Potent, novel in vitro inhibitors of the Pseudomonas aeruginosa deacetylase LpxC

3112

J . Med. Chem. 2002, 45, 3112-3129

P oten t, Novel in Vitr o In h ibitor s of th e P seu d om on a s a er u gin osa Dea cetyla se

Lp xC

Toni Kline,*^,†,‡Niels H. Andersen,*^,§Eric A. Harwood,^†J ason Bowman,^§Andre Malanda,^§Stephanie Endsley,^§

Alice L. Erwin,^†Michael Doyle,^|Susan Fong,^|Alex L. Harris,^|Brian Mendelsohn,^§,‡Khisimuzi Mdluli,^†

Christian R. H. Raetz, C. Kendall Stover,^†,#Pamela R. Witte,^†Asha Yabannavar,^|and Shuguang Zhu^†

Chiron Corporation, 201 Elliot Avenue West, Seattle, Washington, 98119, Department of Chemistry, University of Washington,

Seattle, Washington 98195, Chiron Corporation, 4560 Horton Street, Emeryville, California 94608, and Department of

Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

Received December 21, 2001

Deacetylation of uridyldiphospho-3-O-(R-hydroxydecanoyl)-N-acetylglucosamine by LpxC is the

first committed step in the Pseudomonas aeruginosa biosynthetic pathway to lipid A;

homologous enzymes are found widely among Gram-negative bacteria. As an essential enzyme

for which no inhibitors have yet been reported, the P. aeruginosa LpxC represents a highly

attractive target for a novel antibacterial drug. We synthesized several focused small-molecule

libraries, each composed of a variable aromatic ring, one of four heterocyclic/spacer moieties,

and a hydroxamic acid and evaluated the LpxC inhibition of these compounds against purified

P. aeruginosa enzyme. To ensure that the in vitro assay would be as physiologically relevant

as possible, we synthesized a tritiated form of the specific P. aeruginosa glycolipid substrate

and measured directly the enzymatically released acetate. Several of our novel compounds,

predominantly those having fluorinated substituents on the aromatic ring and an oxazoline as

the heterocyclic moiety, demonstrated in vitro IC₅₀values less than 1 µM. We now report the

synthesis and in vitro evaluation of these P. aeruginosa LpxC inhibitors.

In tr od u ction

P. aeruginosa LpxC by synthesizing novel compounds

of the general motif shown in Figure 2 in which the Zn-

binding hydroxamic acid was conserved and the aryl and

heterocyclic moieties were varied. We particularly fo-

cused on the role of hydrophobic substituents and

aromatic heterocycles in the aromatic moiety. The

acylserine analogues of selected oxazolines were also

synthesized for screening.

Emerging bacterial resistance to current antibiotic

drugs has driven the search for novel prokaryotic targets

and for novel molecules to inhibit the activity of these

targets. The lipid A biosynthetic pathway, which is both

unique and essential to Gram-negative bacteria, pre-

sents several good candidate enzymes.¹LpxC, which

catalyzes the first committed step in the lipid A pathway

(Figure 1) is an essential enzyme²with homologues in

more than 40 Gram-negative species. While there is no

mammalian homologue of LpxC, the fact that it is a Zn-

dependent amidase³places it in a class of enzymes for

which inhibitors have been described.⁴

Merck scientists had previously discovered that aryl-

oxazoline hydroxamic acids were inhibitors of this

enzyme and reported that L-161,240 was a potent

inhibitor of the LpxC from Escherichia coli; this com-

pound also inhibited growth of E. coli.⁵However, this

compound had no effect on the growth of another

pathogenic Gram-negative species, Pseudomonas aeru-

ginosa, and in vitro activity toward P. aeruginosa LpxC

was 50- to 100-fold weaker than toward the E. coli

enzyme.⁶We initiated a discovery project targeting the

Because P. aeruginosa was the target organism, we

required a sensitive in vitro assay specific for the P.

aeruginosa LpxC. To achieve this, we prepared the

native P. aeruginosa substrate with a tritiated N-acetyl

group and assayed the enzymatically released tritium

directly. We now describe the synthesis of three hetero-

cyclic classes of inhibitors (oxazolines, thiazolines, and

oxazines), the development of a radioligand assay for

P. aeruginosa LpxC, and the evaluation of these com-

pounds in our assay.

Resu lts

Ch em ist r y. Oxa zolin es a n d Acylser in es. One

modification that may increase hydrophobicity with

minimal steric perturbation is the replacement of

hydrogens with fluorines. Compounds 35 and 5 were

synthesized as direct analogues of the best reported E.

coli inhibitors of this class shown in Figure 2. Because

the [R] stereochemistry at C-4 was reported to be

significantly preferred,^3,5we synthesized 35 and 5 from

D-serine methyl ester using Burgess reagent at 80 °C

and installed the hydroxamic acid by a stereospecific

route^5b,7as shown in Scheme 1.

* To whom correspondence should be addressed. For T.K.: (address)

Seattle Genetics, 21823 30th Drive SE, Bothell, WA 98021; (phone)

425-527-4728; (fax) 425-527-4109; (e-mail) tkline@seagen.com. For

N.H.A.: (phone) 206-543-7099; (fax) 206-685-8665; (e-mail) andersen@

chem.washington.edu.

^†Chiron Corporation, WA.

^‡Present Address: Seattle Genetics, 21823 30th Drive SE, Bothell,

WA 98021.

^§University of Washington.

^|Chiron Corporation, CA.

Duke University Medical Center.

Present Address: Pfizer Global Research and Development, Ann

We subsequently found that diethylaminosulfur tri-

fluoride (DAST) at low temperatures gave consistently

#

Arbor, MI 48105.

Published on Web 06/07/2002

LpxC Inhibitors

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14 3113

F igu r e 1. LpxC catalyzes the deacetylation of UDP-3-O-(R-3-hydroxydecanoyl)-GlcNAc to the corresponding glucosamine. This

is the first committed step in the biosynthetic pathway to lipid A, an essential component of the Gram-negative outer membrane.

ment on potency. The customized benzoic acids that

were synthesized for conversion to the corresponding

serine derivatives are shown in Scheme 3. Since early

bioassay results indicated that aryl units bearing F,

CF₃, and CF₃O groups had increased potency, these

functions were the focus of the synthetic effort.

Although several solid-phase syntheses of hydroxamic

acids have been reported,⁸the chemistry for solution-

phase parallel synthesis (Scheme 4) that we developed

gave consistently better results over a wide variety of

analogues. The initial condensation of the aromatic acid

with serine methyl ester was straightforward by any of

the three methods used. The choice of hydroxamic acid

installation (methods D-G) was dictated by the func-

tionality of the precursor ester. The original method in

Scheme 1 is designated as method D. Methods E and F

were used with functional groups sensitive to hydro-

genolysis; acid-labile anisyloxamines (method E) were

the most amenable to parallel synthesis. Anisyloxyamine

HCl, while commercially available in small quantities,⁹

was prepared in multigram quantities in three steps

from p-methoxybenzyl chloride (Scheme 4). Methoxide-

promoted ester aminolysis with hydroxylamine (method

F) afforded good yields and simplified purifications but

led to partial racemization at the R carbon of the serine

derivative (C-4 of the oxazolines). CD spectral determi-

nations of the optical purities of selected products

indicated % ee values of 80-95+% for acylserine hy-

droxamic acids and 30-87% for the oxazolines. Methods

D, E, and G, which preserve the stereochemistry, were

used in the analogues in which we wanted to establish

optical purity. The general methods of Scheme 4 were

F igu r e 2. Libraries of LpxC inhibitors targeted against P.

aeruginosa were designed by varying a set of aromatic rings

fused to a set of heterocyclic five- and six-membered rings, all

containing a hydroxamic acid as a Zn-binding group. A related

series of compounds, exemplified by L-161,240, had been

previously reported as inhibitors of the E. coli LpxC by

researchers at Merck.

better results than Burgess reagent at higher temper-

atures, and DAST was used in all subsequent syntheses

(Schemes 2 and 4). Initial efforts focused on solid-phase

parallel synthesis (Scheme 2) for which the aromatic

acids were generally commercially available. As our

SAR developed, the more focused analogues required

specific benzoic acids to address questions concerning

the effects of functional group characteristics and place-

Sch em e 1

3114 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14

Kline et al.

Sch em e 2

Sch em e 3

Sch em e 4

followed for all solution-phase syntheses and, in analogy

to the diversion of intermediates 7 in Scheme 2,

provided for the diversion of intermediates 3 to the

N-aroylserine hydroxamic acids shown in Table 2.

Oxa zin es. Oxazines have been synthesized via the

metal-catalyzed cyclization of â-amino alcohols with

nitriles,¹⁰but we explored a novel and direct route

(Scheme 5) that modified our oxazoline synthesis to

accommodate homoserine as the starting material. Since

for oxazines we had no precedent to follow for preferred

stereochemistry, we used D,L-homoserine. The amino

acid was sequentially protected as the methyl ester, tert-

butyldimethylsilyl ether, then acylated with aromatic

acids that had produced active oxazoline derivatives.

Treatment with 6 equiv of DAST at ambient tempera-

ture for 12 h effected both O-desilylation and cyclization

to the oxazine. The methyl ester was converted to the

hydroxamic acid using method F.

LpxC Inhibitors

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14 3115

Sch em e 5

Sch em e 6

Sch em e 7

Th ia zolin es. Several routes to the thiazolines have

been reported.¹¹In our hands, the method of Wipf¹²

(Scheme 6) gave both good yields and the efficiency of

common oxazoline intermediates for both classes of

compounds. The thiazoline methyl esters could be

converted to the corresponding hydroxamic acids via

either method E or F, but method F gave products

needing minimal purification (typically trituration with

cold methanol) and was therefore selected for our

parallel synthesis.

Assa y Develop m en t. Previously described assays for

E. coli LpxC relied on chromatographic separation of

deacetylated product,^1,3,6,13or on the use of surrogate

substrates.¹⁴While the predicted amino acid sequences

of P. aeruginosa and E. coli LpxC share 82% similarity

and 57% identity, the differences may account for their

observed differences in their sensitivities to inhibitors.¹⁵

Further, the native substrates for these enzymes dif-

fer: the LpxC substrate in E. coli is uridyldiphospho-

3-O-(R-hydroxymyristoyl-N-acetylglucosamine (3-O-

myristoyl UDPGlcNAc), but in P. aeruginosa it is 3-O-

(R-3-hydroxydecanoyl) UDPGlcNAc. We employed a

mixed chemoenzymatic strategy (Scheme 7)^1a,16to pre-

pare [³H-CH₃CONH]-3-O-(R-3-hydroxydecanoyl) UD-

PGlcNAc at a specific activity of 14-17 Ci/mmol. A low

level of the acyl migration (from O3 to O4 and then to

O6 as described by Anderson et al.^1a) occurred during

the isolation of the tritiated substrate (see Experimental

Section) but did not interfere with the assay. Control

experiments with unlabeled substrate acyl migration

products indicate that these were neither substrates nor

inhibitors. The tritiated substrate was used in an assay

that measured the cleaved [³H]-acetate, adapted to a

96-well format from the procedure described by Hyland

et al.¹⁷Incubation of the [³H] substrate with P. aerugi-

nosa LpxC (2 nM) at 22 °C for 90 min, followed by

absorption with charcoal, left a supernatant that con-

tained enzymatically released [³H]-acetate, with a back-

ground as low as e200 cpm, thereby giving us both

sensitivity and specificity. All compounds in Tables 1-4

were evaluated in this assay.

Discu ssion

Oxa zolin es. We explored diversity in the aromatic

ring with emphasis on hydrophobic and heterocyclic

functions. The modest potencies of the p-toluyl deriva-

tive 19 and the p-F derivative 35 indicated that even

simple 2-aryloxazoline hydroxamic acids might be in-

hibitors of the P. aeruginosa LpxC enzyme (Table 1),

with the p-F group being nearly 4-fold more active than

the p-Me species. Consistent beneficial effects of fluorine-

containing groups are demonstrated by the data in

Table 1. The trifluoromethoxy group appears to be the

best substituent and is optimally placed at the meta or

para position. For enhanced activity, it is placed in the

meta position and combined with an alkoxy group at

the para position. The 3-trifluoromethoxy compounds

do not appear to be highly sensitive to the nature of the

4-alkoxy group as long as this complementary group is

present. Analogues 5 and 20-24 all display a similar

improvement over 25. Lone pair orientation may be one

of the factors contributing to enzyme affinity. In the

para-fluoro-substituted series, addition of alkyl or alkoxy

at the meta position had a similar beneficial effect on

the potency (35 versus 36-38 and 44, for example).

3116 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14

Kline et al.

Ta ble 1. Configuration and in Vitro P. aeruginosa LpxC Inhibition of 2-Aryloxazolines

LpxC Inhibitors

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14 3117

Ta ble 1 (Continued)

3118 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14

Ta ble 1 (Continued)

Kline et al.

a

95% and >30% are assigned on the basis of the methods of synthesis and their application to analogues in which optical purity was

b

directly measured. Previously reported by the Merck group (ref 7a).

The meta trifluoromethylated series appears refrac-

tory to the kinds of beneficial modifications seen in the

aryl fluorides or the aryl trifluoromethoxy series. In

analogue 47, there is a detrimental effect upon addition

of a second meta substitution (46). In a comparison of

47 with 48, a hydrophobic ether function at the para

LpxC Inhibitors

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14 3119

Ta ble 2. Configuration and in Vitro P. aeruginosa LpxC

Ta ble 3. In Vitro P. aeruginosa LpxC Inhibition of

Inhibition of Aroylserines

2-Aryloxazines

functional groups are potential hydrogen bond accep-

tors, and hydrogen bond acceptance may contribute to

enzyme affinity.

Heterocyclic aromatic groups in place of phenyl at the

2 position of the oxazoline varied widely in effects. The

pyrazoles 58 and 59 were inactive up to 30 µM, whereas

indole 63 was a good inhibitor whose activity was

enhanced 5-fold upon further halogenation to 64. Benz-

pyrazole 60, a modest inhibitor, however, had its

potency halved upon alkyl substitution at either N1 (61)

or N2 (62). The potency of the meta-CF₃-substituted

phenyl analogue 49 was not affected in the direct pyridyl

homologue 50.

Since Schemes 2 and 4 enabled us to conveniently

obtain the corresponding NR-aroyl-D-serine hydroxamic

acids, these were also evaluated in vitro (Table 2).

Thirty species were examined, with most showing no

activity (IC₅₀> 50 µM). A portion of the data for

acylserines appears in Table 2.

Oxa zin es. Although the trend we generally observed

was that for any given aromatic nucleus the acylserine

was less potent than the corresponding oxazoline, there

were exceptions seen in analogue pairs 66/31 and 69/

26. We considered whether the increased flexibility of

six-membered rather than five-membered ring might

allow optimization of the relationship between the

aromatic and hydroxamic acid moieties. Oxazines were

prepared as direct analogues of a sampling of the

oxazolines. We were disappointed to observe that these

oxazines (Table 3) showed low activities against the P.

aeruginosa LpxC.

