Mechanistic analysis of muraymycin analogues: A guide to the design of MraY inhibitors

Article

pubs.acs.org/jmc

Mechanistic Analysis of Muraymycin Analogues: A Guide to the

Design of MraY Inhibitors

Tetsuya Tanino,^†Bayan Al-Dabbagh,^‡,∥Dominique Mengin-Lecreulx,^‡Ahmed Bouhss,^‡Hiroshi Oyama,^§

Satoshi Ichikawa,*^,†and Akira Matsuda^†

^†Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan

^‡Laboratoire des Enveloppes Bacter

̂

8619 CNRS, Universite Paris-Sud, Batiment 430, Orsay F-91405, France

́ ́

iennes et Antibiotiques, Institut de Biochimie et Biophysique Moleculaire et Cellulaire, UMR

́

^§Shionogi Innovation Center for Drug Discovery, Shionogi & Co., Ltd., Kita-21, Nishi-11, Kita-ku, Sapporo 001-0021, Japan

S

* Supporting Information

ABSTRACT: The systematic structure−activity relationship (SAR) of the muraymycins (MRYs) using an Ugi four-component

reaction (U4CR) was investigated. The impact of the lipophilic substituent on antibacterial activity was significant, and the

analogues 8 and 9 having a lipophilic side chain exhibited good activity against a range of Gram-positive bacterial pathogens,

including MRSA and VRE. Further investigation of compounds 8 and 9 revealed these analogues to be selective inhibitors of the

MraY transferase and nontoxic to HepG2 cells. The SAR of the accessory urea−peptide moiety indicated that it could be

simplified. Our SAR study of the MRYs suggests a probable mechanism for inhibition of the MraY, where the inner moiety of the

urea−dipeptide motif interacts with the carbohydrate recognition domain in the cytoplasmic loop 5. The predicted binding

model would provide further direction toward the design of potent MraY inhibitors. This study has set the stage for the

generation of novel antibacterial “lead” compounds based on MRYs.

MRYs in S. aureus infected mice represents a promising lead for

the development of new antibacterial agents. The extensive use

of antibiotics has raised a serious global public health problem.

Since bacterial pathogens inevitably develop resistance to every

new drug launched in the clinic, the need for new antibiotics to

counteract drug-resistant bacteria such as methicillin-resistant

Staphylococcus aureus (MRSA) and vancomycin-resistant Staph-

ylococcus aureus (VRSA) is critical.⁵Although two new classes

of antibiotics have been launched in the clinic since 2000 to

treat these infections, clinical resistance to both has already

emerged.^6,7In choosing novel antibacterial agents to address

this problem, the target must be essential for growth, the agent

different from existing drugs, and the initial “hit” scaffold

amenable to structural changes that allow for optimization of

the potency and efficacy to generate “lead” compounds.⁸⁻¹⁰

The MRYs inhibit the formation of lipid II and peptidoglycan

INTRODUCTION

■

Natural products are still a rich source for drug development;¹

however, some biologically relevant natural products possess

rather large, complex, or labile chemical structures compared to

synthetic drugs, which limits chemical modification in a process

pursuing a structure−activity relationship (SAR). One of the

solutions to these drawbacks is function-oriented synthesis

(FOS),²namely, the design of less complex targets with

comparable or superior function that could be made in a

practical and even synthetically novel manner. Although

challenging, simpler scaffold designs would provide a practical

synthesis of a set of analogues and synthetic innovation in

current medicinal chemistry.

The muraymycins (MRYs) (Figure 1, 1), isolated from a

culture broth of Streptomyces sp.,³are members of a class of

naturally occurring 6′-N-alkyl-5′-β-O-aminoribosyl-C-glycyluri-

dine antibiotics.⁴The MRYs, having a lipophilic side chain,

have been shown to exhibit excellent antimicrobial activity

against Gram-positive bacteria. In particular, the efficacy of the

Received: July 11, 2011

Published: November 15, 2011

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tide (lipid I) with the subsequent release of UMP.^13,14Since

MraY is an essential enzyme in bacteria,¹⁵it is a potential target

for the development of general antibacterial agents. Because of

these promising biological properties, the MRYs have become

intriguing, challenging synthetic targets.¹⁶Recently, we have

accomplished the first total synthesis of MRY D2 (Figure 3, 1d)

Figure 3. Structures and biological activity of muraymycin D2 and its

epimer.

Figure 1. Structures of muraymycins (MRYs).

and are believed to be inhibitors of phospho-MurNAc-

pentapeptide transferase (MraY), which is responsible for the

formation of lipid I in the peptidoglycan biosynthesis pathway

(Figure 2).¹¹⁻¹⁴The enzymes involved in the biosynthesis of

peptidoglycan are essential for bacterial life and represent

important targets for antibacterial chemotherapy. The MraY

transferase catalyzes the transfer of the phospho-MurNAc-

pentapeptide motif from UDP-MurNAc-pentapeptide onto a

lipid carrier, undecaprenyl phosphate (C₅₅-P), leading to the

formation of undecaprenylpyrophosphoryl-MurNAc-pentapep-

and its epimer (epi-1d).¹⁷However, when 1d and epi-1d were

subjected to a range of Gram-positive bacteria up to 64 μg/mL,

they showed no antibacterial activity whereas against their

target enzyme MraY (IC₅₀= 0.01 and 0.09 μM), respectively,

exhibited potent inhibitory activity. Herein we describe the full

details of our efforts to increase the antibacterial activity and to

simplify the chemical structure based on the FOS of MRYs, as

well as our discovery of simplified analogues that proved to be

effective against a range of bacteria including drug-resistant

Figure 2. Biosynthesis of peptidoglycan precursor.

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Figure 4. Two-directional optimization via U4CR.

strains. Our SAR study provides an insight into a binding model

of the MRYs with the target MraY. The study of the lipophilic

side chain has recently been reported,¹⁸and its details are also

described.

urea−peptide moiety is necessary to pursue FOS. Ideally we

hope to optimize multiple sites by a common synthetic strategy

to chemically supply the analogues. We have accomplished the

total synthesis of 1d and epi-1d,¹⁷which features the

convergent assemblage of isovaleraldehyde (2a), the urea−

dipeptide carboxylic acid 3a, 2,4-dimethoxybenzylamine, and

the isonitrile derivative of the aminoribosyluridine 4 by an Ugi

four-component reaction²²(U4CR) as shown in Figure 4.

Basically, the U4CR is nonstereoselective at the newly formed

stereogenic center, resulting in a mixture of products. From a

medicinal chemistry point of view, this strategy works well for

examining the structure−activity relationship. Thus, the U4CR

gives us two diastereomers, which are useful compounds in

order to examine the structure−activity relationship. In

conjunction with the nature of the multicomponent assem-

blage, this strategy allows us to diversify accessible analogues

simply by altering each component. We planned a two-

directional optimization on both the lipophilic side chain and

the urea−peptide moieties (Figure 4).

Investigation of the Lipophilic Side Chain. The

precursor aldehydes 2c−e, which all contain a biphenyl moiety,

were prepared by a conventional method as shown in Scheme

S1 of Supporting Information. With the components for the

U4CR in hand, we started the optimization from the lipophilic

moiety (Figure 4, left). Because the β-acyloxyleucine moiety

found in the parent MRY A and B classes could be susceptible

to β-elimination or hydrolysis by enzymes such as esterases, we

therefore designed and synthesized analogues such as 8 and 9,

which were linked to a hydrophobic substituent on the MRY

core structure via a C−C bond, as chemically and biologically

stable isosteres of the MRYs (Scheme 1). The molecular design

RESULTS AND DISCUSSION

■

Two-Directional Optimization. The structure of the

muraymycins can be characterized by three components that

are responsible for their biological activity: the amino-

ribosyluridine, the lipophilic side chain, and the accessory

urea−peptide moieties. Previous studies by us¹⁹and others²⁰

associated with antibacterial nucleoside natural products

indicated that the aminoribosyluridine moiety is the essential

structural feature that interacts with the active site of the MraY

enzyme. That the lipophilic side chain is not essential for MraY

inhibition but rather for antibacterial activity clearly indicates

that the side chain contributes to the membrane permeability of

the molecule because MraY is an integral membrane protein

and its active site exists on the cytoplasmic side of the

membrane. Since the lipid bilayer of the cytoplasmic membrane

is thought to be a common barrier in bacteria, a lipophilic side

chain is necessarily attached to the MRY D2 in order to acquire

antibacterial activity. Incorporating a lipophilic side chain onto

molecules that inhibit bacterial cell wall synthesis is a known

concept.²¹However, little is known about the structure−

activity relationship of the lipophilic moiety. The urea−peptide

moiety, the third structural component, is regarded as an

accessory motif linking the aminoribosyluridine moiety to the

lipophilic moiety. Its role and structural requirement for the

biological activity are totally unknown. Therefore, an

investigation of the lipophilic side chain as well as the accessory

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Scheme 1

a

Table 1. Inhibitory Activities of the Synthesized Compounds against MraY

IC₅₀(μM)

mode of inhibition

MraY

WecA

K_i(nM)

UDP-MurNAc-pentapeptide

C₅₅-P

1d

0.01

0.09

0.33

0.74

1230

1560

970

7.6

49

competitive

noncompetitive

epi-1d

8b

247

698

9b

1340

a

The activities of the compounds were tested against purified MraY from B. subtilis. The assay was performed in a reaction mixture of 10 μL

containing, in final concentrations, 100 mM Tris-HCl, pH 7.5, 40 mM MgCl₂, 1.1 mM C₅₅-P, 250 mM NaCl, 0.25 mM UDP-MurNAc-

[¹⁴C]pentapeptide (337 Bq), and 8.4 mM N-lauroylsarcosine. The mixture was incubated for 30 min at 37 °C. The radiolabeled substrate UDP-

MurNAc-pentapeptide and reaction product (lipid I, product of MraY) were separated by TLC on silica gel plates. The radioactive spots were

located and quantified with a radioactivity scanner. IC₅₀values were calculated with respect to a control assay without the inhibitor. Data represent

the mean of independent triplicate determinations. Lineweaver−Burk plots were used to determination of the K_ivalues and the type of inhibition

assuming a ping-pong Bi Bi kinetic mechanism as proposed previously by Heydanek et al.²⁶

was easily transferred to the analogue synthesis by the use of

the simple aldehydes shown in Scheme S1 in the U4CR. Thus,

2b, 3a,¹⁷5, and 6¹⁷were admixed in EtOH to give 7b

containing a pentadecylglycine residue in its structure in 37%

yield as a 1:1 mixture of diastereomers. Deprotection of 7b was

achieved by a two-step sequence (Zn, 1 M aqueous NaH₂PO₄,

THF, then 80% aqueous TFA) to afford the hydrophobic

analogue 8b and its diastereomer 9b, which were easily

separated by reverse-phase HPLC. The newly formed stereo-

genic center at the pentadecylglycine residue of each

diastereomer was determined by conventional amino acid

analysis²³using L- or D-pentadecylglycine as the reference

compound (see Supporting Information). Analogues incorpo-

rating a biphenyl moiety at the end (8c and 9c), in the middle

(8d and 9d), and at the junction (8e and 9e) of the lipophilic

moiety were also synthesized. In HPLC analysis, a similar

behavior was observed between compounds with the R-

configuration (1d and 8b, having shorter retention times)

and those with the S-configuration (epi-1d and 9b, having

longer retention times) at the newly formed stereogenic center

in the U4CR. According to the relative retention time in the

HPLC analysis, the absolute stereochemistries of 8c−e and 9c−

e were tentatively assigned to be the R- and S-configurations,

respectively.

First, the inhibitory activity of 8b and 9b on the purified

MraY enzyme (B. subtilis) was examined by quantifying the

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Table 2. Antibacterial Activity of Lipophilic Side Chain Analogues

a

MIC (μg/mL)

S. aureus

ATCC 29213 (MSSA)

E. faecalis

E. faecium

compd

1d

SR3637 (MRSA)

ATCC 29212

SR7914 (VRE)

ATCC19434

SR7917 (VRE)

>64

2

>64

4

>64

4

>64

4

>64

4

>64

2

epi-1d

8b

9b

2

4

2

4

0.5

4

0.25

8

8c

4

16

64

8

9c

8

16

32

64

8

4

8d

16

64

4

16

32

8

16

4

32

8

9d

8e

4

9e

16

1

16

1

16

1

16

>64

4

8

vancomycin

1

>64

a

MICs were determined by a microdilution broth method as recommended by the NCCLS with cation-adjusted Mueller−Hinton broth (CA-MHB).

Serial 2-fold dilutions of each compound were made in appropriate broth, and the plates were inoculated with 5 × 10⁴CFU of each strain in a

volume of 0.1 mL. Plates were incubated at 35 °C for 20 h, and then MICs were scored.

incorporation of MurNAc-[¹⁴C]pentapeptide by MraY from

UDP-MurNAc-[¹⁴C]pentapeptide into lipid I, the product of

MraY (Table 1).^14,24The lipophilic analogues 8b and 9b were

found to be weaker inhibitors of MraY than 1d but still potent

(IC₅₀of 0.33 and 0.74 μM, respectively). Therefore,

introducing the long lipophilic side chain was still acceptable

for MraY inhibition.

The antibacterial activity of this series of compounds was

then evaluated,²⁷and the results are summarized in Table 2.

The impact of the lipophilic substituent on antibacterial activity

was very good, and both 8b and 9b exhibited good activity

against a range of Gram-positive bacterial pathogens including

S. aureus SR3637 (MRSA) and E. faecium SR7917 (VRE) with

MIC values of 0.25−4 μg/mL. The activity was comparable to

that reported for the MRY A and B classes.³Thus, membrane

permeability plays an important role in terms of the

antibacterial activity among this class of natural products.

Considering the inhibitory properties of these compounds for

MraY, the observed impact of the lipophilic substituent on the

antibacterial activity of 8b in vivo and 9b could be attributed to

its role in facilitating the transfer of the molecule through the

lipophilic membrane (log P values from octanol/water

partitioning calculated with Multiple minimization program in

MacroModel program, version 9.2, are −5.20 for 1d and 0.265

for 8b). Of significance is the discovery of 9b with the

“unnatural” ^D-pentadecylglycine residue, which exhibited 8

times more potent antibacterial activity against E. faecium

SR7917 than 8b with the “natural” stereochemistry. However,

analogues with the “natural” stereochemistry were similar to or

slightly more potent than those with the “unnatural” stereo-

chemistry against Staphylococci. Introducing the rigid biphenyl

group into the lipophilic side chain had no advantage, and the

analogues 8c−e and 9c−e did not improve antibacterial activity

but rather reduced potency in the cases of 8d and 9d, which

contain the biphenyl group in the middle of the side chain.

Thus, the simple long alkyl group proved to be the best

lipophilic side chain among the five groups tested in this study

although further optimization will be necessary.

Next, the type of inhibition of 8b and 9b as well as of 1d

toward the MraY transferase was investigated. In order to

determine the K_ivalues and the type of inhibition, initial rates

were measured at different concentrations of UDP-MurNAc-

[¹⁴C]pentapeptide (0.25−1.5 mM) with a fixed concentration

of C₅₅-P (1 mM) in the presence of different concentrations of

the inhibitors. Similarly, initial rates were also measured at

different concentrations of C₅₅-P (0.1−0.6 mM) with a fixed

concentration of UDP-MurNAc-[¹⁴C]pentapeptide (0.25 mM)

in the presence of different concentrations of the inhibitors. In

each case, Lineweaver−Burk plots revealed that these

compounds present a common behavior with respect to the

substrates of MraY: they were all competitive and non-

competitive inhibitors toward the nucleotide and the lipid

substrates, respectively. This is consistent with the structures of

these compounds that share certain structural moieties with the

nucleotide substrate. However, it is known that liposidomycin

B, a natural inhibitor of MraY, is noncompetitive toward the

nucleotide substrate despite the presence of the uridine

moiety.²⁵Lineweaver−Burk plots were used to determine of

the K_ivalues and the type of inhibition assuming a ping-pong Bi

Bi kinetic mechanism as proposed previously by Heydanek et

al.²⁶Among the muraymycin analogues, the most active

compound is 1d, which has a K_ivalue 6 times lower than

that of epi-1d. The replacement of ^D-leucine with L-leucine

leads to a decrease in its affinity for the MraY target. Moreover,

the substitution of the leucine side chain by a long carbon chain

decreased the K_iby factors of 30 and 90 for compounds 8b and

9b, respectively. Therefore, in each case the compound with the

amino acid residue having the ^L-configuration displayed a

higher affinity for the enzyme target compared to that with the

D-configuration. Furthermore, the extended length of the side

chain limited the binding of the inhibitor in the catalytic site of

the enzyme, whereas the type of inhibition remained

unchanged.

