N-O chemistry for antibiotics: Discovery of N-alkyl-N-(pyridin-2-yl) hydroxylamine scaffolds as selective antibacterial agents using nitroso diels-alder and ene chemistry

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pubs.acs.org/jmc

NꢀO Chemistry for Antibiotics: Discovery of

N-Alkyl-N-(pyridin-2-yl)hydroxylamine Scaffolds as Selective

Antibacterial Agents Using Nitroso DielsꢀAlder and Ene Chemistry

Timothy A. Wencewicz, Baiyuan Yang, James R. Rudloﬀ, Allen G. Oliver, and Marvin J. Miller*

Department of Chemistry and Biochemistry, University of Notre Dame, 251 Nieuwland Science Hall, Notre Dame, Indiana 46556,

United States

S Supporting Information

b

ABSTRACT: The discovery, syntheses, and structureꢀactivity relationships (SAR) of

a new family of heterocyclic antibacterial compounds based on N-alkyl-N-(pyridin-2-

yl)hydroxylamine scaﬀolds are described. A structurally diverse library of ∼100

heterocyclic molecules generated from Lewis acid-mediated nucleophilic ring-opening

reactions with nitroso DielsꢀAlder cycloadducts and nitroso ene reactions with

substituted alkenes was evaluated in whole cell antibacterial assays. Compounds

containing the N-alkyl-N-(pyridin-2-yl)hydroxylamine structure demonstrated selec-

tive and potent antibacterial activity against the Gram-positive bacterium Micrococcus

luteus ATCC 10240 (MIC₉₀= 2.0 μM or 0.41 μg/mL) and moderate activity against

other Gram-positive strains including antibiotic resistant strains of Staphylococcus

aureus (MRSA) and Enterococcus faecalis (VRE). A new synthetic route to the active

core was developed using palladium-catalyzed BuchwaldꢀHartwig amination reac-

tions of N-alkyl-O-(4-methoxybenzyl)hydroxylamines with 2-halo-pyridines that fa-

cilitated SAR studies and revealed the simplest active structural fragment. This work shows the value of using a combination of

diversity-oriented synthesis (DOS) and parallel synthesis for identifying new antibacterial scaﬀolds.

’ INTRODUCTION

isolated from nature or chemically synthesized. Existing com-

pound libraries suﬀer from low structural novelty^5,10,11and often

favor compounds with physical properties suitable for accessing

eukaryotic cellular targets,⁵while prokaryotes require diﬀer-

ent physiochemical properties for cell penetration.^12,13Modern

medicinal chemistry techniques including fragment-based drug

discovery (FBDD),^14,15computational structure-based design

methods,¹⁶high-throughput screening (HTS),¹⁷and other target-

based genomic approaches have been validated for identifying

antibacterial compounds with new biological targets by the so-

called reverse chemical genetic approach.¹⁸Overall, these meth-

ods alone have proven diﬃcult to translate into well estab-

lished whole cell antibiotic susceptibility assays due primarily

to cell permeability issues. Additionally, most compound libraries

used for virtual and in vitro screening lack the structural diversity

for exploring unique chemical space.¹⁹

Traditional combinatorial library synthesis and HTS continue

to play an important role in drug discovery but have yielded

limited success in bringing new chemical entities, including

antibacterials, to market.²⁰This lack of success is often attributed

to the lack of structural diversity found in combinatorial compound

libraries.^19,20Diversity-oriented synthesis (DOS) has emerged as an

important strategy for producing structurally diverse and unbiased

The development of bacterial resistance to current antibacter-

ial chemotherapies is one of modern medicine’s most important

problems.^1ꢀ3The situation is confounded by a complex balance

of medical, ethical, social, political, and economic factors, all

resulting in a decline of antibiotic development and rise in

antibiotic resistance.⁴With only a few last resort drugs available,

such as vancomycin and linezolid, for treating highly resistant

Gram-positive bacterial infections, there is a desperate need for

new antibacterial agents based on new molecular scaﬀolds with

novel modes of action.⁵Historically, the antibiotic arms race has

been maintained through the diligent work of medicinal chemists

synthetically tailoring existing antibacterial scaﬀolds to keep one

step ahead of developing microbial resistance. The evolution of

β-lactam antibiotics into many useful generations is the best

example of this method.^5,6Modiﬁcation of existing scaﬀolds is still

the primary strategy of antibiotic resistance management em-

ployed today,⁷but time is running out, the antibiotic pipeline is

depleted, and a more long-term approach to managing antibiotic

resistance is essential to regaining our medicinal advantage.

One promising approach to managing resistance involves

identifying and developing new small molecules⁸that hit new

antibacterial targets and do not develop cross resistance with

antibiotics acting on existing antibacterial targets.⁹This approach

is challenging because accessing such novel targets will likely

require screening new molecular scaﬀolds that have not been

Received: June 19, 2011

Published: August 22, 2011

r

2011 American Chemical Society

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oxazines, and N-alkyl-N-aryl-hydroxylamines (compounds 1ꢀ5,

Figure 2).

The compounds were screened for biological activity in an

unbiased, target-blind approach using whole cell antibacterial

susceptibility assays. Nitroso ene reactions were used in a parallel

synthesis strategy to explore SAR on the pyridine ring and the

hydroxylamine side chain. A new convergent synthetic route was

developed using palladium-catalyzed BuchwaldꢀHartwig cross-

coupling amination reactions of N-alkyl-O-(4-methoxybenzyl)-

hydroxylamines with 2-halo-pyridines to achieve further SAR

exploration. The simplest active fragment molecule was identi-

ﬁed by synthesizing and testing all major molecular fragments.

The discovery process outlined here highlights the combination

of DOS and parallel synthesis as a useful strategy for identifying

new antibacterial scaﬀolds (Figure 2).

’ RESULTS AND DISCUSSION

Chemistry. The majority of molecules from the structurally

diverse DOS library (compounds 1ꢀ5 generalized in Figure 2

and Table 4; exact structures as shown in the Supporting Informa-

tion Table S1) were synthesized and characterized as described

previously by our group using Lewis acid-mediated nucleophilic

ring-opening reactions of nitroso DielsꢀAlder cycloadducts and

nitroso ene reactions.^24ꢀ26Several important and highly biolo-

gically active compounds (7, 9, and 11) from this library were

resynthesized according to the previously described methods

(Scheme 1). Separable isomers 7a and 7b were prepared using an

indium(III) triflate-mediated nucleophilic ring-opening of 5-bromo-

2-nitrosopyridine DielsꢀAlder cycloadduct 6 with methanol.²⁴

Compounds 9 and 11a,b were synthesized using nitroso ene

reactions with 2-methyl-2-butene and geraniol, respectively.²⁶

The major isomer from the ene reaction with geraniol (11a) was

readily obtained in pure form by standard chromatographic

methods, while the minor isomer (11b) was always isolated as

a mixture with 11a.

As described later, preliminary antibacterial data revealed that

hydroxylamine compounds containing pyridine or isoxazole

heterocycles showed strong antibacterial activity against M. luteus

ATCC 10240, with pyridine being the preferred heterocycle. To

conﬁrm the preference for pyridine, compounds 12, 14, and 16

featuring pyridine, quinoline, and isoxazole heterocycles, respec-

tively, with identical isoprenyl side chains, were synthesized via

ene reactions of the nitroso compounds with 2-methyl-2-butene

(Scheme 2).

Nitroso ene chemistry was also used to rapidly synthesize a

panel of substituted pyridine analogues with an isoprenyl side

chain. Parallel reaction of 2-nitrosopyridine enophiles (17aꢀi)

with 2-methyl-2-butene gave the corresponding racemic N-isoprenyl-

N-(pyridin-2-yl)hydroxylamines (18aꢀi; Table 1). These ana-

logues were designed to probe SAR of the pyridine ring using

substituents with diﬀering electronics, sterics, and substitution

patterns.

Nitroso ene chemistry was also used to generate a small panel

of analogues that probed SAR of the hydroxylamine side chain.

Reaction of 2-nitrosopyridine 10 with a variety of substituted

alkenes gave the corresponding ene products (19aꢀc; Table 2).

Compound 19a has an α-methyl substituted isoprenyl side chain

and is achiral. Compound 19b lacks substitution and chirality at

the carbon α to the hydroxylamine nitrogen and introduces a

styrene unit. Compound 19c retains the isoprenyl stereocenter

and introduces a ketone into the side chain.

Figure 1. Examples of known antibacterial agents containing an NꢀO

bond and the generic N-alkyl-N-(pyridin-2-yl)hydroxylamine scaﬀold

from this work.^34ꢀ38

collections of small molecules covering unique chemical

space.^21,22The use of DOS has already contributed to the

discovery of new antibacterial scaﬀolds using whole cell HTS

assays by the so-called forward chemical genetic approach.^18,23

Inclusion of DOS into antibacterial discovery programs provides

a valuable source of structurally diverse molecular scaﬀolds for

target-blind, unbiased screening.²³

Our group recently published new synthetic methodologies

for the syntheses of highly functionalized hydroxylamine-con-

taining compounds using Lewis acid-mediated nucleophilic ring-

opening reactions of nitroso DielsꢀAlder cycloadducts with

alcohols^24,25and nitroso ene reactions with substituted alkenes.²⁶

These methodologies were an extension of our Modular En-

hancement of Nature’s Diversity (MEND) natural product

derivatization program into a DOS strategy for rapidly pro-

ducing structurally complex small molecules from simple

starting materials.^27ꢀ31Biological screening of the hydroxylamine-

containing compounds revealed selective and potent antibacterial

activity against Micrococcus luteus ATCC 10240 for compounds

with pyridyl or isoxazole heterocycles. Discovery of the anti-

bacterial activity coincided with a report from our group describ-

ing similar activity of pyridyl-hydroxylamines derived from

the ene reaction of 2-nitrosopyridines with the sesquiterpene

natural product caryophyllene.³²Further literature searching

revealed only one other report of N-alkyl-N-(pyridin-2-yl)hydro-

xylamines possessing moderate antibacterial activity against

Staphylococcus aureus SG511, which also came from our group.³³

While other structurally unique hydroxylamine-containing anti-

bacterial scaﬀolds are known (Figure 1),^34ꢀ38the novelty of the

structures and obscurity of documented antibacterial activity

inspired us to pursue this new antibacterial lead further.

