Synthesis and Evaluation of Antibacterial Activity of Bottromycins

Article abstract of DOI:10.1021/acs.joc.8b00045

Total synthesis of bottromycin A₂ can be accomplished through a diastereoselective Mannich reaction of a chiral sulfinamide, mercury-mediated intermolecular amidination, and cyclization of a constrained tetracyclic peptide. Exploitation of this process allowed the synthesis of several novel bottromycin analogs. The antimicrobial activity of these analogs was evaluated in vitro against Gram-positive bacteria, such as methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE). Structure-activity relationships were explored taking into consideration the unique three-dimensional structure of the compounds. Notably, one of the new analogs devoid of a methyl ester, which is known to lower the in vivo efficacy of bottromycin, exhibited antibacterial bioactivity comparable to that of vancomycin.

Full text of DOI:10.1021/acs.joc.8b00045

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Synthesis and Evaluation of Antibacterial Activity of Bottromycins
Takeshi Yamada, Miu Yagita, Yutaka Kobayashi, Goh Sennari, Hiroyuki Shimamura, Hidehito
Matsui, Yuki Horimatsu, Hideaki Hanaki, Tomoyasu Hirose, Satoshi Omura, and Toshiaki Sunazuka
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b00045 • Publication Date (Web): 21 Mar 2018
Downloaded from http://pubs.acs.org on March 21, 2018
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Synthesis and Evaluation of Antibacterial Activity of Bottromycins
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Takeshi Yamada,†, ‡, Miu Yagita,‡ Yutaka Kobayashi,‡ Goh Sennari,‡ Hiroyuki Shimamura,‡ Hidehito
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Matsui,† Yuki Horimatsu,‡ Hideaki Hanaki,† Tomoyasu Hirose,†, ‡ Satoshi Ōmura*, † and Toshiaki Sunazuka*,
†, ‡
†Kitasato Institute for Life Sciences, Kitasato University, 5ꢀ9ꢀ1 Shirokane, Minatoꢀku, Tokyo 108ꢀ8641, Japan
‡Graduate School of Infection Control Sciences, Kitasato University, 5ꢀ9ꢀ1 Shirokane, Minatoꢀku, Tokyo
108ꢀ8641, Japan
Eꢀmail: omuras@insti.kitasatoꢀu.ac.jp,
sunazuka@lisci.kitasatoꢀu.ac.jp
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Table of Contents
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Abstract
Total synthesis of bottromycin A2 can be accomplished through a diastereoselective Mannich
reaction of a chiral sulfinamide, mercuryꢀmediated intermolecular amidination, and cyclization of a
constrained tetracyclic peptide. Exploitation of this process allowed the synthesis of several novel bottromycin
analogs. The antimicrobial activity of these analogs was evaluated in vitro against Gramꢀpositive bacteria,
such as methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE).
Structure activity relationships were explored taking into consideration the unique threeꢀdimensional structure
of the compounds. Notably, one of the new analogs devoid of a methyl ester, which is known to lower the in
vivo efficacy of bottromycin, exhibited antibacterial bioactivity comparable to that of vancomycin.
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Introduction
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Antibioticꢀresistant microbes, such as methicillinꢀresistant Staphylococcus aureus (MRSA) and
vancomycinꢀresistant enterococci (VRE), represent a serious and increasing worldwide problem. Although
Staphylococcus aureus and the enterococci are not always pathogenic, pathogenic strains often infect
immunocompromised patients and can cause a variety of potentially lifeꢀthreatening infections, such as sepsis
and endocarditis. Since MRSA and VRE are multidrug resistant strains, infections with such bacteria can be
almost impossible to treat. Therefore, the development of new antibacterial drugs, especially those with novel
modes of action, is becoming of utmost importance if modern medicine is to overcome the growing
antibacterial resistance problem.
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Our research group has long been investigating the development of new medicines from microbial
metabolite origins.1 In the course of our research, we have been screening for new antiꢀMRSA and antiꢀVRE
agents from our natural product library and previously discovered that bottromycin A2 (1) possessed
antibacterial activity against MRSA and VRE strains (Figure 1).2
Figure 1. Bottromycin A2, B2, C2 and D.
Bottromycin was first isolated from the culture broth of Streptomyces bottropensis by Waisvisz and
coꢀworkers in 1957.3 Several years later, Umezawa’s group isolated bottromycin A2 (1), B2 (2), and C2 (3)
from a culture broth of Streptomyces No. 3668ꢀL2.4 Through a series of intensive structural studies the planar
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structure of 1 was proposed.5 The absolute structure of the compound was finally elucidated by our pioneering
total synthesis of 1.2 Bottromycins are heptapeptides containing a cyclic tetrapeptide and a tripeptide side
chain bounded to the skeletal ring via an amidine functional group. In addition, five of the seven amino acid
residues of 1 are unusual amino acids, namely, (R)ꢀ3ꢀ(thiazolꢀ2ꢀyl)ꢀβꢀalanine (ThiaꢀβꢀAlaꢀOMe),
erythroꢀβꢀmethylꢀLꢀphenylalanine, two Lꢀtertꢀleucines (tꢀLeu) and cisꢀ3ꢀmethylꢀLꢀproline. Instead of the
cisꢀ3ꢀmethylꢀLꢀproline amino acid residue of 1, bottromycins B2 (2) and C2 (3) possess Lꢀproline and
3,3ꢀdimethylꢀLꢀproline, respectively. In 2012, Bugni and coꢀworkers reported the isolation of a new analog,
bottromycin D (4), which contains an Lꢀalanine residue instead of the Lꢀvaline of 1.6 Through our synthetic
studies of bottromycins, we discovered that 1 has an unusual feature in that the three Cꢀterminal residues fold
back on the 12ꢀmembered cyclic skeleton made by the four Nꢀterminal residues (Figure 2).7 Due to the unique
structure of this unprecedented macrocyclic amidine, the rare βꢀmethylated amino acid residues, a terminal
(43R)ꢀThiaꢀβꢀAlaꢀOMe8, and its threeꢀdimensional structure, the biosynthesis of bottromycins has latterly
been studied in depth.9
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Figure 2. Threeꢀdimensional structure of bottromycin A2, which was determined in CDCl3.7
The bottromycins, especially bottromycin A2 (1), display potent antibacterial activity against
Gramꢀpositive bacteria3,4 and mycoplasma.10 In 1991, Yokoyama and coꢀworkers reported the antiꢀMRSA
activity of 1.11 We also found that bottromycin A2 (1) possesses potent antibacterial activity against various
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antimicrobialꢀresistant bacteria, such as MRSA and VRE, with minimum inhibitory concentration (MIC)
values of less than 2.0 ꢀg/mL.2
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The mode of action of 1 has been identified, bottromycin inhibiting bacterial protein synthesis by
interfering with the binding of aminoacylꢀtRNA to the A site on the 50S ribosome. But it does not inhibit
peptide bond formation and translocation steps.12 This is a novel mechanism, different from that seen in
commonly used antibiotics.