Selective toxicity is a key concern in the chemotherapy for

infectious diseases. Consequently, determining the specificity of

the synthesized compounds for inhibition of MraY activity is an

important part of clinical drug development. Among the

paralogues of MraY is WecA, which is an integral membrane

protein that initiates the biosynthesis of the enterobacterial

common antigen and the O-antigen lipopolysaccharide.^28,29

This enzyme catalyzes the transfer of the phospho-GlcNAc

moiety from the nucleotide precursor UDP-GlcNAc onto C₅₅-

P, leading to the formation of C₅₅-PP-GlcNAc (lipid

intermediate I) with the subsequent release of UMP. The

MraY and WecA enzymatic reactions share the same lipid

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Figure 5. Cytotoxicity of selected lipophilic analogues against HepG2 cells. (a) Chemical structure of tunicamycins. HepG2 cells were seeded in 96-

well tissue culture plates at a 1 × 10⁴cells/well. After 24 h, cells were treated with varying concentrations of (b) tunicamycin (a positive control), (c)

9b, (d) 8c, (e) 8d, and (f) 8e for 48 h. After the treatment, the cell viability was measured by WST-8 assay as the absorbance at 450 nm, and

percentage inhibition in growth was calculated against that of cells treated without those compounds. Tunicamycin exhibited cytotoxicity with an

IC₅₀of 26.8 μg/mL.

Scheme 2

substrate, C₅₅-P. However, they differ in the nature of the

nucleotide substrate, which is UDP-MurNAc-pentapeptide and

UDP-GlcNAc for MraY and WecA, respectively. Contrary to

the MraY activity which is specific to bacteria, the reaction

catalyzed by the WecA enzyme is also conserved in eukaryotic

cells. Tunicamycins³⁰(Figure 5) are known to be nonselective

inhibitors of both MraY and WecA and show antibacterial

activity.³¹Tunicamycins also inhibit the eukaryotic UDP-

GlcNAc:dolichol-P GlcNAc-1-P transferase, which transfers N-

acetylglucosamine 1-phosphate (GlcNAc-1-P) from UDP-

GlcNAc to the polyisoprenoid acceptor dolichol phosphate to

form GlcNAc-PP-dolichol,^32,33and are not suitable candidates

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Scheme 3

as antibacterial drugs because of their cytotoxicity. Nonselective

inhibition could be one of the factors for toxicity against host

cells. In order to investigate the selectivity of 8b and 9b for the

MraY enzyme, both compounds were tested on the purified

WecA enzyme from Thermatoga maritima. The standard WecA

assay was performed by quantifying the incorporation of

[¹⁴C]GlcNAc by WecA from UDP-[¹⁴C]GlcNAc into C₅₅-PP-

GlcNAc as previously described.^28,29Residual activities and

IC₅₀values were calculated with respect to a control assay

without inhibitors. Our results showed that the observed

activity of the compounds synthesized in this study was highly

selective toward MraY, since these compounds revealed very

weak inhibitions and IC₅₀values in the millimolar range (Table

1) when tested on the WecA paralogue. Thus, the inhibitory

effects are 3−5 orders of magnitude lower than those observed

in MraY. It is also suspected that the analogues 8 and 9 act as

detergents, which, because of micelle formation, are sometimes

cytotoxic. Accordingly, their cytotoxicity against human

hepatocellular carcinoma (HepG2) cells was then evaluated

(Figure 5). Tunicamycins were also tested as a positive control

and showed cytotoxicity with an IC₅₀of 26.8 μg/mL. Under

these conditions, all the compounds prepared in this study

exhibited no cytotoxicity (IC₅₀> 100 μg/mL), the selected data

of which are shown in Figure 5. In conjunction with the

observed selectivity among the MraY paralogues, these results

clearly indicate that 8 and 9 showed selective toxicity against

bacterial strains. The observed high therapeutic index, together

with the high selectivity against the MraY enzyme, is a desirable

property for antibacterial agents, and analogues 8 and 9 are

suitable candidates for further development.

Investigation of the Accessory Urea−Peptide Moi-

ety. With these results in hands, we next investigated the

impact of the accessory urea−peptide moiety on antibacterial

activity with a pentadecyl group as the lipophilic side chain.

Two sets of analogues were designed to address the structural

requirement of the accessory peptide moiety of the MRYs. One

of the structural features of the MRYs is the ^L-epi-

capreomycidine (Cpm) contained in the accessory moiety.

Thus, several mutated analogues, where the ^L-epi-Cpm was

replaced by the ^L-Cpm, L-Arg, and L-ornithine (Orn),

respectively, were first designed to investigate the role of the

cyclic guanidine functionality (Figure 4, right). The carboxylic

acid containing the ^L-Cpm 3b was prepared as previously

reported.¹⁷The carboxylic acids containing L-Arg 3c or L-Orn

3d were prepared by condensation of the isocyanate of ^L-Val-

O^tBu with either L-Arg or L-Orn (Scheme S2 of Supporting

Information). As for the preparation of the N-protected ^L-epi-

Cpm 3e and 3f, the synthetic route was improved as described

previously.³⁴Truncated analogues at the L-Val residue were also

designed to observe the impact of the C-terminus of the MRYs

on antibacterial activity. These analogues were also prepared by

the U4CR by altering the carboxylic acid component (Scheme

2). With the carboxylic acid components 3b−f in hand, we then

conducted the U4CR assemblage in a manner similar to that for

the preparation of 8 and 9, and the desired mutated and

truncated analogues 10 and 11 were obtained after

deprotection (Zn, THF-NaH₂PO₄buffer (pH 7), then 80%

aqueous TFA) and HPLC separation of diastereomers (>95%

purity). The newly formed stereogenic center was tentatively

assigned as the same as that in 8 and 9. Average overall yields

were ∼30% over three steps. As for the deprotection of 10f and

11f, the 2,4-dimethoxybenzyl group at the newly formed

carboxamide nitrogen atom was surprisingly tolerant, and only

the N-2,4-dimethoxybenzyl derivatives 12f and 13f were

obtained under typical deprotection conditions (80% aqueous

TFA). After extensive efforts to remove the 2,4-dimethox-

ybenzyl group, it was found that using TFA and thioanisole,

known as a push−pull system,³⁵was effective, and the desired

10f and 11f were each obtained in 60% yield (Scheme 3).

The antibacterial activity of this series of compounds was

evaluated, and the results are summarized in Table 3. Overall,

the stereochemistry at the newly formed stereogenic center in

the U4CR did not influence the antibacterial activity, and the

diastereomers exhibited similar antibacterial potency within a

factor of 2. All analogues were also active against drug-resistant

bacteria such as MRSA or VRE as observed for 8 and 9. Both of

the ^L-Cpm and L-Arg mutated analogues 10b,c and 11b,c

exhibited antibacterial activity to a range of Gram-positive

bacteria with MICs of 1−4 μg/mL. These values are

comparable to and, in some cases, twice as strong as those of

the “natural”-type analogues 8b. The results indicate that the

stereochemistry or cyclic structure of the ^L-epi-Cpm residue

found in MRYs is not important for antibacterial activity.

Moreover, the activity of the ^L-Orn mutated analogues 10d and

11d was 2−8 μg/mL, namely, reduced by approximately 2-fold.

Nonetheless, the analogues still showed potent antibacterial

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activity. Although the guanidine functionality is preferred, a less

basic and simple amino functional group is tolerated enough to

exhibit moderate antibacterial activity. From a medicinal

chemical point of view, the presence of the ^L-epi-Cpm residue

is unimportant. It is also noteworthy that the truncated

analogues 10e,f and 11e,f exhibited potent antibacterial activity

with MIC values of 4−8 μg/mL. The ^L-Val residue and/or the

carboxylic acid would not contribute to the activity.

Table 3. Antibacterial Activity of Accessory Peptide

Analogues

a

MIC (μg/mL)

S. aureus

E. faecalis

E. facium

SR7917

(VRE)

ATCC 29213

SR3637

ATCC

SR7914

(VRE)

compd

10b

(MSSA)

(MRSA)

29212

2

4

2

4

2

4

These results prompted us to examine the impact of the

cyclic guanidine moiety of 10f with the analogues replaced with

L-Orn 10g, L-Arg 10h, or L-Met 10i (Scheme 4). In this

particular case, the stereochemistry of the pentadecylglycine

moiety was set as the more active S-configuration, and the

synthetic route was modified as shown in Scheme 4. N-Boc-

allylglycine (14)³⁶was condensed with aminoethanol to give

the carboxamide 15 (N-hydroxysuccinimide, EDCI, CH₂Cl₂,

then aminoethanol, 84% over two steps). The cross-metathesis

of 15 with tetradecene catalyzed by Grubbs second generation

catalyst³⁷in refluxing CH₂Cl₂followed by hydrogenation of the

olefin provided the pentadecylglycin derivative 16 in 57% yield

over two steps. After protecting group manipulation, the

residual primary hydroxyl group of 17 was oxidized (IBX,

MeCN) to give the aldehyde 18. Reductive amination of 18

with 20 provided 21 (NaBH(OAc)₃, 79% over two steps from

19). After removal of the Cbz group (H₂, Pd/C, MeOH), the

resulting secondary amine was coupled with the suitably

protected amino acids (EDCI, CH₂Cl₂) to afford 10g−i,

respectively, after global deprotection (80% aqueous TFA).

The analogues 10g−i exhibited similar or slightly improved

antibacterial activity (Table 3). As indicated by the SAR study

11b

10c

2

1

11c

4

2

10d

11d

10e

4

2

8

4

8

4

8

11e

8

4

10f

8

4

8

11f

8

4

8

10g

4

8

10h

10i

4

8

4

8

23

>64

1

>64

1

32

1

64

>64

64

>64

vancomycin

a

MICs were determined by a microdilution broth method as

recommended by the NCCLS with cation-adjusted Mueller−Hinton

broth (CA-MHB). Serial 2-fold dilutions of each compound were

made in appropriate broth, and the plates were inoculated with 5 × 10⁴

CFU of each strain in a volume of 0.1 mL. Plates were incubated at 35

°C for 20 h, and then MICs were scored.

Scheme 4

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Scheme 5

of 10b−e and 11b−e, the cyclic guanidine of the L-epi-Cpm

residue is not necessary for the antibacterial activity. In order to

see the impact of the accessory motif, the analogue 23, where

the accessory motif was completely removed from 10 or 11,

was prepared from 22¹⁷as shown in Scheme 5,¹⁸and its

biological properties were compared. The truncated analogue

23 was found to be a much weaker MraY inhibitor with an IC₅₀

of 5 μM, which was a 6- to 12-fold reduction of the inhibitory

activity compared to 8b and 9b (Table 2). In addition to and

apart from the binding pocket interacting with the 5′-O-

aminoribosyl-5′-C-glycyluridine moiety, an additional binding

site in MraY presumably exists, which recognizes the accessory

urea−peptide motif. The antibacterial activity of 23 was greatly

decreased with MICs ranging from 32 to 64 μg/mL, although

23 possessed a hydrophobic substituent (Table 3). These

results clearly show that the partial structure of the urea−

peptide accessory motif is a contributing factor in the

interaction with the MraY enzyme to result in strong

antibacterial activity. Two-directional optimization was effec-

tively achieved by the SAR of several analogues resulting from

the U4CR.

Proposed Binding Model of MRY with MraY. Two-

directional optimization was effectively achieved by the SAR of

several analogues, and these results indicate that (i) the

lipophilic side chain is necessary to exhibit antibacterial activity

and the synthetically accessible simple alkyl chain is preferred

and (ii) the stereochemistry or cyclic structure of the ^L-epi-Cpm

residue is not important and the C-terminal ^L-Val is not

necessary. These initial SAR studies provide an insight into a

binding model of the MRYs with the target MraY. The active

conformation when the MRYs bind to the MraY is not yet

known because the three-dimensional structure of this integral

membrane protein has not been reported. However, the

membrane topologies of the MraY transferases from both the

Gram-negative E. coli and Gram-positive S. aureus have been

established to generate a topological model.³⁸The proposed

model indicates that the MraY transferases contain 10

transmembrane domains, with the N- and C-termini exposed

on the periplasmic face of the bacterial membrane. The

topology and distribution are highly conserved in other MraY

subfamily members with five cytoplasmic loops (CL) 1−5 and

four periplasmic loops (PL) 1−4.^12,39,40The five CLs form an

active site. It was revealed that several Asp residues in the CL 2

that are invariant in the whole superfamily are important to the

catalytic activity and the CL 5 is expected to be in proximity to

the CL2 and responsible for the UDP-N-acetyl-^D-hexosamine

substrate specificity as a carbohydrate recognition (CR)

domain.³⁹

ammonium group of the aminoribose moiety of this class of

natural products is important to exhibit antibacterial

activity.^19a,c,20Previous active site mapping of B. subtilis MraY

with 19 mutants strongly suggests that the transfer reaction

would proceed via direct attack of the phosphate group of C₅₅-

P substrate to the diphosphate moiety of UDP-MurNAc-

peptapeptide and the Asp 98 in the CL2 should be involved in

the deprotonation of the phosphate during the catalytic

process.¹⁴Interaction of the Asp residues with the aminoribosyl

moiety of MRYs might be probable, and this arrangement

would block the binding of the UDP-MurNAc pentapeptide to

the active site in a competitive fashion. On the other hand, the

lipophilic side chain is not involved in the interaction with

MraY because the analogues 8 and 9 were noncompetitive to

the other substrate, C₅₅-P. Moreover, that the enzymatic

inhibition by 23, with a partial structure of the urea−peptide

accessory motif, was largely decreased is an additional

contributing factor in the interaction of the inhibitor with the

MraY. This was supported by conformational calculations and

comparison of UDP-MurNAc pentapeptide and the MRY as

follows. A three-dimentional orientation of the MRY and UDP-

MurNAc-pentapeptide was simulated because a solution

structure of these molecules, which are long linear and rather

flexible, was difficult to analyze by NMR. The selected low-

energy conformers of 24 and 25 are shown in Figure 6a,b.⁴¹As

indicated by previous studies, structural comparison of 24 and

25 revealed that the ammonium group of the aminoribose

moiety of 25 was positioned near the diphosphate moiety of 24

(Figure 6c). This is indicative of the interaction of the

aminoribose moiety of MRYs to the Asp residues in the CL2.

The hydroxyl-Leu residue could be freely rotating making the

position of the Val, epi-Cpm, and lipophilic side chain easily

interconvertible. These moieties have a weaker contribution to

enzymatic or antibacterial activity. On the other hand, the inner

moiety of the urea−dipeptide motif (marked by a circle) of 25

is less flexible than the Val, epi-Cpm, and lipophilic side chain

and superimposable on the MurNAc moiety of 24. Since the

MurNAc moiety is recognized by the CR domain in CL5, it is

probable that the inner moiety of the urea−dipeptide motif

interacts with the CR domain. The MraY CR domain contains

a highly conserved motif of 13 amino acids (MAPIHHH-

FELKGW for E. coli) for UDP-MurNAc-pentapeptide recog-

nition. This motif is not found in any of the other subgroups

within the transferase family. The WecA CR domain contains a

different motif (RRxxxGxSPFSPDxxHIHH), which is selec-

tively used for UDP-GlcNAc recognition.^37,38,40,42This is

supported by the high selectivity of 8 and 9 to the MraY

observed in this study. However, the minimum structural

determinant of the accessory motif found in the MRYs for

recognition by the CR domain is not expected to be large

Our SAR study of the MRYs suggests a probable mechanism

for the inhibition of the MraY. It is indicated that the

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Figure 6. Predicted energy-minimum conformations of (a) UDP-MurNAc-Ala-amide (24) and (b) MRY analogue 25. The lipophilic side chain and

Val residue of the urea−peptide moiety were replaced with an octanoyl group and Ala residue, respectively, for the MRY, and the tetrapeptide was

omitted for UDP-MurNAc pentapeptide to simplify the calculations. MacroModel program, version 9.0, was used for conformational search.