Herein is described the discovery and elucidation of the

structureꢀactivity relationships (SAR) of a new class of Gram-

positive selective antibacterial agents based on a N-alkyl-N-

(pyridin-2-yl)hydroxylamine scaﬀold (Figure 1). Application

of a DOS strategy using simple starting materials and the rich

chemistry of nitroso DielsꢀAlder cycloaddition and nitroso

ene reactions generated a structurally diverse library of oxyamino-

containing compounds including hydroxamic acids, bicyclic

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Figure 2. (a) Generalized DOS and combinatorial chemistry strategy for identifying new biologically active lead scaﬀolds. (b) Combination strategy of

DOS and combinatorial chemistry used for discovering the new antibacterial N-alkyl-N-(pyridin-2-yl)hydroxylamine scaﬀold in this work.

Scheme 1. Syntheses of N-Alkyl-N-(pyridin-2-yl)hydroxylamines 7a, 7b, 9, and 11a, and 11b^a

^aReagents and conditions: (a) In(OTf)₃(1.0 equiv), MeOH, 70 °C, 4 h, 95% (1:1 ratio of 7a:7b); (b) 2-methyl-2-butene (2.0 equiv), CH₂Cl₂, 0 °Cꢀrt,

20 min, 38%; (c) geraniol (2.0 equiv), CH₂Cl₂, 0 °Cꢀrt, 25 min, 71% (72:28 ratio of 11a:11b).

To probe deeper into the SAR and identify the simplest active

molecular fragment, a new convergent synthesis of the general

N-alkyl-N-(pyridin-2-yl)hydroxylamine scaﬀold was designed. The

simpliﬁed core structure 20 was viewed retrosynthetically (Figure 3)

by disconnecting the hydroxylamine bond at the 2-position of the

pyridine ring. The CꢀN bond could be formed using Pd(0)-

catalyzed BuchwaldꢀHartwig^39,40cross-coupling amination reac-

tions of commercially available 2-halopyridines 21 with suitable

O-protected hydroxylamines 22. The N-substituted hydroxy-

lamines 22 were available via N-alkylation of N-Boc-O-protected

hydroxylamine 23 with alkyl halides 24 under S_N2 condi-

tions, followed by removal of the N-Boc protecting group

with anhydrous acid.^41,42This synthetic route provided con-

vergent access to the active core 20 where advanced functionality

could be incorporated into both major fragments (21 and 22)

separately.

An O-(4-methoxybenzyl)ether (PMB) protecting group was

chosen for the hydroxylamine because PMB-ethers are easily

removed by exposure to anhydrous acid.⁴³The PMB-protected

hydroxylamine derivative, N-Boc-O-(4-methoxybenzyl)hydroxy-

lamine (BocNHOPMB, 27), was synthesized on multigram

scale (10.5 g) starting from N-hydroxyphthalimide (25) using a

modiﬁed literature protocol (Scheme 3).^44ꢀ46

The target N-alkyl-N-(pyridin-2-yl)hydroxylamines were synthe-

sized using a four-step sequence starting from BocNHOPMB

(27). Deprotonation of BocNHOPMB (27) with sodium

hydride in DMF followed by S_N2 reaction with a variety of alkyl

halides gave N-alkylated hydroxylamines 28aꢀi in excellent yields.

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Scheme 2. Syntheses of Pyridine (12), Quinoline (14), and

Isoxazole (16) Hydroxylamine Analogues with an Isoprenyl

Side Chain^a

Table 2. Syntheses of Pyridine Analogues (19aꢀc) with

Various Hydroxylamine Side Chains

^aReagents and conditions: (a) 2-methyl-2-butene (2.0 equiv), CH₂Cl₂,

0 °Cꢀrt; 15 min, 48% for 12, 1 h, 53% for 14, 10 min, 94% for 16.

Table 1. Syntheses of Substituted Pyridine Analogues

(18aꢀi) with an Isoprenyl Side Chain

Figure 3. Retrosynthetic analysis of N-alkyl-N-(pyridin-2-yl)hydroxyl-

amines (20).

compd

R

time

% yield

18a

18b

18c

18d

18e

18f

6-ethyl

30 min

9 h

46

47

8

Scheme 3. Synthesis of N-Boc-O-(4-methoxybenzyl)-

4-methyl

hydroxylamine (BocNHOPMB, 27)^a

3-methyl

5-chloro

30 min

10 min

1 h

59

64

68

67

62

75

5-bromo

5-iodo

18g

18h

18i

3,5-dichloro

5-bromo-6-methyl

3-chloro-5-CF₃

1 h

30 min

45 min

^aReagents and conditions: (a) 4-(methoxybenzyl)chloride, Et₃N, DMF,

90 °C, 1 h, 71%;(b) NH₂NH₂, DMF:MeOH, 60 °C, 10 min; (c) Boc₂O,

Et₃N, THF:H₂O, 2 h, 90% (steps b,c).

Brief exposure of compounds 28aꢀi to anhydrous triﬂuo-

roacetic acid (TFA) selectively removed the N-Boc protecting

groups, and an aqueous NaHCO₃wash gave the N-alkyl-O-

PMB-hydroxylamines 29aꢀi in good yields. The original aryl

amination conditions reported by Buchwald and co-workers

(Pd₂(dba)₃, (() BINAP, NaO^tBu, toluene, 70 °C)⁴⁷gave suc-

cessful cross-couplings of hydroxylamines 29aꢀi with 2-bromo-

pyridine, yielding N-alkyl-N-(pyridin-2-yl)-O-PMB-hydroxyla-

mines 30aꢀi. Use of hydroxylamines 29aꢀi in crude form

gave the desired products, but chromatographic puriﬁcation of

the hydroxylamines prior to coupling improved yields. Increasing

the size of the hydroxylamine alkyl substituent resulted in

decreased cross-coupling yields following the trend hexyl

(39%) < propyl (51%) < methyl (77%). The ﬁnal deprotection

of the PMB group under acidic conditions (TFA) in the presence

of a cation scavenger (Et₃SiH) proceeded smoothly giving

moderate to good yields (55ꢀ84%) of the desired N-alkyl-

N-(pyridin-2-yl)hydroxylamine products (31aꢀi) after freebas-

ing with aqueous NaHCO₃. The PMB-deprotection reactions

were monitored closely because overexposure to TFA and

Et₃SiH caused reduction of the N-O bond. The choice of Et₃SiH

as a cationscavenger was importantbecause other cation scavengers

such as anisole led to decomposition of the products. The results

from the four-step synthetic sequence are summarized in Table 3.

The analogues generated from the BuchwaldꢀHartwig ami-

nation protocol were designed to investigate some key SAR

points including the presence of a chiral center and allylic

unsaturation. A number of analogues with more drastic structural

changes were also synthesized to address several other important

SAR questions and identify the simplest active molecular frag-

ment. To test the importance of the hydroxyl group an analogue

with the NꢀO bond reduced, N-benzylpyridin-2-amine (32),

was prepared by subjecting compound 31d to Pd-catalyzed

hydrogenolysis. The O-acetyl analogue (33) was prepared by

treatment of 31d with acetic anhydride (Scheme 4). Analogous

NꢀO reduced (34) and O-acetyl (35) compounds were pre-

pared from the more structurally complex compound 7b

(Scheme 4). Selective reduction of the NꢀO bond of compound

7b was achieved using a titanium-mediated reduction procedure,

which gave analogue 34 in good yield.⁴⁸Acetylation of compound

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Scheme 4. Syntheses of NꢀO Reduced Analogues (32 and 34)

and O-Acetylated Analogues (33 and 35)^a

Scheme 5. Synthesis of 3-Pyridyl Analogue 37^a

^aReagents and conditions: (a) 3-bromopyridine, Pd₂(dba)₃, (() BI-

NAP, NaO^tBu, toluene, 70 °C, 67 h, 36%; (b) TFA, Et₃SiH, CH₂Cl₂, rt,

30 h; then aq NaHCO₃, 45%.

KirbyꢀBauer agar diffusion antibiotic susceptibility whole cell

assay (Table 4).^49,50The agar diffusion assay facilitated rapid

identification of active compounds and comparison of relative

potencies (increased size of the growth inhibition zone corre-

sponds to an increase in potency; i.e., a lower minimum inhibitory

concentration value).

The nitroso compounds and hydroxamic acids (1; class

AꢀC), bicyclic DielsꢀAlder cycloadducts (2; class AꢀC),

monocyclic hydroxamate-derived ring opened compounds (3,4;

class A), and acyclic hydroxamate derived ene products (5; class A)

were not active against M. luteus. The pyridyl and isoxazole ring

opened compounds (3,4;classB,C) and ene products (5, classB,C)

all showed strong antibacterial activity against M. luteus (Table 4).

A complete list of biological data for all compounds is provided in

the Supporting Information (Table S2).

^aReagents and conditions: (a) 10% PdꢀC, H₂(1 atm), MeOH, rt, 30

min, 97%; (b) Ac₂O, iPr₂EtN, DMAP, CH₂Cl₂, rt; 12 h, 72% for 33; 9 h,

94% for 35; (c) Cp₂TiCl₂, Zn, THF/MeOH, ꢀ30 °C, 1 h, 73%.

7b was achieved using acetic anhydride which provided analogue

35 in excellent yield.

In general, the N-alkyl-N-(pyridin-2-yl)hydroxylamines (3, 4,

and 5; class B) gave larger zones of growth inhibition in the

agar diﬀusion assay than the N-alkyl-N-(5-methylisoxazol-3-yl)-

hydroxylamines (3, 4, and 5; class C). The most active pyridyl

compounds (7a, 9, and 11a) from the class B N-alkyl-N-(pyridin-

2-yl)hydroxylamines were selected for broader antibacterial test-

ing against a panel of Gram-negative, Gram-positive, and yeast

organisms, including antibiotic resistant strains of S. aureus (MRSA)

and Enterococcus faecalis (VRE). The results of this broad anti-

bacterial screening are summarized in Table 5 using a generalized

scale to clearly show the narrow spectrum of antibacterial activity

of compounds 7a, 9, and 11a. Ciproﬂoxacin was included as a

control antibiotic with known broad-spectrum activity.⁵¹

Compounds 7a, 9, and 11a all displayed a similar narrow

spectrum of antibacterial activity, and the ciproﬂoxacin control

(5 μg/mL in H₂O) showed broad spectrum activity against all

strains except M. luteus. Compounds 7a, 9, and 11a exclusively

inhibited the growth of Gram-positive bacteria with particularly

strong and selective potency against M. luteus ATCC 10240. The

compounds showed comparable, although moderate, activity

against both antibiotic susceptible (S. aureus SG511; E. faecalis

ATCC 49532) and antibiotic resistant (S. aureus 134/94 MRSA;

E. faecalis 1528 VRE) Gram-positive strains. Because of the

highly selective antibacterial activity against Gram-positive or-

ganisms, M. luteus ATCC 10240, S. aureus SG511, and E. faecalis

ATCC 49532 were used as model test organisms to evaluate SAR

of the N-alkyl-N-(pyridin-2-yl)hydroxylamine analogues de-

scribed from this point forward.