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Although 1 has attracted much attention from bioorganic and organic chemists, structure activity
relationship (SAR) studies have proven difficult due to the problem of chemical synthesis and derivatization
of naturallyꢀoccurring 1. Indeed, modification of the terminal methyl ester has only been reported by three
groups, including ours.13,14 In addition, several synthetic studies of 1 have been published,15,16 but our total
synthesis is the only fully successful method reported to date.2
After we discovered the potent antiꢀMRSA and antiꢀVRE activity of 1, we started efforts to
develop a new antibacterial agent using 1 as a lead compound. In this manuscript, we describe the detailed
total synthesis of bottromycin A2 (1) and B2 (2), together with creation of 33ꢀdemethyl analogs17, along with
bottromycin analogs devoid of ThiaꢀβꢀAlaꢀOMe, to study SAR focussing on the unnatural amino acid
residues. All the new analogs were subjected to an in vitro evaluation against Gramꢀpositive bacteria,
including some drugꢀresistant strains.
Results and Discussion
Bottromycins contain a cyclic tetrapeptide which is linked to a tripeptide side chain at the amidine.
A cyclic tetrapeptide is one of the most difficult cyclic systems to prepare by organic chemical synthesis due
to its highly constrained threeꢀdimensional structure, in which the planarity of the amide bonds is usually
twisted.18 In addition, the central amidine acts as a nucleophile to attack the internal electrophilic moiety.
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Therefore, the construction of a tetracyclic skeleton containing an amidine group is a major obstacle and only
Kazmier’s group has accomplished that construction to date.16 There are four possible positions AꢀD to
construct a cyclic tetrapeptide by intramolecular condensation (Scheme 1). We initially considered closing a
tetrapeptide ring at the A bond, namely macroamidination. Even though numerous reaction conditions were
examined, 1 was not detected.19 We next examined cyclization at B. However, the cyclization failed due to the
steric bulkiness.19 Cyclization at C was also unsuccessful due to the undesired dominant cyclization of the
amidine group.19 We eventually decided to cyclize at D, via condensation of the Nꢀterminal proline and the
Cꢀterminal glycine. The corresponding glycinol compound, which the glycine moiety of 5 was reduced, is
identical with the degradation product,2 thus the structure of the synthetic substrate 5 can be confirmed prior
to total synthesis. In addition, it is not necessary to take into consideration the epimerization of the Cꢀterminal
amino acid.
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Scheme 1. Retrosynthetic analysis
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The cyclization precursor 5 can be obtained through peptide synthesis from amidine 7 with
BocꢀValꢀOH and cisꢀ3ꢀmethylꢀLꢀproline 6. Compound 6 is prepared using Sasaki’s method.20 Sauvé and Rao
have previously reported the synthesis of acyclic amidines from a thioamide and a primary amine, such as
hydroxyl amine, hydrazine, and cyanamide, in the presence of Hg(OAc)2 and NEt3 in THF.21 Therefore, we
envisioned the construction of an amidine moiety from the thioamide 82 and tripeptide 9 in the presence of an
appropriate mercury reagent. The tripeptide 9 would be prepared by condensation with Bocꢀt-LeuꢀOH, (2S,
3S)ꢀ2ꢀazidoꢀ3ꢀphenylbutanoic acid 10,22 and (R)ꢀThiaꢀβꢀAlaꢀOMe 11. We anticipated that the compound 11
would arise via a Mannich reaction of Ellman chiral sulfinimine 12 with methyl acetate (13).23
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We synthesized the optically active (R)ꢀThiaꢀβꢀAlaꢀOMe 11 using a diastereoselective Mannich
reaction as shown in Scheme 2. Chiral sulfinimine (S)ꢀ122 was treated with methyl acetate (13) in the presence
o
of sodium hexamethyldisilazane (NaHMDS) at –78 C. The reaction proceeded smoothly, providing the
corresponding Mannich reaction product 15 in excellent yield without diastereoselectivity, dr = 1 : 1. After
examining several nucleophiles, we found that the more sterically bulky malonateꢀtype nucleophiles produced
good diastereoselectivity at the Cꢀ43 stereogenic center.17 Indeed, treatment of chiral sulfinimine (S)ꢀ12 with
allyl methyl malonate 14 in the presence of NaHMDS in THF at –78 oC afforded the corresponding adduct in
quantitative yield. Although the Cꢀ44 stereogenic center was not controlled, the Cꢀ43 stereogenic center was
obtained only as the (R)ꢀisomer. Subsequently, decarboxylation of the allyl ester was performed by treating
with a catalytic amount of Pd(PPh3)4 in the presence of formic acid and NEt3 in THF, to provide the methyl
ester 15 in 76% yield as a single diastereomer.24 The chiral auxiliary was removed by treatment with TFA in
MeOH to provide (R)ꢀThiaꢀβꢀAlaꢀOMe 11 with >99% ee. The optical purity was confirmed by chiral HPLC
analysis. The enantiomer of ThiaꢀβꢀAlaꢀOMe (S)ꢀ11 could be prepared in the same manner from the tertꢀbutyl
sulfinimine 16.
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Scheme 2. Synthesis of optically active ThiaꢀβꢀAlaꢀOMe (11)
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ThiaꢀβꢀAlaꢀOMe (R)ꢀ11 was then condensed with (2S, 3S)ꢀ2ꢀazidoꢀ3ꢀphenylbutanoic acid 10 using
1Hꢀbenzotriazolꢀ1ꢀyloxyꢀtri(pyrrolidino)phosphonium hexafluorophosphate (PyBOP) as a condensation
reagent to provide the dipeptide 17 in quantitative yield (Scheme 3). The azide group in 17 was reduced to an
amine under hydrogenation conditions followed by condensation with Bocꢀt-LeuꢀOH provided the tripeptide
18 in quantitative yield. Removal of the Boc group under acidic conditions afforded an amine 9.
Scheme 3. Synthesis of tripeptide 9
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Next, we examined the intermolecular amidination reaction. Thioamide 82 was treated with
tripeptide 9 in the presence of NEt3 in THF (Table 1, entry 1). The reaction did not proceed, even under reflux
conditions. The reaction in the presence of Hg(OAc)2 did not give the desired amidine product 7, instead
producing the corresponding amide 19 (entry 2).21 Using HgCl2 as a Lewis acid instead of Hg(OAc)2 afforded
7 in 67% yield with 19 and the starting thioamide 8 in 8% and 18% yields, respectively (entry 3). The
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1
existence of the geometrical tautomer of amidine was suggested by its HꢀNMR spectra. Thioamide 8 was
consumed when the more electrophilic Hg(OTf)2 was used as the Lewis acid, although the yield of 7 was not
improved (entry 4). We next examined two other solvents, CH2Cl2 and MeCN, for the amidination reaction
(entry 5, 6). It was found that the polar solvent, MeCN, provided the amidine 7 in a slightly higher yield, 77%
(entry 6). Use of 2,6ꢀlutidine as a base instead of NEt3 improved the yield of 7 to 96% (entry 7).25
Table 1. Amidination of thioamide 8 with tripeptide 9
Yield (%)a
Entry
HgX2
Base
Solvent
7
8
19
No reaction
n.d.c n.d.c 100
67 18
1b
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none
NEt3
NEt3
NEt3
NEt3
NEt3
NEt3
THF
THF
Hg(OAc)2
HgCl2
THF
8
Hg(OTf)2
Hg(OTf)2
Hg(OTf)2
THF
70 n.d.c 29
71 n.d.c 28
77 n.d.c 23
96 n.d.c trace
CH2Cl2
MeCN
Hg(OTf)2 2,6ꢀlutidine MeCN
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a Isolation yield. b Run at reflux condition. c n.d. = not detected.