Ionization status in H₂O under pH 7 ± 1 was predicted by the Epik program, and these structures were used for the following conformational

analysis. Conformational searching was carried out using the Monte Carlo multiple minimum (MCMM) method (100 000 steps), followed by

Polak−Ribiere conjugate gradient (PRCG) minimization with the OPLS 2005 force field. Water was chosen for a solvent with the GB/SA model.

The other settings were used as default. The lowest 1000 conformers were clustered by multiple minimization. Representative conformers were

superimposed by N-1, C-1′, and C-4′ atoms. (c) The representative conformer of 24 or 25 was merged.

considering the simplification at the urea−peptide moiety

provided by the SAR of 10 and 11. Of note is the presumed

interaction of the urea−peptide motif with the CR domain. The

CR domain contains several Lys and His residues and is highly

basic with calculated isoelectric points (pI) ranging from 10−

12.³¹The fact that the analogues 10e−g, which lack the

carboxylate functionality, retained biological activity indicates

that the urea−peptide motif does not interact with the basic

residues in the CR by a simple acid−base interaction. Rather,

one of the possible binding modes of a partial structure of the

urea−peptide is to interact with other residues such as the Phe,

Ile, or Trp by a hydrophobic and/or a cation−π interaction or

with the backbone of the CR domain by hydrogen bonding.

The overall predicted binding mode of the MRYs to MraY is

summarized in Figure 7. An interactive SAR study with both

the MRY analogues and the MraY mutants at the CR domain

would provide further insight into the precise mode of action of

the MRYs and clarify the MraY “machinery”. This proposed

binding mode of the MRYs could be a guide to the rational

design of selective inhibitors for MraY.

CONCLUSION

■

The systematic SAR study of the MRYs was investigated. The

MRY analogues on both the lipophilic side chain and the urea−

peptide moiety were synthesized via the U4CR assemblage. By

virtue of the multicomponent assemblage at the late stage of

the synthesis and despite the challenges this imposes, this

approach was quite effective and has provided ready access to a

range of analogues simply by altering the aldehyde or carboxylic

acid component.

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to establish a synthetic chemical approach based on rational

drug design using FOS. The knowledge obtained from our SAR

study of the MRYs would provide further direction toward the

design of potent MraY inhibitors.

EXPERIMENTAL SECTION

■

General Experimental Methods. NMR spectra were reported in

parts per million (δ) relative to tetramethylsilane (0.00 ppm) as

internal standard unless otherwise noted. Coupling constants (J) were

reported in herz (Hz). Abbreviations of multiplicity were as follows: s,

singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. Data

were presented as follows: chemical shift (multiplicity, integration,

coupling constant). Assignments were based on ¹H−¹H COSY,

HMBC, and HMQC NMR spectra. Purity of all the compounds tested

1

for biological evaluation was confirmed to be >95% by HPLC and H

NMR analyses.

Compounds 8b and 9b (General Procedure of the

Preparation of 8 and 9 by the U4CR). Carboxylic acid 3a (42.7

mg, 0.073 mmol), 2b (89.8 mg, 0.37 mmol), and 5 (53.9 μL, 0.37

mmol) were dissolved in EtOH (1 mL), and the solution was

concentrated in vacuo. The residue was coevaporated with EtOH three

times. The residue and the isonitrile 6¹⁷(56.9 mg, 0.073 mmol) in

EtOH (1 mL) were concentrated in vacuo, and the resulting syrup was

kept at room temperature for 48 h. The mixture was diluted with

AcOEt, and the solution was washed with H₂O, saturated aqueous

NaCl and dried (Na₂SO₄), filtered, and concentrated in vacuo. The

residue was purified by neutral silica gel column chromatography (0.5

cm × 2 cm, 66% AcOEt−hexane) to afford the U4CR product (47.6

mg, 37%) as a white foam. The product (7.0 mg, 0.0040 mmol) in

THF (1 mL) and 1 M aqueous NaH₂PO₄(500 μL) was treated with

Zn (4.3 mg, 0.080 mmol) for 40 h. After the resulting mixture was

concentrated in vacuo, the residue was suspended in AcOEt. The

insoluble portion was filtered off through a short silica gel pad, and the

filtrate was concentrated in vacuo. The residue was treated with 80%

aqueous TFA (2 mL) for 8 h, and the resulting mixture was

concentrated in vacuo. The residue was purified by HPLC (YMC

J'sphere ODS M80, 4.6 mm × 150 mm, 0.1% TFA, a linear gradient

with MeOH from 73% to 80% - 20 min, 7.09 min - 9b, 9.53 min for

8b) to afford 8b (1.5 mg, 35%) and 9b (1.5 mg, 35%) as a white foam.

Figure 7. Schematic model for muraymycin inhibition of MraY

transferase. The Asp residues in cytoplasmic loop (CL) 2 form ion or

hydrogen bonding to muraymycins at aminoribose moiety, occupying

the binding site for the UDP-MurNAc pentapeptide substrate. A

partial urea−peptide motif is recognized by the carbohydrate

recognition (CR) region.

The impact of the lipophilic substituent on antibacterial

activity was very large, and analogues 8b−e and 9b−e exhibited

good activity against a range of Gram-positive bacterial

pathogens, including MRSA and VRE. Other biological

properties of 8b−e and 9b−e were investigated, and it was

found that these analogues are selective inhibitors of the MraY

and are nontoxic to HepG2 cells. The SAR of the accessory

urea−peptide moiety suggested that it could be simplified to a

large extent. On the basis of these SAR studies, the binding

mode of the MRYs was proposed. This could be a guide to the

rational design of selective inhibitors for MraY, whose three-

dimensional structure is not yet known. Since the analogues

prepared in this study are effective against a range of bacterial

pathogens, including MRSA and VRE, selective MraY inhibitors

are promising candidates for antibacterial agents to treat drug-

resistant bacteria.

Compared to synthetic drugs, many biologically relevant

natural products possess large, complex, or labile chemical

structures, which may restrict chemical modifications in a

structure−activity relationship study. Therefore, it is important

to pursue FOS, a strategy for the design of less structurally

complex targets with comparable or superior activity that could

be made in a practical manner. This study revealed that the

molecular complexity of the MRYs could be significantly

reduced (the molecular weight of 10f is two-thirds that of 1a)

with comparable antibacterial activity. This is a good contrast to

the tunicamycins, where the truncation of either the lipophilic

side chain or the GluNAc moiety results in complete loss of

biological activity.³⁰We have also studied a rational and drastic

simplification of the common molecular architecture of MRY

congeners by decoding the role of the aminoribofuranose

moieties, which is another important issue associated with the

simplification of the MRYs.^19aIn summary, a drastic

simplification of the chemical structure of the MRYs is feasible

Data for 8b: [α]²²+294.3 (c 0.053, MeOH); ¹H NMR (D₂O

D

containing CD₃OD, 500 MHz) δ 7.78 (br s, 1H, H-6), 5.91 (br s, 1H,

H-5), 5.85 (br s, 1H, H-1′), 5.25 (br s, 1H, H-1″), 4.65 (br s, 1H, H-

5′), 4.44 (br s, H, H-2′, H-2-epi-Cpm), 4.35 (m, 2H, H-3′ and 4′), 4.20

(m, 4H, H-2″, -3″, -4″, and CHCH₂(CH₂)₁₃CH₃), 4.09 (br s, 1H, H-2-

Val), 3.01 (m, 2H, H-6′ and H-3-epi-Cpm), 3.37 (m, 4H, H-10′ and H-

5-epi-Cpm), 3.23 (m, 4H, H-8′ and 5″), 2.17 (m, 1H, H-3-Val), 2.00

(m, 4H, H-9′ and H-4-epi-Cpm), 1. 72 (m, 2H,

−CHCH₂(CH₂)₁₃CH₃), 1.26 (m, 26H, −CHCH₂(CH₂)₁₃CH₃), 0.97

(m, 3H, H-4-Val), 0.93 (br s, 3H, H-4-Val), 0.86 (s, 3H,

−CHCH₂(CH₂)₁₃CH₃); ¹³C NMR (D₂O, 125 MHz) δ 177.2, 172.2,

166.6, 159.8, 154.9, 152.1, 143.2, 141.9, 109.8, 103.1, 92.6, 85.5, 80.4,

76.8, 75.8, 73.7, 72.8, 70.2, 64.9, 59.7, 56.7, 55.1, 47.6, 47.0, 43.4, 37.1,

32.8, 30.7, 30.6, 30.3, 26.6, 23.5, 20.0, 18.1, 14.7, 9.13; ESIMS-LR m/z

1071 [(M + H)⁺]; ESIMS-HR calcd for C₄₈H₈₄N₁₁O₁₆1070.6098,

1

found 1070.6069. Data for 9b: [α]²²+297.8 (c 0.059, MeOH); H

D

NMR (D₂O, 600 MHz) δ 7.90 (br s, 1H, H-6), 6.04 (br s, 1H, H-5),

6.00 (br s, 1H, H-1′), 5.38 (br s, 1H, H-1″), 4.80 (br s, 1H, H-5′), 4.68

(br s, 1H, H-2′), 4.49 (m, 3H, H-3′, -4′, and H-2-epi-Cpm), 4.33 (m,

4H, H-2″, 3″, 4″, and CHCH₂(CH₂)₁₃CH₃), 4.16 (br s, 1H, H-2-Val),

4.00 (m, 2H, H-6′ and H-3-epi-Cpm), 3.52 (m, 4H, H-10′ and H-5-epi-

Cpm), 3.37 (m, 4H, H-8′ and H-5″), 2.29 (m, 1H, H-3-Val), 2.10 (m,

4H, H-9′ and H-4-epi-Cpm), 1.80 (m, 2H, −CHCH₂(CH₂)₁₃CH₃),

1.37 (m, 26H, −CHCH₂(CH₂)₁₃CH₃), 1.08 (br s, 3H, H-4-Val), 1.03

(br s, 3H, H-4-Val), 0.98 (s, 3H, −CHCH₂(CH₂)₁₃CH₃); ¹³C NMR

(D₂O containing CD₃OD, 150 MHz) δ 177.8, 173.9, 167.2, 160.4,

155.5, 152.6, 143.8, 142.5, 110.4, 103.7, 93.2, 86.1, 80.6, 77.4, 76.4,

74.3, 73.4, 70.7, 65.5, 60.2, 57.3, 55.7, 48.2, 47.6, 43.9, 37.7, 33.4, 31.3,

31.2, 30.9, 27.2, 24.0, 20.6, 18.7, 15.3, 9.7; ESIMS-LR m/z 1070 [(M +

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H)⁺]; ESIMS-HR calcd for C₄₈H₈₄N₁₁O₁₆1070.6098, found

1070.6073.

1H, H-2-4-(4′-butoxybiphenyl-4-yl)ethylglycine), 4.12 (d, 1H, H-2-

Val, J_2,3= 5.2 Hz), 4.06 (m, 1H, H-4″), 4.04 (m, 1H, H-3″), 4.02 (m,

1H, H-2″), 3.97 (t, 2H, OCH₂CH₂CH₂CH₃, J = 7.4 Hz), 3.92 (s, 1H,

H-6′), 3.90 (m, 1H, H-3-epi-Cpm), 3.39 (m, 1H, H-10′a), 3.32 (m, 3H,

H-5″a and H-5a-epi-Cpm), 3.22 (m, 1H, H-5″b), 3.16 (m, 2H, H-8′a

and H-10′b), 3.04 (m, 1H, H-8′b), 2.75 (m, 1H, H-4a-4-(4′-

butoxybiphenyl-4-yl)ethylglycine), 2.64 (m, 1H, H-4b-4-(4′-butoxybi-

phenyl-4-yl)ethylglycine), 2.18 (m, 2H, H-3-Val and H-3a-4-(4′-

butoxybiphenyl-4-yl)ethylglycine), 2.01 (m, 1H, H-3b-4-(4′-butoxybi-

phenyl-4-yl)ethylglycine), 1.87 (m, 4H, H-9′ and H-4-epi-Cpm), 1.75

(m, 2H, OCH₂CH₂CH₂CH₃), 1.51 (m, 2H, OCH₂CH₂CH₂CH₃),

0.99 (d, 3H, H-4a-Val, J_4a,3= 10.6 Hz), 0.96 (m, 3H,

OCH₂CH₂CH₂CH₃), 0.95 (d, 3H, H-4b-Val, J_4b,3= 8.8 Hz); ¹³C

NMR (CD₃OD, 150 MHz) δ 175.8, 173.6, 161.3, 160.9, 156.3, 152.9,

144.2, 141.3, 140.8, 135.2, 130.8, 129.5, 128.4, 116.7, 111.1, 104.0,

94.6, 86.6, 81.3, 78.6, 77.3, 75.2, 74.5, 71.9, 69.6, 57.9, 56.0, 52.2, 48.7,

47.6, 44.7, 38.2, 37.6, 34.6, 33.7, 33.3, 32.3, 27.8, 23.4, 21.1, 20.6, 19.0,

15.0, 10.0; ESIMS-LR m/z 1111 [(M + H)⁺]; ESIMS-HR calcd for

Compounds 8c and 9c. According to the general procedure, 8c

(5.1 mg, 27%) and 9c (5.1 mg, 27%) were obtained as a white foam

from 3 (17.4 mg, 0.03 mmol), 2c (85.0 mg, 0.30 mmol), 5 (45.0 μL,

0.3 mmol), and 6 (23.1 mg, 0.03 mmol) after purification by HPLC

(YMC J'sphere ODS M80, 10 mm × 150 mm, 0.1% TFA, 62%

1

MeOH−H₂O, 8.6 min - 9c, 14.0 min - 8c). Data for 8c: H NMR

(CD₃OD, 500 MHz) δ 7.64 (d, 1H, H-6, J_6,5= 8.0 Hz), 7.56 (d, 2H,

H-1′-biphenyl and H-6′-biphenyl, J = 8.0 Hz), 7.49 (d, 2H, H-2-

biphenyl and H-6-biphenyl, J = 8.0 Hz), 7.39 (dd, 2H, H-3′-biphenyl

and H-5′-biphenyl, J = 7.5, 8.0 Hz), 7.28 (t, 2H, H-4′-biphenyl, J = 7.5

Hz), 7.22 (d, 2H, H-2-biphenyl and H-6-biphenyl, J = 8.0 Hz), 5.75 (d,

1H, H-1′, J₁′_,2′ = 2.3 Hz), 5.70 (d, 1H, H-5, J_5,6= 8.0 Hz), 5.17 (s, 1H,

H-1″), 4.56 (d, 1H, H-5′, J₅′_,4′ = 5.2 Hz), 4.31 (d, 1H, H-2-epi-Cpm, J_2,3

= 7.4 Hz), 4.28 (dd, 1H, H-2′, J₂′_,1′ = 2.3, J₂′_,3′ = 5.2 Hz), 4.23 (m, 2H,

H-3′ and H-4′), 4.15 (m, 1H, H-2-8-(biphenyl-4-yl)heptylglycine),

4.12 (d, 1H, H-2-Val, J_2,3= 4.6 Hz), 4.08−4.02 (m, 3H, H-2″, -3″, and

1

-4″), 3.96 (s, 1H, H-6′), 3.87 (dd, 1H, H-3-epi-Cpm, J_3,2= 4.6, J_3,4

=

C₅₁H₇₄N₁₁O₁₇1111.5259, found 1111.5271. Data for 9d: H NMR

12.3 Hz), 3.40 (m, 1H, H-5a-epi-Cpm), 3.29 (m, 2H, H-10′a and H-

5b-epi-Cpm), 3.18 (m, 4H, H-5″, H-8′a, and H-10′b), 3.04 (m, 1H, H-

8′b), 2.62 (t, 2H, H-9-8-(biphenyl-4-yl)heptylglycine, J = 8.0 Hz), 2.18

(m, 1H, H-3-Val), 1.86 (m, 5H, H-9′, H-4-epi-Cpm and H-3a-8-

(biphenyl-4-yl)heptylglycine), 1.64 (m, 3H, H-3b-8-(4-biphenylyl-4-

yl)heptylglycine and H-4-8-(biphenyl-4-yl)heptylglycine), 1.34 (m,

8H, H-5, H-6, H-7, and H-8-8-(biphenyl-4-yl)heptylglycine), 0.98 (d,

3H, H-4a-Val, J_4a,3= 6.9 Hz), 0.94 (d, 3H, H-4b-Val, J_4b,3= 6.9 Hz);