All the analogues presented thus far featured placement of the

hydroxylamine at the 2-position of pyridine. The same Buchwaldꢀ

Hartwig amination sequence discussed earlier was used to

synthesize an analogue with the hydroxylamine placed at the

3-position of the pyridine ring (compound 37; Scheme 5). The

Pd-catalyzed cross-coupling of hydroxylamine 29d was less

eﬀective at the 3-position of pyridine (compound 36, 36% yield)

relative to coupling at the 2-position (compound 30d, 63% yield;

Table 3) but still aﬀorded the desired product (36). Deprotection

of the PMB group with TFA/Et₃SiH and quenching with aqueous

NaHCO₃provided N-benzyl-N-(pyridin-3-yl)hydroxylamine (37).

The homologated analogue 39 was designed to determine if

direct attachment of the hydroxylamine to the pyridine ring was

essential for activity. Alkylation of hydroxylamine 29d with

2-(bromomethyl)pyridine gave O-PMB-protected hydroxyla-

mine 38. Removal of the PMB group with TFA/Et₃SiH gave

the desired homologated analogue 39 (Scheme 6).

As described later, preliminary testing indicated that nitroso

ene reactions with geraniol generated highly antibacterially active

compounds and that 6-alkyl substitution was favorable. There-

fore, a 6-ethyl geraniol analogue (40a) was synthesized via ene

reaction of 6-ethyl-2-nitrosopyridine with geraniol (Scheme 7).

The major isomer (40a) was isolated in pure form after chromato-

graphic puriﬁcation. A crystal of 40a was obtained from CH₂Cl₂/

hexanes, and X-ray diﬀraction conﬁrmed the structure of the major

isomer (Figure 4; see Supporting Information for experimental

details).

Biology. Selected compounds from the nitroso chemistry

(compounds 1ꢀ5, Figure 2)^24ꢀ26were compiled into a DOS

library of ∼100 small molecules with a high degree of appendage,

functional group, stereochemical, and skeletal diversity.²³The

representative structural classes are given in Table 4. A complete

list of exact structures is given in the Supporting Information

(Table S1). The entire DOS compound library was screened

against Micrococcus luteus ATCC 10240 using an in-house

Two antibacterial susceptibility assays were used to evaluate

the antibacterial activity of all the synthesized analogues, an agar

diﬀusion assay,^49,50and a visual end point broth microdilution

assay.⁵²Data from the agar diﬀusion assay are reported as diameters

of growth inhibition (mm) and data from the broth microdilution

assay are reported as minimum inhibitory concentrations (MIC₉₀;

μM). The ﬂuoroquinolone antibiotic ciproﬂoxacin was included as a

control.⁵¹The results from the two assays are provided in Table 6.

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Table 3. Four-Step Syntheses of Compounds 28aꢀiꢀ31aꢀi^a,b

^aReagents and conditions: (a) NaH, RX (alkyl halide), DMF, 0 °Cꢀrt; (b) TFA, CH₂Cl₂, rt; then aq NaHCO₃; (c) 2-bromopyridine, Pd₂(dba)₃, (()

BINAP, NaO^tBu, toluene, 70 °C; (d) TFA, Et₃SiH, CH₂Cl₂, rt; then aq NaHCO₃. ^bYields are based on isolated, puriﬁed, and characterized material

unless otherwise stated and optimal reaction time listed if reactions were replicated. ^cCrude yield. ^dAverage of two yields. ^eAverage of three yields.

^fCalculated for puriﬁed products only.

Scheme 6. Synthesis of Homologated Analogue 39^a

^aReagents and conditions: (a) 2-(bromomethyl)pyridine hydrobro-

mide, iPr₂EtN, CH₃CN, 50 °C, 17 h, 44%; (b) TFA, Et₃SiH, CH₂Cl₂,

rt; then aq NaHCO₃, 5 h, 73%.

Scheme 7. Synthesis of Geranyl Analogues 40a and 40b^a

Figure 4. X-ray diﬀraction structure of analogue 40a displayed with

50% probability ellipsoids.

selective antibacterial activity against M. luteus. The anti-M. luteus

activity, based on MIC₉₀values, of the heterocylic cores in-

creased in the order of isoxazole (64 μM) < quinoline (8 μM) =

pyridine (8 μM), which conﬁrmed the initial observation of

pyridine as a favorable core.

^aReagents and conditions: (a) geraniol (2.0 equiv), CH₂Cl₂, 0 °Cꢀrt,

The substituted pyridine analogues (18aꢀi) with identical

30 min, 75% (70:30 ratio of 40a:40b).

isoprenyl side chains also preferentially inhibited the growth of

M. luteus relative to S. aureus and E. faecalis. Methyl substitution

at the 4- and 6-positions (compounds 9 and 18b, respectively)

enhanced anti-M. luteus activity (MIC₉₀values of 4 μM)

compared to the 3-position (compound 18c) of the pyridine

ring (MIC₉₀value of 16 μM). There was a noticeable increase in

potency in both assays for the 6-ethyl substituted compound

(18a, 2 μM) compared to the unsubstituted (12, 8 μM) and

The MIC₉₀data for compounds 7a, 9, and 11a mirrored the

agar diﬀusion data and conﬁrmed the selective potency against

M. luteus (MIC₉₀values of 4, 4, and 8 μM, respectively). Geranyl

derivative 11a also had a moderate MIC₉₀of 64 μM against

S. aureus. Similarly, analogues 12, 14, and 16, featuring pyridine,

quinoline, and isoxazole heterocycles, respectively, all displayed

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Table 4. Structure Classes and Antibiotic Activity against M. luteus ATCC 10240 in the Agar Diﬀusion Assay^a,b,c

^aSee Supporting Information for a complete list of structures and antibacterial data (Supporting Information Tables S1 and S2). ^bAll compounds were racemic.

R = alkyl, O-alkyl; R⁰= alkyl, halo, carboxyl; R₁, R₂, R₃, R₄, R₅= alkyl, H; n = 1,2. ^cExactly 50 μL of each compound solution (2.0 mM in 10:1 MeOH:DMSO)

were added to 9 mm wells in agar media (MHII) inoculated with ∼5 ꢁ 10³CFU/mL of M. luteus ATCC 10240. Diameters of growth inhibition zones were

measured (mm) after incubation at 37 °C for 24 h.^{50 d}No: indicates a growth inhibition zone e14 mm. ^eYes: indicates a growth inhibition zone >14 mm.

Table 5. Spectrum of Antibacterial Activity for Compounds 7a, 9, and 11a in the Agar Diﬀusion Assay^a,b,c

^aExactly 50 μL of each compound solution (2.0 mM in 10:1 MeOH:DMSO) were added to 9 mm wells in agar media (MHII) inoculated with ∼5 ꢁ 10³

CFU/mL. Diameters of growth inhibition zones were measured (mm) after incubation at 37 °C for 24 h.^{50 b}See Supporting Information for exact

c

diameters (mm) of inhibition zones (Table S2). Diameters of inhibition zones are generalized as follows: +, inhibition zone of 11ꢀ15 mm; ++,

inhibition zone of 16ꢀ20 mm; +++, inhibition zone of g21 mm; ꢀ, inhibition zone of e10 mm. ^dCiproﬂoxacin was used as a control at 5 μg/mL in

H₂O. ^eAmphotericin B was used as the control (10 μg/mL in 10:1 DMSO:MeOH) instead of ciproﬂoxacin. ^f*Indicates partially unclear inhibition zone.

6-methyl substituted analogues (9, 4 μM). Halogen substitution

(5-Cl, 5-Br, and 5-I) at the 5-position (compounds 18dꢀf) of the

pyridine ring also increased anti-M. luteus activity (MIC₉₀values

from 2 to 4 μM). 3,5-Dichloro substitution (compound 18g)

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Table 6. Antibacterial Activity of Test Compounds in the Agar Diﬀusion Assay^a(Diameter of Growth Inhibition Zones Given in

mm) and the Broth Microdilution Assay^b,c(MIC₉₀Values Given in μM)

test organism

M. luteus ATCC 10240

S. aureus SG511

MIC₉₀(μM)

E. faecalis ATCC 49532

compd

zone (mm)

MIC₉₀(μM)

zone (mm)

MIC₉₀(μM)

7a

34

48

35

31

21

52

29

25

40

35

38

27

35

15

33

20

30

0

4.0

12

18*

12

19

18

13

0

>128

g128

>128

64

16*^e

20*

15*

20*

22*

13*

22*

19*

21*

19*

14*

12*

13*

18*

16*

0

>128

g128

>128

2.0

7b

9

4.0

11a

12

8.0

128

14

8.0

128

16

64

>128

128

18a

18b

18c

18d

18e

18f

18g

18h

18i

19a

19b

19c

30d

31a

31b

31c

31d

31e

31f

31g

31h

31i

32

2.0

11

20

10

18

17*

12

0

4.0

16

>128

128

2.0

4.0

>128

128

2.0

16.0

4.0

13

0

128

4.0

15

19

15*

0

64

32

16

128

>128

g128

>128

128

64

>128

128

18*

13*

15

24

16

14

12*

16

17

0

17*

18

0

16*

20*

15*

12*

0

128

>128

128

>128

128

>128

32

>128

0

13

15*

14*

0

15*

0

33

20

0

12

0

11*

0

34

35

22

15*

0

37

g128

>128

2.0

16*

0

39

0

40a

cipro^d

31

18*

19*

0

8.0

26

0.5

17

^aExactly 50 μL of each compound solution (2.0 mM in 10:1 MeOH:DMSO) were added to 9 mm wells in agar media (MHII) inoculated with ∼5 ꢁ 10³

CFU/mL. Diameters of growth inhibition zones were measured (mm) after incubation at 37 °C for 24 h.^{50 b}MIC₉₀values were determined by the visual

end point broth microdilution method following CLSI guidelines.^{52 c}Each compound was tested in triplicate. ^dCiproﬂoxacin was used as a standard at

5 μg/mL in H₂O in the agar diﬀusion assay. ^e*Indicates partially unclear inhibition zone.

