Development of a novel series of non-natural triaryl agonists and antagonists of the Pseudomonas aeruginosa LasR quorum sensing receptor

Accepted Manuscript

Development of a novel series of non-natural triaryl agonists and antagoinsts of

the Pseudomonas aeruginosa LasR quorum sensing receptor

Joseph N. Capilato, Shane Philippi, Thomas Reardon, Ashleigh McConnell,

Dylan Oliver, Amy Warren, Jill Adams, Chun Wu, Lark J. Perez

PII:

S0968-0896(16)30741-6

DOI:

http://dx.doi.org/10.1016/j.bmc.2016.10.021

BMC 13348

Reference:

Bioorganic & Medicinal Chemistry

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Revised Date:

Accepted Date:

16 September 2016

13 October 2016

17 October 2016

Please cite this article as: Capilato, J.N., Philippi, S., Reardon, T., McConnell, A., Oliver, D., Warren, A., Adams,

J., Wu, C., Perez, L.J., Development of a novel series of non-natural triaryl agonists and antagoinsts of the

Pseudomonas aeruginosa LasR quorum sensing receptor, Bioorganic & Medicinal Chemistry (2016), doi: http://

dx.doi.org/10.1016/j.bmc.2016.10.021

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Development of a Novel Series of Non-natural

Triaryl Agonists and Antagoinsts of the

Pseudomonas aeruginosa LasR Quorum Sensing

Receptor

Joseph N. Capilato, Shane Philippi, Thomas Reardon, Ashleigh McConnell, Dylan Oliver, Amy Warren, Jill

Adams, Chun Wu and Lark J. Perez*

Department of Chemistry and Biochemistry, Rowan University, 201 Mullica Hill Road, Glassboro, NJ 08028,

United States

Bioorganic & Medicinal Chemistry

journal homepage: www.els evier.com

Development of a novel series of non-natural triaryl agonists and antagoinsts of the

Pseudomonas aeruginosa LasR quorum sensing receptor

Joseph N. Capilato, Shane Philippi, Thomas Reardon, Ashleigh McConnell, Dylan Oliver, Amy Warren,

Jill Adams, Chun Wu and Lark J. Perez*

^aDepartment of Chemistry and Biochemistry, Rowan University, 201 Mullica Hill Road, Glassboro, NJ 08028, United States

ARTICLE INFO

ABSTRACT

Article history:

Received

Received in revised form

Accepted

Bacterial chemical communication, through a process called quorum sensing (QS), plays a

central role in infection in numerous bacterial pathogens. Quorum sensing in Pseudomonas

aeruginosa employs a series of small molecule receptors including the master QS regulator,

LasR. In this study we investigate a non-natural triaryl series of LasR ligands using a

combination of structure activity relationship studies and computational modeling. These studies

have enabled the identification of key structural requirements for ligand binding and have

revealed a new strategy for inducing the therapeutically relevant antagonism of LasR.

Available online

Keywords:

Anti-virulence

.

Pseudomonas aeruginosa

Structure-activity relationship

Computational Modeling

Quorum sensing

1. Introduction

treatments provide a considerable growth advantage to bacteria

that can resist the drug. Contrasting this approach, the inhibition

of quorum sensing does not affect cellular viability, decreasing

the growth advantage of resistance.¹³Rather, this approach

targets cellular signaling, inhibiting the expression of virulence

factors necessary for successful infection, rendering the bacteria

Due to the array of medically relevant cellular processes that

are under the control of LuxR-type quorum sensing circuits,

significant effort has been directed toward the identification of

antagonist and agonist molecules in several different bacterial

pathogens.^1-3These efforts have led to the identification of

antagonists and agonists of quorum sensing from natural

sources,⁴diversity-oriented compound libraries,^5,6and focused

library synthesis.^7,8The most broadly explored class of LuxR-

type receptor ligands are based on the structural features found in

the native HSLs (e.g. 1, Figure 1). The wealth of studies in this

area, maintaining the homoserine lactone while changing the

functionality of the acyl-tail is facilitated by ready synthesis of

the HSL compound libraries. Together these investigations

targeting the modulation of quorum sensing in Gram-negative

bacteria have yielded numerous promising results and insightful

structure activity relationships.^9,10

benign until the hosts immune system can clear the pathogen.^2,14-

16

Major challenges that remain in the development of an anti-

quorum sensing strategy targeting the development of antagonists

of LasR and other LuxR-type receptors is ligand potency and the

complications of hydrolytic liability in HSL-based antagonists.¹⁷

Here we advance a strategy to overcome the limitation of

inhibitor potency through the exploration of a novel, non-natural

triaryl series of LasR quorum sensing ligands. Further, many of

the analogs examined in this study are anticipated to overcome

the limitations of the HSL-based agonists and antagonists by

removing the physiologically labile homoserine lactone

functionality.¹⁸

The inhibition of quorum sensing with small molecules offers

a unique strategy for the treatment and prevention of a number of

acute and chronic bacterial infections, a fact that is of particular

significance given the rapid and ongoing spread of antimicrobial-

resistant bacteria.^2,11,12Through the inhibition of quorum sensing,

control over expression of virulence factors and biofilm

formation is achieved, rendering the bacteria benign while not

directly affecting viability. This anti-infective approach is

conceptually distinct from traditional approaches for the

treatment of bacterial infection that typically utilize bactericidal

or bacteriostatic molecules, targeting critical cellular processes.