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Our next effort was directed toward the elongation of the peptide chain. The Phth group was
removed by treatment with hydrazine in EtOH at 60oC to provide the corresponding amine (Scheme 4). The
amine was then condensed with BocꢀValꢀOH to afford the hexapeptide 20 in 89% yield (2 steps). If the
aminoethanol part was oxidized to an ester prior to the removal of the Phth group, an undesired
diketopiperazine was formed quickly. Removal of the Boc protecting group of 20, followed by condensation
with 3ꢀmethylproline 620, provided the heptapeptide 21 in excellent yields. Thus, all amino acid residues
constituted in 1 were connected. The TBDPS group of 21 was removed by treating with TBAF in THF to
provide the primary alcohol 22 in 90% yield.26 The oxidation of 22 to carboxylic acid 23 was problematic due
to the nucleophilicity of the internal amidine. Indeed, all oxidation reactions through a generation of aldehyde
did not provide the carboxylic acid 23. The aldehyde intermediate was trapped by the internal amidine to
provide the corresponding imidazole. Only Jones oxidation successfully provided the corresponding
carboxylic acid 23 in moderate yield (55%). The Boc group on the proline part of 23 was removed by treating
with 4N HCl in dioxane. Crude 24 was used for the final cyclization reaction. In this cyclization event, the
nucleophilic intramolecular cyclization from amidine was also a problem. Through examination of numerous
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reaction
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1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide (EDCI) and iꢀPr2NEt in CH2Cl2 provided bottromycin A2 (1)
1
in moderate yield.27 All spectra data ([α]D, H and 13C NMR, IR and HRMS) were identical to those of the
natural product.28 Thus, we achieved the first total synthesis of bottromycin A2 (1) and determined its absolute
structure.
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Scheme 4. Total syntheses of bottromycin A2 (1)
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To study the SAR, we focused on the influence of the unnatural amino acids, such as
cisꢀ3ꢀmethylꢀLꢀproline, erythroꢀβꢀmethylꢀLꢀphenylalanine and ThiaꢀβꢀAlaꢀOMe, on antibacterial activity
(Figure 3). We chose bottromycin B2 (2),
a naturally occurring demethyl analog at Cꢀ7,
Cꢀ33ꢀdemethylꢀbottromycin A2 (25) and Cꢀ33ꢀdemethylꢀbottromycin B2 (26)17 as initial targets to study
antibacterial impact (Scheme 5, 6).
Figure 3. Envisioned new bottromycin analogs.
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Condensation
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BocꢀProꢀOH
using
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1ꢀ[bis(dimethylamino)methylene]ꢀ1ꢀHꢀbenzotriazolium 3ꢀoxide hexafluorophosphate (HBTU) as
a
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condensation reagent provided heptapeptide 27 in good yield (Scheme 5). Removal of the TBDPS group
followed by oxidation of the resulting alcohol provided carboxylic acid 29. After the removal of the Boc
group, the carboxylic acid 29 was cyclized using EDCI as a condensation reagent to provide bottromycin B2
(2) in 19% yield (2 steps) in a similar manner as the synthesis of 1. The 1H NMR spectra of synthetic 2 was
slightly inconsistent with the reported chemical shifts of naturally occurring 2. This was believed to arise
because the chemical shifts being influenced by salt contaminant and/or substrate concentration. All other
spectral data, including the 13C NMR spectra, IR spectra and melting point, were in good agreement with
1
reported values.4a, 5c, 29 Thus, taking into consideration our total synthesis of bottromycin A2 and its H NMR
behavior28, the synthetic compound was strongly suggested to be bottromycin B2.
Scheme 5. Total synthesis of bottromycin B2 (2)
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The next focus was on the Cꢀ33ꢀdemethyl analogs of 1 and 2 (Scheme 6). The optically active
ThiaꢀβꢀAlaꢀOMe (R)ꢀ11 was condensed with commercially available BocꢀPheꢀOH using PyBOP as a
condensation reagent. The resultant dipeptide 30 was condensed with BocꢀtꢀLeuꢀOH, after the removal of the
Boc group under acidic conditions. Tripeptide 34 was treated with 4N HCl in dioxane, and then reacted with
thioamide 8 in the presence of Hg(OTf)2 and 2,6ꢀlutidine in MeCN, affording the corresponding amidine 36 in
90% yield in 2 steps. The 36 was then condensed with BocꢀValꢀOH and 3ꢀmethylproline 6 to provide a
heptapeptide 40 in good yield. The silyl ether 40 was transformed to carboxylic acid 46 through removal of
the silyl group and Jones oxidation of the primary alcohol. Final cyclization was performed using EDCI as a
condensation reagent to provide Cꢀ33ꢀdemethylꢀbottromycin A2 (25). Cꢀ33ꢀDemethylꢀbottromycin B2 (26)
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1
was prepared from 38 with BocꢀProꢀOH in a manner similar to the synthesis of the bottromycins. The H
NMR spectra of 25 and 26 were complicated because of the existence of conformers, as seen in bottromycin
D.6 This observation suggested the Cꢀ33 methyl group is important for the control of threeꢀdimensional
structure of the bottromycins.
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Scheme 6. Synthesis of Cꢀ33ꢀdemethylꢀbottromycin A2 (25), Cꢀ33ꢀdemethylꢀbottromycin B2 (26) and a
benzyl ester analog 49
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We next decided to focus on the SAR of ThiaꢀβꢀAlaꢀOMe. The methyl ester moiety of
ThiaꢀβꢀAlaꢀOMe is known to affect the antibacterial activity both in vitro and in vivo.14 Therefore, the
efficient synthetic route to access bottromycin analogs containing various functions instead of
ThiaꢀβꢀAlaꢀOMe was useful for finding lead compounds for use in the development of new antibacterial
agents. The synthesis of bottromycin analogs described above started from the condensation of (R)ꢀ11. Thus,
the synthesis of bottromycin analogs devoid of ThiaꢀβꢀAlaꢀOMe was a problematic undertaking.
Consequently, we envisioned the introduction of a Cꢀterminal residue at the end of the analog synthesis.
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2ꢀAzidoꢀ3ꢀphenylbutanoic acid 10 was treated with Cs2CO3 followed by reaction with BnBr to provide the
benzyl ester 31 in 84% yield (Scheme 6). The azide 31 was reduced under Staudinger reaction conditions to
provide the corresponding primary amine 33 in good yield. The 33 was successfully converted to the benzyl
ester analog 49 through an intermolecular amidination of the dipeptide 35 with thioamide 8 and cyclization of
the hexapeptide 48, as described in the above syntheses.
10
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The benzyl ester in 49 could be removed under hydrogenation conditions in the presence of
Pd(OH)2 to afford the carboxylic acid analog 50, which is a key intermediate in the preparation of bottromycin
analogs containing various functions at the Cꢀterminal instead of ThiaꢀβꢀAlaꢀOMe, in 81% yield (Scheme 7).