Th ia zolin es. The thiazoline ring is found in a num-

ber of prokaryotic natural products, particularly where

metal chelation is involved.^11a,e,18We prepared a series

of analogues incorporating aromatic substituents shown

to be beneficial in oxazolines (as shown in Table 1) into

the direct thiazoline analogues. As shown in Table 4,

modest potency (17 µM) was achieved with 80, the sulfur

isostere of 5, but in no case did the thiazolines approach

the potency of the corresponding oxazolines.

position fails to enhance activity significantly, even

allowing for the fact that 48 is partially racemized, and

this is in stark contrast to the 50-fold improvement in

activity when one adds an allyloxy group to the trifluo-

rmethoxy compound 25 to give rise to 23.

In the nonfluorinated analogues, the idea of an

optimal chain length is suggested by comparing 19 (too

short), 30 (too long), and 29, 31 (intermediate length).

Addition of a second aromatic ring is beneficial, with

the benefits somewhat sensitive to the nature of the

linkage (32-34). The idea that a second ring may access

a critical binding site is reinforced by the comparison

of 40 and 41, in which a 10-fold loss of activity occurs

upon shifting the trifluormethyl group in the benzyloxy

ring from meta to ortho.

The similar, low micromolar IC₅₀values of the para

trifluoromethoxy (26), nitro (51), dimethylamino (52),

fluoro (35), bromo (54), and iodo (55) analogues suggest

that the stringency for modest binders such as these is

lower than for relatively strong binders. In this series,

phenol 53 is strikingly inactive. Most of the active para

3120 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14

Kline et al.

Ta ble 4. Configuration and in Vitro P. aeruginosa LpxC

Inhibition of 2-Arylthiazolines

or F substituent, submicromolar potencies (32, 34) could

be achieved by optimizing the hydrophobic group at the

para position. New libraries are under construction to

refine the placement of these contacts.

Our best inhibitors are approaching the 100 nM range

against isolated P. aeruginosa enzyme. Inhibitors in this

range against the E. coli enzyme were reported to

inhibit E. coli growth at MIC values ranging from 1.25

to 12.5 µg/mL.^5bWe are currently evaluating our novel

compounds against a panel of Gram-negative organisms,

both wild-type and selected mutants, to obtain MIC

profiles and thereby to validate the concept of LpxC

inhibitors as therapeutic agents.

Exp er im en ta l Section

Gen er a l. Unless otherwise indicated, all anhydrous sol-

vents were commercially obtained and stored in Sure-seal

bottles under nitrogen. All other reagents and solvents were

purchased as the highest grade available and used without

further purification. Ethereal diazomethane was used for the

production of methyl esters. It was prepared by adding a gram

of N-nitroso-N-methylurea in small portions to an ice-cold

mixture of 4 mL of 40% aqueous KOH and 8 mL of Et₂O. After

5 min, the resulting ether layer was briefly dried over a few

KOH pellets and the solution was added in 1 mL aliquots to

the acid dissolved in ether containing up to 20 vol % MeOH

as required for solubilization. NMR spectra were recorded on

Bruker 400 or 500 MHz instruments. Chemical shifts (δ) are

reported in parts per million (ppm) referenced to ¹H (Me₄Si at

0.00), ¹³C (DMSO at 39.55, CDCl₃at 77.0, and CD₃OD at 49.0),

and ¹⁹F (50 µM TFA at -76.55). All ¹⁹F spectra were recorded

at 470.3 MHz in pH 7 aqueous buffer. Coupling constants (J )

are reported in Hz throughout. Mass spectral data were

acquired on a Esquire LC00066 for low resolution, a Micromass

70 SEQ for high resolution, or a J EOL LC-mate tuned for

either low resolution or high resolution. Optical rotations were

determined on a Perkin-Elmer model 241 polarimeter. Circular

dichroic (CD) spectra over the 325-205 nm wavelength range

were recorded for ca. 1 mM solution in CH₃OH at 20 °C in a

1 mm path length cell using a J ASCO model J 720 spectropo-

larimeter with a nitrogen flow rate of 5 L/min. CD data are

reported as molar ellipticity ([θ]_λin units of deg cm²dmol^-1

)

at the near-UV extremum (λ in nm) corresponding closely to

Con clu sion s

the UV maximum.

All parallel synthesis was conducted on

a Quest 210

Our efforts at expanding the original oxazoline hit

resulted in the first reported inhibitors of the P. aerugi-

nosa LpxC. Our strategy to retain the hydroxamic acid

as the Zn binding function, but vary the heterocyclic and

aromatic rings, enabled an in-depth exploration of the

contribution of these two moieties to overall activity.

The most active heterocyclic ring appeared to be the

oxazoline. Ring expansion (oxazines) abrogated all

activity, and with a few exceptions ring opening

(acylserines) or sulfur substitution (thiazolines) gave

rise to considerably weaker inhibitors. The highest

inhibitory potencies came from aryl oxazolines with 3,4-

disubstituted phenyl rings with fluoro or trifluo-

romethoxy in the para or meta position and a two- to

five-atom hydrophobic group in the complementary

position.

The foregoing would suggest that the electronic

properties of the phenyl ring, the orientation of an

oxygen lone pair, and a certain optimal hydrophobicity

are the chief determinants of good inhibitory potency.

The m-trifluoromethoxy function may be serving some

or all of these functions. The SAR data to date suggest

a somewhat restricted binding site in which the key

contacts, including hydrogen bonding and lipophilic

interactions, need to be made. In the absence of a CF₃O

synthesizer equipped with an automated solvent washing

module and a gaseous reaction and concentration module

(Argonaut Technologies, San Carlos, CA) under an atmosphere

of dry nitrogen. All single syntheses were conducted in

conventional flasks under an atmosphere of dry nitrogen. For

parallel synthesis, evaporation of volatiles in vacuo was done

on a Savant SC210A Speedvac at ca. 0.9 Torr; for single

synthesis, evaporation of volatiles in vacuo was done on a

vacuum-driven Buchi rotavapor at ca. 15 Torr followed by

vacuum-drying at ca. 0.5 Torr. Analytical HPLC was conducted

on a Gilson, Varian, or a Waters instrument using a 2.5 mm

× 250 mm octadecylsilane (ODS) column, 10 µm, 100 Å, at a

flow rate of 1.0 mL/min. Unless otherwise indicated, peaks

were monitored at 254 and 215 nM. Preparative high-

performance liquid chromatography (HPLC) was conducted on

a Varian model 210 using a 10 mm × 250 mm ODS column,

5 µm, 100 Å, at a flow rate of 4.6 mL/min. Peaks were

monitored at 260 and 215 nM. Radial chromatography was

performed on a Chromatotron instrument (Harrison Research,

Palo Alto, CA) using EM Science silica gel as adsorbent;

preparative thin-layer chromatography experiments were done

on VWR GF silica gel plates, and all other preparative normal

phase chromatography experiments were done on either flash

or Biotage systems. Radiochemical synthesis was performed

at the Lawrence Berkeley Laboratory (Berkeley, CA) with the

assistance of the LBL staff.

6-P r op yl-2-tr iflu or om eth oxyp h en ol (1). To a solution of

2-trifluoromethoxyphenol (3.0 g, 16.12 mmol) in freshly dis-

LpxC Inhibitors

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14 3121

tilled acetone (100 mL) was added solid K₂CO₃(4.7 g, 33.7

mmol) and allyl bromide (4.08 g, 33.7 mmol), and the reaction

mixture was refluxed for 3 h, then cooled to ambient temper-

ature and concentrated in vacuo. The residue was dissolved

in CH₂Cl₂, and the organic phase was washed sequentially

with water, saturated Na₂CO₃, and saturated brine, dried, and

concentrated in vacuo to give the allyl ether (3.13 g, 14.4 mmol,

85%) as a light-yellow oil that required no further purifica-

tion: ¹H NMR (500 MHz, CDCl₃) δ 7.24 (H-3, m), 7.22 (H-5,

td, 8, 1.5), 6.98 (H-6, dd, J ) 8, 1.5), 6.94 (H-4, td, 8, 1.5), 6.04

(ddt), 5.44 (dq, J _d) 16), 5.30 (dq, J _d) 10) (-CHdCH₂), and

4.60 (OCH₂); ¹³C NMR (100 MHz, CDCl₃) δ 151.09 (C2), 138.39

(C1), 132.57 (C2′), 127.93 (C4), 123.52 (C5), 120.96 (C6), 120.76

(OCF₃, q, 257.0), 117.51 (C3′), 114.46 (C3), 69.22 (C1′); MS,

m/z 216.9 (M - H)^-.

A solution of the above allyl ether (3.13 g, 14.4 mmol) in

N,N-dimethylaniline (25 mL) was refluxed for 7 h, then cooled

to ambient temperature and poured into a mixture of ethyl

acetate over ice-cold 50% HCl (80 mL). The organic phase was

separated and washed with saturated brine, dried, and

concentrated in vacuo to give the o-allylphenol (3.3 g) as a

yellow oil: ¹H NMR (500 MHz, CDCl₃) δ 7.11 (H-3, d quint, J

) 8, 1), 7.07 (H-5, ∼d, J ) 8), 6.86 (H-4, t, J ) 8), 6.00 (CHd

C, m), 5.42 (OH), 5.09-5.13 (2H, CdCH₂), 3.45 (CH₂, d, J )

7); ¹³C NMR (100 MHz, CDCl₃) δ 145.69 (C1), 136.44 (C2),

135.78 (C1′), 128.46 (C6), 120.49 (OCF₃, q, 258.8), 120.10 (C4),

118.96 (C3), 116.28 (C3′), 34.06 (C1′); MS, m/z 216.8 (M - H)^-.

The Claisen product was dissolved in EtOH (200 mL) and

hydrogenated over 10% Pd-C (300 mg) at 1 atm for 3 h.

Filtration through Celite and concentration of the filtrate in

vacuo gave 1 (3.1 g, 14.1 mmol, 98%): ¹H NMR (500 MHz,

CDCl₃) δ 7.07 (H-3,5, ∼t), 6.84 (H-4, t), 5.32 (OH, br s), 2.65

(2H, t), 1.66 (2H, sextet), 0.96 (CH₃, t); ¹³C NMR (100 MHz,

CDCl₃) δ 145.72 (C1), 136.38 (C2), 131.37 (C5), 128.38 (C6),

120.77 (OCF₃, q, J ) 258.5), 119.74 (C4), 118.29 (C3), 31.85

(C1′), 22.59 (C2′), 13.84 (C3′).

4-Met h oxy-5-p r op yl-3-t r iflu or om et h oxyb en zoic Acid

(2a ). To a -78 °C solution of 1 (3.1 g, 14.1 mmol) in CH₂Cl₂

(200 mL) was added Br₂(2.48 g, 15.5 mmol) dropwise via

syringe. Upon completion of the Br₂addition the reaction

mixture was warmed to - 5 °C, stirred at this temperature

for 4 h, then allowed to warm to ambient temperature

overnight. The reaction mixture was treated with saturated

Na₂S₂O₅(100 mL), stirred for 5 min, then partitioned between

CH₂Cl₂and water. The organic phase was separated, dried,

and concentrated in vacuo to give the 4-bromophenol inter-

mediate (3.5 g, 11.8 mmol, 83%) as a colorless oil that solidified

on standing: ¹H NMR (400 MHz, CDCl₃) δ 7.23, 7.21 (H-3,5),

2.60 (ArCH₂, ∼t), 1.64 (CH₂, sextet), 0.96 (CH₃, t); ¹³C NMR

(100 MHz, CDCl₃) δ 144.96 (C1), 136.41 (C2), 133.17 (C5),

131.00 (C6), 122.50 (C3), 120.58 (OCF₃, q, J ) 260.2), 111.05

(C4), 31.68 (C1′), 22.46 (C2′), 13.79 (C3′); MS, m/z 297.0, 299.0

(M - H)^-.

quenched by the addition of 30 mL of 1 M NaOH followed by

the addition of 30 mL of CH₂Cl₂. The resulting aqueous layer

was washed with an additional 30 mL portion of CH₂Cl₂,

acidified with 6 N HCl, and extracted with EtOAc (3 × 60 mL).

The organic phase was washed with saturated brine, dried,

and concentrated in vacuo, affording acid 2a (520 mg, 42%):

¹H NMR (500 MHz, CDCl₃) δ 7.882 (6H, d, J ) 2), 7.855 (2H,

dq, J ) 2, 1), 3.96 (OMe, s), 2.671 (2H, d, J ) 7.7), 1.65 (2H,

sextet, J ) 7.7), 0.98 (3H, t, J ) 7.5); ¹³C NMR (100 MHz,

CDCl₃) δ 180.45 (COOH), 154.81 (C4), 141.31 (C3), 137.82 (C6),

130.23 (C5), 124.23 (C1), 121.97 (C2), 120.34 (OCF₃, q, J )

258.6), 61.22 (OCH₃), 31.79 (C1′), 23.04 (C2′), 13.45 (C3′); MS,

m/z 276.9 (M - H)^-.

2-(4-Meth oxy-3-p r op yl-5-tr iflu or om eth oxyp h en yl)-4,5-

d ih yd r ooxa zole-4-ca r boxylic Acid Meth yl Ester (4a ). To

a suspension of ^D-serine methyl ester hydrochloride (0.35 g,

2.3 mmol) in CH₂Cl₂(50 mL) was added sequentially 2a (0.5

g, 1.9 mmol), EDCI (0.65 g, 3.4 mmol), and DIEA (0.59 mL,

3.4 mmol) [condensation method B]. The reaction mixture was

stirred at ambient temperature overnight, then washed se-

quentially with water, 0.5 N HCl, saturated NaHCO₃, and

brine. The organic phase was dried and concentrated in vacuo

to give 3a (553 mg, 1.5 mmol, 77%): ¹H NMR (500 MHz,

CDCl₃) δ 7.16-7.20 (H-2,6), 4.867 (HR, dt, J ) 7, 3.5), 4.098/

2

3

4.057 (C_âH₂, AB of ABX, J ) 11.5, J ) 3.8/3.2), 3.91 (OCH₃,

s), 3.83 (OCH₃,s), 2.64 (ArCH₂, AB m), 1.62 (CH₂, m), 0.96

(CH₃, t); ¹³C NMR (100 MHz, CDCl₃) δ 170.83 (CO₂R), 165.93

(CONH), 153.17 (C4), 141.52 (C3), 137.95 (C6), 128.64 (C5),

127.06 (C1), 120.27 (OCF₃, q, J ) 258.6), 119.41 (C2), 62.83

(CH₂OH), 61.16 (Ar-OCH₃), 55.14 (CH), 53.02 (CO-OCH₃),

31.68 (C1′), 23.08 (C2′), 13.69 (C3′).

