Function-oriented synthesis of simplified caprazamycins: Discovery of oxazolidine-containing uridine derivatives as antibacterial agents against drug-resistant bacteria

J. Med. Chem. 2010, 53, 3793–3813 3793

DOI: 10.1021/jm100243n

Function-Oriented Synthesis of Simplified Caprazamycins: Discovery of Oxazolidine-Containing

Uridine Derivatives as Antibacterial Agents against Drug-Resistant Bacteria

Kensuke Ii,^†Satoshi Ichikawa,*^,†Bayan Al-Dabbagh,^‡Ahmed Bouhss,^‡and Akira Matsuda*^,†

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

‡

ꢀ

Laboratoire des Enveloppes Bacteriennes et Antibiotiques, Institut de Biochimie et Biophysique Moleculaire et Cellulaire,

UMR 8619 CNRS, Universite Paris-Sud, Bat. 430, Orsay F-91405, France

ꢀ

^

Received February 24, 2010

The rational simplification of the caprazamycin (CPZ) class of nucleoside natural products was carried

out to address their molecular complexity. First, analogues 6-8, where the diazepanone ring of the CPZ

6⁰

was removed and a lipophilic side chain was attached to either the C-7⁰or N atom, were used to

investigate the conformation-activity relationship. On the basis of this relationship, we designed the

oxazolidine-containing uridine derivatives 18-21 by restricting the conformation of 6-8. As a result,

the Bu ester derivatives 20 were found to be the most active against a range of bacterial strains

t

containing VRE with a potency similar to that of the parent CPZs. This study provides a novel strategy

for the development of a new type of antibacterial agent effective against drug-resistant bacteria.

Introduction

The caprazamycins (CPZs) (Figure 1, 1) were isolated

from a culture broth of the actinomycete strain Strepto-

myces sp. MK730-62F2 in 2003³and represent the newest

members of a class of naturally occurring 6⁰-N-alkyl-5⁰-β-O-

aminoribosyl-C-glycyluridine antibiotics including the lipo-

sidomycins⁴(LPMs, 2). Recently, the genes for CPZ bio-

synthesis in Streptomyces sp. MK730-62F2⁵and a LPM

gene cluster in Streptomyces sp. SN-1061M⁶have been

identified. The CPZs have shown excellent antimycobacter-

ial activity in vitro not only against drug-susceptible (MIC=

3.13 μg/mL) but also multidrug-resistant Mycobacterium

tuberculosis strains (MIC = 3.13 μg/mL) and exhibit no

significant toxicity in mice. A biological target of the 6⁰-N-

alkyl-5⁰-β-O-aminoribosyl- C-glycyluridine class of antibio-

tics is believed to be MraY translocase,⁷and it is known

Natural products can be viewed as a population of privi-

leged structures selected by evolutional pressures to interact

with a variety of biological targets and therefore are still a rich

source for drug development. Roughly 60% of new drug

entries between 1981 and 2006 were natural products and

their derivatives.¹However, some biologically relevant natu-

ral products possess rather large, complex, or labile chemical

structures compared to synthetic drugs, which limits chemical

modification in a process pursuing a structure-activity rela-

tionship (SAR). It is also true that unlike simpler synthetic

compounds, natural products can be limited in supply owing

to sourcing limitations or the impracticality of synthesis. There

are two ways to resolve these problems: one is to develop new

reactions and synthetic strategies that allow for shorter routes

to a target, and the other is to design less complex targets with

comparable or superior function that could be made in a

practical and even synthetically novel manner. Wender et al.

termed this process function-oriented synthesis (FOS^a),²the

centralprincipleofwhich is that“the function ofa biologically

activeleadstructure canbeemulated, tuned, or even improved

by replacement with simpler scaffolds designed to incorporate

the activity-determining structural features of the lead com-

pounds.” Although challenging, simpler scaffold designs

would provide a practical synthesis of a set of analogues and

synthetic innovation in current medicinal chemistry.

that this class of antibiotics strongly inhibits MraY (IC₅₀

=

0.05 μg/mL for LPMs).^4hThis integral membrane enzyme

catalyzes transfer of a phosphomuramoylpentapeptide

moiety onto a undecaprenyl phosphate (C₅₅-P) carrier lipid,

an essential step of peptidoglycan biosynthesis.^8-10Since

MraY is an essential enzyme among bacteria,¹⁰it is poten-

tially a novel target for the development of antibacterial

agents to treat drug resistant bacteria such as methicillin-

resistant Staphylococcus aureus (MRSA), vancomycin-

resistant Enterococcus (VRE), and vancomycin-resistant

Staphylococcus aureus (VRSA).¹¹With such excellent bio-

logical properties, the CPZs themselves are expected to

become promising leads for the development of antibacter-

ial agents with a novel mode of action. However, because

of their molecular complexity, the CPZs are not suitable for

use in preparing a large number of analogues for the study

of a SAR in a lead optimization process. Here we describe

the rational simplification of the CPZ class of nucleoside

natural products to address the issue associated with their

molecular complexity through FOS.

*To whom correspondence should be addressed. For S.I.: phone,

(þ81) 11-706-3763; fax, (þ81) 11-706-4980; e-mail, ichikawa@pharm.

hokudai.ac.jp. For A.M.: phone, (þ81) 11-706-3228; fax, (þ81) 11-706-

4980; e-mail, matuda@pharm.hokudai.ac.jp.

^aAbbreviations: CPZ, caprazamycin; FOS, function-oriented synth-

esis; LPM, liposidomycin; MIC, minimum inhibitory concentration;

MRSA, methicillin-resistant Staphylococcus aureus; SAR, structure-

activity relationship; VRE, vancomycin-resistant Enterococcus; VRSA,

vancomycin-resistant Staphylococcus aureus.

r

2010 American Chemical Society

Published on Web 04/20/2010

pubs.acs.org/jmc

3794 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9

Ii et al.

Figure 1. Structures of caprazamycins, liposidomycins, and their analogues.

Results and Discussion

fatty acyl side chain, was in good accordance with that

observed in the X-ray crystal structure of 5.^3dNext, the

conformations of 5 were searched by molecular mechanics to

determine whether the solution and solid state structures of 5

are correlated to the global-energy minimum conformation

calculated by conventional molecular mechanics. The global

energy-minimized conformations of 5 were calculated by

a conformational search.¹⁸Structural analysis of energy-

minimum conformers revealed two classes of conformers

(Figure 2c). In the first class, the global energy-minimized

conformation was quite similar to that experimentally ob-

served in solution and solid state in terms of the global spatial

orientation of the three key moieties although the conforma-

tion of the diazepanone was distorted. In the second class,

the aminoribose and the diazepanone moieties intercon-

verted by rotation about the C4⁰-C5⁰bond. Similar results

were obtained for palmitoylcaprazol (3), the calculations of

which were actually conducted for 3⁰with a short acyl

(butanoyl) group for the sake of simplification (Figure 2d).

Since the diazepanone moiety is contributory to but not

essential for antibacterial activity, it was predicted that the

characteristic diazepanone ring system of the CPZs must

play a role as a scaffold on which to hang the three key

components mentioned above, thereby allowing them to be

placed in the right orientation to interact with the target

MraY. The diazepanone moiety is also one of the character-

istic structures endowing the CPZs with molecular complex-

ity, and thus, it might be possible to simplify the chemical

structure. To obtain some insight into the rational simplifi-

cation described later, a series of acyclic analogues 6-8,

where the diazepanone ring of the CPZs was removed and

Decoding the Role of the Diazepanone and Aminoribofur-

anose Moiety in Caprazamycins. Recently, we have revealed

that palmitoylcaprazol (3) and its N-desmethyl analogue 4,

which possess a simple fatty acyl side chain at the 3⁰⁰⁰-

position of the diazepanone moiety of the CPZs, exhibited

antibacterial activity with a potency similar to that of the

CPZs (MIC = 6.25 μg/mL against M. smegmatis, 6.25-

12.5 μg/mL against drug-resistant bacteria including MRSA

and VRE strains).¹²Our SAR studies of several key trun-

cated analogues of 3 have also revealed that the uridine, the

aminoribose, and the fatty acyl moieties are crucial structur-

al units of palmitoylcaprazol, and the 5⁰-β-O-aminoribosyl-

C-glycyluridine structure is predicted to be a pharmaco-

phore.¹³On the other hand, the diazepanone moiety was

contributory to but not essential for antibacterial activity.¹⁴

These initial SAR studies prompted us to decode the role of

the diazepanone in the biological activity more precisely.

The active conformation when the CPZs bind to the target

enzyme, MraY, is not yet known because the three-dimen-

sional structure of this integral membrane protein has not

been reported. Therefore, it is important to predict the

interaction of CPZs with MraY by a structure-activity

relationship of CPZ analogues. Although the energy-mini-

mized conformation does not always reflect the biologically

active conformation of a compound, the conformational

analysis of a ligand in free form might sometimes help to

understand its interaction with the target molecule. A solu-

tion structure of synthetic caprazol¹⁵(5, Figure 2), a core

structure of the CPZs, was first measured by several NMR

experiments in D₂O.¹⁶Key features of the conformation are

as follows: the aminoribose moiety is oriented above the 3⁰-β-

face of the uridine moiety, and the 3⁰⁰⁰-hydroxyl group on the

diazepanone, on which a fatty acyl side chain is installed in

the CPZs, is positioned away from the 3⁰-β-face as shown in

Figure 2b. This solution conformation, including the relative

spatial orientations of the uridine, the aminoribose, and the

6⁰

the lipophilic side chain was attached to either the C-7⁰or N

atom with amide or urea linkages, was used to determine the

conformation-activity relationship as shown in Figure 3.

First, the global energy-minimized conformations of these

analogues 6-8 were calculated by a conformational search in

a manner similar to 5. The ionization status for 6-8 in H₂O

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3795

Figure 2. Structure and conformational analysis of 5 and 3⁰: (a) chemical structures of caprazol (5) and butanoylcaprazole (3⁰); (b) solution

structure of 5 in D₂O; (c) calculated two lowest energy-minimum conformers of (c) 5 and (d) 3⁰. Macro-Model program, version 9.0, was

used for conformational search. 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 the

solvent with the GB/SA model. The other settings were used as default.

at pH 7 ( 2 was first predicted by Epik,²⁴which is an

empirically based pK_apredictor and ionization state gen-

and 8 belong to class II. From these calculations, the compounds 6

were expected to have MraY inhibitory activity.

erator based upon the Hammett and Taft methodologies,

On the basis of these predictions, analogues 6-8 were

synthesized. As a lipophilic side chain, linear alkyl and alkyl

groups containing a benzene ring were chosen. Preparation

of the key intermediate 12 was previously reported by our

group¹²(Scheme 1). However there was a problem in base-

catalyzed hydrolysis of the methyl ester 11, which was

sensitive to the basic conditions resulting in partial decom-

position. Moreover, controlling the reaction temperature

was quite important and it was sometimes difficult to main-

tain reproducibility especially in large scale preparations.

Therefore, we sought to improve this step by using LiI in

refluxing AcOEt under near-neutral conditions²⁵to pro-

vide the carboxylic acid 12 in 81% yield. These condi-

tions minimized the decomposition of 11 especially through

β-elimination initiated by deprotonation at the 6⁰-position.

This improvement enabled us to prepare a sufficient quantity

of the material necessary for further derivatization. As for

preparation of the key intermediate 14 for the synthesis of 7

and 8, the carboxyl group of 12 was further protected as a ^tBu

0

generating both a protonated and free form at the N⁶-

nitrogen atom for 6 and only one ionization form for 7 and

8. These structures were used for the following conforma-

tional analysis. The lipophilic side chain of each structure

was reduced to four carbon atoms to simplify the calcula-

tions. Structural analysis of energy-minimum conformers

calculated for each of the 6-8 analogues falls into two

similar classes of conformers, respectively. The classification

was similar to that obtained for 3⁰and 5, and the results are

summarized in Figure 4. In one class, the spatial positions of

the uridine, the aminoribose, and the fatty acyl moiety are

similar to those observed in the conformational analyses of 5

(class I). In the other, the aminoribose moiety and the fatty

acyl side chain interconvert by rotation about the C4⁰-C5⁰

bond (class II). For example, the global energy-minimum

conformer of protonated 6 at the 6⁰⁰-position is in class I

(Figure 4a, left), which is predicted to be 7.312 kJ/mol more

stable than the corresponding class II conformer by comparing the

potential energy of the lowest-energy conformer of each class.

According to this analysis, the global energy-minimum conformers

of the protonated and free 6 belong to class I, and compounds 7

ester to give 13, and hydrogenolysis of the Cbz group at the

N⁶-position was catalyzed by Pd/C to give the amine 14,

which was used for the next step without further purification.

0

3796 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9

Ii et al.

Figure 3. Structures of acyclic analogues.

Figure 4. Two classes of energy-minimum conformation of analogues 6a-8a. Macro-Model program, version 9.0, was used for conforma-

tional search. Ionization status for 6a-8a in H₂O under pH 7 ( 2 was predicted by the Epik program, which generated both a protonated and

0

free form at the N⁶-nitrogen atom for 6a. 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 the solvent with the GB/SA model. The other settings were used as

default. The lipophilic side chain of each structure was reduced to four carbon atoms to simplify the calculations. Shown are two lowest energy-

minimum conformers of (a) 6a, (b) 7a, (c) 8a.

The preparation of 6-8 is described in Scheme 2. The

carboxylic acid 12 was reacted with dodecylamine or

4-octylaniline under the conditions using EDCI and HOBt

in CH₂Cl₂to afford the corresponding amides 15a (93%) or

15b (77%), respectively. A two-step deprotection sequence of

15a or 15b with hydrogenolysis of the Cbz group and acid-

catalyzed hydrolysis of the isopropylidene, 3-pentylidene,

and Boc groups provided the N-amide derivatives 6a (95%

over two steps from 15a) or 6b (89% over two steps from 15b)

0

as TFA salts. In a similar manner, the N⁶-acyl derivatives

16a,b were prepared by acylation of 14 with either dodeca-

noic acid or 4-octylbenzoic acid (80% over two steps for 16a,

82% over two steps for 16b) by using EDCI and NaHCO₃in

CH₂Cl₂. The urea derivatives 17a,b were also prepared by

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3797

Scheme 1

Scheme 2

Table 1. Inhibitory Activities of the Synthesized Compounds against

MraY^a

C-amide

6b

46

N-amide

7b

256

urea

6a

7a

8a

8b

IC₅₀(μM)

18

63

36

75

^aThe activities of the compounds were tested against purified MraY

from B. subtilis. The assay was performed in a reaction mixture of 10 mL

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-Mur-

NAc-[¹⁴C]pentapeptide (337 Bq), and 8.4 mM N-lauroyl sarcosine.

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.

are shown in Table 1. The assay was performed by quantify-

ing the incorporation of MurNAc-[¹⁴C]pentapeptide by

MraY from UDP-MurNAc-[¹⁴C]pentapeptide into lipid I.

The acyclic analogues 6-8 exhibited relatively moderate

MraY inhibitory effects. Compounds 6, which have the class

I global energy-minimum conformation, were more active

compared to 7 and 8, compounds with the class II global

energy-minimum conformation. Thus, the MraY inhibitory

activity of a set of analogues was correlated to some extent to

the type of conformers classified by global energy-minimum

calculation by these results. It is noteworthy that the pre-

sence of a benzene ring attached to the lipophilic side chain

led to a decrease in inhibitory efficiency. Antibacterial

activity of the analogues was evaluated against a range of

bacterial strains including MRSA and VRE, and the mini-

mum inhibitory concentrations (MICs, μg/mL) are sum-

marized in Table 2. The C-amide derivatives 6, categorized

as class I according to the global energy-minimum con-

former, exhibited antibacterial activity against not only

drug-susceptible but also drug-resistant Enteroccoci strains

(VRE). On the other hand, analogues 7 and 8, which were in

the class II conformation, lost antibacterial activity, which

was correlated to the MraY inhibitory activity. Although

active, both MraY inhibitory and antibacterial activities of

treatment of 14 with either dodecyl isocyanate or 4-octyl-

phenyl isocyanate (77% over two steps for 17a, 58% over two

steps for 17b), respectively. Global deprotection of 16 and 17

with 80% aqueous TFA successfully afforded the desired

analogues 7 and 8 in good yield as shown in Scheme 2.

The inhibitory effects of a series of acyclic compounds on

purified MraY activity were first evaluated,⁷and the results

3798 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9

Table 2. Antibacterial Activity of 6-8

Ii et al.

MIC (μg/mL)^a

E. faecalis ATCC E. faecalis SR7914 E. facium ATCC E. faecium SR7917

S. aureus ATCC 29213

(MSSA)

S. aureus SR3637

(MRSA)

compd

29212

(VRE)

19434

(VRE)

6a

6b

7a

7b

8a

8b

64

16

32

64

16

32

16

32

>64

1

>64

1

>64

64

>64

64

>64

1

>64

64

>64

vancomycin

2

^aMICs 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

Figure 5. Proposed binding models.

Figure 6. Structural comparison in terms of the rotatable bonds.

analogues 6-8 were largely reduced compared to the parent

natural products, indicating that the diazepanone moiety

was not essential for but contributory to antibacterial acti-

vity. Together with studies previously reported,²⁶it was

revealed experimentally that the characteristic diazepanone

ring system of the CPZs plays a role as a scaffold on which to

hang three key components, thus allowing them to be placed

in the right orientation to interact with the target MraY

exhibiting antibacterial activity. We further predicted the

interaction of the CPZs with MraY. MraY catalyzes the first

membrane step of peptidoglycan biosynthesis, where UDP-

MurNAc-pentapeptide is attacked by the undecaprenyl

phosphate in the bacterial cell membrane leading to the

formation of lipid I. Although the three-dimensional struc-

ture is not yet available, extensive studies to elucidate the

molecular basis of MraY suggested that several Asp residues

are essential for the catalytic activity.¹⁰It was proposed that

in B. subtilis, residues H45, D174, and D177 are involved in

Figure 7. Structures of oxazolidine analogues.

the binding with Mg^2þ, forming a salt bridge with the

diphosphate moiety of the UDP-MurNAc-pentapeptide

(Figure 5a). It was also reported that the amino group at

the 5⁰⁰-position of 6⁰-N-alkyl-5⁰-β-O-aminoribosyl-C-glycy-

luridine class of antibiotics is necessary for antibacterial

activity.^26cConsidering the structural similarity of the

UDP-MurNAc-pentapeptide and the CPZs that share a

uridine moiety, it is suggested that the ammonium group of

the aminoribose moiety could form an ionic bond with the

essential residues involved in the binding of the metal cation

(Figure 5b). From this model, we speculated that the ribo-

furanose ring system might also behave as a scaffold linking

the terminal ammonium group and the uridine moiety in a

proper spatial position.

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3799

Scheme 3

Design, Synthesis, and Biological Evaluation of Oxazoli-

could restrict the number of possible conformers and there-

fore reduce the molecular complexity and retain the relative

spatial orientation of the three key components of the CPZs.

Incorporating sequential rotatable bonds through N-6⁰-C-

6⁰-C-5⁰-O-5⁰-C-1⁰⁰into a single ring scaffold would result

in simultaneous restriction of all the dihedral angles χ₂-χ₆.

The oxazolidine ring was selected to be constructed by

linking a nitrogen atom at the 6⁰-position and a carbon atom

at the 1⁰⁰-position of either 6a or 7a as shown in Figure 7. The

oxazolidine ring formation might be enough to restrict the

three key components, and the ribofuranose moiety and

the diazepanone ring system could then be removed accord-

ing to our model for the molecular basis of the activity of the

CPZs. Likewise we designed the oxazolidine-containing

dine-Containing Uridine Derivatives. The acyclic analogues

6-8 are rather flexible compounds with a number of rota-

table bonds. As shown in Figure 6, the dominant determi-

nants of the conformation of 6a or 7a are the dihedral angles

χ₁-χ₇. Retrospectively considering the conformation of the

CPZs compared to the acyclic analogues 6-8, the number of

the rotatable dihedral angles is reduced resulting in restric-

tion of the conformation because the CPZs possess the

diazepanone ring. This is presumably one of the reasons

for the reduced biological activity of the acyclic analogues

6-8 with the increased number of the rotatable dihedral

angles. We hypothesized that by reducing the number of the

rotatable dihedral angles in the acyclic analogues 6-8, we

3800 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9

Ii et al.

Scheme 4

uridine derivatives 18-20 from 7a and 21 from 6a. This

design allowed us to simplify drastically the molecular archi-

tecture of the parent CPZs.

Syntheses of the oxazolidine-containing uridine deriva-

tives 18-20 and 21 are summarized in Schemes 3 and 4,

respectively. The isopropylidene protecting group of 10 was

removed by acid-catalyzed hydrolysis, and the resulting triol

was selectively protected by a TBS group to give the 2⁰,3⁰-di-

O-TBS derivative 22 in 75% yield over two steps. Hydro-

genolysis of the Cbz group at the N⁶-position in 22 cleanly

provided the amino alcohol 23. An oxazolidine ring was

constructed simply by mixing 23 with azidoacetaldehyde²⁷to

give 24 as a mixture of diastereomers at the newly formed

stereogenic center (1⁰⁰-position) judging by TLC and ¹H

NMR analysis of the reaction mixture (about 2:1). Upon

treatment of the crude oxazolidine 24 with palmitoyl chlor-

ide, the N-palmitoyloxazolidine derivative 25a with the

R-configuration at the aminal center was selectively obtained

in 76% yield from 23.²⁸Determination of the stereochemis-

try of the newly formed stereogenic center (1⁰⁰) was con-

firmed to be R (described later). The reaction of 23 with

3-azidopropanal²⁷or 4-azidobutanal²⁷also gave 25b (79%

over two steps) and 25c (87% over two steps), respectively,

after palmitoylation.

