Dissecting the structure of thiopeptides: Assessment of thiazoline and tail moieties of baringolin and antibacterial activity optimization

Article abstract of DOI:10.1021/jm500062g

Several analogues of baringolin (1) were prepared to evaluate the role of its characteristic thiazoline ring and pentapeptidic tail with the aim of defining structure-activity relationships for these moieties. The thiazoline ring appeared as a crucial moiety to maintain a broad scope of activities against different Gram-positive bacteria. Further modifications were performed to simplify the structure of the natural product and assess the role of its tail, resulting in an enhanced in vitro performance. Analogue 25, with the thiazole-containing macrocycle and a 4-aminocyclohexane-1-carboxylic acid moiety in place of the pentapeptidic tail, was identified as a much more potent analogue, capable of overcoming the absence of the thiazoline ring and performing extraordinarily well against all strains tested. This is the first library of thiopeptide analogues produced by chemical synthesis alone, which demonstrates the robustness and convenience of the synthetic strategy used.

Full text of DOI:10.1021/jm500062g

Article
pubs.acs.org/jmc
Dissecting the Structure of Thiopeptides: Assessment of Thiazoline
and Tail Moieties of Baringolin and Antibacterial Activity
Optimization
Xavier Just-Baringo,†,‡,∇,†P,‡,a#olo Bruno,†,‡,○ Cristina Pitart,§ Jordi Vila,§ Fernando Albericio,†,‡,∥,⊥
́
and Mercedes Alvarez*
†Institute for Research in Biomedicine, Barcelona Science Park, University of Barcelona, Baldiri Reixac 10, 08028 Barcelona, Spain
‡CIBER-BBN, Networking Centre on Bioengineering Biomaterials and Nanomedicine, 08028 Barcelona, Spain
§Center for International Health Research, CRESIB, Hospital Clinic, University of Barcelona, 08036 Barcelona, Spain
∥Department of Organic Chemistry, University of Barcelona, E-08028 Barcelona, Spain
⊥School of Chemistry, University of KwaZulu−Natal, 4001 Durban, South Africa
#Laboratory of Organic Chemistry, Faculty of Pharmacy, University of Barcelona, 08028 Barcelona, Spain
S
* Supporting Information
ABSTRACT: Several analogues of baringolin (1) were
prepared to evaluate the role of its characteristic thiazoline
ring and pentapeptidic tail with the aim of defining structure−
activity relationships for these moieties. The thiazoline ring
appeared as a crucial moiety to maintain a broad scope of
activities against different Gram-positive bacteria. Further
modifications were performed to simplify the structure of
the natural product and assess the role of its tail, resulting in an
enhanced in vitro performance. Analogue 25, with the thiazole-
containing macrocycle and a 4-aminocyclohexane-1-carboxylic
acid moiety in place of the pentapeptidic tail, was identified as
a much more potent analogue, capable of overcoming the absence of the thiazoline ring and performing extraordinarily well
against all strains tested. This is the first library of thiopeptide analogues produced by chemical synthesis alone, which
demonstrates the robustness and convenience of the synthetic strategy used.
(Figure 1). Alternative approaches to the preparation of
modified thiopeptides have been previously explored, all taking
advantage of the biosynthetic pathway that produces the parent
natural products.12 On one hand, engineering of the
biosynthetic pathway has shown its potential for obtaining
analogues arising from different kinds of modifications: residue
replacement, enzyme knockout, and feeding with non-natural
precursors.13 Replacement of putative Ile, which is oxidized to
form 2-hydroxy-4-methylpyrrolidine in the parent peptide
sequence of GE37468A (3),14 with Pro produced the
mutasynthetic analogue 4,15 which has a macrocycle identical
to that of baringolin (1) (Figure 1). On the other hand,
semisynthesis permits chemical modification of the product at
its most reactive sites, giving rise to a wide variety of
transformations such as the conversion of thiomuracin A
(5)16 into its derivative 6.17
INTRODUCTION
■
Antibiotic resistance to marketed drugs is an increasing concern
in the clinic and requires the development of new compounds
that can overcome this phenomenon.1 The discovery of new
molecules with new modes of action is key to avoid cross-
resistance. In this context, thiopeptide antibiotics have arisen as
promising candidates due to their good performance in in vitro
assays against various microorganisms. Despite the good
activities reported, their lack of aqueous solubility has limited
their use to the treatment of skin infections, regardless of the
huge efforts carried out for the synthesis of more soluble
analogues.2−4
The complex architecture of thiopeptides5 has prompted
many groups to develop sophisticated and robust synthetic
strategies to achieve the total synthesis of many members of
this family of antibiotics.6 These approaches have scarcely been
applied to the synthesis of analogues, with most of the reports
focusing on fragment and synthetic intermediates screen-
ing.7−10 In this communication we report the first de novo
synthesis of a library of thiopeptide analogues11 to assess the
impact of different moieties in baringolin (1), leading to the
preparation of various derivatives with general structure 2
Despite the very limited presence of fully synthetic
thiopeptide derivatives in the literature, our recent studies on
the total synthesis of 1 aimed to develop a modular and
Received: January 14, 2014
Published: April 16, 2014
© 2014 American Chemical Society
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Figure 1. Thiopeptide antibiotics of the d series (top) and analogues of diverse origin derived from them (bottom). Baringolin analogues with
general structure 2, reported herein, were produced by chemical synthesis alone, 4 was produced by mutasynthesis, and 6 was obtained by
semisynthesis.
a
Scheme 1. Synthesis of 11
a
Reagents and conditions: (a) allyl chloroformate, NEt3, CH2Cl2, 0 °C, 2 h, 86%; (b) Lawesson’s reagent, THF, rt, 4.5 h, 88%; (c) (i) ethyl
bromopyruvate, KHCO3, DME, 0 °C, 2.5 h, (ii) TFAA, 2,6-lutidine, DME, −20 °C, 2.5 h, 99% (95% ee); (d) LiOH, H2O, THF, rt, 15 h, 86%. DME
= dimethoxyethane, TFAA = trifluoroacetic anhydride.
convergent strategy that should facilitate the preparation of its
analogues.18 In the present study, our goal was to assess the
role of both the thiazoline ring and the pentapeptidic tail of 1.
To do so, we first aimed at the synthesis of a new macrocycle in
which a thiazole moiety replaces the naturally occurring
thiazoline. Second, shorter peptidic tails were to be introduced
to assess their impact in antibacterial activity. Some of these
modifications led to analogues with improved activity. Herein
we present the first library of thiopeptide analogues obtained
solely by chemical synthesis.
the same fully unsaturated thiazole on the equivalent position
of their similar macrocycle.16 To obtain the desired analogue, a
suitable building block was synthesized (Scheme 1). Phenyl-
alaninamide (7) was protected with the Alloc group, yielding 8,
which was then converted into the corresponding thioamide
(9) and subsequently transformed into the desired thiazole
(10) by means of a two-step Hantzsch cyclization.19−21 Ester
hydrolysis produced carboxylic acid (11), a suitably function-
alized fragment for further condensation. The use of Alloc-
protected fragment 11 will facilitate the deprotection step prior
to macrocyclization in subsequent stages of the synthesis.
To evaluate the role of the dehydroaminoacid tail of
baringolin, the precursor tri- and tetrapeptides were synthesized
to be used in the preparation of analogues of various tail
lengths. The phenylselecnocysteine-containing peptides, 12 and
13, were synthesized by solid-phase peptide synthesis (SPPS)
on a Rink-Amide/Chem-Matrix resin22 using the methodology
described by van der Donk et al.23 The corresponding C-
terminal amides were thus obtained using the same method-
ology described for the preparation of the baringolin
pentapeptide precursor (14)18,24 (Scheme 2).
RESULTS AND DISCUSSION
■
Substitution of the thiazoline moiety of the macrocycle in 1
with the corresponding thiazole was regarded as a modification
likely to introduce rigidity and stability to the macrocyclic
scaffold.15 Furthermore, this modification would substitute a
thiazoline building block with a more robust thiazole and
should also enhance the chemical stability of the final
compound. Such a modification was expected to retain activity
against Staphylococcus aureus because the thiomuracins display
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a
Scheme 2. Solid Phase Synthesis of Tri-, Tetra-, and Pentapeptide Precursors, 12, 13, and 14, Respectively
a
Reagents and conditions: (a) (i) Fmoc-AA-OH, DIPCDI, OxymaPure, DMF, rt, 1.5 h, (ii) 20% piperidine in DMF, rt (4 treatments); (b) 95% TFA
in CH2Cl2, rt (4 treatments). Yields: 12 (quant), 13 (quant), 14 (89%). DIPCDI = N,N′-diisopropylcarbodiimide.
a
Scheme 3. Synthesis of Analogues 19−21, 23, and 25−30
a
Reagents and conditions: (a) 11 or 16, EDC, HOAt, DIPEA, DMF, 0 °C to rt, 3 h, 85% (17), 68% (18); (b) Pd(PPh3)4, PhSiH3, CH2Cl2, rt; (c)
EDC, HOAt, DMF (1 mM), rt, 61% (19), 30% (20); (d) Me3SnOH, ClCH2CH2Cl, 60 °C, 19 h; (e) LiOH, H2O/THF, rt, 17 h, 99% (21); (f) 24,
EDC, HOAt, DIPEA, DMF, 0 °C to rt, 5 h; (g) Pd(PPh3)4, PhSiH3, CH2Cl2, rt, 2 h, 39% (2 steps); (h) NH4HCO3, EDC, HOAt, DIPEA, DMF, 0
°C to rt, 28 h, 68%; (i) 12, 13, or 14, EDC, HOAt, DIPEA, DMF, 0 °C to rt; (j) tBuOOH, CH2Cl2, rt, 39% (26, 2 steps), 63% (27, 3 steps), 33%
(28, 2 steps), 55% (29, 3 steps), 50% (30, 2 steps), 53% (1, 3 steps).