To a solution of 3a (553 mg, 1.5 mmol) in anhydrous THF

(12 mL) in a pressure tube was added Burgess reagent (383

mg, 1.60 mmol). The tube was flushed with nitrogen, sealed,

and heated to 70 °C for 2.5 h. The reaction was cooled to

ambient temperature, and the solution was concentrated in

vacuo to an oily residue. Chromatography on silica (90:10

hexane/ethyl acetate as eluant) gave ester 4a (254 mg, 0.7

mmol, 49%): ¹H NMR (500 MHz, CDCl₃) δ 7.778 (H-5, d, J )

2), 7.710 (H-2, dq), 4.944 (1H, dd, J ) 10.5, 8), 4.694 (1H, ∼t,

J ) 8.3), 4.589 (1H, dd, J ) 10.5, 8.5), 2.635 (2H, AB m), 1.627

(2H, sextet), 0.956 (3H, t, J ) 7.5); ¹³C NMR (100 MHz, CDCl₃)

δ 169.54 (CO₂R), 163.12/66.71/67.79 (oxaz-C2,4,5), 151.45 (C4),

139.68 (C3), 136.12 (C6), 126.93 (C5), 120.93 (C1), 119.89 (C2),

119.58 (OCF3, q), 59.50 (Ar-OCH₃), 50.83 (CO-OCH₃), 30.02

(C1′), 21.66 (C2′), 12.0 (C3′); MS, m/z 362.1 (M + H)⁺.

2-(4-Meth oxy-5-p r op yl-3-tr iflu or om eth oxyp h en yl)-4,5-

d ih yd r ooxa zole-4-ca r boxylic Acid Hyd r oxa m id e (5). To

an ice-cooled suspension of benzyloxyamine hydrochloride (146

mg, 0.97 mmol) in anhydrous benzene (1.5 mL) was added a 2

M solution of trimethylaluminum in toluene (0.44 mL, 0.88

mmol). The resulting solution was stirred at ambient temper-

ature for 90 min, then transferred via cannula to a solution of

4a (254 mg, 0.70 mmol) in anhydrous benzene (2 mL). The

reaction mixture was heated to 50 °C for 5.5 h, then cooled to

ambient temperature, quenched by addition of water (200 µL),

and stirred for 10 min. After the insoluble material was filtered

off, the filtrate was partitioned between ethyl acetate and

water, and the organic phase was dried and concentrated in

vacuo to give the benzyloxy intermediate (280 mg, 0.62 mmol,

89%, MS, m/z 453.2), which was dissolved in 22 mL of

methanol and hydrogenated over 20% Pd(OH)₂(50 mg) at 1

atm for 1 h. The catalyst was filtered off, and the filtrate was

concentrated in vacuo to give 250 mg of the crude product as

an off-white solid. Purification on silica gel (60:40 hexane/ethyl

acetate as eluant) followed by precipitation with hexane gave

Solid K₂CO₃(1.62 g, 11.8 mmol) was flame-dried under

nitrogen in a flask. The product of the above reaction in freshly

distilled acetone (100 mL) was added, followed by addition of

methyl iodide (1.7 g, 11.8 mmol). The reaction mixture was

refluxed for 6 h, stirred at ambient temperature overnight,

then filtered and concentrated in vacuo. The residue was

partitioned between CHCl₃and water, and the organic phase

was separated, dried, and concentrated in vacuo to give the

methyl ether (3.4 g, 10.9 mmol, 93%) as a yellow oil: ¹H NMR

(400 MHz, CDCl₃) δ 7.25 (H-2,6, s), 3.85 (OCH₃s), 2.58 (ArCH₂,

∼t), 1.61 (CH₂, sextet), 0.97 (CH₃, t); ¹³C NMR (100 MHz,

CDCl₃) δ 149.99 (C4), 142.88 (C3), 140.08 (C6), 131.65 (C5),

122.15 (C2), 119.58 (OCF₃, q), 115.73 (C1), 61.75 (OCH₃), 32.11

(C1′), 23.89 (C2′), 14.28 (C3′); MS, m/z 311.2, 313.1 (M - H)^-.

To a 78 °C solution of the above intermediate (1.40 g, 4.47

mmol) in anhydrous ether (45 mL), a 1.6 M solution of

n-butyllithium (3.2 mL, 5.12 mmol) in hexane was added

dropwise. The reaction mixture was stirred at - 78 °C for 1 h

before the cooling bath was removed. Carbon dioxide gas was

bubbled vigorously through the reaction mixture for 1.5 h as

the reaction mixture warmed to -10 °C. The reaction was

5 (85 mg, 0.24 mmol, 39%): mp 140-143 °C; [R]²⁵-49.00° (c

D

0.1, CH₃OH), CD [θ]₂₅₃) -12 700; ¹H NMR (400 MHz, CDCl₃)

δ 9.4-9.5 (NH, br), 7.6-7.7 (H-2,6), 4.8-4.9 (HR, ∼t), 4.46-

4.7 (CâH₂, AB pattern), 3.9 (OCH₃, s), 2.6-2.7 (ArCH₂, t), 1.6

(CH₂, sextet), 0.9 (CH₃, t); ¹³C NMR (100 MHz, CDCl₃) δ 170.33

(CONHOH), 167.73 /69.23/72.23 (oxaz-C2,4,5), 155.56 (C4),

143.53 (C3), 140.13 (C6), 123.62 (C1), 122.28 (C2), 63.35

(OCH₃), 33.47 (C1′), 24.35 (C2′), 15.80 (C3′); ¹⁹F NMR δ -58.92;

3122 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14

Kline et al.

MS, m/z 363 (M + H)⁺. Anal. found C 49.50, H 4.30, N 7.53;

calcd for C₁₅H₁₇F₃N₂O₅, C 49.73, H 4.73, N 7.73.

s), 7.23-7.53 (H_phenyl, m), 4.48-4.87 (CH₂â and CHR, m); MS,

m/z 341.0 (M + H)⁺.

Gen er a l P r oced u r e for th e Solid -P h a se P a r a llel Syn -

th esis of Acylser in e Hyd r oxa m ic Acid s. To obtain the

acyclic analogues, the resin-bound acylserine, prepared as

described above, was subjected directly to the TFA cleavage

following the washing protocol. Purification of the crude

acylserine hydroxamic acids was by radial or preparative thin-

layer chromatography using 3% MeOH in CH₂Cl₂as eluant.

Determinations of purity and structure were the same as for

the oxazolines.

Gen er a l P r oced u r e for th e Solid -P h a se P a r a llel Syn -

th esis of Oxa zolin e Hyd r oxa m ic Acid s. Into 10 10 mL

reaction vessels was dispensed fluorenemethyleneoxycarbonyl

(Fmoc)-aminoxy-o-chlortritylpolystyrene (0.58 g, 0.39 mmol per

reaction vessel, from a 0.6 mmol/g, Advanced ChemTech

batch), and the resins were treated three successive times with

20% piperidine in DMF for 1 h, each time followed by a DMF

wash, to remove the Fmoc group. The resins were washed one

final time with, successively, DMF (3×), MeOH (3×), and CH₂-

Cl₂(3×). The reaction block was cooled to 0 °C, and to each

reaction vessel was added a solution of either Fmoc-^D-serine

(0.13 g, 0.4 mmol) or Fmoc-^D-threonine (0.14 g, 0.4 mmol) in

2 mL of anhydrous DMF, followed by a solution of HATU (0.27

g, 0.7 mmol) and DIEA (0.14 mL, 0.8 mmol) in 2 mL of

anhydrous DMF. The reaction block was equilibrated to

ambient temperature, and the vessels were agitated for 16 h,

then washed using the three-solvent wash protocol described

above. The Fmoc group was removed under the standard

reaction and wash conditions described above, followed by

treatment of each individual reaction vessel with a solution

of an arylcarboxylic acid (0.42 mmol), HATU (0.16 g, 0.42

mmol), and DIEA (0.09 mL, 0.5 mmol) in 4 mL of anhydrous

DMF, agitation for 16 h at ambient temperature, and washing

as described above. The resin was suspended in CH₂Cl₂, the

reaction block cooled to -15 °C, and DAST (0.22 mL, 1.7 mmol)

added via syringe. Agitation was continued for 1 h at -15 °C,

and the reaction was then quenched by addition of excess

aqueous NH₄OH. The reaction block was equilibrated to

ambient temperature, and the resins were washed as above.

To release the products from the resin, 10% TFA in CH₂Cl₂

(5.0 mL) was added and the resins were agitated for 90 min.

Filtrates from each reaction vessel were drained into vials and

concentrated in vacuo. The crude oxazoline hydroxamic acids

were purified by preparative thin-layer chromatography using

3% MeOH in CH₂Cl₂as eluant. Yields were ca. 50%. All

products from the solid-phase library were characterized by

proton NMR and mass spectrometry and were shown to be

>90% pure by reverse-phase HPLC.

Rep r esen ta tive Nr-Ar oylser in e Hyd r oxa m ic Acid s

P r ep a r ed by Solid -P h a se Meth od s. N-(4-Meth ylben zoyl)-

1

ser in e h yd r oxa m ic a cid (65): H NMR (400 MHz, CD₃OD)

δ 7.71 (2H, d, J ) 8.1), 7.19 (2H, d, J ) 8.1), 4.61-4.44 (1H,

m), 3.95-3.78 (2H, m), 2.25 (3H, s); MS, m/z 239.0 (M + H)⁺.

N-[4-(Bu t-3-en yloxy)ben zoyl]ser in e h yd r oxa m ic a cid

(66): ¹H NMR (400 MHz, CD₃OD) δ 7.84 (2H, d, J ) 8.7), 6.98

(2H, d, J ) 8.7), 5.96-5.88 (1H, m), 5.17 (1H, dd, J ) 17.2,

1.6), 5.09 (1H, dd, J ) 11.7, 1.6), 4.57 (1H, bs), 4.08 (2H, t, J

) 6.6), 3.86 (2H, d, J ) 4.7), 2.54 (2H, q, J ) 6.6); MS, m/z

295.1 (M + H)⁺.

N-(7 Ben zp yr a zolyloyl)ser in e h yd r oxa m ic a cid (72): ¹H

NMR (400 MHz, CD₃OD) δ 8.20 (1H, d, J ) 8.2), 7.61 (1H, d,

J ) 8.5), 7.45 (1H, t, J ) 7.2), 7.29 (1H, t, J ) 7.6), 4.65 (1H,

bs), 3.95-3.81 (2H, m); MS, m/z 265.1 (M + H)⁺.

Br om op h en ols 9 (Sch em e 3). The example R_F) CF₃O,

R ) H is representative of the general bromination procedure.

To a solution of 2-trifluoromethoxyphenol (7.40 g, 41.5 mmol)

in CH₂Cl₂(100 mL) at -78 °C was added a bromine solution

(45 mL, 1.0 M, 45 mmol) in CH₂Cl₂dropwise over 20 min. The

reaction was allowed to warm to ambient temperature and

stirred for 48 h. Saturated Na₂SO₃(100 mL) was added, and

the reaction mixture was stirred vigorously until disappear-

ance of the orange color was observed. The biphasic mixture

was diluted with CH₂Cl₂(200 mL), and the organic layer was

separated, washed with brine (100 mL), dried over MgSO₄,

and concentrated in vacuo to give 9.80 g (93%) of a white

solid: mp 50-52 °C (sublimed); ¹H NMR (500 MHz, CDCl₃) δ

7.38 (H-3, dq, J ) 2.0, 1.1), 7.32 (H-5, dd, J ) 8.9, 2.0), 6.95

(H-6, d, J ) 8.9).

If the bromination was carried out with added KOAc to

accelerate the process or with excess bromine, 4,6-dibromo-2-

trifluoromethoxyphenol was obtained as a major byproduct:

mp 39-41 °C; ¹H NMR (500 MHz, CDCl₃) δ 7.615 (H-5, d, J )

1.9), 7.38 (H-3, dq, J ) 2, 1). For phenols with only a single

available ortho or para position, the addition of KOAc allowed

the reaction to go to completion in a few hours at ambient

temperature. 4-Bromo-2-fluoro-, 4-bromo-2-trifluoromethyl-,

and 4-bromo-2-fluoro-5-methylphenol were prepared in the

same manner.

5-Br om o-3-flu or o-4-m eth oxyben zoic Acid (13, R_F) F ,

R ) H). Bromine (3.1 mmol, 1.05 equiv) was added dropwise

with stirring over 30 min to an ice-cooled solution of methyl

3-fluoro-4-hydroxybenzoate¹⁹(0.5 g, 2.94 mmol) and 6 mL of

1:1 CH₂Cl₂/AcOH. After being stirred overnight at room

temperature, the mixture was diluted with the ethyl acetate

(20 mL) and the resulting solution was aqueous Na₂SO₃, water,

and brine and was dried over MgSO₄. Evaporation in vacuo

afforded (84% yield) methyl 5-bromo-3-fluoro-4-hydroxyben-

zoate as a white solid: ¹H NMR (500 MHz, CDCl₃) δ 7.97 (H-

6, dd J ) 1.8, 1.5), 7.76 (H-2, dd, J ) 10.3, 1.5), 6.01(OH),

3.88 (3H, s). Methylation by the standard procedure: ¹H NMR

(500 MHz, CDCl₃) δ 7.97 (H-6, dd, J ) 1.8, 1.4), 7.67 (H-2, dd,

J ) 11.7, 1.8), 4.03 (ArOCH₃, d, J _F) 2.5), 3.88 (COOCH₃, s).

Methylation followed by saponification afforded the title acid

in 71% overall yield: mp 158-160 °C; ¹H NMR (500 MHz,

DMSO-d₆) δ 7.93 (H-6), 7.76 (H-2, dd, J ) 11.6, 2), 3.98 (3H,

bs).

Rep r esen ta tive 2-Ar yloxa zolin e-4-h yd r oxa m ic Acid s

P r ep a r ed by Solid -P h a se Meth od s. 2-p-Tolyloxa zolin e-

4-h yd r oxa m ic a cid (19): ¹H NMR (400 MHz, CDCl₃, CD₃-

OD) δ 7.82 (2H, d, J ) 8.1), 7.25 (2H, d, J ) 8.1), 4.76 (1H, t,

J ) 10.3), 4.65 (1H, t, J ) 9.8), 4.57 (1H, t, J ) 10.3), 2.40

(3H, s); MS, m/z 221.0 (M + H)⁺.

2-(3-Tr iflu or om eth oxyp h en yl)oxa zolin e-4-h yd r oxa m -

ic a cid (25): ¹H NMR (400 MHz, acetone-d₆) δ 7.40-7.92 (4H,

m), 4.73-4.78 (1H, m) 4.33-4.60 (2H, m); MS, m/z 291.0 (M

- H)^-.

2-(4-Tr iflu or om eth oxyp h en yl)oxa zolin e-4-h yd r oxa m -

ic a cid (26): CD [θ]₂₄₆) -12 400; ¹H NMR (DMSO-d₆) δ 10.9

and 9.03 (CONHOH), 8.01 (H-3,5, d, J ) 8.7), 7.49 (H-2,6, d),

4.70/4.59/4.52 (CHCH₂); MS, m/z 291.0623 (M + H)⁺, calcd for

C

₁₁H₁₀F₃N₂O₄, 291.0593; [R]²⁵-46.62° (c 0.21, CH₃OH).