¹³C NMR (CD₃OD, 150 MHz) δ 176.4, 175.2, 172.5, 166.3, 166.0,

160.4, 155.6, 152.1, 143.3, 143.1, 142.4, 139.9, 130.1, 129.9, 129.8,

128.0, 127.8, 127.8, 110.2, 103.1, 93.9, 85.7, 80.4, 77.6, 76.4, 74.4, 73.9,

73.8, 71.1, 60.1, 59.2, 56.9, 55.7, 43.9, 37.5, 36.7, 36.4, 32.6, 32.4, 31.5,

30.7, 30.6, 30.4, 30.2, 27.2, 22.5, 19.8, 18.2; ESIMS-LR m/z 1110 [(M

+ H)⁺]; ESIMS-HR calcd for C₅₂H₇₆N₁₁O₁₆1110.5466, found

(CD₃OD, 500 MHz) δ 7.62 (d, 1H, H-6, J_6,5= 8.1 Hz), 7.46 (m, 4H,

H-3, H-5, H-2′, and H-6′-bihenyl), 7.23 (d, 2H, H-2-biphenyl and H-6-

biphenyl, J = 8.6 Hz), 6.94 (d, 2H, H-3′-biphenyl and H-5′-biphenyl, J

= 9.2 Hz), 5.73 (d, 1H, H-1′, J₁′_,2′ = 2.8 Hz), 5.70 (d, 1H, H-5, J_5,6= 8.1

Hz), 5.17 (s, 1H, H-1″), 4.57 (d, 1H, H-5′, J₅′_,4′ = 5.7 Hz), 4.45 (d, 1H,

H-2-epi-Cpm, J_2,3= 8.6 Hz), 4.30 (dd, 1H, H-2′, J₂′_,1′ = 2.8, J₂′_,3′ = 5.2

Hz), 4.23 (m, 3H, H-3′, H-4′ and H-2-4-(4′-butoxybiphenyl-4-

yl)ethylglycine), 4.18 (d, 1H, H-2-Val, J_2,3= 4.5 Hz), 4.04 (m, 3H,

H-2″, H-3″ and H-4″), 3.99 (t, 2H, OCH₂CH₂CH₂CH₃, J = 6.9), 3.93

(s, 1H, H-6′), 3.75 (dd, 1H, H-3-epi-Cpm, J_3,4= 5.7, J_3,2= 8.6 Hz), 3.44

(m, 1H, H-5a-epi-Cpm), 3.36 (m, 1H, H-5b-epi-Cpm), 3.35 (m, 1H,

H-10′a), 3.28 (m, 1H, H-10′b), 3.21−3.15 (m, 4H, H-8′a, 10′b, and H-

5″), 3.06 (m, 1H, H-8′b), 2.78 (m, 1H, H-4a-4-(4′-butoxybiphenyl-4-

yl)ethylglycine), 2.66 (m, 1H, H-4b-4-(4′-butoxybiphenyl-4-yl)-

ethylglycine), 2.16 (m, 1H, H-3-Val), 2.07 (m, 2H, H-3-4-(4′-

butoxybiphenyl-4-yl)ethylglycine), 1.96 (m, 2H, H-4-epi-Cpm), 1.88

(m, 2H, H-9′), 1.75 (m, 2H, OCH₂CH₂CH₂CH₃), 1.51 (m, 2H,

OCH ₂CH ₂CH ₂CH₃), 0. 99 (m, 6H, H-4a-Val and

OCH₂CH₂CH₂CH₃), 0.92 (d, 3H, H-4b-Val, J_4b,3= 6.8 Hz); ¹³C

NMR (CD₃OD, 150 MHz) δ 176.7, 176.1, 173.3, 166.8, 161.0, 160.9,

156.4, 152.9, 144.3, 141.2, 140.9, 135.3, 130.8, 129.6, 128.4, 116.7,

111.1, 104.0, 94.5, 86.6, 81.2, 78.6, 77.3, 75.0, 74.5, 72.0, 69.6, 60.5,

57.7, 56.1, 52.9, 48.0, 44.7, 38.2, 35.2, 33.5, 33.3, 32.6, 28.0, 23.2, 21.1,

20.5, 18.8, 15.0; ESIMS-LR m/z 1111 [(M + H)⁺]; ESIMS-HR calcd

for C₅₁H₇₄N₁₁O₁₇1111.5259, found 1111.5272.

1

1110.5465. Data for 9c: H NMR (CD₃OD, 500 MHz) δ 7.64 (d,

1H, H-6, J_6,5= 8.0 Hz), 7.55 (d, 2H, H-1′ and 6′-biphenyl, J = 8.0 Hz),

7.49 (d, 2H, H-2 and 6-biphenyl, J = 8.0 Hz), 7.39 (dd, 2H, H-3′ and

5′-biphenyl, J = 7.5, 8.0 Hz), 7.28 (t, 2H, H-4′-biphenyl, J = 7.5 Hz),

7.22 (d, 2H, H-2 and 6-biphenyl, J = 8.0 Hz), 5.74 (d, 1H, H-1′, J₁′_,2′ =

2.8 Hz), 5.71 (d, 1H, H-5, J_5,6= 8.0 Hz), 5.18 (s, 1H, H-1″), 4.58 (d,

1H, H-5′, J₅′_,4′ = 4.9 Hz), 4.43 (d, 1H, H-2-epi-Cpm, J_2,3= 5.5 Hz), 4.30

(dd, 1H, H-2′, J₂′_,1′ = 2.8, J₂′_,3′ = 5.5 Hz), 4.24 (m, 2H, H-3′ and H-4′),

4.16 (d, 2H, H-2-8-(biphenyl-4-yl)heptylglycine and H-2-Val, J_2,3= 5.2

Hz), 4.08−4.00 (m, 3H, H-2″, -3″, and -4″), 3.93 (s, 1H, H-6′), 3.70

(dd, 1H, H-3-epi-Cpm, J_3,2= 5.5, J_3,4= 13.6 Hz), 3.40 (m, 2H, H-5a-

epi-Cpm), 3.29 (m, 2H, H-10′a), 3.18 (m, 4H, H-5″, 8′a, and H-10′b),

3.06 (m, 1H, H-8′b), 2.62 (t, 2H, H-9-8-(biphenyl-4-yl)heptylglycine, J

= 8.1 Hz), 2.15 (m, 1H, H-3-Val), 1.93 (m, 4H, H-9′ and H-4-epi-

Cpm), 1.69 (m, 3H, H-3-8-(biphenyl-4-yl)heptylglycine and H-4-8-

(biphenyl-4-yl)heptylglycine), 1.34 (m, 8H, H-5, -6, -7, and H-8-8-

(biphenyl-4-yl)heptylglycine), 0.95 (d, 3H, H-4a-Val, J_4a,3= 6.9 Hz),

0.91 (d, 3H, H-4b-Val, J_4b,3= 6.9 Hz); ¹³C NMR (CD₃OD, 150 MHz)

δ 189.2, 176.4, 173.1, 166.8, 161.0, 156.4, 152.9, 144.2, 143.9, 143.2,

140.6, 130.7, 130.6, 128.8, 128.6, 128.6, 111.0, 104.0, 98.5, 94.8, 93.6,

86.6, 81.2, 77.3, 75.1, 74.6, 72.0, 57.5, 56.7, 53.0, 44.7, 38.2, 37.8, 37.2,

33.5, 33.4, 32.6, 31.2, 31.0, 27.9, 23.1, 20.5, 18.8; ESIMS-LR m/z 1110

[(M + H)⁺]; ESIMS-HR calcd for C₅₂H₇₆N₁₁O₁₆1110.5466, found

1110.5466.

Compounds 10b and 11b. Compound 3b (113 mg, 0.194

mmol), hexadecanal (46.6 mg, 0.194 mmol), and 5 (29.1 μL, 0.194

mmol) were dissolved in EtOH (1.0 mL), and the solution was

concentrated in vacuo. The residue was coevaporated with EtOH (1.0

mL), and this was repeated 3 times. The residue and 6 (150 mg, 0.194

mmol) in EtOH (2.0 mL) were stirred at 50 °C for 12 h. Hexadecanal

(46.6 mg, 0.194 mmol) and 2,4-dimethoxybenzylamine (29.1 μL,

0.194 mmol) were added to the solution, and the mixture was stirred

further 18 h. The organic phase was diluted with AcOEt and washed

with 1 M aqueous HCl, saturated aqueous NaHCO₃, H₂O, saturated

aqueous NaCl, dried (Na₂SO₄), and concentrated in vacuo. The

residue was purified by neutral silica gel column chromatography (2

cm × 10 cm, 40% AcOEt−hexane) to afford a mixture of U4CR

products (114 mg, 37%) as a white foam. The products (114 mg,

0.065 mmol) in THF (1.6 mL) and 1 M aqueous NaH₂PO₄(400 μL)

were treated with Zn (204 mg, 3.25 mmol) for 12 h. After the resulting

mixture was concentrated in vacuo, the residue was suspended in

AcOEt. The insoluble was filtered off through a short silica gel pad,

and the filtrate was concentrated in vacuo. The residue was treated

with 80% aqueous TFA containing 5% Et₃SiH (2 mL) for 24 h. The

solution was concentrated in vacuo, and the residue was purified by

HPLC (YMC J'sphere ODS M80, 10 mm × 150 mm, 0.1% TFA, a

linear gradient from 70% to 75% MeOH−H₂O for 20 min, 10.9 min−

11b, 15.0 min−10b) to afford lipophilic MRY derivative 10b (33.0 mg,

Compounds 8d and 9d. According to the general procedure, 8d

(4.8 mg, 24%) and 9d (4.8 mg, 24%) were obtained as a white foam

from 3 (17.4 mg, 0.03 mmol), 2d (84.6 mg, 0.30 mmol), 5 (45.0 μL,

0.3 mmol), and 6 (23.1 mg, 0.03 mmol) after purification by HPLC

(YMC J'sphere ODS M80, 10 mm × 150 mm, 0.1% TFA, 60%

1

MeOH−H₂O, 5.8 min - 9d, 10.2 min - 8d). Data for 8d: H NMR

(CD₃OD, 500 MHz) δ 7.65 (d, 1H, H-6, J_6,5= 8.0 Hz), 7.48 (m, 4H,

H-3, 5, 2′ and H-6′-biphenyl), 7.24 (d, 2H, H-2-biphenyl and H-6-

biphenyl, J = 8.6 Hz), 6.94 (d, 2H, H-3′-biphenyl and H-5′-biphenyl, J

= 6.9 Hz), 5.76 (d, 1H, H-1′, J₁′_,2′ = 2.3 Hz), 5.70 (d, 1H, H-5, J_5,6= 8.0

Hz), 5.16 (s, 1H, H-1″), 4.57 (d, 1H, H-5′, J₅′_,4′ = 5.7 Hz), 4.27 (dd,

1H, H-2′, J₂′_,1′ = 2.3, J₂′_,3′ = 5.2 Hz), 4.24 (m, 1H, H-2-epi-Cpm), 4.23

(dd, 1H, H-4′, J₄′_,5′ = 5.7, J₄′_,3′ = 9.8 Hz), 4.21 (m, 1H, H-4′), 4.19 (m,

1

46%) and 11b (33.0 mg, 46%) as a white foam. Data for 10b: H

NMR (CD₃OD, 500 MHz) δ 7.64 (d, 1H, H-6, J_6,5= 8.0 Hz), 5.74 (d,

8432

dx.doi.org/10.1021/jm200906r | J. Med. Chem. 2011, 54, 8421−8439

Journal of Medicinal Chemistry

Article

1H, H-1′, J₁′_,2′ = 2.3 Hz), 5.73 (d, 1H, H-5, J_5,6= 8.0 Hz), 5.18 (s, 1H,

H-1″), 4.57 (d, 1H, H-5′, J₅′_,4′ = 5.7 Hz), 4.44 (d, 1H, H-2-epi-Cpm, J_2,3

= 5.7 Hz), 4.31 (dd, 1H, H-4′, J₄′_{, 3}′ = 2.4, J₄′_{, 5}′ = 5.7 Hz), 4.28 (dd, 1H,

H-2′, J₂′_,1′ = 2.3, J₂′_,3′ = 5.8 Hz), 4.23 (d, 1H, H-3′, J₃′_{, 2}′ = 5.8 Hz), 4.18

(d, 1H, H-2-Val, J_2,3= 4.6 Hz), 4.16 (m, 1H, −CHCH₂(CH₂)₁₃CH₃),

4.06 (2H, H-2″ and H-4″), 4.01 (s, 1H, H-3″), 3.99 (s, 1H, H-6′), 3.79

(dt, 1H, H-3-epi-Cpm, J_3,2= J_3,4a= 5.7, J_3,4b= 8.6 Hz), 3.43 (m, 1H, H-

10′a), 3.32 (m, 3H, H-10′b and H-5-epi-Cpm), 3.25−3.18 (m, 3H, H-

8′a and H-5″), 3.06 (m, 1H, H-8′b), 2.17 (dt, 1H, H-3-Va, J_3,2= 4.6,

1H, H-5′), 4.11−3.83 (m, 10H, H-2-Val, H-2-Val,

−CHCH₂(CH₂)₁₃CH₃, 2′, 3′, 4′, 6′, 2″, 3″, and H-4″), 1.98 (s, 1H,

H-3-Va), 1.73−1.49 (m, 8H, H-9′a, −CHCH₂(CH₂)₁₃CH₃, 3-Arg, and

H-4-Arg), 1.12 (m, 26H, −CHCH₂(CH₂)₁₃CH₃), 0.72 (m, 9H, H-4a-

Val and −CHCH₂(CH₂)₁₃CH₃); ¹³C NMR (D₂O, 150 MHz) δ 177.1,

167.7, 162.0, 160.3, 160.2, 153.7, 145.0, 112.0, 104.7, 95.5, 87.2, 82.0,

79.4, 78.0, 75.9, 75.2, 72.6, 61.4, 56.8, 49.4, 48.2, 45.4, 43.5, 38.2, 34.6,

34.0, 33.2, 32.3, 32.2, 32.2, 32.1, 32.0, 31.8, 28.8, 28.6, 27.8, 25.2, 21.3,

19.7, 16.0; ESIMS-LR m/z 1072 [(M + H)⁺]; ESIMS-HR calcd for

C₄₈H₈₆N₁₁O₁₆1072.6249, found 1072.6259.