gave decreased anti-M. luteus activity (16 μM) relative to

5-chloro substitution (compound 18d, 2 μM). Anti-M. luteus

activity was not improved by the use of mixed alkyl-halogen

substitution (5-Br-6-Me, 18h; MIC₉₀value of 4 μM) relative to

monohalogen (5-Br, 18e; MIC₉₀value of 4 μM) or alkyl

substitution alone (6-Me, 9; MIC₉₀value of 4 μM). Substitution

of the pyridine using 3-Cl-5-CF₃substituents (compound 18i)

was detrimental (MIC₉₀value of 128 μM against M. luteus). As

supported by compounds 18c, 18g, and 18i, any substitution at

the 3-position of the pyridine ring resulted in decreased anti-

M. luteus activity. Most of the analogues had no signiﬁcant activity

against E. faecalis or S. aureus, but the 4-methyl (18b) and 5-bromo

(18e) derivatives showed a noticeable increase in activity against

S. aureus relative to the other analogues. In general, for the sub-

stituted pyridine analogues (18aꢀi), 5-halo and 6-alkyl substitu-

tion was favored for increased anti-M. luteusactivity and the 6-ethyl

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Figure 5. General antibacterial SAR trends for N-alkyl-N-(pyridin-2-yl)hydroxylamines.

analogue (18a) emerged as the most potent compound in both

assays (inhibition zone of 52 mm; MIC₉₀value of 2 μM). The

preference for 6-ethyl substitution was conﬁrmed by the 6-ethyl

substituted geranyl derivative 40a, which showed enhanced anti-

M. luteus activity (MIC₉₀value of 2 μM) relative to the unsub-

stituted geranyl derivative 11a (MIC₉₀value of 8 μM).

oﬀered a versatile point of derivatization. Preliminary SAR studies

around the phenyl ring of benzyl derivative 31d were performed

using 3-phenoxy (31g), 4-triﬂuoromethyl (31h), and 4-triﬂuor-

omethoxy (31i) substituted compounds generated from the

BuchwaldꢀHartwig cross-coupling chemistry. The phenyl sub-

stituted analogues were less active than the parent benzyl

compound, and the anti-M. luteus activity increased in the order

of 3-(PhO)benzyl (31g) < 4-(CF₃)benzyl (31h) ≈ 4-(CF₃O)-

benzyl (31i) < benzyl (31d). Electron withdrawing groups

appeared to be tolerated better than electron donating groups,

but more data is needed to conﬁrm this trend.

Comparison of the anti-M. luteus activity of allyl derivative 31f

(inhibition zone of 14 mm; MIC₉₀value of >128 μM) to isopre-

nyl derivative 12 (inhibition zone of 31 mm; MIC₉₀value of

8 μM) revealed a drastic diﬀerence in activity for only subtle

diﬀerences in structure and molecular weight. The same trend

was apparent when comparing all the simpliﬁed N-alkyl-N-

(pyridin-2-yl)hydroxylamines 31aꢀi to the unsubstituted pyr-

idyl compounds with isoprenyl or substituted isoprenyl side

chains generated from nitroso ene chemistry (11a, 12, 19a, and

19c). The increased activity associated with the isoprenyl side

chains must be attributed to substitution at the carbon α to the

hydroxylamine or alkyl branching from the double bond, which

are the only clear structural diﬀerences.

The scaﬀold SAR was expanded by evaluating compounds

with more drastic structural modiﬁcations (32ꢀ35, 37, and 39).

The NꢀO reduced analogues 32 and 34 had no antibacterial

activity in both susceptibility assays. Additionally, testing of

O-PMB protected compound 30d revealed no antibacterial activity.

These observations conﬁrmed that the presence of the NꢀOH

group is critical to activity. The O-acetyl analogues 33 and 35 had

somewhat reduced activity against M. luteus (MIC₉₀values of 128

and 32 μM, respectively) compared to the parent compounds

31d and 7b (MIC₉₀values of 64 and 4 μM, respectively). The

acetylated derivatives likely acted as prodrugs for the parent

hydroxylamine compounds triggered by hydrolysis in the testing

media. The 3-substituted pyridine analogue 37 had reduced

activity against M. luteus relative to 2-substituted pyridine analogue

31d. The homologated analogue 39 showed no antibacterial

activity. The anti-M. luteus data for compounds 37 and 39

suggested that N-arylation of the hydroxylamine is essential for

activity andthatN-arylation atthe 2-position of pyridine isoptimal.

Overall, the agar diﬀusion data and the MIC₉₀data in Table 6

correlated nicely. Compounds with the largest diameter growth

inhibition zones (mm) in the agar diﬀusion assay gave the lowest

MIC₉₀values (μM) in the broth microdilution assay. Com-

pounds with the N-alkyl-N-(pyridin-2-yl)hydroxylamine core

Evaluation of the pyridine analogues with diﬀering hydroxy-

lamine alkyl side chains (19aꢀc) showed that substitution of the

hydroxylamine nitrogen is tolerated but does noticeably inﬂu-

ence the potency and selectivity. Compound 19a had a gem-

dimethyl isoprene unit and maintained strong potency against

M. luteus (MIC₉₀value of 4 μM) and weak anti-S. aureus activity

(MIC₉₀value of 128 μM). Compound 19b featured a styrene

side chain and showed decreased anti-M. luteus activity (MIC₉₀

value of 64 μM) and increased anti-S. aureus activity (MIC₉₀

value of 32 μM). Compound 19c gave MIC₉₀values of 16 μM

against M. luteus and 128 μM against S. aureus, which showed

that an alkyl ketone was tolerated as a side chain. Antibacterial

data from compounds 19a and 19c and the other unsubstituted

pyridyl nitroso ene products 11a and 12 showed that high

substitution is tolerated on the α carbon of the isoprenyl

hydroxylamine side chain. Data from compound 19b indicated

that alkyl substitution of the α carbon is important for enhancing

the potency against M. luteus and that modiﬁcation of the

hydroxylamine side chain can enhance potency against S. aureus.

The N-alkyl-N-(pyridin-2-yl)hydroxylamine products 31aꢀi

from the BuchwaldꢀHartwig chemistry revealed some interest-

ing SAR trends. Compound 31a with a simple N-methyl side

chain showed weak but noticeable activity against M. luteus. This

observation was important because compound 31a represents

the simplest active molecular fragment with all the critical

structural components (pyridine heterocycle, NꢀOH at 2-position,

and alkyl substitution of the hydroxylamine nitrogen). There was

no drastic change in anti-M. luteus activity observed when the size

of the alkyl side chain was increased from Me to Pr to n-hexyl

(compounds 31aꢀc). However, an N-benzyl group was found to

be a suitable side chain, and compound 31d was the most active

analogue generated from the BuchwaldꢀHartwig chemistry

against both M. luteus (MIC₉₀value of 64 μM) and S. aureus

(MIC₉₀value of 128 μM). The benzyl derivative 31d showed

increased activity relative to the phenethyl derivative (31e)

where the unsaturated carbon system was homologated. Com-

pound 31f had a simple N-allyl side chain and showed only weak

anti-M. luteus activity in the agar diﬀusion assay.

The presence of an allylic unsaturated system appeared to be

important for antibacterial activity and a benzyl side chain

seemed to be a good choice especially because the phenyl ring

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Figure 6. Structural similarities of N-benzyl-N-(pyridin-2-yl)hydroxylamine (31d) to known DXR inhibitors (fosmidomycin and compounds 41ꢀ44).

structure had selectively potent activity against M. luteus (MIC₉₀

values as low as 2 μM) and weak to moderate activity against

S. aureus (MIC₉₀values as low as 32 μM). Although many

compounds gave larger diameter zones of inhibition against

E. faecalis compared to S. aureus in the agar diﬀusion assay, these

zones were hazy and correlated to high MIC₉₀values of >128

μM. Although the N-alkyl-N-(pyridin-2-yl)hydroxylamines only

have weak to moderate activity against S. aureus and E. faecalis,

the agar diﬀusion antibacterial data indicated that the compounds

had comparable activity against antibiotic resistant strains (MRSA

and VRE, Table 5). The SAR studies showed that the structure can

be optimized to enhance activity against these clinically relevant

Gram-positive pathogens. The SAR trends for anti-M. luteus

activity of the N-alkyl-N-(pyridin-2-yl)hydroxylamines revealed

in this study are summarized in Figure 5.

Representative compounds were also evaluated for growth

inhibition of cancer cell lines (MCF-7, PC-3, and HeLa). None of

the compounds showed signiﬁcant toxicity toward the mamma-

lian cell lines, which indicated a potentially favorable therapeutic

index. Experimental details and results from the anticancer assay

are given in the Supporting Information (Table S2).

discrete, conserved cellular target. This hypothesis was also

supported by the selective biological activity against Gram-

positive bacteria because a generally toxic molecule should show

a broader spectrum of activity.

While any discussion of potential modes of action for these

compounds is speculation at this point, some key structural

features of the N-alkyl-N-(pyridin-2-yl)hydroxylamines showed

striking similarities to known antibacterial compounds in the

literature. The presence of a metal chelating group, the 2-pyr-

idylhydroxylamine functionality,^65,66was similar to other known

antibiotics containing NꢀOH bonds (Figure 1). For these

known antibacterial agents, the NꢀOH bond played a key

role by aiding in chelation of a metal in the active site of the

target enzyme.^34ꢀ37,67Recent literature reports of fosmidomycin

analogues^68ꢀ71revealed many structural similarities of N-alkyl-

N-(pyridin-2-yl)hydroxylamines to known inhibitors (41ꢀ44)

of 1-deoxy-^D-xylulose-5-phosphate reductoisomerase (DXR) in

the methylerythritol phosphate (MEP) isoprene biosynthesis

pathway,⁷²a validated target for antibacterial agents (Figure 6).⁷³

The preference for isoprenyl and geranyl hydroxylamine side

chains of the most active N-alkyl-N-(pyridin-2-yl)hydroxylamine

analogues found in this work might aid in binding to enzymes

along terpenoid biosynthetic pathways. This might explain

the selective antibacterial activity of these compounds against

M. luteus, which is known to express high levels of terpenoid

biosynthesis related enzymes in its cell membrane. Furthermore,

M. luteus is known to possess a more promiscuous set of

magnesium-dependent prenylation enzymes, relative to Gram-

negative (E. coli) and other Gram-positive (S. aureus) strains.⁵⁵

Therefore, it is important to investigate these compounds for

inhibition of isoprenoid and lipid biosynthetic pathways.^73,74

’ FURTHER DISCUSSION AND PERSPECTIVE

The selective antibacterial activity observed for the N-alkyl-

N-(pyridin-2-yl)hydroxylamines against M. luteus was intriguing.