D

Prepared also by Scheme 4 (methods C and F): mp 181-

182°C; CD [θ]₂₄₆) -16 000; ¹³C NMR δ 166.94 (CONHOH),

163.06/66.91/69.98 (oxaz-C2,4,5), 163.06 (CONHOH), 150.67

1

(C4), 130.37 (C2,6), 126.09 (C1), 120.00 (OCF₃, q, J ) 257.7),

120.99 (C3,5); ¹⁹F NMR δ -58.65; MS, m/z 291.02 (M + H)⁺,

313.0 (M + Na)⁺. Anal. found C 45.45, H 2.89, N 10.22; calcd

for C₁₁H₉F₃N₂O₄, C 45.53, H 3.13, N 9.65. Compound 26 was

also prepared by methods B and D: CD [θ]₂₄₆) -18 600.

2-(2-Tr iflu or om eth oxyp h en yl)oxa zolin e-4-h yd r oxa m -

ic a cid (28): ¹H NMR (400 MHz, CD₃OD) δ 8.26 (1H, m), 7.92

(1H, m), 7.69-7.76 (2H, m), 4.96-5.10 (1H, m), 4.89-4.94 (2H,

m); MS, m/z 291.1 (M + H)⁺.

2-(5-Meth yl-1-p h en yl-(1H)-p yr a zol-4-yl)oxa zolin e-4-h y-

d r oxa m ic a cid (58): ¹H NMR (400 MHz, CD₃OD) δ 8.10 (1H,

s), 7.63-7.45 (5H, m), 5.21-5.13 (1H, m), 4.96-4.82 (2H, m),

2.57 (3H, s); MS, m/z 287.2 (M + H)⁺.

3-Alk yl-4-flu or oben zoic Acid s 14 (R′ ) P r op yl Is Rep -

r esen ta tive). A solution of methyl 3-bromo-4-fluorobenzoate

(1.080 g, 4.634 mmol) in DMF (13 mL) was added to a pressure

tube containing tetrakis(triphenylphosphine)palladium(0) (350

mg, 0.302 mmol). Allyltributyltin (1.8 mL, 5.8 mmol) was

2-(5-Tr iflu or om et h yl-1-p h en yl-(1H )-p yr a zol)-4-ylh y-

d r oxa m ic a cid (59): ¹H NMR (400 MHz, CD₃OD) δ 8.10 (H_pyr

,

LpxC Inhibitors

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14 3123

added, the solution was purged with a stream of nitrogen for

5 min, and the pressure tube was sealed. The reaction mixture

was heated at 85 °C for 40 h, cooled, and filtered through a

plug of SiO₂eluting with Et₂O (200 mL). The organic layer

was washed with H₂O (5 × 80 mL) and brine (80 mL), dried

over MgSO₄, and concentrated in vacuo. Chromatography on

silica gel (100% hexanes) afforded 1.46 g of a yellow oil

consisting of methyl 3-allyl-4-fluorobenzoate and Bu₃SnBr,

which was dissolved in EtOH (15 mL), treated with 10% Pd-C

(310 mg), and stirred under a hydrogen atmosphere (ambient

pressure) for 16 h. The reaction mixture was filtered through

a pad of Celite eluting with CH₂Cl₂(100 mL), concentrated,

and then filtered through a plug of silica gel eluting with 10:1

hexanes/EtOAc (150 mL). The organic layer was concentrated

in vacuo to give a colorless oil, which was dissolved in a

mixture of THF (14 mL), MeOH (2 mL), and H₂O (6 mL).

Aqueous KOH (2.5 mL, 6 M, 15 mmol) was added, and the

reaction was stirred overnight. The solution was concentrated

to a volume of ca. 5 mL, H₂O (45 mL) was added, and the

aqueous layer was washed with Et₂O (2 × 50 mL). The organic

layer was discarded, and the aqueous layer was acidified with

6 M HCl (5 mL) and extracted with CH₂Cl₂(3 × 30 mL), and

the organic layer was dried over MgSO₄. Concentration in

vacuo gave 648 mg (77%, three steps) of acid 14 (R′ ) Pr), a

white powder: ¹H NMR (500 MHz, CDCl₃) δ 7.99-7.93 (H-

2,6, m), 7.09 (H-5, t, J ) 9.0), 2.67 (ArCH₂, t, J ) 7.5), 1.68

(CH₂, sextet, 8.0), 0.97 (CH₃, t, J ) 7.5).

added oxalyl chloride (188 µL, 2.15 mmol) and DMF (7 µL).

The reaction mixture was warmed gradually to ambient

temperature over 1 h and then recooled to 0 °C. A solution of

D-serine methyl ester hydrochloride (334 mg, 2.15 mmol) and

DIEA (1.1 mL, 6.3 mmol) in CH₂Cl₂was added dropwise over

5 min, and the reaction mixture was stirred 16 h, slowly

warming to ambient temperature. The reaction mixture was

concentrated and filtered through a plug of silica gel, eluting

with CH₂Cl₂/MeOH (15:1) and concentrated to give a 560 mg

of a yellow oil that was used directly in the cyclization reaction.

Gen er a l P r oced u r e for Cycliza tion . 2-(3-P r op yl-4-flu o-

r op h en yl)-4,5-d ih yd r ooxa zole-4-ca r boxylic Acid Meth yl

Ester (4, Ar ) 3-P r -4-F -p h en yl). The acylserine intermedi-

ate described immediately above was dissolved in CH₂Cl₂(6

mL) and cooled to -20 °C. A solution of DAST (2.0 mL, 1.0 M,

2.0 mmol) in CH₂Cl₂was added, and the solution was stirred

for 30 min at -20 °C. One additional equivalent of DAST (2.0

mL, 1.0 M, 2.0 mmol) was added, and the reaction mixture

was stirred for 1 h. Saturated NaHCO₃(15 mL) was added,

the solution warmed to ambient temperature, and the reaction

mixture diluted with CH₂Cl₂(60 mL). The organic layer was

separated, washed with 1 M HCl (30 mL), dried over MgSO₄,

and concentrated in vacuo to give a yellow oil. Purification by

silica gel chromatography (100:1 CH₂Cl₂/MeOH) afforded 475

mg (90% over two steps) of the methyl oxazoline-4-carboxylate

as a pale-yellow oil: ¹H NMR (500 MHz, CDCl₃) δ 7.87 (H-2,

dd, J ) 2.5, 7.5), 7.79 (H-6, ddd, J ) 2.5, 5.0, 9.0), 7.03 (H-5,

t, J ) 10.0), 4.94 (1H, dd, J ) 8.0, 10.5), 4.69 (1H, t, J ) 9.0),

4.59 (1H, dd, J ) 8.5, 10.5), 3.85 (3H, s), 2.64 (2H, m), 1.64

(2H, sextet, J ) 7.5), 0.94 (3H, t, J ) 7.5).

In a subsequent reaction from methyl 3-bromo-4-fluoroben-

zoate (11 mmol), the saponification was performed on the

3-allylbenzoate, affording 1.77 g (89% in three steps) of acid

1

14 (R′ ) allyl) as a white solid: mp 95-96 °C; H NMR (500

In like manner, N-(3-ethoxy-4-fluorobenzoyl)-^D-serine meth-

yl ester (161 mg, 0.564 mmol) was cyclized affording 147 mg

(98%, crude) of the oxazoline carboxylate: ¹H NMR (500 MHz,

CDCl₃) δ 7.60 (H-2, dd, J ) 2.0, 8.0), 7.53 (H-6, ddd, J ) 2.0,

4.5, 8.5), 7.10 (H-5, dd, J ) 8.5, 11.0), 4.94 (1H, dd, J ) 7.5,

10.5), 4.69 (1H, t, J ) 8.3), 4.59 (1H, dd, J ) 9.0, 10.5), 4.15

(2H, m), 3.83 (3H, s), 1.46 (3H, 7.0).

MHz, CDCl₃) δ 8.01-7.97 (H-2,6, m), 7.12 (H-5, t, J ) 9.0),

6.01-5.92 (CHdC, m), 5.16-5.09 (CdCH₂, m), 3.46 (ArCH₂,

d, J ) 6.0).

Rep r esen ta tive P r oced u r es for Solu tion -P h a se Syn -

th eses of 2-P h en yloxa zolin e Hyd r oxa m ic Acid s (Sch em e

4). Meth od A. N-(3-Eth oxy-4-flu or oben zoyl)-^D-ser in e Meth -

yl Ester (3, Ar ) 3-OEt-4-F -p h en yl). To a solution of

3-ethoxy-4-fluorobenzoic acid (177 mg, 0.961 mmol), d-serine

methyl ester hydrochloride (179 mg, 1.15 mmol), HATU (420

mg, 1.10 mmol), and HOAt (131 mg, 0.962 mmol) in DMF (3

mL) at 0 °C was added DIEA (370 µL, 2.2 mmol), and the

solution was stirred for 16 h, slowly warming to ambient

temperature. The reaction mixture was diluted with ethyl

acetate (75 mL) and washed with 1 M HCl (2 × 20 mL),

saturated NaHCO₃(2 × 20 mL), and H₂O (4 × 20 mL). The

organic layer was dried over MgSO₄, filtered, and concentrated

in vacuo to afford 245 mg (89%) of a pale-yellow oil, which

slowly solidified to give a white solid: mp 111-112°C; ¹H NMR

(500 MHz, CDCl₃) δ 7.52 (H-2, dd, J ) 2.0, 8.0), 7.32 (H-6,

ddd, J ) 2.0, 4.0, 8.5), 7.12 (H-5, dd, J ) 8.5, 10.5), 7.02 (NH,

d, J ) 6.5), 4.86 (1H, dt, J ) 3.5, 7.0), 4.17 (2H, q, J ) 7.0),

4.12-4.03 (2H, m), 3.84 (3H, s), 2.39 (OH, t, J ) 6.0), 1.47

(3H, t, J ) 7.0).

Meth od B. N-[3-Flu or o-4-(3′-tr iflu or om eth ylben zyloxy)-

ben zoyl]-D-ser in e Meth yl Ester [3, Ar ) 3-F , 4-(3′-Tr if-

lu or om eth ylben zyloxy)p h en yl]. To a solution of 4-(3′-

trifluoromethylbenzyloxy)-3-fluorobenzoic acid (200 mg, 0.63

mmol) in 4 mL of DMF was added, sequentially, ^D-serine

methyl ester HCl (90 mg, 0.58 mmol), EDCI (120 mg, 0.63

mmol), HOAt (77 mg, 0.57 mmol), and DIEA (0.23 mL, 13.3

mmol). The mixture was stirred at room temperature for 16

h, then diluted with ethyl acetate (15 mL) and washed

sequentially with 1 M HCl (3 × 5 mL), saturated NaCO₃(3 ×

5 mL), water (5 mL), and brine (5 mL). The combined organic

extracts were dried (MgSO₄) and filtered, and the solvent was

evaporated to give 210 mg (79%) of the title ester as an oil

that required no further purification: ¹H NMR (500 MHz,

CDCl₃) δ 7.72 (1H, s), 7.62-7.67 (2CH, NH), 7.59 (1H, ddd),

7.55 (1H, t), 7.04 (2H, ∼t), 5.25 (2H, s), 4.87 (HR, dt, J ) 7.1,

3.6), 4.08 (AB of ABX, ∆δ_AB) 0.042, J _AB) 11.2), 3.84 (3H, s).

Meth od F (Illu str a ted w ith th e Syn th esis of Hyd r ox-

a m ic Acid s 38 a n d 44). 2-(3-P r op yl-4-flu or op h en yl)-4,5-

d ih yd r ooxa zole-4-ca r boxylic Acid Hyd r oxa m ic Acid (38).

To a solution of hydroxylamine hydrochloride (138 mg, 1.99

mmol) in anhydrous MeOH (2.0 mL) at 0 °C was added sodium

methoxide (737 mg, 25 wt % in MeOH, 3.4 mmol). Methyl ester

4 (Ar ) 3-Pr-4-F-phenyl, 471 mg, 1.78 mmol) in MeOH (2.0

mL) was added, and the solution was stirred at 0 °C for 5 h.

The reaction mixture was quenched with H₂O (4 mL) and 1

M HCl (5.0 mL), cooled to 0 °C, and filtered to give 343 mg

(73%) of partially racemized 38 as a white solid: mp 163-

165°C; CD [θ]₂₄₆) -10 800 (74% ee); MS, m/z 267 (M + H)⁺;

¹H NMR (500 MHz, DMSO-d₆) δ 10.87 and 9.01 (CONHOH, 2

s), 7.82 (H-2, dd, J ) 2.0, 7.5), 7.78-7.74 (H-6, m), 7.26 (H-5,

t, J ) 9.0), 4.66 (1H, dd, J ) 8.0, 9.5), 4.55 (1H, t, J ) 8.5),

4.48 (1H, t, J ) 8.0), 2.63 (2H, t, J ) 7.0), 1.58 (2H, sextet, J

) 7.0), 0.90 (3H, t, J ) 7.0); ¹³C NMR (125.7 MHz, DMSO-d₆)

δ 167.08 (CONHOH), 163.39/66.83/69.75 (oxaz-C2,4,5), 162.61

1

3

2

(C4, d, J ) 248.9), 130.95 (C2, d, J ) 5.0), 129.20 (C3, d, J

) 17.6), 128.11 (C6, d, ³J ) 8.8), 123.31 (C1), 115.63 (C5, d, ²J

) 23.9), 30.05 (C1′), 22.73 (C2′), 13.51 (C3′); ¹⁹F NMR δ

-113.18.

In like manner, 142 mg of ester 4 (Ar ) 3-EtO-4-F-phenyl)

afforded 61 mg (43%) of 44 as a white solid: mp 160-162 °C;

¹H NMR (500 MHz, DMSO-d₆) δ 10.82 and 9.01 (CONHOH),

7.60 (H-2, dd, J ) 2.0, 8.0), 7.46 (H-6, ddd, J ) 2.0, 4.5, 8.5),

7.33 (H-5, dd, J ) 8.5, 11.0), 4.68 (1H, dd, J ) 7.5, 10.0), 4.56

(1H, dd, J ) 8.5, 10.0), 4.49 (1H, t, J ) 8.0), 4.15 (2H, q, J )

7.0), 1.37 (3H, t, J ) 7.0); MS, m/z 269.0948 (M + H)⁺, calcd

for C₁₂H₁₄FN₂O₄, 269.0938.

In a subsequent experiment, the optically pure R enanti-

omer, (-)-38, was prepared from 14 (R′ ) allyl) using the same

cyclization method but installing the hydroxamate function

by method D with hydrogenation of the allyl group occurring

during the hydrogenolysis of the benzyloxyamide: CD [θ]₂₄₆

) -14 600; MS, m/z 267.1146 (M + H)⁺, calcd for C₁₃H₁₆FN₂O₃,

Meth od C. N-[3-P r opyl-4-flu or oben zoyl]-^D-ser in e Meth -

yl Ester (3, Ar ) 3-P r -4-F -p h en yl). To a suspension of

14(R′ ) Pr) (364 mg, 2.00 mmol) in CH₂Cl₂(4 mL) at 0 °C was

3124 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14

Kline et al.