J

_3,4a= J_3,4b= 6.8 Hz), 2.05 (m, 1H, H-4a-epi-Cpm), 1.88 (m, 3H, H-9′

and H-4b-epi-Cpm), 1.73 (m, 2H, −CHCH₂(CH₂)₁₃CH₃), 1.26 (m,

Compounds 10d and 11d. A solution of 3d (56.0 mg, 0.13

mmol), hexadecanal (31.2 mg, 0.13 mmol), 5 (19.5 μL, 0.13 mmol),

and ammonium chloride (20 mg, 0.39 mmol) were suspended in

toluene (2.0 mL), and the suspension was concentrated in vacuo. The

residue was coevaporated with EtOH (1.0 mL), and this was repeated

3 times. The residue and 6 (100 mg, 0.13 mmol) in toluene (2.0 mL)

were stirred at 50 °C for 12 h. Hexadecanal (31.2 mg, 0.13 mmol) and

5 (19.5 μL, 0.13 mmol) was added to the solution, and the mixture

was stirred a further 36 h. The organic phase was diluted with AcOEt

and washed with 1 M aqueous HCl, saturated aqueous NaHCO₃,

H₂O, saturated aqueous NaCl, dried (Na₂SO₄), and concentrated in

vacuo. The residue was purified by neutral silica gel column

chromatography (2 cm × 10 cm, 40% AcOEt−hexane) to afford a

mixture of U4CR products (119 mg, 56%) as a white foam. The

products (119 mg, 0.074 mmol) were treated with 80% aqueousTFA

containing 5% Et₃SiH (2 mL) for 8 h. The solution was concentrated

in vacuo, and the residue was purified by HPLC (YMC J'sphere ODS

M80, 10 mm × 150 mm, 0.1% TFA, a linear gradient from 70% to

75% MeOH−H₂O for 20 min, 11.3 min−11d, 15.3 min−10d) to

afford lipophilic MRY derivative 10d (33.0 mg, 45%) and 11d (33.0

26H, −CHCH₂(CH₂)₁₃CH₃), 0.97 (d, 3H, H-4a-Val, J_4a,3= 6.9 Hz),

0.92 (d, 3H, H-4b-Val, J_4b,3

= 6.9 Hz), 0.87 (t, 3H,

−CHCH₂(CH₂)₁₃CH₃, J = 7.2 Hz); ¹³C NMR (CD₃OD, 125 MHz)

δ 174.7, 171.0, 170.2, 164.6, 161.3, 158.9, 154.7, 150.8, 142.1, 108.7,

101.8, 92.9, 84.3, 80.6, 78.9, 76.3, 75.1, 72.8, 72.6, 69.9, 63.5, 58.2,

55.6, 54.2, 50.9, 47.1, 42.7, 36.6, 35.3, 31.7, 31.6, 30.4, 29.4, 29.3, 29.1,

26.0, 25.8, 22.8, 22.4, 18.4, 16.5, 13.1; ESIMS-LR m/z 1068 [(M −

H)⁻]; ESIMS-HR calcd for C₄₈H₈₂N₁₁O₁₆1068.5947, found

1

1068.5956. Data for 11b: H NMR (CD₃OD, 500 MHz) δ 7.64 (d,

1H, H-6, J_6,5= 8.0 Hz), 5.76 (d, 1H, H-1′, J₁′_,2′ = 2.9 Hz), 5.70 (d, 1H,

H-5, J_5,6= 8.0 Hz), 5.18 (s, 1H, H-1″), 4.57 (d, 1H, H-5′, J₅′_,4′ = 5.2

Hz), 4.39 (d, 1H, H-2-epi-Cpm, J_2,3= 4.6 Hz), 4.29 (m, 2H, H-2′ and

H-4′), 4.20 (m, 2H, H-3′ and −CHCH₂(CH₂)₁₃CH₃), 4.14 (d, 1H, H-

2-Val, J_2,3= 4.6 Hz), 4.07 (m, 2H, H-3″ and H-4″), 4.03 (s, 1H, H-2″),

3.94 (s, 1H, H-6′), 3.78 (dt, 1H, H-3-epi-Cpm, J_3,2= 4.6, J_3,4a= J_3,4b

=

9.2 Hz), 3.42 (m, 2H, H-5-epi-Cpm), 3.29−3.18 (m, 5H, H-8′a, 10′,

and H-5″), 3.04 (m, 1H, H-8′b), 2.17 (dt, 1H, H-3-Va, J_3,4a= J_3,4b= 6.9,

J_3,2= 12.0 Hz), 2.00 (m, 1H, H-4a-epi-Cpm), 1.91 (m, 1H, H-4b-epi-

Cpm), 1.84 (m, 3H, H-9′a and −CHCH₂(CH₂)₁₃CH₃), 1.64 (m, 1H,

H-9′b), 1.26 (m, 26H, −CHCH₂(CH₂)₁₃CH₃), 0.97 (d, 3H, H-4a-Val,

J_4a,3= 6.9 Hz), 0.93 (d, 3H, H-4b-Val, J_4b,3= 6.9 Hz), 0.87 (t, 3H,

−CHCH₂(CH₂)₁₃CH₃, J = 6.9 Hz); ¹³C NMR (CD₃OD, 125 MHz) δ

174.8, 173.9, 170.9, 170.3, 164.7, 159.1, 154.6, 150.7, 142.0, 108.7,

101.8, 92.7, 84.1, 79.0, 76.3, 75.1, 74.5, 73.0 72.6, 69.9, 63.5, 58.4, 55.8,

54.0, 51.1, 45.3, 42.7, 36.7, 35.3, 36.7, 35.3, 31.7, 31.4, 30.3, 29.5, 29.3,

29.1, 29.0, 26.0, 25.8, 22.9, 22.4, 18.4, 16.7, 13.1; ESIMS-LR m/z 1068

[(M − H)⁻]; ESIMS-HR calcd for C₄₈H₈₂N₁₁O₁₆1068.5947, found

1068.5957.

1

mg, 45%) as a white foam. Data for 10d: H NMR (CD₃OD, 500

MHz) δ 7.65 (d, 1H, H-6, J_6,5= 8.8 Hz), 5.76 (d, 1H, H-1′, J₁′_,2′ = 2.3

Hz), 5.70 (d, 1H, H-5, J_5,6= 8.8 Hz), 5.17 (s, 1H, H-1″), 4.57 (d, 1H,

H-5′, J₅′_,4′ = 4.0 Hz), 4.26 (m, 2H, H-2′ and H-4′), 4.22 (m, 1H, H-3′),

4.17 (m, 2H, H-2-Orn and −CHCH₂(CH₂)₁₃CH₃), 4.11 (d, 1H, H-2-

Val, J_2,3= 4.6 Hz), 4.07 (m, 2H, H-2″ and H-4″), 4.04 (m, 2H, H-6′

and H-3″), 3.33 (m, 1H, H-10′a), 3.24 (m, 4H, H-10′b, 8′a, and H-5″),

3.07 (m, 1H, H-8′b), 2.95 (t, 2H, H-5-Orn), 2.15 (dt, 1H, H-3-Va, J_3,2

= 4.6, J_3,4a= J_3,4b= 6.9 Hz), 1.86 (m, 5H, H-9′a, Orn, and H-4-Orn),

1.69 (m, 3H, H-9′b and −CHCH₂(CH₂)₁₃CH₃), 1.26 (m, 26H,

−CHCH₂(CH₂)₁₃CH₃), 0.96 (d, 3H, H-4a-Val, J_4a,3= 6.9 Hz), 0.93

(d, 3H, H-4b-Val, J_4b,3= 6.9 Hz), 0.87 (t, 3H, −CHCH₂(CH₂)₁₃CH₃, J

= 7.5 Hz); ¹³C NMR (CD₃OD, 125 MHz) δ 174.8, 174.3, 173.9,

170.0, 164.7, 159.1, 150.7, 142.1, 109.0, 101.7, 92.8, 84.1, 79.1, 76.3,

75.0, 73.0, 72.4, 69.8, 62.9, 58.4, 54.0, 53.3, 45.2, 42.7, 38.9, 35.2, 31.7,

31.3, 30.4, 29.5, 29.3, 29.2, 29.1, 29.0, 25.8, 23.7, 22.4, 18.4, 16.7, 13.1;

ESIMS-LR m/z 1028 [(M − H)⁻]; ESIMS-HR calcd for C₄₇H₈₂N₉O₁₆

1028.5885, Ffound 1028.5903. Data for 11d: ¹H NMR (CD₃OD, 500

MHz) δ 7.65 (d, 1H, H-6, J_6,5= 8.1 Hz), 5.74 (d, 1H, H-1′, J₁′_,2′ = 2.3

Hz), 5.72 (d, 1H, H-5, J_5,6= 8.1 Hz), 5.17 (s, 1H, H-1″), 4.58 (d, 1H,

H-5′, J₅′_,4′ = 5.2 Hz), 4.31 (dd, 1H, H-2′, J₂′_,1′ = 2.3, J₂′_,3′ = 5.8 Hz), 4.27

(d, 1H, H-4′, J₄′_,5′ = 5.2 Hz), 4.23 (d, 2H, H-3′, J₃′_,2′ = 5.8 Hz), 4.20 (m,

1H, H-2-Orn), 4.17 (d, 2H, H-2-Val and −CHCH₂(CH₂)₁₃CH₃), 4.06

(m, 2H, H-3″ and H-4″), 4.03 (m, 2H, H-6′ and H-2″), 3.29 (m, 5H,

H-8′a, 10′, and H-5″), 3.06 (m, 1H, H-8′b), 2.96 (t, 2H, H-5-Orn), 2.16

(dt, 1H, H-3-Va, J_3,2= 5.2, J_3,4a= J_3,4b= 6.9, Hz), 1.86 (m, 3H, H-9′

and H-4a-Orn), 1.74 (m, 5H, H-4b, 3-Orn, and

−CHCH₂(CH₂)₁₃CH₃), 1.26 (m, 26H, −CHCH₂(CH₂)₁₃CH₃), 0.96

(d, 3H, H-4a-Val, J_4a,3= 6.9 Hz), 0.92 (d, 3H, H-4b-Val, J_4b,3= 6.9 Hz),

0.87 (t, 3H, −CHCH₂(CH₂)₁₃CH₃, J = 7.5 Hz); ¹³C NMR (CD₃OD,

125 MHz) δ 174.6, 174.2, 173.8, 170.0, 164.7, 159.1, 150.8, 142.3,

108.9, 101.8, 93.0, 84.1, 79.0, 76.4, 75.0, 72.8, 72.5, 69.9, 63.0, 58.2,

54.2, 53.3, 45.6, 42.7, 38.9, 35.4, 31.7, 31.4, 30.6, 29.5, 29.4, 29.3, 29.1,

29.1, 25.8, 23.5, 22.4, 18.4, 16.6, 13.1; ESIMS-LR m/z 1028 [(M −

H)⁻]; ESIMS-HR calcd for C₄₇H₈₂N₉O₁₆1028.5885, found

1028.5904.

Compounds 10c and 11c. A solution of 3c (32.2 mg, 0.05

mmol), hexadecanal (12.4 mg, 0.05 mmol), 5 (7.8 μL, 0.05 mmol),

and ammonium chloride (4.0 mg, 0.075 mmol) in toluene (600 μL)

were stirred at room temperature for 30 min. The isonitrile 6 (40 mg,

0.05 mmol) was added to the solution, and the suspension was stirred

at 50 °C for 48 h. The organic phase was diluted with AcOEt and

washed with 1 M aqueous HCl, saturated aqueous NaHCO₃, H₂O,

saturated aqueous NaCl, dried (Na₂SO₄), and concentrated in vacuo.

The residue was purified by neutral silica gel column chromatography

(2 cm × 10 cm, 40% AcOEt−hexane) to afford a mixture of U4CR

products (40 mg, 47%) as a white foam. The products (40 mg, 0.022

mmol) were treated with 80% aqueous TFA containing 5% Et₃SiH (2

mL) for 8 h. The solution was concentrated in vacuo, and the residue

was purified by HPLC (YMC J'sphere ODS M80, 10 mm × 150 mm,

0.1% TFA, a linear gradient from 70% to 75% MeOH−H₂O for 20

min, 10.1 min−11c, 14.0 min−10c) to afford lipophilic MRY

derivative 10c (7.5 mg, 32%) and 11c (7.5 mg, 32%) as a white

1

foam. Data for 10c: [α]²³+1.59 (c 0.34, MeOH); H NMR (D₂O,

D

500 MHz) δ 7.60 (s, 1H, H-6), 5.73 (s, 1H, H-5), 5.67 (s, 1H, H-1′),

5.08 (s, 1H, H-1″), 4.49 (s, 1H, H-5′), 4.25−3.85 (m, 10H, H-2-Val,

−CHCH₂(CH₂)₁₃CH₃, 2′, 3′, 4′, 6′, 2″, 3″, and H-4″), 1.99 (s, 1H, H-3-

Va), 1.68−1.53 (m, 8H, 9′a, −CHCH₂(CH₂)₁₃CH₃, 3-Arg and H-4-

Arg), 1.13 (m, 26H, −CHCH₂(CH₂)₁₃CH₃), 0.70 (m, 9H, H-4a-Val

and −CHCH₂(CH₂)₁₃CH₃); ¹³C NMR (D₂O, 150 MHz) δ 177.8,

177.0, 167.6, 162.2, 160.2, 153.7, 145.2, 111.9, 104.7, 95.7, 87.3, 82.0,

79.5, 78.0, 75.8, 75.3, 72.7, 57.1, 56.6, 48.6, 45.5, 43.5, 38.3, 34.6, 34.1,

33.4, 32.3, 32.3, 32.2, 32.1, 32.0, 32.0, 31.9, 28.6, 27.6, 25.2, 21.3, 19.6,

16.0; ESIMS-LR m/z 1072 [(M + H)⁺]; ESIMS-HR calcd for

Compounds 10e and 11e. A solution of 3e (15.3 mg, 0.3

mmol), hexadecanal (71.3 mg, 3.0 mmol), and 5 (44.6 μL, 3.0 mmol)

in toluene (2.0 mL) was stirred at room temperature for 30 min. The

isonitrile 6 (22.8 mg, 0.03 mmol) was added to the solution and stirred

C₄₈H₈₆N₁₁O₁₆1072.6249, found 1072.6264. Data for 11c: [α]²³

D

+4.34 (c 0.34, MeOH); ¹H NMR (D₂O, 600 MHz) δ 7.54 (s, 1H, H-

6), 5.63 (s, 1H, H-5), 5.58 (s, 1H, H-1′), 5.03 (s, 1H, H-1″), 4.44 (s,

8433

dx.doi.org/10.1021/jm200906r | J. Med. Chem. 2011, 54, 8421−8439

Journal of Medicinal Chemistry

Article

at 50 °C for a week. The reaction mixture was partitioned between

AcOEt and 1 M aqueous HCl, and the organic phase was washed with

saturated aqueous NaHCO₃, H₂O, saturated aqueous NaCl, dried

(Na₂SO₄), and concentrated in vacuo. The residue was purified by

neutral silica gel column chromatography (2 cm × 6 cm, 40% AcOEt−

hexane) to afford a mixture of U4CR products (16 mg, 31%) as a

white foam. The products (16 mg, 0.009 mmol) in THF (2.0 mL) and

1 M aqueous NaH₂PO₄(500 μL) were treated with Zn (59.8 mg, 0.92

mmol) for 48 h. After the resulting mixture was concentrated in vacuo,

the residue was suspended in AcOEt. The insoluble portion was

filtered off through a short silica gel pad, and the filtrate was

concentrated in vacuo. The residue was treated with 80% aqueous

TFA for 18 h. The solution was concentrated in vacuo, and the residue

was purified by HPLC (YMC J'sphere ODS M80, 10 mm × 150 mm,

0.1% TFA, a linear gradient from 70% to 85% MeOH−H₂O for 20

min, 13.2 min−11e, 15.7 min−10e) to afford lipophilic MRY

derivative 10e (2.2 mg, 23%) and 11e (2.2 mg, 23%) as a white

foam. Data for 10e: ¹H NMR (CD₃OD, 500 MHz) δ 7.60 (d, 1H, H-

6, J_6,5= 8.0 Hz), 7.33 (m, 5H, phenyl), 5.74 (s, 1H, H-1′), 5.70 (d, 1H,

H-5, J_5,6= 8.0 Hz), 5.17 (s, 1H, H-1″), 5.10 (d, 2H, CH₂Ph, J = 12.8

(d, 1H, H-1′, J₁′_,2′ = 2.3 Hz), 5.69 (d, 1H, H-5, J_5,6= 8.0 Hz), 5.17 (s,

1H, H-1″), 4.80 (d, 1H, H-5′, J₅′_,4′ = 4.5 Hz), 4.70 (d, 1H, CH₂Ph, J =

16.0 Hz), 4.56 (d, 1H, H-2-epi-Cpm, J_2,3= 4.1 Hz), 4.50 (d, 1H,

CH₂Ph, J = 16.0 Hz), 4.30 (m, 2H, H-2′ and −CHCH₂(CH₂)₁₃CH₃,

J

_2,3a= 5.2, J_2,3b= 10.6 Hz), 4.22 (m, 2H, H-3′ and 4′), 4.17 (m, 1H, H-

3-epi-Cpm), 4.06 (m, 1H, H-3″), 4.04 (m, 1H, H-4″), 4.01 (t, 1H, H-

2″, J = 2.3 Hz), 3.88 (s, 3H, OMe), 3.79 (s, 3H, OMe), 3.42 (m, 1H,

H-10′a), 3.38 (m, 1H, H-5a-epi-Cpm), 3.29 (m, 3H, H-5″a and H-5b-

epi-Cpm), 3.25 (m, 2H, H-5″b and H-10′b), 3.16 (m, 1H, H-8′a), 3.03

(m, 1H, H-8′b), 1.99 (m, 2H, H-4-epi-Cpm), 1.89 (m, 1H, H-9′a), 1.82

(m, 1H, H-9′b), 1.79 (m, 2H, −CHCH₂(CH₂)₁₃CH₃), 1.30 (m, 26H,

−CHCH₂(CH₂)₁₃CH₃), 0.88 (m, 3H, −CHCH₂(CH₂)₁₃CH₃, J = 6.9

Hz); ESIMS-LR m/z 1077 [(M + H)⁺]; ESIMS-HR calcd for

1

C₅₁H₈₅N₁₀O₁₅1077.6190, found 1077.6182. Data for 13f: H NMR

(CD₃OD, 500 MHz) δ 7.66 (d, 1H, H-6, J_6,5= 8.1 Hz), 7.21 (d, 1H,

aromatic, J = 8.6 Hz), 6.58 (s, 1H, aromatic), 6.54 (d, 1H, aromatic, J =

8.6 Hz), 5.73 (d, 1H, H-1′, J₁′_,2′ = 2.3 Hz), 5.72 (d, 1H, H-5, J_5,6= 8.0

Hz), 5.16 (s, 1H, H-1″), 4.95 (m, 1H, H-2-epi-Cpm), 4.57 (d, 1H, H-

5′, J₅′_,4′ = 4.6 Hz), 4.28 (m, 1H, H-2′), 4.22 (m, 2H, H-3′ and H-4′),

4.17 (m, 1H, CH₂Ph), 4.12 (m, 1H, H-3-epi-Cpm), 4.07 (m, 1H, H-

4″), 4.04 (m, 2H, H-3″ and CH₂Ph), 4.01 (m, 1H, H-2″), 3.92 (m, 2H,

H-6′ and −CHCH₂(CH₂)₁₃CH₃), 3.87 (s, 3H, OMe), 3.79 (s, 3H,

OMe), 3.45 (m, 3H, H-10′a and H-5a-epi-Cpm), 3.29 (m, 1H, H-

10′b), 3.20 (m, 2H, H-5″), 3.07 (m, 2H, H-8′), 2.13 (m, 1H, H-4a-epi-

Cpm), 2.02 (m, 1H, H-4b-epi-Cpm), 1.86 (m, 2H, H-9′), 1.78 (m, 2H,

−CHCH₂(CH₂)₁₃CH₃), 1.27−0.99 (m, 26H, −CHCH₂(CH₂)₁₃CH₃),

0.88 (t, 3H, −CHCH₂(CH₂)₁₃CH₃, J = 6.8 Hz); ESIMS-LR m/z 1077

[(M + H)⁺]; ESIMS-HR calcd for C₅₁H₈₅N₁₀O₁₅1077.6190, found

1077.6184.