While M. luteus is a commonly encountered Gram-positive

bacterium that is part of normal human ﬂora, it has a unique

biochemistry^53ꢀ55and pattern of antibiotic susceptibility (nitro-

furantoin and ciproﬂoxacin resistant).^51,56Consequently, M. luteus

is commonly used in antibacterial screening programs to identify

compounds with potential activity against resistant Gram-positive

strains.⁵⁷Beyond its use as a model laboratory organism,

M. luteus has clinical relevance as an infectious pathogen. While

this bacterium was long considered nonpathogenic, recent

literature recognizes M. luteus and other Microccoci as commonly

misdiagnosed opportunistic pathogens that cause a variety of

infections in immunocompromised patients that have been

documented to be lethal in some cases.^58,59Clearly, Microccoci

are clinically relevant pathogens that merit the development of

new eﬀective antibiotics especially because there is renewed

interest in ﬁnding markets for species selective antimicrobial

agents as a means of managing the development of resistance.^5,60

Initially it was suspected that the N-alkyl-N-(pyridin-2-yl)-

hydroxylamines might be acting as prodrugs for releasing a toxic

nitroso compound inside of the bacterial cells⁶¹or possibly

generating reactive oxygen species from the aryl hydroxylamine.^62,63

However, the parent nitroso compounds (1, class B,C; Table 4)

and DielsꢀAlder cycloadducts (2, class AꢀC; Table 4), which

can undergo retro DielsꢀAlder reactions to generate a nitroso

species,⁶⁴had no signiﬁcant antibacterial activity. Additionally,

establishment of clear SAR trends suggested the existence of a

’ SUMMARY AND CONCLUSIONS

A DOS library of compounds was generated using nitroso

DielsꢀAlder and nitroso ene chemistry which led to the dis-

covery of N-alkyl-N-(pyridin-2-yl)hydroxylamine scaﬀolds as

selective antibacterial agents against Micrococcus luteus ATCC

10240. The SAR was investigated using parallel synthesis with

nitroso ene chemistry and a new BuchwaldꢀHartwig amination

cross-coupling reaction of O-PMB-protected hydroxylamines

with 2-bromopyridine. From these SAR studies, the simplest

active fragment molecule, N-methyl-N-(pyridin-2-yl)hydroxy-

lamine (31a), was determined. New lead structures (11a, 18a,

18d, 31d, and 40a) were identiﬁed with potent activity against M.

luteus (MIC₉₀values as low as 2.0 μM) that will direct the

production of future analogues. Structural similarities of N-alkyl-

N-(pyridin-2-yl)hydroxylamines to known inhibitors of the MEP

isoprenoid biosynthesis pathway (41ꢀ44) have inspired the

direction for future mode of action studies and the design of

new analogues. This work demonstrated that an interdisciplinary

combination of DOS, parallel synthesis, and whole-cell antibacterial

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susceptibility assays is an eﬀective strategy for the discovery of

new, structurally novel antibacterial scaﬀolds.

0.13 mmol), and a mixed fraction of 7a/7b (20.0 mg, 0.07 mmol) as a

clear, viscous oil (95% overall yield). (7a) HPLC retention time 6.86

min. (7b) HPLC retention time 7.32 min. All characterization data

matched the previously reported data.²⁴

’ EXPERIMENTAL SECTION

Typical Procedure for Nitroso Ene Reactions. (()N-(6-Ethylpyridin-

2-yl)-N-(3-methylbut-3-en-2-yl)hydroxylamine (18a). This procedure

followed a general method reported previously.²⁶6-Ethyl-2-nitrosopyr-

idine (17a; 271.0 mg, 1.99 mmol) was added as a solid to a solution of

2-methyl-2-butene (0.42 mL, 3.97 mmol) in 4 mL of anhydrous CH₂Cl₂

at 0 °C (ice bath temperature), and the solution turned orange. After 30

min, no starting nitroso compound (17a) was detected by TLC (20%

EtoAc in hexanes). The solution was warmed to rt, and the CH₂Cl₂was

evaporated under reduced pressure. The crude product was purified by

column chromatography (1.25 ꢁ 7.5 in silica gel; 5%ꢀ30% EtOAc in

hexanes) to give the desired ene adduct (18a) in 46% yield as a tan solid

(190.0 mg, 0.92 mmol); mp 64ꢀ65 °C. ¹H NMR (600 MHz, DMSO-

d₆) δ 8.68 (br s, NꢀOH, 1 H), 7.48 (dd, J = 8.2, 7.3 Hz, 1 H), 6.85 (d, J =

8.2 Hz, 1 H), 6.57 (d, J = 7.0 Hz, 1 H), 5.12 (q, J = 6.5 Hz, 1 H),

4.88ꢀ4.86 (m, 1 H), 4.84ꢀ4.82 (m, 1 H), 2.59 (q, J = 7.6 Hz, 3 H), 1.71

(s, 3 H), 1.18ꢀ1.15 (m, 6 H). ¹³C NMR (150 MHz, DMSO-d₆) δ 162.5,

160.2, 146.1, 137.8, 112.2, 111.6, 105.8, 58.2, 30.5, 21.5, 13.7, 13.6.

HRMS-ESI (m/z): [M + H]⁺calcd for C₁₂H₁₉N₂O: 207.1492, found

207.1488. IR (neat): 3139.7 (br), 3092.3, 1589.6, 1572.1, 1441.2,

1108.9, 968.9 cm^ꢀ1. HPLC retention time 8.34 min.

Chemistry. General Materials and Methods. All reactions were

performed under a dry argon atmosphere unless otherwise stated. All

solvents and reagents were obtained from commercial sources and used

without further purification unless otherwise stated. Dichloromethane

(CH₂Cl₂) was distilled from calcium hydride. Tetrahydrofuran (THF)

was distilled from Na/benzophenone. Dimethylformamide (DMF),

diisopropylethylamine (iPr₂EtN), acetonitrile (CH₃CN), and triethyl-

silane (Et₃SiH) were used from Acros Seal anhydrous bottles. All

nitroso compounds were prepared from aminoheterocyclic precursors

by a literature protocol⁷⁵and were stored at ꢀ20 °C. Cycloadduct 6 was

obtained from previous work published by our group.²⁴Silica gel column

chromatography was performed using Sorbent Technologies silica gel

60 (32ꢀ63 μm). ¹H NMR and ¹³C NMR spectra were obtained on a

300 MHz, MHz, or MHz Varian DirectDrive spectrometer and FIDs

were processed using ACD/ChemSketch version 10.04. Chemical shifts

(δ) are given in parts per million (ppm) and are referenced to residual

nondeuterated solvent. Coupling constants (J) are reported in hertz

(Hz). High resolution, accurate mass measurements were obtained with

a Bruker micrOTOF II electrospray ionization time-of-flight mass

spectrometer in positive ion mode. Sample was introduced via flow

injection at a rate of 4 μL/min, and mass spectra were accumulated from

50 to 3000 m/z for 2 min. X-ray diffraction analysis was performed on a

Bruker APEX-II diffractometer (full experimental details and data

processing are given in the Supporting Information). High-performance

liquid chromatography (HPLC) was performed on a Waters 1525

Binary HPLC Pump instrument with a Waters 2487 Dual λ absorbance

detector set at 254 nm operated by Breeze version 3.30 software. A YMC

Pro C18 reverse phase column (3.0 mm ꢁ 50 mm) fit with precolumn

frit (0.5 μm) and YMC Pro C18 guard column (2.0 mm ꢁ 10 mm) was

used for all analyses. Mobile phases used were 10 mM ammonium

acetate in HPLC grade water (A) and HPLC grade acetonitrile (B). A

gradient was formed from 5%ꢀ80% of B in 10 min, then 80%ꢀ95% of B

in 2 min, and then 95%ꢀ5% of B in 3 min at a flow rate of 0.7 mL/min

(total run time of 15 min). Thin layer chromatography (TLC) was

performed with aluminum-backed Merck 60-F₂₅₄silica gel plates using a

254 nm lamp, aq FeCl₃, aq KMnO₄, or iodine vapor for visualization.

Infrared (IR) spectra were recorded on a Bruker Tensor series FT-IR

spectrometer using a diamond ATR accessory and signals are reported as

wavenumbers in reciprocal centimeters (cm^ꢀ1). Melting points were

determined in capillary tubes using a ThomasꢀHoover melting point

apparatus and are uncorrected. The purity of compounds tested in

biological assays was evaluated by analytical HPLC and verified to be

g95%, unless otherwise noted.

N-Boc-O-(4-Methoxybenzyl)hydroxylamine (BocNHOPMB, 27).

This compound was prepared in 64% overall yield following a modified

literature procedure.⁴⁴N-Hydroxyphthalimide (10.53 g, 64.5 mmol),

25, was treated with 4-methoxybenzyl chloride (8.9 mL, 65.4 mmol) and

triethylamine (22.0 mL, 158.0 mmol), respectively, in 200 mL of DMF.

The dark-red solution was heated to 90 °C (oil bath temperature) for

1 h. The mixture was cooled to ∼45 °C and poured over iceꢀwater.

Product 26 precipitated as a light-brown solid and was isolated via

vacuum filtration and dried overnight under vacuum (12.99 g, 71%). ¹H

NMR (500 MHz, CDCl₃) δ 7.80ꢀ7.75 (m, 2 H), 7.73ꢀ7.68 (m, 2 H),

7.43 (d, J = 8.4 Hz, 2 H), 6.86 (d, J = 8.4 Hz, 2 H), 5.13 (s, 2 H), 3.78

(s, 3 H). ¹³C NMR (125 MHz, CDCl₃) δ 163.4, 160.3, 134.3, 131.5, 128.7,

125.7, 123.3, 113.8, 79.4, 55.1. The solid was dissolved in 188 mL of

DMF:MeOH (12:25) while at 60 °C (oil bath temperature) and treated

with hydrazine hydrate (65% v/v; 7.0 mL, 146.5 mmol). After 10 min,

the mixture was cooled to rt and diluted with 100 mL of H₂O. The

MeOH was removed under reduced pressure and the DMF/H₂O

mixture was extracted with EtOAc (4 ꢁ 100 mL). The combined

EtOAc layers were dried over anhydrous MgSO₄, filtered, and concen-

trated to give 11.0 g of a faint, orange liquid. This liquid was immediately

dissolved in 100 mL of THF:H₂O (1:1) and treated with triethylamine

(6.5 mL, 46.6 mmol) and Boc anhydride (12.0 g, 55.0 mmol),

respectively. After 2 h, the THF was removed under reduced pressure,

and the H₂O was extracted with EtOAc (2 ꢁ 50 mL). The combined

EtOAc layers were washed with 5% aq citric acid (1 ꢁ 25 mL) and water

(1 ꢁ 25 mL), dried over anhydrous MgSO₄, filtered, and concentrated

to give 14.7 g of a clear, orange liquid. Purification by column

chromatography (3 ꢁ 6 in silica gel; 20% EtOAc in hexanes) provided

the desired product (27) in 90% yield (2 steps) as a clear, colorless liquid

(10.49 g, 41.4 mmol). ¹H NMR (600 MHz, CDCl₃) δ7.35ꢀ7.32 (m, 2 H),

7.07 (s, 1 H), 6.91ꢀ6.88 (m, 2 H), 4.80 (s, 2 H), 3.82 (s, 3 H), 1.49 (s, 9 H).