As exemplified by the spread of bacterial resistance, these

Recently a highly potent agonist of quorum-sensing in P.

aeruginosa was identified from a high-throughput screen (2,

Figure 2).⁵While the activation of virulence factor expression in

this bacterial pathogen is not therapeutically desirable our

rationale to obtain the desired inhibitory function for this potent

agonist through analog synthesis is based on precedent. Most

notably, a large number of inhibitors have been discovered by

systematic modification of HSLs, molecules which serve as

natural QS activators. The agonist discovered (2) bears many

structural and pharmacological properties that potentially make it

superior to HSLs as a lead compound for the development of an

anti-virulence therapy, including the absence of the metabolically

labile homoserine lactone. Additionally, the potency of this

molecule for activation of LasR is comparable to the potency of

the natural signaling molecule; however, compounds of this

structure type have never been systematically analyzed for the

effect of their structure-activity relationships (SARs).^5,19Here we

describe the first systematic evaluation of the structure-activity

relationships of this novel class of LasR QS modulators. Our

studies reveal critical details of the activity profile of this new

ligand class for LasR activity and using computational modeling

we provide insight into the molecular interactions with LasR that

contribute to agonist and antagonist activity.

(Figure 1, highlighted in blue) may be tolerated. Indeed,

examination of the scaffold resulting from the inversion of the

amide linker between the “B-ring” and “C-ring” (highlighted in

red, 3) more closely maps onto the orientation of this structure in

the native LasR agonist, 3-oxo-C12HSL (1).

Based on these target scaffolds, we have successfully

identified two efficient and modular synthetic routes (Figure 1) to

analogs containing the core scaffold as in 2 (Scaffold #1) analogs

containing the core scaffold as in 3 (Scaffold #2). In our

optimized six step synthesis of 2 and analogs of this structure,

bromination and benzyl protection of salicylamide is followed by

reduction of the amide and introduction of the “C-ring” through

amidation with 11. Final incorporation of the “A-ring” is

achieved through deprotection of the phenol and esterification

with 8. This route is modular, enabling ready diversification of

the “A-ring” and “C-ring” of the ligand and proceeds in 35%

yield overall from salicylamide. The synthesis of 3 and related

analogs involves initial coupling of 4 and 2-nitro aniline (5).

Deprotection of the phenol and bromination of the more electron

rich “B-ring” proceeds smoothly to provide 7 which can be

directly esterified with 8. Overall, this route allows rapid

diversification of the “A-ring” and “C-ring” of 3, proceeding in

four steps with 52% overall yield from 4.

2. Library Synthesis.

Challenging our efforts toward the goal of identifying

inhibitors of LasR based on the structure of 2 is the number of

synthetic steps required for its assembly (Figure 1).¹⁹Previous

studies had described an eight step synthesis of 2, however, to

prepare a library of molecules related to 2 ideally required a more

efficient synthetic route. Identification of an alternative scaffold

to target for structure-activity relationship studies was informed

by examination of the recently published crystal structure of the

QS receptor in P. aeruginosa, LasR, in the presence of 2.²⁰

Analysis of the binding pocket for the ligand suggested that a

subtle structural change in the orientation of the amide bond in 2

3. Library Design and SAR.

Analysis of the crystal structure of LasR complexed to 2²⁰

suggests that the two terminal aromatic rings (Figure 1, labeled

“A” and “C”) play an important role by interacting with LasR

whereas the central ring appears to play primarily a scaffolding

role in ligand binding. Therefore, we have focused our initial

efforts on identifying the structure activity correlations of the

pendant ring structures (“A-ring” and “C-ring”) while

maintaining the structure of the central ring (“B-ring”). Our

strategy involved initial broad investigation of structure-activity

relationships in the more readily synthesized ligand series

(Scaffold #2, e.g. 3) before returning to investigate key analogs

prepared based on Scaffold #1. While the two scaffolds differ

only in the orientation of one amide bond, we have observed

some notable differences in biological activity for analogs

prepared on the two different scaffolds.

3.1. “A-ring” SAR and computational analysis.

Our initial investigations of the structure activity relationships

involved modulation of the structure of the “A-ring” in Scaffold

#2. From this series of structural analogs, we observe a consistent

dependence of substitution in this ring structure on the potency of

the molecules (Table 1). We discovered that substitution in the

ortho-position of the arene (entries 1, 4, 7, 9, 10, and 13-14) is

critical for agonist activity. Interestingly, the activity of the

analogs bearing ortho-substitution appears to be largely

independent of the nature of the substituent. Analogs with

electon-withdrawing substituents (halogen, nitro, trifluoromethyl)

and electron-donating groups (methyl, methoxy) in the ortho-

position all are agonists with EC₅₀values between 2.4 and 15.8

µM. By contrast to substitution in the ortho-position of the “A-

ring”, substitution in the meta- or para-position of the “A-ring”

(entries 2-3, 5-7 and 11-12) leads to the complete loss of activity.

In support of the significance of ortho-substitution, an analog

was prepared containing chlorine atoms at both of the ortho-

positions of the “A-ring” (entry 16) fully retains the activity of

the mono-ortho-chloro LasR agonist 3. We additionally find that

the carbonyl of the ester is not significant for agonist activity as

an analog removing this functionality, linking the “A-ring”

through an ether linker (28, entry 17) retains full agonist activity.

Figure 1. Top; the structures of the native agonist of the QS receptor, LasR,

3-oxo-C12HSL and the triaryl scaffolds evaluated as LasR agonists and

antagonists in this study. Bottom; a description of the modular chemical

synthesis pathways for library assembly.

Table 1. Structure-activity relationships of “A-ring” analogs.

a

EC₅₀values are reported as the mean of triplicate analysis with the range defining the standard deviation. The native

LasR agonist (1) was used as a control with an EC₅₀=72.9 24.6nM. ^bMaximal percent activation of GFP at 50 µM with

respect to 3-oxo-C12 HSL (1) as a positive control for LasR agonism, set at 100% activation.