267.1145. NMR spectra matched those previously recorded for

the partially racemized sample.

Gen er a l P r oced u r es for th e Solu tion -P h a se P a r a llel

Syn th esis of Oxa zolin e Hyd r oxa m ic Acid s. All of the

methods shown in Scheme 4 were amenable to parallel

synthesis. The following examples are illustrative.

dropwise to each reaction vessel, and the solutions were

agitated at 5 °C for 30 min and then warmed to ambient

temperature for 14 h. Each reaction mixture was drained,

filtered through silica gel (1 g) eluting with CH₂Cl₂/THF (7:

1), and concentrated under a stream of nitrogen to a volume

of 5 mL. The crude acylserines 3 were dispensed into fresh

reaction vessels, returned to the Quest, and cyclized using the

standard DAST procedure. Crude oxazoline methyl esters 4

were dissolved in MeOH (3 mL), cooled to 3 °C, treated

sequentially with hydroxylamine hydrochloride (104 mg, 1.5

mmol), and sodium methoxide (1.4 mL, 2.05 M, 2.9 mmol) in

MeOH, and agitated at 3 °C for 14 h. Water (2.5 mL) and 1 M

HCl (2 mL) were added, and the resulting precipitates were

agitated for 20 min. The solutions were drained, leaving the

crude hydroxamic acids in the reaction vessels. The hydrox-

amic acids were washed with H₂O (5 mL) and Et₂O (7 mL),

dried under high vacuum, and used without further purifica-

tion.

Note that the use of oxalyl chloride in the coupling reaction

also resulted in introduction of a chlorine at the C-3 position

of the indole ring in compound 64.

2-Ar yloxa zolin e-4-h yd r oxa m ic Acid s. All solution-phase

library compounds were characterized by, at a minimum,

proton NMR and mass spectrometry. Purity was determined

by reverse-phase HPLC (g90% unless otherwise indicated) or

thin-layer chromatography (single spot in two systems, unless

otherwise indicated). Analytical data for selected compounds

are given below.

The 2-aryloxazoline-2-hydroxamic acids and N-benzoylserine

hydroxamic acids produced displayed nearly invariant ¹H

NMR (500 MHz) data in DMSO-d₆. For the oxazolines the

NCHCH₂O unit appears as a dd (J ) 10, 8.5 Hz) at 4.69 (

0.03 and an AB pattern centered at 4.54 ( 0.04 (J _AB) 9 Hz,

∆δ_AB) 0.078 ( 0.015 ppm), and the CONHOH function is

characterized by singlets at 9.03 ( 0.03 and 10.84 ( 0.04 ppm.

In the N-benzoylserines, the characterizing pattern for the

NCRH-CâH₂OH unit was 4.96 (OH, t), ∼4.42 (HR, dt, J ) 7,

3.5 Hz), and 3.67 ppm (âCH₂, AB of ABX, J _AB) 11 Hz) with

the CONHOH function signals slightly upfield (10.69 for the

NH, 8.84 for the OH) relative to the values for the oxazoline

series. These signals are not reported in detail for the

compounds listed below when the spectra were recorded in

DMSO-d₆at 11.4 T; when this is the case, the solvent and

reference frequency are not listed. The specific methods used

for each hydroxamic acid are listed.

Meth od s A a n d E. Individual aromatic carboxylic acids (2.2

mmol acid) were dispensed into 10 mL reaction vessels, and

to each was added 2.4 mL of a DMF solution that was 0.83 M

in ^D-serine methyl ester HCl and 0.92 M in HOAt (or HOBt).

The reaction vessels were positioned on the Quest, flushed with

nitrogen, and cooled to 4 °C, and to each was added, with

gentle agitation, a 0.55 M solution of HATU in DMF (4 mL)

followed by DIEA (4.4 mmol, 766 µL). The reaction vessels

were brought to ambient temperature and agitated under a

nitrogen atmosphere for 16 h. Each reaction vessel was drained

into a vial, the volatiles were removed in vacuo, and the

residues were dissolved in ethyl acetate and individually

washed successively with 0.5 N HCl, saturated NaHCO₃, and

saturated brine. Each ethyl acetate solution was dried and

concentrated in vacuo to give the individual intermediates 3.

The crude acylserines 3 were dissolved in CH₂Cl₂(3 mL) and

returned to fresh reaction vessels on the Quest. The reaction

block was cooled to -7 °C, and 1.1 equiv of a 2 M solution of

DAST in CH₂Cl₂was added to each reaction vessel with gentle

agitation. After 30 min of agitation, a second portion of 1.1

equiv of the DAST solution was added, and the reaction

mixture was allowed to proceed for an additional 30 min. A

solution of saturated NaHCO₃was added to quench the

reaction, and the block was warmed to ambient temperature.

The organic phases were drained, dried over Na₂SO₄, and

concentrated in vacuo to give the individual oxazoline methyl

esters 4.

Each ester 4 was dissolved in 1 mL of anhydrous benzene

and returned to fresh reaction vessels. To each was added, with

gentle agitation, 1.25 equiv of a 1 M benzene solution of

dimethyl(p-anisyloxyamino)aluminum chloride (prepared from

anisyloxyamine HCl and trimethylaluminum according to ref

7b). The reaction block was heated to 50 °C for 5 h, then cooled

to ambient temperature. Each reaction vessel was treated with

water (100 equiv per mmol of aluminum complex), agitated

for 10 min, diluted with ethyl acetate, and filtered through a

bed of Celite to remove the aluminum salts. The organic phases

were separated, dried over Na₂SO₄, and concentrated in vacuo

to give the individual oxazoline carboxylic acid p-anisyloxya-

mides, which were purified by radial chromatography using

step gradients of 30-100% ethyl acetate in hexane as eluant.

Each was treated with 1.6 mL of 30% TFA in CH₂Cl₂at

ambient temperature for 90 min and concentrated in vacuo,

and the residue was triturated with cold diethyl ether to

precipitate the hydroxamic acid. Purification of the crude

hydroxamic acid was performed on preparative thin-layer

chromatography plates (3:97 MeOH/CH₂Cl₂as eluant).

Meth od B. To a solution of aromatic acid (1 mmol) in 4 mL

of solvent (CH₂Cl₂with DMF added as required for solubility

or DMF alone) was added sequentially serine methyl ester HCl

(0.98 mmol), EDCI (1 mmol), HOBt (0.95 mmol), and DIEA

(2.25 mmol). The reaction mixture was stirred (or agitated)

at room temperature for 16 h. The solution was then diluted

with ethyl acetate and washed sequentially with 1 M HCl,

saturated sodium bicarbonate, water, and brine. The organic

layer was dried over magnesium sulfate and concentrated to

give acylserines 3 in yields and purity comparable to those of

the product via method A.

Meth od s C a n d F (w ith P a r tia l Ra cem iza tion a t Cr,

Oxa zolin e-C₄). Individual carboxylic acids (1.5 mmol) were

dispensed into 10 mL reaction vessels and dissolved in CH₂-

Cl₂(3 mL). If necessary, THF (1-2 mL) was added for complete

dissolution of the acid. The reaction vessels were purged with

nitrogen, cooled to 5 °C, and treated with oxalyl chloride (140

µL, 1.60 mmol) and catalyst DMF (5 µL). The reaction vessels

were agitated at 5 °C for 30 min, warmed to 20 °C for 1 h, and

recooled to 5 °C. A solution (2.0 mL) of ^D-serine methyl ester

hydrochloride (0.79 M) and DIEA (2.2 M) in CH₂Cl₂was added

4-Meth oxy-3-tr iflu or om eth oxyp h en yl (20) (m eth od s C

a n d F ): mp 165-166 °C; CD [θ]₂₅₅) -3300 (30% ee); ¹H NMR

δ 9.01 (s), 7.87 (H-6, dd, J ) 1.5, 8.5), 7.79 (H-2), 7.36 (H-5, d,

J ) 8.5), 4.67/4.56/4.485 (CHCH₂), 3.93 (3H, s); ¹³C NMR δ

167.05 (CONHOH), 162.77/66.80/69.94 (oxaz-C2,4,5), 162.77

(CONHOH), 154.24 (C4), 136.52 (C3), 128.82 (C6), 122.24 (C2),

1

120.25 (OCF₃, q, J ) 256.4), 119.57 (C1), 113.83 (C5), 56.52

(OCH₃); ¹⁹F NMR δ -59.43; MS, m/z 321.1 (M + H)⁺, 343.0

(M + Na)⁺.

4-(3′-F lu or oben zyloxy)-3-tr iflu or om eth oxyp h en yl (21)

(m eth od s C a n d F ): CD [θ]₂₅₇) -4600 (40% ee); ¹H NMR δ

10.9 and 9.01 (CONHOH), 7.87 (H-6, dd, J ) 1.5, 8.5), 7.82

(H-2, s), 7.47 (H-5′, td, J ) 8.2, 5.5), 7.43 (H-5 or -6′, d, J )

8.5), 7.29 (H-6′ or -5, d, J ) 8.5), 7.27 (H-2′, ∼d), 7.20 (H-4′,

∼td, J ) 9.0, 1.5), 5.33 (2H, s), 4.67/4.57/4.48 (CHCH₂); ¹³C

NMR δ 166.11 (CONHOH), 161.81/65.88/69.06 (oxaz-C2,4,5),

1

161.28 (C3′, d, J ) 244.2), 152.12 (C4), 137.93 (C1′, d, ³J )

6.8), 135.80 (C3), 129.77 (C5′, d, ³J ) 8.4), 127.87 (C6), 122.45

1

(C6′), 121,70 (C2), 119.33 (OCF₃, q, J ) 256.8), 119.06 (C1),

114.06 (C2′, d, ²J ) 21.2), 114.03 (C5), 113.16 (C4′, d, ²J )

22.0), 68.48 (CH₂); MS, m/z 415 (M + H)⁺, 437.0732 (M + Na)⁺,

437.0737 calcd for C₁₈H₁₄F₄N₂O₅Na.

4-(Bu t-3′-en yloxy)-3-tr iflu or om eth oxyph en y (22) (m eth -

od s C a n d F ): mp 124-126 °C; CD [θ]₂₆₀) -4320 (35% ee);

¹H NMR δ 9.01 (s), 7.85 (H-6, dd, J ) 2.0, 8.5), 7.78 (H-2, s),

7.37 (H-5, d, J ) 8.5), 5.91-5.83 (1H, m), 5.17 (1H, dd, J )

1.5, 17.0), 5.09 (1H, d, J ) 10.5), 4.67/4.56/4.48 (CHCH₂), 4.20

(2H, t, J ) 6.5), 2.53-2.50 (2H, m); ¹³C NMR δ 166.12

(CONHOH), 161.84/65.85/68.99 (oxaz-C2,4,5), 152.62 (C4),

LpxC Inhibitors

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14 3125

1

135.65 (C3), 133.32 (C3′), 127.86 (C6), 119.29 (OCF₃, q, J )

256.9), 121.56 (C2), 118.55 (C1), 116.40 (C4′), 113.54 (C5),

67.05 (C1′), 31.86 (C2′); ¹⁹F NMR δ -59.36; MS, m/z 361 (M +

H)⁺, 383.0827 (M + Na)⁺, 383.0831 calcd for C₁₅H₁₅F₃N₂O₅-

Na.

δ 166.83 (CONHOH), 162.20/66.99/70.46 (oxaz-C2,4,5), 160.74

(C4, d, ¹J ) 258.9), 135.03 (C6, d, ³J ) 10.1), 127.11 (C2),

124.06 (C1), 122.19 (CF₃, q, ¹J ) 272.7), 118.15 (C5, d, ²J )

21.4), 117.15 (C3, dq, J _C3-CF3) 32.7, J _C3-CF) 13.8); ¹⁹F NMR

δ -62.65 (CF₃), -109.99 (F); MS, m/z 293.0 (M + H)⁺.

4-F lu or o-3-m eth ylp h en yl (37) (m eth od s C a n d F ): mp

3-Tr iflu or om eth oxy-4-a llyloxyp h en yl ((()-23) (m eth -

od s A a n d F ): MS, m/z 347 (M + H)⁺; 400 MHz NMR

spectrum was identical with the spectrum of (+)-23, prepared

for comparison (methods B and E). (+)-23: ¹H NMR δ 10.83

and 9.01 (CONHOH), 7.855 (H-6, dd, J ) 2, 8.5), 7.80 (H-2, s),

7.355 (H-5, d, J ) 8.5), 6.045 (dCH, m), 5.42 (dCH₂, 1H, dd,

J ) 17, 1), 5.34 (dCH₂, 1H, dd, J ) 10, 1), 4.765 (OCH_2′, d, J

) 5), 4.67/4.57/4.49 (CHRCH₂â); MS, m/z 346.9 (M + H)⁺. The

1

169-171°C; H NMR δ 9.01 (OH, s), 7.84 (H-2, bd, J ) 6.5),

7.74 (H-6, ddd), 7.25 (H-5, ∼t, J ) 9.5), 4.66/4.55/4.48 (CHCH₂),

2.28 (3H, s); ¹³C NMR δ 167.08 (CONHOH), 163.36/66.82/69.73

1

(oxaz-C2,4,5), 163.36 (CONHOH), 162.74 (C4, d, J ) 247.6),

3

131.70, (C2, d, J ) 5.0), 127.97 (C6, d, J ) 8.8), 124.92 (C3,

d, ²J ) 17.6), 123.23 (C1), 115.40 (C5, d, ²J ) 22.6), 14.07

(CH₃); ¹⁹F NMR δ -111.70; MS, m/z 221, 239.0 (M + H)⁺, 261.0

(M + Na)⁺.

S enantiomer displayed a positive CD extremum ([θ]₂₅₈

+16 100) in analogy to (+)-5 (the S isomer of 5) and an IC₅₀of

)

Ra cem ic 3-F lu or o-4-a llyloxyp h en yl (39) (m eth od s A

1

13 µM (versus 0.12 µM for (()-23).

a n d F ): H NMR (400 MHz, DMSO-d₆) δ 10.83 (1H, s), 9.02

(1H, s), 7.85 (1H, dd, J ) 8.7, 2.1), 7.80 (1H, s), 7.35 (1H, d, J

) 8.8), 6.08-6.01 (1H, m), 5.42 (1H, dd, J ) 17.24, 1.7), 5.31

(1H, dd, J ) 10.6, 1.6), 4.77-4.46 (5H, m); MS, m/z 346.8 (M

+ H)⁺.