Figure 8. Key NOE correlations observed in CD₃OD.

sterically hindered ester at the 7⁰-position might restrict

the rotation around the C4⁰-C5⁰σ-bond linking the two

rings, the ribofuranose and the oxazolidine. Thus, one of the

remaining dihedral angles, χ₁, could also be modulated by

the choice of a substituent at the carboxyl terminal of the

oxazolidine derivatives. With this in mind, we planned to

synthesize the ^tBu ester analogues 20 in addition to 19. The

methyl esters 28a-c were obtained simply by deprotection of

the TBS groups of 25 (90% for 28a, 89% for 28b, 92% for

28c) followed by catalytic hydrogenation of the azide group

in the presence of HCl (54% for 19a, 57% for 19b, 56% for

19c). In the absence of HCl, catalytic hydrogenation of 28b

gave the bicyclic lactam 29 in 53% yield, which was produced

by cyclization upon generation of the free amine. This result

clearly indicated that the relative configuration of the ami-

noalkyl group on the oxazolidine ring was cis to the methyl

ester group. The formation of 29 unambiguously determined

The methyl ester of 25 was converted to the carboxylic acid

26 (78% for 26a, 67% for 26b, 74% for 26c) with LiI in

refluxing AcOEt. Finally, deprotection of the TBS groups

with 3HF Et₃N in MeCN-CH₂Cl₂afforded 27 (89% for

3

27a, 92% for 27b, 95% for 27c), which was followed by

reduction of the azide group to the amine by hydrogenation

catalyzed by Pd(OH)₂/C to afford the target oxazolidine-

containing uridine derivatives 18 (58% for 18a, 68% for 18b,

68% for 18c). The 7⁰-carboxyl group was not necessary for

antibacterial activity, since the C-amide derivatives 6a,b

showed antibacterial activity. Therefore, the methyl ester

derivatives 19a-c were also prepared in order to observe the

impact of the carboxyl group attached to the oxazolidine ring

on the antibacterial activity. It was also thought that a more

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3801

Table 3. Antibacterial Activity of Oxazolidine Analogues

MIC (μg/mL)^a

E. faecalis ATCC E. faecalis SR7914 E. facium ATCC E. faecium SR7917

S. aureus ATCC 29213

(MSSA)

S. aureus SR3637

(MRSA)

compd

I8a

18b

29212

(VRE)

19434

(VRE)

>64

8

>64

13

>64

32

8

>64

64

>64

32

>64

16

18c

19a

19b

19c

8

16

4

8

16

8

4

20a

20b

8

4

2

4

8

16

32

2

16

2

16

8

16

21a

21b

32

16

32

0.5

1

16

32

16

21c

28b

>64

0.5

1

>64

0.5

1

>64

2

64

28c

4

vancomycin

>64

1

>64

^aMICs 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

the stereochemistry of the aminal moiety of 18 and 19 to be

the R-configuration. The tert-butyl esters 20a,b were also

synthesized from 26a,b. Protection of the carboxylic acid 26

with N,N⁰-diisopropyl-O-tert-butylisourea²⁹in the presence

of ammonium chloride in ^tBuOH-CH₂Cl₂gave the ^tBu ester

30 (73% for 30a, 76% for 30b). The TBS groups of 30 were

removed in a similar manner to give 31 (79% for 31a, 80%

for 31b) followed by reduction of the azide group to give 20a

(54%) and 20b (50%), respectively. Analogues 21a-c, which

possess an N-hexadecylcarbamoyl group at the 6⁰-position,

were synthesized as shown in Scheme 4. Oxazolidine formation

of the amine 23 followed by acetylation of the resulting

secondary amine selectively gave the N-acetyloxazolidines

32a-c with the R-configuration at the aminal center (86%

for 32a, 85% for 32b, 88% for 32c). The methyl esters 32a-c

were converted to the carboxylic acids 33a-c (73% for 33a,

75% for 33b, 81% for 33c), which were condensed with

hexadecylamine using EDCI and HOBt to provide 34a-c

(71% for 34a, 70% for 34b, 84% for 34c). Finally, the target

analogues 21a-c were obtained by deprotection of the TBS

groups (81% for 35a, 76% for 35b, 88% for 35c) followed by

catalytic hydrogenation of the azide group (69% for 21a,

72% for 21b, 51% for 21c). All the analogues were purified

by C18 HPLC (80-90% aqueous MeOH, >98% purity)

and used for the evaluation of the antibacterial activity.

With the analogues in hand, the conformation of the

synthesized oxazolidine derivatives was then analyzed by

NMR experiments. For the sake of the molecular structure

of the oxazolidine analogues, where the tetrahydrofuran and

oxazolidine rings are connected through a single σ-bond, the

conformations of the analogues 18-21 are rather restricted

and are largely governed by the rotatable dihedral angle of

the σ-bond linking the two rings (χ₁). This property allowed

Table 4. Inhibitory Activities of the Oxazolidine Derivatives against

MraY^a

18a

18b

18c

19a

19b

19c

IC₅₀(μM)

740

451

360

980

920

1200

^aExperiments were conducted using the same procedure as in Table 1.

observed upon irradiation at H-1⁰⁰. These data indicated that

the dihedral angles χ₁were estimated to be approximately

60° (clockwise from H-4⁰to H-5⁰) with the two endocyclic

oxygen atoms of the tetrahydrofuran and oxazolidine rings

in a gauche relationship. As a result, the aminoalkyl group

attached to the oxazolidine ring at the 1⁰⁰-position with the

R-configuration is oriented above the 3⁰-β-face of the uridine

moiety with the amino group vertical to the tetrahydrofuran

face, and the lipophilic side chain is positioned away from the

3⁰-β-face. The overall conformation of the oxazolidine ana-

logues resembles that observed and calculated for caprazol

(5) or the class I conformers of 6-8.

The antibacterial activity of three sets of the oxazolidine

analogues was evaluated, and the data are shown in Table 3.

Analogues with the carboxyl group 18a-c showed no anti-

bacterial activity with MIC up to 64 μg/mL. The correspond-

ing ester analogues 19 and 20, on the other hand, exhibited

antibacterial activity against drug-susceptible S. aureus,

E. faecalis, and E. faecium with MICs of 2-16 μg/mL. These

analogues were also active against drug-resistant bacterial

strains such as MRSA and VRE. It is noteworthy that the

antibacterial activity of the tert-butyl ester analogues 20a,b

was most potent against both the drug-susceptible and drug-

resistant E. faecalis with a potency similar to that of des-

methylpalmitoylcaprazol (4). The large contrast between

analogues with the carboxylic acid 18 and the esters 19 and

20 in terms of antibacterial activity may be attributed to the

permeability of the analogues with regard to the bacterial cell

membrane. The amino function at the oxazolidine moiety

plays an important role in antibacterial activity by the fact

that azide derivatives 28b and 28c showed reduced antibac-

terial activity to a large extent. The analogues 21a-c, which

possess an N-hexadecylcarbamoyl group at the 6⁰-position,

also exhibited moderate activity. Although FOS of the CPZs

was successfully achieved as a function of antibacterial

us to predict the conformations of 18-20 simply by compar-

1

ing the coupling constant of H-4⁰and H-5⁰(J_{4 ,5}) from H

NMR and NOE experiments in D₂O (Figure 8). The overall

0

data of 18-21 were similar. For example, the J_{4 ,5}values

0

were relatively small (J_{4 ,5}=3.4 Hz for 18b, 2.9 Hz for 19b,

2.9 Hz for 20b, and 2.3 Hz for 21b). In addition, a strong

NOE correlation was observed for both H-3⁰and H-4⁰upon

irradiation at H-5⁰although only for H-5⁰upon irradiation at

H-6⁰(Figure 8). Characteristic correlation for H-6 was also

3802 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9

Ii et al.

activity, these analogues were revealed to be weak MraY

inhibitors with IC₅₀of 920-1200 μM (Table 4). There could

be different target(s) of the oxazolidine analogues, such as 19

and 20, and further studies are needed to elucidate their

mode of action. In summary, a novel class of molecules with

a different mode of action from the parent natural products

was generated through FOS of the CPZs in this study.

MeOH-CHCl₃) to afford 12 (6.97 g, 81%) as a white foam,

properties of which were identical in all respects to those

1

previously reported.¹²[R]²¹þ21.6 (c 1.00, CHCl₃); H NMR

D

(CDCl₃, 500 MHz) δ 7.64 (br d, 1H, H-6, J_6,5= 8.0 Hz),

7.38-7.27 (m, 5H, phenyl), 5.74 (br s, 1H, H-1⁰), 5.65 (br d,

1H, H-5, J_5,6=8.0 Hz), 5.22 (s, 1H, H-1⁰⁰), 5.16 (d, 1H, benzyl,

J=12.5 Hz), 5.11 (m, 1H, H-2⁰), 5.02 (d, 1H, benzyl, J=12.5 Hz),

4.98 (m, 1H, H-3⁰), 4.90 (m, 1H, H-2⁰⁰), 4.65 (m, 1H, H-6⁰), 4.56

0

(d, 1H, H-3⁰⁰, J_{3 ,2}=5.8 Hz), 4.50 (m, 1H, H-5 ), 4.18-4.11 (m,

00 00

2H, H-4⁰, H-4⁰⁰), 3.13 (m, 2H, H-5⁰⁰a, H-5⁰⁰b), 1.60 (m, 4H,

CH₂CH₃ꢀ 2), 1.49 (s, 3H, acetonide), 1.48 (s, 9H, tert-butyl),

1.31 (s, 3H, acetonide), 0.79 (m, 6H, CH₂CH₃ꢀ 2); ¹³C NMR

(CDCl₃, 125 MHz) δ 177.5, 166.2, 158.5, 152.0, 138.3, 130.3,

129.7, 129.2, 128.7, 117.4, 115.6, 88.4, 87.9, 83.8, 83.4, 80.5, 57.4,

44.5, 30.5, 29.0, 28.6, 28.2, 27.8, 27.3, 25.8, 8.6, 7.9, 7.6;

FABMS-HR (NBA) m/z calcd for C₃₇H₄₉N₄O₁₅789.3194,

found 789.3194.

Conclusions

Compared to synthetic drugs, many biologically relevant

natural products possess large, complex, or labile chemical

structures that may restrict chemical modifications in a struc-

ture-activity relationship study. Therefore, it is quite impor-

tant to pursue FOS, a strategy for the design of less complex

structured targets with comparable or superior activity that

could be made in a practical manner.

6-Benzyloxycarbonylamino-5-O-[5-tert-butoxycarbonylamino-

5-deoxy-2,3-O-(3-pentylidene)-β-^D-ribofuranosyl]-6-deoxy-N-

dodecyl-2,3-O-isopropylidene-1-(uracil-1-yl)-β-^D-glycero-L-talo-

heptofuranuronamide (15a). A mixture of 12 (79 mg, 0.10 mmol)

and dodecylamine (27.8 mg, 0.30 mmol) in CH₂Cl₂(1 mL) was

treated with EDCI (57.5 mg, 0.30 mmol) and HOBt (40 mg,

0.30 mmol) at room temperature for 12 h. The reaction mixture

was partitioned between AcOEt and 0.5 N aqueous HCl, and the

organic phase was washed with saturated aqueous NaHCO₃and

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

was purified by silica gel column chromatography (2 cm ꢀ 6 cm,

60% AcOEt-hexane) to give 15a (89 mg, 93%) as a white foam:

¹H NMR (CD₃OD, 500 MHz) δ 7.68 (d, 1H, H-6, J_6,5=8.0 Hz),

7.43-7.31 (m, 5H, phenyl), 5.75 (s, 1H, H-1⁰), 5.69 (d, 1H, H-5,

J_5,6=8.0 Hz), 5.21 (d, 1H, benzyl, J=12.6 Hz), 5.19 (s, 1H, H-

Of significance in this study is a rational and drastic

simplification of the CPZ’s molecular architecture by decod-

ing the role of the diazepanone and aminoribofuranose

moieties in the CPZs and designing the oxazolidine scaffold

assisted by conformational considerations. It is important to

simplify the hydrophilic core structures of the CPZs in order

to reduce the size of the molecules and to stabilize the

chemically labile structure. Although this aspect of the research

is likely the most difficult to accomplish, a full chemical syn-

thetic approach based on rational drug design considering

FOS led tothe discovery of theoxazolidine-containing uridine

derivatives as the lead compounds active against drug-resistant

bacterial pathogens. Oxazolidine-containing uridine deriva-

tives possess a simple chemical structure that can be easily

prepared. The simpler scaffold design would provide a prac-

ticalsynthesisofa setofanaloguesand synthetic innovation in

current medicinal chemistry. Indeveloping novel antibacterial

agents to treat drug-resistant pathogens, 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.^30-32This study provides a novel

strategy for the development of a new type of antibacterial

agent effective against drug-resistant bacteria.

1⁰⁰), 5.11-5.09 (m, 2H, benzyl, H-2⁰), 4.94 (dd, 1H, H-3⁰, J_{3 ,2}

0

=

00

0

00 00

4.6, J_{3 ,4}= 9.2 Hz), 4.70 (d, 1H, H-2 , J_{2 ,3}₀= 5.8 Hz), 4.56

(d, 1H, H-3⁰⁰, J_{3 ,2}=5.8 Hz), 4.42 (m, 2H, H-5 , H-6 ), 4.20 (dd,

0

00 00

1H, H-4⁰, J_{4 ,3}=9.2, J_{3 ,5}=4.6 Hz), 4.11 (t, 1H, H-4 , J_{4 ,5}

=

00

0

00 00

7.5 Hz), 3.08 (m, 1H, CH₃(CH₂)₁₀CH₂NH), 3.17 (m, 2H, CH₃-

(CH₂)₁₀CH₂NH, H-5⁰⁰a), 3.00 (m, 1H, H-5⁰⁰b), 1.57-1.32 (m,

39H, CH₃(CH₂)₁₀CH₂NH, tert-butyl, CH₂CH₃ꢀ 2, acetonide),

0.93 (t, 3H, CH₃(CH₂)₁₀CH₂NH, J=6.9 Hz), 0.85, 0.84 (each t,

each 3H, CH₂CH₃, J=7.4 Hz); ¹³C NMR (CD₃OD, 125 MHz) δ

204.6, 198.8, 191.0, 190.9, 184.5, 177.9, 170.6, 162.1, 161.8,

161.7, 149.9, 148.2, 145.2, 135.5, 128.4, 121.2, 120.2, 120.0,

118.0, 116.0, 115.3, 112.9, 112.8, 100.6, 89.7, 76.8, 73.4, 65.6,

63.4, 63.3, 63.2, 63.1, 63.0, 62.9, 62.4, 61.4, 60.6, 60.1, 58.2, 56.3,

47.0, 41.3, 40.3; ESIMS-HR m/z calcd for C₄₉H₇₉N₅NaO₁₄

(M þ Na)^þ980.5208, found 980.5222.

Experimental Section

6-Benzyloxycarbonylamino-5-O-[5-tert-butoxycarbonylamino-

5-deoxy-2,3-O-(3-pentylidene)-β-^D-ribofuranosyl]-6-deoxy-2,3-

O-isopropylidene-N-(4-octylphenyl)-1-(uracil-1-yl)-β-^D-glycero-

L-talo-heptofuranuronamide (15b). Compound 15b was prepared

from12 (79mg, 0.10 mmol) and4-octylaniline (69μL, 0.30 mmol)

as described above for the synthesis of 15a. Purification by silica

gel column chromatography (2 cm ꢀ 6 cm, 66% AcOEt/hexane)

afforded 15b (75 mg, 77%) as a white foam: ¹H NMR (CD₃OD,

500 MHz) δ 7.67 (d, 1H, H-6, J_6,5=8.0 Hz), 7.49 (d, 2H, CH₃-

(CH₂)₆CH₂C₆H₄NH, J= 8.0 Hz), 7.43-7.31 (m, 5H, phenyl),

7.49 (d, 2H, CH₃(CH₂)₆CH₂C₆H₄NH, J=8.0 Hz), 5.74 (s, 1H,

H-1⁰), 5.67 (d, 1H, H-5, J_5,6=8.0 Hz), 5.23 (d, 1H, benzyl, J=12.6

General Experimental Methods. NMR spectra are reported in

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

internal standard otherwise noted. Coupling constants (J) are

reported in hertz (Hz). Abbreviations of multiplicity are as

follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multi-

plet; br, broad. Data are presented as follows: chemical shift

(multiplicity, integration, coupling constant). Assignment was

1

based on H-¹H COSY, HMBC, and HMQC NMR spectra.

MS data were obtained on a JEOL JMS-HX101 or JEOL JMS-

700TZ. Purity of all the compounds tested for biological evalua-

tion was confirmed to be >95% by HPLC and ¹H NMR

analyses.

Hz), 5.21 (s, 1H, H-1⁰⁰), 5.14-5.09 (m, 2H, benzyl, H-2⁰), 5.05 (m,

6-Benzyloxycarbonylamino-5-O-[5-tert-butoxycarbonylamino-

5-deoxy-2,3-O-(3-pentylidene)-β-^D-ribo-pentofuranosyl]-6-deoxy-

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

anuronate (12). Lithium iodide (22.0 g, 163 mmol) was added to

a solution of 11¹²(8.80 g, 10.9 mmol) in AcOEt (100 mL) at

room temperature, and the resulting mixture was heated under

refluxed for 4 h. The reaction mixture was cooled to room

temperature and diluted with AcOEt (300 mL). The solution

was washed with 0.3 M aqueous HCl, brine, dried (Na₂SO₄),

filtered, and concentrated in vacuo. The residue was purified by

silica gel column chromatography (5 cm ꢀ 17 cm, 10%

0

1H, H-3⁰), 4.71 (d, 1H, H-2⁰⁰, J_{2 ,3}=6.3Hz), 4.60 (br s, 1H, H-6 ),

00 00

4.56 (d, 1H, H-3⁰⁰, J_{3 ,2}=6.3 Hz), 4.51 (d, 1H, H-5 , J_{5 ,4}=8.6

0

00 00

0

Hz), 4.28 (m, 1H, H-4⁰), 4.07 (t, 1H, H-4⁰⁰, J_{4 ,5}=6.9 Hz), 3.13

(dd, 1H, H-5⁰⁰a, J_{5 a,4}=6.9, J_{5 a,5 b}=13.8 Hz), 2.99 (dd, 1H, H-

00 00

00

5⁰⁰b, J_{5 b,4}= 6.9, J_{5 b,5 a}= 13.8 Hz), 2.60 (t, 2H, CH₃-

(CH₂)₆CH₂C₆H₄NH, J = 6.9 Hz), 1.67-1.31 (m, 31H, CH₃-

(CH₂)₆CH₂C₆H₄NH, tert-butyl, CH₂CH₃ꢀ 2, acetonide), 0.92

(t, 3H, CH₃(CH₂)₆CH₂C₆H₄NH, J=6.9 Hz), 0.85, 0.84 (each t,

each 3H, CH₂CH₃, J=7.4 Hz); ¹³C NMR (CD₃OD, 125 MHz) δ

170.4, 166.2, 158.4, 158.3, 152.0, 145.6, 140.4, 138.0, 137.0, 129.7,

00

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3803

129.5, 129.2, 122.0, 117.4, 115.5, 112.9, 102.9, 96.5, 90.0, 87.8,

87.5, 85.5, 83.4, 82.8, 80.7, 80.3, 68.1, 57.6, 54.8, 44.2, 36.4, 33.0,

32.7, 30.6, 30.4, 30.3, 29.8, 28.8, 27.5, 25.6, 23.7, 14.5, 8.8,

7.7; ESIMS-HR m/z calcd for C₅₇H₇₁N₅NaO₁₄(M þ Na)^þ

1000.4895, found 1000.4889.

(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

14. The amine 14 in CH₂Cl₂(1 mL) was treated with dodecyl

isocyanate (240 mg, 1.0 mmol) at room temperature for 3 h. The

reaction mixture was concentrated in vacuo, and the residue was

purified by silica gel column chromatography (2 cm ꢀ 10 cm,

66% AcOEt-hexane) to give 17a (72 mg, 77% over two steps) as

a white foam: ¹H NMR (CD₃OD, 500 MHz) δ 7.71 (d, 1H, H-6,

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

pentylidene)-β-^D-ribofuranosyl]-6-deoxy-6-dodecanoylamino-2,3-

O-isopropylidene-1-(uracil-1-yl)-β-D-glycero-L-talo-heptofuranuro-

nate (16a). A mixture of 13 (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

14. The amine 14 in CH₂Cl₂(1 mL) was treated with EDCI

(57.5 mg, 0.30 mmol) and dodecanoic acid (60 mg, 0.30 mmol) at

room temperature for 30 min. The reaction mixture was diluted

with AcOEt (50 mL), which was washed with 0.3 N aqueous

HCl, saturated aqueous NaHCO₃, and brine. The organic phase

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

purified by silica gel column chromatography (2 cm ꢀ 10 cm,

66% AcOEt-hexane) to give 16a (72 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), 5.71 (d, 1H, H-1⁰, J_{1 ,2}=1.7 Hz), 5.68 (d, 1H, H-5,

0 0

_5,6=8.0 Hz), 5.22 (dd, 1H, H-2⁰, J_{2 ,1}=1.7, J_{2 ,3}=6.3 Hz), 5.08

0

(s, 1H, H-1⁰⁰), 4.86 (dd, 1H, H-3⁰, J_{3 ,2}=6.3, J_{3 ,4}=4.0 Hz), 4.71

0

00

0

(d, 1H, H-2⁰⁰, J_{2 ,3}=5.7 Hz), 4.55 (d, 1H, H-3 , J_{3 ,2}=5.7 Hz),

00 00

4.46 (m, 2H, H-5⁰, H-6⁰), 4.20 (dd, 1H, H-4⁰, J_{4 ,3}=9.2, J_{4 ,5}=3.5

0

Hz), 4.10 (t, 1H, H-4⁰⁰, J_{4 , 5}= 7.5 Hz), 3.28 (dd, 1H, H-5

00

a, J_{5 a,4}= 6.9, J_{5 a,5 b}= 14.3 Hz), 3.16 (m, 2H, CH₃-

00

(CH₂)₁₀CH₂NH), 2.99 (dd, 1H, H-5⁰⁰b, J_{5 b,4}=8.0, J_{5 b,5 a}

00

=

14.3 Hz), 1.59-1.32 (m, 39H, CH₃(CH₂)₁₀CH₂NH, tert-butyl,

CH₂CH₃ꢀ 2, acetonide), 0.93 (t, 3H, CH₃(CH₂)₁₀CH₂NH, J=

6.9 Hz), 0.84, 0.83 (each t, each 3H, CH₂CH₃, J=7.4 Hz); ¹³

C

NMR (CD₃OD, 125 MHz) δ 172.1, 166.4, 160.6, 158.5, 152.1,

146.1, 117.3, 115.4, 113.8, 102.8, 97.3, 89.0, 87.6, 87.2, 85.7, 83.6,

83.3, 82.1, 80.3, 55.2, 44.4, 40.1, 33.1, 31.3, 30.8, 30.7, 30.6, 30.5,

30.4, 29.8, 28.8, 28.5, 27.9, 27.5, 25.5, 23.7, 14.5, 8.8, 7.6;

J

_6,5=8.0 Hz), 5.70 (s, 1H, H-1⁰), 5.68 (d, 1H, H-5, J_5,6=8.0 H₀z₀),

5.23 (dd, 1H, H-2⁰, J_{2 ,1}=1.8, J_{2 ,3}=6.3 Hz), 5.08 (s, 1H, H-1 ),

0

4.83 (dd, 1H, H-3⁰, J_{3 ,2}=6.3, J_{3 ,4}=4.0 Hz), 4.76 (br d, 1H, H-

0

ESIMS-HR m/z calcd for C₄₄H₇₇N₅NaO₁₄(M

946.5365, found 946.5343.

þ

H)^þ

6⁰, J_{6 ,5}=5.7 Hz), 4.63 (d, 1H, H-2 , J_{2 ,3}₀=5.7 Hz), 4.55 (d, 1H,

00

0

00 00

H-3⁰⁰, J_{3 ,2}=5.7 Hz), 4.51 (d, 1H, H-5 , J_{5 ,4}=5.7 Hz), 4.14

00 00

0

(m, 2H, H-4⁰, H-4⁰⁰), 3.26 (dd, 1H, H-5⁰⁰a, J_{5 a,4}=6.9, J_{5 a,5 b}

00

=

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

pentylidene)-β-^D-ribofuranosyl]-6-deoxy-2,3-O-isopropylidene-6-

(4-octylphenylureido)-1-(uracil-1-yl)-β-^D-glycero-L-talo-heptofura-

nuronate (17b). Compound 17b was prepared from 13 (85 mg,

0.10 mmol) and 4-octylphenyl isocyanate (244 mg, 1.0 mmol) as

described above for the synthesis of 17a. Purification by silica gel

column chromatography (2 cm ꢀ 10 cm, 66% AcOEt/hexane)

afforded 17b (76 mg, 58% over two steps) as a white foam: ¹H

NMR (CD₃OD, 500 MHz) δ 7.71 (d, 1H, H-6, J_6,5=7.9 Hz),

7.28 (d, 2H, CH₃(CH₂)₆CH₂C₆H₄NH, J=8.0 Hz), 7.09 (d, 2H,

00 00

14.3 Hz), 3.05 (dd, 1H, H-5⁰⁰b, J_{5 b,4}=8.0, J_{5 b,5 a}=14.3 Hz),

2.36 (t, 1H, CH₃(CH₂)₉CH₂CO, J=7.5 Hz), 2.30 (t, 1H, CH₃-

(CH₂)₉CH₂CO, J = 7.5 Hz), 1.67-1.32 (m, 37H, CH₃-

(CH₂)₉CH₂CO, tert-butyl, CH₂CH₃ꢀ 2, acetonide), 0.93

(t, 3H, CH₃(CH₂)₉CH₂CO, J=6.9 Hz), 0.84, 0.82 (each t, each

3H, CH₂CH₃, J=7.4 Hz); ¹³C NMR (CD₃OD, 125 MHz) δ

177.7, 176.8, 170.6, 166.4, 158.4, 152.0, 146.2, 117.2, 115.4,

114.1, 102.8, 97.6, 89.4, 87.6, 87.3, 85.9, 83.9, 83.3, 81.6, 80.2,

54.9, 44.2, 36.5, 35.0, 33.1, 30.8, 30.7, 30.6, 30.5, 30.4, 30.2, 29.8,

28.8, 28.5, 28.3, 27.5, 27.1, 26.1, 25.4, 23.7, 14.5, 8.8, 8.7, 7.6;

ESIMS-HR m/z calcd for C₄₅H₇₄N₄NaO₁₄(M þ H)^þ917.5099,

found 917.5097.

