66.6, 63.1, 56.6, 45.4, 40.5, 39.7, 31.3, 31.2, 31.0, 28.4, 24.8, 19.0, 17.4, 17.3, –1.5; IR
(neat) 3393, 2960, 1736, 1684, 1508, 1446, 1390, 1249, 1171, 859; HRMS (ESI) m/z calcd for
C52H66N4NaO7S2Si [M+Na]+ 973.4040, found 973.4042; [ꢁ]D = –19.0 (c 1.11, CHCl3).
22

(S,E)-2-(Trimethylsilyl)ethyl 3-(((S)-2-((R)-2-(2-(((tert-butoxycarbonyl)amino)methyl)pyridin-4-yl)-
4-methyl-4,5-dihydrothiazole-4-carboxamido)-3-methylbutanoyl)oxy)-7-(tritylthio)hept-4-enoate
(38): To a solution of Fmoc protected amine 23 (1.55 g, 1.84 mmol) in acetonitrile (125 mL) at ambient
temperature was added diethylamine (10.0 mL). After 2 h, the reaction mixture was concentrated under
reduced vacuum, taken back up in ethyl acetate (50 mL), and concentrated again. In a separate flask,
acid 36 (0.650 g, 1.84 mmol), PyBOP (1.92 g, 3.68 mmol), and DIPEA (0.96 mL, 5.52 mmol) was
combined in CH2Cl2 (125 mL). The freshly deprotected amine was added via CH2Cl2 (10 mL) to the
flask containing the activated acid at ambient temperature. After 3 h, the resulting mixture was
concentrated under reduced pressure. The crude residue was purified using flash chromatography (5%
to 40% ethyl acetate in hexanes) to obtain the desired amide (1.42 g, 81%): 1H-NMR (CDCl3, 400 MHz)
ꢂ 8.51-8.52 (m, 1H), 7.72-7.73 (m, 1H), 7.15-7.38 (m, 15H), 7.11 (d, J = 8.8 Hz, 1H), 5.69 (dt, J = 15.2,
6.8 Hz, 1H), 5.60-5.65 (m, 1H), 5.36 (dd, J = 15.2, 7.6 Hz, 1H), 4.54-4.56 (m, 2H), 4.48 (dd, J = 8.8, 4.4
Hz, 1H), 4.14 (dd, J = 10.0, 8.0 Hz, 2H), 3.84 (d, J = 11.6 Hz, 1H), 3.41 (d, J = 11.6 Hz, 1H), 2.67 (dd, J
= 15,6, 8.0 Hz, 1H), 2.54 (dd, J = 16.0, 5.6 Hz, 1H), 2.03-2.18 (m, 5H), 1.56 (s, 3H), 1.44 (s, 9H), 0.92-
0.97 (m, 2H), 0.81 (d, J = 6.8 Hz, 3H), 0.74 (d, J = 6.8 Hz, 3H), 0.01 (s, 9H); 13C-NMR (CDCl3, 100
MHz) ꢂ 173.8, 170.3, 169.6, 167.0, 158.7, 155.9, 149.7, 144.8, 140.3, 133.9, 129.5, 127.8, 127.7,
126.6, 120.3, 119.9, 85.4, 79.6, 71.8, 66.6, 63.1, 56.8, 45.7, 41.7, 39.7, 31.3, 31.1, 31.0, 28.4, 24.5,
19.0, 17.4, 17.2, –1.5; IR (neat) 3390, 2960, 1737, 1681, 1593, 1504, 1444, 1248, 1170, 836, 744;
HRMS (ESI) m/z calcd for C52H66N4NaO7S2Si [M+Na]+ 973.4040, found 973.4058; [ꢁ]D = –32.5 (c 1.03,
CHCl3).