3-Tr iflu or om eth oxy-4-p r en yloxyp h en yl ((()-24) (m eth -

od s A/F ): ¹H NMR δ 10.8 and 9.02 (CONHOH), 7.86 (H-6, dd,

J ) 2, 8.5), 7.78 (H-2, s), 7.34 (H-5, d, J ) 8.5), 5.46 (dCH, t,

J ) 6.5), 4.73 (OCH_2′, d, J ) 6), 4.67/4.57/4.49 (CHRCH₂â),

1.80 (3H, s), 1.76 (3H, s); MS, m/z 375 (M + H)⁺.

4-(3′-Tr iflu or om et h ylben zyloxy)-3-flu or op h en yl (40)

1

(m eth od s B a n d F ): mp 173-175 °C; H NMR δ 10.81 and

3-Ch lor o-4-t r iflu or om et h oxyp h en yl (27) (m et h od s C

a n d F ): ¹H NMR δ 9.034 (CONHOH, d, J ) 1), 8.135 (H-2, d,

J ) 2), 7.96 (H-6, dd, J ) 8, 2), 7.715 (H-5, dq, J ) 8, 1), 4.736

(dd, J ) 10.3, 7.7), 4.622 (∼t), 4.529 (∼t); ¹³C NMR δ 166.77

(CONHOH), 162.11/66.77/67.0/70.42 (oxaz-C2,4,5), 146.29 (C4),

130.46 (C2), 128.76 (C6), 127.55 (C3), 126.30 (C1), 123.16 (C5),

119.95 (OCF₃, q, ¹J ) 258.9); ¹⁹F NMR δ -58.72; MS, m/z 325.0

and 327.0 (M + H)⁺, 346.9 and 349.0 (M + Na)⁺.

9.01 (CONHOH), 7.85 (H-2′, s), 7.79 and 7.74 (H-6, H-4′, d’s),

7.68-7.71 (2H, m), 7.39 (H-5, t), 5.37 (CH₂, s), 4.66/4.55/4.47

(CHCH₂); MS, m/z 398.8 (M + H)⁺, 420.7 (M + Na)⁺, 397.5

(M^•)⁺, and 795.5 (dimer, M^•)⁺.

4-(2′-Tr iflu or om eth oxyben zyloxy)-3-flu or op h en yl (41)

1

(m eth od s A a n d F ): mp >200°C; H NMR δ 10.81 and 9.01

(CONHOH), 7.73-7.68 (H-2, 6, 6′, m), 7.55 (1H, ∼t), 7.50-

7.42 (3H, m), 5.30 (CH₂, s), 4.67/4.56/4.47 (CHCH₂); MS, m/z.

415.0 (M + H)⁺, 437.0 (M + Na)⁺.

4-Bu tylp h en yl (29) (m eth od s A a n d E): ¹H NMR (400

MHz, CDCl₃, CD₃OD) δ 7.85 (2H, d, J ) 8.1), 7.26 (2H, d, J )

8.1), 4.79 (1H, t, J ) 9.0), 4.64 (2H, q, J ) 8.6), 2.67 (2H, t, J

) 7.6), 1.66-1.58 (2H, m), 1.39-1.34 (2H, m), 0.94 (3H, t, J )

7.3); MS, m/z 263.1 (M + H)⁺.

5-Br om o-4-m eth oxy-3-flu or oph en yl (42) (m eth ods B/F):

1

mp 178-180°C; H NMR δ 10.81 and 9.02 (CONHOH), 7.92

(H-6, s), 7.74 (H-2, d, J ) 11.4), 4.70/4.59/4.50 (CHCH₂), 3.97

(3H, s); MS, m/z 333.0 and 335.0 (M + H)⁺, 331 and 333 (M -

H)^-.

4-(Hep t-1-yloxy)p h en yl (30) (m eth od s A/E): ¹H NMR

(400 MHz, CDCl₃) δ 7.87 (2H, d, J ) 8.7), 6.93 (2H, d, J )

8.7), 4.81 (1H, dd, J ) 9.5, 8.9), 4.69-4.64 (2H, m), 4.02 (2H,

t, J ) 6.4), 1.82-1.77 (2H, m), 1.48-1.43 (2H, m), 1.39-1.31

(6H, m), 0.90 (3H, t, J ) 6.4); MS, m/z 321.1 (M + H)⁺.

4-(Bu t-3-en yloxy)p h en yl (31) (m eth od s A a n d D): ¹H

NMR (400 MHz, CD₃OD) δ 7.89 (2H, d, J ) 8.6), 6.96 (2H, d,

J ) 8.6), 5.97-5.86 (1H, m), 5.17 (1H, dd, J ) 18.8, 1.6), 5.10

(1H, dd, J ) 10.3, 1.4), 4.80-4.56 (3H, m), 4.09 (2H, t, J )

6.6), 2.56 (2H, q, J ) 5.0); MS, m/z 277.0 (M + H)⁺.

4′-P r op ylbip h en -4-yl (32) (m eth od s A a n d E): ¹H NMR

(400 MHz, DMSO-d₆) δ 10.86 (1H, s), 9.03 (1H, s), 7.95 (2H,

d, J ) 8.4), 7.78 (2H, d, J ) 8.4), 7.65 (2H, d, J ) 8.2), 7.31

(2H, d, J ) 8.2), 4.69 (1H, dd, J ) 9.9, 7.8), 4.60-4.48 (2H,

m), 2.60 (2H, t, J ) 7.4), 1.67-1.58 (2H, m), 0.92 (3H, t, J )

7.3); MS, m/z 325.1 (M + H)⁺.

3-F lu or o-4-m eth oxy-5-a llyl-6-m eth ylp h en yl (43) (m eth -

od s C a n d F ): ¹H NMR δ 9.01 (CONHOH), 7.462 (H-2, s),

5.90 (dCH, m), 5.01 (dCH₂, 1H, dd, J ) 10, 1), 4.84 (dCH₂,

1H, dd, J ) 17, 1), 4.69/4.50/4.41 (CHCH₂), 3.865 (3H, d, J )

1), 3.45 (CH₂, d, J ) 5.5), 2.39, (Me, s); MS, m/z. 309.1 (M +

H)⁺, 331.1 (M + Na)⁺.

4-F lu or o-3-br om op h en yl (45) (m eth od s C a n d F ): mp

1

171-172 °C; H NMR δ 9.02 (d, J ) 1.0), 8.18 (H-2, dd, J )

2.0, 6.5), 7.92 (H-6, ddd, J ) 2.0, 5.0, 8.5), 7.52 (H-5, ∼t, J )

8.5), 4.71/4.595/4.505 (CHCH₂); MS, m/z 285, 287, 303.0, and

304.9 (M + H)⁺, 325.0 and 327.0 (M + Na)⁺.

5-F lu or o-3-tr iflu or om eth ylp h en yl (46) (m eth od s C a n d

F ): mp 164-166°C; ¹H NMR δ 9.05 (s), 8.025 (H-2, s), 7.98

(H-4, d, J ) 8.0), 7.96 (H-6, d, J ) 9.0), 4.775/4.66/4.56

(CHCH₂); MS, m/z 275, 293.0 (M + H)⁺.

4-(3′-Nitr oben zyloxy)p h en yl (33) (m eth od s B a n d F ):

3-Tr iflu or om eth ylp h en yl (47) (m eth od s A a n d E): ¹H

NMR (400 MHz, Me₂CO-d₆) δ 8.47 (H_Ar, s), 8.42 (H_Ar, d, J )

8.1), 8.11 (H_Ar, d, J ) 7.6), 7.96 (H_Ar, t, J ) 8.0), 5.08-5.1 (CR,

m), 4.85-4.91 (CH₂â, AB of ABX); MS, m/z 275.0 (M + H)⁺.

3-Tr iflu or om et h yl-4-a llyloxyp h en yl (48) (m et h od s C

a n d F ): ¹H NMR δ 8.94 (1H, d), 8.10 (H-2,6, m), 7.38 (H-5, d,

J ) 9.5), 6.04 (1H, m), 5.43 (1H, dd, J ) 17.5, 1.5), 5.30 (1H,

dd, J ) 9, 1.5), 4.81 (CH₂, s), 4.69/4.59/4.50 (CHCH₂); MS, m/z

331.1 (M + H)⁺, 353.1 (M + Na)⁺.

1

mp 178-179 °C; H NMR δ 10.81 and 9.00 (CONHOH), 8.34

(H-2′, s), 8.22 (H-4′, dd, J ) 1.5, 8.0), 7.93 (H-6′, d, J ) 8.0),

7.85 (H-2,6, d, J ) 8.5), 7.72 (H-5′, t, J ) 8.0), 7.15 (H-3,5, d,

J ) 8.5), 5.35 (2H, s), 4.64/4.53/4.46 (CHCH₂); MS, m/z 358.0

(M + H)⁺.

4-(3′-Nitr op h en oxym eth yl)p h en yl (34) (m eth od s C a n d

1

F ): H NMR δ 9.02 (CONHOH), 7.92 (H-2,6, d, J ) 8), 7.85-

7.77 (H-2′,4′, overlapped), 7.59 (H-3,5, d, J ) 8) overlapped

with 7.60 (H-5′, t), 7.51 (H-6′, dt), 5.34 (2H, s), 4.68/4.57/4.49

(CHCH₂); MS, m/z 358 (M + H)⁺, 380 (M + Na)⁺.

4-Tr iflu or om eth ylp h en yl (49) (m eth od s C a n d F ): mp

1

4-F lu or op h en yl (35) (m eth od s B a n d D): [R]²⁵-68.9°

194-195 °C; H NMR δ 9.04 (OH, d, J ) 1.5), 8.09 (H-3,5, d,

D

(c 0.16, CH₃OH); CD [θ]₂₄₄) -20 200; ¹H NMR (400 MHz, CD₃-

OD) δ 7.85 (H-3,5, dd, J ) 8. 5), 7.05 (H-2,6, ∼t), 4.9-5.0 (HR,

m), 4.5-4.9 (CH₂â, AB of ABX); ¹³C NMR (100 MHz, CD₃OD)

δ 167.07 (CONHOH), 163.26/66.85/69.84 (oxaz-C2,4,5), 164.19

J ) 8.0), 7.88 (H-2,6, d, J ) 8.0), 4.735/4.62/4.54 (CHCH₂);

¹³C NMR δ 166.81 (CONHOH), 163.07/66.96/70.07 (oxaz-

2

C2,4,5), 163.07 (CONHOH), 131.62 (C4, q, J ) 31.4), 130.76

(C1), 128.92 (C2,6), 125.69 (C3,5), 123.88 (CF₃, q, ¹J ) 271.5);

¹⁹F NMR δ -63.92; MS, m/z 275.0 (M + H)⁺, 297.0 (M + Na)⁺.

6-Tr iflu or om eth ylp yr id -3-yl (50) (m eth od s C a n d F ):

¹H NMR δ 10.9 and 9.07 (CONHOH), 9.21 (H-2, bs), 8.50 (H-

5, d), 8.06 (H-4, d, J ) 8.0), 4.78/4.66/4.57 (CHCH₂); MS, m/z

276.0 (M + H)⁺, 298.0 (M + Na)⁺.

1

3

(C4, d, J ) 249.6), 130.70 (C2,6, d, J ) 9.3), 123.57 (C1, d,

⁴J ) 2.8), 115.83 (C3,5, d, ²J ) 22.0); ¹⁹F NMR δ -107.57; MS,

m/z 225.0675 (M + H)⁺, calcd for C₁₀H₁₀FN₂O₃225.0674. Anal.

found C 52.98, H 3.69, N 12.16; calcd for C₁₀H₉FN₂O₃, C 53.57,

H 4.05, N 12.50.

4-F lu or o-3-tr iflu or om eth ylp h en yl (36) (m eth od s C a n d

F ): mp 169-171°C; ¹H NMR δ 9.03 (OH, s), 8.23-8.21 (H-2,6,

m), 7.68 (H-5, ∼t, J ) 10), 4.74/4.63/4.54 (CHCH₂); ¹³C NMR

Ra cem ic 4-Nitr op h en yl (51) (m eth od s A a n d F ): ¹H

NMR (400 MHz, DMSO-d₆) δ 10.92 (NH, s), 9.07 (OH, s), 8.34

(2H, d, J ) 9.0), 8.14 (2H, d, J ) 9.0), 4.76 (1H, dd, J ) 10.0,

3126 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14

Kline et al.

7.8), 4.63 (1H, dd, J ) 10.0, 8.2), 4.56 (1H, dd, J ) 8.2, 7.8);

MS, m/z 251.9 (M + H)⁺.

Meth od G. To a solution of N-benzoyl-^D-serine methyl ester

3 (0.40 mmol) in methanol (2 mL) was added a solution of

lithium hydroxide (50 mg, 3 equiv) in water (1 mL). After being

stirred for 12 h at room temperature, the mixture was diluted

with water (5 mL), the aqueous mixture was washed with CH₂-

Cl₂(2 × 5 mL), and the acid (typically a white solid) was

isolated by extraction with CHCl₃(2 × 10 mL) after acidifica-

tion with 1 N HCl. The entire acid sample, in 3 mL of DMF,

was treated sequentially with 60 mg of O-benzylhydroxylamine‚

HCl (0.37 mmol), 80 mg of EDCI (0.41 mmol), 60 mg of HOAt

(0.40 mmol), and 0.15 mL of DIEA (0.86 mmol) and was then

stirred for 16 h at room temperature. The resulting solution

was diluted with 15 mL of EtOAc, washed with 1 N HCl (3 ×

3 mL), saturated aqueous NaHCO₃(3 × 3 mL), water, and

brine. The benzyloxyamide obtained by evaporating the dried

solution was hydrogenated at 1 atm in 5 mL of EtOH over 50

mg of 20% Pd(OH)₂for 6-16 h. Filtration of the catalyst

followed by concentration in vacuo afforded a residue that

crystallized upon trituration with ether (overall yield, 45-

65%). The following N-benzoylserine hydroxamates are rep-

resentative.

4-Dim eth yla m in op h en yl (52) (m eth od s A a n d F ): ¹H

NMR (400 MHz, CDCl₃) δ 7.85 (2H, d, J ) 9.0), 6.85(2H, d, J

) 9.0), 4.69/4.59/4.50 (CHCH₂), 3.00 (6H, s, N(CH₃)₂); MS, m/z

250.0 (M + H)⁺.

4-Hyd r oxyp h en yl (53) (m eth od s A a n d F ): ¹H NMR (400

MHz, CDCl₃/CD₃OD) δ 7.81 (2H, d, J ) 8.8), 6.85 (2H, d, J )

8.8), 4.75 (1H, dd, J ) 10.1, 8.1), 4.61-4.56 (2H, m); MS, m/z

223.0 (M + H)⁺.

Ra cem ic 4-Br om op h en yl (54) (m eth od s A a n d F ): ¹H

NMR (400 MHz, DMSO-d₆) δ 10.85 (1H, s), 9.02 (1H, s), 7.81,

(2H, d, J ) 8.6), 7.71 (2H, d, J ) 8.6), 4.68 (1H, dd, J ) 9.9,

7.7), 4.57 (1H, dd, J ) 9.9, 8.0), 4.49 (1H, dd, J ) 8.0, 7.7);

MS, m/z 284.8 (M + H)⁺.