We set bottromycin A2 (1) as the initial synthetic target to confirm the absolute structure of 50. The carboxylic
acid
50
was
treated
with
(R)ꢀ11
in
the
presence
of
1ꢀ[bis(dimethylamino)methylene]ꢀ1Hꢀ1,2,3ꢀtriazolo[4,5ꢀb]pyridinium 3ꢀoxide hexafluorophosphate (HATU)
and DIPEA in a mixed solvent (CH2Cl2 : DMF = 3 : 1). The reaction proceeded smoothly, providing 1
together with an unexpected bicyclic product 51 in 7% and 80% yield, respectively. Although the yield of 1
was low, the absolute structure of 50 was confirmed as described. To study the reaction mechanism of the
final condensation reaction, 1 was treated under the same condensation conditions. As expected, the reaction
did not proceed and 1 was recovered quantitatively. Carboxylic acid 50 was treated under the same
condensation condition without ThiaꢀβꢀAlaꢀOMe (R)ꢀ11, the reaction being analysed by LCꢀMS. As a result,
starting material 50 was completely consumed and a mass peak identical to the dehydrated activating ester 54
was detected as a major component after stirring for 3 min. This result suggested that the reaction mechanism
was as described in Scheme 8. In this mechanism, the starting carboxylic acid 50 was converted to the
activated ester 52, which was trapped by the internal amide moiety to afford an azlactone 53. The azlactone in
53 was also attacked by internal nucleophilic amidine, followed by isomerization to the more stable imidazole
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to provide a dehydrated product 54. The bicyclic product 51 was obtained following condensation with
(R)ꢀ11.
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49
50
51
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Scheme 7. Total synthesis of bottromycin A2 (1) from a benzyl ester 49
Scheme 8. Plausible reaction mechanism to generate bicyclic product 51
We examined various condensation conditions (Table 2). Use of carbodiimideꢀtype reagents, such
as DCC and EDCI, did not improve the yields (entry 2, 3). Common condensation reagents, such as PyBOP,
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diphenylphosphoryl azide (DPPA), and propylphosphoric acid anhydride (T3P), were not effective and
resulted in a complex mixture (entry 4ꢀ6). We next considered using a protic polar solvent, which may change
the conformation of 50 to prevent its undesired cyclization. Thus, Kunishima’s condensation condition,30
reaction with 4ꢀ(4,6ꢀdimethoxyꢀ1,3,5ꢀtriazinꢀ2ꢀyl)ꢀ4ꢀmethylmorpholinium chloride (DMTꢀMM) in methanol,
was tried (entry 7). Unfortunately, the yield of 1 was not improved and the bicyclic product 51 was obtained
in 80% yield. We also tried azeotropic conditions in the presence of boric acid in toluene (entry 8).31 The
reaction did not proceed at all and the starting material 50 was recovered. We considered that preventing the
10
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formation
of
the
azlactone
53
would
increase
the
yield
of
1.
Therefore,
1ꢀ[(1ꢀ(cyanoꢀ2ꢀethoxyꢀ2ꢀoxoethylideneaminooxy)dimethylaminomorpholino)]uranium hexafluorophosphate
(COMU) was used as the condensation reagent (entry 9).32 However, 1 was not detected at all. Eventually, we
found that the use of an excess amounts of 1ꢀhydroxyꢀ7ꢀazabenzotriazole (HOAt) with HATU was the most
effective way of generating 1 in 22% yield together with 51 in 75% yield (entry 10).
Table 2. Condensation of 50 with (R)ꢀ11
Entry
Reagent
HATU
Solvent
1a
51a
1
2
3
4
CH2Cl2, DMF
7%
80%
DCC, DMAP, CSA CH2Cl2
No reaction
8% Trace
n.d.b 34%
EDCI
CH2Cl2
CH2Cl2
PyBOP
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DPPA
CH2Cl2
Trace 33%
T3P
CH2Cl2, DMF
MeOH
7% Trace
7% 80%
7
DMTꢀMM
B(OH)3
COMU
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11
12
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50
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8
Toluene, 120 oC n.d.b n.d.b
9
CH2Cl2, DMF
CH2Cl2, DMF
n.d.b 97%
22% 75%
10
HATU, HOAt
a Isolation yield. b n.d. = not detected.
Under the optimized reaction conditions, several appropriate amines were condensed with
carboxylic acid 50 to clarify the requirement for ThiaꢀβꢀAlaꢀOMe, Rꢀconfiguration, Cꢀacetyl group and
thiazole group at Cꢀ43 for a preliminary SAR study (Table 3). Condensation with (S)ꢀThiaꢀβꢀAlaꢀOMe 11,
which was prepared from (R)ꢀtꢀbutylsulfinimine 16 (Scheme 2), successfully provided the corresponding
43ꢀepiꢀbottromycin A2 (55), a postulated biosynthetic intermediate of 1.9 Other amines such as benzylamine,
2ꢀaminomethylthiazole, and βꢀalanine methyl ester also provided the corresponding analogs 56ꢀ58, in a
manner similar to the synthesis of 1. In each case, a bicyclic product was also generated.
Table 3. Condensation of 50 with amines
Entry
1
R
Yield (%)
27
2
42
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All synthetic bottromycins and their analogs were evaluated in vitro for their antibacterial activity
against five Gramꢀpositive bacteria [S. aureus FDA209P, S. aureus Smith, MRSA70, MRSA 92ꢀ1191, VRE
(Enterococcus faecalis NCTC12201)], using standard serialꢀdilution techniques specified by National
Committee for Clinical Laboratory Standards (Table 4).33 Synthetic bottromycin A2 (1) displayed potent
antiꢀMRSA and antiꢀVRE activity, as did naturally occurring 1 (MIC 1ꢀ2 ꢀg/mL). Synthetic bottromycin B2
(2), a natural demethyl analog at Cꢀ7, revealed slightly reduced antibacterial activity (MIC 4 ꢀg/mL). The
antibacterial activity of the Cꢀ33ꢀdemethyl analogs 25 and 26 was dramatically reduced (MIC >32 ꢀg/mL).
The Cꢀ33 methyl group is likely to be important in the controlling the threeꢀdimensional structure, as
suggested by the 1H NMR described above (Figure 2). The bicyclic analog 51 and a synthetic intermediate19,
which did not contain a cyclic peptide, did not show notable antibacterial activity, probably due to the
difference of threeꢀdimensional conformation compared with 1. A carboxylic acid analog 50, which did not
contain a Cꢀterminal residue, similarly did not demonstrate any antibacterial properties. It has been reported
that a ThiaꢀβꢀAlaꢀOH analog is devoid of antibacterial activity.13a,14 Therefore, it might be a decrease of
hydrophobicity which led to the reduced antibacterial activity. Unexpectedly, the 43ꢀepiꢀbottromycin A2 (55)
revealed comparable antibacterial activity to 1 (MIC 2 ꢀg/mL). Surprisingly, although the benzylamine analog
56 showed comparable antibacterial activity to 1 (MIC 2 ꢀg/mL against MRSA and VRE strains), the benzyl
ester analog 49 was not very effective (MIC >32 ꢀg/mL). The Cꢀ43ꢀdeacetyl analog 57 and Cꢀ43ꢀdethiazolyl
analog 58 revealed slightly weaker antibacterial activity than 1 (MIC 2ꢀ4 ꢀg/mL for 57, MIC 8 ꢀg/mL for 58).