Trityl protected depsipeptide pyridyl “IN” macrocycle (39): To a solution of depsipeptide 37 (2.10
g, 2.21 mmol) in CH2Cl2 (100 mL) at 0 °C was treated with TFA (20.0 mL) dropwise. The reaction
mixture was allowed to stir for 18 h. The mixture was concentrated under reduced pressure, taken back
up in toluene (50 mL), and concentrated again. The remaining residue was taken up in acetonitrile
(2300 mL) and was treated with HATU (1.68 g, 4.42 mmol), HOBt (0.597 g, 4.42 mmol), and DIPEA
23

(2.32 mL, 13.3 mmol) at room temperature. After 18 h, the reaction mixture was concentrated under
reduced pressure. The crude residue was passed through a silica plug using an eluent of 10% MeOH in
CH2Cl2. The filtrate was concentrated and taken up in ethyl acetate (150 mL). The solution was washed
with a saturated solution of NaHCO3 (100 mL). The aqueous layer was extracted ethyl acetate (3 X 50
mL) and then the combined organic layers were washed with a saturated solution of NaCl (100 mL).
After drying over Na2SO4 and concentrating under reduced vacuum, the crude mixture was purified
1
using flash chromatography (1% to 6% MeOH in DCM) to obtain the macrocycle (0.334 g, 20%): H-
NMR (CDCl3, 400 MHz) ꢂ 7.78 (t, J = 7.6 Hz, 1H), 7.62 (d, J = 7.6 Hz, 1H), 7.15-7.37 (m, 16H), 7.06 (d,
J = 4.8 Hz, 1H), 5.74 (dtd, J = 15.6, 6.8, 1.2 Hz, 1H), 5.62 (q, J = 6.0 Hz, 1H), 5.38 (dd, J = 15.6, 6.4
Hz, 1H), 4.97 (dd, J = 18.0, 6.8 Hz, 1H), 4.66 (dd, J = 10.0, 4.0 Hz, 1H), 4.29 (dd, J = 18.0, 2.0 Hz, 1H),
4.07 (d, J = 11.2 Hz, 1H), 3.36 (d, J = 11.6 Hz, 1H), 2.68-2.70 (m, 2H), 2.08-2.17 (m, 3H), 2.02 (q, J =
7.2 Hz, 2H), 1.82 (s, 3H), 0.73 (d, J = 7.2 Hz, 3H), 0.52 (d, J = 6.8 Hz, 3H); 13C-NMR (CDCl3, 100 MHz)
ꢂ 173.4, 170.8, 168.9, 168.7, 156.5, 149.4, 144.8, 137.7, 133.0, 129.4, 127.8, 127.3, 126.5, 124.1,
123.1, 84.9, 72.9, 66.5, 57.4, 43.8, 43.5, 41.4, 33.5, 31.5, 31.1, 24.7, 18.9, 16.7; IR (neat) 3368, 2960,
1735, 1675, 1575, 1512, 1444, 1242, 1037, 974, 744; HRMS (ESI) m/z calcd for C42H44N4NaO4S2
[M+Na]+ 755.2702, found 755.2696; [ꢁ]D = +27.4 (c 0.57, CHCl3).
Trityl protected depsipeptide pyridyl “OUT” macrocycle (40): To a solution of depsipeptide 38
(2.00 g, 2.10 mmol) in CH2Cl2 (100 mL) at 0 °C was treated with TFA (20.0 mL) dropwise. The reaction
mixture was allowed to stir for 18 h. The mixture was concentrated under reduced pressure, taken back
up in toluene (50 mL), and concentrated again. The remaining residue was taken up in acetonitrile
(2100 mL) and was treated with HATU (1.60 g, 4.20 mmol), HOBt (0.570 g, 4.20 mmol), and DIPEA
(2.19 mL, 12.6 mmol) at room temperature. After 18 h, the reaction mixture was concentrated under
reduced pressure. The crude residue was passed through a silica plug using an eluent of 10% MeOH in
CH2Cl2. The filtrate was concentrated and taken up in ethyl acetate (150 mL). The solution was washed
with a saturated solution of NaHCO3 (100 mL). The aqueous layer was extracted ethyl acetate (3 X 50
mL) and then the combined organic layers were washed with a saturated solution of NaCl (100 mL).