Hz), 4.55 (d, 1H, H-5′, J₅′_,4′ = 4.0 Hz), 4.33 (d, 1H, H-2-epi-Cpm, J_2,3

=

8.6 Hz), 4.25 (m, 3H, H-2′, -3′, and -4′), 4.15 (dd, 1H,

−CHCH₂(CH₂)₁₃CH₃, J = 5.2, J = 10.6 Hz), 4.07 (m, 1H, H-4″),

4.02 (m, 2H, H-2′ and H-3″), 3.87 (s, 1H, H-6′), 3.75 (m, 1H, H-3-epi-

Cpm), 3.40 (m, 2H, H-5-epi-Cpm), 3.29 (m, 3H, H-5″a and H-10′a),

3.19 (m, 2H, H-10′b and H-8′a), 3.07 (m, 1H, H-8′b), 1.93 (m, 4H, H-

9′ and −CHCH₂(CH₂)₁₃CH₃), 1.71 (m, 2H, H-4-epi-Cpm), 1.35 (m,

26H, −CHCH₂(CH₂)₁₃CH₃), 0.86 (t, 3H, −CHCH₂(CH₂)₁₃CH₃, J =

6.9 Hz); ESIMS-LR m/z 1061 [(M + H)⁺]; ESIMS-HR calcd for

1

C₅₀H₈₁N₁₀O₁₅1061.5880, found 1061.5886. Data for 11e: H NMR

Compound 10f. A solution of 12f (3.3 mg, 0.0031 mmol) and

thioanisole (100 μL, 0.085 mmol) was treated with 80% aqueous TFA

for 48 h. The solution was concentrated in vacuo. The residue was

diluted with H₂O, and the aqueous phase was washed with AcOEt.

The solution was concentrated in vacuo. The residued was purified by

HPLC (YMC J'sphere ODS M80, 10 mm × 150 mm, 0.1% TFA, 71%

MeOH−H₂O for 20 min, 6.3 min) to afford lipophilic MRY derivative

(CD₃OD, 500 MHz) δ 7.57 (d, 1H, H-6, J_6,5= 8.0 Hz), 7.34 (m, 5H,

phenyl), 5.74 (d, 1H, H-1′, J₁′_,2′ = 2.3 Hz), 5.69 (d, 1H, H-5, J_5,6= 8.0

Hz), 5.17 (s, 2H, H-1″ and CH₂Ph), 5.10 (d, 1H, CH₂Ph, J = 12.6 Hz),

4.57 (d, 1H, H-5′, J₅′_,4′ = 5.7 Hz), 4.34 (d, 1H, H-2-epi-Cpm, J_2,3= 7.4

Hz), 4.24 (dd, 1H, H-2′, J₂′₁′ = 2.3, J₂′_,3′ = 5.2 Hz), 4.22 (dd, 1H, H-4′,

J₄′_,5′ = 5.1, J₄′_,3′ = 5.7 Hz), 4.18 (dd, 1H, H-3′, J₃′_,2′ = 5.1, J₃′_,4′ = 5.1 Hz),

4.13 (dd, 1H, −CHCH₂(CH₂)₁₃CH₃, J = 5.5, J = 8.3 Hz), 4.07 (dd,

1H, H-4″, J₄″_,3″ = 2.8, J₄″_,5″ = 6.3 Hz), 4.05 (d, 1H, H-2″, J₂″_,3″ = 4.0

Hz), 4.01 (dd, 1H, H-3″, J₃″_,4″ = 2.8, J₃″_,2″ = 4.0 Hz), 3.87 (m, 2H, H-6′

and H-3-epi-Cpm), 3.48 (m, 1H, H-5a-epi-Cpm), 3.39 (m, 1H, H-5b-

epi-Cpm), 3.30 (m, 2H, H-10′a and H-5″a), 3.23 (m, 1H, H-10′b), 3.14

(m, 2H, H-8′a and H-5″b), 3.04 (m, 1H, H-8′b), 1.91 (m, 3H, H-9′a

and H-4-epi-Cpm), 1.80 (m, 2H, H-9′b and −CHCH₂(CH₂)₁₃CH₃),

1.26 (m, 26H, −CHCH₂(CH₂)_{1 3}CH₃), 0.88 (t, 3H,

−CHCH₂(CH₂)₁₃CH₃, J = 6.8 Hz); ESIMS-LR m/z 1061 [(M +

H)⁺]; ESIMS-HR calcd for C₅₀H₈₁N₁₀O₁₅1061.5877, found

1061.5886.

Compounds 12f and 13f. A solution of 3f (20.0 mg, 0.042

mmol), hexadecanal (99.6 mg, 0.42 mmol), and 5 (62.3 μL, 0.42

mmol) in EtOH (2.0 mL) was stirred at room temperature for 30 min.

The isonitrile 6 (32.0 mg, 0.042 mmol) was added to the solution and

stirred at 50 °C for 24 h. The reaction mixture was partitioned

between AcOEt and 1 M aqueous HCl, and the organic phase was

washed with saturated aqueousNaHCO₃, H₂O, saturated aqueous

NaCl, dried (Na₂SO₄), and concentrated in vacuo. The residue was

purified by neutral silica gel column chromatography (2 cm × 6 cm,

40% AcOEt−hexane) to afford a mixture of U4CR products (18 mg,

26%) as a white foam. The products (18 mg, 0.011 mmol) in THF

(2.0 mL) and 1 M aqueous NaH₂PO₄(500 μL) was treated with Zn

(74.8 mg, 1.15 mmol) for 48 h. After the resulting mixture was

concentrated in vacuo, the residue was suspended in AcOEt. The

insoluble was filtered off through a short silica gel pad, and the filtrate

was concentrated in vacuo. The residue was treated with 80% aqueous

TFA for 18 h. The solution was concentrated in vacuo, and the residue

was purified by HPLC (YMC J'sphere ODS M80, 10 mm × 150 mm,

0.1% TFA, 71% MeOH−H₂O for 20 min, 16.7 min−13f, 18.5 min−

12f) to afford lipophilic MRY derivative 12f (3.3 mg, 29%) and 13f

(3.3 mg, 29%) as a white foam. Data for 12f: ¹H NMR (CD₃OD, 500

MHz) δ 7.65 (d, 1H, H-6, J_6,5= 8.0 Hz), 7.16 (d, 1H, aromatic, J = 8.6

Hz), 6.60 (s, 1H, aromatic), 6.56 (d, 1H, aromatic, J = 8.6 Hz), 5.72

1

10f (1.8 mg, 60%) as a white foam. H NMR (CD₃OD, 500 MHz) δ

7.65 (d, 1H, H-6, J_6,5= 8.0 Hz), 5.74 (d, 1H, H-1′, J₁′_,2′ = 2.3 Hz), 5.72

(d, 1H, H-5, J_5,6= 8.0 Hz), 5.17 (s, 1H, H-1″), 4.57 (d, 1H, H-5′, J₅′_,4′ =

4.6 Hz), 4.29 (m, 1H, H-2′), 4.23 (m, 2H, H-3′ and 4′), 4.19 (dd, 1H,

−CHCH₂(CH₂)₁₃CH₃, J = 5.8, 8.6 Hz), 4.07 (m, 1H, H-4″), 4.04 (m,

1H, H-3″), 4.01 (m, 2H, H-2″ and H-2-epi-Cpm), 3.89 (m, 2H, H-6′

and H-3-epi-Cpm), 3.48 (m, 1H, H-10′a), 3.42 (m, 2H, H-5-epi-Cpm),

3.19 (m, 2H, H-5″), 3.15 (m, 2H, H-8′), 3.11 (m, 1H, H-10′b), 2.12

(m, 1H, H-4a-epi-Cpm), 1.97 (m, 1H, H-4b-epi-Cpm), 1.90 (m, 2H,

H-9′), 1.73 (m, 2H, −CHCH₂(CH₂)₁₃CH₃), 1.30 (m, 26H,

−CHCH₂(CH₂)₁₃CH₃), 0.88 (t, 3H, −CHCH₂(CH₂)₁₃CH₃, J = 6.3

Hz); ESIMS-LR m/z 927 [(M + H)⁺]; ESIMS-HR calcd for

C₄₂H₇₅N₁₀O₁₃927.5510, found 927.5517.

Compound 11f. A solution of 13f (3.3 mg, 0.0031 mmol) and

thioanisole (100 μL, 0.085 mmol) was treated with 80% aqueous TFA

for 48 h. The solution was concentrated in vacuo. The residue was

diluted with H₂O, and the aqueous phase was washed with AcOEt.

The solution was concentrated in vacuo. The residue was purified by

HPLC (YMC J'sphere ODS M80, 10 mm × 150 mm, 0.1% TFA, 71%

MeOH−H₂O for 20 min, 6.1 min) to afford lipophilic MRY derivative

1

11f (1.8 mg, 60%) as a white foam. H NMR (CD₃OD, 500 MHz) δ

7.65 (d, 1H, H-6, J_6,5= 8.0 Hz), 5.74 (d, 1H, H-1′, J₁′_,2′ = 2.9 Hz), 5.71

(d, 1H, H-5, J_5,6= 8.0 Hz), 5.19 (s, 1H, H-1″), 4.57 (d, 1H, H-5′, J₅′_,4′ =

4.6 Hz), 4.29 (m, 1H, H-2′), 4.23 (m, 2H, H-3′ and -4′), 4.18 (d, 1H,

H-2-epi-Cpm, J = 4.6 Hz), 4.12 (dd, 1H, −CHCH₂(CH₂)₁₃CH₃, J =

6.8, 8.4 Hz), 4.07 (m, 1H, H-3″), 4.05 (m, 1H, H-2″), 4.00 (m, 2H, H-

4″ and H-3-epi-Cpm), 3.85 (s, 1H, H-6′), 3.48 (m, 2H, H-10′), 3.32

(m, 2H, H-5-epi-Cpm), 3.28 (m, 2H, H-5″), 3.11 (m, 2H, H-8′), 2.01

(m, 2H, H-9′a and H-4a-epi-Cpm), 1.90 (m, 1H, H-4b-epi-Cpm), 1.81

(m, 1H, H-9′b), 1.74 (m, 1H, −CHCH₂(CH₂)₁₃CH₃), 1.69 (m, 1H,

−CHCH₂(CH₂)₁₃CH₃), 1.29 (m, 26H, −CHCH₂(CH₂)₁₃CH₃), 0.88

(t, 3H, −CHCH₂(CH₂)₁₃CH₃, J = 6.8 Hz); ESIMS-LR m/z 927 [(M

+ H)⁺]; ESIMS-HR calcd for C₄₂H₇₅N₁₀O₁₃927.5510, found

927.5519.

8434

dx.doi.org/10.1021/jm200906r | J. Med. Chem. 2011, 54, 8421−8439

Journal of Medicinal Chemistry

Article

(2S)-2-tert-Butoxycarbonylamino-N′-3-hydroxylpropylpent-

4-enamide (15). A mixture of 14⁴¹(3.8 g, 17.5 mmol) and N-

hydroxysuccinimide (3.0 g, 26.2 mmol) in CH₂Cl₂(100 mL) and THF

(50 mL) at 0 °C was treated with EDCI (5.0 g, 26.2 mmol), and the

mixture was stirred at the same temperature for 12 h. The reaction was

quenched with H₂O, and the mixture was partitioned between AcOEt

and H₂O. The organic phase was washed with saturated aqueous

NaCl, dried (Na₂SO₄), filtered, and concentrated in vacuo. The

residue in CH₂Cl₂(100 mL) was treated with 3-aminopropanol (2.0

mL, 26.2 mmol) at 0 °C and stirred at room temperature for 8 h. The

resulting mixture was partitioned between AcOEt and H₂O. The

organic phase was washed with H₂O (twice), saturated aqueous NaCl,

dried (Na₂SO₄), filtered, and concentrated in vacuo. The residue was

purified by silica gel column chromatography (5 cm × 10 cm, 40%

AcOEt−hexane) to afford 15 as a colorless syrup (4.0 g, 84% over two

steps). [α]²¹_D−5.94 (c 2.41, MeOH); ¹H NMR (CD₃OD, 500 MHz)

δ 6.97 (br s, 1H, NH-1′), 5.69 (m, 1H, H-4), 5.24 (br d, 1H, NH-Boc,

J_NH,2= 7.5 Hz), 5.11 (dd, 1H, H-5a, J_5a,5b= 3.8, J_5a,4= 14.5 Hz), 5.09

(dd, 1H, H-5b, J_5b,5a= 3.8, J_5b,4= 5.2 Hz), 4.12 (m, 1H, H-2), 3.58 (m,

2H, H-3′), 3.36 (m, 2H, H-1′), 2.48 (dt, 1H, H-3a, J = 6.3, 14.3 Hz),

2.42 (dt, 1H, H-3b, J = 7.6, 14.3 Hz), 1.65 (t, 2H, H-2′, J = 5.3 Hz),

δ 179.9, 172.4, 156.4, 137.6, 128.8, 128.3, 128.2, 65.8, 58.9, 55.2, 36.2,

32.9, 32.6, 31.8, 30.1, 29.6, 29.6, 29.5, 29.3, 29.2, 25.9,22.6, 14.5;

ESIMS-LR m/z 499 [(M + Na)⁺]; ESIMS-HR calcd for

C₂₈H₄₈N₂O₄Na 499.3506, found 499.3510.

tert-Butyl 5-O-[5-tert-Butoxycarbonylamino-5-deoxy-2,3-O-

(3-pentylidene)-β-^D-ribo-pentofuranosyl]-6-N-[3-(2S-

benzyloxycarbonylaminohexadecanoyl)aminopropyl]amino-

6-deoxy-2,3-O-isopropylidene-1-(uracil-1-yl)-β-^D-glycelo-L-

talo-heptofuranuronate (21). Aldehyde 18 was prepared as

follows. A mixture of compound 17 (309 mg, 0.70 mmol) and Et₃N

(389 μL, 2.8 mmol) in CH₂Cl₂(4 mL) and DMSO (4 mL) was

treated with pyridine−sulfur trioxide complex (35.0 mg, 1.4 mmol) at

room temperature for 2 h. The mixture was partitioned between

AcOEt and 0.1 M aqueous HCl. The organic phase was washed with

saturated aqueous NaHCO₃and saturated aqueous NaCl, dried

(Na₂SO₄), filtered, and concentrated in vacuo to afford 18 (308 mg,

quantitative) as a white wax. The compound was used in the next

1

reaction without further purification. H NMR (CDCl₃, 500 MHz) δ

9.77 (s, 1H, formyl), 7.34 (m, 5H, phenyl), 6.37 (br s, NH-1′), 5.20

(br s, NH-Cbz), 5.08 (s, 2H, CH₂Ph), 4.04 (m, 1H, H-2), 3.52 (m,

2H, H-1′), 2.74 (m, 2H, H-2′), 2.04 (m, 1H, H-3a), 1.56 (m, 1H, H-

3a), 1.24 (m, 26H, CHCH₂(CH₂)₁₃CH₃), 0.86 (t, 3H,

CHCH₂(CH₂)₁₃CH₃, J = 11.5 Hz); ESIMS-LR m/z 497 [(M +

Na)⁺]. A mixture of 19⁴(692 mg, 0.70 mmol) and 10% Pd/C (70 mg)

in MeOH (7 mL) was vigorously stirred under H₂atmosphere at room

temperature for 30 min. The catalyst was filtered off through Celite

pad, and the filtrate was concentrated in vacuo to give a crude amine

20. A solution of 20 (500 mg, 0.70 mmol) and 18 (308 mg, 0.70

mmol) and AcOH (384 μL, 7.0 mmol) in CH₂Cl₂(20 mL) was stirred

at room temperature for 30 min. The mixture was treated with

NaBH(OAc)₃(443 mg, 2.1 mmol) at room temperature for 1.5 h. The

reaction was quenched by saturated aqueous NaHCO₃(7 mL), and

the whole mixture was partitioned between AcOEt and saturated

aqueous NaHCO₃. The organic phase was washed with saturated

aqueous NaCl, dried (Na₂SO₄), filtered, and concentrated in vacuo.