Generally we find that analogs with substituents in the ortho-

position are potent agonists whereas the meta- or para-

substituted analogs are universally not active. This may suggest

that the binding pocket in LasR for this portion of the molecule is

not able to accommodate substituents in either the meta- or para-

position. Further, the comparable activities of the analogs

containing electronically and sterically diverse substituents in the

ortho-position suggests that this substitution does not have a

direct role interacting with the receptor (no specific hydrogen-

bonding or electronic interactions with the receptor) but rather

may simply play an anchoring role for the overall positioning of

the “A-ring”. In support of this hypothesis, analog 26, containing

an ortho-bromo substituent and a para-fluoro substituent is active

as a LasR agonist (entry 15), while the analog containing the

para-fluoro substituent alone is inactive (entry 8). Further

supporting the significance of maintaining an ortho-substituent

on the “A-ring” is the lack of agonist activity found in analog 29

(entry 18) that possesses an unsubstituted phenyl ring at this

position.

The ligands that were inactive as agonists in this series were

further evaluated as antagonists and were found to have no

antagonist activity. This observation suggests that the presence of

minimally one ortho substituent on the “A-ring” of ligands in this

series is required for ligand binding to LasR in the cell.

Figure 2. Computational binding contributions of individual amino acids in

LasR with the ligands in Table 1. Energies are described in kcal/mol.

To further investigate the strict requirement for the presence

of an ortho-substituent on the “A-ring” for LasR agonist activity

we performed computational binding studies using the

Schrodinger Drug Discovery Suite. We were pleased to find that

the MMGBSA scores (measure of binding affinity) from this

computational binding analysis for the full series of compounds

in Table 1 was strongly correlated to the observed biological

activity of the analogs with the ortho-substituted analogs

showing the strongest binding and the unsubstituted phenyl “A-

ring” analog (29) predicted to be a poor LasR ligand (Table 2).

HSL-binding proteins, Y56 and D73 (Figure 3, yellow), display

different patterns of binding between the analogs dependent on

the substitution of the “A-ring”. While Y56 shows strongly

stabilizing interactions with all analogs, by virtue of a hydrogen

bond to the carbonyl oxygen of the linker amide, this stabilizing

interaction appears to be slightly weakened in the binding of the

para-substituted ligands (Figure 2). In contrast, the interactions

between the ligand and D73 are calculated to be generally low in

energy with ortho- and meta-substituted analogs however

becomes significantly destabilizing in interactions with ligands

Table 2. Calculated binding energies for “A-ring” analogs.

MMGBSA Binding

bearing

a para-substituent in the “A-ring”. Apparently,

substitution in the para-position of the distant “A-ring” is

effectively propagated through the ligand to disrupt the hydrogen

bonding interactions between LasR and the linker amide of the

ligand.

Energy (kcal/mol)

Ortho-Substituted Analogs^a

Meta-Substituted Analogs^b

Para-Substituted Analogs^c

–87.12 20.87^d

–37.35 6.15^d

–18.86 27.79^d

–11.05

Not surprisingly, a series of amino acids in the binding pocket

proximal to the “A-ring” were found to be most significantly

influenced by the structural changes investigated in this ligand

series. While much of this large portion of the HSL binding

pocket in LasR is hydrophobic, key amino acids displaying

significant differences in binding to the structural variations in

the “A-ring” include Y47, R61, D65, V76, G126 and A127

(Figure 3, magenta). Two primary trends are noted in the binding

contribution of amino acids in this region of LasR. Firstly, in

stabilizing interactions the ortho-substituted analogs show greater

stabilization then meta- or para-substituted analogs. This trend is

Unsubstituted Phenyl Analog 29

a

Average calculated binding energy of analogs 3, 15, 18, 20, 21,

24 and 25. ^bAverage calculated binding energy of analogs 13, 16

and 22. ^cAverage calculated binding energy of analogs 14, 17, 19

and 23. MMGBSA is reported as the average binding energy

d

with the error representing standard deviation.

Building on this result we computationally investigated the

relative contributions for each of the amino acids within the LasR

binding pocket that, in sum, contribute to the overall MMGBSA

score for the compound. We hypothesized that this analysis may

enable the identification of key residues in LasR that contribute

to ligand binding rationalizing that the meta- and para-

substituted “A-ring” analogs would lack these stabilizing

interactions or would display destabilizing interactions. We

identified several individual amino acids that showed variations

in their extracted contribution to the computational binding

energies (Figure 2).

Generally, amino acids residues proximal to the 2-nitro aniline

“C-ring” of the analog, not varied in this series of analogs,

displayed similar binding energies to each of the ligands,

irrespective of the substitution pattern on the “A-ring”. For

example, W60 and F101 (Figure 3, light blue) independently

contribute stabilizing binding interactions to all of the analogs

evaluated (Figure 2).

Figure 3. Key residues in LasR shown with 3 docked. W60 and F101 are

shown in light blue, Y56 and D73 are shown in yellow and Y47, R61, D65,

V76, G126 and A127 are shown in magenta. Inset: a schematic

representation highlighting these amino acids in proximity to three regions

of the ligand.

Some variations in the extracted binding energies are noted in

the amino acid residues responsible for interactions with the

amide linker between the “C-ring” and “B-ring” of the analogs.

Amino acid residues which are highly conserved among soluble

exemplified in the binding contributions of Y47, V76 and G126

(Figure 2). Secondly, two residues show strong destabilizing

interactions uniquely with the para-substituted analogs, D65 and

A127 (Figure 3). These residues are located on opposing sides of

the binding pocket possibly suggesting that a steric clash on one

side of the ligand binding pocket pushes the ligand toward the

opposing side of the binding pocket resulting in destabilizing

interactions with these two spatially separated amino acids.

Lastly, it should be noted that not all amino acids showed a

preference for the ortho-substituted analogs. One residue

analyzed, R61 shows significant destabilizing interactions with

both ortho- and meta-substituted analogs that is alleviated in the

para-substituted series of compounds.

Table 3. Structure-activity relationships of “C-ring”

analogs.

3.2. “C-ring” SAR and computational analysis.