The promising antibacterial activity of the benzyl amine analog 56 and dethiazolyl analog 58 indicates that the
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thiazole moiety of 1 is not essential for the antiꢀMRSA and antiꢀVRE activity. However, the aromatic
substituent at Cꢀ43 seems to be responsible for potent antibacterial activity. All of the thiazolyl modified
analogs likely adopted the folded structure, as described above, indicated by the characteristic upfield shift of
the valine αꢀproton in 1H NMR spectra.2,7 SAR studies are summarized in figure 4. Changing substituents on
proline, valine and ThiaꢀβꢀAlaꢀOMe has a corresponding impact on the antibacterial activity of 1. These
residues locate on the same side of the threeꢀdimensional structure (Figure 2). Therefore, the binding site of 1
to the 50Sꢀribosome may be on the side constituted of proline, valine and ThiaꢀβꢀAlaꢀOMe. Two sterically
bulky tꢀleucines on the opposite side may not bind to the key receptor but are also important in determining
the folded threeꢀdimensional structure.
10
11
12
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42
43
44
45
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48
49
50
51
52
53
54
55
56
57
58
59
60
Table 4. Minimum inhibitory concentrations (MICs) of the bottromycin derivatives (µg/mL)
S. aureus
S. aureus Smitha
MRSA70b
MRSA 92ꢀ1191b VRE NCTC12201c
FDA209Pa
1d
1e
1
1
1
1
1
2
2
2
1
1
2e
4
4
4
4
4
32
>32
>32
>32
>32
2
>32
>32
32
>32
>32
2
>32
>32
32
>32
>32
2
>32
>32
>32
>32
>32
2
32
>32
32
>32
>32
2
25
26
49
50
51
55
56
57
2
4
2
2
2
4
4
4
4
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58
VCMf
2
0.5
>128
7
8
9
a
b
Staphylococcus aureus FDA209P and Smith: susceptible strains. MRSA70 and 92ꢀ1191: MRSA strains
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c
d
isolated from clinical patients. Enterococcus faecalis NCTC12201: encoded by van A gene. Naturally
occurring compound. e Synthetic compounds. f Vancomycin.
Figure 4. Summary of SAR of 1.
Conclusions
In conclusion, we achieved the first total synthesis of bottromycin A2 (1) through a
diastereoselective Mannich reaction, mercuryꢀmediated amidination of thioamide and amine, and cyclization
of the tetrapeptide including amidine. Based on this synthetic process, we synthesized bottromycin B2 (2),
Cꢀ33ꢀdemethylꢀbottromycin A2 (25), and Cꢀ33ꢀdemethylꢀbottromycin B2 (26). In addition, we developed a
new synthetic route to introduce a Cꢀterminal residue at the end of the analog synthesis. This method allowed
us to prepare several bottromycin analogs containing several functions instead of ThiaꢀβꢀAlaꢀOMe. All
synthetic bottromycins and related analogs were subjected to in vitro evaluation of their antibacterial
characteristics. It was found that the featured threeꢀdimensional structure and Cꢀ33ꢀmethyl group of
bottromycins was essential for their antibacterial activity. The ThiaꢀβꢀAlaꢀOMe moiety could be replaced with
other aromatic compounds without losing the antibacterial activity. Notably, a new analog devoid of the
methyl ester, which is known to lower in vivo efficacy of bottromycins, showed comparable activity to that of
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vancomycin. Therefore, the newly developed synthetic route will likely be important for further SAR studies
focused on the Cꢀterminal residue. These findings offer the possibility of accelerating the development of a
new class of antibiotics, particularly with potential to combat multidrugꢀresistant bacteria.
10
11
12
13
14
15
16
17
18
19
20
21
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24
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26
27
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50
51
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60
EXPERIMENTAL SECTION
General Information
All reagents and solvents were purchased from commercial suppliers and were used without further
purification. Flash chromatography was carried out with Kanto Chemical silica gel (Kanto Chemical Co., Inc.,
silica gel 60N, spherical neutral, 0.040ꢀ0.050 mm). Preparative thinꢀlayer chromatography (Prep. TLC) was
carried out with preꢀcoated silica gel plates with a fluorescent indicator (Merck 60 F254 0.25 or 0.50 mm,
Merck KGaA). Nuclear magnetic resonance (NMR) spectra were determined using the JEOL JNMꢀECAꢀ500
(1H NMR (500 MHz), 13C NMR (125 MHz)) spectrometer. The chemical shifts are expressed in ppm
referenced to the residual solvent peaks of CDCl3 (7.26 ppm, 1H NMR) and CD3OD (3.31 ppm, 1H NMR) and
coupling constant (J values) are given in Hertz. Chemical shifts for 13CꢀNMR were reported in ppm relative to
the center line at 77.0 ppm (CDCl3) and 49.0 ppm (CD3OD). All infrared (IR) spectra were measured on a
Horiba FTꢀ210 spectrometer and were reported in wavenumbers (cmꢀ1). Optical rotations were measured with
a Jasco P1010 polarimeter. Melting points were measured using the micro melting point apparatus (Yanaco
New Science Inc., MPꢀS3). Highꢀresolution mass spectra (HRMS) were measured on a Micromass LCT
spectrometer with a timeꢀof flight (TOF) analyzer.
(R)-N-{N-tert-butoxycarbonyl-
L-prolyl-L-valyl-[L-(2-tert-butyldiphenylsilyloxyethyl)imino-tert-le
ucyl]-
L
-tert-leucyl-(erythro-3-methyl- -phenylalanyl)}-3-amino-3-(thiazol-2-yl)-propanoic acid methyl ester
L
(27). To a solution of 20 (368 mg, 0.35 mmol) in CH2Cl2 (4.0 mL) was added TFA (2.5 mL) at 0 oC under an
argon atmosphere. After being stirred at room temperature for 2 h, the reaction mixture was concentrated
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under reduced pressure. The residue was dissolved in ethyl acetate, then basified by 30% aqueous NH3 at 0 oC,
after which the mixture was extracted with ethyl acetate (10 mL x 3). The combined organic extracts were
washed with brine (30 mL), dried over Na2SO4, and concentrated under reduced pressure. The crude product
was used for the next reaction without further purification. To a solution of crude mixture and BocꢀProꢀOH
(83.0 mg, 0.39 mmol) in CH2Cl2 (3.5 mL) was added DIPEA (183 ꢁL, 1.05 mmol) and HBTU (172 mg, 0.46
mmol) in DMF (1.8 mL) at 0 oC under an argon atmosphere. After being stirred at room temperature for 10 h,
the reaction mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (20
mL), followed by washing with 10% aqueous citric acid (20 mL), saturated aqueous NaHCO3 (20 mL) and
brine (40 mL), then dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by
flash column chromatography on silica gel (Hexane/CHCl3 = 2/1 to 2/3) to give heptapeptide 27 (282 mg,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
70% in 2 steps) as white solid. The H and 13C NMR signals of 27 were broad and complicated due to the
existence of amidine tautomers and rotamers. Therefore, NMR data assignments are not given for 27. The
actual NMR spectra are shown in Supporting Information Sꢀ7 and Sꢀ8, respectively. IR (Diamond prism) 3278,
2954, 2870, 1651, 1512, 1396, 1257, 1165 cmꢀ1; HRMS (ESIꢀTOF) m/z [M+H]+ calcd for [C62H91N8O9S1Si]+
1151.6399, found 1151.6406; [α]D24 −70.3 (c 1.0, CHCl3); mp 167 oC.