¹³C NMR (150 MHz, CDCl₃) δ 159.8, 156.7, 130.8, 127.8, 113.8, 81.6,

78.0, 55.3, 28.2. HRMS-ESI (m/z): [M + Na]⁺calcd for C₁₃H₁₉NNaO₄:

276.1206, found 276.1202. IR (neat): 3285.8 (br), 1715.8, 1245.3, 1162.8,

(()N-(5-Bromopyridin-2-yl)-N-((1S/R,4S/R)-4-methoxycyclohex-2-

enyl)hydroxylamine (7a) and N-(5-Bromopyridin-2-yl)-N-((1S/R,2S/

R)-2-methoxycyclohex-3-enyl)hydroxylamine (7b). The synthesis of

compounds 7a and 7b was replicated from work reported by Yang and

Miller.²⁴A solution of nitroso DielsꢀAlder cycloadduct 6 (103.0 mg,

0.39 mmol) in 3 mL of MeOH was treated with In(OTf)₃(216.7 mg,

0.39 mmol) and heated to 70 °C (oil bath temperature). After 4 h, TLC

(20% EtOAc in hexanes) showed complete consumption of starting

material. The solution was cooled to rt and concentrated under reduced

pressure. The resulting oil was partitioned between EtOAc (20 mL) and

water (10 mL). The layers were separated, and the water was extracted

with EtOAc (1 ꢁ 10 mL). The EtOAc layers were combined, dried over

anhydrous MgSO₄, filtered, and concentrated. The crude product (∼1:1

mixture of 7a:7b) was purified by column chromatography (0.75 ꢁ 8 in

silica gel; 10%ꢀ25% EtOAc in hexanes) to give isomer 7a as an off-white

solid (49.2 mg, 0.16 mmol), isomer 7b as a clear, viscous oil (40.0 mg,

1099.8, 1032.4 cm^ꢀ1

.

Typical Procedure for the Alkylation of BocNHOPMB: N-Boc-N-

Benzyl-O-(4-methoxybenzyl)hydroxylamine (28d). Sodium hydride

(60% in mineral oil; 400 mg, 10.00 mmol) solid was added to a solution

of compound 27 (1.955 g, 7.72 mmol) in 20 mL of anhydrous DMF at

0 °C (ice bath temperature). The solution bubbled and turned a faint

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Journal of Medicinal Chemistry

ARTICLE

yellow. After stirring for 30 min, benzylbromide (1.1 mL, 9.26 mmol)

was added dropwise over 5 min, and the mixture was warmed to rt. After

6 h, no starting material (27) was detected by TLC (10% EtOAc in

hexanes). The off-white, opaque mixture was cooled to 0 °C, and 10% aq

NaHCO₃(10 mL) was added slowly. The DMF and H₂O were removed

by high vacuum rotary evaporation (∼1 mmHg). The resulting slurry

was partitioned between EtOAc (30 mL) and H₂O (30 mL). The layers

were separated, and the H₂O was extracted with EtOAc (1 ꢁ 30 mL).

The EtOAc layers were combined, washed with brine (1 ꢁ 30 mL),

dried over anhydrous MgSO₄, filtered, and concentrated. The crude

product was purified by column chromatography (1 ꢁ 6 in silica gel;

10%ꢀ20% EtOAc in hexanes) to give the desired product (28d) in 99%

yield as a clear, colorless liquid (2.64 g, 7.69 mmol). ¹H NMR (600 MHz,

CDCl₃) δ 7.38ꢀ7.28 (m, 5 H), 7.21ꢀ7.18 (m, 2 H), 6.87ꢀ6.84 (m,

2 H), 4.63 (s, 2 H), 4.55 (s, 2 H), 3.81 (s, 3 H), 1.52 (s, 9 H). ¹³C NMR

(150 MHz, CDCl₃) δ 159.7, 156.6, 136.9, 131.0, 128.7, 128.3, 127.7,

127.4, 113.7, 81.4, 76.8, 55.2, 53.9, 28.3. HRMS-ESI (m/z): [M + H]⁺

calcd for C₂₀H₂₆NO₄: 344.1856, found 344.1851. IR (neat): 3064.4,

Typical Procedure for the PMB-Deprotection Reactions: N-Benzyl-

N-(pyridin-2-yl)hydroxylamine (31d). Anhydrous TFA (3 mL) was

added slowly to a solution of compound 30d (100.0 mg, 0.31 mmol) and

triethylsilane (0.10 mL, 0.63 mmol) in 7 mL of anhydrous CH₂Cl₂. After

11 h, a small aliquot of the solution was quenched with satd aq NaHCO₃,

and no starting material (30d) was detected by TLC (20% EtOAc in

hexanes). The solution was diluted with CH₂Cl₂(20 mL) and washed

with satd aq NaHCO₃(1 ꢁ 30 mL). The CH₂Cl₂was separated, dried

over anhydrous MgSO₄, filtered, and concentrated to give a faint-green

liquid. The desired product (31d) was precipitated by trituration with

cold pentanes and isolated in 82% yield as white crystals (51.0 mg, 0.26

1

mmol); mp 116ꢀ118 °C. H NMR (600 MHz, CDCl₃) δ 8.25 (d,

J = 4.7 Hz, 1 H), 7.64ꢀ7.58 (m, 1 H), 7.41 (d, J = 7.0 Hz, 2 H), 7.35 (t,

J = 7.3 Hz, 2 H), 7.32ꢀ7.28 (m, 1 H), 7.13 (d, J = 8.5 Hz, 1 H), 6.83 (dd,

J = 7.0, 5.3 Hz, 1 H), 4.84 (s, 2 H). ¹³C NMR (150 MHz, CDCl₃) δ

162.1, 146.9, 137.8, 137.0, 129.0, 128.4, 127.5, 116.3, 109.7, 58.7.

HRMS-ESI (m/z): [M + H]⁺calcd for C₁₂H₁₃N₂O: 201.1022, found

201.1019. IR (neat): 3120.2 (br), 3068.3, 3029.9, 1591.2, 1432.2,

1213.4, 986.2 cm^ꢀ1. HPLC retention time 6.30 min.

3033.1, 3003.0, 1697.9, 1247.4, 1157.1, 1089.4, 1032.2 cm^ꢀ1

.

Typical Procedure for the N-Boc-deprotections of N-Boc-N-alkyl-

O-(4-methoxybenzyl)hydroxylamines: N-Benzyl-O-(4-methoxyben-

zyl)hydroxylamine (29d). Anhydrous TFA (2 mL) was added slowly

to a solution of compound 28d (1.14 g, 3.32 mmol) in 8 mL of

anhydrous CH₂Cl₂. The clear, colorless solution bubbled upon addition

of TFA. After 15 min, the TFA and CH₂Cl₂were evaporated under

reduced pressure, giving a clear, colorless liquid. This material was

dissolved in CHCl₃(25 mL) and washed with satd aq NaHCO₃(1 ꢁ

50 mL). The CHCl₃was separated, dried over anhydrous MgSO₄,

filtered, and concentrated. The crude product was purified by column

chromatography (1.25 ꢁ 6 in silica gel; 10%ꢀ20% EtOAc in hexanes) to

give the desired product (29d) in 77% yield as a clear, colorless liquid

(623.8 mg, 2.56 mmol). ¹H NMR (600 MHz, CDCl₃) δ 7.38ꢀ7.28 (m,

5 H), 7.27ꢀ7.24 (m, 2 H), 6.89ꢀ6.86 (m, 2 H), 5.69 (br s, NH, 1 H),

4.60 (s, 2 H), 4.05 (s, 2 H), 3.81 (s, 3 H). ¹³C NMR (150 MHz, CDCl₃)

δ 159.3, 137.6, 130.1, 129.9, 129.0, 128.4, 127.4, 113.7, 75.9, 56.5, 55.2.

HRMS-ESI (m/z): [M + H]⁺calcd for C₁₅H₁₈NO₂: 244.1332, found

244.1320. IR (neat): 3260.3 (br), 3062.0, 3030.5, 3001.4, 1611.0,

N-Benzylpyridin-2-amine (32). Compound 31d (2.7 mg, 0.013

mmol) was dissolved in 3 mL of MeOH in a round-bottom flask. The

flask was charged with 10% PdꢀC (0.2 mg) and sealed under argon. The

flask was flushed several times with hydrogen gas using intermediate

vacuum evacuations. The mixture was then left to stir at rt under a

hydrogen atmosphere (1 atm). After 30 min, no starting material (31d)

was detected by TLC (20% EtOAc in hexanes). The flask was flushed

with argon, and the mixture was vacuum filtered through Celite.

Evaporation of the MeOH gave pure product (32) in 97% yield as a

1

white solid (2.4 mg, 0.013 mmol). H NMR (300 MHz, CDCl₃) δ

ppm 8.04 (ddd, J = 5.0, 2.0, 1.0 Hz, 1 H), 7.40ꢀ7.16 (m, 5 H), 6.53 (ddd,

J = 7.1, 5.0, 0.8 Hz, 1 H), 6.31 (dt, J = 8.4, 1.1 Hz, 1 H), 4.87 (br s, NH, 1

H), 4.44 (d, J = 5.8 Hz, 2 H). HRMS-ESI (m/z): [M + H]⁺calcd for

C₁₂H₁₃N₂: 185.1073, found 185.1066. HPLC retention time 6.42 min.