00

CH₃(CH₂)₆CH₂C₆H₄NH, J=8.0 Hz), 5.71 (d, 1H, H-1⁰, J_{1 ,2}

=

1.7 Hz), 5.67 (d, 1H, H-5, J_5,6=7.9 Hz), 5.24 (dd, 1H, H-2⁰₀,

00

0

J_{2 ,1}=1.7, J_{2 ,3}=6.9 Hz), 5.11 (s, 1H, H-1 ), 4.90 (dd, 1H, H-3 ,

0

00

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

(3-pentylidene)-β-^D-ribofuranosyl]-6-deoxy-2,3-O-isopropylidene-

6-(4-octylbenzoylamino)-1-(uracil-1-yl)-β-D-glycero-L-talo-hepto-

furanuronate (16b). Compound 16b was prepared from 13

(85 mg, 0.10 mmol) and 4-octylbenzoic acid (70 mg, 0.30 mmol)

as described above for the synthesis of 16a. Purification by silica

gel column chromatography (2 cm ꢀ 10 cm, 66% AcOEt/

hexane) afforded 16b (76 mg, 82% over two steps) as a white

foam: ¹H NMR (CD₃OD, 500 MHz) δ 7.75 (d, 2H, CH₃-

0

00 00

0

J_{3 ,2}=6.9, J_{3 ,4}=4.0 Hz), 4.73 (d, 1H, H-2 , J_{2 ,3}=6.3 Hz), 4.56

00 00

(d, 1H, H-3⁰⁰, J_{3 ,2}=6.3 Hz), 4.53 (br s, 1H, H-6 ), 4.83 (br s, 1H,

H-5⁰), 4.23 (dd, 1H, H-4⁰, J_{5 ,4}=9.2, J_{5 ,6}=4.5 Hz), 4.12 (t, 1H,

H-4⁰⁰, J_{4 ,5}=7.4 Hz), 3.30 (dd, 1H, H-5 a, J_{5 a,4}=7.5, J_{5 a,5 b}

0

00

00 00

00

=

13.8 Hz), 3.01 (dd, 1H, H-5⁰⁰b, J_{5 b,4}=7.5, J_{5 b,5 a}=13.8 Hz),

2.57 (t, 2H, CH₃(CH₂)₆CH₂C₆H₄NH, J=8.0 Hz), 1.61-1.31

(m, 31H, CH₃(CH₂)₆CH₂C₆H₄NH, tert-butyl, CH₂CH₃ꢀ 2,

acetonide), 0.92 (t, 3H, CH₃(CH₂)₆CH₂C₆H₄NH, J=6.9 Hz),

0.84, 0.83 (each t, each 3H, CH₂CH₃, J=7.4 Hz); ¹³C NMR

(CD₃OD, 125 MHz) δ 171.9, 166.4, 158.5, 157.8, 152.0, 146.2,

138.4, 138.1, 129.7, 120.3, 117.3, 115.5, 113.9, 102.8, 97.5, 89.2,

87.6, 87.2, 85.8, 83.9, 83.4, 83.3, 82.1, 80.4, 55.1, 44.5, 36.2, 33.0,

32.8, 30.6, 30.4, 30.3, 29.8, 28.8, 28.5, 28.4, 27.4, 25.5, 23.7, 14.5,

8.9, 7.6; ESIMS-HR m/z calcd for C₄₈H₇₃N₅NaO₁₄(M þ Na)^þ

966.5052, found 966.4919.

00

(CH₂)₆CH₂C₆H₄CO, J = 8.0 Hz), 7.71 (d, 1H, H-6, J_6,5

8.0 Hz), 7.32 (d, 2H, CH₃(CH₂)₆CH₂C₆H₄CO, J=8.0 Hz), 5.73

=

(d, 1H, H-1⁰, J_{1 ,2}=1.7 Hz), 5.69 (d, 1H, H-5, J_5,6=8.0 Hz), 5.22

00 00

00

(dd, 1H, H-2⁰, J_{2 ,1}=1.7, J_{2 ,3}=6.3 Hz), 5.14 (s, 1H, H-1 ), 4.90

0

(m, 1H, H-3⁰), 4.83 (br s, 1H, H-6⁰), 4.81 (d, 1H, H-2⁰⁰, J_{2 ,3}=5.8

00 00

Hz), 4.61-4.59 (m, 2H, H-3⁰⁰, H-5⁰), 4.25 (dd, 1H, H-4⁰, J_{5 ,4}

0

=

00

0

00 00

9.2, J_{5 ,6}=4.0 Hz), 4.13 (t, 1H, H-4 , J_{4 ,5}=7.4 Hz), 3.34 (m,

1H, H-5⁰⁰a), 3.11 (m, 1H, H-5⁰⁰b), 2.71 (t, 2H, CH₃-

(CH₂)₆CH₂C₆H₄CO, J = 7.5 Hz), 1.67-1.31 (m, 31H, CH₃-

(CH₂)₆CH₂C₆H₄CO, tert-butyl, CH₂CH₃ꢀ 2, acetonide), 0.92

(t, 3H, CH₃(CH₂)₆CH₂C₆H₄CO, J = 6.9 Hz), 0.84 (t, 6H,

CH₂CH₃ꢀ 2, J=7.4 Hz); ¹³C NMR (CD₃OD, 125 MHz) δ

170.7, 170.4, 166.3, 152.0, 148.6, 145.9, 132.7, 129.7, 128.6,

117.3, 115.5, 114.0, 102.9, 97.0, 89.2, 87.5, 87.4, 85.8, 84.1,

83.4, 83.1, 81.5, 80.2, 55.4, 44.3, 36.8, 33.0, 32.4, 30.6, 30.5,

30.4, 30.3, 29.8, 28.8, 28.5, 28.3, 27.4, 25.5, 23.7, 14.5, 8.8, 7.7;

ESIMS-HR m/z calcd for C₄₈H₇₂N₄NaO₁₄(M þ Na)^þ

951.4943, found 951.4939.

6-Amino-5-O-(5-amino-5-deoxy-β-^D-ribofuranosyl)-6-deoxy-

N-dodecyl-1-(uracil-1-yl)-β-^D-glycero-L-talo-heptofuranuronamide

Trifluoroacetic Salt (6a). A mixture of 15a (50 mg, 0.060 mmol)

and 10% Pd/C (10 mg) in MeOH (1 mL) was vigorously stirred

for 1 h under H₂atmosphere. The catalyst was filtered off

through a Celite pad, and the filtrate was concentrated in vacuo

to give the free amine. The amine 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 6a (35 mg,

95% overtwo steps) as a TFAsalt: ¹H NMR (CD₃OD, 500MHz)

δ 7.74 (d, 1H, H-6, J_6,5=8.0 Hz), 5.86 (s, 1H, H-1⁰), 5.76 (d, 1H,

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

pentylidene)-β-^D-ribofuranosyl]-6-deoxy-6-dodecylureido-2,3-O-

isopropylidene-1-(uracil-1-yl)-β-^D-glycero-L-talo-heptofuranuro-

nate (17a). A mixture of 13 (85 mg, 0.1 mmol) and 10% Pd/C

H-5, J_5,6=8.0 Hz), 5.15 (d, 1H, H-1⁰⁰, J_{1 ,2}=2.3 Hz), 4.38 (br s,

00 00

1H, H-5⁰), 4.30 (m, 2H, H-3⁰, H-3⁰⁰), 4.25 (d, 1H, H-2⁰, J_{2 ,3}

0

=

5.2 Hz), 4.16-4.11 (m, 4H, H-4⁰, H-6⁰, H-2⁰⁰, H-4⁰⁰), 3.39 (m, 1H,

3804 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9

Ii et al.

CH₃(CH₂)₉CH₂CH₂NH), 3.26 (br s, 2H, H-5⁰⁰a, H-5⁰⁰b), 3.24

(m, 1H, CH₃(CH₂)₉CH₂CH₂NH), 1.61 (m, 2H, CH₃(CH₂)₉-

CH₂CH₂NH), 1.35-1.32 (m, 18H, CH₃(CH₂)₉CH₂CH₂NH),

0.93 (t, 3H, CH₃(CH₂)₉CH₂CH₂NH, J = 6.9 Hz); ¹³C NMR

(CD₃OD, 125 MHz) δ 168.7, 166.0, 152.1, 142.6, 112.2, 102.9,

93.2, 93.1, 85.4, 81.6, 79.4, 76.5, 74.7, 72.8, 70.3, 56.8, 43.1, 41.2,

33.0, 30.8, 30.7, 30.5, 30.4, 30.0, 28.1, 23.7, 14.4; ESIMS-HR m/z

calcd for C₂₈H₅₀N₅O₁₀(M þ H)^þ616.3479, found 616.3723.

6-Amino-5-O-(5-amino-5-deoxy-β-^D-ribofuranosyl)-6-deoxy-1-

N-(4-octylphenyl)(uracil-1-yl)-β-^D-glycero-L-talo-heptofuranuro-

namide Trifluoroacetic Salt (6b). Compound 6b was prepared

from 15b (30 mg, 0.030 mmol) as described above for the

synthesis of 6a. Purification by C18 reverse phase column

chromatography (1.5 cm ꢀ 10 cm, 80% aqueous MeOH con-

taining 0.5% TFA) afforded 6b (17 mg, 89% over two steps) as a

1H, H-5⁰⁰a, J_{5 a,5 b}=12.9 Hz), 3.23 (dd, 1H, H-5⁰⁰b, J_{5 b,4}=9.2,

00

J_{5 b,5 a}=12.9 Hz), 2.71 (t, 2H, CH₃(CH₂)₅CH₂CH₂C₆H₄CO,

J = 7.8 Hz), 1.67 (m, 2H, CH₃(CH₂)₅CH₂CH₂C₆H₄CO),

1.36-1.31 (m, 10H, CH₃(CH₂)₅CH₂CH₂C₆H₄CO), 0.92 (t, 3H,

CH₃(CH₂)₅CH₂CH₂C₆H₄CO, J=6.9 Hz); ¹³C NMR (CD₃OD,

125 MHz) δ 173.7, 170.5, 166.1, 152.1, 148.8, 142.9, 132.5, 129.7,

128.7, 111.1, 102.6, 92.3, 84.8, 80.0, 79.8, 76.3, 74.7, 73.9, 71.6, 55.8,

44.3, 36.8, 33.0, 32.4, 30.5, 30.4, 30.3, 23.7, 14.4; ESIMS-HR m/z

calcd for C₃₁H₄₄N₄NaO₁₂(M þ Na)^þ687.2852, found 687.2845.

5-O-(5-Amino-5-deoxy-β-^D-ribofuranosyl)-6-deoxy-6-dodecy-

lureido-1-(uracil-1-yl)-β-D-glycero-L-talo-heptofuranuronic Acid

Trifluoroacetic Salt (8a). Compound 8a was prepared from

17a (50 mg, 0.056 mmol) as described above for the synthesis

of 7a. Purification by C18 reverse phase column chromatogra-

phy (1.5 cm ꢀ 10 cm, 80% aqueous MeOH containing 0.5%

TFA) gave 8a (34 mg, 96%) as a TFA salt: ¹H NMR (CD₃OD,

500 MHz) δ 7.87 (d, 1H, H-6, J_6,5=8.0 Hz), 5.79 (d, 1H, H-1⁰,

1

TFA salt: H NMR (CD₃OD, 500 MHz) δ 7.72 (d, 1H, H-6,

J_6,5=8.0 Hz), 7.47 (d, 2H, CH₃(CH₂)₅CH₂CH₂C₆H₄NH, J=

J

_{1 ,2}=4.0 Hz), 5.76 (d, 1H, H-5, J_5,6=8.0 Hz), 5.20 (₀s, 1H, H-1⁰⁰),

8.6 Hz), 7.22 (d, 2H, CH₃(CH₂)₅CH₂CH₂C₆H₄NH, J=8.6 Hz),

5.83 (d, 1H, H-1⁰, J_{1 ,2}=2.3 Hz), 5.73 (d, 1H, H-5, J_5,6=8.0 Hz),

0

4.80 (d, 1H, H-6⁰, J_{6 ,5}=2.3 Hz), 4.33 (br s, 1H, H-5 ), 4.20-4.17

0

5.17 (d, 1H, H-1⁰⁰, J_{1 ,2}=2.3 Hz), 4.55 (br s, 1H, H-5 ), 4.43 (d,

(m, 4H, H-2⁰, H-3⁰, H-4⁰, H-2⁰⁰), 4.13 (br d, 1H, H-4⁰⁰, J=3.6 Hz),

00 00

1H, H-3⁰⁰, J_{3 ,2}=6.0 Hz), 4.31 (dd, 1H, H-3 , J_{3 ,2}=5.1, J_{3 ,4}

=

0

00

4.04 (d, 1H, H-3⁰⁰, J_{3 ,2}=4.0 Hz), 3.32 (m, 1H, H-5 a), 3.22 (m,

1H, H-5⁰⁰b), 3.15 (t, 2H, CH₃(CH₂)₉CH₂CH₂NH, J=6.9 Hz),

1.49 (m, 2H, CH₃(CH₂)₉CH₂CH₂NH), 1.32 (m, 18H, CH₃-

(CH₂)₉CH₂CH₂NH), 0.93 (t, 3H, CH₃(CH₂)₉CH₂CH₂NH, J=

6.9 Hz); ¹³C NMR (CD₃OD, 125 MHz) δ 174.4, 166.1, 160.4,

152.2, 142.8, 110.7, 102.6, 91.6, 84.8, 80.2, 79.8, 76.3, 74.7, 74.0,

71.4, 55.4, 44.4, 40.9, 33.1, 31.2, 30.8, 30.5, 28.0, 23.7, 14.4;

ESIMS-HR m/z calcd for C₂₉H₄₉N₅NaO₁₂(M þ Na)^þ

682.3275, found 682.3285.

00 00

0

8.0 Hz), 4.26 (dd, 1H, H-2⁰, J_{2 ,1}=2.3, J_{2 ,3}=5.1 Hz),₀₀4.18 (dd,

0

1H, H-4⁰, J_{4 ,3}=8.0, J_{4 ,5}=5.3 Hz), 4.04 (dd, 1H, H-2 , J_{2 ,1}

=

0

00 00

0

00 00

0

2.3, J_{2 ,3}=6.0 Hz), 4.04 (t, 1H, H-6 , J_{6 ,5}=5.2 Hz), 4.04 (dd,

1H, H-4⁰⁰, J_{4 ,5 a}=6.0, J_{4 ,5 b}=6.9 Hz), 3.05 (br s, 2H, H-5⁰⁰a,

H-5⁰⁰b), 2.60 (t, 2H, CH₃(CH₂)₅CH₂CH₂C₆H₄NH), 1.61 (m, 2H,

CH₃(CH₂)₅CH₂CH₂C₆H₄NH), 1.32-1.28 (m, 10H, CH₃(CH₂)₅-

CH₂CH₂C₆H₄NH), 0.89 (t, 3H, CH₃(CH₂)₅CH₂CH₂C₆H₄NH,

J=6.9 Hz); ¹³C NMR (CD₃OD, 125 MHz) δ 167.0, 166.1, 152.1,

142.7, 141.8, 140.3, 136.1, 130.2, 122.1, 111.7, 102.9, 85.3, 81.1,

78.6, 76.4, 74.6, 72.8, 70.5, 57.3, 43.1, 36.4, 33.0, 32.7, 30.6, 30.4,

30.3, 23.7, 14.4; ESIMS-HR m/z calcd for C₃₀H₄₆N₅O₁₀(M þ

H)^þ636.3166, found 636.3231.

00 00

5-O-(5-Amino-5-deoxy-β-^D-ribofuranosyl)-6-deoxy-1-6-(4-

octylphenylureido)(uracil-1-yl)-β-^D-glycero-L-talo-heptofuranuronic

Acid Trifluoroacetic Salt (8b). Compound 8b was prepared from

17b (61 mg, 0.065 mmol) as described above for the synthesis of

7a. Purification by C18 reverse phase column chromatography

(1.5 cm ꢀ 10 cm, 80% aqueous MeOH containing 0.5% TFA)

gave 8b (41 mg, 93%) as a TFA salt: ¹H NMR (CD₃OD,

500 MHz) δ 7.86 (d, 1H, H-6, J_6,5= 8.0 Hz), 7.28 (d, 2H,

CH₃(CH₂)₅CH₂CH₂C₆H₄NH, J=8.1 Hz), 7.10 (d, 2H, CH₃-

5-O-(5-Amino-5-deoxy-β-^D-ribofuranosyl)-6-deoxy-6-dodeca-

noylamino-1-(uracil-1-yl)-β-D-glycero-L-talo-heptofuranuronic Acid

Trifluoroacetic Salt (7a). Compound 16a (50 mg, 0.056 mmol)

was treated with 80% aqueous TFA (1 mL) at room tempera-

ture 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 con-

taining 0.5% TFA) to afford 7a (33 mg, 94%) as a TFA salt: ¹H

NMR (CD₃OD, 500 MHz) δ 7.85 (d, 1H, H-6, J_6,5=8.1 Hz),

(CH₂)₅CH₂CH₂C₆H₄NH, J=8.1 Hz), 5.80 (d, 1H, H-1⁰, J_{1 ,2}

0

=

3.7 Hz), 5.76 (d, 1H, H-5, J_5,6=8.0 Hz), 5.23 (s, 1H, H-1⁰⁰),

4.87 (br s, 1H, H-6⁰), 4.42 (br s, 1H, H-5⁰), 4.23-4.21 (m, 3H,

H-2⁰, H-4⁰, H-2⁰⁰), 4.15 (br s, 1H, H-4⁰⁰), 4.07 (br s, 1H, H-3⁰⁰),

3.34 (m, 1H, H-5⁰⁰a), 3.22 (m, 1H, H-5⁰⁰b), 2.57 (t, 2H, CH₃-

(CH₂)₅CH₂CH₂C₆H₄NH, J=8.0 Hz), 1.62 (m, 2H, CH₃(CH₂)₅-

CH₂CH₂C₆H₄NH), 1.36-1.31 (m, 10H, CH₃(CH₂)₅CH₂CH₂-

C₆H₄NH), 0.93 (t, 3H, CH₃(CH₂)₅CH₂CH₂C₆H₄NH, J=6.9 Hz);

¹³C NMR (CD₃OD, 125 MHz) δ 176.6, 166.1, 157.8, 152.2,

142.9, 138.6, 138.0, 129.8, 127.6, 120.4, 110.8, 102.7, 91.9, 84.8,

80.1, 79.9, 76.3, 74.7, 74.0, 71.4, 55.4, 44.3, 36.2, 33.0, 32.8, 30.6,

30.4, 30.3, 23.7, 14.4; ESIMS-HR m/z calcd for C₃₁H₄₅N₅NaO₁₂

(M þ Na)^þ702.2962, found 702.2965.

5.76 (s, 1H, H-1⁰), 5.75 (d, 1H, H-5, J_5,6=8.1 Hz), 5.20 (s, 1H, H-

0

1⁰⁰), 5.00 (d, 1H, H-6⁰, J_{6 ,5}=2.9 Hz₀), 4.32 (dd, 1H, H-5 , J_{5 ,4}

0

=

0

2.9, J_{5 ,6}=4.6 Hz), 4.20 (t, 1H, H-2 , J_{2 ,3}=4.0 Hz), 4.33-4.17

(m, 5H, H-3⁰, H-4⁰, H-2⁰⁰, H-3⁰⁰, H-4⁰⁰), 4.06 (d, 1H, H-3⁰⁰, J_{3 ,2}

0

=

4.0 Hz), 3.30 (br d, 1H, H-5⁰⁰a, J_{5 a,5 b}=12.6 Hz), 3.24 (dd, 1H,

00

H-5⁰⁰b, J_{5 b,4}=9.2, J_{5 b,5 a}=12.6 Hz), 3.34 (t, 2H, CH₃(CH₂)₈-

CH₂CH₂CO, J=7.5 Hz), 1.64 (m, 2H, CH₃(CH₂)₈CH₂CH₂CO),

1.32 (br s, 16H, CH₃(CH₂)₈CH₂CH₂CO), 0.93 (t, 3H, CH₃-

(CH₂)₈CH₂CH₂CO, J=6.9 Hz); ¹³C NMR (CD₃OD, 125 MHz)

δ 176.6, 173.7, 166.1, 152.1, 142.9, 111.0, 102.6, 101.3, 92.2, 84.8,

79.9, 79.8, 76.3, 74.8, 73.9, 71.5, 55.0, 44.3, 36.6, 33.1, 30.8, 30.7,

30.6, 30.5, 30.4, 30.3, 26.9, 25.1, 23.7, 14.4; ESIMS-HR m/z

calcd for C₂₈H₄₇N₄O₁₂(M þ H)^þ613.3112, found 613.3177.