The preparation of the macrocycle-containing analogues
proceeded from 15, an intermediate in the total synthesis of
baringolin.18 The condensation of 15 with either thiazole
building block 11 or the corresponding thiazoline (16)18
furnished the protected macrocycle precursors 17 and 18,
respectively (Scheme 3). Next, removal of all allyl-based
protecting groups and macrocyclization under diluted con-
ditions (1 mM) afforded the desired macrocycles 19 and 20. At
this point, different conditions were required for hydrolysis of
the ethyl ester of the two macrocycles. While the ester in 19
was hydrolyzed under more conventional basic conditions to
obtain carboxylic acid 21, 20 contains a thiazoline ring and
necessitated the use of trimethyltin hydroxide to ensure a mild
and epimerization-free saponification to yield 22.25 With both
21 and 22 in hand, manipulation of the carboxylic acids was
performed to introduce a series of substituents. The amide of
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a
Scheme 4. Synthesis of 31
a
Reagents and conditions: (a) (i) Fmoc-AA-OH, DIPCDI, OxymaPure, DMF, rt, 1.5 h, 20% piperidine in DMF, rt (4 treatments), (ii) tBuOOH,
CH2Cl2, rt; (b) 95% TFA in CH2Cl2, rt (4 treatments), 36% (2 steps); (c) (i) Fmoc-AA-OH, N,N′-diisopropylcarbodiimide, OxymaPure, DMF, rt,
1.5 h, 20% piperidine in DMF, rt (4 treatments), (ii) 95% TFA in CH2Cl2, rt (4 treatments), 47%; (d) tBuOOH, CH2Cl2, rt, 72%. DIPCDI = N,N′-
diisopropylcarbodiimide.
the thiazole−macrocycle analogue was also synthesized in an
analogous manner, giving rise to 23. Moreover, allyl trans-4-
aminocyclohexanoate (24) was also condensed with 21 to
generate the corresponding cyclohexanoic acid derivative 25
after deprotection. The cyclohexanoic acid moiety has been
previously installed into other thiopeptides through different
linkers with satisfactory results.3 Peptides 12−14 were
condensed with acids 21 and 22 to obtain analogues 25−29
and 118 in order to assess the impact of both thiazoline and
thiazole rings as well as the role of the different peptidic tails.
In addition to the macrocyclic analogues, fragment 31,
consisting of the pentapeptide present in baringolin bearing a
C-terminal 4-thiazolecarboxylic acid, was synthesized in order
to assess whether the peptidic tail possess antibiotic activity in
the absence of the macrocyclic scaffold. Two alternative
approaches were used for the preparation of 31: a solid-phase
strategy analogous to the one described above (Scheme 4) and
also the solution-phase oxidation-elimination step of the solid-
support-cleaved phenylselenocysteine peptide (32). The two
methods were equally efficient, and 31 was obtained in 34−
36% overall yield.
Table 1. Antibacterial Activity and Solubility of Baringolin
Analogues
b
solubility
a
MIC (μg/mL)
(mg/mL)
S.
aureus
P.
acnes
B.
subtilis
M.
luteus
compd
H2O
PB 0.1 M
c
c
c
c
c
c
c
c
c
c
c
c
c
c
baringolin (1)
0.25
>8
0.125
0.25
>8
8
0.5
2
BLD
BLD
BLD
BLD
BLD
BLD
BLD
BLD
BLD
BLD
BLD
BLD
BLD
BLD
19
20
21
23
25
26
27
28
29
30
31
4
>8
4
4
2
2
8
>8
>8
0.5
2
0.023
0.007
0.018
1
8
8
0.03
0.5
0.25
0.5
0.5
0.5
>8
0.06
4
0.03
8
c
BLD
c
0.125
8
0.25
0.5
0.5
1
0.5
2
BLD
c
BLD
c
0.5
8
1
BLD
c
2
BLD
8
>8
2
4.661
6.654
a
b
MIC = minimum inhibitory concentration. Solubility was
determined by measuring the concentration of a saturated solution
c
of compounds. BLD = below limit of detection.
The antibacterial activity of all prepared baringolin analogues
was evaluated in vitro against different strains of Gram-positive
bacteria: Staphylococcus aureus, Propionibacterium acnes, Bacillus
subtilis, and Micrococcus luteus. 1 displayed good potency against
all strains (Table 1), while analogues with shorter tails, such as
27 and 29, performed similarly to 1, indicating that the
unusually long tail of baringolin is not essential for its activity.
Interestingly, analogues 26, 28, and 30 that possess the same
variable-length peptidic tails but that incorporate a thiazole ring
instead of a thiazoline, retained potency against S. aureus but
exhibited generally diminished activity. Such results indicate
that the more conformationally flexible analogues incorporating
a thiazoline ring might be more readily accommodated into the
binding site of their biological target, presumably elongation
factor Tu (EF-Tu).26 While ethyl esters 19 and 20 were devoid
of any remarkable activity, carboxylic acid 21 and amide 23
retained theirs against S. aureus. Such behavior was in
accordance with the results obtained for the thiazole series
analogues mentioned above. Surprisingly, and to our delight,
analogue 25 showed an improved profile when compared to
baringolin. Despite the presence of the thiazole ring in place of
thiazoline, 25 remained active toward all tested strains and
showed higher potencies against S. aureus, P. acnes, and B.
subtilis. These results point out to key interactions of the newly
introduced carboxylic acid,2,3 which were able to overcome the
presumably increased rigidity of the non-natural macrocycle.
Despite the higher solubility of 31 (Table 1), its poor biological
profile reinforces the hypothesis of the limited impact of the
peptidic tail and the otherwise key role of the macrocyclic
scaffold to exert its antibacterial activity.
A robust and convergent strategy consisting of a combination
of solution and solid-phase modes has facilitated the
construction of the first fully synthetic library of thiopeptide
analogues. The modifications introduced have helped us
identify the thiazoline moiety as responsible for the broader
activity profile of baringolin when compared to its less saturated
analogue 29. Moreover, the role of the tail region has also been
evaluated, showing a very limited impact of tail length on
activity and potency. Using the thiazole-containing macrocycle
analogue as a more robust and accessible platform, the trans-4-
aminocyclohexanoic acid moiety was introduced to furnish 25;
this modification restored the activity profile and highly
improved the potency of baringolin toward most strains.
The use of a fully synthetic approach such as the one
presented herein could be used to further assess the role of
other regions of thiopeptides not easily modified by alternative
methods of analogue production.
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employed: (s) singlet, (d) doublet, (t) triplet, and (q) quartet. The
same abbreviations were also used for the multiplicity of signals in 1H
NMR, plus: (m) multiplet, (dd) double doublet, (ddd) double doublet
of doublets, (dq) double quartet and (bs) broad singlet. Spectra were
referenced to appropriate residual solvent peaks (CDCl3, DMSO-d6,
acetone-d6, or pyridine-d5). High-resolution mass spectroscopy
(HRMS) was performed on either a LTQ-FT Ultra (Thermo
Scientific) or a LCT-Premier (Waters) high resolution mass
spectrometer by the Mass Spectrometry Service of the Institute for
Research in Biomedicine (IRB). Purity of tested compounds was
assessed by HPLC to be >95%.
(S) N-(Allyloxycarbonyl)phenylalaninamide (8). Allyl chlorofor-
mate (1.2 mL, 10.96 mmol) was added slowly to a stirring suspension
of hydrochloride salt of 7 (2.0 g, 9.97 mmol) in dry CH2Cl2 (200 mL)
cooled in an ice bath. Next, NEt3 (3.1 mL, 21.93 mmol) was added
dropwise. After stirring for 2 h at 0 °C, the reaction mixture was
poured onto brine (200 mL), fractions were separated and the
aqueous layer extracted with CH2Cl2 (2 × 100 mL). Combined
organic fractions were dried (Na2SO4) and concentrated in vacuo. The
crude product was purified by silica flash column chromatography
(hexanes/EtOAc, 3:7). The title product was obtained as a white solid
(2.13 g, 86%). The product obtained in this manner was identical to
the one described in the literature.27 1H NMR (400 MHz, CDCl3) δ =
3.06 (dd, J = 14.0, 6.8 Hz, 1 H), 3.12 (dd, J = 13.6, 6.8 Hz, 1 H), 4.38−
4.48 (m, 1 H), 4.53−4.57 (m, 2 H), 5.21 (ddd, J = 10.4, 2.8, 1.2 Hz, 1
H), 5.27 (ddd, J = 17.2, 2.8, 1.6 Hz, 1 H), 5.36 (bs, 1 H), 5.53 (bs, 1
H), 5.77 (bs, 1 H), 5.82−5.93 (m, 1 H), 7.20−7.28 (m, 3 H), 7.29−
7.34 (m, 2 H) ppm. HRMS m/z calcd for C13H17O3N2 (M + H)
249.1234, found 249.1234.
(S) N-(Allyloxycarbonyl)phenylalanine thioamide (9). A solution
of 8 (1.83 g, 7.37 mmol) and Lawesson’s reagent (1.49 g, 3.69 mmol)
in dry THF (10 mL) was stirred at rt. After 4.5 h, saturated aq
NaHCO3 (50 mL) was added to the reaction vessel. After 1 h, the
reaction mixture was poured into saturated aq NaHCO3 (100 mL).
Layers were separated, and the aqueous phase was extracted with
CH2Cl2 (3 × 150 mL). Combined organic fractions were dried
(Na2SO4) and concentrated in vacuo. The crude product was purified
by silica flash column chromatography (hexanes/EtOAc, 1:1). The
title product was obtained as a colorless oil (1.71 g, 88%). [α]D +30.6
(c = 1.00, CH2Cl2). IR (film) 3302, 3206, 2943, 1700, 1623, 1502,
1438, 1152, 1041 cm−1. 1H NMR (400 MHz, CDCl3) δ = 3.12 (dd, J =
13.6, 8.2 Hz, 1 H), 3.21 (dd, J = 13.6, 8.2 Hz, 1 H), 4.50−4.56 (m, 2
H), 4.62−4.70 (m, 1 H), 5.21 (ddd, J = 10.4, 2.8, 1.2 Hz, 1 H), 5.28
(ddd, J = 17.2, 2.8, 1.6 Hz, 1 H), 5.58 (bs, 1 H), 5.82−5.93 (m, 1 H),
7.14 (bs, 1 H), 7.22−7.38 (m, 6 H) ppm. 13C NMR (100 MHz,
CDCl3) δ = 42.2 (t), 61.6 (d), 66.4 (t), 118.3 (t), 127.4 (d), 128.9 (d),
129.6 (d), 132.5 (d), 136.5 (s), 156.2 (s) ppm. HRMS m/z calcd for
C13H17O2N2S (M + H) 265.1005, found 265.1018.
CONCLUSION
■
Two of the most characteristic moieties of baringolin were
modified to assess their structure−activity profile. Thiazoline
was substituted by its aromatic counterpart using the
corresponding Phe-derived thiazole. Peptidic tail variants were
obtained using solid-phase synthesis. Several thiazole-4-
carboxylic acid derivatives were also synthetized in the absence
of a peptidic tail. Testing of all analogues against various Gram-
positive bacterial strains showed that substitution of the
thiazoline ring in the macrocycle with thiazole could affect
the scope of antibacterial activity. By contrast, the peptidic tail
did not appear as a crucial moiety, and its substitution for a
trans-4-aminocyclohexane-1-carboxilyc acid moiety in com-
pound 25 improved the antibacterial potency against most
strains and overcame the restrictions of the thiazole series of
analogues.
Notably, this is the first fully synthetic library of thiopeptide
analogues ever reported. This fact, combined with the good
activity results obtained, are excellent evidence to validate the
synthetic strategy as a suitable one for the assessment of
structure−activity relationships of such complex molecules.