While most analogs that we have analyzed with changes in the

structure of the “A-ring” were found to be either agonists or to

have no activity, analogs with structural changes in the “C-ring”

have both changes in potency and changes in function (agonism

vs. antagonism). We first assessed the impact of the position of

the nitro-substituent on the activity of the molecules (Table 3,

entries 1-3), revealing the importance of an ortho-nitro

substituent. We then prepared a series of analogs in which the

ortho-nitro group on the “C-ring” of the parent compound (3)

was exchanged for functionality that had been previously found

to be a suitable bioisostere of an aromatic nitro group.²¹While

the 2-pyridine was observed to have no agonist activity (entry 4)

the di-fluoro bioisostere was found to show enhanced activity as

a LasR agonist (entry 5). Introducing additional electron-

withdrawing substituents onto the ring (entry 6) had minimal

effect on activity however, interestingly, the pyrimidine analog

(entry 7) was found to be active as an agonist. Combining the 2-

nitro substitution with a ring nitrogen at the 4-position (entry 8)

also provided an agonist of comparable activity to the parent

compound. Therefore, within the series of aromatic and hetero-

aromatic analogs of this ring highest agonist activity was noted

with the di-fluoro analog (entry 5) and the 2-pyridine and meta-

and para-nitro analogs were found to be inactive. Based on

published crystal-structure analysis from which the ortho-nitro

ring serves the role of a mimic of the homoserine lactone

characteristic of this class of signaling molecules having direct

interactions with a conserved tryptophan (W60) in the LasR

binding pocket. Accordingly, we replaced the 2-nitro aniline “C-

ring” with a homoserine lactone (37). Notably, this analog

displays significantly enhanced activity to 3 and the native LasR

signaling agonist, 3-oxo-C12-HSL, with an EC₅₀value of 0.9 nM

(Entry 9).

3.3. Evaluation of the orientation of the central amide linker.

We next turned our attention to an evaluation of selected

analogs identified as significant in our investigation of the SAR

of Scaffold #2 as described in Tables 1 and 2. Accordingly we

prepared a series of analogs based on Scaffold #1, maintaining

the ortho-chloro substitutent on the “A-ring” and incorporating

three selected “C-ring” motifs. We selected the new 2-pyridine,

di-fluoro and pyrimidine “C-ring” analogs for this analysis as

these analogs displayed a range of agonist activities in our initial

library. Therefore, we evaluated analogs 32, 33, 35 and 38-40 for

both their agonist and antagonist activity in the LasR quorum

sensing reporter strain (Figure 4). In our analysis, we found that

2-pyridine analog 32, earlier observed to show no agonist activity

against LasR (Table 3, entry 4) possessed potent antagonist

activity with an IC₅₀of 42µM. We additionally prepared this

analog with the amide linker between the “B-ring” and the “C-

ring” reversed and observed that this analog (38) had similarly

high LasR antagonist activity (Figure 4). We next prepared an

^aEC₅₀values are reported as the mean of triplicate analysis with the range

defining the standard deviation. The native LasR agonist (1) was used as a

b

control with an EC₅₀=72.9 24.6nM. Maximal percent activation of GFP

at 50 µM with respect to 3-oxo-C12 HSL (1) as a positive control for LasR

agonism, set at 100% activation.

analog containing a reversed amide corresponding to the potent

difluoro arene full LasR agonist 33 (Figure 4, EC₅₀= 46nM). To

substituted “A-ring” analogs based on MMGBSA scores, in this

analysis the ligands shown in Figure 5 are universally predicted

to have show comparable binding to LasR. As before, we

therefore evaluated the independent contributions of amino acid

residues within the LasR binding pocket (Figure 5). Importantly,

all of the residues that are proximal to the “A-ring” and “B-ring”,

which are unchanged in this series of ligands show minimal

changes in their binding energies (e.g. Y47, R61, D65, V76,

G126 and A127, Figure 5). One significant change in binding

energies at the individual amino acid level is noted and occurs

with a residue predicted to be close to the “C-ring” of the ligand.

The pyridine analog 32, an antagonist, shows a stronger binding

interaction with Y56 along with diminished binding to W60

when compared to 3 and 33, two agonists. Evaluation of the

docking poses for these ligands reveals a notable reorganization

of the ligand which likely leads to the above changes in the

binding energies for Y56 and W60 (Figure 6). In these poses

there is a clear shift in protein binding involving rotation of the

“C-ring” from interaction between the 2-nitro substituent of the

agonist 3 with W60 (light blue, Figure 6A) to an interaction

between the 2-pyridine of the antagoinst 32 with Y56 (yellow,

Figure 6B).

We hypothesized that this subtle change in protein binding

Figure 4. The effect of amide bond orientation on the activity of selected

LasR ligands. Bioassay and data fitting was performed according to the

standard procedure. EC₅₀and IC₅₀values are reported as the mean of

triplicate analysis with the range defining the standard deviation.

Figure 6. Comparison of the docking poses for LasR agonist 3 (panel A)

and LasR antagonist 32 (panel B).

may represent a novel trigger to shift LasR from an agonized to

an antagonized state. To test this hypothesis we prepared an

analog containing a 3-pyridine ring in leiu of the 2-pyridine ring

contained in the antagonist 32. Due to the placement of the ring

nitrogen, the pyridine should be prevented from interacting with

Y56 and we hypothesized would therefore lose its antagonist

activity. We accordingly evaluated 41 (Figure 7) and found this

analog to show no antagonist activity, in striking contrast to the

2-pyridine antagonist (32), this analog acts as an agonist of LasR.

our surprise, the analog containing the reversed amide as in

Scaffold #1 (39) was not an agonist of LasR, instead it displayed

a change in function, instead acting as an partial antagonist of

LasR QS with an IC₅₀of 65uM (80% maximal inhibition). In

contrast to this finding, when we prepared the reversed amide

analog of the weak agonist pyrimidine 34 (EC₅₀= 0.6µM) we

found the new analog (40) to similarly be a weak agonist of LasR

with an EC₅₀of 1.6 µM. Therefore, while in most cases we

evaluated changing the orientation of the amide linker results in

small (2-3 fold) changes in the potency of the analog, in certain

cases simply reversing this amide linkage appears to result in a

shift from LasR agonism to antagonism.