5-O-(5-Amino-5-deoxy-β-^D-ribofuranosyl)-6-deoxy-1-6-(4-oct-

ylbenzolyamino)-1-(uracil-1-yl)-β-D-glycero-L-talo-heptofuranuronic

Acid Trifluoroacetic Salt (7b). Compound 7b was prepared from

16b (50 mg, 0.056 mmol) as described above for the synthesis of

7a. Purification by C18 reverse phase column chromatography

(1.5 cm ꢀ 10 cm, 80% aqueous MeOH containing 0.5% TFA)

00

Methyl 6-Benzyloxycarbonylamino-2,3-di-O-(tert-butyldimet-

hylsilyl)-6-deoxy-1-(uracil-1-yl)-β-^D-glycelo-L-talo-heptofuranuro-

nate (22). Compound 10 (1.50 g, 3.0 mmol) was treated with

80% aqueous TFA (30 mL) for 1 h. The solvent was removed in

vacuo. The residue in DMF (30 mL) was treated with TBSCl

(1.40 g, 9.0 mmol) and imidazole (1.22 g, 18.0 mmol), and the

mixture was stirred for 60 h. After the reaction was quenched

with H₂O (10 mL), the mixture was partitioned between AcOEt

and H₂O, and the organic layers were washed H₂O, 1 N aqueous

HCl, saturated aqueous NaHCO₃and brine, dried (Na₂SO₄),

and concentrated in vacuo. The residue was purified by silica gel

column chromatography (5 cm ꢀ 13 cm, 33% AcOEt/hexane) to

give 22 (1.56 g, 75% over two steps) as a white solid: ¹H NMR

(CD₃OD, 500 MHz) δ 8.10 (d, 1H, H-6, J_6,5=8.0 Hz), 7.32 (m,

1

gave 7b (34 mg, 95%) as a TFA salt: H NMR (CD₃OD, 500

MHz) δ 7.86 (d, 1H, H-6, J_6,5= 8.0 Hz), 7.78 (d, 2H, CH₃-

(CH₂)₅CH₂CH₂C₆H₄CO, J = 8.3 Hz), 7.32 (d, 2H, CH₃-

(CH₂)₅CH₂CH₂C₆H₄CO, J=8.3 Hz), 5.77 (d, 1H, H-1⁰, J_{1 ,2}

0

=

3.7 Hz), 5.76 (d, 1H, H-5, J_5,6=8.0 Hz), 5.25 (s, ₀1H, H-1⁰⁰), 5.19

5H, phenyl), 5.88 (d, 1H, H-1⁰, J_{1 ,2}=5.7 Hz), 5.70 (d, 1H, H-5,

0

(d, 1H, H-6⁰, J_{6 ,5}=3.2 Hz), 4.47 (br s, 1H, H-5 ), 4.26 (dd, 1H,

J

_5,6=8.0 Hz), 5.15 (d, 1H, benzyl, J=12.6 Hz), 5.01 (d, 1H,

0

H-4⁰, J_{4 ,3}=4.1, J_{4 ,5}=6.0 Hz), 4.24 (dd, 1H, H-2 , J_{2 ,1}=3.7,

benzyl, J=12.6 Hz), 4.51 (d, 1H, H-4⁰, J_{4 ,3}=5.2 Hz), 4.36 (dd,

0

J

1H, H-2⁰, J_{2 ,1}=5.7, J_{2 ,3}=3.6 Hz), 4.16 (m, 1H, H-5 ), 4.15 (m,

0

_{2 ,3}=5.5 Hz), 4.19 (dd, 1H, H-3 , J_{3 ,2}=5.5, J_{3 ,4}=4.1 Hz), 4.16

0

(m, 2H, H-2⁰⁰, H-4⁰⁰), 4.10 (d, 1H, H-3⁰, J_{3 ,2}=3.7 Hz), 3.30 (br d,

1H, H-6⁰), 4.12 (d, 1H, H-3⁰, J_{3 ,4}= 5.2 Hz), 3.72 (s, 3H,

0

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3805

-CO₂Me), 0.93, 0.88 (each s, each 9H, tert-butyl), 0.12, 0.12,

0.06, 0.01, (each s, each 3H, SiMe₃); ¹³C NMR (CD₃OD, 125

MHz) δ 172.4, 166.1, 158.6, 152.4, 143.1, 138.1, 129.4, 129.1,

128.8, 103.1, 89.7, 87.8, 76.3, 75.2, 70.9, 67.7, 58.5, 52.9, 26.4,

18.9, 18.9, -4.2, -4.4, -4.5, -4.6; ESIMS-HR calcd for

C₃₂H₅₁N₃NaO₁₀Si₂(M þ Na)^þ716.3011, found 716.2993.

(2R,4S,5S)-Methyl 2-Azidomethyl-5-[(1R,2R,3R,4R)-2,3-di-

tert-butyldimethylsilyloxy-4-(uracil-1-yl)]tetrahydrofuryl-3-pal-

mitoyl-(1,3)-oxazolidine-4-carboxylate (25a). Azidoacetoalde-

hyde was prepared as follows. A solution of azidoethanol³³

(626 mg, 7.2 mmol) in CH₂Cl₂(70 mL) was treated by

Dess-Martin periodinane³⁴(3.97 g, 9.36 mmol) at room tem-

perature for 1 h. The insoluble was filtered off through a Celite

pad, and the filtrate was concentrated in vacuo. The residue was

semipurified by silica gel column chromatography (3 cm ꢀ 10 cm,

25% Et₂O-pentane) to give azidoacetoaldehyde (620 mg,

quant). A solution of 22 (1.00 g, 1.45 mmol) and 10% Pd-

(OH)₂/C (66 mg) in MeOH (15 mL) was vigorously stirred at

room temperature under H₂atmosphere for 2 h. The catalyst

was filtered off through a Celite pad, and the filtrate was

concentrated in vacuo to give the free amine 23. The amine,

azidoacetoaldehyde (245 mg, 2.89 mmol), and MS4A (1.0 g) in

CH₂Cl₂(15 mL) were stirred at room temperature for 3 h. After

the reaction mixture was cooled to 0 °C, palmitoyl chloride

(792 mg, 2.89 mmol) and Et₃N (292 mg, 2.89 mmol) were added,

and the whole mixture was stirred at 0 °C for 12 h. The mixture

was diluted with CHCl₃(15 mL), which was washed with 0.1 N

aqueous HCl, saturated aqueous NaHCO₃, and brine. The

organic layer was dried (Na₂SO₄), filtered, and concentrated

in vacuo. The residue was purified by silica gel column chroma-

tography (4 cm ꢀ 15 cm, 33% AcOEt-hexane) to give 25a

(949 mg, 76%) as a yellow syrup: ¹H NMR (CD₃OD, 500 MHz,

3:2 mixture of rotamers at 20 °C, data for the major rotamer) δ

1.29 (br s, 24H, CH₃(CH₂)₁₂CH₂CH₂CO), 0.95, 0.91 (each s, each

9H, tert-butyl), 0.90 (t, 3H, CH₃(CH₂)₁₂CH₂CH₂CO, J=6.9 Hz),

0.17, 0.15, 0.09, 0.06 (each s, each 3H, SiCH₃);¹³CNMR(CD₃OD,

125 MHz) δ174.0, 171.3, 165.6, 152.2, 141.2, 103.6, 90.3, 89.6, 85.0,

80.3, 76.3, 73.4, 60.0, 53.8, 34.9, 33.4, 33.1, 30.8, 30.7, 30.6, 30.5,

30.3, 30.2, 30.1, 26.4, 26.0, 25.7, 23.8, 19.0, 18.9, 14.5, -4.1, -4.4,

-4.5, -4.6; ESIMS-HR m/z calcd for C₄₃H₇₈N₆NaO₉Si₂(M þ

Na)^þ901.5267, found 901.5249.

(2R,4S,5S)-Methyl 2-Azidopropyl-5-[(1R,2R,3R,4R)-2,3-di-

tert-butyldimethylsilyloxy-4-(uracil-1-yl)]tetrahydrofuryl-3-pal-

mitoyl-(1,3)-oxazolidine-4-carboxylate (25c). 4-Azidobutanal

was prepared as follows. A solution of 4-azido-1-butanol³⁶

(828 mg, 7.20 mmol) in CH₂Cl₂(70 mL) was treated by Dess-

Martin periodinane (3.97 g, 9.36 mmol) at room temperature for

1 h. The insoluble was filtered off through a Celite pad, and the

filtrate was concentrated in vacuo. The residue was semipurified

by silica gel column chromatography (1.5 cm ꢀ 10 cm, 25%

Et₂O-pentane) to give 4-azidobutanal (798 mg, 98%). Com-

pound 25c was prepared from 22 (1.00 g, 1.44 mmol) and 4-

azidopropanal (332 mg, 2.89 mmol) as described above for the

synthesis of 25a. Purification by silica gel column chromato-

graphy (3 cmꢀ12 cm, 33% AcOEt-hexane) afforded 25c (1.12 g,

87%) as a yellow syrup: ¹H NMR (CD₃OD, 500 MHz, 3:2

mixture of rotamers at 20 °C, data for the major rotamer) δ

7.59 (d, 1H, H-6, J_6,5=8.0 Hz), 5.95 (d, 1H, H-1⁰, J_{1 ,2}=5.2 Hz),

0

5.82 (dd, 1H, H-1⁰⁰, J_{1 ,2 a}=4.6, J_{1 ,2 b}=6.9 Hz), 5.73 (d, 1H,

00 00

H-5, J_5,6=8.0 Hz), 4.85 (d, 1H, H-6⁰, J_{6 ,5}=5.7 Hz), 4.73 (dd, 1H,

0

H-5⁰, J_{5 ,4}=1.2, J_{5 ,6}=5.7 Hz), 4.30 (m, 3H, H-2 , H-3 , H-4 ),

0

3.84 (s, 3H, CO₂CH₃), 3.36 (t, 2H, H-4⁰⁰, J_{4 ,3}=6.9 Hz), 2.44-2.20

00 00

(m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO), 1.98-1.80 (m, 2H, H-3⁰⁰),

1.80-1.68 (m, 2H, H-2⁰⁰), 1.59 (m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO),

1.29 (br s, 24H, CH₃(CH₂)₁₂CH₂CH₂CO), 0.95, 0.90 (each s,

each 9H, tert-butyl), 0.90 (t, 3H, CH₃(CH₂)₁₂CH₂CH₂CO, J=6.9

Hz), 0.17, 0.15, 0.09, 0.07 (each s, each 3H, SiCH₃); ¹³C NMR

(CD₃OD, 125 MHz) δ 174.1, 171.8, 165.7, 152.2, 141.2, 103.4,

91.8, 89.8, 84.7, 79.9, 76.4, 73.3, 60.0, 53.7, 34.9, 33.1, 31.4,

30.8, 30.7, 30.6, 30.6, 30.5, 30.2, 30.1, 26.4, 26.1, 25.8, 25.4,

23.8, 20.9, 19.0, 14.5, -4.1, -4.4, -4.5, -4.6; ESIMS-HR m/z

calcd for C₄₄H₈₀N₆NaO₉Si₂(M þ Na)^þ915.5423, found

915.5417.

7.56 (d, 1H, H-6, J_6,5=8.0 Hz), 5.96 (d, 1H, H-1⁰, J_{1 ,2}=5.8 Hz),

0

5.85 (dd, 1H, H-1⁰⁰, J_{1 ,2 a}=2.3, J_{1 ,2 b}=5.2 Hz), 5.75 (d, 1H,

00 00

H-5, J_5,6=8.0 Hz), 4.94 (d, 1H, H-6⁰, J_{6 ,5}=7.5 Hz), 4.₀88 (dd,

0

1H, H-5⁰, J_{5 ,4}=1.2, J_{5 ,6}=7.5 Hz), 4.31 (m, 3H, H-2 , H-3 ,

0

H-4⁰), 3.84 (s, 3H, CO₂CH₃), 3.46 (t, 2H, H-2⁰⁰, J_{2 ,1}=2.3 Hz),

00 00

2.40-2.24 (m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO), 1.61 (m, 2H,

CH₃(CH₂)₁₂CH₂CH₂CO), 1.29 (br s, 24H, CH₃(CH₂)₁₂-

CH₂CH₂CO), 0.96, 0.90 (each s, each 9H, tert-butyl), 0.90

(t, 3H, CH₃(CH₂)₁₂CH₂CH₂CO, J=6.9 Hz), 0.18, 0.15, 0.10,

0.07 (each s, each 3H, SiCH₃); ¹³C NMR (CD₃OD, 125 MHz)

δ 174.3, 171.2, 165.6, 152.1, 141.4, 103.6, 90.8, 90.1, 84.8, 80.7,

76.1, 73.1, 60.5, 53.0, 34.8, 33.1, 30.8, 30.8, 30.6, 30.5, 30.3, 30.2,

30.1, 26.4, 26.0, 25.7, 23.8, 19.0, 18.9, 14.5, -4.1, -4.3, -4.4,

-4.5; ESIMS-HR m/z calcd for C₄₂H₇₆N₆NaO₉Si₂(M þ Na)^þ

887.5110, found 887.5133.

(2R,4S,5S)-Methyl 2-Azidoethyl-5-[(1R,2R,3R,4R)-2,3-di-tert-

butyldimethylsilyloxy-4-(uracil-1-yl)]tetrahydrofuryl-3-palmitoyl-

(1,3)-oxazolidine-4-carboxylate (25b). 3-Azidopropanal was pre-

pared as follows. A solution of 3-azido-1-propanol³⁵(727 mg,

7.20 mmol) in CH₂Cl₂(70 mL) was treated by Dess-Martin

periodinane (3.97 g, 9.36 mmol) at room temperature for 1 h.

The insoluble was filtered off through a Celite pad, and the filtrate

was concentrated in vacuo. The residue was semipurified by silica

gel column chromatography (1.5 cmꢀ10 cm, 25% Et₂O-pentane)

to give 3-azidopropanal (713 mg, quant). Compound 25b was

prepared from 22 (998 mg, 1.44 mmol) and 3-azidopropanol (285

mg, 2.88 mmol) as described above for the synthesis of 25a.

Purification by silica gel column chromatography (3 cmꢀ12 cm,

33% AcOEt-hexane) afforded 25b (1.00 g, 79%) as a yellow

syrup: ¹H NMR (CD₃OD, 500 MHz, 3:2 mixture of rotamers at 20

(2R,4S,5S)-2-Azidomethyl-5-[(1R,2R,3R,4R)-2,3-di-tert-but-

yldimethylsilyloxy-4-(uracil-1-yl)]tetrahydrofuryl-3-palmitoyl-

(1,3)-oxazolidine-4-carboxylic Acid (26a). A mixture of 25a

(47 mg, 0.054 mmol) and LiI (87 mg, 0.65 mmol) in AcOEt

(1 mL) was heated under reflux for 5 h. The mixture was cooled

to room temperature and diluted with AcOEt. The mixture was

washed with 0.1 N aqueous HCl, saturated aqueous NaHCO₃,

and brine, dried (Na₂SO₄), filtered, and concentrated in vacuo.

The residue was purified by silica gel column chromatography

(1.5 cmꢀ8 cm, 2% MeOH-CHCl₃) to give 26a (36 mg, 78%) as

1

a yellow syrup: H NMR (CD₃OD, 500 MHz, 3:1 mixture of

rotamers at 20 °C, data for the major rotamer) δ 7.63 (d, 1H,

H-6, J_6,5=8.0 Hz), 6.00 (d, 1H, H-1⁰, J_{1 ,2}=4.6 Hz), 5.82 (dd,

0

1H, H-1⁰⁰, J_{1 ,2 a}=2.3, J_{1 ,2 b}=6.9 Hz), 5.76 (d, 1H, H-₀5, J_5,6

=

00 00

8.0 Hz), 4.80 (d, 1H, H-5⁰, J_{5 ,6}=6.4 Hz), 4.65 (d, 1H, H-6 , J_{6 ,5}

6.4 Hz), 4.31 (m, 3H, H-2⁰, H-3⁰, H-4⁰), 3.43 (t, 2H, H-2⁰⁰, J_{2 ,1}

0

00 00

4.6 Hz), 2.45-2.26 (m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO), 1.58 (m,

2H, CH₃(CH₂)₁₂CH₂CH₂CO), 1.28 (br s, 24H, CH₃(CH₂)₁₂-

CH₂CH₂CO), 0.95, 0.90 (each s, each 9H, tert-butyl), 0.89 (t,

3H, CH₃(CH₂)₁₂CH₂CH₂CO, J=6.9 Hz), 0.17, 0.14, 0.09, 0.07

(each s, each 3H, SiCH₃); ¹³C NMR (CD₃OD, 125 MHz) δ

174.4, 165.6, 152.2, 141.3, 103.5, 90.7, 89.5, 85.1, 81.9, 76.4, 73.7,

61.7, 52.7, 35.1, 33.0, 30.7, 30.7, 30.5, 30.4, 30.4, 30.2, 26.4, 26.1,

25.7, 23.6, 18.9, 18.9, 14.3, -4.1, -4.3, -4.4, -4.5; ESIMS-HR

(negative mode) m/z calcd for C₄₁H₇₃N₆O₉Si₂(M - H)^-849.4983,

found 849.5005.

°C, data for the major rotamer) δ 7.56 (d, 1H, H-6, J_6,5=8.0 Hz),

00

5.99 (d, 1H, H-1⁰, J_{1 ,2}=5.2 Hz), 5.89 (dd, 1H, H-1 , J_{1 ,2 a}=4.6,

00 00

0

00 00

J_{1 ,2 b}=8.0 Hz), 5.74 (d, 1H, H-5, J_5,6=8.0 Hz), 4.89 (d, 1H, H-6⁰,

0

(2R,4S,5S)-2-Azidoethyl-5-[(1R,2R,3R,4R)-2,3-di-tert-butyl-

dimethylsilyloxy-4-(uracil-1-yl)]tetrahydrofuryl-3-palmitoyl-(1,3)-

oxazolidine-4-carboxylic Acid (26b). Compound 26b was pre-

pared from 25b (53 mg, 0.06 mmol) as described above for the

J_{6 ,5}=6.0 Hz), 4.76 (dd, 1H, H-5 , J_{5 ,4}=1.2, J_{5 ,6}=6.0 Hz), 4.30

(m, 3H, H-2⁰, H-3⁰, H-4⁰), 3.84 (s, 3H, CO₂CH₃), 3.44 (t, 2H, H-3⁰⁰,

00 00

J_{3 ,2}= 6.3 Hz), 2.40-2.25 (m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO),

2.10-1.87 (m, 2H, H-2⁰⁰), 1.60 (m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO),

3806 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9

Ii et al.

synthesis of 26a. Purification by silica gel column chromato-

graphy(1.5 cmꢀ10 cm, 2% MeOH-CHCl₃) afforded26b (35mg,

67%) as a yellow syrup: ¹H NMR (CD₃OD, 500 MHz, 3:1

mixture of rotamers at 20 °C, data for the major rotamer) δ

(27 mg, 0.031 mmol) as described above for the synthesis of 27a.

Purification by silica gel column chromatography (1.5 cmꢀ8 cm,

10% MeOH-CHCl₃) afforded 27b (18 mg, 92%) as a yellow

syrup: ¹H NMR (CD₃OD, 500 MHz, 4:1 mixture of rotamers at

7.65 (d, 1H, H-6, J_6,5=8.0 Hz), 5.98 (d, 1H, H-1⁰, J_{1 ,2}=4.6 Hz),

20 °C, data for the major rotamer) δ 7.64 (d, 1H, H-6, J_6,5

=

00

0

5.84 (dd, 1H, H-1⁰⁰, J_{1 ,2 a}=4.0, J_{1 ,2 b}=8.0 Hz), 5.74 (d, 1H, H-

8.0 Hz), 5.99 (d, 1H, H-1⁰, J_{1 ,2}=4.6 Hz), 5.72 (m, 1H, H-1 ),

00 00

0

5.71 (d, 1H, H-5, J_5,6=8.0 Hz), 4.60 (dd, 1H, H-5⁰, J_{5 ,4}=1.2,

5, J_5,6=8.0 Hz), 4.66 (dd, 1H, H-5⁰, J_{5 ,4}=1.2, J_{5 ,6}=6.4₀Hz), 4.58

0

⁰0

0

(d, 1H, H-6⁰, J_{6 5}=6.4 Hz), 4.32(m, 3H, H-2 , H-3 , H-4 ), 3.44 (t,

0

J_{5 ,6}=5.7 Hz), 4.42 (d, 1H, H-6 , J_{6 ,5}=5.7 Hz), 4.27 (m, 2H, H-

0

3⁰, H-4⁰), 4.20 (dd, 1H, H-2⁰, J_{2 ,1}=4.6, J_{2 ,3}=2.9 Hz), 3.45 (t,

2H, H-3⁰⁰, J_{3 ,2}=6.3 Hz), 2.44-2.22 (m, 2H, CH₃(CH₂)₁₂CH₂-

0

00 00

2H, H-3⁰⁰, J_{3 ,2}= 6.9 Hz), 2.34-2.22 (m, 2H, CH₃(CH₂)₁₂-

CH₂CO), 2.20-1.96 (m, 2H, H-2⁰⁰), 1.60 (m, 2H, CH₃(CH₂)₁₂-

CH₂CH₂CO), 1.28 (br s, 24H, CH₃(CH₂)₁₂CH₂CH₂CO), 0.95,

0.90 (each s, each 9H, tert-butyl), 0.89 (t, 3H, CH₃(CH₂)₁₂-

CH₂CH₂CO, J = 6.9 Hz), 0.16, 0.14, 0.09, 0.07 (each s, each

3H, SiCH₃); ¹³C NMR (CD₃OD, 125 MHz) δ 174.0, 165.8 152.2,

141.2, 103.3, 90.0, 89.4, 85.0, 81.3, 76.6, 73.5, 61.8, 52.4, 35.1, 33.3,

33.1, 30.8, 30.8, 30.6, 30.5, 30.5, 30.2, 26.4, 26.1, 25.8, 23.8, 19.0,

18.9, 14.5, -4.1, -4.3, -4.4, -4.5; ESIMS-HR (negative mode)

m/z calcd for C₄₂H₇₅N₆O₉Si₂(M - H)^-863.5134, found

863.5142.

00 00

CH₂CH₂CO), 2.10 (m, 2H, H-2⁰⁰), 1.56 (m, 2H, CH₃(CH₂)₁₂-

CH₂CH₂CO), 1.28 (br s, 24H, CH₃(CH₂)₁₂CH₂CH₂CO), 0.90,

(t, 3H, CH₃(CH₂)₁₂CH₂CH₂CO, J=6.9 Hz); ¹³C NMR (CD₃OD,

125 MHz) δ 173.9, 165.9, 152.4, 141.7, 103.2, 90.0, 89.5, 85.1,

82.4, 75.3, 71.8, 62.5, 52.2, 35.3, 33.5, 33.1, 30.8, 30.8, 30.6, 30.6,

30.5, 30.3, 25.8, 23.7, 14.5, 9.2; ESIMS-HR (negative mode) m/z

calcd for C₃₀H₄₇N₆O₉(M - H)^-635.3405, found 635.3396.

(2R,4S,5S)-2-Azidopropyl-5-[(1R,2R,3R,4R)-2,3-dihydroxy-4-

(uracil-1-yl)]tetrahydrofuryl-3-palmitoyl-(1,3)-oxazolidine-4-

carboxylic Acid (27c). Compound 27c was prepared from 26c

(44 mg, 0.050 mmol) as described above for the synthesis of 27a.

Purification by silica gel column chromatography (1.5 cmꢀ8 cm,

10% MeOH-CHCl₃) afforded 27c (31 mg, 95%) as a yellow

syrup: ¹H NMR (CD₃OD, 500 MHz, 4:1 mixture of rotamers at

20 °C, data for the major rotamer) δ 7.65 (d, 1H, H-6, J_6,5=8.0

(2R,4S,5S)- 2-Azidopropyl-5-[(1R,2R,3R,4R)-2,3-di-tert-bu-

tyldimethylsilyloxy-4-(uracil-1-yl)]tetrahydrofuryl-3-palmitoyl-(1,3)-

oxazolidine-4-carboxylic Acid (26c). Compound 26c was pre-

pared from 25c (69 mg, 0.077 mmol) as described above for the

synthesis of 26a. Purification by silica gel column chromatog-

raphy (1.5 cmꢀ10 cm, 2% MeOH-CHCl₃) afforded 26c (50 mg,

74%) as a yellow syrup: ¹H NMR (CD₃OD, 500 MHz, 2:1

mixture of rotamers at 20 °C, data for the major rotamer) δ 7.67

Hz), 5.99 (d, 1H, H-1⁰, J_{1 ,2}=5.2 Hz), 5.71 (d, 1H, H-5, J_{5, 6}=8.0

0

Hz), 5.63 (dd, 1H, H-1⁰⁰, J_{1 ,2 a}=4.0, J_{1 ,2 b}=8.0 Hz), 4.59 (dd,

00 00

0

1H, H-5⁰, J_{5 ,4}=2.3, J_{5 ,6}=5.8 Hz), 4.42 (d, 1H, H-6 , J_{6 ,5}

=

0

(d, 1H, H-6, J_6,5=8.0 Hz), 5.96 (d, 1H, H-1⁰, J_{1 ,2}=4.0 Hz), 5.77

0

00 00

5.8 Hz), 4.28 (dd, 1H, H-3⁰, J_{3 ,2}=5.2, J_{5 ,6}=4.6 Hz), 4.27 (dd,

(dd, 1H, H-1⁰⁰, J_{1 ,2 a}=4.6, J_{1 ,2 b}=8.0 Hz), 5.73 (d, 1H, H-5,

0

1H, H-4⁰, J_{5 ,6}=4.6, J_{5 ,6}=2.3 Hz), 4.22 (t, 1H, H-2 , J_{2 ,1}

=

_5,6=8.0 Hz), 4.65 (dd, 1H, H-5⁰, J_{5 ,4}=1.2, J_{5 ,6}=5.8 Hz Hz),

0

J

00

0

4.63 (d, 1H, H-6⁰, J_{6 ,5}=5.8 Hz), 4.32 (m, 3H, H-2 , H-3 , H-4 ),

0

00 00

0

J

_{2 ,3}=5.2 Hz), 3.35 (t, 2H, H-4 , J_{4 ,3}=6.9 Hz), 2.34-2.26 (m,

00 00

2H, CH₃(CH₂)₁₂CH₂CH₂CO), 1.88 (m, 2H, H-3⁰⁰), 1.73 (m, 2H,

H-2⁰⁰), 1.57 (m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO), 1.28 (br s, 24H, CH₃-

(CH₂)₁₂CH₂CH₂CO), 0.90 (t, 3H, CH₃(CH₂)₁₂CH₂CH₂CO, J=

6.9 Hz); ¹³C NMR (CD₃OD, 125 MHz) δ 173.9, 165.9, 152.4,

141.8, 103.2, 91.4, 90.0, 85.0, 82.0, 75.3, 71.9, 62.2, 52.2, 35.3,

33.1, 31.3, 30.8, 30.8, 30.6, 30.6, 30.5, 30.3, 25.8, 25.5, 23.7, 14.5,

9.2; ESIMS-HR (negative mode) m/z calcd for C₃₁H₄₉N₆O₉

(M - H)^-649.3567, found 649.3574.