(R)-N-{N-tert-butoxycarbonyl-
L-prolyl-L-valyl-[L-(2-hydroxyethyl)imino-tert-leucyl]-L-tert-leucyl
-(erythro-3-methyl- -phenylalanyl)}-3-amino-3-(thiazol-2-yl)propanoic acid methyl ester (28). To a solution
L
of 27 (261 mg, 0.226 mmol) in THF (2.3 mL) was added TBAF (1.0 M in THF, 454 ꢁL, 0.454 mmol) at room
temperature under an argon atmosphere. After being stirred at the same temperature for 3 h, the reaction
mixture was quenched with saturated aqueous NH4Cl (6.0 mL). The mixture was extracted with ethyl acetate
(10 mL) and washed with brine (20 mL), then dried over Na2SO4 and concentrated under reduced pressure.
The residue was purified by flash column chromatography on silica gel (CHCl3/MeOH = 9/1) to give 28 (205
1
mg, 99%) as a white solid. The H and 13C NMR signals of 28 were broad and complicated due to the
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existence of amidine tautomers and rotamers. Therefore, NMR data assignments are not given for 28. The
actual NMR spectra are shown in Supporting Information Sꢀ9 and Sꢀ10, respectively. IR (Diamond prism)
3302, 2962, 2360, 1658, 1512, 1396, 1165, 702 cmꢀ1; HRMS (ESIꢀTOF) m/z [M+H]+ calcd for
[C46H73N8O9S1]+ 913.5221, found 913.5220; [α]D23 −67.5 (c 1.0, CHCl3); mp 128ꢀ130 oC.
10
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12
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27
28
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(R)-N-[N-tert-Butoxycarbonyl-L-prolyl-L-valyl-(L-carboxymethylimino-tert-leucyl)-L-tert-leucyl-(
erythro-3-methyl-
L-phenylalanyl)]-3-amino-3-(thiazol-2-yl)propanoic acid methyl ester (29). To a solution
o
of 28 (50.0 mg, 54.8 ꢁmol) in acetone (550 ꢁL) was added dropwise Jones reagent (2.8 M, 824 ꢁL) at 0 C.
After being stirred at the same temperature for 25 min, the reaction was quenched with 2ꢀpropanol (0.3 mL)
and neutralized with saturated aqueous NaHCO3 (8 mL). The resulting mixture was extracted with ethyl
acetate (10 mL x 3) and washed with brine (10 mL), then dried over anhydrous Na2SO4 and concentrated
under reduced pressure. The residue was purified by preparative TLC (CHCl3/MeOH/AcOH = 9:1:0.1,
elution: CHCl3/MeOH = 9/1) to give carboxylic acid 29 (16.3 mg, 32%) as a white solid: 1H NMR (500 MHz,
CD3OD) δ 7.72 (d, J = 3.3 Hz, 1H), 7.51 (d, J = 3.3 Hz, 1H), 7.30ꢀ7.15 (4H), 7.10 (m, 1H), 5.77 (m, 1H), 4.87
(m, 1H), 4.57 (d, J = 11.0 Hz, 1H), 4.44 (m, 1H), 4.37 (brs, 1H), 4.27 (dd, J = 8.3, 3.3 Hz, 1H), 4.07 (s, 1H),
3.70 (s, 3H), 3.70ꢀ3.63 (1H), 3.55ꢀ3.43 (2H), 3.38 (m, 1H), 3.27 (dd, J = 16.4, 4.8 Hz, 1H), 3.20ꢀ3.10 (2H),
3.02 (dd, J = 16.4, 9.5 Hz, 1H), 2.24ꢀ1.77 (4H), 1.48ꢀ1.34 (6H), 1.26 (d, J = 6.5 Hz, 3H), 0.98 (s, 9H), 0.91 (s,
9H); The 13C NMR signals of 29 were broad and complicated due to the existence of amidine tautomers and
rotamers. Therefore, 13C NMR data assignments are not given for 29. The actual 13CꢀNMR spectra are shown
in Supporting Information Sꢀ12. IR (Diamond prism) 3302, 2962, 2330, 1651, 1520, 1381, 1165, 756 cmꢀ1;
HRMS (ESIꢀTOF) m/z [M+Na]+ calcd for [C46H70N8NaO10S1]+ 949.4833, found 949.4831; [α]D23 −55.4 (c 1.3,
CHCl3); mp 119 oC.
Bottromycin B2 (2). A solution of 29 (21 mg, 22.6 ꢁmol) in 4 M HCl/dioxane (1.0 mL) was stirred
at room temperature under a nitrogen atmosphere for 1 h. The reaction mixture was concentrated under
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reduced pressure and the residue was azeotroped with toluene to afford the crude product. To a solution of
crude mixture in CH2Cl2 (12 mL) was added DIPEA (16 ꢁL, 92 ꢁmol) and EDCI•HCl (44 mg, 0.230 mmol) at
room temperature under an argon atmosphere. After being stirred at the same temperature for 10 h, the
reaction mixture was concentrated under reduced pressure. The residue was dissolved in EtOAc (10 mL). The
solution was washed with H2O (5 mL) and brine (5 mL), then dried over anhydrous Na2SO4 and concentrated
under reduced pressure. The residue was purified by preparative TLC (CHCl3/MeOH /30% aqueous NH3 =
20/1/0.1, elution: CHCl3/MeOH = 10/1) to give 2 (3.5 mg, 19%) as a white solid: 1H NMR (500 MHz, CDCl3)
δ 7.66 (d, J = 3.3 Hz, 1H), 7.47 (d, J = 6.0 Hz, 1H), 7.36 (d, J = 7.5 Hz, 2H), 7.32 (t, J = 7.5 Hz, 2H), 7.24 (d,
J = 3.3 Hz, 1H), 7.21 (m, 1H), 7.10 (d, J = 10.5 Hz, 1H), 6.92 (d, J = 8.1 Hz, 1H), 6.73 (m, 1H), 5.61 (m, 1H),
4.87 (dd, J = 8.1, 4.2 Hz, 1H), 4.62 (d, J = 10.8 Hz, 1H), 4.21 (dd, J = 9.5, 2.1 Hz, 1H), 4.05 (app t, 1H), 3.91
(s, 1H), 3.83 (dd, J = 12.2, 4.1 Hz, 1H), 3.68 (s, 3H), 3.75ꢀ3.59 (2H), 3.40 (m, 1H), 3.01 (dd, J = 16.9, 6.5 Hz,
1H), 2.86 (dd, J = 16.9, 5.5 Hz, 1H), 2.78 (m, 1H), 2.42 (m, 1H), 2.31 (m, 1H), 2.02ꢀ1.91 (2H), 1.34 (d, J =
7.1 Hz, 3H), 0.99 (s, 9H), 0.95 (s, 9H), 0.75 (d, J = 6.5 Hz, 3H), 0.73 (d, J = 6.5 Hz, 3H); 13C NMR (125 MHz,
CDCl3) δ 176.7, 172.6, 171.7, 171.1, 170.5, 169.6, 169.1, 157.2, 142.7, 141.3, 128.6 (2C), 128.1 (2C), 127.1,
119.9, 70.3, 68.4, 61.1, 57.5, 54.0, 52.1, 48.1, 48.0, 47.5, 41.6, 39.2, 35.4, 32.9 (2C), 27.6 (6C), 26.9, 22.8,
19.5 (2C), 15.4; IR (Diamond prism) 3286, 2962, 1735, 1643, 1504, 1365, 1226, 1164 cmꢀ1; HRMS
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59
60
23
(ESIꢀTOF) m/z [M+Na]+ calcd for [C41H60N8NaO7S1]+ 831.4203, found 831.4214; [α]D −57.7 (c 0.20,
CHCl3); mp 140ꢀ143 oC.