The residue was purified by silica gel column chromatography (5 cm ×

10 cm, 50% AcOEt−hexane) to afford 21 (629 mg, 79%) as a white

foam. [α]²¹_D+15.7 (c 1.40, MeOH); ¹H NMR (CD₃OD, 500 MHz) δ

7.64 (d, 1H, H-6, J_6,5= 8.0 Hz), 7.33 (m, 5H, phenyl), 5.65 (s, 1H, H-

1′), 5.61 (d, 1H, H-5, J_5,6= 8.0 Hz), 5.17 (d, 1H, H-2′, J₂′_,3′ = 6.3 Hz),

5.09 (d, 1H, CH₂Ph, J = 12.0 Hz), 5.04 (d, 1H, CH₂Ph, J = 12.0 Hz),

4.99 (s, 1H, H-1″), 4.85 (m, 1H, H-6′), 4.81 (t, 1H, H-3′, J₃′_{, 2}′ = J₃′_,4′ =

6.3 Hz), 4.62 (d, 1H, H-5′, J₅′_,4′ = 5.7 Hz), 4.49 (d, 1H, H-2″, J₂″_{, 3}″ =

9.3 Hz), 4.45 (dd, 1H, H-4′, J₄′_,5′ = 5.7, J₄′_,3′ = 6.3 Hz), 4.18 (d, 1H, H-

3″, J₃″_,2″ = 9.3 Hz), 4.09 (t, 1H, H-4″, J₄″_,5″_a= J₄″_,5′_b′ = 6.9 Hz,), 4.01 (m,

1H, −CHCH₂(CH₂)₁₃CH₃), 3.28 (m, 2H, H-10′), 3.17 (m, 1H, H-

5″a), 2.99 (m, 1H, H-5″b), 2.75 (m, 1H, H-8′a), 2.37 (m, 1H, H-8′b),

1.60 (m, 2H, H-9′), 1.52 (m, 15H, (CH₃CH₂)₂CH, ^tBu and

t

1.40 (s, 9H, Bu); ¹³C NMR (CD₃OD, 125 MHz) δ 172.8, 155.8,

133.1,119.0, 80.3, 59.3, 54.1, 37.0, 36.3, 32.2, 28.4; ESIMS-LR m/z 295

[(M + H)⁺]; ESIMS-HR calcd for C₁₃H₂₄N₂O₄Na 295.1628, found

295.1629.

(2S)-2-tert-Butoxycarbonylamino-N′-3-hydroxylpropylhexa-

decanamide (16). A mixture of tetradecene (37 mL, 147 mmol) and

15 (4.3 g, 14.7 mmol) and Grubbs catalyst second generation (623

mg, 0.74 mmol) in CH₂Cl₂(150 mL) were refluxed at 40 °C for 92 h.

The mixture was concentrated in vacuo, and the residue was purified

by silica gel column chromatography (10 cm × 10 cm, 40% AcOEt−

hexane) to afford cross-metathesis products as a brown syrup (5.0 g,

77%, 1.7:1 mixture of geometric isomers). ¹H NMR (CDCl₃, 500

MHz) δ 7.25 (br s, 1H, NH-1′), 5.43 (m, 3H, H-3 and H-4), 4.04 (m,

1H, H-2), 3.95 (m, 2H, NH-Boc and OH), 3.51 (m, 2H, H-3′), 3.28

(m, 2H, H-1′), 2.33 (m, 1H, H-6a), 2.26 (m, 2H, H-6b), 1.87 (m, 2H,

H-7), 1.58 (m, 2H, H-2′), 1.56 (s, 9H, ^tBu), 1.33 (m, 18H,

C H C H ₂C H C H C H ₂( C H ₂) _{1 0}C H ₃) , 0 . 7 7 ( t , 3 H ,

CHCH₂CHCHCH₂(CH₂)₁₀CH₃, J = 6.9 Hz); ESIMS-LR m/z 463

[(M + Na)⁺]. The olefins and 10% Pd/C (500 mg) in MeOH (5 mL)

were vigorously stirred under H₂atmosphere at room temperature for

24 h. The insoluble was filtered off through Celite pad, and the filtrate

was concentrated in vacuo. The residue was purified by silica gel

column chromatography (10 cm × 10 cm, 40% AcOEt−hexane) to

afford 16 (3.7 g, 57% over two steps) as a colorless oil. [α]²¹_D−7.05 (c

1.80, MeOH); ¹H NMR (CDCl₃, 500 MHz) δ 6.92 (br s, 1H, NH-1′),

5.27 (br d, 1H, NH-Boc, J_NH,2= 7.4 Hz), 4.02 (d, 1H, H-2, J_2,3= 6.3

Hz), 3.58 (m, 3H, H-3′ and OH), 3.38 (m, 2H, H-1′), 1.74 (m, 1H, H-

t

CHCH₂(CH₂)₁₃CH₃), 1.42 (m, 12H, Bu and acetonide), 1.26 (m,

3a), 1.66 (m, 2H, H-2′), 1.56 (m, 1H, H-3b), 1.40 (s, 9H, Bu), 1.33

29H, acetonide and CHCH₂(CH₂)₁₃CH₃), 0.88 (t, 3H,

CHCH₂(CH₂)₁₃CH₃, J = 6.9 Hz), 1.26 (m, 29H, acetonide and

CHCH₂(CH₂)₁₃CH₃), 0.88 (t, 3H, CHCH₂(CH₂)₁₃CH₃, J = 6.9 Hz),

0.76 (m, 6H, (CH₃CH₂)₂CH); ¹³C NMR (CD₃OD, 125 MHz) δ

173.5, 172.5, 165.1, 157.0, 156.9, 150.7, 145.0, 136.9, 128.2, 127.7,

127.6, 115.8, 114.2, 112.9, 101.4, 96.1, 88.0, 86.5, 86.1, 84.9, 82.4, 82.2,

82.0, 78.9, 66.3, 61.7, 55.3, 45.3, 43.1, 37.1, 32.2, 31.8, 29.5, 29.5, 29.4,

29.3, 29.2, 29.0, 28.9, 28.5, 27.5, 27.4, 26.3, 25.6, 24.4, 22.5, 13.2, 7.6,

6.3; ESIMS-LR m/z 1171 [(M + H)⁺]; ESIMS-HR calcd for

C₆₁H₉₉N₆O₁₆1171.7112, found 1171.7094.

Compound 10g. Compound 21 (17.0 mg, 0.014 mmol) and 10%

Pd(OH)₂/C (6 mg) in MeOH (1 mL) were vigorously stirred under

H₂atmosphere at room temperature for 5 h. The insoluble was filtered

off through Celite pad, and the filtrate was concentrated in vacuo to

afford the crude amine. Boc-Orn(Cbz)-OH (6.36 mg, 0.017 mmol)

and HOSu (2.0 mg, 0.014 mmol) in CH₂Cl₂(1 mL) were treated with

EDCI (5.36 mg, 0.028 mmol) at 0 °C for 1 h. The amine in CH₂Cl₂(1

mL) was added to the solution of the amino acid at 0 °C, and the

mixture was stirred at room temperature for 8 h. The reaction mixture

was partitioned between AcOEt and 1 M aqueous HCl. The organic

phase was washed with H₂O, saturated aqueous NaHCO₃, and

(m, 26H, CHCH₂(CH₂)₁₃CH₃), 0.85 (t, 3H, CHCH₂(CH₂)₁₃CH₃, J =

6.8 Hz); ¹³C NMR (CD₃OD, 125 MHz) δ 178.3, 173.8, 156.0, 80.1,

59.1, 54.9, 36.1, 32.7, 32.2, 32.0, 29.8, 29.7, 29.7, 29.6, 29.4, 28.4, 25.8,

22.8, 14.2; ESIMS-LR m/z 465 [(M + Na)⁺]; ESIMS-HR calcd for

C₂₅H₅₀N₂O₄Na 465.3663, found 465.3671.

(2S)-2-Benzyloxycarbonylamino-N′-3-hydroxylpropylhexa-

decanamide (17). Compound 16 (2.0 g, 4.52 mmol) was treated

with 4.0 M HCl in AcOEt (40 mL) at room temperature for 3 h. The

resulting mixture was concentrated in vacuo. The residue in AcOEt

(100 mL) was treated with saturated aqueous NaHCO₃(100 mL) and

benzyl chloroformate (760 μL, 5.3 mmol) at 0 °C for 8 h. The organic

phase was washed with saturated aqueous NaCl, dried (Na₂SO₄),

filtered, and concentrated in vacuo. The residue was purified by silica

gel column chromatography (5 cm × 10 cm, 67% AcOEt−hexane) to

afford 17 (1.6 g, 74% over two steps) as a white wax. [α]²¹+4.47 (c

D

0.37, MeOH); ¹H NMR (DMSO-d₆, 500 MHz) δ 7.82 (t, 1H, NH-1′,

J = 5.5 Hz), 7.30 (m, 6H, phenyl and NH-Cbz), 4.98 (m, 2H, CH₂Ph),

4.39 (br s, 1H, OH), 3.88 (dd, 1H, H-2, J_2,3a= 8.6, J_2,3b= 13.8 Hz),

3.35 (m, 2H, H-3′), 3.04 (m, 2H, H-1′), 1.50 (m, 4H, H-3′ and H-3),

1.21 (m, 26H, CHCH₂(CH₂)_{1 3}CH₃), 0.80 (t, 3H,

CHCH₂(CH₂)₁₃CH₃, J = 7.5 Hz); ¹³C NMR (DMSO-d₆, 125 MHz)

8435

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Journal of Medicinal Chemistry

Article

saturated aqueous NaCl, dried (Na₂SO₄), filtered, and concentrated in

vacuo. The residue was treated with 80% aqueous TFA (2 mL) for 8 h,

and the solution was concentrated in vacuo. The residue was triturated

from cyclopentyl methyl ether to afford 10g (15.4 mg, quantitative) as

a white foam. Compound for biological assays was purified by HPLC

(YMC J'sphere ODS M80, 10 mm × 150 mm, 0.1% TFA, a linear

CHCH₂(CH₂)₁₃CH₃), 0.86 (t, 3H, CHCH₂(CH₂)₁₃CH₃, J = 7.0

Hz); ESIMS-LR m/z 904 [(M + H)⁺]; ESIMS-HR calcd for

C₄₁H₇₃N₇O₁₃S 904.5060, found 904.5081.

tert-Butyl 5-O-[5-tert-Butoxycarbonylamino-5-deoxy-2,3-O-

(3-pentylidene)-β-^D-ribofuranosyl]-6-deoxy-6-dodecylamino-

2,3-O-isopropylidene-1-(uracil-1-yl)-β-^D-glycero-L-talo-hepto-

furanuronate. A mixture of 22¹²(85 mg, 0.1 mmol) and 10% Pd/C

(10 mg) in MeOH (3 mL) was vigorously stirred for 1 h under H₂

atmosphere. The catalyst was filtered off through a Celite pad, and the

filtrate was concentrated in vacuo to give the free amine. The amine

and dodecanal (24 μL, 0.1 mmol) in CH₂Cl₂(1 mL) were treated with

AcOH (28 μL) and NaBH(OAc)₃(84 mg, 0.40 mmol) at room

temperature for 1 h. The reaction mixture was diluted with AcOEt (50

mL), which was washed with saturated aqueous NaHCO₃and brine.

The organic phase was dried (Na₂SO₄) and concentrated in vacuo.

The residue was purified by silica gel column chromatography (2 cm ×

10 cm, 50% AcOEt/hexane) to give the title compound (71 mg, 80%

over two steps) as a white foam. ¹H NMR (CD₃OD, 500 MHz) δ 7.70

(d, 1H, H-6, J_6,5= 8.0 Hz), 5.70 (d, 1H, H-1′, J₁″_,2″ = 1.7 Hz), 5.66 (d,

1H, H-5, J_5,6= 8.0 Hz), 5.21 (dd, 1H, H-2′, J₂′_,1′ = 1.7, J₂′_,3′ = 6.3 Hz),

5.04 (s, 1H, H-1″), 4.83 (dd, 1H, H-3′, J₃′_,2′ = 6.3, J₃′_,4′ = 4.0 Hz), 4.67

(d, 1H, H-2″, J₂″_,3″ = 5.7 Hz), 4.55 (d, 1H, H-3″, J₃″_,2″ = 5.7 Hz), 4.48

(dd, 1H, H-4′, J₄′_,3′ = 9.7, J₄′_,5′ = 4.6 Hz), 4.42 (d, 1H, H-5′, J₅′_,4′ = 4.6

Hz), 4.14 (t, 1H, H-4″, J₄″_,5″ = 6.3 Hz), 3.25 (br s, 1H, H-6′), 3.21 (dd,

1H, H-5″a, J₅″_a,4″ = 5.7, J₅″_a,5″_b= 14.3 Hz), 3.05 (dd, 1H, H-5″b, J₅″_b,4″ =

7.5, J₅″_b,5″_a= 14.3 Hz), 2.68 (m, 1H, CH₃(CH₂)₁₀CH₂NH), 2.42 (m,

1H, CH₃(CH₂)₁₀CH₂NH), 1.58−1.32 (m, 39H, CH₃(CH₂)₁₀CH₂NH,

1

gradient from 60% to 75% MeOH−H₂O for 20 min, 10.5 min). H

NMR (CD₃OD, 400 MHz) δ 7.67 (d, 1H, H-6, J_6,5= 8.1 Hz), 5.75 (d,

1H, H-1′), 5.71 (d, 1H, H-5, J_5,6= 8.1 Hz), 5.15 (s, 1H, H-1″), 4.55 (m,

1H, H-5′), 4.26 (m, 3H, H-2′, H-3′ and H-4′), 4.04 (m, 5H, H-2″, H-3″,

H-4″, H-2-Orn, and CHCH₂(CH₂)₁₃CH₃), 3.89 (s, 1H, H-6′), 3.45

(m, 1H, H-10′a), 3.23−2.97 (m, 7H, H-8′, H-10′b, H-5″, and H-5-

Orn), 1.93 (m, 4H, H-9′ and CHCH₂(CH₂)₁₃CH₃), 1.68 (m, 2H, H-3-

Orn), 1.25 (m, 26H, CHCH₂(CH₂)₁₃CH₃), 0.87 (t, 3H,

CHCH₂(CH₂)₁₃CH₃, J = 7.0 Hz); ESIMS-LR m/z 887 [(M + H)⁺];

ESIMS-HR calcd for C₄₁H₇₅N₈O₁₃887.5454, found 887.5456.