3.36 (t, 2H, H-4⁰⁰, J_{4 ,3}= 6.9 Hz), 2.42-2.24 (m, 2H, CH₃-

(CH₂)₁₂CH₂CH₂CO), 2.03-1.85 (m, 2H, H-3⁰⁰), 1.84-1.70 (m,

2H, H-2⁰⁰), 1.60 (m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO), 1.28 (br s,

24H, CH₃(CH₂)₁₂CH₂CH₂CO), 0.95, 0.90 (each s, each 9H, tert-

butyl), 0.90 (t, 3H, CH₃(CH₂)₁₂CH₂CH₂CO, J=6.9 Hz), 0.16,

0.14, 0.09, 0.09 (each s, each 3H, SiCH₃); ¹³C NMR (CD₃OD,

125 MHz) δ 174.0, 165.8, 152.2, 141.2, 103.2, 91.8, 89.6, 84.7,

80.7, 76.6, 73.4, 60.9, 52.1, 35.1, 33.1, 31.3, 30.8, 30.8, 30.6, 30.5,

30.5, 30.2, 26.4, 26.1, 25.8, 25.5, 23.8, 18.9, 18.9, 14.5, -4.1,

-4.3, -4.4, -4.5; ESIMS-HR m/z calcd for C₄₃H₇₈N₆NaO₉Si₂

(M þ Na)^þ901.5267, found 901.5282.

(2R,4S,5S)-2-Aminomethyl-5-[(1R,2R,3R,4R)-2,3-dihydroxy-

4-(uracil-1-yl)]tetrahydrofuryl-3-palmitoyl-(1,3)-oxazolidine-4-

carboxylic Acid (18a). A solution of 27a (6.1 mg, 0.0098 mmol)

and 10% Pd(OH)₂/C (1 mg) in MeOH (1 mL) was vigorously

stirred at room temperature under H₂atmosphere for 3 h. The

Pd(OH)₂/C was filtered off through a Celite pad, and the filtrate

was concentrated in vacuo. The residue was purified by C18

HPLC (80% aqueous MeOH) to give 18a (3.4 mg, 58%) as a

white foam: ¹H NMR (CD₃OD, 500 MHz) δ 7.56 (d, 1H, H-6,

(2R,4S,5S)-2-Azidomethyl-5-[(1R,2R,3R,4R)-2,3-dihydroxy-

4-(uracil-1-yl)]tetrahydrofuryl-3-palmitoyl-(1,3)-oxazolidine-4-

carboxylic Acid (27a). A solution of 26a (27 mg, 0.032 mmol) in

CH₃CN-CH₂Cl₂(1:1, 1 mL) was treated with 3HF Et₃N

3

(51 mg, 0.32 mmol) at room temperature for 80 h. The mixture

was partitioned between CHCl₃and 0.1 N aqueous HCl, and the

organic phase was washed with saturated aqueous NaHCO₃and

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

residue was purified by silica gel column chromatography

(1.5 cm ꢀ 8 cm, 10% MeOH-CHCl₃) to give 27a (18 mg,

89%) as a yellow syrup: ¹H NMR (CD₃OD, 500 MHz, 4:1

mixture of rotamers at 20 °C, data for the major rotamer) δ 7.63

J

_6,5=8.0 Hz), 5.71 (d, 1H, H-1⁰, J_{1 ,2}=4.0 Hz), 5.70 (d, 1H, H-5,

0 0

_5,6=8.0 Hz), 5.61 (m, 1H, H-1₀⁰⁰), 4.61 (dd, 1H, H-5⁰, J_{5 ,4}=4.0,

0

_{5 ,6}=2.3 Hz), 4.53 (d, 1H, H-6 , J_{6 ,5}=2.3 Hz), 4.35 (dd, 1H, H-

0

3⁰, J_{3 ,2}=6.3, J_{3 ,4}=6.9 Hz), 4.28 (dd, 1H, H-2 , J_{2 ,1}=4.0, J_{2 ,3}

=

0

6.3 Hz), 3.99 (dd, 1H, H-4⁰, J_{4 ,3}=6.9, J_{4 ,5}=4.0 Hz), 3.56-3.22

(m, 2H, H-2⁰⁰), 2.38-2.21 (m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO),

1.59 (m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO), 1.29 (br s, 24H, CH₃-

(CH₂)₁₂CH₂CH₂CO), 0.90 (t, 3H, CH₃(CH₂)₁₂CH₂CH₂CO, J=

6.9 Hz); ¹³C NMR (CD₃OD, 125 MHz) δ 177.3, 174.7, 166.0,

152.0, 143.6, 103.2, 93.6, 88.7, 85.4, 83.4, 74.2, 70.5, 63.0, 49.6,

40.7, 36.0, 33.1, 30.8, 30.8, 30.6, 30.5, 30.5, 30.3, 25.4, 23.8, 14.5;

ESIMS-HR m/z calcd for C₂₉H₄₇N₄O₉(M - H)^-595.3343,

found 595.3331.

0

(d, 1H, H-6, J_6,5=8.0 Hz), 5.99 (d, 1H, H-1⁰, J_{1 ,2}=5.2 Hz), 5.73

0

(d, 1H, H-5, J_5,6=8.0 Hz), 5.69 (dd, 1H, H-1⁰⁰, J_{1 ,2 a}=2.3,

00 00

J_{1 ,2 b}=8.2 Hz), 4.66 (dd, 1H, H-5⁰, J_{5 ,4}=1.2, J_{5 ,6}=5.8 Hz),

00 00

0

4.43 (d, 1H, H-6⁰, J_{6 ,5}=5.8 Hz), 4.27 (m, 2H, H-3 , H-4 ), 4.23

0

00

(dd, 1H, H-2⁰, J_{2 ,1}=5.2, J_{2 ,3}=2.9 Hz), 3.39 (m, 2H, H-2 ),

2.38-2.26 (m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO), 1.56 (m, 2H,

CH₃(CH₂)₁₂CH₂CH₂CO), 1.28 (br s, 24H, CH₃(CH₂)₁₂-

CH₂CH₂CO), 0.90, (t, 3H, CH₃(CH₂)₁₂CH₂CH₂CO, J = 6.9

Hz); ¹³C NMR (CD₃OD, 125 MHz) δ 174.2, 165.9, 152.4, 141.8,

103.3, 90.5, 90.1, 85.0, 82.8, 75.1, 71.8, 62.7, 52.4, 35.2, 33.1,

30.8, 30.8, 30.6, 30.5, 30.5, 30.3, 25.7, 23.8, 14.5, 9.2. ESIMS-HR

(negative mode) m/z calcd for C₂₉H₄₅N₆O₉(M - H)^-621.3254,

found 621.3263.

0

(2R,4S,5S)-2-Aminoethyl-5-[(1R,2R,3R,4R)-2,3-dihydroxy-4-

(uracil-1-yl)]tetrahydrofuryl-3-palmitoyl-(1,3)-oxazolidine-4-

carboxylic Acid (18b). Compound 18b was prepared from 27b

(8.1 mg, 0.013 mmol) as described above for the synthesis of 18a.

Purification by C18 HPLC (80% aqueous MeOH) afforded 18b

(5.3 mg, 68%) as a white foam: ¹H NMR (CD₃OD, 500 MHz) δ

7.60 (d, 1H, H-6, J_6,5=8.0 Hz), 5.85 (d, 1H, H-1⁰, J_{1 ,2}=4.0 Hz),

0

(2R,4S,5S)-2-Azidoethyl-5-[(1R,2R,3R,4R)-2,3-dihydroxy-4-

(uracil-1-yl)]tetrahydrofuryl-3-palmitoyl-(1,3)-oxazolidine-4-

carboxylic Acid (27b). Compound 27b was prepared from 26b

5.70 (d, 1H, H-5, J_5,6=8.0 Hz), 5.65 (dd, 1H, H-1⁰⁰, J_{1 ,2 a}=4.6,

00 00

J_{1 ,2 b}=5.2 Hz), 4.63 (dd, 1H, H-5⁰, J_{5 ,4}=3.4, J_{5 ,6}=4.6 Hz),

00 00

0

Article

4.48 (d, 1H, H-6⁰, J_{6 ,5}=4.6 Hz), 4.28 (t, 1H, H-3 , J_{3 ,2}=J_{3 ,4}

=

0

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3807

0

CH₃(CH₂)₁₂CH₂CH₂CO), 1.29 (br s, 24H, CH₃(CH₂)₁₂-

CH₂CH₂CO), 0.90 (t, 3H, CH₃(CH₂)₁₂CH₂CH₂CO, J=6.9 Hz);

¹³C NMR (CD₃OD, 125 MHz) δ 174.1, 171.9, 165.9, 152.3,

142.0, 103.4, 90.9, 90.0, 84.5, 80.8, 74.8, 71.3, 60.2, 53.8, 35.1,

33.8, 33.1, 30.8, 30.8, 30.6, 30.5, 30.2, 30.1, 26.0, 25.7, 23.7, 14.5;

ESIMS-HR m/z calcd for C₃₁H₅₀N₆NaO₉(M þ Na)^þ673.3532,

found 673.3531.

5.8 Hz), 4.23 (dd, 1H, H-2⁰, J_{2 ,1}=4.0, J_{2 ,3}=5.8 Hz), 4.13 (dd,

0

1H, H-4⁰, J_{4 ,3}= 5.8, J_{4 ,5}= 3.4 Hz), 3.07 (m, 2H, H-3⁰⁰),

2.38-2.24 (m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO), 2.30-2.15 (m,

2H, H-2⁰⁰), 1.58 (m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO), 1.28 (br s,

24H, CH₃(CH₂)₁₂CH₂CH₂CO), 0.90 (t, 3H, CH₃(CH₂)₁₂CH₂-

CH₂CO, J=6.9 Hz); ¹³C NMR (CD₃OD, 125 MHz) δ 176.7,

175.2, 166.0, 152.2, 142.6, 103.1, 91.7, 89.5, 85.0, 82.6, 74.8, 71.2,

63.1, 49.6, 35.9, 35.6, 33.1, 31.7, 30.8, 30.8, 30.6, 30.5, 30.5, 30.3,

25.7, 23.8, 14.5; ESIMS-HR (negative mode) m/z calcd for

C₃₀H₄₉N₄O₉(M - H)^-609.3500, found 609.3502.

0

(2R,4S,5S)-Methyl 2-Azidopropyl-5-[(1R,2R,3R,4R)-2,3-di-

hydroxy-4-(uracil-1-yl)]tetrahydrofuryl-3-palmitoyl-(1,3)-oxazo-

lidine-4-carboxylate (28c). Compound 28c was prepared from

25c (93 mg, 0.10 mmol) as described above for the synthesis of

27a. Purification by silica gel column chromatography (2 cmꢀ

8 cm, 5% MeOH-CHCl₃) afforded 28c (64 mg, 92%) as a

(2R,4S,5S)-2-Aminopropyl-5-[(1R,2R,3R,4R)-2,3-dihydroxy-

4-(uracil-1-yl)]tetrahydrofuryl-3-palmitoyl-(1,3)-oxazolidine-4-

carboxylic Acid (18c). Compound 18c was prepared from 27c

(8.4 mg, 0.013 mmol) as described above for the synthesis of 18a.

Purification by C18 HPLC (80% aqueous MeOH) afforded 18c

(5.5 mg, 68%) as a white foam: ¹H NMR (CD₃OD, 500 MHz) δ

1

colorless syrup: H NMR (CD₃OD, 500 MHz, 3:2 mixture of

rotamers at 20 °C, data for the major rotamer) δ 7.56 (d, 1H,

H-6, J_6,5=8.0 Hz), 5.92 (d, 1H, H-1⁰, J_{1 ,2}=4.6 Hz), 5.69 (d, 1H,

0

H-5, J_5,6=8.0 Hz), 5.67 (dd, 1H, H-1⁰⁰, J_{1 ,2 a}=4.0, J_{1 ,2 b}

=

0

00 00

6.9 Hz), 4.86 (d, 1H, H-6⁰, J_{6 ,5}=6.9 Hz), 4.75 (dd, 1H, H-5 ,

7.61 (d, 1H, H-6, J_6,5=8.0 Hz), 5.94 (d, 1H, H-1⁰, J_{1 ,2}=4.6 Hz),

0

5.69 (d, 1H, H-5, J_5,6=8.0 Hz), 5.66 (dd, 1H, H-1⁰⁰, J_{1 ,2 a}=5.2,

0

J_{5 ,4}=2.3, J_{5 ,6}=6.9 Hz), 4.27 (dd, 1H, H-3 , J_{3 ,2}=5.2, J_{3 ,4}

0

=

00 00

4.6 Hz), 4.21 (dd, 1H, H-2⁰, J_{2 ,1}= 4.6, J_{2 ,3}= 5.2 Hz), 4.16

J

_{1 ,2 b}=5.7 Hz), 4.60 (dd, 1H, H-5⁰, J_{5 ,4}=2.3, J_{5 ,6}=5.7 Hz),

0

00 00

0

(dd, 1H, H-4⁰, J_{4 ,3}=4.6, J_{4 ,5}=2.3 Hz), 3.84 (s, 3H, CO₂CH₃),

4.46 (d, 1H, H-6⁰, J_{6 ,5}=5.7 Hz), 4.22 (dd, 1H, H-3 , J_{3 ,2}=5.2,

0

3.34 (t, 2H, H-4⁰⁰, J_{4 ,3}= 6.9 Hz), 2.38-2.18 (m, 2H, CH₃-

0

00 00

0

J

_{3 ,4}=5.8 Hz), 4.21 (dd, 1H, H-4 , J_{4 ,3}=5.8, J_{4 ,5}=2.3 Hz), 4.19

(CH₂)₁₂CH₂CH₂CO), 1.97-1.85 (m, 2H, H-3⁰⁰), 1.80-1.68

(m, 2H, H-2⁰⁰), 1.58 (m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO), 1.28

(br s, 24H, CH₃(CH₂)₁₂CH₂CH₂CO), 0.90 (t, 3H, CH₃(CH₂)₁₂-

CH₂CH₂CO, J = 6.9 Hz); ¹³C NMR (CD₃OD, 125 MHz) δ

174.1, 171.9, 165.9, 152.3, 142.1, 103.3, 91.7, 90.8, 84.5, 80.6,

74.9, 71.4, 60.2, 53.7, 35.1, 33.1, 31.7, 30.8, 30.8, 30.6, 30.5, 30.2,

30.1, 26.0, 25.7, 25.3, 23.8, 14.4; ESIMS-HR m/z calcd for

C₃₂H₅₂N₆NaO₉(M þ Na)^þ687.3688, found 687.3692.

(dd, 1H, H-2⁰, J_{2 ,1}=4.6, J_{2 ,3}=5.2 Hz), 2.99-2.93 (m, 2H, H-

4⁰⁰), 2.36-2.24 (m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO), 1.93-1.80

(m, 2H, H-3⁰⁰), 1.79 (m, 2H, H-2⁰⁰), 1.59 (m, 2H, CH₃(CH₂)₁₂-

CH₂CH₂CO), 1.28 (br s, 24H, CH₃(CH₂)₁₂CH₂CH₂CO), 0.90

(t, 3H, CH₃(CH₂)₁₂CH₂CH₂CO, J=6.9 Hz); ¹³C NMR (CD₃OD,

125 MHz) δ 176.4, 174.3, 166.1, 152.4, 141.9, 103.2, 90.9, 90.4,

85.1, 82.2, 75.2, 71.8, 62.6, 49.8, 40.4, 35.4, 33.1, 31.2, 30.8, 30.8,

30.6, 30.6, 30.5, 30.3, 25.8, 24.2, 23.7, 14.5; ESIMS-HR (negative

mode) m/z calcd for C₃₁H₅₁N₄O₉(M - H)^-623.3656, found

623.3664.

0

(2R,4S,5S)-Methyl 2-Aminomethyl-5-[(1R,2R,3R,4R)-2,3-di-

hydroxy-4-(uracil-1-yl)]tetrahydrofuryl-3-palmitoyl-(1,3)-oxazo-

lidine-4-carboxylate HCl Salt (19a). Compound 19a was pre-

pared from 28a (7.8 mg, 0.012 mmol) in the presence of 1 N

aqueous HCl (0.5 mL) as described above for the synthesis of

18a. Purification by C18 HPLC (85% aqueous MeOH contain-

ing 0.5% HCl) afforded 19a (4.2 mg, 54%) as a white foam: ¹H

NMR (CD₃OD, 500 MHz, 9:1 mixture of rotamers at 20 °C,

data for the major rotamer) δ 7.53 (d, 1H, H-6, J_6,5=8.0 Hz),

(2R,4S,5S)-Methyl 2-Azidomethyl-5-[(1R,2R,3R,4R)-2,3-di-

hydroxy-4-(uracil-1-yl)]tetrahydrofuryl-3-palmitoyl-(1,3)-oxazo-

lidine-4-carboxylate (28a). Compound 28a was prepared from

25a (70 mg, 0.080 mmol) as described above for the synthesis of

27a. Purification by silica gel column chromatography (2 cmꢀ

8 cm, 5% MeOH-CHCl₃) afforded 28a (46 mg, 90%) as a

1

colorless syrup: H NMR (CD₃OD, 500 MHz, 2:1 mixture of

5.70 (d, 1H, H-5, J_5,6=8.0 Hz), 5.69 (d, 1H, H-1⁰J_{1 ,2}=3.4 Hz),

0 0

rotamers at 20 °C, data for the major rotamer) δ 7.54 (d, 1H, H-

6, J_6,5=8.0 Hz), 5.89 (d, 1H, H-1⁰, J_{1 ,2}=4.6 Hz), 5.76 (dd, 1H,

5.68 (m, 1H, H-1⁰⁰), 5.05 (d, 1H, H-6⁰, J_{6 ,5}=2.4 Hz), 4.81 (dd,

0

H-1⁰⁰, J_{1 ,2 a}=5.2, J_{1 ,2 b}=6.4 Hz), 5.73 (d, 1H, H-5, J_5,6

=

0

1H, H-5⁰, J_{5 ,4}=3.4, J_{5 , 6}=2.4 Hz), 4.36 (dd, 1H, H-3 , J_{3 ,2}

=

0

00 00

0

8.0 Hz), 4.95 (d, 1H, H-6⁰, J_{6 ,5}=6.9 Hz), 4.94 (dd, 1H, H-5 ,

6.4, J_{3 ,4}=6.9 Hz), 4.31 (dd, 1H, H-2 , J_{2 ,1}=3.4, J_{2 ,3}=6.4 Hz),

0

J

4.03 (dd, 1H, H-4⁰, J_{4 ,3}= 6.9, J_{4 ,5}= 3.4 Hz), 3.87 (s, 3H,

CO₂CH₃), 3.35 (m, 2H, H-2⁰⁰), 2.38-2.24 (m, 2H, CH₃-

(CH₂)₁₂CH₂CH₂CO), 1.59 (m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO),

1.28 (br s, 24H, CH₃(CH₂)₁₂CH₂CH₂CO), 0.90 (t, 3H, CH₃-

(CH₂)₁₂CH₂CH₂CO, J = 6.9 Hz); ¹³C NMR (CD₃OD, 125

MHz) δ 175.1, 172.7, 165.9, 152.0, 143.6, 103.3, 94.0, 88.6,

84.7, 81.7, 74.0, 70.5, 60.6, 54.0, 35.4, 33.1, 30.8, 30.8, 30.6,

30.5, 30.5, 30.1, 25.3, 23.7, 14.4; ESIMS-HR m/z calcd for

C₃₀H₅₁N₄O₉(M þ H)^þ611.3651, found 611.3651.

0

_{5 ,4}=2.3, J_{5 ,6}=6.9 Hz), 4.31 (t, 1H, H-3 , J_{3 ,2}=J_{3 ,4}=5.8 Hz),

0

4.24 (dd, 1H, H-2⁰, J_{2 ,1}=4.6, J_{2 ,3}=5.8 Hz), 4.13 (dd, 1H, H-4 ,

0

0 0 0 0

J_{4 ,3}=5.8, J_{4 ,5}=2.3 Hz), 3.84 (s, 3H, CO₂CH₃), 3.48 (br s, 2H,

H-2⁰⁰), 2.40-2.27 (m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO), 1.58 (m,

2H, CH₃(CH₂)₁₂CH₂CH₂CO), 1.28 (br s, 24H, CH₃(CH₂)₁₂-

CH₂CH₂CO), 0.90 (t, 3H, CH₃(CH₂)₁₂CH₂CH₂CO, J=6.9 Hz);

¹³C NMR (CD₃OD, 125 MHz) δ 174.5, 171.5, 165.9, 152.2,

142.2, 103.4, 91.2, 90.8, 84.8, 81.0, 74.6, 71.1, 60.1, 53.4, 35.0,

33.1, 30.8, 30.8, 30.6, 30.5, 30.2, 30.1, 25.9, 25.6, 23.8, 14.5;

ESIMS-HR m/z calcd for C₃₀H₄₈N₆NaO₉(M þ Na)^þ659.3375,

found 659.3371.