EXPERIMENTAL SECTION
■
MIC Assays. MIC assays were performed using Staphylococcus
aureus and Propionibacterium acnes from our collection, isolated from
clinical samples, Microccus luteus ATCC 9341 and Bacillus subtilis
ATCC 6633. Isolates were taken from the freezer and transferred at
least twice on supplemented Brucella agar for anaerobes and on sheep
blood agar for aerobes to ensure purity and good growth. Anaerobes
were incubated for 48 h and aerobes for 24 h prior to testing. Inocula
were prepared by direct suspensions of cells into saline solution to
achieve the turbidity of the 0.5 McFarland standard. For facultative
and aerobic bacteria (S. aureus, B. subtilis, and M. luteus), MIC was
performed by microdilution method in Mueller−Hinton broth
according to CLSI guideleness (M7-A9)28 incubated at 35 °C for
24h. For anaerobic bacteria (P. acnes), MIC was performed in Brucella
broth supplemented with hemin (5 μg/mL), vitamin K1 (1mcg/mL),
and lysed horse blood (5%) as described in CLSI-M11-A829 incubated
at anaerobic conditions, at 35 °C for 24h.
Solubility Determination. An amount of approximately 1 mg of
compound was weighed, and a known volume of the solvent was
added to ensure a saturated solution would result. Vigorous vortexing
for 1 min and shaking for 48 h followed. After centrifugation at 10000
rpm during 3 min, the supernatant was analyzed using a
spectrophotometer at a reading wavelength of 304 nm.
(S)-Ethyl 2-(1-(Allyloxycarbonylamino)-2-phenylethyl)thiazole-4-
carboxylate (10). A mixture of 9 (1.41 g, 5.35 mmol) and KHCO3
(5.9 g, 58.85 mmol) in dry DME (13.4 mL) was stirred at rt. After 15
min, the mixture was placed in an ice bath and ethyl bromopyruvate
(2.0 mL, 16.05 mmol) was added dropwise, and the resulting mixture
was stirred at 0 °C. After 20 h, the mixture was allowed to reach rt,
filtered through celite, and washed with Et2O. Volatiles were removed,
and the crude hydroxythiazoline was redissolved in dry DME (13.4
mL) and cooled at −20 °C. A preformed mixture of trifluoroacetic
anhydride (3.0 mL, 21.4 mmol) and 2,6-lutidine (5.6 mL, 48.15
mmol) was added dropwise to the stirring solution. After 2.5 h at the
same temperature, the mixture was diluted with CH2Cl2 (250 mL),
washed with 1 N HCl (250 mL) and saturated aq NaHCO3 (300 mL),
dried (Na2SO4), and concentrated in vacuo. The crude product was
purified by silica flash column chromatography (hexanes/tBuOMe,
1:1). The title product was obtained as a yellowish oil (1.91 g, 99%).
95% ee; H2O (0.045% TFA):MeCN (0.036% TFA), 50% MeCN (tR =
10.30 min); detected at 254 nm. [α]D −15.4 (c = 1.00, CH2Cl2). IR
(KBr) 3327, 2982, 2933, 1715, 1237, 1212 cm−1. 1H NMR (400 MHz,
CDCl3) δ = 1.42 (t, J = 7.6 Hz, 3 H), 3.34 (d, J = 6.4 Hz, 2 H), 4.44
(q, J = 7.6 Hz, 2 H), 4.54 (d, J = 5.6 Hz, 2 H), 5.19 (d, J = 10.4 Hz, 1
H), 5.26 (d, J = 16.8 Hz, 1 H), 5.30−5.38 (m, 1 H), 5.51 (bs, 1 H),
Synthesis. Tetrahydrofuran (THF) and N,N-dimethylformamide
(DMF) were dried using a PureSolv solvent purification system. All
other solvents and reagents were used as purchased without further
purification. Flash column chromatography was performed on SDS
silica gel (60A 35−70 μm) as stationary phase. Analytical thin layer
chromatography was performed using aluminum backed plates coated
with Merck Kieselgel 60 F254; compounds were visualized under a UV
lamp (254 nm). Melting points were determined in a Buchi melting
point B540 apparatus in open capillaries. Reverse-phase analytical
HPLC was performed on a Waters Alliance separation module 2695
equipped with a Waters XBridge C18 column (4.6 mm × 75 mm, 2.5
μm) and a Waters 996 PDA with a photodiode array detector, using
MeCN (0.036% TFA) and H2O (0.045% TFA) as mobile phases in 8
min runs. Enantiomeric excess (ee) was determined by HPLC on the
same separation module with a chiral stationary phase Chiralpak IA
250 mm × 4.6 mm 5 μm analytical column, flow rate 1 mL min−1 in 40
min runs. Polarimetry studies were performed on a Perkin−Elmer 241
or Jasco P-2000 polarimeter. IR spectra were recorded on a Thermo
1
Nicolet FT-IR Nexus spectrometer. H NMR and 13C NMR spectra
were recorded on a Varian Mercury 400 MHz or Bruker 600 MHz
spectrometer. Multiplicity of the carbons was assigned with gHSQC
experiments. Standard abbreviations for off-resonance decoupling were
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5.77−5.94 (m, 1 H), 7.07−7.12 (m, 2 H), 7.19−7.29 (m, 3 H), 8.04
(s, 1 H) ppm. 13C NMR (100 MHz, CDCl3) δ = 16.7 (q), 43.9 (t),
56.6 (d), 63.8 (t), 68.3 (t), 120.2 (t), 129.5 (d), 129.6 (d), 131.0 (d),
131.7 (d), 134.8 (d), 138.3 (s), 149.7 (s), 157.8 (s), 163.6 (s)174.5 (s)
ppm. HRMS m/z calcd for C18H21O4N2S (M + H) 361.1217, found
361.1218.
(S)-2-(1-(Allyloxycarbonylamino)-2-phenylethyl)thiazole-4-car-
boxylic Acid (11). Aqueous 2 N LiOH (5 mL, 10.18 mmol) was added
to a stirring solution of 10 (1.84 g, 5.09 mmol) in THF (57 mL). The
mixture was stirred at rt under air. After 15 h, EtOAc (250 mL) and
H2O (250 mL) were added and the layers were separated. Starting
material was recovered from the organic fraction (257 mg, 14%).
Aqueous 2N HCl was added to the aqueous layer until it reached a pH
of 2−3, and it was extracted with EtOAc (3 × 250 mL). The title
product was obtained as a white solid (1.46 g, 86%). The crude
product was used in following reactions without further purification or
characterization.
General Method for Solid-Phase Peptide Synthesis. The Rink-
Amide Chem-Matrix resin (loading = 0.52 mmol/g) was swollen in
MeOH, then in DMF, and finally in CH2Cl2. Fmoc-AA-OH (3 equiv),
preactivated by vigorous shaking for 4 min with DIPCDI (3.3 equiv)
and Oxyma Pure (3.3 equiv) in DMF, was poured onto the resin and
the resulting mixture was gently shaken for 1 h. The resin was then
washed with DMF and CH2Cl2 (5× each). The N-terminus was
deprotected using 20% piperidine in DMF (treatments of 2 × 1 min,
then 2 × 5 min). The resin was then washed with CH2Cl2 and DMF
(5× each). Loading onto the resin (0.249 mmol/g, 48%) was
determined through measuring dibenzofulvene absorbance at 290 nm
of cleavage solutions and washings.
Elongation of the peptide proceeded as follows; the number of
equivalents refers to the original functionalization, not the loading.
Fmoc-AA-OH (2.3 equiv) was preactivated by vigorous shaking for 4
min in the presence of DIPCDI (2.5 equiv) and Oxyma Pure (2.5
equiv) in DMF and was then poured onto the resin. The resulting
mixture was gently shaken for 1.5 h. Deprotection and coupling cycles
were repeated with the appropriate amino acids to provide the desired
peptide. The peptide was cleaved from the resin by treatment with
95% TFA in CH2Cl2 (4 × 20 min) at rt followed by filtration and
collection of the filtrate. Next, washing of the resin with CH2Cl2 (×6)
was performed. Most TFA was removed under vacuum, and the
resulting concentrated solution was poured into cold Et2O.
Centrifugation and pouring off the solvent yielded the desired peptide
as TFA salt. Purity was determined by HPLC (linear gradient: 0−
100% MeCN in H2O over 8 min; flow rate = 1.0 mL/min).
H-Sec(Ph)-Ala-Pro-NH2 (12). Tripeptide 12 was prepared according
to the general method for solid-phase peptide synthesis, starting from
1.0 g of resin. The title product was obtained as a pale powder (131
mg, quant based on calculated loading of the resin). HPLC purity:
100%; H2O (0.045% TFA):MeCN (0.036% TFA), 0−100% MeCN
(tR = 3.91 min); detected at 254 nm; mp (Et2O) decomposes above
125 °C. [α]D −22.2 (c = 0.50, CH2Cl2). IR (KBr) 1675, 1630, 1207,
1130 cm−1. 1H NMR (400 MHz, DMSO-d6) δ = 1.26 (d, J = 7.2 Hz, 3
H), 1.78−2.11 (m, 4 H), 3.23−3.37 (m, 2 H), 3.55−3.64 (m, 2 H),
4.08−4.16 (m, 1 H), 4.25 (dd, J = 8.4, 3.6 Hz, 1 H), 4.47−4.58 (m, 1
H), 6.94 (bs, 2 H), 7.29−7.36 (m, 3 H), 7.51−7.57 (m, 2 H), 7.58−
7.62 (m, 1 H), 8.51 (bs, 3 H), 8.85 (d, J = 7.6 Hz, 1 H) ppm. 13C
NMR (100 MHz, DMSO-d6) δ = 18.1 (q), 25.3 (t), 28.1 (t), 30.3 (t),
47.5 (d), 47.6 (t), 52.7 (d), 60.5 (d), 128.2 (d), 130.0 (s), 130.2 (d),
132.9 (d), 167.3 (s), 170.4 (s), 174.4 (s) ppm. HRMS m/z calcd for
C17H25N4O3Se (M + H) 413.1086, found 413.1087
4.52−4.63 (m, 1 H), 7.23−7.39 (m, 8 H), 7.49−7.59 (m, 4 H), 8.01−
8.09 (m, 1 H), 8.51 (bs, 3 H), 8.90−8.97 (m, 1 H) ppm. 13C NMR
(100 MHz, DMSO-d6) δ = 18.1 (q), 25.4 (t), 28.1 (t), 29.8 (t), 29.9
(t), 47.6 (d), 47.8 (t), 52.7 (d), 53.4 (d), 61.1 (d), 127.6 (d), 128.2
(d), 129.9 (s), 130.2 (d), 130.2 (d), 131.2 (s), 132.4 (d), 132.9 (d),
167.3 (s), 171.1 (s), 172.2 (s); 172.8 (s) ppm. HRMS m/z calcd for
C26H34N5O4Se2 (M + H) 640.0936, found 640.0951.