Data was consistent with material from commercial sources (Sigma-

Aldrich Co.).

Typical Procedure for the O-Acetylation of N-Alkyl-N-(pyridin-2-

yl)hydroxylamines: O-Acetyl-N-benzyl-N-(pyridin-2-yl)hydroxylamine

(33). Compound 31d (8.0 mg, 0.04 mmol), Ac₂O (4.9 mg, 0.05 mmol),

iPr₂EtN (7.8 mg, 0.06 mmol), and catalytic DMAP (1.0 mg, 0.01 mmol)

were dissolved in 4 mL of anhydrous CH₂Cl₂, respectively. After 12 h, no

starting material (31d) was detected by TLC (20% EtOAc in hexanes).

The CH₂Cl₂was evaporated, and the crude product was purified by

column chromatography (0.25 ꢁ 5 in silica gel; 10%ꢀ20% EtOAc in

hexanes) to give the desired product (33) in 72% yield as a clear, faint-

yellow oil (7.0 mg, 0.03 mmol). ¹H NMR (300 MHz, CDCl₃) δ 8.36

(ddd, J = 4.9, 1.7, 0.8 Hz, 1 H), 7.65ꢀ7.57 (m, 1 H), 7.42ꢀ7.25 (m, 5 H),

6.94 (ddd, J = 7.2, 4.9, 0.8 Hz, 1 H), 6.80 (dt, J = 8.5, 0.9 Hz, 1 H), 5.01

(s, 2 H), 2.04 (s, 3 H). ¹³C NMR (75 MHz, CDCl₃) δ 176.3, 160.4,

147.7, 137.9, 135.9, 129.3, 128.3, 127.6, 117.8, 109.9, 57.4, 18.8. HRMS-ESI

(m/z): [M + H]⁺calcd for C₁₄H₁₅N₂O₂: 243.1128, found 243.1137. IR

(neat): 3063.1, 3031.2, 3008.4, 1780.6, 1590.0, 1467.2, 1434.7,

1184.1 cm^ꢀ1. HPLC retention time 7.19 min.

(()5-Bromo-N-((1S/R,4S/R)-4-methoxycyclohex-2-enyl)pyridin-

2-amine (34). This compound was prepared following a general method

reported previously.⁴⁸Activated zinc (65 mg, 1.00 mmol) was added to a

solution of Cp₂TiCl₂(124.0 mg, 0.50 mmol) in 3 mL of anhydrous THF

in a flame-dried round-bottom flask purged with argon. This mixture was

stirred at rt for 45 min. The reaction mixture changed color from dark-

red to olive-green. The mixture was cooled to ꢀ30 °C, and a solution of

7b (60.0 mg, 0.20 mmol) in 2 mL of MeOH was added dropwise over 3

min. The mixture was stirred for 1 h, while the bath temperature was

maintained between ꢀ10 and ꢀ30 °C. The mixture was warmed to rt

and partitioned between satd aq K₂CO₃(5 mL) and EtOAc (15 mL).

The organic layer was separated and filtered through a Whatman glass

1511.7, 1244.7, 1031.9 cm^ꢀ1

.

Typical Procedure for the BuchwaldꢀHartwig Amination Cross-

Coupling Reactions: N-Benzyl-O-(4-methoxybenzyl)-N-(pyridin-2-

yl)hydroxylamine (30d). This procedure followed a general method

reported previously by Buchwald and co-workers.⁴⁷Hydroxylamine 29d

(300.0 mg, 1.23 mmol), 2-bromopyridine (0.10 mL, 1.00 mmol),

Pd₂(dba)₃(18.0 mg, 0.02 mmol), (() BINAP (25.0 mg, 0.04 mmol),

and NaO^tBu (134.0 mg, 1.40 mmol) were dissolved in 9 mL of

anhydrous toluene, respectively, in an oven-dried, argon-flushed Schlenk

tube. The brown mixture was heated to 70 °C (oil bath temperature)

overnight. The mixture turned darker brown over the course of the

reaction. After 64 h, the mixture was cooled to rt, diluted with Et₂O

(20 mL), and filtered through Celite. The clear, orange solution was

washed with brine (1 ꢁ 20 mL), dried over anhydrous MgSO₄, filtered,

and concentrated. The crude product was purified by column chroma-

tography (1 ꢁ 6 in silica gel; CH₂Cl₂as eluent) to give the desired

product (30d) in 63% yield as a clear, colorless liquid (201.2 mg, 0.63

mmol). ¹H NMR (600 MHz, CDCl₃) δ 8.31 (ddd, J = 4.8, 1.9, 0.9 Hz,

1 H), 7.60ꢀ7.57 (m, 1 H), 7.46ꢀ7.44 (m, 2 H), 7.36ꢀ7.32 (m, 2 H),

7.32ꢀ7.28 (m, 1 H), 7.19ꢀ7.16 (m, 2 H), 7.10 (dt, J = 8.2, 0.9 Hz, 1 H),

6.87ꢀ6.85 (m, 2 H), 6.82 (ddd, J = 7.3, 4.8, 0.9 Hz, 1 H), 4.87 (s, 2 H),

4.56 (s, 2 H), 3.81 (s, 3 H). ¹³C NMR (150 MHz, CDCl₃) δ 162.4,

159.6, 147.8, 137.9, 137.6, 130.8, 129.7, 128.1, 128.0, 127.2, 116.3,

113.8, 109.2, 76.2, 57.4, 55.2. HRMS-ESI (m/z): [M + H]⁺calcd for

C₂₀H₂₁N₂O₂: 321.1598, found 321.1599. IR (neat): 3061.7, 3031.0,

3004.1, 1587.0, 1432.8, 1247.3, 1030.9 cm^ꢀ1. HPLC retention time

9.78 min.

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dx.doi.org/10.1021/jm200794r |J. Med. Chem. 2011, 54, 6843–6858

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ARTICLE

microfiber filter (type GF/F). The aq layer was extracted with EtOAc

(2 ꢁ 10 mL), and the separated organic layer was filtered through a

Whatman glass microfiber filter (type GF/F) each time. The combined,

filtered organics were dried over Na₂SO₄, filtered through a Whatman

glass microfiber filter (type GF/F), and concentrated. The crude

product was purified by column chromatography (0.5 ꢁ 6 in silica

gel; 25% EtOAc in hexanes) to give the desired product (34) in 73%

yield as a white solid (41.6 mg, 0.15 mmol); mp 50ꢀ52 °C. ¹H NMR

(600 MHz, DMSO-d₆) δ 8.01 (dd, J = 2.5, 0.4 Hz, 1 H), 7.48 (dd, J = 8.9,

2.5 Hz, 1 H), 6.74 (d, J = 6.5 Hz, 1 H), 6.51 (dd, J = 8.9, 0.7, 1 H),

5.86ꢀ5.81 (m, 1 H), 5.74ꢀ5.70 (m, 1 H), 3.99ꢀ3.93 (m, 1 H), 3.71 (td,

J = 4.4, 2.0 Hz, 1 H), 3.28 (s, 3 H), 2.12ꢀ2.05 (m, 2 H), 1.89ꢀ1.81

(m, 1 H), 1.55ꢀ1.46 (m, 1 H). ¹³C NMR (150 MHz, DMSO-d₆) δ

157.1, 147.4, 138.8, 129.9, 126.3, 110.7, 104.7, 77.2, 55.4, 49.1, 25.7, 23.2.

HRMS-ESI (m/z): [M + H]⁺calcd for C₁₂H₁₆BrN₂O: 283.0441, found

283.0455. IR (neat): 3301.6 (br), 3161.9, 3131.0, 3071.3, 3032.0, 3001.8,

1593.8, 1498.8, 1099.3, 1085.8 cm^ꢀ1. HPLC retention time 7.48 min.

N-(4-Methoxybenzyloxy)-N-benzyl(pyridin-2-yl)methanamine (38).

Compound 29d (36.0 mg, 0.15 mmol), 2-(bromomethyl)pyridine hydro-

bromide (56.2 mg, 0.22 mmol), and iPr₂EtN (0.10 mL, 0.57 mmol) were

dissolved in 6 mL of anhydrous CH₃CN, respectively. This clear, red

solution was heated to 50 °C (oil bath temperature) overnight. After 17 h,

only trace starting material (29d) was detected by TLC (33% EtOAc in

hexanes). The reaction mixture was cooled to rt, and the CH₃CN was

evaporated under reduced pressure. The resulting material was purified by

silica gel column chromatography (0.75 ꢁ 4 in silica gel; 20%ꢀ40%

EtOAc inhexanes) to give thedesired product (38) in 44% yield as a clear,

faint-yellow oil (22.0 mg, 0.07 mmol). ¹H NMR (600 MHz, CDCl₃) δ

8.61 (ddd, J = 4.9, 1.8, 0.9 Hz, 1 H), 7.67 (td, J = 7.6, 1.8 Hz, 1 H),

7.47ꢀ7.43 (m, 3 H), 7.37ꢀ7.33(m, 2 H), 7.33ꢀ7.28(m, 1H), 7.21 (ddd,

J = 7.6, 4.9, 1.3 Hz, 1 H), 6.88ꢀ6.84 (m, 2 H), 6.76ꢀ6.73 (m, 2 H), 4.15

(s, 2 H), 4.07 (s, 2 H), 3.96 (s, 2 H), 3.76 (s, 3 H). ¹³C NMR (150 MHz,

CDCl₃) δ 159.2, 157.8, 149.2, 137.4, 136.1, 130.5, 130.0, 129.1, 128.1,

127.3, 124.2, 122.2, 113.5, 75.4, 64.7, 63.2, 55.2. HRMS-ESI (m/z):

[M + H]⁺calcd for C₂₁H₂₃N₂O₂: 335.1754, found 335.1738. IR (neat):

for 18ꢀ24 h, and the inhibition zone diameters were measured (mm)

with an electronic caliper after 24ꢀ48 h.

Antibiotic Susceptibility Testing by the Broth Microdilution Method.