24

After drying over Na2SO4 and concentrating under reduced vacuum, the crude mixture was purified
1
using flash chromatography (1% to 6% MeOH in DCM) to obtain the macrocycle (0.350 g, 23%): H-
NMR (CDCl3, 400 MHz) ꢂ 8.65 (d, J = 5.2 Hz, 1H), 7.77 (s, 1H), 7.15-7.40 (m, 16H), 6.97 (d, J = 9.2 Hz,
1H), 6.33 (dd, J = 8.0, 5.2 Hz, 1H), 5.78 (dtd, J = 15.6, 6.4, 0.8 Hz, 1H), 5.71 (q, J = 6.0 Hz, 1H), 5.50
(dd, J = 15.6, 6.0 Hz, 1H), 4.97 (dd, J = 17.6, 8.0 Hz, 1H), 4.52 (dd, J = 8.8, 4.0 Hz, 1H), 4.02 (dd, J =
18.0, 5.2 Hz, 1H), 3.99 (d, J = 11.6 Hz, 1H), 3.38 (d, J = 11.6 Hz, 1H), 2.74-2.78 (m, 2H), 2.01-2.29 (m,
5H), 1.74 (s, 3H), 0.76 (d, J = 6.8 Hz, 3H), 0.66 (d, J = 6.8 Hz, 3H); 13C-NMR (CDCl3, 100 MHz) ꢂ
173.1, 169.9, 168.4, 168.2, 159.3, 150.5, 144.6, 140.0, 132.6, 129.4, 128.1, 127.9, 126.7, 120.8, 116.9,
84.5, 71.5, 66.7, 58.0, 44.5, 43.5, 40.8, 33.4, 31.2, 31.0, 24.9, 18.9, 17.2; IR (neat) 3405, 2962, 1739,
1683, 1552, 1508, 1404, 1255, 1183, 1029, 748; HRMS (ESI) m/z calcd for C42H44N4NaO4S2 [M+Na]+
755.2702, found 755.2694; [ꢁ]D = +27.6 (c 0.58, CHCl3).
Depsipeptide pyridyl “IN” thiol (6): To a solution of trityl protected thiol 39 (300 mg, 0.409 mmol) in
CH2Cl2 (55 mL) at 0 °C was treated with TFA (2.25 mL) and triisopropylsilane (0.17 mL, 0.818 mmol).
The reaction mixture was warmed to ambient temperature and stirred for 2 h. The mixture was
concentrated and purified using flash chromatography (1% to 6% MeOH in CH2Cl2) to obtain the
desired thiol (164 mg, 82%): 1H-NMR (CDCl3, 400 MHz) ꢂ 7.86 (t, J = 7.6 Hz, 1H), 7.68 (d, J = 7.6 Hz,
1H), 7.42 (d, J = 8.0 Hz, 1H), 7.35 (bs, 1H), 7.25 (d, J = 9.6 Hz, 1H), 5.85 (dtd, J = 15.2, 7.2, 1.2 Hz,
1H), 5.64-5.69 (m, 1H), 5.54 (ddt, J = 15.6, 6.8, 1.2 Hz, 1H), 5.11 (dd, J = 18.0, 7.6 Hz, 1H), 4.69 (dd, J
= 10.0, 4.0 Hz, 1H), 4.34 (dd, J = 18.0, 1.6 Hz, 1H), 4.14 (d, J = 11.2 Hz, 1H), 3.46 (d, J = 11.6 Hz, 1H),
2.87 (dd, J = 15.2, 10.0 Hz, 1H), 2.71 (dd, J = 15.2, 2.4 Hz, 1H), 2.53 (qd, J = 7.6, 0.8 Hz, 1H), 2.31-
2.37 (m, 2H), 2.09-2.17 (m, 1H), 1.91 (s, 3H), 1.40 (t, J = 8.0 Hz, 1H), 0.71 (d, J = 6.8 Hz, 3H), 0.49 (d,
J = 6.8 Hz, 3H); 13C-NMR (CDCl3, 100 MHz) ꢂ 174.8, 172.6, 169.7, 168.5, 156.9, 148.0, 138.3, 132.8,
128.4, 125.1, 123.7, 83.5, 72.6, 57.5, 43.7, 43.5, 41.2, 36.4, 33.3, 23.8, 23.8, 18.6, 16.5; IR (neat)
3369, 2962, 1735, 1670, 1572, 1515, 1426, 1295, 1174, 989, 799; HRMS (ESI) m/z calcd for
C23H31N4O4S2 [M+H]+ 491.1787, found 491.1791; [ꢁ]D = +58.5 (c 0.53, CHCl3).