Protected Open Macrocycle Thiazole Analogue (17). A mixture of
EDC·HCl (69 mg, 0.359 mmol), HOAt (49 mg, 0.359 mmol), and
DIPEA (60 μL, 0.359 mmol) was added to a solution of 1518 (310 mg,
0.299 mmol) and 11 (119 mg, 0.359 mmol) in dry DMF (6 mL)
cooled in an ice bath. The resulting solution was stirred at 0 °C for 2.5
h, then was allowed to reach rt and stirred for another 2 h. The
reaction mixture was diluted with EtOAc (150 mL), washed with
saturated aq NH4Cl (100 mL), NaHCO3 (100 mL), and H2O (100
mL), dried (Na2SO4), and concentrated in vacuo. The crude product
was purified by silica flash column chromatography (CH2Cl2/EtOAc,
2:8 to EtOAc). The title compound was obtained as a white solid (343
mg, 85%), mp (EtOAc) 135−138 °C. [α]D +9.6 (c = 0.33, CH2Cl2).
1
IR (KBr) 3440, 2922, 1719, 1643, 1510, 1424, 1239 cm−1. H NMR
(400 MHz, CDCl3) δ = 1.44 (t, J = 7.0 Hz, 3 H), 1.98−2.32 (m, 4 H),
2.36−2.60 (m, 3 H), 2.69−3.41 (m, 6 H), 3.46−4.06 (m, 2 H), 4.26−
4.62 (m, 6 H), 4.72−4.84 (m, 2 H), 4.91−5.42 (m, 8 H), 5.43−5.50
(m, 1 H), 5.58 (bs, 1 H), 5.68−6.10 (m, 4 H), 6.62−6.94 (m, 2 H),
6.98−7.25 (m, 7 H), 7.41 (s, 1 H), 7.81 (bs, 1 H), 8.02−8.08 (m, 1
H), 8.11−8.15 (m, 1 H), 8.25 (d, J = 8.2 Hz, 1 H), 8.28−8.33 (m, 1
H), 8.37 (d, J = 8.2 Hz, 1 H), 8.59−8.68 (m, 3 H) ppm. 13C NMR
(100 MHz, CDCl3) δ = 12.0 (q), 14.7 (q), 24.8 (t), 32.2 (t), 37.5 (t),
38.9 (t), 40.6 (t), 47.8 (t), 48.1 (d), 53.1 (d), 54.2 (d), 59.2 (d), 62.0
(t), 66.2 (t), 66.2 (t), 69.0 (t), 115.2 (d), 115.4 (d), 117.8 (t), 118.3
(t), 119.0 (d), 119.2 (t), 121.8 (d), 123.0 (s), 124.1 (d), 127.2 (d),
128.5 (d), 128.9 (d), 129.7 (s), 129.9 (d), 130.2 (s), 130.6 (d), 130.6
(d), 130.8 (s), 132.1 (d), 132.9 (d), 133.6 (d), 136.6 (s), 140.3 (d),
146.8 (s), 149.0 (s), 149.1 (s), 149.3 (s), 149.6 (s), 151.3 (s), 151.4
(s), 154.1 (s), 154.3 (s), 154.6 (s), 155.9 (s), 157.5 (s), 157.9 (s),
161.2 (s), 161.6 (s), 161.8 (s), 161.9 (s), 162.1(s), 169.1 (s), 172.4 (s),
173.3 (s) ppm. HRMS m/z calcd for C64H61O12N12S5 (M + H)
1349.3130, found 1349.3196.
Thiazole Analogue Ethyl Ester (19). A solution of Pd(PPh3)4 (64
mg, 0.055 mmol) in dry CH2Cl2 (7 mL) was added to a stirring
solution of 17 (745 mg, 0.552 mmol) in dry CH2Cl2 (30 mL). PhSiH3
(340 μL, 2.760 mmol) was subsequently added. The resulting mixture
was stirred at rt. After 7 h, more Pd(PPh3)4 (32 mg, 0.028 mmol) in
dry CH2Cl2 (3.5 mL) was added and the reaction mixture stirred for
another 2 h. Upon consumption of all starting material, volatiles were
evaporated under reduced pressure. The flask was filled with N2 and
the crude dissolved in dry DMF (550 mL). Addition of EDC·HCl
(131 mg, 0.662 mmol) and HOAt (90 mg, 0.662 mmol) followed.
After 3 days, a further amount of EDC·HCl (131 mg, 0.662 mmol)
and HOAt (90 mg, 0.662 mmol) were added. After three more days,
all starting material was consumed and the DMF volume was reduced
to approximately 100 mL under reduced pressure. H2O (250 mL) was
added and the mixture extracted with CH2Cl2 (3 × 250 mL).
Combined organics were dried (Na2SO4) and concentrated in vacuo.
The crude product was purified by silica flash column chromatography
(CH2Cl2/MeOH, 98:2 to 95:5). The title product was obtained as a
white solid (392 mg, 61%). HPLC: 30−80% MeCN (tR = 7.70 min);
mp (CH2Cl2) decomposes above 120 °C. [α]D +117.8 (c = 1.00,
CH2Cl2). IR (KBr) 3383, 3118, 2924, 2852, 1781, 1728, 1655, 1546,
1497, 1211, 1168 cm−1. 1H NMR (400 MHz, CDCl3) δ = 0.65 (d, J =
16.8, 1 H), 1.44 (t, J = 7.1 Hz, 3 H), 1.97−2.08 (m, 1 H), 2.10−2.22
(m, 1 H), 2.26−2.40 (m, 2 H), 2.45−2.58 (m, 1 H), 2.83 (s, 3 H), 3.09
(dd, J = 14.0, 4.8 Hz, 1 H), 3.18 (dd, J = 14.0, 3.0 Hz, 1 H), 3.35 (d, J
= 5.2 Hz, 2 H), 3.95−4.05 (m, 2 H), 4.47 (q, J = 7.1 Hz, 2 H), 5.02−
5.10 (m, 1 H), 5.29−5.44 (m, 3 H), 5.60 −5.67 (m, 1 H), 6.60 (d, J =
8.4 Hz, 2 H), 6.82−6.97 (m, 4 H), 7.18−7.24 (m, 3 H), 7.57 (s, 1 H),
7.73 (d, J = 10.0 Hz, 1 H), 7.86 (s, 1 H), 7.93 (s, 1 H), 8.09 (s, 1 H),
8.27−8.33 (m, 2 H), 8.41 (d, J = 8.0 Hz, 1 H), 8.62 (d, J = 7.2 Hz, 1
H), 8.70 (d, J = 9.6 Hz, 1 H) ppm. 13C NMR (100 MHz, CDCl3) δ =
12.4 (q), 14.7 (q), 25.8 (t), 34.2 (t), 37.2 (t), 38.5 (t), 43.5 (t), 48.2
H-Sec(Ph)-Ala-Pro-Sec(Ph)-NH2 (13). Tetrapeptide 13 was pre-
pared according to the general method for solid-phase peptide
synthesis, starting from 1.0 g of resin. The title product was obtained
as a white powder (187 mg, quant based on calculated loading of the
resin). HPLC purity: 100%; H2O (0.045% TFA):MeCN (0.036%
TFA), 0−100% MeCN (tR= 5.21 min); detected at 254 nm; mp
(Et2O) 87−90 °C. [α]D −26.8 (c = 1.00, CH2Cl2). IR (KBr) 1662,
1201, 1130 cm−1. 1H NMR (400 MHz, DMSO-d6) δ = 1.28 (d, J = 7.2
Hz, 3 H), 1.78−2.15 (m, 4 H), 3.12−3.40 (m, 4 H), 3.50−3.69 (m, 2
H), 4.07−4.16 (m, 1 H), 4.27−4.35 (m, 1 H), 4.36−4.45 (m, 1 H),
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(d), 48.5 (t), 51.0 (d), 53.3 (d), 61.7 (d), 62.0 (t), 114.3 (d), 117.2
(d), 119.2 (d), 122.7 (d), 122.8 (d), 122.9 (s); 124.7 (d), 125.5 (s),
127.4 (d), 128.6 (d), 130.2 (d), 130.3 (s), 130.6 (d), 131.6 (d), 135.6
(s), 138.8 (d), 148.6 (s), 148.9 (s), 148.9 (s), 149.2 (s), 151.0 (s),
151.9 (s), 154.5 (s), 154.6 (s), 156.0 (s), 156.3 (s), 160.0 (s), 160.3
(s), 160.7 (s), 161.6 (s), 163.3 (s), 169.0 (s), 169.2 (s), 171.0 (s),
172.4 (s), 173.1 (s); 173.8 (s) ppm. HRMS m/z calcd for
C54H47O9N12S5 (M + H) 1167.2187, found 1167.2190.
mixture was then diluted with EtOAc (250 mL), washed with H2O
(250 mL) and brine (2 × 100 mL), dried (Na2SO4), and then
concentrated in vacuo. The desired product was obtained as a colorless
oil (1.13 g, 97%). IR (film) 3378, 2976, 2936, 2862, 1733, 1713m
1
1519, 1173 cm−1. H NMR (400 MHz, CDCl3) δ = 1.05−1.17 (m, 2
H), 1.43 (s, 9 H), 1.47−1.63 (m, 2 H), 1.98−2.12 (m, 4 H), 2.25 (tt, J
= 12.0, 3.6 Hz, 1 H), 3.41 (bs, 1 H), 4.37 (bs, 1 H), 4.56 (dt, J = 5.6,
1.2 Hz, 2 H), 5.20−5.33 (m, 2 H), 5.84−5.96 (m, 1 H) ppm. 13C
NMR (100 MHz, CDCl3) δ = 28.2 (t), 28.8 (q), 32.9 (t), 42.8 (d),
49.3 (d), 65.3 (t), 79.6 (s), 118.4 (t), 132.6 (d), 155.5 (s), 175.4 (s)
ppm. HRMS m/z calcd for C15H26O4N (M + H) 284.1856, found
2841858.
Allyl trans-4-Aminocyclohexanoate Hydrochloride (24). A sol-
ution of 4 M HCl in 1,4-dioxane (18 mL, 70.2 mmol) was added to a
stirring solution of S1 (996 mg, 3.51 mmol) in dry 1,4-dioxane (18
mL). After 4 h, the reaction mixture was diluted with CH2Cl2 and then
concentrated in vacuo. The title product was obtained as a white solid
(747 mg, 97%). The crude product was used in following reactions
without further purification or characterization.