(R)-N-(N-tert-Butoxycarbonyl-phenylalanyl)-3-amino-3-(thiazol-2-yl)propanoic acid methyl
ester (30). To a solution of ThiaꢀβꢀAlaꢀOH (R)ꢀ11 (760 mg, 4.09 mmol) and BocꢀPheꢀOH (1.08 g, 4.41 mmol)
in CH2Cl2 (41 mL) was added DIPEA (2.20 mL, 12.3 mmol) and PyBOP (2.76 g, 5.31 mmol) at 0 oC under an
argon atmosphere. After being stirred at room temperature for 2 h, the reaction mixture was concentrated
under reduced pressure. The residue was dissolved in EtOAc (100 mL). The solution was washed with 10%
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aqueous citric acid (40 mL), saturated aqueous NaHCO3 (50 mL), H2O (50 mL) and brine (50 mL), then dried
over anhydrous Na2SO4, and concentrated under reduced pressure. The crude product was purified by flash
column chromatography on silica gel (hexane/EtOAc = 2/1 to 1/1) to give 30 (1.77 g, quant.) as a white solid:
1H NMR (500 MHz, CDCl3) δ 7.64 (d, J = 3.5 Hz, 1H), 7.31ꢀ7.26 (2H), 7.25ꢀ7.19 (4H), 5.62 (dd, J = 5.2, 4.3
Hz, 1H), 4.41 (m, 1H), 3.59 (s, 3H), 3.15 (dd, J = 17.0, 4.3 Hz, 1H), 3.15 (m, 1H), 3.05 (dd, J = 14.0, 7.5 Hz,
1H), 2.67 (dd, J = 17.0, 5.2 Hz, 1H), 1.41 (s, 9H); 13C NMR (125 MHz, CDCl3) δ 171.3, 170.6, 170.0, 155.2,
142.4, 136.5, 129.3 (2C), 128.7 (2C), 127.0, 119.5, 80.2, 55.9, 51.8, 47.1, 38.4, 37.5, 28.2 (3C); IR (Diamond
prism) 3262, 2946, 1743, 1697, 1658, 1519, 1241, 1164 cmꢀ1; HRMS (ESIꢀTOF) m/z [M+Na]+ calcd for
[C21H27N3NaO5S1]+ 456.1569, found 456.1599; [α]D17 −18.8 (c 0.68, CHCl3). mp: 110ꢀ112 oC.
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(R)-N-[(N-tert-Butoxycarbonyl-L-tert-leucyl)-L-phenylalanyl]-3-amino-3-(thiazol-2-yl)propanoic
acid methyl ester (34). To a solution of 30 (1.30 g, 3.00 mmol) in EtOAc (5.0 mL) was added 4 M
HCl/EtOAc (15 mL) at room temperature. After being stirred at the same temperature for 30 min, the reaction
mixture was concentrated under reduced pressure. The residue was azeotroped with toluene. The crude
product was used for the next reaction without further purification. To a solution of crude product and
BocꢀtꢀLeuꢀOH (760 mg, 3.00 mmol) in CH2Cl2 (15 mL) was added DIPEA (1.00 mL, 5.98 mmol) and HBTU
o
(1.46 g, 3.88 mmol) in DMF (15 mL) at 0 C under an argon atomosphere. After being stirred at room
temperature for 2 h, the reaction mixture was concentrated under reduced pressure. The residue was dissolved
in EtOAc (50 mL). The solution was washed with 10% aqueous citric acid (30 mL), saturated aqueous
NaHCO3 (30 mL), H2O (30 mL) and brine (30 mL), then dried over anhydrous Na2SO4 and concentrated
under reduced pressure. The crude product was purified by flash column chromatography on silica gel
1
(hexane/EtOAc = 2/1) to give 34 (1.51 g, 93% in 2 steps) as a white amorphous solid: H NMR (500 MHz,
CDCl3) δ 7.61 (d, J = 3.0 Hz, 1H), 7.29ꢀ7.24 (2H), 7.23ꢀ7.18 (4H), 7.11 (brd, J = 8.0 Hz, 1H), 6.48 (m, 1H),
5.59 (m, 1H), 5.19 (brd, J = 7.5 Hz, 1H), 4.69 (m, 1H), 3.84 (d, J = 8.5 Hz, 1H), 3.59 (s, 3H), 3.16 (dd, J =
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13.5, 6.0 Hz, 1H), 3.07 (dd, J = 17.0, 4.8 Hz, 1H), 3.00 (dd, J = 13.5, 8.8 Hz, 1H), 2.63 (dd, J = 17.0, 5.5 Hz,
1H), 1.39 (s, 9H), 0.94 (s, 9H); 13C NMR (125 MHz, CDCl3) δ 171.0, 170.6, 170.0, 169.8, 155.8, 142.3, 136.3,
129.2 (2C), 128.7 (2C), 127.0, 119.5, 79.8, 62.6, 54.6, 51.8, 47.2, 38.5, 37.4, 34.4, 28.3 (3C), 26.5 (3C); IR
(Diamond prism) 3286, 2962, 1735, 1643, 1504, 1365, 1226, 1164 cmꢀ1; HRMS (ESIꢀTOF) m/z [M+Na]+
calcd for [C27H38N4NaO6S1]+ 569.2409, found 569.2425; [α]D23 −23.1 (c 1.0, CHCl3).
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(R)-N-{[N-Phthaloyl-L-(2-tert-butyldiphenylsilyloxyethyl)imino-tert-leucyl]-L-phenylalanyl}-3-a
mino-3-(thiazol-2-yl)propanoic acid methyl ester (36). To a solution of 34 (1.14 g, 2.09 mmol) in dioxane
(3.0 mL) was added 4 M HCl/dioxane (15 mL) at room temperature. After being stirred at the same
temperature for 1 h, the reaction mixture was diluted with EtOAc basified by 30% aqueous NH3 at 0 oC. The
mixture was extracted with EtOAc (10 mL x 3) washed with brine (30 mL), dried over anhydrous Na2SO4,
and concentrated under reduced pressure. The crude product was used for the next reaction without further
purification. To a solution of crude product and thioamide 8 (970 mg, 1.74 mmol) in CH3CN (20 mL) was
added 2,6ꢀlutidine (810 ꢀL, 6.96 mmol) and Hg(OTf)2 (1.03 g, 2.09 mmol) at room temperature under an
argon atmosphere. After being stirred at the same temperature for 1 h, the reaction was quenched with 5%
aqueous Na2S2O3 (8.0 mL), filtered through a pad of Celite. The filter cake was washed with EtOAc (40 mL)
and the filtrate was washed with 1 M aqueous HCl (20 mL), saturated aqueous NaHCO3 (20 mL) and brine
(20 mL). The organic solution was dried over anhydrous Na2SO4 and concentrated under reduced pressure.