Antibacterial activity of the compounds was determined by measuring

their minimum inhibitory concentrations (MIC₉₀s) using the broth

microdilution method according to the Clinical and Laboratory Stan-

dards Institute (CLSI, formerly the NCCLS) guidelines.⁵²Each well of a

96-well microtiter plate was filled with 50 μL of sterile MHII broth. Each

test compound was dissolved in DMSO making a 20 mM solution, then

diluted with sterile MHII broth to 512 μM. Exactly 50 μL of the

compound solution was added to the first well of the microtiter plate,

and 2-fold serial dilutions were made down each row of the plate. Exactly

50 μL of bacterial inoculum (5 ꢁ 10⁵CFU/mL in MHII broth) was then

added to each well, giving a total volume of 100 μL/well and a final

compound concentration gradient of 128 μMꢀ0.0625 μM. The plate

was incubated at 37 °C for 18 h, and then each well was examined for

bacterial growth. The MIC₉₀was recorded as the lowest compound

concentration (μM) required to inhibit 90% of bacterial growth as

judged by turbidity of the culture media relative to a row of wells filled

with a DMSO standard. Ciprofloxacin was included in a control row at a

concentration gradient of 32 μMꢀ0.0156 μM.

’ ASSOCIATED CONTENT

S

Supporting Information. Complete list of compounds

b

subjected to biological testing. Complete list of biological data

collected during this study. Complete list of strains, markers, and

origins for microorganisms used in this work. Experimental

procedures for the preparation of unpublished compounds not

given in main article text. Copies of H NMR and ¹³C NMR

1

spectra for all unpublished compounds (9,11a, 12, 14, 16, 18aꢀi,

19aꢀc, 27, 28cꢀi, 29aꢀi, 30aꢀi, 31aꢀi, 33ꢀ40a). Copies of

HPLC chromatograms for compounds subjected to biological

testing (7a, 7b, 9, 11a, 12, 14, 16, 18aꢀi, 19aꢀc, 30aꢀi, 31aꢀi,

32ꢀ40a). Crystal data, images of unit cell, and tables of atom

coordinates and thermal parameters for the X-ray diﬀraction

analysis of compound 40a. CCDC 824255 contains the supple-

mentary crystallographic data for this paper. These data can be

obtained free of charge from The Cambridge Crystallographic

Data Centre via www.ccdc.cam.ac.uk/data_request/cif. This

material is available free of charge via the Internet at http://

pubs.acs.org.

3062.3, 3031.4, 3007.4, 1611.97, 1588.8, 1513.3, 1247.4, 1032.5 cm^ꢀ1

HPLC retention time 8.81 min.

.

Microbiology. General Materials and Methods. All liquids and

media were sterilized by autoclaving (121 °C, 15 min) before use. All aq

solutions and media were prepared using distilled, deionized, and filtered

water (Millipore Milli-Q Advantage A10 Water Purification System).

Luria broth (LB) was purchased from VWR. Mueller-Hinton no. 2 broth

(MHII broth; cation adjusted) was purchased from Sigma-Aldrich

(St. Louis, MO). Mueller-Hinton no. 2 agar (MHII agar; HiMedia

Laboratories) was purchased from VWR. McFarland BaSO₄turbidity

standards were purchased from bioMꢀerieux, Inc. Sterile plastic Petri

dishes (145 mm ꢁ 20 mm; Greiner Bio-One) were purchased from

VWR. Ciprofloxacin was purchased from Sigma-Aldrich (St. Louis,

MO). Amphotericin B was purchased from Amresco (Solon, OH). All

test organisms used in this work were from sources given in the

Supporting Information (Table S3).

’ AUTHOR INFORMATION

Corresponding Author

*Phone: (574) 631-7571. Fax: (574) 631-6652. E-mail: mmiller1@

nd.edu.

’ ACKNOWLEDGMENT

We gratefully acknowledge the use of NMR facilities provided

by the Lizzadro Magnetic Resonance Research Center at The

University of Notre Dame (UND) and the mass spectrometry

services provided by The UND Mass Spectrometry & Proteo-

mics Facility (N. Sevova, Dr. W. Boggess, and Dr. M. V. Joyce;

supported by the National Science Foundation under CHE-

0741793). We thank Patricia A. Miller (UND) for anticancer

testing and assistance with antibacterial susceptibility testing. We

thank Dr. Ute M€ollmann (HKI) for antibacterial testing against

the resistant strains S. aureus 134/94 (MRSA) and E. faecalis 1528

(VRE) and for many helpful discussions. We thank Dr. Viktor

Krchꢁnꢀak (UND) for assistance with analytical and preparative

Antibiotic Susceptibility Testing by the Agar Diffusion Method.

Antibacterial activity of the compounds was determined by a modified

KirbyꢀBauer agar diffusion assay.⁵⁰Overnight cultures of test organ-

isms were grown in LB broth for 18ꢀ24 h and standard suspensions of

1.5 ꢁ 10⁶CFU/mL were prepared in saline solution (0.9% NaCl)

according to a 0.5 BaSO₄McFarland Standard.⁷⁶Each standardized

suspension (0.1 mL) was added to 34 mL of sterile, melted MHII agar

tempered to 47ꢀ50 °C. After gentle mixing, the inoculated agar media

was poured into a sterile plastic Petri dish (145 mm ꢁ 20 mm) and

allowed to solidify near a flame with the lid cracked for ∼30 min. Wells of

9.0 mm diameter were cut from the Petri dish agar and filled with exactly

50 μL of the test sample solution. The Petri dish was incubated at 37 °C

6855

dx.doi.org/10.1021/jm200794r |J. Med. Chem. 2011, 54, 6843–6858

Journal of Medicinal Chemistry

ARTICLE

HPLC and for helpful discussions. T.A.W. gratefully acknowl-

edges The UND ChemistryꢀBiochemistryꢀBiology Interface

(CBBI) Program funded by NIH (T32GM075762) for three

years of fellowship support. T.A.W. also thanks The UND

Department of Chemistry and Biochemistry (Grace Fellowship),

the Center for Environmental Science and Technology (CEST),

and Bayer for additional fellowship support. B.Y. acknowledges a

Reilly Graduate Fellowship, and J.R.R. acknowledges a College of

Science Summer Undergraduate Research Fellowship (COS-

SURF), both sponsored by UND. We acknowledge The Uni-

versity of Notre Dame and the NIH (GM 075855) for support-

ing this work.

Kamovsky, A.; Kuhn, M.; Limberakis, C.; Liu, J. Y.; Mehrens, S.; Mueller,

W. T.; Narasimhan, L.; Ogden, A.; Ohren, J.; Prasad, J. V. N. V.; Shelly,

J. A.; Skerlos, L.; Sulavik, M.; Thomas, V. H.; VanderRoest, S.; Wang, L.;

Wang, Z.; Whitton, A.; Zhu, T.; Stover, C. K. A Class of Selective

Antibacterials Derived from a Protein Kinase Inhibitor Pharmacophore.

Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 1737–1742.

(11) Walsh, C. T.; Fischbach, M. A. Repurposing Libraries of Eukar-

yotic Protein Kinase Inhibitors for Antibiotic Discovery. Proc. Natl. Acad.

Sci. U.S.A. 2009, 106, 1689–1690.

(12) Silver, L. L. Are Natural Products Still the Best Source for

Antibacterial Discovery? The Bacterial Entry Factor. Expert Opin. Drug

Discovery 2008, 3, 487–500.

(13) O’Shea, R.; Moser, H. E. Physiochemical Properties of Anti-

bacterial Compounds: Implications in Drug Discovery. J. Med. Chem.

2008, 51, 2871–2878.

(14) For reviews of FBDD see:(a) Murray, C. W.; Rees, D. C. The

Rise of Fragment-Based Drug Discovery. Nature Chem. 2009,

1, 187–192. (b) Congreve, M.; Chessari, G.; Tisi, D.; Woodhead, A. J.

Recent Developments in Fragment-Based Drug Discovery. J. Med.

Chem. 2008, 51, 3661–3680. (c) Erlanson, D. A.; McDowell, R. S.;

O’Brien, T. Fragment-Based Drug Discovery. J. Med. Chem. 2004, 47,

3463–3482.

(15) For successful application of FBDD for antibacterialdiscovery see:

(a) Mochalkin, I.; Miller, J. R.; Narasimhan, L.; Thanabal, V.; Erdman, P.;

Cox, P. B.; Prasad, J. V. N. V.; Lightle, S.; Huband, M. D.; Stover, C. K.

Discovery of Antibacterial Biotin Carboxylase Inhibitors by Virtual Screen-

ing and Fragment-Based Approaches. ACS Chem. Biol. 2009, 4, 473–483.

(b) Waldrop, G. L. Smaller is Better for Antibiotic Discovery. ACS Chem.

Biol. 2009, 4, 397–399.

(16) For successful application of computational structure-based

design methods for antibacterial discovery, see:Shen, Y.; Liu, J.; Estiu,

G.; Isin, B.; Ahn, Y.-Y.; Lee, D.-S.; Barabꢀasi, A.-L.; Kapatral, V.; Wiest,

O.; Oltvai, Z. N. Blueprint for Antimicrobial Hit Discovery Targeting

Metabolic Networks. Proc. Natl. Acad. Sci. U.S.A. 2010, 107,

1082–1087.

’ ABBREVIATIONS USED

Ac, acetyl; ATCC, American Type Culture Collection; BINAP,

2,2⁰-bis(diphenylphosphino)-1,1⁰-binaphthyl; Bn, benzyl; Boc, tert-

butoxycarbonyl; CFU, colony forming unit; Cp, cyclopenta-

dienyl; dba, dibenzylideneacetone; DMAP, 4-dimethylaminopyr-

idine;DMF, dimethylformamide;DMSO, dimethylsulfoxide;DOS,

diversity-oriented synthesis; DXR, 1-deoxy-^D-xylulose-5-phos-

phate reductoisomerase; ESI, electrospray ionization; FBDD,

fragment-based drug design; HPLC, high-performance liquid

chromatography; HRMS, high-resolution mass spectrometry;

HTS, high-throughput screening; IR, infrared; MEND, modular

enhancement of nature’s diversity; MEP, methylerythritol phos-

phate; MHII, MuellerꢀHinton media no. II; MIC, minimum

inhibitory concentration; MRSA, methicillin-resistant Staphylococcus

aureus; NMR, nuclear magnetic resonance; PG, protecting group;

PMB, 4-methoxybenzyl; SAR, structureꢀactivity relationship; TFA,

trifulouroacetic acid; THF, tetrahydrofuran; TLC, thin layer chroma-

tography; VRE, vancomycin-resistant Enterococci

(17) For successful applications of target-based HTS for antibacter-

ial discovery see: (a) Jarvest, R. L.; Berge, J. M.; Berry, V.; Boyd, H. F.;

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N-O chemistry for antibiotics: Discovery of N-alkyl-N-(pyridin-2-yl) hydroxylamine scaffolds as selective antibacterial agents using nitroso diels-alder and ene chemistry

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