Thiazole Analogue Carboxylic Acid (21). Aqueous 3 N LiOH (340
μL, 1.028 mmol) was added to a stirring solution of 19 (200 mg, 0.171
mmol) in THF (1.7 mL). The resulting mixture was stirred at rt under
air. After 17 h, the reaction was diluted with THF/CH2Cl2 (25 mL,
1:1) and 2 M HCl (25 mL) were added. Layers were separated, and
the aqueous phase was extracted with THF/CH2Cl2 (2 × 25 mL, 1:1).
Combined organic fractions were dried (Na2SO4) and concentrated in
vacuo. The title product was obtained as a white solid (193 mg, 99%).
HPLC: 50−100% MeCN (tR = 2.68 min); mp (CH2Cl2) decomposes
above 200 °C. [α]D +119.5 (c = 1.00, CH2Cl2/MeOH, 96:4). IR
(KBr) 3385, 2917, 2847, 1649, 1549, 1489, 1425, 1201 cm−1. 1H NMR
(400 MHz, pyridine-d5) δ = 1.15−1.42 (m, 1 H), 1.66−2.28 (m, 5 H),
2.81 (s, 3 H), 3.17−3.85 (m, 6 H), 5.38−5.54 (m, 1 H), 5.63−5.73
(m, 1 H), 5.76−5.85 (m, 1 H), 5.88−6.07 (m, 1 H), 7.04−7.13 (m, 2
H), 7.15−7.22 (m, 5 H), 7.39 (d, J = 8.4 Hz, 2 H), 7.64 (bs, 1 H), 7.98
(bs, 1 H), 8.04 (s, 1 H), 8.22 (s, 1 H), 8.28 (s, 1 H), 8.32 (d, J = 8.0
Hz, 1 H), 8.37 (s, 1 H), 8.38−8.42 (m, 1 H), 8.52 (d, J = 8.0 Hz, 1 H),
8.76 (s, 1 H), 9.08 (d, J = 7.2 Hz, 1 H), 9.46 (d, J = 8.4 Hz, 1 H) ppm.
13C NMR (100 MHz, pyridine-d5) δ = 11.9 (q), 25.2 (t), 33.6 (t), 37.7
(t), 39.0 (t), 43.5 (t), 48.0 (t), 49.5 (d), 52.2 (d), 53.6 (d), 61.3 (d),
115.6 (d), 116.6 (d), 118.9 (d), 122.9 (d), 123.0 (d), 124.8 (d), 126.4
(s), 127.3 (d), 128.7 (d), 130.2 (d), 131.0 (s), 131.5 (d), 136.7 (d),
139.3 (d), 149.3 (s), 149.3 (s), 151.3 (s), 151.6 (s), 152.2 (s), 154.0
(s), 154.9 (s), 156.0 (s), 158.1 (s), 160.1 (s), 160.5 (s), 161.3 (s),
162.8 (s), 164.0 (s), 168.4 (s), 169.2 (s), 171.7 (s), 173.1 (s), 175.0
(s), 175.1 (s) ppm. HRMS m/z calcd for C52H43O9N12S5 (M + H)
1139.1874, found 1139.1888.
Thiazole Cyclohexanoic Acid Analogue (25). 24 (14 mg, 65.8
μmol), EDC·HCl (10 mg, 52.7 μmol), HOAt (7 mg, 52.7 μmol), and
DIPEA (12 μL, 65.8 μmol) were added to a stirring solution of 21 (50
mg, 43.9 μmol) in dry DMF (0.9 mL) cooled in an ice bath. The
reaction mixture was allowed to reach rt. After 5 h, CH2Cl2 (25 mL)
was added and the resulting solution was washed with saturated aq
NH4Cl (25 mL) and saturated aq NaHCO3 (25 mL), dried (Na2SO4),
and then concentrated in vacuo. The crude product was purified by
silica flash column chromatography (CH2Cl2/MeOH, 98:2 to 96:4).
The condensation product was obtained as a white solid (36 mg,
63%). A solution of Pd(PPh3)4 (3 mg, 2.8 μmol) in dry CH2Cl2 (1.9
mL) and PhSiH3 (17 μL, 0.138 mmol) was added to a flask charged
with the condensation product (36 mg, 27.6 μmol). The resulting
solution was stirred at rt for 2 h. Volatiles were removed under
reduced pressure and the crude product purified by preparative
reverse-phase column (C18, 30− 70% MeCN in H2O). The title
product was obtained as a white solid (21 mg, 39% for two steps).
HPLC: 30−80% MeCN (tR = 6.26 min); mp (CH2Cl2) decomposes
above 140 °C. [α]D +92.7 (c = 1.00, CH2Cl2/MeOH, 96:4). IR (KBr)
3385, 2917, 2841, 1656, 1534, 1495, 1419, 1194 cm−1. 1H NMR (400
MHz, pyridine-d5) δ = 1.18−2.05 (m, 8 H), 2.12−2.36 (m, 5 H),
2.42−2.52 (m, 1 H), 2.79 (s, 3 H), 3.19−3.62 (m, 5 H), 3.66−3.84
(m, 2 H), 4.33−4.46 (m, 1 H), 5.38−5.46 (m, 1 H), 5.63−5.72 (m, 1
H), 5.74−5.83 (m, 1 H), 5.98−6.06 (m, 1 H), 7.04−7.10 (m, 2 H),
7.16−7.22 (m, 5 H), 7.38 (d, J = 8.4 Hz, 2 H), 7.64 (bs, 1 H), 7.95−
8.03 (m, 3 H), 8.21 (s, 1 H), 8.23−8.28 (m, 2 H), 8.36 (s, 1 H), 8.39
(d, J = 10.0 Hz, 1 H), 8.66 (s, 1 H), 8.71−8.76 (d, J = 8.8 Hz, 1 H),
9.08 (d, J = 6.8 Hz, 1 H), 9.46 (d, J = 8.8 Hz, 1 H) ppm. 13C NMR
(100 MHz, pyridine-d5) δ = 11.8 (q), 25.2 (t); 28.9 (t), 32.4 (t), 33.7
(t), 37.7 (t); 39.1 (t), 43.2 (d), 43.5 (t), 48.0 (t), 48.6 (d), 49.5 (d),
52.2 (d), 53.6 (d), 61.3 (d), 115.6 (d), 116.6 (d), 118.7 (d), 122.9 (d),
123.0 (d), 124.8 (d), 126.4 (s), 127.0 (s), 127.3 (d), 128.7 (d), 130.2
(d), 131.0 (s), 131.5 (d), 136.7 (d), 139.1 (d), 149.2 (s), 149.4 (s),
151.4 (s), 152.2 (s), 153.1 (s), 153.9 (s), 154.8 (s), 156.0 (s), 158.1
(s), 160.1 (s), 160.5 (s), 160.6 (s), 161.3 (s), 162.8 (s), 168.0 (s),
169.2 (s), 171.7 (s), 173.1 (s), 175.1 (s), 175.1 (s), 177.8 (s) ppm.
HRMS m/z calcd for C59H54O10N13S5 (M + H) 1264.2715, found
1264.2741.
Thiazole Amide Analogue (23). A mixture of NH4HCO3 (10 mg,
132.0 μmol), EDC·HCl (10 mg, 52.7 μmol), HOAt (7 mg, 52.7
μmol), and DIPEA (24 μL, 132.0 μmol) was added to a stirring
solution of 21 (50 mg, 43.9 μmol) in dry DMF (0.9 mL) cooled in an
ice bath. The reaction mixture was allowed to reach rt. After 28 h,
CH2Cl2 (25 mL) was added and the resulting solution was washed
with saturated aq NH4Cl (25 mL) and saturated aq NaHCO3 (25
mL), dried (Na2SO4), and concentrated in vacuo. The crude product
was purified by silica flash column chromatography (CH2Cl2/MeOH,
99:1 to 96:4). The title product was obtained as a white solid (34 mg,
68%). HPLC: 50−100% MeCN (tR = 2.60 min); mp (CH2Cl2)
decomposes above 180 °C. [α]D +114.4 (c = 1.00, CH2Cl2/MeOH,
1
96:4). IR (KBr) 3385, 2917, 2847, 1649, 1534, 1489, 1419 cm−1. H
NMR (400 MHz, DMSO-d6) δ = 1.92−2.27 (m, 5 H), 2.39−2.51 (m,
1 H), 2.78 (s, 3 H), 2.82−2.97 (m, 2 H), 3.10−3.26 (m, 2 H), 3.71−
3.92 (m, 2 H), 5.03−5.13 (m, 1 H), 5.30−5.42 (m, 2 H), 5.63−5.72
(m, 1 H), 6.68 (d, J = 8.4 Hz, 2 H), 6.83 (bs, 1 H), 7.12 (d, J = 8.4 Hz,
2 H), 7.17−7.38 (6 H), 7.74−7.81 (m, 2 H), 8.00 (d, J = 9.2 Hz, 1 H),
8.10 (bs, 1 H), 8.13 (s, 1 H), 8.23 (s, 1 H), 8.37 (s, 1 H), 8.50 (d, J =
8.2 Hz, 1 H), 8.52 (s, 1 H), 8.56 (d, J = 8.2 Hz, 1 H), 8.70−8.80 (m, 2
H), 9.25 (s, 1 H) ppm. 13C NMR (100 MHz, DMSO-d6) δ = 12.6 (q),
25.5 (t), 33.6 (t), 38.0 (t), 39.0 (t), 42.6 (t), 48.2 (t), 49.4 (d), 52.7
(d), 53.4 (d), 60.4 (d), 116.1 (d), 117.2 (d), 119.6 (d); 123.2 (d),
123.3 (s), 124.5 (d), 125.8 (d), 127.4 (d), 127.7 (s), 128.3 (d), 129.2
(d), 130.4 (d), 131.3 (d), 137.4 (s), 140.9 (d), 148.5 (s), 149.8 (s),
151.8 (s), 152.1 (s), 152.7 (s), 154.0 (s), 154.6 (s), 156.6 (s), 157.0
(s), 160.0 (s), 161.1 (s), 161.7 (s), 162.0 (s), 163.0 (s), 167.6 (s),
170.3 (s), 171.2 (s), 172.6 (s), 174.5 (s), 175.3 (s) ppm. HRMS m/z
calcd for C52H44O8N13S5 (M + H) 1138.2034, found 1138.2043.
Allyl trans-4-(tert-butoxycarbonylamino)cyclohexanoate (S1).