Ra cem ic 4-Iod op h en yl (55) (m eth od s A a n d F ): ¹H NMR

(400 MHz, DMSO-d₆) δ 10.85 (1H, d, J ) 1.5), 9.02 (1H, d, J

) 1.5), 7.88 (2H, d, J ) 8.5), 7.65 (2H, d, J ) 8.5), 4.67 (1H,

dd, J 9.9, 7.7), 4.56 (1H, dd, J ) 9.9, 8.1), 4.48 (1H, dd, J )

8.1, 7.7); MS, m/z 332.8 (M + H)⁺.

1

4-Acetylp h en yl (56) (m eth od s A a n d F ): H NMR (400

4-F lu or o (67) (m eth od s B a n d F ): mp 178-180 °C; CD

MHz, DMSO-d₆) δ 10.89 (1H, s), 9.05 (1H, s), 8.06 (2H, d, J )

8.6), 8.01 (2H, d, J ) 8.6), 4.72 (1H, dd, J ) 10.0, 7.9), 4.60

(1H, dd, J ) 10.0, 8.2), 4.53 (1H, dd, J ) 8.2, 7.9), 2.62 (3H, s);

MS, m/z 249.0 (M + H)⁺.

1

[θ]₂₃₇) -4500; H NMR δ 10.71 and 8.82 (CONHOH), 8.32

(NH, d, J ) 7.7), 7.98 (H-2,6, dd, J ) 7.8, 5.7), 7.30 (H-3,5, dd,

J ) 7.8, 9.2), 4.96 (OH, t, J ) 5.6), 4.42 (H, ∼q, J ) 6.4), 3.68

(2H, AB of ABX); MS, m/z 243.0 (M + H)⁺, 265.0 (M + Na)⁺.

4-F lu or o-3-eth oxy (68) (m eth od s A a n d F ): mp 152-153

4-Aceta m id op h en y (57) (m eth od s A a n d F ): ¹H NMR

(400 MHz, DMSO-d₆) δ 10.81 (1H, d, J ) 1.4), 10.21 (1H, s),

9.00 (1H, d, J ) 1.4), 7.81 (2H, d, J ) 8.7), 7.68 (2H, d, J )

8.7), 4.63 (1H, dd, J ) 9.9, 7.8), 4.52 (1H, dd, J ) 9.9, 7.8),

4.45 (1H, dd, J ) 8.1, 7.8), 2.01 (3H, s); MS, m/z 264.0 (M +

H)⁺.

1

°C; H NMR δ 10.68 and 8.84 (CONHOH), 8.35 (NH, d, J )

8.0), 7.66 (H-2, dd, J ) 2.0, 8.5), 7.51 (H-6, ddd, J ) 2.0, 4.5,

8.5), 7.30 (H-5, dd, J ) 8.5, 11.0), 4.96 (OH, t, J ) 5.8), 4.42

(HR, q, J ) 6.5), 4.18 (2H, q, J ) 7.0), 3.67 (âCH₂, AB of ABX),

1.38 (3H, t, J ) 7.0); MS, m/z 309.0858 (M + Na)⁺, calcd for

4,5-Ben z-(1H)-p yr a zol-3-yl (60) (m eth od s A a n d D): ¹H

NMR (400 MHz, CD₃OD) δ 8.18 (1H, d, J ) 8.2), 7.60 (1H, d,

J ) 8.5), 7.45 (1H, t, J ) 7.2), 7.28 (1H, t, J ) 7.6), 4.92-4.81

(1H, m), 4.75-4.65 (2H, m); MS, m/z 246.9 (M + H)⁺.

1-Bu tyl-4,5-ben z-(1H)-p yr a zol-3-yl (61) (m eth od s A a n d

E): ¹H NMR (400 MHz, CDCl₃, CD₃OD) δ 8.16 (1H, d, J )

8.2), 7.50-7.43 (2H, m), 7.30 (1H, dt J ) 7.6, 1.3), 4.97 (1H,

dd, J ) 10.3, 8.6), 4.73 (2H, q, J ) 8.8), 4.46 (2H, t, J ) 7.1),

1.94 (2H, q, J ) 7.4), 1.38-1.30 (2H, m), 0.94 (3H, t, J ) 7.3);

MS, m/z 303.1 (M + H)⁺.

C

₁₂H₁₅FN₂O₅Na, 309.0863.

4-Tr iflu or om eth oxy (69) (m eth od s C a n d F ): mp 161-

162 °C; CD [θ]₂₃₈) -2600; ¹H NMR δ 10.73 and 8.84

CONHOH), 8.43 (NH, d), 8.02 (H-2,6, d, J ) 8), 7.49 (H-3,5,

d, J ) 8), 4.96 (OH, t), 4.42 (1HR, ∼q), 3.68 (2H, AB of ABX);

MS, m/z - 276.0 (M - NHOH)⁺, 309.0692 (M + H)⁺, calcd

C

₁₀H₁₂F₃N₂O₅, 309.0698, 331.0518, calcd C₁₀H₁₁F₃N₂O₅Na,

331.0518.

3-Tr iflu or om eth oxy-4-a llyloxy (70) (m eth od s A a n d F ):

mp 123-124 °C; CD [θ]₂₄₉) -7500; ¹H NMR δ 10.68 and 8.83

(CONHOH), 8.38 (NH, d, 8.0), 7.94 (H-6, d, J ) 8.5), 7.92 (H-

2, s), 7.32 (H-5, d, J ) 8.5), 6.04 (1H, m), 5.42 (1H, dd, J )

1.5, 17.5), 5.30 (1H, d, J ) 11.0), 4.94 (OH, t, J ) 5.8), 4.75

(2H, d, J ) 4.5), 4.41 (HR, q, J ) 7.0), ∼3.66 (âCH₂, AB of

ABX); MS, m/z 365 (M + H)⁺, 387 (M + Na)⁺.

1-Bu tyl-3,4-ben z-(1H)-p yr a zol-5-yl (62): ¹H NMR (400

MHz, DMSO-d₆) δ 10.91 (1H, s), 9.06 (1H, s), 8.18 (1H, d, J )

8.2), 7.80 (1H, d, J ) 8.6), 7.48 (1H, t, J ) 7.2), 7.29 (1H, t, J

) 7.5), 4.75 (1H, dd, J ) 9.9, 7.8), 4.56 (1H, dd, J ) 9.7, 8.1),

4.51-4.47 (3H, m), 1.87-1.80 (2H, m), 1.26-1.20 (2H, m), 0.88

(3H, t, J ) 7.4); MS, m/z 303.1 (M + H)⁺.

3-Tr iflu or om eth oxy-4-m eth oxy-5-p r op yl (71) (m eth od s

C a n d G): mp 139-141 °C; CD [θ]₂₄₅) -6350; ¹H NMR δ

10.69 and 8.85 (CONHOH), 8.45 (NH, d, 7.7), 7.84 and 7.76

(H6,-2), 4.96 (OH, t, 5.6), 4.42 (HR, ∼q, ∼6.4), 3.84(3H, s), 3.67

(âCH₂, AB of ABX), 2.64 (CH₂, ∼t), 1.61 (CH₂, ∼sextet), 0.94

(CH₃, t); MS, m/z 381.0 (M + H)⁺, 403.1 (M + Na)⁺.

Syn th esis of Oxa zin e Hyd r oxa m ic Acid s. O-ter t-Bu -

tyld im eth ylsilyl-^DL-h om oser in e Meth yl Ester 15. To a

solution of tert-butylmethylsilyl chloride (21.5 g, 143 mmol)

and DBU (20.5 mL, 137 mmol) in acetonitrile (40 mL) was

added ^DL-homoserine (4.9 g, 41 mmol) in portions over 30 min.

The solvent was removed in vacuo, and the residue was

extracted into diethyl ether, washed with water, and allowed

to stand. A white precipitate formed and was filtered to give

the silyl ether in a state suitable for the next reaction (5.3 g,

22.6 mmol, 55%): ¹H NMR (400 MHz, CD₃OD) δ 4.8 (NH₂, s),

3.8 (OCH₂, t), 3.6 (HR, m), 2.1-1.8 (CH_2′, m), 0.8 (C(CH₃)₃, s),

0.1 (Si(CH₃)₂, s); ¹³C NMR (100.6 MHz, CD₃OD) δ 173.88

(COOH), 62.05 (OCH₂), 54.92 (CH), 34.36 (CH₂), 26.40 ((CH₃)₃),

19.10 (SiC(CH₃)₃), -5.39 ((CH₃)₂Si); MS (APCI⁺), m/z 234 (M

+ H)⁺.

Ra cem ic 5-F lu or oin d ol-2-yl (63) (m eth od s A a n d F ): ¹H

NMR δ 10.80 and 9.10 (CONHOH), 7.37-7.40 (2H, m), 7.07-

7.08(1H, m), 6.95 (1H, s), 4.49-4.70 (3H, m); MS, m/z 264.0703

(M + H)⁺, calcd for C₁₂H₁₁FN₃O₃, 264.0785.

5-F lu or o-2-ch lor oin d ol-2-yl (64) (m eth od s C a n d F ): ¹H

NMR δ 10.90 and 9.10 (CONHOH), 7.47 (H-7, dd, J ) 4.5,

9.0), 7.32 (H-4, dd, J ) 2.0, 9.0), 7.19 (H-6, dt, J ) 2.5, 9.0),

4.71/4.62/4.53 (CHCH₂); MS, m/z 298.00 and 299.99 (M + H)⁺,

320.03 and 322.01 (M + Na)⁺.

Gen er a l P r oced u r es for th e Solu tion -P h a se P a r a llel

Syn th esis of N-Acylser in e Hyd r oxa m ic Acid s. Meth od

F . N-Benzoyl-^D-serine methyl ester 3 (0.4 mmol, in 4 mL

MeOH) from methods A, B, or C was added to an ice-cold

solution of hydroxylamine hydrochloride (0.42 mmol) in metha-

nol (2 mL) to which 80 µL of 25% methanolic NaOMe had been

added. An additional 60 µL portion of methoxide solution was

added, and the resulting cloudy solution was stirred for 4-10

h at 0 °C. The reaction mixture was then diluted with water

(3 mL) and washed with CH₂Cl₂(5 mL). The aqueous layer

was cooled to 0 °C and acidified (1 N HCl) to pH 2. At this

point, the desired hydroxamic acid precipitated out and was

collected by filtering the thick suspension. If no, or a modest

quantity of, precipitate formed, the hydroxamic acid was

obtained by CHCl₃extraction. In either case, the resulting solid

was washed with a limited amount of water and several

portions of ether to give a white solid.

To a solution of O-tert-butyldimethylsilyl-^DL-homoserine

(250 mg, 1.1 mmol) in methanol (3 mL) and benzene (10.5 mL)

was added trimethylsilyldiazomethane (2.7 mL, 5.4 mmol). The

mixture was stirred at ambient temperature overnight. The

solvents were removed in vacuo to give the methyl ester (280

mg, 1.1 mmol, 100%): ¹H NMR (400 MHz, CDCl₃) δ 3.8-3.7

LpxC Inhibitors

J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14 3127

(OCH₂, m), 3.7 (OCH₃, s), 3.6-3.55 (CH, m), 2.0-1.9 (0.5 CH₂,

m), 1.8 (NH₂, s), 1.7-1.65 (0.5 CH₂, m), 0.8 (C(CH₃)₃, s), 0.1

(Si(CH₃)₂, s); ¹³C NMR (100.6 MHz, CDCl₃) δ 175.95 (COOCH₃),

59.83 (OCH₂), 51.90 (CH), 36.57 (CH₂), 25.80 ((CH₃)₃), 18.17

(SiC(CH₃)₃); MS (APCI⁺), m/z 248 (M + H)⁺.

a closed system for 48 h, repeating the H₂S treatment at 24 h.

The volatiles were removed in vacuo, and the residue was

dissolved in anhydrous CH₂Cl₂(5 mL/mmol) and cooled to -10

°C. A freshly prepared 2 M solution of DAST in CH₂Cl₂(1.1

equiv) was added, and the reaction mixture was stirred or

agitated for 90 min at -10 °C, with a second 1.1 equiv of the

DAST solution being added at 45 min. The reaction was

quenched by addition of excess saturated aqueous NaHCO₃,

and the mixture was stirred or agitated for 30 min while

warming to ambient temperature. The organic phase was

separated and dried over Na₂SO₄, and the volatiles were

removed in vacuo to give the crude thiazoline methyl esters

18. The hydroxamic acids were prepared by direct conversion

of the methyl esters according to method E or F. The structure

of each thiazoline was determined by proton NMR and mass

spectrometry, and purity was g90% by reverse-phase HPLC.

2-p -Tolyl-4,5-d ih yd r ot h ia zole-4-ca r b oxylic a cid h y-

d r oxa m id e (76): ¹H NMR (400 MHz, CD₃OD) δ 7.55 (H-2,6,

d, J ) 8.1), 6.99 (H-3,5, d, J ) 8.1), 5.15 (HR, t, J ) 9.4) 3.40

(CâH₂, AB of ABX), 2.40 (CH₃, s); ¹³C NMR (100 MHz, CD₃-

OD) δ 173.60 (CONHOH), 170.31/78.93/36.15 (thiaz-C2,4,5),

143.76 (C4), 131.35 (C1), 130.29 (C3,5), 129.60 (C2,6), 21.48

(CH₃); MS, m/z 236.9 (M + H)⁺. Mass calcd for C₁₁H₁₃N₂O₂S,

237.0698; found 237.0751.

Gen er a l P r oced u r e for th e P r ep a r a tion of 2-Ar yl-4,5-

d ih yd r ooxa zin e-4-ca r boxylic Acid Hyd r oxa m id es 73-75.

Substituted benzoic acid (1.2 mmol), O-tert-butyldimethylsilyl-

DL-homoserine methyl ester (1 mmol), HATU (1.2 mmol), HOAt

(1.2 mmol), and DIEA (2.5 mmol) were dissolved in dry DMF

(5 mL). The resulting yellow-orange solution was stirred at

ambient temperature under N₂overnight. Ethyl acetate (15

mL) and water (10 mL) were added to the reaction mixture.

The organic layer was separated and washed with saturated

NaHCO₃solution (3 × 10 mL) and brine (10 mL), dried over

MgSO₄, and concentrated in vacuo to give a yellow oil. The

crude products were purified on a silica gel flash column (47-

73% yield, 5:6 EtOAc/hexanes as eluant).

To each of the resulting amides (0.5 mmol) dissolved in dry

CH₂Cl₂(2 mL) was added DAST (2 mmol), and the reaction

mixtures were stirred at ambient temperature under N₂for 4

h. Saturated NaHCO₃solution was added, and the products

were extracted into CH₂Cl₂(2 mL). In all cases, the organic

layer was washed with water, dried over MgSO_4,and concen-

trated in vacuo to give the crude products as yellow oils that

were purified on a silica gel flash column (35:65 EtOAc/

hexanes as eluant), affording the oxazine methyl esters 17

(54-77% yield).