25

Depsipeptide pyridyl “OUT” thiol (8): To a solution of trityl protected thiol 40 (320 mg, 0.437 mmol) in
CH2Cl2 (60 mL) at 0 °C was treated with TFA (2.30 mL) and triisopropylsilane (0.18 mL, 0.873 mmol).
The reaction mixture was warmed to ambient temperature and stirred for 2 h. The mixture was
concentrated and purified using flash chromatography (1% to 6% MeOH in CH2Cl2) to obtain the
1
desired thiol (188 mg, 89%): H-NMR (CDCl3, 400 MHz) ꢂ 8.70 (d, J = 5.2 Hz, 1H), 7.89 (s, 1H), 7.45
(dd, J = 5.2, 1.6 Hz, 1H), 6.92 (d, J = 9.2 Hz, 1H), 6.36-6.39 (m, 1H), 5.87 (dt, J = 15.6, 6.4 Hz, 1H),
5.73-5.78 (m, 1H), 5.67 (ddt, J = 15.6, 6.4, 1.2 Hz, 1H), 5.09 (dd, J = 17.6, 8.0 Hz, 1H), 4.57 (dd, J =
9.2, 4.0 Hz, 1H), 4.37 (dd, J = 17.6, 4.8 Hz, 1H), 4.05 (d, J = 11.6 Hz, 1H), 3.40 (d, J = 11.6 Hz, 1H),
2.76-2.89 (m, 2H), 2.50-2.64 (m, 2H), 2.30-2.44 (m, 2H), 2.11-2.20 (m, 1H), 1.78 (s, 3H), 1.38 (t, J = 7.6
Hz, 1H), 0.75 (d, J = 6.8 Hz, 3H), 0.66 (d, J = 6.8 Hz, 3H); 13C-NMR (CDCl3, 100 MHz) ꢂ 173.1, 169.3,
168.7, 168.4, 158.7, 149.4, 141.1, 132.4, 128.8, 121.3, 117.8, 84.5, 72.2, 57.8, 43.6, 43.6, 40.3, 36.1,
33.6, 24.9, 23.7, 18.9, 17.0; IR (neat) 3319, 2962, 1736, 1670, 1551, 1508, 1242, 1184, 1027, 971,
730; HRMS (ESI) m/z calcd for C23H31N4O4S2 [M+H]+ 491.1787, found 491.1795; [ꢁ]D = +68.3 (c 0.74,
CHCl3).
Depsipeptide pyridyl “IN” (5): To a solution of depsipeptide pyridine “IN” thiol 6 (20.0 mg, 0.041
mmol) in THF (0.8 mL) at ambient temperature was treated with N-octanoyl–imidazole (15.8 mg, 0.082
mmol), imidazole (5.6 mg, 0.082 mmol), and DMAP (0.5 mg, 0.004 mmol). The reaction mixture was
warmed to 50 °C and stirred for 6 h. The mixture was cooled and concentrated and purified using flash
chromatography (0.5% to 4% MeOH in CH2Cl2) to obtain the desired octanoyl masked thiol (17.4 mg,
1
69%): H-NMR (CDCl3, 400 MHz) ꢂ 7.82 (t, J = 7.6 Hz, 1H), 7.65 (d, J = 7.6 Hz, 1H), 7.39 (d, J = 8.0
Hz, 1H), 7.30 (d, J = 9.6 Hz, 1H), 5.85 (dt, J = 15.6, 6.8 Hz, 1H), 5.63 (td, J = 16.0, 2.0 Hz, 1H), 5.52
(dd, J = 15.2, 6.8 Hz, 1H), 5.06-5.12 (m, 1H), 4.67 (dd, J = 9.6, 3.6 Hz, 1H), 4.32 (d, J = 18.0 Hz, 1H),
4.15 (d, J = 11.6 Hz, 1H), 3.42 (d, J = 11.6 Hz, 1H), 2.81-2.90 (m, 2H), 2.63 (dd, J = 14.0, 1.2 Hz, 1H),
2.47 (t, J = 7.6 Hz, 2H), 2.27 (q, J = 7.6 Hz, 2H), 2.06-2.13 (m, 1H), 1.92 (s, 3H), 1.56-1.61 (m, 2H),
1.23-1.27 (m, 10H), 0.84 (t, J = 6.8 Hz, 3H), 0.69 ( d, J = 6.8 Hz, 3H), 0.49 (d, J = 6.8 Hz, 3H); 13C-NMR
(CD3OD, 100 MHz) ꢂ 199.4, 174.2, 173.7, 170.7, 168.7, 157.5, 147.7, 138.7, 132.3, 128.7, 125.0,
26

123.2, 83.4, 72.9, 54.1, 43.4, 42.8, 42.7, 40.2, 33.1, 31.9, 31.4, 28.6, 28.5, 27.4, 25.3, 23.0, 22.2, 18.1,
15.3, 13.0; IR (neat) 2959, 2928, 2856, 1737, 1682, 1571, 1419, 1239, 990; HRMS (ESI) m/z calcd for
C31H45N4O5S2 [M+H]+ 617.2826, found 617.2832; [ꢁ]D = +125 (c 0.80, CHCl3).