Dry DMF (23 mL) was added to a flask charged with 4-trans-(tert-
butoxycarbonylamino)cyclohexanoic acid (1.00 g, 4.11 mmol) and
NaHCO3 (2.49 g, 29.59 mmol). The mixture was stirred at rt under
inert atmosphere. After 10 min, allyl bromide (7.5 mL, 86.31 mmol)
was added and the mixture stirred for another 24 h at rt. The reaction
Thiazole Tripeptide Analogue (26). 12 (25 mg, 0.047 mmol),
EDC·HCl (9 mg, 0.047 mmol), HOAt (6 mg, 0.047 mmol), and
DIPEA (8 μL, 0.047 mmol) were added to a stirring solution of 21 (45
mg, 0.039 mmol) in dry DMF (0.8 mL) cooled in an ice bath. The
reaction mixture was allowed to reach rt. After 5 h, CH2Cl2 (15 mL)
was added and the resulting solution was washed with saturated aq
NH4Cl (10 mL), saturated aq NaHCO3 (10 mL), and brine (10 mL),
dried (Na2SO4), and then concentrated in vacuo. The crude product
was purified by silica flash column chromatography (CH2Cl2/MeOH,
95:5 to 90:10). The condensation product was obtained as white solid
(32 mg, 53%). Then 5.5 M tBuOOH in decane (38 μL, 0.209 mmol)
was added to a solution of the condensation product (32 mg, 20.9
μmol) in dry CH2Cl2 (7 mL), and the resulting solution was stirred at
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Article
rt. Then 5.5 M tBuOOH in decane (86 μL, 0.474 mmol) was added
twice more after 5 and 22 h after the first addition. Upon consumption
of the starting material after 48 h, CH2Cl2 (5 mL) and a mixture of
saturated aq Na2S2O3/NaHCO3 (1:1, 10 mL) were added to the
reaction mixture and the aqueous layer was extracted with more
CH2Cl2 (3 × 10 mL). Combined organic fractions were dried
(Na2SO4) and concentrated in vacuo. The crude product was purified
by silica flash column chromatography (CH2Cl2 to CH2Cl2/MeOH
90:10). The title product was obtained as a white solid (21 mg, 73%).
HPLC: 40−60% MeCN (tR = 5.11 min); mp (CH2Cl2) decomposes
above 200 °C. [α]D +42.0 (c = 1.00, CH2Cl2/MeOH, 96:4). IR (KBr)
3340, 2911, 2847, 1681, 1643, 1515, 1201, 1124 cm−1. 1H NMR (400
MHz, pyridine-d5) δ = 1.14−1.31 (m, 2 H), 1.59 (d, J = 6.8 Hz, 3 H),
1.64−2.25 (m, 7 H), 2.80 (s, 3 H), 3.19−3.31 (m, 2 H), 3.36 (dd, J =
14.6, 4.6 Hz, 1 H), 3.47 (dd, J = 16.6, 3.0 Hz, 1 H), 3.56 (dd, J = 13.4,
4.2 Hz, 1 H), 3.64−3.94 (m, 4 H), 4.91 (dd, J = 8.2, 3.4 Hz, 1 H),
5.10−5.25 (m, 1 H), 5.38−5.47 (m, 1 H), 5.37−5.72 (m, 1 H), 5.76−
5.83 (m, 1 H), 5.97−6.04 (m, 1 H), 6.13 (s, 1 H), 7.03−7.11 (m, 3 H),
7.13−7.22 (m, 5 H), 7.38 (d, J = 8.0 Hz, 2 H), 7.64 (bs, 1 H), 7.89−
8.00 (m, 2 H), 8.03 (s, 1 H), 8.15−8.30 (m, 5 H), 8.33−8.42 (m, 2 H),
8.63 (s, 1 H), 9.08 (d, J = 6.8 Hz, 1 H), 9.45 (d, J = 8.4 Hz, 1 H), 9.70
(d, J = 7.2 Hz, 1 H), 10.64 (s, 1 H) ppm. 13C NMR (100 MHz,
pyridine-d5) δ = 11.8 (q), 17.3 (q), 25.2 (t), 29.4 (t), 30.0 (t), 33.7 (t),
37.6 (t), 39.0 (t), 43.5 (t), 47.4 (t), 48.0 (t), 48.5 (d), 49.5 (d), 52.2
(d), 53.6 (d), 60.5 (d), 61.3 (d), 103.2 (t), 115.6 (d), 116.6 (d), 118.8
(d), 123.0 (s), 123.3 (d), 123.5 (d), 124.9 (d), 126.4 (s), 127.3 (d),
128.0 (d), 128.7 (d), 130.2 (d), 130.9 (s), 131.5 (d), 136.7 (s), 139.4
(d), 149.2 (s), 149.3 (s), 150.8 (s), 152.0 (s), 152.1 (s), 154.0 (s),
154.7 (s), 155.9 (s), 158.1 (s), 159.7 (s), 160.1 (s), 160.4 (s), 161.2
(s), 162.7 (s), 164.7 (s), 168.3 (s), 169.2 (s), 171.7 (s), 171.8 (s),
173.1(s), 174.5 (s), 175.0 (s), 175.1 (s) ppm. HRMS m/z calcd for
C63H59O11N16S5 (M + H) 1375.3147, found 1375.3247.
Thiazoline Tripeptide Analogue (27). Trimethyltin hydroxide (47
mg, 0.256 mmol) was added to a solution of 2018 (50 mg, 42.8 μmol)
in dry 1,2-dichloroethane (0.85 mL), and the reaction mixture was
then stirred at 60 °C. After 15 h, the mixture was diluted in CH2Cl2
(40 mL), washed with 6% HCl (30 mL), dried (Na2SO3), and
concentrated in vacuo. EDC·HCl (10 mg, 51.4 μmol), HOAt (7 mg,
51.4 μmol), and DIPEA (9 μL, 51.4 μmol) were added to a stirring
solution of the crude carboxylic acid (22)18 and 12 (27 mg, 51.4
μmol) in DMF (0.9 mL) at 0 °C. After 17 h, the mixture was diluted in
CH2Cl2 (20 mL), washed with 1 M NaHCO3 (20 mL), dried
(Na2SO3), and concentrated in vacuo with the aid of toluene to
remove DMF traces. The crude product was purified by flash column
chromatography (CH2Cl2/MeOH, 98:2 to 90:10). The condensation
product was obtained as a white solid (47 mg, 72%). Then 5.5 M
tBuOOH in decane (56 μL, 0.306 mmol) was added to a solution of
the condensation product (47 mg, 30.6 μmol) in dry CH2Cl2 (10 mL),
and the resulting solution was stirred at rt. Then 5.5 M tBuOOH in
decane (70 μL, 0.384 mmol) was added twice more after 5 and 22 h
after the first addition. Upon consumption of the starting material after
46 h, CH2Cl2 (10 mL) and a mixture of saturated aq Na2S2O3/
NaHCO3 (1:1, 10 mL) were added to the reaction mixture and the
aqueous layer was extracted with more CH2Cl2 (2 × 10 mL).
Combined organic fractions were dried (Na2SO4) and concentrated in
vacuo. The crude product purified by preparative reverse-phase
column (C18, 40−50% MeCN in H2O). The title product was
obtained as a white solid (37 mg, 88%). HPLC: 40−60% MeCN (tR =
5.23 min); mp (CH2Cl2) decomposes above 130 °C. [α]D +3.2 (c =
0.5, CH2Cl2/MeOH, 96:4). IR (KBr) 3327, 2917, 2841, 1649, 1502,
1457 cm−1. 1H NMR (600 MHz, DMSO-d6) δ = 1.23−1.32 (m, 2 H),
1.38 (d, J = 6.6 Hz, 3 H), 1.81−2.34 (m, 7 H), 2.39−2.50 (m, 1 H),
2.74 (s, 3 H), 2.79−3.06 (m, 2 H), 3.09−3.45 (m, 4 H), 3.48−3.72
(m, 2 H), 4.26−4.31 (m, 1 H), 4.72−4.89 (m, 2 H), 4.90−5.11 (m, 2
H), 5.27−5.50 (m, 2 H), 5.97 (s, 1 H), 6.48−6.68 (m, 3 H), 6.83−7.15
(m, 3H), 7.16−7.46 (m, 5 H), 7.62−7.77 (m, 1 H), 7.86−8.08 (m, 1
H), 8.31−8.75 (m, 4 H), 8.76−8.87 (m, 4 H), 8.98 (d, J = 9.0 Hz, 1
H), 9.13 (d, J = 8.4 Hz, 1 H), 10.04−10.11 (m, 2 H) ppm. 13C NMR
(150 MHz, DMSO-d6) δ = 12.5 (q), 17.4 (q), 25.4 (t), 25.6 (t), 29.9
(t), 30.1 (t), 33.5 (t), 37.1 (t), 37.4 (t), 39.1 (t), 39.1 (t), 47.5 (t), 48.3
(d), 49.3 (d), 53.2 (d), 54.9 (d), 60.3 (d), 60.6 (d), 78.2 (d), 104.3 (t),
115.9 (d), 117.4 (d), 119.1 (d), 123.3 (d), 124.8 (d), 127.5 (d), 129.2
(d), 129.6 (d), 130.2 (d), 130.5 (s), 131.4 (d), 134.5 (s), 138.1 (s),
141.8 (d), 148.8 (s), 150.2 (s), 150.8 (s), 151.5 (s), 152.5 (s), 153.8
(s), 154.6 (s), 156.9 (s), 157.2 (s), 158.9 (s), 159.5 (s), 160.2 (s),
163.7 (s), 168.4 (s), 170.1 (s), 171.0 (s), 172.3 (s), 174.0 (s), 174.4
(s), 175.3 (s), 176.0 (s) ppm. HRMS m/z calcd for C63H61O11N16S5
(M + H) 1377.3304, found 1377.3327.
Thiazole Tetrapeptide Analogue (28). A mixture of 13 (35 mg,
0.047 mmol), EDC·HCl (9 mg, 0.047 mmol), HOAt (6 mg, 0.047
mmol), and DIPEA (8 μL, 0.047 mmol) was added to a stirring
solution of 21 (45 mg, 0.039 mmol) in dry DMF (0.8 mL) and cooled
in an ice bath. Next, the reaction mixture was allowed to reach rt. After
4 h, CH2Cl2 (15 mL) was added and the resulting solution was washed
with saturated aq NH4Cl (10 mL), saturated aq NaHCO3 (10 mL)
and brine (10 mL), dried (Na2SO4), and then concentrated in vacuo.