We had previously identified a novel series of potent

We turned to computational modeling in an effort to further

investigate these observations. In contrast to the ligands in Table

1, where a clear binding preference was noted for ortho-

Figure 7. The 3-pyridine analog is a LasR agonist. Bioassay and data fitting

was performed according to the standard procedure. EC₅₀and IC₅₀values are

reported as the mean of triplicate analysis with the range defining the standard

deviation.

maleimide containing LasR antagoinsts that act to irreversibly

inhibit LasR through covalent modification of Cys79 in the LasR

binding pocket.²²Structurally, the maleimide functionality we

identified as being central to the LasR antagonist activity maps

onto the “A-ring” of the current ligand series. Accordingly we

investigated a series of hybrid analogs containing a 2-pyridine or

a homoseriene lactone in the “C-ring” position and a pendant

Figure 5. Computational binding contributions of individual amino acids in

LasR with agonist ligands 3 (dark grey) and 33 (light grey) and the antagonist

ligand 32 (medium grey).

maleimide as the “A-ring” structure (Figure 9). Upon analysis of

this series of compounds we found that all fully antagonized

LasR, however with considerably different levels of activity

(Figure 9). Analog 42, combining the 2-pyridine “C-ring” with a

pendant maleimide was found to be a weak antagonist of LasR. It

appears that combining the two structural features we had

identified to independently lead to LasR antagonism, through two

distinct mechanisms of interaction with LasR, do not work

together in this ligand in an additive manner. Analogs 43 and 44

both containing a homoseriene lactone in place of the 2-nitro

aniline “C-ring” display a striking dependence of activity on the

linker length to the maleimide. Consistent with our previous

studies for ligands containing this amide isomer²², the maleimide

linker containing two methylene units (44) was more potent with

an IC₅₀of 23.8µM. This analog represents one of the most potent

antagonists of LasR discovered, being comparable in activity to

the most potent analogs we had previously identified.²²

NMR spectra were recorded using a Varian Mercury Plus

1

spectrometer (400 MHz for H-NMR; 100 MHz for ¹³C-NMR).

Chemical shifts are reported in parts per million (ppm) and were

calibrated according to residual protonated solvent. Mass

spectroscopy data was collected using an Agilent 1100-Series

LC/MSD Trap LC-MS or a Micromass Quattromicro with a

Waters 2795 Separations Module LC-MS with acetonitrile

containing 0.1% formic acid as the mobile phase in positive

ionization mode. Purity was determined on a Agilent 1100 series

equipped with a Phenomenex Kinetex 2.6 µm C18-UPLC

column using a gradient of water to acetonitrile with 0.1% TFA.

All final compounds were evaluated to be of greater then 90%

1

purity by analysis of H-NMR, ¹³C-NMR, and analytical HPLC.

1

Full H-NMR and ¹³C-NMR spectra are included in the online

supporting information.

5.1.2. 2-(Benzyloxy)-3,5-dibromobenzamide, 10.

Salicylamide (10.0 g, 72.9 mmol) was dissolved in glacial acetic

acid (146 mL, 0.5 M) and Br₂(11.2 mL, 218.7 mmol) was added

dropwise. The reaction stirred overnight at room temperature and

then was quenched with water (500 mL). Product was filtered

and washed with a small amount of water and then dried in a

dessicator, affording 20.2 g of the desired product, which was

used crude in the subsequent step, 93.5% yield. 3,5-Dibromo-2-

hydroxybenzamide (10.0 g, 33.7 mmol) was dissolved in acetone

(337 mL, 0.1 M) and K₂CO₃(5.58 g, 40.4 mmol) was added,

followed by benzyl bromide (4.80 mL, 40.4 mmol) dropwise.

The mixture was stirred at reflux overnight and then was cooled

to room temperature. Potassium carbonate was filtered off

(filtrate was washed with more acetone) and the solvent was

removed in vacuo. The product was recrystallized in acetone,

affording 11.3 g of the desired product, 87.1% yield. Spectral

data was consistent with previous report.²²

Figure 8. Hybrid analogs combining optimized structural features

identified in this study with a maleimide for LasR irreversible inhibition.

Bioassay and data fitting was performed according to the standard

procedure. EC₅₀and IC₅₀values are reported as the mean of triplicate

analysis with the range defining the standard deviation.

4. Conclusions.

We have evaluated a series of synthetic libraries of non-

natural modulators of LasR-mediated QS in P. aeruginosa. The

compounds prepared are significant as they represent a novel

drug-like scaffold for the preparation of inhibitors of bacterial

virulence. We have identified several notable stricture-activity

relationships and in so doing have paved the way for the rational

design of potent drug-like inhibitors of LasR QS. Using a

combination of experimental and computational investigations,

our studies have unveiled a new potential strategy for inducing

LasR antagonism and have defined critical structural features of

this novel ligand class for receptor binding.

5.1.3. N-(2-(Benzyloxy)-3,5-dibromobenzyl)-2-

nitrobenzamide, 12.

2-(Benzyloxy)-3,5-dibromobenzamide (1.0 g, 2.58 mmol) was

.

dissolved in BH₃THF (13.24 mL, 13.24 mmol) under N₂and the

mixture was heated to reflux. The reaction stirred for 48 h at 75

°C and was monitored by TLC (10 mL additional BH₃^.THF was

added at 30 h). Methanol (10 mL x 2) was added dropwise to

quench the reaction, and then the solvent was removed in vacuo.

The product was used crude in the subsequent step without

1

purification. H NMR (400 MHz, DMSO-d₆) δ 7.95 (d, J = 2.5

Hz, 1H), 7.91 (s, 1H), 7.74 (s, 1H), 7.60 (d, J = 2.4 Hz, 1H), 7.48

– 7.42 (m, 2H), 7.40 – 7.29 (m, 3H), 4.95 (s, 2H), 4.36 (t, 2H).