Depsipeptide pyridyl “OUT” (7): To a solution of depsipeptide pyridine “OUT” thiol 8 (85.0 mg, 0.179
mmol) in CH2Cl2 (15.0 mL) at ambient temperature was treated with octanoyl chloride (.153 mL, 0.894
mmol) and triethylamine (0.050 mL, 0.357 mmol). The reaction mixture was stirred for 2 h before
cooling to 0 °C and quenching with methanol (5.0 mL). The mixture was cooled and concentrated and
purified using flash chromatography (0.5% to 4% MeOH in CH2Cl2) to obtain the desired octanoyl
masked thiol (54.0 mg, 49%): 1H-NMR (CDCl3, 400 MHz) ꢂ 8.67 (d, J = 5.2 Hz, 1H), 7.91 (s, 1H), 7.45
(d, J = 4.8 Hz, 1H), 6.89 (d, J = 8.8 Hz, 1H), 6.57-6.60 (m, 1H), 5.85 (dt, J = 14.8, 7.2 Hz, 1H), 5.62-
5.73 (m, 2H), 5.10 (dd, J = 18.0, 8.0 Hz, 1H), 4.56 (dd, J = 8.8, 3.6 Hz, 1H), 4.42 (dd, J = 17.6, 3.6 Hz,
1H), 4.05 (d, J = 11.6 Hz, 1H), 3.40 (d, J = 11.6 Hz, 1H) 2.74-2.90 (m, 3H), 2.49 (t, J = 7.2 Hz, 2H),
2.28-2.34 (m, 1H), 2.12-2.19 (m, 1H), 1.78 (s, 3H), 1.58-1.63 (m, 2H), 1.24-1.26 (m, 10H), 0.84 (t, J =
13
6.8 Hz, 3H), 0.74 (d, J = 6.8 Hz, 3H), 0.65 (d, J = 6.8 Hz, 3H); C-NMR (CD3OD, 100 MHz) ꢂ 199.4,
173.9, 170.2, 169.6, 168.2, 158.6, 149.0, 141.7, 132.0, 128.5, 121.5, 117.9, 84.3, 72.4, 57.7, 43.4,
42.7, 42.7, 39.1, 33.4, 32.0, 31.4, 28.6, 28.5, 27.4, 25.3, 23.6, 22.2, 18.1, 16.1, 13.0; IR (neat) 2959,
2929, 2856, 1740, 1684, 1553, 1420, 1234, 987; HRMS (ESI) m/z calcd for C31H45N4O5S2 [M+H]+
617.2826, found 617.2827; [ꢁ]D = +94.3 (c 0.62, CHCl3).