The crude product was purified by silica flash column chromatography
(CH2Cl2/MeOH, 95:5 to 90:10). The condensation product was
obtained as white solid (36 mg, 52%). Then 5.5 M tBuOOH in decane
(56 μL, 0.306 mmol) was added to a solution of the condensation
product (36 mg, 20.4 μmol) in dry CH2Cl2 (7 mL), and the resulting
solution was stirred at rt. Then 5.5 M tBuOOH in decane (86 μL,
0.474 mmol) was added twice more after 5 and 22 h after the first
addition. Upon consumption of the starting material after 47 h,
CH2Cl2 (5 mL) and a mixture of saturated aq Na2S2O3/NaHCO3 (1:1,
10 mL) were added to the reaction mixture and the aqueous layer was
extracted with more CH2Cl2 (3 × 10 mL). Combined organic fractions
were dried (Na2SO4) and concentrated in vacuo. The crude product
was purified by silica flash column chromatography (CH2Cl2 to
CH2Cl2/MeOH 90:10). The title product was obtained as a white
solid (19 mg, 64%). HPLC: 40−60% MeCN (tR= 5.93 min); mp
(CH2Cl2) decomposes above 200 °C. [α]D +63.7 (c = 1.00, CH2Cl2/
MeOH, 96:4). IR (KBr) 3334, 2917, 2847, 1643, 1515, 1419 cm−1. 1H
NMR (400 MHz, pyridine-d5) δ = 1.67 (d, J = 6.8 Hz, 3 H), 1.70−2.02
(m, 7 H), 2.14−2.26 (m, 2 H), 2.80 (s, 3 H), 3.23−3.32 (m, 2 H), 3.37
(dd, J = 14.6, 4.6 Hz, 1 H), 3.48 (dd, J = 16.4, 3.2 Hz, 1 H), 3.56 (dd, J
= 13.2, 4.0 Hz, 1 H), 3.65−3.84 (m, 3 H), 3.85−3.94 (m, 1 H), 4.94−
5.00 (m, 1 H), 5.15−5.25 (m, 1 H), 5.40−5.47 (m, 1 H), 5.64−5.74
(m, 1 H), 5.77−5.85 (m, 1 H), 5.96 (s, 1 H), 5.97−6.05 (m, 1 H), 6.15
(s, 1 H), 6.85 (s, 1 H), 7.04−7.12 (m, 3 H), 7.14−7.22 (m, 5 H), 7.38
(d, J = 8.4 Hz, 2 H), 7.63 (bs, 1 H), 7.97 (bs, 1 H), 8.03 (s, 1 H), 8.21
(s, 1 H), 8.24 (s, 1 H), 8.27 (d, J = 8.0 Hz, 1 H), 8.30 (d, J = 8.0 Hz, 1
H), 8.37 (s, 1 H), 8.49 (bs, 1 H), 8.63 (s, 1 H), 8.86−8.94 (m, 1 H),
9.09 (d, J = 6.8 Hz, 1 H), 9.46 (d, J = 8.8 Hz, 1 H), 9.77 (d, J = 6.8 Hz,
1 H), 9.85 (s, 1 H), 10.63 (s, 1 H) ppm. 13C NMR (100 MHz,
pyridine-d5) δ = 11.8 (q), 17.5 (q), 25.3 (t), 25.3 (t), 28.8 (t), 33.7 (t),
37.7 (t), 39.1 (t), 43.5 (t), 47.5 (t), 48.1 (t), 48.5 (d), 49.5 (d), 52.2
(d), 53.6 (d), 61.3 (d), 61.7 (d), 102.8 (t), 103.3 (t), 115.6 (d), 116.6
(d), 118.9 (d), 123.0 (s), 123.4 (d), 123.6 (d), 124.0 (s), 124.9 (d),
126.4 (s), 127.3 (d), 128.0 (d), 128.8 (d), 130.2 (d), 130.9 (s), 131.6
(d), 135.0 (s), 136.7 (s), 139.4 (d), 149.2 (s), 149.3 (s), 149.3 (s),
150.8 (s), 152.0 (s), 152.1 (s), 154.0 (s); 154.8 (s), 155.9 (s), 158.1
(s), 159.7 (s), 160.1 (s), 160.5 (s), 161.2 (s), 162.8 (s), 164.8 (s),
166.8 (s), 168.4 (s), 169.2 (s), 171.0 (s), 171.8 (s), 172.9 (s), 173.1
(s), 175.0 (s), 175.2 (s) ppm. HRMS m/z calcd for C66H62O12N17S5
(M + H) 1444.3362, found 1444.3399.
Thiazoline Tetrapeptide Analogue (29). Trimethyltin hydroxide
(47 mg, 0.256 mmol) was added to a solution of 2018 (50 mg, 42.8
μmol) in dry 1,2-dichloroethane (0.85 mL), and the reaction mixture
was then stirred at 60 °C. After 15 h, the mixture was diluted in
CH2Cl2 (40 mL), washed with 6% HCl (30 mL), dried (Na2SO3), and
concentrated in vacuo. EDC·HCl (10 mg, 51.4 μmol), HOAt (7 mg,
51.4 μmol), and DIPEA (9 μL, 51.4 μmol) were added to a stirring
solution of the crude 2218 and 13 (39 mg, 51.4 μmol) in DMF (0.9
mL) at 0 °C. After 17 h, the mixture was diluted in CH2Cl2 (20 mL),
washed with 1 M NaHCO3 (20 mL), dried (Na2SO3), and then
concentrated in vacuo with the aid of toluene to remove DMF traces.
The crude product was purified by flash column chromatography
(CH2Cl2/MeOH, 95:5 to 90:10). The condensation product was
obtained as a white solid (45 mg, 60%). Then 5.5 M tBuOOH in
4192
dx.doi.org/10.1021/jm500062g | J. Med. Chem. 2014, 57, 4185−4195

Journal of Medicinal Chemistry
Article
decane (70 μL, 0.384 mmol) was added to a solution of the
condensation product (45 mg, 25.6 μmol) in dry CH2Cl2 (8.5 mL),
and the resulting solution was stirred at rt. Then 5.5 M tBuOOH in
decane (70 μL, 0.384 mmol) was added twice more after 5 and 22 h
after the first addition. Upon consumption of the starting material after
45 h, CH2Cl2 (10 mL) and a mixture of saturated aq Na2S2O3/
NaHCO3 (1:1, 10 mL) were added to the reaction mixture and the
aqueous layer was extracted with more CH2Cl2 (2 × 10 mL).
Combined organic fractions were dried (Na2SO4) and concentrated in
vacuo. The crude product was purified by preparative reverse-phase
column (C18, 40−50% MeCN in H2O). The title product was
obtained as a white solid (34 mg, 92%). HPLC: 40−60% MeCN (tR =
5.47 min); mp (CH2Cl2) decomposes above 200 °C. [α]D +12.4 (c =
0.50, CH2Cl2/MeOH, 96:4). IR (KBr) 3430, 2917, 2841, 1739, 1649,
1444, 1041 cm−1. 1H NMR (400 MHz, pyridine-d5) δ = 1.58−1.70 (m,
2 H), 1.88−2.06 (m, 4 H), 2.08−2.41 8m, 6 H), 2.52−2.66 (m, 2 H),
3.11−3.20 (m, 3 H), 3.36−3.43 (m, 1 H), 3.53−3.58 (m, 1 H), 3.73−
3.78 (m, 1 H), 3.96−4.17 (m, 5 H), 4.22−4.30 (m, 1 H), 5.31−5.46
(m, 2 H), 5.50−5.66 (m, 2 H), 5.73−5.80 (m, 2 H), 6.23−6.28 (m, 1
H), 6.33 (s, 1 H), 6.52 (s, 1 H), 7.22 (s, 1 H), 7.40−7.58 (m, 8 H),
7.64−7.77 (m, 2 H), 7.97 (bs, 1 H), 8.09 (d, J = 9.0 Hz, 1 H), 8.27 (bs,
1 H), 8.35 (s, 1 H), 8.52−8.62 (m, 4 H), 8.86 (bs, 1 H), 8.99 (s, 1 H),
9.12 (d, J = 6.6 Hz, 1 H), 9.29 (bs, 1 H), 9.70 (d, J = 9.0 Hz, 1 H),
10.12 (d, J = 6.6 Hz, 1 H), 10.23 (d, J = 6.6 Hz, 1 H),10.94−11.00 (m,
1 H) ppm. 13C NMR (150 MHz, pyridine-d5) δ = 11.8 (q), 17.5 (q),
25.2 (t), 25.5 (t), 28.8 (t), 30.0 (t), 34.3 (t), 36.2 (t), 36.8 (t), 38.4 (t),
39.9 (t), 47.5 (t), 48.5 (d), 49.2 (d), 52.3 (d), 54.1 (d), 61.7 (d), 61.9
(d), 78.8 (d), 102.8 (t), 103.3 (t), 116.2 (d), 116.8 (d), 118.6 (d),
123.0 (d), 123.1 (d), 125.8 (s), 127.3 (d), 127.9 (d), 128.7 (d), 130.2
(s), 130.2 (d), 131.0 (s), 131.7 (d), 136.9 (s), 139.2 (d), 148.9 (s),
150.8 (s), 150.9 (s), 152.0 (s), 153.9 (s), 154.7 (s), 155.9 (s), 157.9
(s), 159.7 (s), 160.5 (s), 161.2 (s), 162.7 (s), 164.8 (s), 166.8 (s),
168.3 (s), 169.1 (s), 171.0 (s), 171.2 (s), 172.6 (s), 173.0 (s), 173.2
(s), 174.8 (s), 175.1 (s) ppm. HRMS m/z calcd for C66H64O12N17S5
(M + H) 1446.3518, found 1446.3603.
9.52 (s, 1 H), 10.09 (s, 1 H) ppm. 13C NMR (100 MHz, DMSO-d6) δ
= 12.6 (q), 17.3 (q), 25.5 (t), 25.6 (t), 29.9 (t), 29.9 (t), 33.5 (t), 37.9
(t), 42.6 (t), 47.7 (t), 48.3 (t), 48.3 (d), 49.4 (d), 52.7 (d), 53.4 (d),
60.4 (d), 61.0 (d), 104.3 (t), 105.3 (t), 107.9 (t), 116.1 (d), 117.2 (d),
119.3 (d), 123.4 (d), 123.5 (s), 124.5 (d), 125.8 (d), 127.4 (s), 127.7
(d), 129.1 (s), 129.2 (d), 129.6 (d), 130.1 (s); 130.4 (d); 131.3 (d),
134.4 (s), 135.6 (s), 137.3 (s), 137.4 (s), 141.3 (d), 148.5 (s), 149.8
(s), 151.2 (s), 151.5 (s), 152.2 (s), 153.9 (s), 154.7 (s), 156.5 (s),
157.0 (s), 159.5 (s), 160.0 (s), 161.1 (s), 161.8 (s), 162.0 (s); 163.2
(s), 163.8 (s), 166.0 (s), 168.4 (s), 170.3 (s), 171.2 (s), 171.5 (s),
172.2 (s), 172.6 (s), 174.6 (s), 175.3 (s) ppm. HRMS m/z calcd for
C69H65O13N18S5 (M + H) 1513.3577, found 1513.3673.
Thz-Dha-Ala-Pro-Dha-Dha-NH2 (31). The title peptide could be
obtained by two different methods: (A) on-resin oxidation/
elimination, (B) oxidation/elimination after cleavage.
Method A. Prepared according to the general method for solid-
phase peptide synthesis, starting from 1.06 g of resin. Prior to cleavage
from the resin, an overnight treatment with 3 M tBuOOH in isooctane
(6 mL, 18 mmol, 32 equiv) in CH2Cl2 (3 mL) at rt was performed.