(2R,4S,5S)-Methyl 2-Aminoethyl-5-[(1R,2R,3R,4R)-2,3-di-

hydroxy-4-(uracil-1-yl)]tetrahydrofuryl-3-palmitoyl-(1,3)-oxazo-

lidine-4-carboxylate HCl Salt (19b). Compound 19b was pre-

pared from 28b (8.2 mg, 0.013 mmol) in the presence of 1 N

aqueous HCl (0.5 mL) as described above for the synthesis of

18a. Purification by C18 HPLC (85% aqueous MeOH contain-

ing 0.5% HCl) afforded 19b (4.9 mg, 57%) as a white foam: ¹H

NMR (CD₃OD, 500 MHz, 9:1 mixture of rotamers at 20 °C,

data for the major rotamer) δ 7.55 (d, 1H, H-6, J_6,5=8.0 Hz),

(2R,4S,5S)-Methyl 2-Azidoethyl-5-[(1R,2R,3R,4R)-2,3-dihy-

droxy-4-(uracil-1-yl)]tetrahydrofuryl-3-palmitoyl-(1,3)-oxazoli-

dine-4-carboxylate (28b). Compound 28b was prepared from

25b (87 mg, 0.10 mmol) as described above for the synthesis of

27a. Purification by silica gel column chromatography (2 cmꢀ

8 cm, 5% MeOH-CHCl₃) afforded 28b (57 mg, 89%) as a

1

colorless syrup: H NMR (CD₃OD, 500 MHz, 3:2 mixture of

5.77 (d, 1H, H-1⁰, J_{1 ,2}=4.0 Hz), 5.70 (d, 1H, H-5, J_5,6=8.0 Hz),

0 0

rotamers at 20 °C, data for the major rotamer) δ 7.55 (d, 1H,

H-6, J_6,5=8.0 Hz), 5.92 (d, 1H, H-1⁰, J_{1 ,2}=4.6 Hz), 5.75 (dd,

5.68 (dd, 1H, H-1⁰⁰, J_{1 ,2 a}=5.2, J_{1 ,2 b}=6.3 Hz), 4.99 (d, 1H, H-

0

00 00

0

1H, H-1⁰⁰, J_{1 ,2 a}=3.4, J_{1 ,2 b}=9.2 Hz), 5.71 (d, 1H, H-5, J_{5, 6}

=

0

6⁰, J_{6 ,5}=3.5 Hz), 4.79 (dd, 1H, H-5 , J_{5 ,4}=2.9, J_{5 ,6}=3.5 Hz),

00 00

0

8.0 Hz), 4.87 (d, 1H, H-6⁰, J_{6 ,5}=4.6 Hz), 4.77 (dd, 1H, H-5 ,

4.32 (dd, 1H, H-3⁰, J_{3 ,2}=5.8, J_{3 ,4}=6.3 Hz),₀4.27 (dd, 1H, H-2 ,

0

_{5 ,4}=2.3, J_{5 ,6}=4.6 Hz), 4.28 (dd, 1H, H-3 , J_{3 ,2}=5.2, J_{3 ,4}

0

J

=

J

_{2 ,1}=4.0, J_{2 ,3}=5.8 Hz), 4.08 (dd, 1H, H-4 , J_{4 ,3}=6.3, J_{4 ,5}=

4.0 Hz), 4.21 (dd, 1H, H-2⁰, J_{2 ,1}=4.6, J_{2 ,3}=5.2 Hz), 4.16 (dd,

2.9 Hz), 3.85 (s, 3H, CO₂CH₃), 3.07 (m, 2H, H-3⁰⁰), 2.35-2.25

(m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO), 2.17-2.08 (m, 2H, H-2⁰⁰),

1.56 (m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO), 1.29 (br s, 24H, CH₃-

(CH₂)₁₂CH₂CH₂CO), 0.90 (t, 3H, CH₃(CH₂)₁₂CH₂CH₂CO,

0

1H, H-4⁰, J_{4 ,3}=4.0, J_{4 ,5}=2.3 Hz), 3.84 (s, 3H, CO₂CH₃), 3.42

0

(t, 2H, H-3⁰⁰, J_{3 ,2}= 6.9 Hz), 2.40-2.24 (m, 2H, -CH₃-

00 00

(CH₂)₁₂CH₂CH₂CO), 2.12-1.88 (m, 2H, H-2⁰⁰), 1.58 (m, 2H,

3808 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9

Ii et al.

J=6.9 Hz); ¹³C NMR (CD₃OD, 125 MHz) δ 174.8, 172.0, 165.9,

152.2, 143.0, 103.2, 90.1, 89.9, 84.5, 81.1, 74.4, 70.9, 60.4, 53.8,

36.9, 35.2, 33.1, 30.8, 30.8, 30.6, 30.5, 30.5, 30.1, 25.6, 23.7, 14.4;

ESIMS-HR m/z calcd for C₃₁H₅₃N₄O₉(M þ H)^þ625.3807,

found 625.3803.

24H, CH₃(CH₂)₁₂CH₂CH₂CO), 0.94, 0.91 (each s, each 9H, tert-

butyl), 0.90 (t, 3H, CH₃(CH₂)₁₂CH₂CH₂CO, J=6.9 Hz), 0.15,

0.14, 0.10, 0.09 (each s, each 3H, SiCH₃); ¹³C NMR (CD₃OD,

125 MHz) δ 174.4, 169.7, 165.9, 152.1, 141.5, 103.3, 93.9, 90.3,

84.8, 81.0, 76.3, 72.8, 60.9, 53.4, 34.9, 33.1, 30.8, 30.7, 30.5,

30.5, 30.4, 30.3, 30.1, 30.1, 28.2, 26.4, 26.4, 26.0, 25.6, 23.7, 18.9,

18.9, 14.5, -4.1, -4.4, -4.4, -4.5; ESIMS-HR m/z calcd for

C₄₅H₈₂N₆NaO₉Si₂(M þ Na)^þ929.5574, found 929.5583.

(2R,4S,5S)-tert-Butyl 2-Azidoethyl-5-[(1R,2R,3R,4R)-2,3-di-

tert-butyldimethylsilyloxy-4-(uracil-1-yl)]tetrahydrofuryl-3-

palmitoyl-(1,3)-oxazolidine-4-carboxylate (30b). Compound 30b

was prepared from 26b (171 mg, 0.20 mmol) as described above

for the synthesis of 30a. Purification by silica gel column

chromatography (2 cmꢀ10 cm, 33% AcOEt-hexane) afforded

30b (138 mg, 76%) as a yellow syrup: ¹H NMR (CD₃OD, 500

MHz, 2:1 mixture of rotamers at 20 °C, data for the major

(2R,4S,5S)-Methyl 2-Aminopropyl-5-[(1R,2R,3R,4R)-2,3-di-

hydroxy-4-(uracil-1-yl)]tetrahydrofuryl-3-palmitoyl-(1,3)-oxazo-

lidine-4-carboxylate HCl Salt (19c). Compound 19c was pre-

pared from 28c (8.7 mg, 0.013 mmol) in the presence of 1 N

aqueous HCl (0.5 mL) as described above for the synthesis of

18a. Purification by C18 HPLC (85% aqueous MeOH contain-

ing 0.5% HCl) afforded 19c (4.9 mg, 56%) as a white foam: ¹H

NMR (CD₃OD, 500 MHz, 9:1 mixture of rotamers at 20 °C,

data for the major rotamer) δ 7.55 (d, 1H, H-6, J_6,5=8.0 Hz),

5.84 (d, 1H, H-1⁰, J_{1 ,2}=4.0 Hz), 5.69 (d, 1H, H-5, J_5,6=8.0 Hz),

0

5.63 (dd, 1H, H-1⁰⁰, J_{1 ,2 a}=4.6, J_{1 ,2 b}=5.2 Hz), 4.93 (d, 1H, H-

00 00

0

6⁰, J_{6 ,5}=4.0 Hz), 4.76 (dd, 1H, H-5 , J_{5 ,4}=2.9, J_{5 ,6}=4.0 Hz),

rotamer) δ 7.63 (d, 1H, H-6, J_6,5=8.0 Hz), 5.95 (d, 1H, H-1⁰,

0

4.28 (dd, 1H, H-3⁰, J_{3 ,2}=5.7, J_{3 ,4}=5.2 Hz),₀4.24 (dd, 1H, H-2 ,

00

0

00 00

J

_{1 ,2}=4.6 Hz), 5.89 (dd, 1H, H-1 , J_{1 ,2 a}=4.6, J_{1 ,2 b}=8.0 Hz),

0

5.74 (d, 1H, H-5, J_5,6=8.0 Hz), 4.72 (d, 1H, H-6⁰, J_{6 ,5}=6.3 Hz),

0

J_{2 ,1}=4.0, J_{2 ,3}=5.7 Hz), 4.12 (dd, 1H, H-4 , J_{4 ,3}=5.2, J_{4 ,5}=

0

2.9 Hz), 3.84 (s, 3H, CO₂CH₃), 2.96 (m, 2H, H-4⁰⁰), 2.36-2.21

(m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO), 1.89-1.76 (m, 2H, H-2⁰⁰),

1.74 (m, 2H, H-3⁰⁰), 1.58 (m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO), 1.28

(br s, 24H, CH₃(CH₂)₁₂CH₂CH₂CO), 0.90 (t, 3H, CH₃(CH₂)₁₂-

CH₂CH₂CO, J = 6.9 Hz); ¹³C NMR (CD₃OD, 125 MHz) δ

174.4, 171.9, 165.9, 152.2, 142.5, 103.2, 91.4, 91.3, 84.5, 80.7,

74.7, 71.4, 60.3, 53.7, 35.2, 33.1, 31.6, 30.8, 30.8, 30.6, 30.6,

30.5, 30.2, 25.7, 24.0, 23.8, 14.5; ESIMS-HR m/z calcd for

C₃₂H₅₅N₄O₉(M þ H)^þ639.3964, found 639.3964.

4.63 (dd, 1H, H-5⁰, J_{5 ,4}=1.2, J_{5 ,6}=6.3 Hz), 4.29 (t, 1H, H-3 ,

0

J_{3 ,2}=J_{3 ,4}=4.0 Hz), 4.28 (dd, 1H, H-2 , J_{2 ,1}=4.6, J_{2 ,3}

0

=

4.0 Hz), 4.26 (dd, 1H, H-4⁰, J_{4 ,3}=4.0, J_{4 ,5}=1.2 Hz), 3.46 (t, 2H,

0

H-3⁰⁰, J_{3 ,2}= 6.9 Hz), 2.42-2.15 (m, 2H, CH₃(CH₂)₁₂CH₂-

00 00

CH₂CO), 2.10-1.89 (m, 2H, H-2⁰⁰), 1.62 (m, 2H, CH₃(CH₂)₁₂-

CH₂CH₂CO), 1.53 (s, 9H, tert-butyl), 1.28 (br s, 24H, CH₃-

(CH₂)₁₂CH₂CH₂CO), 0.95, 0.91 (each s, each 9H, tert-butyl),

0.88 (t, 3H, CH₃(CH₂)₁₂CH₂CH₂CO, J=6.9 Hz), 0.17, 0.15,

0.10, 0.09 (each s, each 3H, SiCH₃); ¹³C NMR (CD₃OD, 125

MHz) δ 173.9, 170.4, 165.9, 152.2, 141.2, 103.3, 93.9, 89.8, 84.9,

80.5, 76.3, 73.1, 60.6, 35.0, 33.4, 33.1, 30.8, 30.8, 30.7, 30.5, 30.5,

30.4, 30.2, 30.1, 28.2, 26.4, 26.4, 26.1, 25.7, 23.7, 19.0, 18.9, 14.5,

-4.1, -4.4, -4.4, -4.5; ESIMS-HR m/zcalcdfor C₄₆H₈₄N₆NaO₉Si₂

(M þ Na)^þ943.5731, found 943.5734.

Bicyclic Lactam (29). A solution of 28b (7.9 mg, 0.012 mmol)

and 10% Pd(OH)₂/C (1.0 mg) in MeOH (1 mL) was vigorously

stirred at room temperature under H₂atmosphere for 4 h. The

catalyst was filtered off through a Celite pad, and the filtrate was

concentrated in vacuo. The residue was purified by C18 HPLC

(80% aqueous MeOH) to give 29 (3.9 mg, 53%) as a white foam:

¹H NMR (500 MHz, CD₃OD, 5:1 mixture of rotamers at 20 °C,

data for the major rotamer) δ 7.42 (d, 1H, H-6, J_6,5=8.0 Hz),

(2R,4S,5S)-tert-Butyl 2-Azidomethyl-5-[(1R,2R,3R,4R)-2,3-

dihydroxy-4-(uracil-1-yl)]tetrahydrofuryl-3-palmitoyl-(1,3)-oxa-

zolidine-4-carboxylate (31a). Compound 31a was prepared from

30a (150 mg, 0.17 mmol) as described above for the synthesis of

37a. Purification by silica gel column chromatography (1.5 cmꢀ

8 cm, 75% AcOEt-hexane) afforded 31a (89 mg, 79%) as a

6.24 (dd, 1H, H-1⁰⁰, J_{1 ,2 a}=1.7, J_{1 ,2 b}=4.0 Hz), 5.93 (d, 1H, H-

00 00

1⁰, J_{1 ,2}=5.2 Hz), 5.71 (d, 1H, H-5, J_5,6=8.0 Hz), 4.91 (t, 1H, H-

0

5⁰, J_{5 ,4}=J_{5 ,6}=2.3 Hz), 4.72 (d, 1H, H-5 , J_{6 ,5}=2.3 Hz), 4.25 (t,

0

1

0

1H, H-3⁰, J_{3 ,2}=J_{3 ,4}=5.2 Hz), 4.15 (t, 1H, H-2 , J_{2 ,1}=J_{2 ,3}

=

0

colorless syrup: H NMR (CD₃OD, 500 MHz, 2:1 mixture of

5.2 Hz), 4.10 (dd, 1H, H-3⁰, J_{4 ,3}=5.2, J_{4 , 5}=2.3 Hz), 3.59-3.18

(m, 2H, H-3⁰⁰), 2.40-2.29 (m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO),

2.05-1.93 (m, 2H, H-2⁰⁰), 1.55 (br s, 2H, CH₃(CH₂)₁₂-

CH₂CH₂CO), 1.29 (br s, 24H, CH₃(CH₂)₁₂CH₂CH₂CO), 0.90

(t, 3H, CH₃(CH₂)₁₂CH₂CH₂CO, J=6.9 Hz); ¹³C NMR (CD₃OD,

125 MHz) δ 172.0, 165.9, 141.5, 103.4, 89.9, 88.6, 86.5, 80.8,

74.8, 71.2, 63.6, 52.1, 37.7, 34.9, 34.8, 33.1, 30.8, 30.8, 30.8, 30.6,

30.6, 30.5, 30.4, 30.2, 25.6, 23.8, 14.5; ESIMS-HR m/z calcd for

C₃₀H₄₈N₄NaO₈(M þ Na)^þ615.3364, found 615.3372.

0

rotamers at 20 °C, data for the major rotamer) δ 7.54 (d, 1H,

H-6, J_6,5=8.0 Hz), 5.89 (d, 1H, H-1⁰, J_{1 ,2}=4.6 Hz), 5.70 (d, 1H,

0

H-5, J_5,6=8.0Hz),5.69(dd, 1H,H-1⁰⁰,J_{1 ,2 a}=4.6, J_{1 ,2 b}=6.9 Hz),

00 00

4.87 (dd, 1H, H-5⁰, J_{5 ,4}= 2.3, J_{5 , 6}= 4.6 Hz), 4.80 (d, 1H,

0

H-6⁰, J_{6 ,5}=4.6 Hz), 4.31 (t, 1H, H-3 , J_{3 ,2}=J_{3 ,4}=5.2 Hz), 4.2₀3

0

(dd, 1H, H-2⁰, J_{2 ,1}=4.6, J_{2 ,3}=5.2 Hz), 4.10 (m, 1H, H-4 ,

0

00

0

00 00

J_{4 ,3}=5.2, J_{4 ,5}=2.3 Hz), 3.49 (t, 2H, H-2 , J_{2 ,1}=3.5 Hz),

2.40-2.25 (m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO), 1.59 (m, 2H,

CH₃(CH₂)₁₂CH₂CH₂CO), 1.54 (s, 9H, tert-butyl), 1.29 (br s,

24H, CH₃(CH₂)₁₂CH₂CH₂CO), 0.90 (t, 3H, CH₃(CH₂)₁₂-

CH₂CH₂CO, J = 6.9 Hz); ¹³C NMR (CD₃OD, 125 MHz)

δ 174.4, 169.9, 165.9, 152.3, 142.2, 103.4, 91.1, 90.8, 84.8,

81.3, 74.6, 71.2, 61.3, 53.4, 35.1, 33.1, 30.8, 30.8, 30.7, 30.6,

30.5, 30.4, 30.2, 30.2, 28.1, 26.0, 25.6, 23.7, 14.5; ESIMS-HR

m/z calcd for C₃₃H₅₄N₆NaO₉(M þ Na)^þ701.3845, found

701.3859.

(2R,4S,5S)-tert-Butyl 2-Azidomethyl-5-[(1R,2R,3R,4R)-2,3-

di-tert-butyldimethylsilyloxy-4-(uracil-1-yl)]tetrahydrofuryl-3-

palmitoyl-(1,3)-oxazolidine-4-carboxylate (30a). A solution of

t

26a (212 mg, 0.25 mmol) in BuOH-CH₂Cl₂(1:1, 4 mL) was

treated with N,N⁰-diisopropyl-O-tert-butylisourea (251 mg,

1.25 mmol) and NH₄Cl (41 mg, 1.25 mmol) at 0 °C for 20 h.

The mixture was diluted with CHCl₃(200 mL), which was

washed with saturated aqueous NaHCO₃and brine. The org-

anic layer was dried (Na₂SO₄), filtered, and concentrated in

vacuo. The residue was purified by silica gel column chromato-

graphy (2 cmꢀ10 cm, 33% AcOEt-hexane) to give 30a (165 mg,

73%) as a white foam: ¹H NMR (CD₃OD, 500 MHz, 3:2

mixture of rotamers at 20 °C, data for the major rotamer) δ

(2R,4S,5S)-tert-Butyl 2-Azidoethyl-5-[(1R,2R,3R,4R)-2,3-di-

hydroxy-4-(uracil-1-yl)]tetrahydrofuryl-3-palmitoyl-(1,3)-oxazo-

lidine-4-carboxylate (31b). Compound 31b was prepared from

30b (121 mg, 0.13 mmol) as described above for the synthesis of

27a. Purification by silica gel column chromatography (1.5 cmꢀ

8 cm, 75% AcOEt-hexane) afforded 31b (73 mg, 80%) as a

1

7.63 (d, 1H, H-6, J_6,5=8.0 Hz), 5.91 (d, 1H, H-1⁰, J_{1 ,2}=4.0 Hz),

0

colorless syrup: H NMR (CD₃OD, 500 MHz, 2:1 mixture of

5.83 (dd, 1H, H-1⁰⁰, J_{1 ,2 a}=2.9, J_{1 ,2 b}=5.2 Hz), 5.75 (d, 1H,

00 00

rotamers at 20 °C, data for the major rotamer) δ 7.57 (d, 1H,

H-6, J_6,5=8.0 Hz), 5.93 (d, 1H, H-1⁰, J_{1 ,2}=5.2 Hz), 5.74 (dd,

H-5, J_5,6=8.0 Hz), 4.84 (dd, 1H, H-5⁰, J_{5 ,4}=1.2, J_{5 ,6}=6.3 Hz),

0

1H, H-1⁰⁰, J_{1 ,2 a}=4.0, J_{1 ,2 b}=7.5 Hz), 5.71 (d, 1H, H-5, J_5,6

=

0

4.78 (d, 1H, H-6⁰, J_{6 ,5}=6.3 Hz), 4.30 (dd, 1H, H-3 , J_{3 ,2}=4.0,

00 00

0

8.0 Hz), 4.73 (d, 1H, H-6⁰, J_{6 ,5}=5.2 Hz), 4.69 (dd, 1H, H-5 ,

0

J_{3 ,4}=4.6 Hz), 4.28 (t, 1H, H-2 , J_{2 ,1}=J_{2 ,3}=4.0 Hz), 4.24 (dd,

0

1H, H-4⁰, J_{4 ,3}= 4.6, J_{4 ,5}= 1.2 Hz), 3.53 (m, 2H, H-2⁰⁰),

2.44-2.18 (m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO), 1.60 (m, 2H,

CH₃(CH₂)₁₂CH₂CH₂CO), 1.53 (s, 9H, tert-butyl), 1.28 (br s,

0

J

_{5 ,4}=2.3, J_{5 ,6}=5.2 Hz), 4.28 (dd, 1H, H-3 , J_{3 ,2}=4.6, J_{3 ,4}

0

=

5.2 Hz), 4.21 (dd, 1H, H-2⁰, J_{2 ,1}= 5.2, J_{2 ,3}= 4.6 Hz), 4.14

0

00

(dd, 1H, H-4⁰, J_{4 ,3}= 5.2, J_{4 ,5}= 2.3 Hz), 3.43 (t, 2H, H-3 ,

0

Article

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3809

00 00

J_{3 ,2}=6.4 Hz), 2.40-2.18 (m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO),

-4.5; ESIMS-HR m/z calcd for C₂₈H₄₈N₆NaO₉Si₂(M þ Na)^þ

691.2914, found 691.2909.

2.12-1.88 (m, 2H, H-2⁰⁰), 1.61 (m, 2H, CH₃(CH₂)₁₂CH₂-

CH₂CO), 1.53 (s, 9H, tert-butyl), 1.29 (br s, 24H, CH₃-

(CH₂)₁₂CH₂CH₂CO), 0.90 (t, 3H, CH₃(CH₂)₁₂CH₂CH₂CO,

J = 6.9 Hz); ¹³C NMR (CD₃OD, 125 MHz) δ 174.0, 170.5,

165.9, 152.3, 142.0, 103.3, 90.8, 89.9, 84.6, 81.1, 74.9, 71.4, 60.9,

35.2, 33.9, 33.1, 30.8, 30.7, 30.6, 30.6, 30.5, 30.4, 30.2, 30.2, 28.2,

26.1, 25.7, 23.8, 14.5; ESIMS-HR m/z calcd for C₃₄H₅₆N₆NaO₉

(M þ Na)^þ715.4001, found 715.4009.

(2R,4S,5S)-Methyl 3-Acetyl-2-azidoethyl-5-[(1R,2R,3R,4R)-

2,3-di-tert-butyldimethylsilyloxy-4-(uracil-1-yl)]tetrahydrofuryl-

(1,3)-oxazolidine-4-carboxylate (32b). Compound 32b was pre-

pared from 22 (500 mg, 0.72 mmol), 3-azidopropanal (143 mg,

1.4 mmol), and acetyl chloride (113 mg, 1.4 mmol) as described

above for the synthesis of 25a. Purification by silica gel column

chromatography (2 cmꢀ10 cm, 33% AcOEt-hexane) afforded

1

(2R,4S,5S)-tert-Butyl 2-Aminomethyl-5-[(1R,2R,3R,4R)-2,3-

dihydroxy-4-(uracil-1-yl)]tetrahydrofuryl-3-palmitoyl-(1,3)-oxa-

zolidine-4-carboxylate (20a). Compound 20a was prepared from

31a (11 mg, 0.016 mmol) as described above for the synthesis of

18a. Purification by C18 HPLC (90% aqueous MeOH) afforded

20a (5.6 mg, 54%) as a white foam: ¹H NMR (500 MHz,

CD₃OD, 2:1 mixture of rotamers at 20 °C, data for the major

diastereomer) δ 7.54 (d, 1H, H-6, J_6,5=8.2 Hz), 5.70 (d, 1H, H-5,

32b (417 mg, 85%) as a yellow foam: H NMR (CD₃OD, 500

MHz, 3:2 mixture of rotamers at 20 °C, data for the major

rotamer) δ 7.59 (d, 1H, H-6, J_6,5=8.0 Hz), 5.98 (d, 1H, H-1⁰,

00

0 0 00 00 00 00

_{1 ,2}=5.2 Hz), 5.85 (dd, 1H, H-1 , J_{1 ,2 a}=4.0, J_{1 ,2 b}=8.0 Hz),

J

5.74 (d, 1H, H-5, J_5,6=8.0 Hz), 4.86 (d, 1H, H-6⁰, J_{6 ,5}=5.8 Hz),

0

4.74 (dd, 1H, H-5⁰, J_{5 ,4}=1.2, J_{5 , 6}=5.8 Hz), 4.31 (dd, 1H, H-2 ,

0

J_{2 ,1}=5.2, J_{2 , 3}=4.6 Hz), 4.30 (dd, 1H, H-3 , J_{3 ,2}=4.6, J_{3 , 4}

0

=

4.0 Hz), 4.27 (dd, 1H, H-4⁰, J_{4 ,3}=4.0, J_{4 , 5}=1.2 Hz), 3.85 (s,

0

J_5,6=8.2 Hz), 5.67 (m, 1H, H-1⁰⁰), 5.66 (d, 1H, H-1⁰, J_{1 ,2}

3.2 Hz), 4.93 (d, 1H, H-6⁰, J_{6 ,5}=2.8 Hz), 4.72 (dd, 1H, H-5 ,

=

0

00 00

3H, CO₂CH₃), 3.44 (t, 2H, H-3⁰⁰, J_{3 ,2}=6.3 Hz), 2.16-2.01 (m,

0

2H, H-2⁰⁰), 2.03 (s, 3H, acetyl), 0.95, 0.90 (each s, each 9H, tert-

butyl), 0.16, 0.15, 0.09, 0.07 (each s, each 3H, SiCH₃); ¹³C NMR

(CD₃OD, 125 MHz) δ 171.5, 171.2, 170.0, 165.5, 152.1, 141.2,

103.4, 90.2, 89.8, 84.5, 80.2, 76.2, 73.2, 60.5, 53.8, 33.3, 26.4,

22.3, 18.9, 18.9, -4.2, -4.3, -4.3, -4.6; ESIMS-HR m/z calcd

for C₂₉H₅₀N₆NaO₉Si₂(M þ Na)^þ705.3070, found 705.3077.