¹³C NMR (101 MHz, DMSO-d₆) δ 151.79, 136.32, 136.20,

134.68, 131.19, 128.40, 128.39, 119.03, 116.67, 60.79, 29.25. 2-

Nitrobenzonic acid (534.8 mg, 3.20 mmol) was dissolved in

CH₂Cl₂and the solution was cooled to 0 °C under N₂. Oxalyl

chloride (0.550 mL, 6.40 mmol) was added dropwise, followed

by DMF (1 drop). The reaction was allowed to warm to room

temperature as it stirred for 2 h. Solvent and excess oxalyl

chloride was removed in vacuo and then the residue was

resuspended in CH₂Cl₂. This acid chloride was added dropwise to

a solution of (2-(benzyloxy)-3,5-dibromophenyl)methanamine

(655 mg, 1.60 mmol) in CH₂Cl₂(32 mL, 0.1 M). Triethylamine

(1.56 mL, 11.2 mmol) was added, followed by DMAP (cat.) and

the mixture stirred overnight at room temperature. After

quenching the reaction with water (25 mL), more CH₂Cl₂was

added (50 mL) and the organic layer was washed with HCl (50

mL, 1 M), sat. NaHCO₃(50 mL) and then brine (50 mL), and

then was dried over Na₂SO₄. Purification via silica-gel column

chromatography (product elutes around 30% EtOAc) afforded

5. Experimental section.

5.1. Chemistry

5.1.1. General Experimental.

Unless otherwise noted, all reactions were performed in

flame-dried glassware under an atmosphere of nitrogen or argon

using dried reagents and solvents. All chemicals were purchased

from commercial vendors and used without further purification.

Anhydrous solvents were purchased from commercial vendors.

Flash chromatography was performed using standard grade

silica gel 60 230-400 mesh from SORBENT Technologies or was

performed using a Biotage Flash Purification system equipped

with Biotage SNAP columns. All purifications were performed

using gradients of mixtures of ethyl acetate and hexanes.

Analytical thin-layer chromatography was carried out using

Silica G TLC plates, 200 µm with UV₂₅₄fluorescent indicator

(SORBENT Technologies), and visualization was performed by

staining and/or by absorbance of UV light.

1

354 mg of the desired product, 42.5% yield. H NMR (400 MHz,

CDCl₃) δ 8.04 (dd, 1H), 7.69 (d, J = 2.4 Hz, 1H), 7.66 – 7.59 (m,

1H), 7.59 – 7.53 (m, 2H), 7.44 – 7.40 (m, 2H), 7.37 – 7.29 (m,

3H), 7.26 – 7.20 (m, 1H), 5.81 (t, J = 6.1 Hz, 1H), 5.05 (s, 2H),

4.46 (d, J = 6.1 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 166.46,

153.03, 146.43, 136.07, 135.70, 134.99, 133.83, 132.54, 132.45,

130.73, 129.03, 128.89, 128.87, 128.75, 124.72, 118.29, 117.99,

75.85, 39.21.

calculated for C₂₁H₁₄Br₂ClN₂O₅, 566.89 [M+H], observed

566.90. HPLC purity 92.2%.

5.1.7. 2,4-Dibromo-6-(2-((2-nitrophenyl)amino)-2-

oxoethyl)phenyl 4-chlorobenzoate, 14.

Prepared according to the procedure provided for 2,4-dibromo-6-

(2-((2-nitrophenyl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate

using the appropriate acid chloride, affording 15.8 mg of the

desired product (70% yield). ¹H-NMR (500MHz, CDCl₃) δ 10.33

(s, 1H), 8.67 (dd, J = 8.5, 1.1 Hz, 1H), 8.15 (dd, J = 8.5, 1.5 Hz,

1H), 8.08-8.03 (m, 2H), 7.79 (d, J = 2.2 Hz, 1H), 7.62 (ddd, J =

8.6, 7.2, 1.4 Hz, 1H), 7.57 (d, J = 2.2 Hz, 1H), 7.43-7.37 (m, 2H),

7.18 (ddd, J = 8.5, 7.2, 1.2 Hz, 1H), 3.71 (s, 3H). ¹³C-NMR

(125MHz, CDCl₃) δ 167.9, 163.0, 146.7, 141.2, 136.4, 136.2,

135.8, 134.5, 133.7, 132.0, 130.8, 129.4, 126.4, 126.0, 123.9,

122.2, 120.5, 118.8, 41.0. ESI-MS calculated for

C₂₁H₁₄Br₂ClN₂O₅, 566.89 [M+H], observed 566.80. HPLC purity

91.7%.

5.1.4. 2,4-Dibromo-6-((2-

nitrobenzamido)methyl)phenyl 2-chlorobenzoate, 2.

N-(2-(Methoxy)-3,5-dibromobenzyl)-2-nitrobenzamide (354 mg,

0.681 mmol) was dissolved in CH₂Cl₂(4.54 mL, 0.15 M) and

cooled to -78 °C in a dry ice/acetone bath. BBr₃(65 µL, 0.681

mmol) was added dropwise and the mixture stirred for 3 h while

the temperature was kept between -20 and -60 °C. The reaction

was quenched with sat. NaHCO₃(25 mL) and was extracted with

CH₂Cl₂(50 mL). This organic layer was washed with brine and

then dried over Na₂SO₄. Purification via silica-gel column

chromatography (product elutes around 40% EtOAc) afforded

1

129.5 mg of the desired product, 30.2% yield. H NMR (400

5.1.8. 2,4-Dibromo-6-(2-((2-nitrophenyl)amino)-2-

oxoethyl)phenyl 2-bromobenzoate, 15.