To a 0 °C solution of hydroxylamine hydrochloride (0.3

mmol) in dry methanol (1 mL) was added sodium methoxide

(25% methanolic solution, 1.5 mmol), and the solution was

stirred at 0 °C under N₂. A solution of each oxazine methyl

ester 17 (0.3 mmol) in methanol (1 mL) was cooled at 0 °C

and added via syringe to the hydroxylamine solution. The

mixture was stirred at 0-5 °C under N₂overnight, then

acidified to pH 4.0 with dilute HCl. The precipitated products

were collected by filtration and dried under vacuum. The

structure of each oxazine was determined by proton NMR and

mass spectrometry, and purity was g90% by reverse-phase

HPLC.

2-(4-Bu t-3′-en yloxyp h en yl)-4,5-d ih yd r oth ia zole-4-ca r -

1

boxylic a cid h yd r oxa m id e (77): H NMR (400 MHz, CD₃-

OD) δ 7.90 (H-2,6, d, J ) 7.7), 7.05 (H-3,5, d, J ) 8.8), 5.99

(dCH, m), 5.03-5.27 (CH₂d, m over HR, m), 4.17 (OCH₂, t, J

) 6.6), 3.64-3.77 (CâH₂, AB of ABX), 2.60-2.63 (CH₂, m); MS,

m/z. 293.0 (M + H)⁺.

2-(4-F lu or op h en yl)-4,5-d ih yd r ot h ia zole-4-ca r b oxylic

a cid h yd r oxa m id e (78): ¹H NMR (400 MHz, DMSO-d₆) δ

7.90 (H-3,5, dd, J ) 5.5, 6.0), 7.34 (H-2,6, t, J ) 8.9), 5.10 (HR,

t, J ) 9.08), 3.58-3.71 (CâH₂, AB of ABX); ¹³C NMR (100 MHz,

DMSO) δ 166.87 (CONHOH), 166.62/77.54/35.13 (thiaz-

1

C2,4,5), 164.04 (C4, d, J ) 249.8), 130.80 (C2,6, d, J ) 9.0),

128.95 (C1), 115.85 (C3,5, d, J ) 22.0); HPLC t_R33.43 (97.9%);

MS, m/z 241.2 (M + H)⁺; HPLC t_R33.43 (97.9%). Mass calcd

for C₁₀H₁₀N₂O₂FS, 241.0447; found 241.0471

2-(4-Tr iflu or om eth oxyp h en yl)-4,5-d ih yd r oth ia zole-4-

ca r boxylic a cid h yd r oxa m id e (79): ¹H NMR (400 MHz,

DMSO-d₆) δ 10.81 and 9.06 (CONHOH), 7.97 (H-3, 5, d, J )

8.8), 7.49 (H-2,6, d, J ) 8.0), 5.14 (HR, dd, J ) 9.0, 9.2), 3.65

(CâH₂, m); ¹³C NMR (100 MHz, DMSO-d₆) δ 166.82 (CON-

HOH), 166.51/77.61/35.22 (thiaz-C2,4,5), 150.48 (C4), 131.37

(C1), 130.47 (C2,6), 121.09 (C3,5); MS, m/z 307.04 (M + H)⁺;

HPLC t_R37.76 (97.9%). Mass calcd for C₁₁H₁₀N₂O₃F₃S, 307.0364;

found 307.0350.

2-(4-Meth oxy-5-p r op yl-3-tr iflu or om eth oxyp h en yl)-4,5-

d ih yd r oth ia zole-4-ca r boxylic a cid h yd r oxa m id e (80): ¹H

NMR (400 MHz, CD₃OD) δ 7.52-7.60 (H-2,6, m), 5.18 (HR,

m), 3.79 (OCH₃, s), 3.73-3.78 (CâH₂, AB of ABX), 2.64-2.80

(ArCH₂, m,), 1.23-1.28 (CH₂, m), 0.98 (CH₃, t); MS, m/z 379

(M + H)⁺.

2-(5-Meth yl-1-p h en yl-1H-p yr a zol-4-yl)-4,5-d ih yd r oth ia -

zole-4-ca r boxylic a cid h yd r oxa m id e (81): ¹H NMR (400

MHz, CDCl₃) δ 7.38-7.89 (ArH, m), 5.18 (HR, m), 3.60-3.79

(CâH₂, AB of ABX), 2.61 (CH₃, s); MS, m/z. 303.0 (M + H)⁺.

Mass calcd for C₁₁H₁₀N₂O₃F₃S, 307.0364; found 307.0350.

P r ep a r a tion of [³H-CH₃CONH]-UDP -3-O-(R-3-h yd r oxy-

d eca n oyl) GlcNAc. The coding region of the lpxC gene was

amplified from P. aeruginosa genomic DNA using the poly-

merase chain reaction and cloned into a T7 expression vector,

pET21b (Novagen), using E. coli DH5R as host strain. Over-

expression was achieved by inducing with IPTG. The overex-

pressed protein was purified by DEAE-Sepharose chroma-

tography followed by phenyl-Sepharose and Q Sepharose

chromatography in the presence of EDTA. Activity of the

enzyme was restored by the addition of zinc, and the final step

of enzyme purification was achieved by passage over a Super-

dex 75 column.

2-(4-Met h ylp h en yl)-4,5-d ih yd r ooxa zin e-4-ca r b oxylic

a cid h yd r oxa m id e (73): (24% yield for the last step) ¹H NMR

(400 MHz, CDCl₃) δ 7.9 (Ar-H, d), 7.2 (Ar-H, d), 4.4-4.6

(CH₂, m), 4.3-4.2 (CH, m), 2.4 (CH₃, s), 2.0-2.3 (CH₂, m); ¹³

C

NMR (100.6 MHz, CD₃OD) δ 171.66 (CONHOH), 158.87/55.12/

25.65/65.42 (oxaz-C2,4,5,6), 142.50 (C4), 131.88 (C1), 129.72

(C3,5), 128.46 (C2,6), 21.42 (CH₃); MS, m/z 235.1082 (M + H)⁺

(calcd 235.1083), 257.0903 (M + Na)⁺(calcd 257.0902).

2-(3-Tr iflu or om et h yl-4-flu or op h en yl)-4,5-d ih yd r oox-

a zin e-4-ca r boxylic a cid h yd r oxa m id e (74): (18% yield for

1

the last step) H NMR (400 MHz, DMSO-d₆) δ 10.7 and 8.9

(CONHOH), 8.3 (Ar-H, m), 7.6 (Ar-H, t), 4.6-4.4 (CH₂, m),

4.2-4.1 (CH, m), 2.2-1.9 (CH₂, m); ¹³C NMR (100.6 MHz,

DMSO-d₆) δ 167.81 (CONHOH), 160.25 (C4, d, ¹J ) 257.6),

152.65/53.27/23.93/64.47 (oxaz-C2,4,5,6), 133.96 (C6, d, ³J )

3

1

10.1), 130.18 (C2, d, J ) 3.0), 126.11 (C1), 122.49 (CF₃, q, J

) 272.7), 117.25 (C5, d, ³J ) 21.1), 116.47 (C3, dq); MS

(APCI⁺), m/z 307.0683 (M + H)⁺(calcd 307.0706).

2-(5-F lu or oin d ol-2-yl)-4,5-d ih yd r ooxa zin e-4-ca r boxyl-

1

ic a cid h yd r oxa m id e (75): (12% yield for the last step) H

NMR (400 MHz, DMSO-d₆) δ 10.6 and 9.0 (CONHOH), 7.4

(Ar-H, m), 7.3 (Ar-H, d), 7.0 (Ar-H, t), 6.8 (Ar-H, s), 4.2-

4.3 (CH₂, m), 4.15-4.10 (CH, m), 2.2-1.7 (CH₂, m); ¹³C NMR

(100.6 MHz, DMSO-d₆) δ 167.67 (CONHOH), 157.04 (C5, d,

¹J ) 232.4), 150.09/53.11/ 24.46/64.72 (oxaz-C2,4,5,6), 133.31

3

(C7a), 133.0 (C2), 127.41 (C3a, d, J ) 11.1), 112.76 (C7, d, J

) 10.1), 111.77 (C6, d, ²J ) 26.2), 105.62 (C4, d, ²J ) 23.1),

102.73 (C3); MS (APCI⁺), m/z 278.1005 (M + H) (calcd

278.0941).

Gen er a l P r oced u r e for th e Syn th esis of Th ia zolin e

Hyd r oxa m ic Acid s. Oxazoline methyl esters 4 were dissolved

in 2:1 MeOH/NEt₃(15 mL/mmol), and the solution was

saturated with H₂S gas, then stirred (single synthesis) or

agitated (Quest parallel synthesis) at ambient temperature in

UDP-3-O-(R-3-hydroxydecanoyl)-GlcNAc (38 mg, 0.049 mmol)

was incubated with a purified preparation of P. aeruginosa

LpxC enzyme (3.3 mg, specific activity 500 nmol min^-1mg^-1

)

3128 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 14

Kline et al.

at 30 °C and pH 6.7 for 60 min with a further addition of

enzyme (1.5 mg) at 30 min. The reaction was stopped with

ice-cold ethanol to 80% final concentration, and the mixture

was then chilled on ice for 15 min and centrifuged at 15000g

for 15 min. The supernatant was filtered through 38 g of silica

previously equilibrated with ice-cold 80% aqueous EtOH, and

the filter cake was washed with an additional 100 mL of the

same eluant. The eluted glycolipid solution was concentrated

in vacuo to ca. 25 mL, diluted with 75 mL of ice-cold 0.05%

pH 5.6 NH₄OAc, and lyophilized. It was determined by

analytical HPLC that 58% conversion had occurred, with

minimal (<5%) rearrangement of the nascent amine. Purifica-

tion by preparative reverse-phase HPLC enabled the recovery

of both unconverted starting N-acetyl compound (eluting

between 20 and 25 min) and the primary amine product

(eluting between 26 and 34 min). The yield of purified amine

[MS, m/z 736.2 (M + H)⁺] was 15.1 mg (16%), and 9.2 mg of

N-acetyl starting material was recovered.

sodium phosphate buffer, pH 7.5, containing 1 mg/mL BSA.

Reactions were stopped by the addition of 180 µL of a 3%

suspension of activated charcoal powder in 100 mM sodium

acetate, pH 7.5. Supernatants were clarified by centrifugation.

A portion of the clarified supernatant, containing the enzy-

matically released [³H]-acetate, was transferred to opaque

white 96-well plates containing scintillation fluid. The radio-

activity was measured in a Perkin-Elmer/Wallac Trilux Mi-

crobeta counter. Control reactions to which 5 mM EDTA had

been added were included with each run to determine non-

specific tritium release.

Ack n ow led gm en t. The authors thank David Myles

and Susan Kaufman for their contributions to assay

development. We are grateful for the fine analytical

support from J oseph Therrien, Matthew Bailey, Mal-

colm Reider, and J ulie Bukowski and for informatics

and sample management from, respectively, Dennis

Kowalik and Tat Leung. We especially thank the Cystic

Fibrosis Foundation for the Therapeutics Discovery

Grant KLINE99X0 that provided financial support for

this work.

Analytical HPLC: 250 mm × 4.6 mm C18 Luna column,

100 Å, 5 µm column at 1 mL/min, with a linear gradient of

10% to 40% B in A over 40 min, A ) 0.05% NH₄OAc in water,

pH 5.6, B ) acetonitrile, monitoring λ ) 260 nm. Preparative

HPLC: 250 mm × 10 mm C18 Luna column, 5 µm column at

4.7 mL/min, with a linear gradient of 10% to 40% B in A over

40 min, A ) 0.05% NH₄OAc in water, pH 5.6, B ) acetonitrile,

monitoring λ ) 260 nm.

Su p p or tin g In for m a tion Ava ila ble: Chemical synthesis

and characterization details for representative benzoic acids

and their respective intermediates. This material is available

free of charge via the Internet at http://pubs.acs.org.

R ea cet yla t ion w it h N-([³H ]-Acet oxy)p h t h a lim id e.

Freshly prepared N-([³H]-acetoxy)phthalimide (50 µmol, ca. 4

equiv per equivalent of amine substrate, 20 Ci/mmol) was

dissolved in 600 µL of dry acetonitrile and was added to a

reaction vessel containing UDP-3-O-(R-3-hydroxydecanoyl)-

glucosamine (8.94 mg, 12 µmol) suspended in ca. 1 mL of dry

acetonitrile. To this suspension was added dropwise triethyl-

amine (10 equiv per equivalent of amine, 120 µmol, 12.1 mg,

17 µL) dissolved in 183 µL of dry acetonitrile. The reaction

mixture was flushed with dry nitrogen and warmed to 35 °C

overnight, during which time a strong yellow color developed.

Analytical HPLC indicated disappearance of the amine peak

at ca. 24 min and concomitant appearance of the N-acetylated

peak at ca. 20 min under the conditions described above. The

volatiles were removed in vacuo, and the residue was dissolved

in HPLC buffer A and purified directly by preparative HPLC,

with the product collected between 17 and 19 min: HPLC t_R

18.53 min (81.2 area %) for both ³H and UV₂₆₀detection,

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3

specific activity 14 Ci/mmol. Diagnostic peaks in the H NMR

1

spectra (with and without H decoupling) and H NMR spectra

revealed the product and an acyl migration byproduct; these

were analyzed by reference to 1D and 2D spectra of the cold

material. ³H NMR (D₂O, proton undecoupled) δ 1.90 (t) and

1

1.85 (t); H NMR (300 MHz, D₂O) δ 7.80 (H-6^U, d), 5.81 (H-

1′^U, d), 5.78 (H-5^U, d), 5.40 (H-1, dd, major), 5.33 (H-1, dd,

minor), 5.04 (H-3, t, major), 4.24-4.17 (H-2′^U+ H-6ab minor),

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C³-H), 3.69 (glc-C⁶H₂), 2.50 (CH₂CO, minor), 2.48 (CH₂CO,

major), 1.32 and 1.12 (methylenes), 0.7 (Me). The spectra

demonstrate the correct anomeric stereochemistry and the

correct regiochemistry for the hydroxydecanoyl group in the

major product.

Control experiments with the cold substrate in D₂O buffer

(pH* ) 6.3) following the slow acyl migration over several

weeks revealed that the glc-H-1 at 5.40 ppm correlated with

4.03 (H-2), 5.04 (H-3), 3.59 (H-4), 3.83 (H-5), and ∼3.69 ppm

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3.67 (H-2), 3.84 (H-3), 3.44 (H-4, t), 3.96 (H-5, dt), and 4.23

ppm (H-6ab). A minor peak at 4.85 ppm (H-4, t, minor)

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GlcNAc. These acyl migrations have been observed previously.^1a

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J M010579R

Article Doi

Potent, novel in vitro inhibitors of the Pseudomonas aeruginosa deacetylase LpxC

DOI: 10.1021/jm010579r

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Authors:

Article abstract of DOI:10.1021/jm010579r

Full text of DOI:10.1021/jm010579r

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