(2R,4S,5S)-Methyl 3-Acetyl-2-azidopropyl-5-[(1R,2R,3R,4R)-

2,3-di-tert-butyldimethylsilyloxy-4-(uracil-1-yl)]tetrahydrofuryl-

(1,3)-oxazolidine-4-carboxylate (32c). Compound 32c was pre-

pared from 22 (300 mg, 0.43 mmol), 4-azidobutanal (98 mg,

0.87 mmol), and acetyl chloride (68 mg, 0.87 mmol) as described

above for the synthesis of 25a. Purification by silica gel column

chromatography (2 cmꢀ8 cm, 33% AcOEt-hexane) afforded

32c (265 mg, 88%) as a yellow foam: ¹HNMR(CD₃OD, 500 MHz,

3:2 mixture of rotamers at 20 °C, data for the major rotamer) δ 7.65

0

J_{5 ,4}=3.2, J_{5 ,6}=2.8 Hz), 4.37 (dd, 1H, H-3 , J_{3 ,2}=6.9, J_{3 ,4}

0

=

6.9 Hz), 4.32 (dd, 1H, H-2⁰, J_{2 ,1}=3.2, J_{2 ,3}=6.9 Hz),₀4₀.02 (dd,

0

1H, H-4⁰, J_{4 ,3}=6.9, J_{4 ,5}=3.2 Hz), 3.32 (m, 2H, H-2 ), 2.41-

2.16 (m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO), 1.59 (m, 2H, CH₃(CH₂)₁₂-

CH₂CH₂CO), 1.56 (s, 9H, tert-butyl), 1.29 (br s, 24H, CH₃-

(CH₂)₁₂CH₂CH₂CO), 0.90 (t, 3H, CH₃(CH₂)₁₂CH₂CH₂CO, J=

6.9 Hz); ¹³C NMR (CD₃OD, 125 MHz) δ 174.8, 171.7, 165.9,

152.0, 143.7, 103.2, 94.3, 88.6, 84.7, 82.1, 74.1, 70.6, 60.8, 42.1,

35.5, 33.0, 30.8, 30.8, 30.6, 30.5, 30.4, 30.3, 30.2, 30.2, 28.1, 25.3,

23.8, 14.5; ESIMS-HR calcd for C₃₃H₅₇N₄O₉(M þ H)^þ

653.4120, found 653.4140.

0

(2R,4S,5S)-tert-Butyl 2-Aminoethyl-5-[(1R,2R,3R,4R)-2,3-

dihydroxy-4-(uracil-1-yl)]tetrahydrofuryl-3-palmitoyl-(1,3)-oxa-

zolidine-4-carboxylate (20b). Compound 20b was prepared from

31b (7.9 mg, 0.011 mmol) as described above for the synthesis of

18a. Purification by C18 HPLC (90% aqueous MeOH) afforded

20b (3.8 mg, 50%) as a white foam: ¹H NMR (500 MHz,

CD₃OD, 3:2 mixture of rotamers at 20 °C, data for the major

diastereomer) δ 7.56 (d, 1H, H-6, J_6,5=8.0 Hz), 5.77 (d, 1H,

(d, 1H, H-6, J_6,5=8.0 Hz), 5.95 (d, 1H, H-1⁰, J_{1 ,2}=5.2 Hz), 5.80 (d,

0

1H, H-5, J_5,6=8.0 Hz), 5.79 (m, 1H, H-1⁰⁰), 4.85(d, 1H, H-6⁰, J_{6 ,5}

0

=

5.7 Hz), 4.70 (dd, 1H, H-5⁰, J_{5 ,4}=1.2, J_{5 ,6}=5.7₀Hz), 4.31 (dd, 1H,

0

H-3⁰, J_{3 ,2}=5.2, J_{3 ,4}=4.0 Hz), 4.29 (dd, 1H, H-2 , J_{2 ,1}=1.2, J_{2 ,3}

=

0

5.2 Hz), 4.24 (dd, 1H, H-4⁰, J_{4 ,3}=4.0, J_{4 ,5}=1.2 Hz), 3.85 (s, 3H,

0

H-1⁰, J_{1 ,2}=4.0 Hz), 5.69 (d, 1H, H-5, J_5,6=8.0 Hz), 5.67 (t, 1H,

CO₂CH₃), 3.36 (t, 2H, H-4⁰⁰, J_{4 ,3}=6.9 Hz), 2.04 (s, 3H, acetyl),

0

00 00

H-1⁰⁰, J_{1 ,2}=5.2 Hz), 4.82 (d, 1H, H-6 , J_{6 ,5}=4.0 Hz), 4.71 (d,

0

1.95-1.70 (m, 4H, H-2⁰, H-3⁰⁰), 0.95, 0.91 (each s, each 9H, tert-

butyl), 0.16, 0.15, 0.11, 0.09 (each s, each 3H, SiCH₃); ¹³C NMR

(CD₃OD, 125 MHz) δ 171.5, 171.1, 165.5, 152.2, 141.1, 103.2, 91.8,

89.9, 84.1, 79.8, 76.3, 73.1, 60.4, 53.7, 51.9, 31.3, 26.4, 25.4, 22.3,

18.9, 18.9, -4.1, -4.3, -4.4, -4.5; ESIMS-HR m/z calcd for

C₃₀H₅₂N₆NaO₉Si₂(M þ Na)^þ719.3227, found 719.3223.

00 00

0

1H, H-5⁰, J_{5 ,4}=2.9, J_{5 ,6}=4.0 Hz), 4.32 (t, 1H, H-3 , J_{3 ,2}

=

0

J

_{3 ,4}=5.7 Hz), 4.27 (dd, 1H, H-2 , J_{2 ,1}=4.0, J_{2 ,3}=5.7 Hz), 4.07

(dd, 1H, H-4⁰, J_{4 ,3}=5.7, J_{4 ,5}=2.9 Hz), 3.16-3.04 (m, 2H, H-

3⁰⁰), 2.32 (m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO), 2.27-2.14 (m, 2H,

H-2⁰⁰), 1.57 (m, 2H, CH₃(CH₂)₁₂CH₂CH₂CO), 1.54 (s, 9H, tert-

butyl), 1.28 (br s, 24H, CH₃(CH₂)₁₂CH₂CH₂CO), 0.90 (t, 3H,

CH₃(CH₂)₁₂CH₂CH₂CO, J=6.9 Hz); ¹³C NMR (CD₃OD, 125

MHz) δ 174.6, 170.4, 165.9, 152.2, 142.9, 103.2, 90.8, 89.9, 84.5,

81.3, 74.5, 71.1, 61.0, 39.0, 37.1, 35.3, 33.1, 30.8, 30.8, 30.8, 30.6,

30.6, 30.5, 30.4, 30.2, 28.2, 25.9, 23.7, 14.4; ESIMS-HR calcd for

C₃₄H₅₉N₄O₉(M þ H)^þ667.4277, found 667.4284.

0

(2R,4S,5S)-3-Acetyl-2-azidomethyl-5-[(1R,2R,3R,4R)-2,3-di-

tert-butyldimethylsilyloxy-4-(uracil-1-yl)]tetrahydrofuryl-(1,3)-

oxazolidine-4-carboxylic Acid (33a). Compound 33a was pre-

pared from 32a (101 mg, 0.15 mmol) as described above for the

synthesis of 26a. Purification by silica gel column chromatog-

raphy (2 cmꢀ10 cm, 5% MeOH-CHCl₃) afforded 33a (72 mg,

73%) as a yellow syrup: ¹H NMR (CD₃OD, 500 MHz, 3:1

mixture of rotamers at 20 °C, data for the major rotamer) δ 7.69

(2R,4S,5S)-Methyl 3-Acetyl-2-azidomethyl-5-[(1R,2R,3R,4R)-

2,3-di-tert-butyldimethylsilyloxy-4-(uracil-1-yl)]tetrahydrofuryl-

(1,3)-oxazolidine-4-carboxylate (32a). Compound 32a was pre-

pared from 22 (500 mg, 0.72 mmol), azidoacetoaldehyde (123 mg,

1.4 mmol), and acetyl chloride (110 mg, 1.4 mmol) as described

above for the synthesis of 25a. Purification by silica gel column

chromatography (2 cmꢀ8 cm, 33% AcOEt-hexane) afforded 32a

(414 mg, 86%) as a yellow foam: ¹HNMR(CD₃OD, 500 MHz, 3:2

mixture of rotamers at 20 °C, data for the major rotamer) δ 7.60

(d, 1H, H-6, J_6,5=8.0 Hz), 6.01 (d, 1H, H-1⁰, J_{1 ,2}=4.6 Hz), 5.77

0

(dd, 1H, H-1⁰⁰, J_{1 2 a}=4.0, J_{1 2 b}=8.2 Hz), 5.76, (d, 1H, H-5,

00 00

_5,6=8.0 Hz), 4.68 (dd, 1H, H-5⁰, J_{5 ,4}=1.2, J_{5 ,6}=6.9 Hz), 4.44

0

J

0

(d, 1H, H-6⁰, J_{6 ,5}=6.9 Hz), 4.37 (dd, 1H, H-4 , J_{4 ,3}=4.0, J_{4 ,5}

=

0

1.2 Hz), 4.31 (dd, 1H, H-2⁰, J_{2 ,1}=4.6, J_{2 ,3}=4.0 Hz), 4.30 (t, 1H,

0

00

H-3⁰, J_{3 ,2}=J_{3 ,4}=4.0 Hz), 3.67-3.40 (m, 2H, H-4 ), 2.04 (s, 3H,

acetyl), 1.90 (m, 2H, H-2⁰⁰), 1.79 (m, 2H, H-3⁰⁰), 0.94, 0.90 (each

s, each 9H, tert-butyl), 0.16, 0.13, 0.09, 0.09 (each s, each 3H,

SiCH₃); ¹³C NMR (CD₃OD, 125 MHz) δ 176.2, 171.9, 165.8,

152.2, 141.4, 103.4, 90.5, 89.3, 84.6, 82.0, 76.4, 73.7, 62.8, 52.0,

26.4, 22.4, 18.9, 18.9, -4.2, -4.4, -4.4, -4.5; ESIMS-HR (negative

mode) m/z calcd for C₂₇H₄₅N₆O₉Si₂(M - H)^-653.2792, found

653.2798.

0

(d, 1H, H-6, J_6,5=8.0 Hz), 5.96 (d, 1H, H-1⁰, J_{1 ,2}=5.7 Hz), 5.82

0

(dd, 1H, H-1⁰⁰, J_{1 ,2 a}=4.6, J_{1 ,2 b}=7.5 Hz), 5.76 (d, 1H, H-5,

00 00

J_5,6= 8.0 Hz), 4.93 (br s, 1H, H-6⁰), 4.71 (d, 1H, H-5⁰, J_{5 ,4}

0

=

1.2 Hz), 4.32 (dd, 1H, H-2⁰, J_{2 ,1}=5.7, J_{2 ,3}=4.6 Hz),₀4.29 (dd, 1H,

0

H-3⁰, J_{3 ,2}=4.6, J_{3 ,4}=3.5 Hz), 4.25 (dd, 1H, H-4 , J_{4 ,3}=3.5,

0

00

0

J_{4 ,5}=1.2 Hz), 3.85 (s, 3H, CO₂CH₃), 3.46 (t, 2H, H-2 ), 2.07 (s,

3H, acetyl), 0.95, 0.91 (each s, each 9H, tert-butyl), 0.18, 0.15, 0.10,

0.07 (each s, each 3H, SiCH₃); ¹³C NMR (CD₃OD, 125 MHz) δ

172.1, 171.1, 170.5, 165.7, 152.2, 141.5, 103.5, 90.8, 90.4, 84.6, 80.6,

76.1, 73.1, 61.0, 53.8, 53.1, 26.4, 22.2, 18.9, 18.9, -4.1, -4.3, -4.4,

(2R,4S,5S)-3-Acetyl-2-azidoethyl-5-[(1R,2R,3R,4R)-2,3-di-

tert-butyldimethylsilyloxy-4-(uracil-1-yl)]tetrahydrofuryl-(1,3)-

oxazolidine-4-carboxylic Acid (33b). Compound 33b was pre-

pared from 32b (120 mg, 0.18 mmol) as described above for the

3810 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9

Ii et al.

synthesis of 26a. Purification by silica gel column chromato-

graphy (2 cmꢀ10 cm, 5% MeOH-CHCl₃) afforded 33b (88 mg,

75%) as a yellow syrup: ¹H NMR (CD₃OD, 500 MHz, 3:1

mixture of rotamers at 20 °C, data for the major rotamer) δ 7.71

ound 34b was prepared from 33b (29 mg, 0.044 mmol) as

described above for the synthesis of 34a. Purification by

silica gel column chromatography (1.5 cm ꢀ 8 cm, 50%

AcOEt-hexane) afforded 34b (27 mg, 70%) as a yellow

syrup: ¹H NMR (CD₃OD, 500 MHz, 1:1 mixture of rotamers

(d, 1H, H-6, J_6,5=8.2 Hz), 5.97 (d, 1H, H-1⁰, J_{1 ,2}=3.6 Hz), 5.80

0

(dd, 1H, H-1⁰⁰, J_{1 ,2 a}=3.7, J_{1 ,2 b}=4.4 Hz), 5.74 (d, 1H, H-5,

00 00

at 20 °C, data for one rotamer) δ 7.78 (d, 1H, H-6, J_6,5

=

00

0

=

8.0 Hz), 5.95 (d, 1H, H-1⁰, J_{1 ,2}=4.6 Hz), 5.83 (m, 1H, H-1 ),

_5,6=8.2 Hz), 4.61 (dd, 1H, H-5⁰, J_{5 ,4}=1.4, J_{5 ,6}=6.4 H), 4.45

0

J

5.80 (d, 1H, H-5, J_5,6= 8.0 Hz), 4.52 (d, 1H, H-6⁰, J_{6 ,5}

0

(d, 1H, H-6⁰, J_{6 ,5}=6.4 Hz), 4.37 (d, 1H, H-4 , J_{4 ,5}=1.4 Hz),

4.31 (m, 2H, H-2⁰, H-3⁰), 3.47 (m, 2H, H-3⁰⁰), 2.20-1.98 (m, 2H,

H-2⁰⁰), 2.04 (s, 3H, acetyl), 0.94, 0.91 (each s, each 9H, tert-

butyl), 0.15, 0.14, 0.09, 0.08 (each s, each 3H, SiCH₃); ¹³C NMR

(CD₃OD, 125 MHz) δ 176.5, 171.6, 165.9, 152.2, 141.3, 103.2,

89.8, 89.5, 84.7, 81.6, 76.6, 73.5, 62.7, 33.2, 26.4, 22.4, 19.0, 18.9,

-4.1, -4.3, -4.4, -4.5; ESIMS-HR (negative mode) m/z calcd

for C₂₈H₄₇N₆O₉Si₂(M - H)^-667.2949, found 667.2956.

(2R,4S,5S)-3-Acetyl-2-azidopropyl-5-[(1R,2R,3R,4R)-2,3-di-

tert-butyldimethylsilyloxy-4-(uracil-1-yl)]tetrahydrofuryl-(1,3)-

oxazolidine-4-carboxylic Acid (33c). Compound 33c was pre-

pared from 32c (115 mg, 0.18 mmol) as described above for the

synthesis of 26a. Purification by silica gel column chromatog-

raphy (2 cmꢀ10 cm, 5% MeOH-CHCl₃) afforded 33c (91 mg,

81%) as a yellow syrup: ¹H NMR (CD₃OD, 500 MHz, 2:1

mixture of rotamers at 20 °C, data for the major rotamer) δ 7.75

0

7.5 Hz), 4.50 (dd, 1H, H-5⁰, J_{5 ,4}=1.2, J_{5 ,6}=7.5 ₀Hz), 4.30 (t,

0

1H, H-2⁰, J_{2 ,1}=J_{2 ,3}=4.6 Hz), 4.29 (dd, 1H, H-3 , J_{3 ,2}=4.6,

0

J_{3 ,4}=4.0 Hz), 4.22 (dd, 1H, H-4 , J_{4 ,3}=4.0, J_{4 ,5}=1.2 Hz),

00 00

3.54 (t, 2H, H-3⁰⁰, J_{3 ,2}=5.2 Hz), 3.36-3.24 (m, 2H, CH₃-

(CH₂)₁₃CH₂CH₂NHCO), 2.28-2.05 (m, 2H, H-2⁰⁰), 2.14 (s,

3H, acetyl), 1.51 (m, 2H, CH₃(CH₂)₁₃CH₂CH₂NHCO), 1.29

(br s, 26H, CH₃(CH₂)₁₃CH₂CH₂NHCO), 0.94, 0.91 (each s,

each 9H, tert-butyl), 0.89 (t, 3H, CH₃(CH₂)₁₃CH₂CH₂NHCO,

J=6.9 Hz), 0.14, 0.14, 0.10, 0.09 (each s, each 3H, SiCH₃);

¹³C NMR (CD₃OD, 125 MHz) δ 171.5, 171.1, 170.9, 166.0,

152.3, 141.4, 103.2, 90.6, 90.5, 83.5, 81.5, 76.4, 73.2, 62.3,

52.8, 40.8, 33.6, 33.1, 30.8, 30.7, 30.5, 28.0, 26.4, 23.8, 22.3,

22.1, 19.0, 19.0, 14.5, -4.0, -4.0, -4.4, -4.5; MS-HR m/z

calcd for C₄₄H₈₁N₇NaO₈Si₂(M þ Na)^þ914.5577, found

914.5580.

(d, 1H, H-6, J_6,5=8.0 Hz), 5.94 (d, 1H, H-1⁰, J_{1 ,2}=4.0 Hz), 5.74

(2R,4S,5S)-3-Acetyl-2-azidopropyl-5-[(1R,2R,3R,4R)-2,3-di-

tert-butyldimethylsilyloxy-4-(uracil-1-yl)]tetrahydrofuryl-N-

hexadecyl-(1,3)-oxazolidine-4-carboxylamide (34c). Compound

34c was prepared from 33c (40 mg, 0.059 mmol) as described

above for the synthesis of 34a. Purification by silica gel column

chromatography (1.5 cm ꢀ 20 cm, 50% AcOEt-hexane) af-

forded 34c (45 mg, 84%) as a yellow syrup: ¹H NMR (CD₃OD,

500 MHz, 1:1 mixture of rotamers at 20 °C, data for one

rotamer) δ 7.82 (d, 1H, H-6, J_6,5=8.0 Hz), 5.92 (d, 1H, H-1⁰,

0

(d, 1H, H-5, J_5,6=8.0 Hz), 5.70, (dd, 1H, H-1⁰⁰, J_{1 ,2 a}=4.0,

00 00

_{1 ,2 b}=7.5 Hz), 4.60 (dd, 1H, H-5⁰, J_{5 ,4}=1.7, J_{5 ,6}=6.9 Hz),

00 00

0

J

0

4.45 (d, 1H, H-6⁰, J_{6 ,5}=6.9 Hz), 4.33 (dd, 1H, H-4 , J_{4 ,3}=4.0,

0

J

_{4 ,5}=1.7 Hz), 4.29 (dd, 1H, H-2 , J_{2 ,1}=4.0, J_{2 ,3}=5.2 H₀z₀), 4.28

(dd, 1H, H-3⁰, J_{3 ,2}=5.2, J_{3 ,4}=4.0 Hz), 3.36 (m, 2H, H-4 ), 2.05

(s, 3H, acetyl), 1.93-1.71 (m, 2H, H-2⁰⁰), 1.79 (m, 2H, H-3⁰⁰),

0.94, 0.90 (each s, each 9H, tert-butyl), 0.15, 0.14, 0.09, 0.08

(each s, each 3H, SiCH₃); ¹³C NMR (CD₃OD, 125 MHz) δ

176.7, 171.5, 165.9, 152.1, 141.3, 103.0, 91.5, 89.6, 84.3, 81.0,

76.7, 73.3, 62.5, 52.1, 31.1, 26.4, 25.6, 22.5, 18.9, 18.9, -4.1,

-4.3, -4.4, -4.5; ESIMS-HR (negative mode) m/z calcd for

C₂₉H₄₉N₆O₉Si₂(M - H)^-681.3105, found 681.3114.

0

J

_{1 ,2}=4.0 Hz), 5.79 (d, 1H, H-5, J_5,6=8.0 Hz), 5.78 (m, 1H, H-

0

1⁰⁰), 4.52 (d, 1H, H-6⁰, J_{6 ,5}=7.5 Hz)₀, 4.50 (dd, 1H, H-5 , J_{5 ,4}

0

=

1.2, J_{5 ,6}=7.5 Hz), 4.31 (dd, 1H, H-2 , J_{2 ,1}=4.0, J_{2 ,3}=5.7 Hz),

0

4.27 (dd, 1H, H-3⁰, J_{3 ,2}=5.7, J_{3 ,4}=4.6 Hz),₀4₀.24 (dd, 1H, H-4 ,

0

00 00

J_{4 ,3}=4.6, J_{4 ,5}=1.2 Hz), 3.38 (t, 2H, H-4 , J_{4 ,3}=6.9 Hz),

3.36-3.24 (m, 2H, CH₃(CH₂)₁₃CH₂CH₂NHCO), 2.14 (s, 3H,

acetyl), 1.91 (m, 2H, H-2⁰⁰), 1.80 (m, 2H, H-3⁰⁰), 1.52 (m, 2H,

CH₃(CH₂)₁₃CH₂CH₂NHCO), 1.29 (br s, 26H, CH₃(CH₂)₁₃-

CH₂CH₂NHCO), 0.94, 0.91 (each s, each 9H, tert-butyl), 0.90

(t, 3H, CH₃(CH₂)₁₃CH₂CH₂NHCO, J = 6.9 Hz), 0.15, 0.14,

0.11, 0.11 (each s, each 3H, SiCH₃); ¹³C NMR (CD₃OD, 125 MHz)

δ 171.5, 171.1, 170.6, 165.9, 152.2, 141.4, 103.0, 92.3, 90.5, 83.4,

80.1, 76.5, 73.0, 60.8, 52.1, 40.7, 32.3, 31.6, 30.8, 30.7, 30.5, 28.0,

26.5, 25.7, 25.6, 23.8, 22.4, 22.1, 19.0, 19.0, 14.5, -4.0, -4.0,

-4.4, -4.5; ESIMS-HR m/z calcd for C₄₅H₈₃N₇NaO₈Si₂(M þ

Na)^þ928.5734, found 928.5735.