MHz, DMSO-d₆) δ 9.25 (t, J = 6.2 Hz, 1H), 7.99 (d, J = 8.0 Hz,

1H), 7.75 (t, J = 7.5 Hz, 1H), 7.70 – 7.56 (m, 3H), 7.34 (d, J =

2.2 Hz, 1H), 4.37 (d, J = 5.1 Hz, 2H). ¹³C NMR (101 MHz,

DMSO-d₆) δ 166.33, 151.20, 147.18, 133.78, 133.02, 131.85,

131.14, 130.27, 130.03, 129.20, 124.28, 112.36, 110.97, 38.51.

ESI-MS calculated for C₁₄H₁₁Br₂N₂O₄428.9 [M+H], observed

428.9. This product was reacted with 2-chlorobenzoyl chloride

Prepared according to the procedure provided for 2,4-dibromo-6-

(2-((2-nitrophenyl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate

using the appropriate acid chloride, affording 14.3 mg of the

desired product (63% yield). ¹H-NMR (500MHz, CDCl₃) δ 10.32

(s, 1H), 8.68 (dd, J = 8.5, 1.0 Hz, 1H), 8.17-8.11 (m, 2H), 7.81

(d, J = 2.2 Hz, 1H), 7.71-7.66 (m, 1H), 7.63-7.57 (m, 2H), 7.41-

7.36 (m, 2H), 7.16 (ddd, J = 8.5, 7.2, 1.2 Hz, 1H), 3.79 (s, 2H).

¹³C-NMR (125MHz, CDCl₃) δ 167.8, 162.7, 146.6, 136.6, 136.1,

135.9, 135.1, 134.5, 134.1, 133.8, 132.7, 130.7, 129.5, 127.6,

126.0, 123.9, 123.1, 122.4, 120.6, 118.8, 40.8. ESI-MS calculated

for C₂₁H₁₄Br₃N₂O₅, 610.84 [M+H], observed 610.79. HPLC

purity 91.2%.

following

the

procedure

for

2,4-dibromo-6-(2-((2-

nitrophenyl)amino)-2-oxoethyl)phenyl

2-chlorobenzoate to

provide the desired product, 72.1 mg (92% yield). Spectral data

was consistent with previous report.¹⁹

5.1.5. 2,4-Dibromo-6-(2-((2-nitrophenyl)amino)-2-

oxoethyl)phenyl 2-chlorobenzoate, 3.

To

2-(3,5-dibromo-2-hydroxyphenyl)-N-(2-

5.1.9. 2,4-Dibromo-6-(2-((2-nitrophenyl)amino)-2-

oxoethyl)phenyl 3-bromobenzoate, 16.

nitrophenyl)acetamide²²(53.0 mg, 0.123 mmol) in CH₂Cl₂(2.46

mL, 0.05 M) under N₂was added 2-chlorobenzoyl chloride (19

µL, 0.148 mmol), followed by Et₃N (41 µL, 0.295 mmol) and

then DMAP (1.5 mg, 0.0123 mmol). The mixture was allowed to

stir overnight at room temperature and then was diluted with

CH₂Cl₂(50 mL) and washed with HCl (25 mL, 1 M), sat.

NaHCO₃(25 mL) and then brine (25 mL). After drying over

Na₂SO₄, the material was purified via silica-gel flash

chromatography, affording 12.4 mg of the desired product (55%

Prepared according to the procedure provided for 2,4-dibromo-6-

(2-((2-nitrophenyl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate

using the appropriate acid chloride, affording 16.3 mg of the

desired product (67% yield). ¹H-NMR (500MHz, CDCl₃) δ 10.35

(s, 1H), 8.68 (dd, J = 8.5, 1.0 Hz, 1H), 8.22 (t, J = 1.0 Hz, 1H),

8.15 (dd, J = 8.4, 1.0 Hz, 1H), 8.06 (d, J = 8.5 Hz, 1H), 7.79 (d, J

= 2.2 Hz, 1H), 7.75-7.71 (m, 1H), 7.64-7.61 (m, 1H), 7.57 (d, J =

2.1 Hz, 1H), 7.33 (t, J = 8.7 Hz, 1H), 7.18 (t, J = 8.6 Hz, 1H),

3.71 (s, 2H). ¹³C-NMR (125MHz, CDCl₃) δ 167.8, 162.5, 146.6,

137.5, 136.3, 135.8, 134.5, 133.8, 133.5, 130.8, 130.5, 129.9,

129.3, 126.0, 124.0, 123.0, 122.2, 120.6, 118.7, 41.0. ESI-MS

calculated for C₂₁H₁₄Br₃N₂O₅, 610.84 [M+H], observed 610.81.

HPLC purity 98.1%.

1

yield). H-NMR (500MHz, CDCl₃) δ 10.32 (s, 1H), 8.67 (dd, J =

8.5, 1.1 Hz, 1H), 8.17-8.10 (m, 2H), 7.81 (d, J = 2.2 Hz, 1H),

7.63-7.56 (m, 2H), 7.50-7.45 (m, 2H), 7.36-7.30 (m, 1H), 7.16

(ddd, J = 8.5, 7.3, 1.3 Hz, 1H), 3.78 (s, 2H). ¹³C-NMR (125MHz,

CDCl₃) δ 169.8, 162.2, 146.6, 136.6, 136.2, 135.9, 135.1, 134.5,

134.2, 133.8, 132.6, 131.7, 130.7, 127.6, 127.1, 125.9, 123.9,

122.4, 120.6, 118.9, 40.8. ESI-MS calculated for

C₂₁H₁₄Br₂ClN₂O₅, 566.89 [M+H], observed 566.82. HPLC purity

90.1%.

5.1.10. 2,4-Dibromo-6-(2-((2-nitrophenyl)amino)-2-

oxoethyl)phenyl 4-bromobenzoate, 17.