The residue was purified by flash column chromatography on silica gel (CHCl3/MeOH = 100/1 to 60/1) to
1
give 36 (1.74 g, 90%) as a colorless amorphous solid. The H and 13C NMR signals of 36 were broad and
complicated due to the existence of amidine tautomers and rotamers. Therefore, NMR data assignments are
not given for 36. The actual NMR spectra are shown in Supporting Information Sꢀ19 and Sꢀ20, respectively.
IR (neat) 3382, 3070, 2958, 2860, 1712, 1497, 1384, 1110 cmꢀ1; HRMS (ESIꢀTOF) m/z [M+Na]+ calcd for
[C54H66N6NaO7S1Si]+ 993.4381, found 993.4366; [α]D25 −96.2 (c 1.00, CHCl3).
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(R)-N-{N-tert-Butoxycarbonyl-L-valyl-[L-(2-tert-butyldiphenylsilyloxyethyl)imino-tert-leucyl]-L-p
5
6
7
8
henylalanyl}-3-amino-3-(thiazol-2-yl)propanoic acid methyl ester (38). To a solution of 36 (970 mg, 1.00
mmol) in EtOH (10 mL) was added N2H4•H2O (145 ꢁL, 3.00 mmol) at room temperature under an argon
atmosphere. After being stirred at 70 ºC for 2 h, the reaction mixture was concentrated under reduced pressure.
The residue was dissolved in EtOAc (30 mL) then the solution was washed with 15% aqueous NH3 (20 mL),
H2O (20 mL), and brine (20 mL) then dried over anhydrous Na2SO4 and concentrated under reduced pressure
to afford the crude product. To a solution of the crude mixture (800 mg, 0.951 mmol) and BocꢀValꢀOH (227
mg, 1.04 mmol) in CH2Cl2 (5.0 mL) was added DIPEA (415 ꢁL, 2.37 mmol) and HBTU (467 mg, 1.23 mmol)
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º
in DMF (5.0 mL) at 0 C under an argon atmosphere. After being stirred at room temperature for 3 h, the
reaction mixture was concentrated under reduced pressure. The residue was dissolved in EtOAc (30 mL). The
solution was washed with 10% aqueous citric acid (20 mL), a saturated aqueous NaHCO3 (20 mL) and brine
(40 mL), then dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was
purified by flash column chromatography on silica gel (CHCl3/MeOH = 150/1 to 80/1) to give 38 (791 mg,
78% in 2 steps) as a white amorphous solid. The 1H and 13C NMR signals of 38 were broad and complicated
due to the existence of amidine tautomers and rotamers. Therefore, NMR data assignments are not given for
38. The actual NMR spectra are shown in Supporting Information Sꢀ21 and Sꢀ22, respectively. IR (Diamond
prism) 2954, 2861, 1735, 1635, 1504, 1241, 1164, 1103 cmꢀ1; HRMS (ESIꢀTOF) m/z [M+Na]+ calcd for
[C56H81N7NaO8S1Si]+ 1062.5534, found 1062.5515; [α]D24 −52.3 (c 1.00, CHCl3).
(R)-N-{N-tert-Butoxycarbonyl-(cis-3-methyl-L-prolyl)-
L-valyl-[L-(2-tert-butyldiphenylsilyloxyeth
yl)imino-tert-leucyl]- -tert-leucyl- -phenylalanyl}-3-amino-3-(thiazol-2-yl)propanoic acid methyl ester (40).
L
L
To a solution of 38 (320 mg, 0.308 mmol) in CH2Cl2 (1.5 mL) was added TFA (300 ꢁL) at 0 ºC under an argon
atmosphere. After being stirred at room temperature for 3 h, the reaction mixture was concentrated under
reduced pressure. The residue was dissolved in EtOAc (10 mL), then neutralized with 10% aqueous NH3 (10
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mL) and the organic layer separated. The aqueous phase was extracted with EtOAc (10 mL x 3). The
combined organic extracts were washed with brine (30 mL), dried over anhydrous Na2SO4 and concentrated
under reduced pressure to afford the crude product. To a solution of the crude product and
Bocꢀcisꢀ3ꢀmethylproline 6 (78.0 mg, 0.339 mmol) in CH2Cl2 (1.5 mL) was added DIPEA (160 ꢁL, 0.921
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mmol) and HBTU (151 mg, 0.401 mmol) in DMF (1.5 mL) at 0 C under an argon atmosphere. After being
stirred at room temperature for 1 h, the reaction mixture was concentrated under reduced pressure. The residue
was dissolved in EtOAc (20 mL). The solution was washed with 10% aqueous citric acid (20 mL), a saturated
aqueous NaHCO3 (20 mL) and brine (40 mL), then dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The residue was purified by flash column chromatography on silica gel (CHCl3/MeOH =
100/1 to 60/1) to give 40 (265 mg, 75% in 2 steps) as a white solid. The 1H and 13C NMR signals of 40 were
broad and complicated due to the existence of amidine tautomers and rotamers. Therefore, NMR data
assignments are not given for 40. The actual NMR spectra are shown in Supporting Information Sꢀ23 and
Sꢀ24, respectively. IR (Diamond prism) 3301, 2962, 2877, 1743, 1666, 1511, 1365, 1110 cmꢀ1; HRMS
(ESIꢀTOF) m/z [M+Na]+ calcd for [C62H90N8NaO9S1Si]+ 1173.6218, found 1173.6226; [α]D25 −44.1 (c 1.00,
CHCl3); mp 90ꢀ92 oC.
(R)-N-{N-tert-Butoxycarbonyl-(cis-3-methyl-
L-prolyl)-L-valyl-[L-(2-hydroxyethyl)imino-tert-leuc
yl]-
L
-tert-leucyl- -phenylalanyl}-3-amino-3-(thiazol-2-yl)propanoic acid methyl ester (43). To a solution of
L
40 (330 mg, 0.286 mmol) in THF (3.0 mL) was added TBAF (1.0 M in THF, 573 ꢁL, 0.573 mmol) at room
temperature under an argon atmosphere. After being stirred at the same temperature for 2 h, the reaction was
quenched with saturated aqueous NH4Cl (6.0 mL). The mixture was extracted with EtOAc (10 mL) and
washed with brine (20 mL), then dried over anhydrous Na2SO4 and concentrated under reduced pressure. The
residue was purified by flash column chromatography on silica gel (CHCl3/MeOH = 80/1 to 10/1) to give 43
(243 mg, 93%) as white solid. The H and 13C NMR signals of 43 were broad and complicated due to the
1
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