(S,E)-Methyl
3-((S)-2-((R)-2-(6-(((tert-butoxycarbonyl)amino)methyl)pyridin-2-yl)-4-methyl-4,5-
dihydrothiazole-4-carboxamido)-3-methylbutanamido)-7-(tritylthio)hept-4-enoate (41): To
a
solution of Boc protected amine 28 (500 mg, 0.793 mmol) in CH2Cl2 (30 mL) at 0 °C was treated with
TFA (3.0 mL). After 2 h, the reaction mixture was concentrated under vacuum, taken back up in toluene
(15 mL), and concentrated again. In another flask, acid 32 (278 mg, 0.793 mmol), PyBOP (822 mg,
1.58 mmol), and DIPEA (0.41 mL, 2.37 mmol) were combined in CH2Cl2 (35 mL). The freshly
deprotected amine was added via CH2Cl2 (10 mL) to the flask containing the activated acid at ambient
27

temperature. After 3 h, the resulting mixture was concentrated under reduced pressure. The crude
residue was purified using flash chromatography (20% to 90% ethyl acetate in hexanes) to obtain the
1
desired amide (570 mg, 84%): H-NMR (CDCl3, 400 MHz) ꢂ 8.00 (d, J = 7.6 Hz, 1H), 7.73 (t, J = 7.6
Hz, 1H), 7.34-7.37 (m, 7H), 7.30 (d, J = 8.8 Hz, 1H), 7.15-7.26 (m, 9H), 6.59 (d, J = 10.0 Hz, 1H), 5.47
(dtd, J = 15.6, 6.4, 0.8 Hz, 1H), 5.36 (dd, J = 15.2, 6.0 Hz, 1H), 4.70-4.74 (m, 1H), 4.46 (d, J = 5.2 Hz,
2H), 4.17 (dd, J = 8.8, 6.0 Hz, 1H), 3.67 (d, J = 11.6 Hz, 1H), 3.61 (s, 3H), 3.28 (d, J = 11.6 Hz, 1H),
2.57 (d, J = 5.6 Hz, 2H), 2.08-2.16 (m, 3H) 2.01 (q, J =6.8 Hz, 2H), 1.56 (s, 3H), 1.45 (s, 9H), 0.82 (d, J
= 6.8 Hz, 3H), 0.79 (d, J = 6.8 Hz, 3H); 13C-NMR (CDCl3, 100 MHz) ꢂ 174.7, 171.5, 171.1, 169.7, 157.7,
156.0, 149.8, 144.8, 137.3, 130.6, 129.5, 127.8, 126.5, 123.8, 120.1, 85.6, 79.6, 66.5, 58.2, 51.7, 47.4,
45.4, 40.6, 38.7, 31.4, 31.3, 31.1, 28.4, 24.7, 19.3, 17.9; IR (neat) 2969, 1654, 1508, 1444, 1365, 1169,
1031, 751; HRMS (ESI) m/z calcd for C48H57N5NaO6S2 [M+Na]+ 886.3648, found 886.3639; [ꢁ]D = –30.8
(c 0.67, CHCl3).
(S,E)-Methyl
3-((S)-2-((R)-2-(2-(((tert-butoxycarbonyl)amino)methyl)pyridin-4-yl)-4-methyl-4,5-
dihydrothiazole-4-carboxamido)-3-methylbutanamido)-7-(tritylthio)hept-4-enoate (42): To
a
solution of Boc protected amine 28 (500 mg, 0.793 mmol) in CH2Cl2 (30 mL) at 0 °C was treated with
TFA (3.0 mL). After 2 h, the reaction mixture was concentrated under vacuum, taken back up in toluene
(15 mL), and concentrated again. In another flask, acid 36 (278 mg, 0.793 mmol), PyBOP (822 mg,
1.58 mmol), and DIPEA (0.41 mL, 2.37 mmol) were combined in CH2Cl2 (35 mL). The freshly
deprotected amine was added via CH2Cl2 (10 mL) to the flask containing the activated acid at ambient
temperature. After 3 h, the resulting mixture was concentrated under reduced pressure. The crude
residue was purified using flash chromatography (20% to 100% ethyl acetate in hexanes) to obtain the
1
desired amide (630 mg, 92%): H-NMR (CDCl3, 400 MHz) ꢂ 8.61 (dd, J = 4.8, 0.8 Hz, 1H), 7.59-7.61
(m, 2H), 7.15-7.38 (m, 16H), 6.59 (d, J = 8.8 Hz, 1H), 5.47 (dtd, J = 15.6, 6.4, 0.8 Hz, 1H), 5.35 (dd, J =
15.2, 6.0 Hz, 1H), 4.69-4.75 (m, 1H), 4.48 (d, J = 5.2 Hz, 2H), 4.15 (dd, J = 8.8, 6.4 Hz, 1H), 3.79 (d, J
= 11.6 Hz, 1H), 3.61 (s, 3H), 3.37 (d, J = 11.2 Hz, 1H), 2.57 (d, J = 5.2 Hz, 2H), 2.08-2.17 (m, 3H), 2.01
13
(q, J = 6.8 Hz, 2H), 1.55 (s, 3H), 1.45 (s, 9H), 0.82 (d, J = 6.4 Hz, 3H), 0.79 (d, J = 6.8 Hz, 3H); C-
28

NMR (CDCl3, 100 MHz) ꢂ 174.1, 171.6, 169.5, 167.2, 158.8, 155.9, 149.7, 144.8, 140.2, 130.6, 129.5,
129.5, 127.8, 126.5, 120.3, 120.0, 85.3, 79.6, 66.5, 58.4, 51.7, 47.3, 45.7, 41.7, 38.6, 31.4, 31.1, 31.0,
28.4, 24.4, 19.3, 17.9; IR (neat) 2969, 1654, 1511, 1443, 1390, 1366, 1168, 846, 750; HRMS (ESI) m/z
calcd for C48H57N5NaO6S2 [M+Na]+ 886.3648, found 886.3661; [ꢁ]D = –29.9 (c 0.64, CHCl3).