After cleavage, the crude product was further purified by silica flash
column chromatography (EtOAc/THF, 9:1 to 8:2). The title product
was obtained as a white solid (102 mg, 36% based on functionalization
of the resin).
Method B. First, 3 M tBuOOH in isooctane (3.5 mL, 10.5 mmol, 34
equiv) was added to a solution of 32 (304 mg, 0.311 mmol) in dry
CH2Cl2 (9 mL). The resulting mixture was stirred at rt for 7 h . The
crude product was purified by silica flash column chromatography
(EtOAc/THF, 9:1 to 8:2). The title product was obtained as a white
solid (112 mg, 72%). HPLC: 50−100% MeCN (tR = 1.18 min); mp
(EtOAc) decomposes above 100 °C. [α]D −85.9 (c = 1.00, CH2Cl2).
1
IR (KBr) 3334, 2981, 2911, 2873, 1630, 1515 cm−1. H NMR (400
MHz, acetone-d6) δ = 1.56 (d, J = 6.8 Hz, 3 H), 2.12−2.30 (m, 4 H),
3.77−3.85 (m, 1 H), 3.88−3.96 (m, 1 H), 4.62−4.69 (m, 1 H), 4.90
(q, J = 3.0, 1 H), 5.61 (bs, 1 H), 5.68 (bs, 1 H), 5.72 (bs, 1 H),6.24
(bs, 1 H), 6.31 (d, J = 1.2 Hz, 1 H), 6.40 (d, J = 1.2 Hz, 1 H), 6.68 (d,
J = 1.6 Hz, 1 H), 6.96 (bs, 1 H), 7.66 (d, J = 6.4 Hz, 1 H), 8.37 (d, J =
2.0 Hz, 1 H), 8.79 (bs, 1 H), 8.94 (bs, 1 H), 9.10 (d, J = 2.0 Hz, 1 H),
10.05 (bs, 1 H) ppm. 13C NMR (100 MHz, acetone-d6) δ = 16.9 (q),
25.2 (t), 28.6 (t), 47.6 (t), 48.1 (d), 61.3 (d), 102.0 (t), 104.5 (t),
105.0 (t), 124.7 (d), 134.4 (s), 134.4 (s), 136.0 (s), 151.2 (s), 154.8
(d), 159.5 (s), 162.6 (s), 163.5 (s), 165.7 (s), 171.3 (s), 172.4 (s)
ppm. HRMS m/z calcd for C21H26O6N7S (M + H) 504.1660, found
504.1663.
Thiazole Pentapeptide Analogue (30). A mixture of 1418 (36 mg,
42.1 μmol), EDC·HCl (8 mg, 42.1 μmol), HOAt (6 mg, 42.1 μmol),
and DIPEA (8 μL, 42.1 μmol) was added to a stirring solution of 21
(40 mg, 35.1 μmol) in dry DMF (0.7 mL) and cooled in an ice bath.
The reaction mixture was allowed to reach rt. After 4 h, CH2Cl2 (25
mL) was added and the resulting solution was washed with saturated
aq NH4Cl (25 mL), saturated aq NaHCO3 (25 mL) and brine (25
mL), dried (Na2SO4), and concentrated in vacuo. The crude product
was purified by silica flash column chromatography (CH2Cl2/MeOH,
95:5 to 90:10). The condensation product was obtained as white solid
(49 mg, 70%). Then 5.5 M tBuOOH in decane (86 μL, 0.474 mmol)
was added to a solution of the condensation product (47 mg, 23.7
μmol) in dry CH2Cl2 (8 mL), and the resulting solution was stirred at
rt. Then 5.5 M tBuOOH in decane (86 μL, 0.474 mmol) was added
twice more after 5 and 26 h after the first addition. Upon consumption
of the starting material after 32 h, CH2Cl2 (5 mL) and a mixture of
saturated aq Na2S2O3/NaHCO3 (1:1, 10 mL) were added to the
reaction mixture and the aqueous layer was extracted with more
CH2Cl2 (4 × 10 mL). Combined organic fractions were dried
(Na2SO4) and concentrated in vacuo. The crude product was purified
by silica flash column chromatography (CH2Cl2/MeOH, 99:1 to
90:10). The title product was obtained as a white solid (26 mg, 72%).
HPLC: 40−60% MeCN (tR = 5.67 min); mp (CH2Cl2) decomposes
above 120 °C. [α]D +20.6 (c = 1.00, CH2Cl2/MeOH, 96:4). IR (KBr)
3443, 2917, 2853, 1649, 1489, 1419 cm−1. 1H NMR (400 MHz,
DMSO-d6) δ = 1.24−1.31 (m, 1 H), 1.40 (d, J = 6.8 Hz, 3 H), 1.85−
2.26 (m, 8 H), 2.40−2.51 (m, 1 H), 2.78 (s, 3 H), 2.83−2.97 (m, 2 H),
3.09−3.27 (m, 2 H), 3.34−3.44 (m, 1 H), 3.62−3.91 (m, 3 H), 4.52−
4.60 (m, 1 H), 4.71−4.82 (m, 1 H), 5.02−5.13 (m, 1 H), 5.30−5.43
(m, 2 H), 5.58 (s, 1 H), 5.62−5.72 (m, 2 H), 5.90 (s, 1 H), 5.98 (s, 1
H), 6.14 (s, 1 H), 6.56 (s, 1 H), 6.68 (d, J = 8.6 Hz, 2 H), 6.83 (bs, 1
H), 7.11 (d, J = 8.6 Hz, 2 H), 7.20 (d, J = 6.8 Hz, 2 H), 7.24−7.39 (m,
4 H), 7.57 (bs, 1 H), 7.76 (s, 1 H), 7.96−8.04 (m, 2 H), 8.13 (s, 1 H),
8.23 (s, 1 H), 8.32−8.40 (m, 2 H), 8.58 (d, J = 8.4 Hz, 1 H), 8.71 (s, 1
H), 8.76 (d, J = 7.6 Hz, 2 H), 8.85 (d, J = 6.4 Hz, 1 H), 9.13 (s, 1 H),
Thz-Sec(Ph)-Ala-Pro-Sec(Ph)-Sec(Ph)-NH2 (32). Compound 32 was
prepared according to the general method for solid-phase peptide
synthesis, starting from 1.35 g of resin. The crude product was further
purified by silica flash column chromatography (CH2Cl2/MeOH,
95:5). The title product was obtained as a pale solid (330 mg, 47%
based on functionalization of the resin). HPLC: 0−100% MeCN (tR =
7.29 min); mp (CH2Cl2) 185−188 °C. [α]D −116.6 (c = 0.33,
1
CH2Cl2). IR (KBr) 3436, 3295, 2924, 1668, 1623, 1041 cm−1. H
NMR (400 MHz, DMSO-d6) δ = 1.26 (d, J = 6.8 Hz, 3 H), 1.77−2.12
(m, 4 H), 3.08−3.19 (m, 2 H), 3.24−3.50 (m, 4 H), 3.54−3.68 (m, 2
H), 4.28−4.47 (m, 3 H), 4.56−4.65 (m, 1 H), 4.76−4.86 (m, 1 H),
7.21−7.36 (m, 9 H), 7.46−7.56 (m, 6 H), 8.15 (d, J = 8.4 Hz, 1 H),
8.22 (d, J = 7.2 Hz, 1 H), 8.39 (d, J = 2.0 Hz, 1 H), 8.48−8.58 (m, 2
H), 9.22 (d, J = 2.0 Hz, 1 H) ppm. 13C NMR (100 MHz, DMSO-d6) δ
= 17.9 (q), 25.4 (t), 29.4 (t), 29.6 (t), 29.9 (t), 30.4 (t), 47.5(d), 47.7
(t), 53.4 (d); 53.8 (d), 54.1 (d), 60.9 (d), 125.7 (d), 127.6 (d), 127.6
(d), 127.8 (d), 130.0 (d), 130.2 (d), 130.2 (d), 130.7 (s), 130.8 (s),
131.0 (s), 132.4 (d), 132.7 (d), 132.7 (d), 150.1 (s), 155.9 (d), 161.0
(s), 170.0 (s), 170.8 (s), 171.5 (s), 172.4 (s), 172.7 (s) ppm. HRMS
m/z calcd for C39H44N7O6SSe3 (M + H) 998.0392, found 998.0454.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra images of new compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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(11) During the preparation of this manuscript, we became aware of
the synthesis and testing of an analogue of amythiamicin D, a
protected intermediate and and epimer of the former: Gross, S.;
Nguyen, F.; Bierschenk, M.; Sohmen, D.; Menzel, T.; Antes, I.;
Wilson, D. N.; Bach, T. Amythiamicin D and related thiopeptides as
inhibitors of the bacterial elongation factor EF-Tu: modification of the
amino acid at carbon atom C2 of ring C dramatically influences
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4022.
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their pathway engineering. Nat. Prod. Rep. 2013, 30, 218−226.
(14) Ferrari, P.; Colombo, L.; Stella, S.; Selva, E.; Zerilli, L. F.
Antibiotic GE37468 A: a novel inhibitor of bacterial protein synthesis.
II. Structure elucidation. J. Antibiot. 1995, 48, 1304−1311.
(15) Young, T. S.; Dorrestein, P. C.; Walsh, C. T. Codon
randomization for rapid exploration of chemical space in thiopeptide
antibiotic variants. Chem. Biol. 2012, 19, 1600−1610.
(16) Morris, R. P.; Leeds, J. A.; Naegeli, H. U.; Oberer, L.; Memmert,
K.; Weber, E.; LaMarche, M. J.; Parker, C. N.; Burrer, N.; Esterow, S.;
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AUTHOR INFORMATION
Corresponding Author
*Phone: + 34 93 403 70 86. Fax: + 34 93 403 71 26. E-mail:
mercedes.alvarez@irbbarcelona.org.
■
Present Addresses
∇For X.J.-B.: School of Chemistry, University of Manchester,
Oxford Road, Manchester M13 9PL, United Kingdom.
○For P.B.: Aptuit Verona, Srl, Via Alessandro Fleming 4, 37135
Verona, Italy.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge support from the Spanish Science
and Innovation Ministry, CICYT (CTQ2012-30930), and the
Generalitat de Catalunya (2009SGR 1024). X.J. thanks ISCIII
for a PFIS grant.
ABBREVIATIONS USED
■
TFAA, trifluoroacetic anhydride; DME, dimethoxyethane;
DIPCDI, N,N′-diisopropylcarbodiimide; EDC, N-(3-dimethy-
laminopropyl)-N′-ethylcarbodiimide hydrochloride; HOAt, 1-
hydroxy-7-azabenzotriazole; DIPEA, diisopropylethylamine;
TFA, trifluoroacetic acid; MIC, minimal inhibitory concen-
tration; BLD, below limit of detection
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