(2R,4S,5S)-3-Acetyl-2-azidomethyl-5-[(1R,2R,3R,4R)-2,3-di-

tert-butyldimethylsilyloxy-4-(uracil-1-yl)]tetrahydrofuryl-N-

hexadecyl-(1,3)-oxazolidine-4-carboxylamide (34a). A mix-

ture of 33a (40 mg, 0.061 mmol), hexadecylamine (44 mg,

0.18 mmol), and HOBt (25 mg, 0.18 mmol) in CH₂Cl₂(1 mL)

was treated with EDCI (35 mg, 0.18 mmol) at room tepera-

ture for 12 h. The mixture was diluted with AcOEt (50 mL),

which was washed with 0.1 N aqueous HCl, saturated aqu-

eous NaHCO₃, and brine. The organic layer was dried

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

was purified by silica gel column chromatography (1.5 cmꢀ8

cm, 50% AcOEt-hexane) to give 34a (38 mg, 71%) as a

1

(2R,4S,5S)-3-Acetyl-2-azidomethyl-5-[(1R,2R,3R,4R)-2,3-di-

hydroxy-4-(uracil-1-yl)]tetrahydrofuryl-N-hexadecyl-(1,3)-oxazo-

lidine-4-carboxylamide (35a). Compound 35a was prepared

from 34a (26 mg, 0.030 mmol) as described above for the

synthesis of 27a. Purification by silica gel column chromatog-

raphy (1.5 cmꢀ8 cm, 2% MeOH-CHCl₃) afforded 35a (16 mg,

81%) as a yellow syrup: ¹H NMR (CD₃OD, 500 MHz, 3:2

mixture of rotamers at 20 °C, data for the major rotamer) δ 7.63

yellow syrup: H NMR (CD₃OD, 500 MHz, 1:1 mixture of

rotamers at 20 °C, data for one rotamer) δ 7.75 (d, 1H, H-6,

J

_6,5=8.0 Hz), 5.98 (d, 1H, H-1⁰, J_{1 ,2}=4.6 Hz), 5.92 (dd, 1H,

0 0

H-1⁰⁰, J_{1 ,2 a}=4.6, J_{1 ,2 b}=7.5 Hz), 5.82 (d, 1H, H-5, J_5,6

=

0

00 00

8.0 Hz), 4.59 (d, 1H, H-6⁰, J_{6 ,5}=6.9 Hz),₀4.54 (d, 1H, H-5 ,

0

_{5 ,4}=1.2, J_{5 ,6}=8.0 Hz), 4.33 (dd, 1H, H-2 , J_{2 ,1}=4.6, J_{2 ,3}=

0

J

4.0 Hz), 4.30 (dd, 1H, H-3⁰, J_{3 ,2}=4.0, J_{3 ,4}=4.6 Hz), 4.21

0

(dd, 1H, H-4⁰, J_{4 ,3}=4.6, J_{4 ,5}=1.2 Hz), 3.85-3.71 (m, 2H,

H-2⁰⁰), 3.36-3.25 (m, 2H, CH₃(CH₂)₁₃CH₂CH₂NHCO), 2.07

(s, 3H, acetyl), 1.52 (m, 2H, CH₃(CH₂)₁₃CH₂CH₂NHCO),

1.29 (br s, 26H, CH₃(CH₂)₁₃CH₂CH₂NHCO), 0.94, 0.89

(each s, each 9H, tert-butyl), 0.89 (t, 3H, CH₃-

(CH₂)₁₃CH₂CH₂NHCO, J = 6.9 Hz), 0.16, 0.14, 0.09, 0.07

(each s, each 3H, SiCH₃); ¹³C NMR (CD₃OD, 125 MHz) δ

171.9, 170.9, 170.6, 165.9, 152.2, 141.6, 103.4, 91.1, 90.3,

83.5, 81.9, 76.1, 73.2, 62.4, 52.8, 40.7, 33.1, 30.8, 30.7, 30.5,

28.0, 26.5, 23.8, 22.3, 22.2, 19.0, 19.0, -4.2, -4.4, -4.4, -4.5;

ESIMS-HR m/z calcd for C₄₃H₇₉N₇NaO₈Si₂(M þ Na)^þ

900.5421, found 900.5428.

0

(d, 1H, H-6, J_6,5=8.0 Hz), 5.90 (d, 1H, H-1⁰, J_{1 ,2}=4.0 Hz), 5.73

0

(d, 1H, H-5, J_5,6=8.0 Hz), 5.67 (dd, 1H, H-1⁰⁰, J_{1 ,2 a}=2.3,

00 00

J_{1 ,2 b}=6.3 Hz), 4.66 (dd, 1H, H-5⁰, J_{5 ,4}=2.3, J_{5 ,6}=7.5 Hz),

00 00

0

4.61 (d, 1H, H-6⁰, J_{6 ,5}=7.5 Hz), 4.31 (dd, 1H, H-3 , J_{3 ,2}=5.2,

0

J_{3 ,4}=5.7 Hz), 4.25 (dd, 1H, H-2 , J_{2 ,1}=4.0, J_{2 ,3}=5.2 Hz), 4.12

00

(dd, 1H, H-4⁰, J_{4 ,3}=5.7, J_{4 ,5}=2.3 Hz), 3.65 (m, 2H, H-2 ),

3.36-3.14 (m, 2H, CH₃(CH₂)₁₃CH₂CH₂NHCO), 2.01 (s, 3H,

acetyl), 1.53 (m, 2H, CH₃(CH₂)₁₃CH₂CH₂NHCO), 1.29 (br s,

26H, CH₃(CH₂)₁₃CH₂CH₂NHCO), 0.90 (t, 3H, CH₃(CH₂)₁₃-

CH₂CH₂NHCO, J=6.9 Hz); ¹³C NMR (CD₃OD, 125 MHz) δ

172.3, 171.1, 170.9, 165.9, 152.3, 142.3, 103.3, 91.3, 91.1, 84.0,

82.2, 74.7, 71.3, 62.6, 52.7, 40.9, 33.1, 30.8, 30.7, 30.7, 30.5, 30.4,

30.4, 30.3, 28.0, 23.8, 22.2, 14.5; ESIMS-HR m/z calcd for

C₃₁H₅₁N₇NaO₈(M þ Na)^þ672.3691, found 672.3697.

0

(2R,4S,5S)-3-Acetyl-2-azidoethyl-5-[(1R,2R,3R,4R)-2,3-di-

tert-butyldimethylsilyloxy-4-(uracil-1-yl)]tetrahydrofuryl-N-

hexadecyl-(1,3)-oxazolidine-4-carboxylamide (34b). Comp-

Article

(2R,4S,5S)-3-Acetyl-2-azidoethyl-5-[(1R,2R,3R,4R)-2,3-di-

Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3811

for the synthesis of 18a. Purification by C18 HPLC (85%

aqueous MeOH containing 0.5% HCl) afforded 21b (6.4 mg,

72%) as a white foam: ¹H NMR (CD₃OD, 500 MHz, 5:1

mixture of rotamers at 20 °C, data for the major rotamer) δ

hydroxy-4-(uracil-1-yl)]tetrahydrofuryl-N-hexadecyl-(1,3)-oxazo-

lidine-4-carboxylamide (35b). Compound 35b was prepared

from 34b (25 mg, 0.028 mmol) as described above for the

synthesis of 27a. Purification by silica gel column chromatog-

raphy (1.5 cmꢀ8 cm, 2% MeOH-CHCl₃) afforded 35b (14 mg,

76%) as a yellow syrup: ¹H NMR (CD₃OD, 500 MHz, 1:1

mixture of rotamers at 20 °C, data for one rotamer) δ 7.70 (d,

7.63 (d, 1H, H-6, J_6,5=8.0 Hz), 5.80 (d, 1H, H-1⁰, J_{1 ,2}=3.5 Hz),

0

5.71 (d, 1H, H-5, J_5,6=8.0 Hz), 5.65 (t, 1H, H-1⁰⁰, J_{1 ,2}=5.2 Hz),

00 00

0

4.62 (d, 1H, H-6⁰, J_{6 ,5}=5.2 Hz), 4.57 (dd, 1H, H-5 , J_{5 ,4}=2.3,

0

J_{5 ,6}=5.2 Hz), 4.32 (dd, 1H, H-3 , J_{3 ,2}=5.8, J_{3 ,4}=6.3 Hz), 4.2₀8

1H, H-6, J_6,5=8.0 Hz), 5.93 (d, 1H, H-1⁰, J_{1 ,2}=4.6 Hz), 5.76 (d,

(dd, 1H, H-2⁰, J_{2 ,1}=3.5, J_{2 ,3}=5.8 Hz), 4.08 (dd, 1H, H-4 ,

0

1H, H-5, J_5,6=8.0 Hz), 5.75 (m, 1H, H-1⁰⁰), 4.57 (d, 1H, H-6⁰,

0

J_{4 ,3}= 6.3, J_{4 ,5}= 2.3 Hz), 3.34-3.18 (m, 2H, CH₃(CH₂)₁₃-

0

CH₂CH₂NHCO), 3.11 (t, 2H, H-3⁰⁰, J_{3 ,2}=6.3 Hz), 2.26 (br s,

0

J

_{6 ,5}=6.9 Hz), 4.56 (dd, 1H, H-5 , J_{5 ,4}=1.7, J_{5 ,6}=6.9 Hz), 4.2₀7

00 00

(dd, 1H, H-3⁰, J_{3 ,2}=5.7, J_{3 ,4}=5.2 Hz), 4.22 (dd, 1H, H-2 ,

0

2H, H-2⁰⁰), 2.02 (s, 3H, acetyl), 1.56 (br s, 2H, CH₃(CH₂)₁₃-

CH₂CH₂NHCO), 1.29 (br s, 26H, CH₃(CH₂)₁₃CH₂CH₂NHCO),

0.90 (t, 3H, CH₃(CH₂)₁₃CH₂CH₂NHCO, J=6.9 Hz); ¹³C NMR

(CD₃OD, 125 MHz) δ 172.2, 171.3, 165.9, 152.2, 142.9, 103.2,

92.7, 90.4, 83.9, 82.0, 74.5, 70.9, 62.5, 41.0, 33.1, 32.2, 30.8, 30.8,

30.7, 30.5, 30.4, 30.3, 28.1, 23.7, 22.6, 14.5; ESIMS-HR m/z

calcd for C₃₂H₅₆N₅O₈(M þ H)^þ638.4123, found 638.4123.

(2R,4S,5S)-3-Acetyl-2-aminopropyl-5-[(1R,2R,3R,4R)-2,3-

dihydroxy-4-(uracil-1-yl)]tetrahydrofuryl-N-hexadecyl-(1,3)-

oxazolidine-4-carboxylamide HCl Salt (21c). Compound 21c

was prepared from 35c (9.7 mg, 0.014 mmol) as described above

for the synthesis of 18a. Purification by C18 HPLC (85% aqu-

eous MeOH containing 0.5% HCl) afforded 21c (4.8 mg, 51%)

as a white foam: ¹H NMR (CD₃OD, 500 MHz, 2:1 mixture of

rotamers at 20 °C, data for the major rotamer) δ 7.65 (d, 1H,

0

J

_{2 ,1}=4.6, J_{2 ,3}=5.7 Hz), 4.13 (dd, 1H, H-4 , J_{4 ,3}=5.2, J_{4 ,5}

=

1.7 Hz), 3.53 (t, 2H, H-3⁰⁰, J_{3 ,2}=6.9 Hz), 3.36-3.22 (m, 2H,

CH₃(CH₂)₁₃CH₂CH₂NHCO), 2.12 (s, 3H, acetyl), 2.05 (m, 2H,

H-2⁰⁰), 1.54 (m, 2H, CH₃(CH₂)₁₃CH₂CH₂NHCO, 1.29 (br s,

26H, CH₃(CH₂)₁₃CH₂CH₂NHCO), 0.90 (t, 3H, CH₃(CH₂)₁₃-

CH₂CH₂NHCO), J=6.9 Hz); ¹³C NMR (CD₃OD, 125 MHz) δ

171.9, 171.1, 170.8, 165.9, 152.3, 142.1, 103.3, 91.0, 90.3, 83.9,

81.7, 74.9, 71.4, 62.5, 40.8, 33.9, 33.1, 30.8, 30.7, 30.7, 30.5, 30.4,

30.4, 30.3, 28.0, 23.8, 22.1, 14.5; ESIMS-HR m/z calcd for

C₃₂H₅₃N₇NaO₈(M þ Na)^þ686.3848, found 686.3851.

00 00

(2R,4S,5S)-3-Acetyl-2-azidopropyl-5-[(1R,2R,3R,4R)-2,3-di-

hydroxy-4-(uracil-1-yl)]tetrahydrofuryl-N-hexadecyl-(1,3)-oxazo-

lidine-4-carboxylamide (35c). Compound 35c was prepared from

34c (39 mg, 0.043 mmol) as described above for the synthesis of

27a. Purification by silica gel column chromatography (1.5 cmꢀ

8 cm, 2% MeOH-CHCl₃) afforded 35c (26 mg, 88%) as a

yellow syrup: ¹H NMR (CD₃OD, 500 MHz, 1:1 mixture of

rotamers at 20 °C, data for one rotamer) δ 7.71 (d, 1H, H-6,

H-6, J_6,5=8.0 Hz), 5.87 (d, 1H, H-1⁰, J_{1 ,2}=3.5 Hz), 5.71 (d, 1H,

0

H-5, J_5,6=8.0 Hz), 5.60 (dd, 1H, H-1⁰⁰,J_{1 ,2 a}=3.4, J_{1 ,2 b}=6.3 Hz),

00 00

0

4.58 (d, 1H, H-6⁰, J_{6 ,5}=5.8 Hz), 4.55 (dd, 1H, H-5 , J_{5 ,4}=1.7,

0

J

_{5 ,6}=5.8 Hz), 4.27 (dd, 1H, H-3 , J_{3 ,2}=5.2, J_{3 ,4}=5.8 Hz), 4.2₀3

J_6,5=8.0 Hz), 5.92 (d, 1H, H-1⁰, J_{1 ,2}=4.6 Hz), 5.75 (d, 1H, H-5,

0

(dd, 1H, H-2⁰, J_{2 ,1}=3.5, J_{2 ,3}=5.2 Hz), 4.11 (dd, 1H, H-4 ,

0

J_5,6=8.0 Hz), 5.64 (m, 1H, H-1⁰⁰), 4.57 (d, 1H, H-6⁰, J_{6 ,5}

0

=

0

J_{4 ,3}= 5.8, J_{4 ,5}= 1.7 Hz), 3.33-3.15 (m, 2H, CH₃(CH₂)₁₃-

6.9 Hz), 4.56 (dd, 1H, H-5⁰, J_{5 ,4}=1.7, J_{5 ,6}=6.9 Hz), 4.26 (t, 1H,

CH₂CH₂NHCO), 2.99 (t, 2H, H-4⁰⁰, J_{4 ,3}=6.9 Hz), 2.00 (s, 3H,

0

00 00

H-3⁰, J_{3 ,2}=J_{3 ,4}=5.7 Hz), 4.22 (dd, 1H, H-2 , J_{2 ,1}=4.6, J_{2 ,3}

=

0

acetyl), 1.84 (m, 4H, H-2⁰⁰, H-3⁰⁰), 1.54 (m, 2H, CH₃(CH₂)₁₃-

CH₂CH₂NHCO), 1.29 (br s, 26H, CH₃(CH₂)₁₃CH₂CH₂NHCO),

0.90 (t, 3H, CH₃(CH₂)₁₃CH₂CH₂NHCO, J=6.9 Hz); ¹³C NMR

(CD₃OD, 125 MHz) δ 171.8, 171.0, 166.0, 152.2, 142.5, 103.1,

91.8, 91.8, 83.5, 81.7, 74.9, 71.3, 62.5, 40.4, 33.1, 31.5, 30.8, 30.7,

30.5, 30.4, 30.3, 28.0, 24.2, 23.8, 22.6, 14.5; ESIMS-HR m/z

calcd for C₃₃H₅₈N₅O₈(M þ H)^þ652.4280, found 652.4281.

Enzymatic Evaluation. The activities of the compounds were

tested against purified MraY from B. subtilis.⁷The 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]pentapep-

tide (337 Bq), and 8.4 mM N-lauroyl sarcosine. 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-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.

0

5.7 Hz), 4.13 (dd, 1H, H-4⁰, J_{4 ,3}=5.7, J_{4 ,5}=1.7 Hz), 3.42 (t, 2H,

0

H-4⁰⁰, J_{4 ,3}= 6.9 Hz), 3.35-3.24 (m, 2H, CH₃(CH₂)₁₃-

00 00

CH₂CH₂NHCO), 2.11 (s, 3H, acetyl), 1.92 (m, 2H, H-2⁰⁰), 1.76

(m, 2H, H-3⁰⁰), 1.53 (m, 2H, CH₃(CH₂)₁₃CH₂CH₂NHCO), 1.29

(br s, 26H, CH₃(CH₂)₁₃CH₂CH₂NHCO), 0.90 (t, 3H, CH₃-

(CH₂)₁₃CH₂CH₂NHCO, J = 6.9 Hz); ¹³C NMR (CD₃OD,

125 MHz) δ 171.9, 171.1, 170.9, 165.9, 152.3, 142.2, 103.2, 92.2,

90.8, 83.8, 81.5, 74.9, 71.4, 62.5, 52.2, 40.8, 33.1, 31.9, 30.8, 30.8, 30.7,

30.5, 30.4, 30.4, 30.4, 28.0, 25.5, 23.8, 22.1, 14.5; ESIMS-HR m/z

calcd for C₃₃H₅₅N₇NaO₈(M þ Na)^þ700.4004, found 700.4006.

(2R,4S,5S)-3-Acetyl-2-aminomethyl-5-[(1R,2R,3R,4R)-2,3-

dihydroxy-4-(uracil-1-yl)]tetrahydrofuryl-N-hexadecyl-(1,3)-

oxazolidine-4-carboxylamide HCl Salt (21a). Compound 21a

was prepared from 35a (9.7 mg, 0.015 mmol) as described above

for the synthesis of 18a. Purification by C18 HPLC (85%

aqueous MeOH containing 0.5% HCl) afforded 21a (6.4 mg,

69%) as a white foam: ¹H NMR (CD₃OD, 500 MHz, 9:1

mixture of rotamers at 20 °C, data for the major rotamer) δ

7.57 (d, 1H, H-6, J_6,5=8.0 Hz), 5.71 (d, 1H, H-5, J_5,6=8.0 Hz),

00

5.67 (d, 1H, H-1⁰, J_{1 ,2}=3.5 Hz), 5.62 (dd, 1H, H-1 , J_{1 ,2 a}

0

00 00

=

2.3, J_{1 ,2 b}=2.9 Hz), 4.69 (d, 1H, H-6⁰, J_{6 ,5}=2.3₀Hz), 4.61 (t,

00 00

0

1H, H-5⁰, J_{5 ,4}=J_{5 ,6}=2.3 Hz), 4.41 (dd, 1H, H-3 , J_{3 ,2}=6.3,

0

J_{3 ,4}=6.9 Hz), 4.32 (dd, 1H, H-2 , J_{2 ,1}=3.5, J_{2 ,3}=6.3 Hz), 4.04

(dd, 1H, H-4⁰, J_{4 ,3}=6.9, J_{4 ,5}=2.3 Hz), 3.51 (m, 2H, H-2 ),

3.36-3.18 (m, 2H, CH₃(CH₂)₁₃CH₂CH₂NHCO), 2.03 (s, 3H,

acetyl), 1.57 (br s, 2H, CH₃(CH₂)₁₃CH₂CH₂NHCO), 1.29 (br s,

26H, CH₃(CH₂)₁₃CH₂CH₂NHCO), 0.90 (t, 3H, CH₃-

(CH₂)₁₃CH₂CH₂NHCO, J=6.9 Hz); ¹³C NMR (CD₃OD, 125

MHz) δ 172.6, 172.2, 165.9, 152.1, 143.8, 103.4, 93.9, 89.2, 85.1,

82.6, 73.9, 70.3, 61.9, 41.0, 33.1, 30.8, 30.7, 30.7, 30.4, 30.4, 30.2,

28.0, 23.7, 23.0, 14.4; ESIMS-HR m/z calcd for C₃₁H₅₄N₅O₈

(M þ H)^þ624.3967, found 624.3964.

00

0

Antibacterial Activity Evaluation. Vancomycin-resistant Ente-

rococcus 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).³⁷

MICs 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 Muel-

ler-Hinton broth (CA-MHB) (Becton Dickinson, Sparks, MD).

Serial 2-fold dilutions of each compound were made in appro-

priate broth, and the plates were inoculated with 5ꢀ10⁴CFU of

(2R,4S,5S)-3-Acetyl-2-aminoethyl-5-[(1R,2R,3R,4R)-2,3-

dihydroxy-4-(uracil-1-yl)]tetrahydrofuryl-N-hexadecyl-(1,3)-

oxazolidine-4-carboxylamide HCl Salt (21b). Compound 21b

was prepared from 35b (9.2 mg, 0.014 mmol) as described above

3812 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9

Ii et al.

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

for 20 h, and then MICs were scored.

(9) Bouhss, A.; Trunkfield, A. E.; Bugg, T. D.; Mengin-Lecreulx, D.

The biosynthesis of peptidoglycan lipid-linked intermediates.

FEMS Microbiol. Rev. 2008, 32, 208–33.

(10) Al-Dabbagh, B.; Henry, X.; El Ghachi, M.; Auger, G.; Blanot, D.;

Parquet, C.; Mengin-Lecreulx, D.; Bouhss, A. Active site mapping

of MraY, a member of the polyprenyl-phosphate N-acetylhexosa-

mine 1-phosphate transferase superfamily, catalyzing the first

membrane step of peptidoglycan biosynthesis. Biochemistry 2008,

47, 8919–8928.

(11) (a) Dini, C. MraY inhibitors as novel antibacterial agents. Curr.

Top. Med. Chem. 2005, 5, 1221–1236. (b) Kimura, K.; Bugg, T. D. H.

Recent advances in antimicrobial nucleoside antibiotics targeting cell

wall biosynthesis. Nat. Prod. Rep. 2003, 20, 252–273. (c) Ichikawa, S.;

Matsuda, A. Nucleoside natural products and related analogs with

potential therapeutic properties as antibacterial and antiviral agents.

Expert Opin. Ther. Pat. 2007, 17, 487–498.

Acknowledgment. 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) for measurement of the mass

spectra. We also acknowledge the CNRS (Grant UMR 8619).

Supporting Information Available: HPLC purity data and

NOE data. This material is available free of charge via the

Internet at http://pubs.acs.org.

(12) Hirano, S.; Ichikawa, S.; Matsuda, A. Synthesis of caprazamycin

analogues and their structure-activity relationship for antibacter-

ial activity. J. Org. Chem. 2008, 73, 569–577.

(13) Hirano, S.; Ichikawa, S.; Matsuda, A. Structure-activity relation-

ship of truncated analogs of caprazamycins as potential anti-

tuberculosis agents. Bioorg. Med. Chem. 2008, 16, 5123–5133.

(14) Hirano, S.; Ichikawa, S.; Matsuda, A. Design and synthesis of

diketopiperazine and acyclic analogs related to the caprazamycins

and liposidomycins as potential antibacterial agents. Bioorg. Med.

Chem. 2008, 16, 428–436.

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Article Doi

Function-oriented synthesis of simplified caprazamycins: Discovery of oxazolidine-containing uridine derivatives as antibacterial agents against drug-resistant bacteria

DOI: 10.1021/jm100243n

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