Trityl protected peptide isostere pyridyl “IN” macrocycle (43): To a solution of methyl ester 41 (530
mg, 0.613 mmol) in THF/H2O (15 mL, 2:1, v/v) at ambient temperature was added lithium hydroxide
monohydrate (77.2 mg, 1.84 mmol). After 2 h, the reaction was cooled to 0 °C and acidified to a pH of
~3 using 1.0 M HCl. The mixture was extracted with CH2Cl2 (3 x 15 mL), dried over Na2SO4, and
concentrated under vacuum. The freshly furnished acid was taken up in CH2Cl2 (20 mL) and cooled to
0 °C. The flask was treated with TFA (2.0 mL) and warmed to ambient temperature. After 2 h, the
mixture was concentrated under reduced pressure, taken back up in toluene (25 mL), and concentrated
again. The crude TFA salt was taken up in acetonitrile (20 mL) and was treated with DIPEA (0.68 mL,
3.90 mmol). The mixture was allowed to stir for 30 min. In another flask containing acetonitrile (610 mL)
was added HATU (488 mg, 1.28 mmol), HOBt (173 mg, 1.28 mmol), and DIPEA (0.68 mL, 3.90 mmol)
at room temperature. The flask containing the peptide was added to the flask containing the coupling
reagents dropwise over 12 h using a syringe pump. After another 12 h, the reaction mixture was
concentrated under reduced pressure. The crude residue was taken up in CH2Cl2 (15 mL) and the
resulting solid was removed using filtration. The filtrate was concentrated and purified using flash
1
chromatography (1% to 6% MeOH in DCM) to obtain the macrocycle (146 mg, 33%): H-NMR (CDCl3,
400 MHz) ꢂ 7.81 (t, J = 7.6 Hz, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.16-7.37 (m, 16H), 6.92 (d, J = 6.4 Hz,
1H), 6.73 (d, J = 10.8 Hz, 1H), 6.42 (d, J = 8.4 Hz, 1H), 5.47 (dt, J = 15.6, 6.4 Hz, 1H), 5.34 (dd, J =
15.6, 5.6 Hz, 1H), 5.03 (dd, J = 18.0, 7.2 Hz, 1H), 4.83-4.90 (m, 1H), 4.60 (dd, J = 10.8, 3.2 Hz, 1H),
4.24 (dd, J = 17.6, 2.0 Hz, 1H), 3.95 (d, J = 12.0 Hz, 1H), 3.45 (d, J = 12.0 Hz, 1H), 2.67 (dd, J = 14.0,
3.6 Hz, 1H), 2.48-2.52 (m, 1H), 2.44 (dd, J = 14.4, 10.8 Hz, 1H), 2.16 (t, J = 8.0 Hz, 2H), 1.99-2.06 (m,
2H), 1.87 (s, 3H), 0.74 (d, J = 6.8 Hz, 3H), 0.32 (d, J = 6.8 Hz, 3H); 13C-NMR (CDCl3, 100 MHz) ꢂ
173.8, 172.8, 169.8, 169.8, 157.5, 149.2, 144.8, 138.0, 130.3, 129.6, 129.5, 127.8, 126.6, 124.6, 122.9,
29