Compound 10h. Compound 21 (30 mg, 0.025 mmol) and 10%

Pd(OH)₂/C (10 mg) in MeOH (1 mL) were vigorously stirred under

H₂atmosphere at room temperature for 5 h. The insoluble was filtered

off through Celite pad, and the filtrate was concentrated in vacuo. Boc-

Arg-Pbf-OH (13.2 mg, 0.025 mmol) and HOBt (3.4 mg, 0.025 mmol)

in DMF (1 mL) was treated with EDCI (7.8 mg, 0.050 mmol) at 0 °C

and stirred at the same temperature for 1 h. The amine in DMF (1

mL) was added to the solution of the amino acid at 0 °C, and the

mixture was stirred at room temperature for 8 h. The mixture was

partitioned between AcOEt and H₂O. The organic phase was washed

with H₂O and saturated aqueous NaCl, dried (Na₂SO₄), filtered, and

concentrated in vacuo. The residue was treated with 80% aqueous

TFA (2 mL) for 8 h, and the solution was concentrated in vacuo. The

residue was triturated from MeOH to afford 10h (19.8 mg, 85%) as a

white foam. Compound for biological assays was purified by HPLC

(YMC J'sphere ODS M80, 10 mm × 150 mm, 0.1% TFA, a linear

tert-butyl, CH₂CH₃

× 2, and acetonide), 0.93 (t, 3H,

CH₃(CH₂)₁₀CH₂NH, J = 6.9 Hz), 0.83, 0.81 (each t, each 3H,

CH₂CH₃, J = 7.4 Hz); ¹³C NMR (CD₃OD, 125 MHz) δ 174.0, 166.4,

158.2, 152.2, 146.3, 117.1, 115.4, 114.2, 102.7, 97.3, 89.1, 87.7, 87.4,

86.2, 83.7, 83.6, 83.4, 83.3, 80.2, 62.9, 44.4, 33.1, 30.8, 30.7, 30.6, 30.5,

30.4, 29.7, 28.8, 28.6, 28.5, 28.3, 27.6, 25.7, 23.8, 14.5, 8.9, 7.6; ESIMS-

HR m/z calcd for C₄₅H₇₇N₄O₁₃881.5409, found 881.5480.

1

gradient from 60% to 75% MeOH−H₂O for 20 min, 10.6 min). H

NMR (D₂O, 500 MHz) δ 7.59 (d, 1H, H-6, J_6,5= 8.0 Hz), 5.75 (d, 1H,

H-5, J_5,6= 8.0 Hz), 5.66 (s, 1H, H-1′), 5.07 (s, 1H, H-1″), 4.47 (d, 1H,

H-5′, J₅′_,4′ = 9.7 Hz), 4.28 (m, 1H, H-2′), 4.17 (m, 2H, H-3′ and H-4′),

4.01 (m, 4H, H-2″, H-3″, H-4″ and CHCH₂(CH₂)₁₃CH₃), 3.91 (m,

1H, H-2-Arg), 3.80 (s, 1H, H-6′), 3.20−2.95 (m, 8H, H-8′, H-10′, H-

5″, and H-3-Arg), 1.57 (m, 4H, H-9′ and CHCH₂(CH₂)₁₃CH₃), 1.10

(m, 26H, CHCH₂(CH₂)₁₃CH₃), 0.70 (s, 3H, CHCH₂(CH₂)₁₃CH₃);

ESIMS-LR m/z 929 [(M + H)⁺]; ESIMS-HR calcd for C₄₂H₇₇N₁₀O₁₃

929.5666, found 929.5664.

5-O-(5-Amino-5-deoxy-β-^D-ribofuranosyl)-6-deoxy-6-dode-

cylamino-1-(uracil-1-yl)-β-^D-glycero-L-talo-heptofuranuronic

Acid Trifluoroacetic Salt (23). tert-Butyl 5-O-[5-tert-butoxycarbo-

nylamino-5-deoxy-2,3-O-(3-pentylidene)-β-^D-ribofuranosyl]-6-deoxy-

6-dodecylamino-2,3-O-isopropylidene-1-(uracil-1-yl)-β-^D-glycero-L-

talo-heptofuranuronate (50 mg, 0.057 mmol) was treated with 80%

aqueous TFA (1 mL) at room temperature for 6 h. The reaction

mixture was concentrated in vacuo, and the residue was purified by

C18 reverse phase column chromatography (1.5 cm × 10 cm, 80%

aqueous MeOH containing 0.5% TFA) to afford 23 (33 mg, 94%) as a

TFA salt. ¹H NMR (CD₃OD, 500 MHz) δ 7.69 (d, 1H, H-6, J_6,5= 8.0

Hz), 5.77 (s, 1H, H-1′), 5.76 (d, 1H, H-5, J_5,6= 8.0 Hz), 5.23 (s, 1H,

H-1″), 4.61 (br s, 1H, H-5′), 4.33 (br s, 1H, H-2′), 4.27 (m, 2H, H-6′

and H-3″), 4.21 (br s, 1H, H-4′), 4.14−4.06 (m, 3H, H-3′, H-2″, and

H-4″), 3.24 (br s, 2H, H-5″a and H-5″b), 3.22−3.11 (m, 2H,

CH₃(CH₂)₉CH₂CH₂NH), 1.75 (m, 2H, CH₃(CH₂)₉CH₂CH₂NH),

1.36−1.32 (m, 18H, CH₃(CH₂)₉CH₂CH₂NH), 0.93 (t, 3H,

CH₃(CH₂)₉CH₂CH₂NH, J = 6.9 Hz); ¹³C NMR (CD₃OD, 125

MHz) δ 170.9, 165.9, 152.0, 143.5, 110.1, 103.1, 94.5, 85.3, 80.3, 77.7,

76.3, 74.2, 73.7, 71.2, 63.8, 44.0, 33.1, 30.7, 30.6, 30.5, 30.2, 28.2, 27.6,

26.8, 23.7, 14.4; ESIMS-HR m/z calcd for C₂₈H₄₉N₄O₁₁617.3320,

found 617.3381.

MraY Enzymatic Assay. The activities of the compounds were

tested against purified MraY from B. subtilis.²³The assay was

performed in a reaction mixture (10 μL) containing, in final

concentrations, 100 mM Tris-HCl, pH 7.5, 40 mM MgCl₂, 1.1 mM

C₅₅-P, 250 mM NaCl, 0.25 mM UDP-MurNAc-[¹⁴C]pentapeptide

(337 Bq), and 8.4 mM N-lauroylsarcosine. The reaction was initiated

by the addition of MraY enzyme, and the mixture was incubated for 30

min at 37 °C under shaking with a thermomixer (Eppendorf). The

reaction was stopped by heating at 100 °C for 1 min. The radiolabeled

substrate UDP-MurNAc-pentapeptide and reaction product (lipid I,

product of MraY) were separated by TLC on silica gel plates LK6D

(Whatman) using 2-propanol/concentrated ammonium hydroxide/

water (6:3:1; v/v/v) as a mobile phase. The radioactive spots were

located and quantified with a radioactivity scanner (model Multi-

Compound 10i. Compound 21 (29.3 mg, 0.025 mmol) and 10%

Pd(OH)₂/C (10 mg) in MeOH (3 mL) were vigorously stirred under

H₂atmosphere at room temperature for 5 h. The insoluble was filtered

off through Celite pad, and the filtrate was concentrated in vacuo. The

residue was suspended in AcOEt, and the insoluble was filtered off

through a short silica gel pad. The filtrate was concentrated in vacuo to

afford the crude amine. Boc-Met-OH (6.86 mg, 0.025 mmol) and

HOBt (3.38 mg, 0.025 mmol) in DMF (2 mL) were treated with

EDCI (5.36 mg, 0.028 mmol) at 0 °C, and the mixture was stirred at

the same temperature for 1 h. The crude amine in DMF (1 mL) was

added to the solution of the amino acid at 0 °C, and the mixture was

stirred at room temperature for 8 h. The reaction mixture was

partitioned between AcOEt and 1 M aqueous HCl. The organic phase

was washed with H₂O, saturated aqueous NaHCO₃, and saturated

aqueous NaCl, dried (Na₂SO₄), filtered, and concentrated in vacuo.

The residue was treated with 80% aqueous TFA (2 mL) for 8 h, and

the solution was concentrated in vacuo. The residue was triturated

from diethyl ether to afford 10i (16.2 mg, 72%) as a white foam.

Compound for biological assays was purified by HPLC (YMC J'sphere

ODS M80, 10 mm × 150 mm, 0.1% TFA, a linear gradient from 60%

1

to 80% MeOH−H₂O for 14 min). H NMR (CD₃OD, 400 MHz) δ

7.65 (d, 1H, H-6, J_6,5= 8.0 Hz), 5.76 (d, 1H, H-1′, J₁′_{, 2}′ = 2.3 Hz), 5.71

(d, 1H, H-5, J_5,6= 8.0 Hz), 5.17 (s, 1H, H-1″), 4.56 (d, 1H, H-5′, J₅′_{, 4}′

= 5.7 Hz), 4.27 (m, 3H, H-2′, H-4′ and CHCH₂(CH₂)₁₃CH₃), 4.16

(m, 1H, H-3′), 4.03 (m, 4H, H-2″, H-3″, H-4″, H-2-Met), 3.89 (s, 1H,

H-6′), 3.33 (m, 1H, H-10′a), 3.24 (m, 4H, H-8′a, H-10′b, H-5″), 2.61

(m, 2H, H-4-Met), 2.11 (m, 5H, H-3-Met, SMe), 1.87 (m, 2H, H-9′),

1.71 (m, 2H, CHCH₂(CH₂)_{1 3}CH₃), 1.26 (m, 26H,

8436

dx.doi.org/10.1021/jm200906r | J. Med. Chem. 2011, 54, 8421−8439

Journal of Medicinal Chemistry

Article

Tracemaster LB285; EG&G Wallac/Berthold). IC₅₀values were

calculated with respect to a control assay without the inhibitor. Data

represent the mean of independent triplicate determinations.

ASSOCIATED CONTENT

■

S

* Supporting Information

Full experimental procedures, compound purities by HPLC,

and NMR data for all new compounds. This material is

available free of charge via the Internet at http://pubs.acs.org.

WecA Enzymatic Assay. A standard WecA assay^26,27was

performed in a reaction mixture (10 μL) containing, in final

concentrations, 100 mM Tris-HCl buffer, pH 8, 10 mM MgCl₂, 1.1

mM C₅₅-P, 0.16 mM UDP-[¹⁴C]GlcNAc (550 Bq), and 92.7 mM

Triton X-100. The reaction was initiated by the addition of WecA

enzyme, and the mixture was incubated for 30 min at 65 °C. The

reaction was stopped by heating at 100 °C for 1 min, and the

radiolabeled substrate and product, UDP-GlcNAc and C₅₅-PP-

GlcNAc, were separated by thin-layer chromatography on silica gel

plates LK6D (Whatman) using 2-propanol−ammonium hydroxide−

water (6:3:1; v/v/v) as a mobile phase. The radioactive spots were

located and quantified with a radioactivity scanner (model Multi-

Tracemaster LB285; Berthold- France). For WecA activity, residual

activities and IC₅₀values were calculated with respect to a control

assay without the inhibitors. Data represent the mean of independent

triplicate determinations, and the standard deviation was less than

20%.

AUTHOR INFORMATION

■

Corresponding Author

*Phone: (+81) 11-706-3229. Fax: (+81) 11-706-4980. E-mail:

ichikawa@pharm.hokudai.ac.jp.

Present Address

^∥Department of Internal Medicine, Faculty of Medicine and

Health Sciences, United Arab Emirates University, P.O. Box

17666, Al Ain, United Arab Emirates.

ACKNOWLEDGMENTS

■

We acknowledge a Grant-in-Aid for Young Scientists (A)

(support of T.T. and S.I.) and Grant CNRS UMR 8619

(support of B.A.-D. and A.B.). We thank Kouichi Uotani

(Discovery Research Laboratories, Shionogi & Co., Ltd.) for

evaluating the antibacterial activities of the synthesized

analogues. We thank S. Oka and A. Tokumitsu (Center for

Instrumental Analysis, Hokkaido University, Japan) for

measurement of the mass spectra.

Antibacterial Activity Evaluation. Vancomycin-resistant Enter-

ococcus faecalis SR7914 (VanA) and Entercoccus faecium SR7917

(VanA) and methicillin-resistant Staphylococcus aureus SR3637 were

clinical isolates collected from hospitals of Japan and kindly provided

by Shionogi & Co., Ltd. (Osaka, Japan).²⁵MICs were determined by a

microdilution broth method as recommended by the NCCLS

(National Committee for Clinical Laboratory Standards, 2000,

National Committee for Clinical Laboratory Standards, Wayne, PA)

with cation-adjusted Mueller−Hinton broth (CA-MHB) (Becton

Dickinson, Sparks, MD). Serial 2-fold dilutions of each compound

were made in appropriate broth, and the plates were inoculated with 5

× 10⁴CFU of each strain in a volume of 0.1 mL. Plates were incubated

at 35 °C for 20 h, and then MICs were scored.

ABBREVIATIONS USED

■

Cpm, L-epi-capreomycidine; CR, carbohydrate recognition; CL,

cytoplasmic loop; FOS, function-oriented synthesis; HepG2,

human hepatocellular liver carcinoma; MRY, muraymycin; PL,

periplasmic loop; MraY, phospho-MurNAc-pentapeptide trans-

ferase; MRSA, Staphylococcus aureus; SAR, structure−activity

relationship; U4CR, Ugi four-component reaction; C₅₅-P,

undecaprenyl phosphate; VRSA, vancomycin-resistant Staph-

ylococcus aureus

Assay of Cytotoxicity. HepG2 cells were suspended in

Dulbecco's modified Eagle’s medium containing 10% fetal bovine

serum and then seeded in 96-well tissue culture plates at 1 × 10⁴cells/

well. After 24 h, cells were treated with varying concentrations of 8b−

e, 9b−e, or tunicamycin (as a positive control) for 48 h. After the

treatment, WST-8 reagent (Kishida Chemical) was added to each well,

and cells were incubated for 1.5 h at 37 °C. The cell viability was

measured as the absorbance at 450 nm, and percentage inhibition in

growth was calculated against that of cells treated without those

compounds. Tunicamycin exhibited cytotoxicity with an IC₅₀of 26.8

μg/mL. IC₅₀values of all the compounds tested were >100 μg/mL.

Conformation Analysis. The number of carbon atom of the

lipophilic side chain was reduced for the MRY, and the tetrapeptide

was omitted for UDP-MurNAc to simplify the calculation. The energy-

minimized conformations of 24 and 25 were calculated by a

conformational search by a MacroModel program, version 9.2.⁴³

The ionization status in H₂O at pH 7 ± 1 was first predicted by Epik,⁴⁴

which is an empirically based pK_apredictor and ionization state

generator based upon the Hammett and Taft methodologies. These

structures were used for the following conformational analysis.

Conformational searching was carried out using the Monte Carlo

multiple minimum (MCMM) method⁴⁵(100 000 steps), followed by

Polak−Ribiere conjugate gradient (PRCG) minimization⁴⁶with the

OPLS 2005 force field. Water was chosen for a solvent with the GB/

SA model.⁴⁷The other settings were used as default. Structural

analysis of energy-minimum conformers calculated for 24 and 25

indicates several conformers within 25 kJ/mol (6.2 kcal/mol). Among

them, the calculated conformers where the uracil base interacts with

the remaining residue in the molecule were not used for further

analysis because the MraY should include the nucleotide-binding motif

considering the reaction mechanism and the uracil base posed at the

clef of the motif. Finally the calculated conformers were refined by

density functional theory (DFT) quantum mechanical calculations at

the BL3LYP/6-31G* level.⁴⁸

REFERENCES

■

(1) (a) Newman, D. J.; Cragg, G. M. Natural products as sources of

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(b) O'Shea, R.; Moser, H. E. Physicochemical properties of

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