Prepared according to the procedure provided for 2,4-dibromo-6-

(2-((2-nitrophenyl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate

using the appropriate acid chloride, affording 15.1 mg of the

desired product (62% yield). ¹H-NMR (500MHz, CDCl₃) δ 10.34

(s, 1H), 8.68 (dd, J = 8.5, 0.9 Hz, 1H), 8.16 (dd, J = 8.4, 1.0 Hz,

1H), 7.98 (d, J = 8.5 Hz), 7.79 (d, J = 2.3 Hz, 1H), 7.65-7.54 (m,

3H), 7.18 (t, J = 8.4 Hz, 1H), 3.71 (s, 2H). ¹³C-NMR (125MHz,

CDCl₃) δ 167.9, 163.1, 146.7, 136.3, 135.8, 134.5, 133.7, 132.4,

132.1, 130.8, 130.0, 126.8, 126.0, 123.9, 122.2, 120.6, 118.8,

41.0. ESI-MS calculated for C₂₁H₁₄Br₃N₂O₅, 610.84 [M+H],

observed 610.82. HPLC purity 93.6%.

5.1.6. 2,4-Dibromo-6-(2-((2-nitrophenyl)amino)-2-

oxoethyl)phenyl 3-chlorobenzoate, 13.

Prepared according to the procedure provided for 2,4-dibromo-6-

(2-((2-nitrophenyl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate

using the appropriate acid chloride, affording 11.7 mg of the

desired product (52% yield). ¹H-NMR (500MHz, CDCl₃) δ 10.35

(s, 1H), 8.68 (d, J = 8.4 Hz, 1H), 8.15 (dd, J = 8.5, 1.3 Hz, 1H),

8.09-8.05 (m, 1H), 8.02 (d, J = 7.9 Hz, 1H), 7.79 (d, J = 2.2Hz,

1H), 7.63 (ddd, J = 8.5, 7.2, 1.0 Hz, 1H), 7.60-7.54 (m, 2H), 7.39

(t, J = 7.9 Hz, 1H), 7.18 (ddd, J = 8.4, 7.3, 1.1 Hz, 1H), 3.71 (s,

2H). ¹³C-NMR (125MHz, CDCl₃) δ 167.8, 162.6, 146.6, 136.4,

136.3, 135.8, 135.2, 134.6, 134.5, 133.8, 130.8, 130.6, 130.3,

129.7, 128.8, 126.0, 123.9, 122.2, 120.6, 118.8, 41.0. ESI-MS

5.1.11. 2,4-Dibromo-6-(2-((2-nitrophenyl)amino)-2-

oxoethyl)phenyl 2-fluorobenzoate, 18.

Prepared according to the procedure provided for 2,4-dibromo-6-

(2-((2-nitrophenyl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate

using the appropriate acid chloride, affording 15.4 mg of the

desired product (70% yield). ¹H-NMR (500MHz, CDCl₃) δ 10.32

(s, 1H), 8.67 (dd, J = 8.4, 0.9 Hz, 1H), 8.15 (dd, J = 8.5, 1.0 Hz,

1H), 8.06 (dt, J = 8.4, 0.9 Hz, 1H), 7.79 (d, J = 2.2 Hz, 1H), 7.64-

7.55 (m, 2H), 7.24-7.11 (m,2H), 3.75 (s, 2H). ¹³C-NMR

(125MHz, CDCl₃) δ167.8, 163.7, 161.6, 161.2, 161.1, 136.3,

136.2, 136.2, 135.8, 134.5, 133.7, 133.0, 130.7, 125.9, 124.5,

124.5, 123.9, 122.4, 120.5, 118.8, 117.6, 117.4, 116.7, 116.6,

40.8. ESI-MS calculated for C₂₁H₁₄Br₂FN₂O₅, 550.93 [M+H],

observed 550.91. HPLC purity 95.8%.

NMR (125MHz, CDCl₃) δ 167.9, 163.9, 146.9, 138.9, 136.4,

136.2, 135.7, 135.4, 134.6, 133.7, 131.1, 130.9, 128.8, 127.9,

127.8, 125.9, 123.8, 122.2, 120.3, 118.9, 40.9, 21.4. ESI-MS

calculated for C₂₂H₁₇Br₂N₂O₅, 546.95 [M+H], observed 546.96.

HPLC purity 94.4%.

5.1.16. 2,4-Dibromo-6-(2-((2-nitrophenyl)amino)-2-

oxoethyl)phenyl 4-methylbenzoate, 23.

Prepared according to the procedure provided for 2,4-dibromo-6-

(2-((2-nitrophenyl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate

using the appropriate acid chloride, affording 17.1 mg of the

desired product (78% yield). ¹H-NMR (500MHz, CDCl₃) δ 10.32

(s, 1H), 8.69 (dd, J = 8.4, 0.9 Hz, 1H), 8.13 (dd, J = 8.4, 0.9 Hz,

1H), 8.00 (d, J = 8.5 Hz, 2H), 7.78 (d, J = 2.1 Hz, 1H), 7.61 (t, J

= 8.3 Hz, 1H), 7.56 (d, J = 2.1 Hz, 1H), 7.20 (d, J = 8.5 Hz, 2H),

7.17 (t, J = 8.4 Hz, 1H), 3.71 (s, 2H), 2.40 (s, 3H). ¹³C-NMR

(125MHz, CDCl₃) δ 168.0, 163.8, 146.9, 145.6, 136.4, 136.2,

135.7, 134.6, 133.6, 131.0, 130.8, 129.6, 125.9, 125.1, 123.8,

122.3, 120.2, 118.9, 40.9, 22.1. ESI-MS calculated for

C₂₂H₁₇Br₂N₂O₅, 546.95 [M+H], observed 546.92. HPLC purity

91.7%.

5.1.12. 2,4-Dibromo-6-(2-((2-nitrophenyl)amino)-2-

oxoethyl)phenyl 4-fluorobenzoate, 19.

Prepared according to the procedure provided for 2,4-dibromo-6-

(2-((2-nitrophenyl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate

using the appropriate acid chloride, affording 12.7 mg of the

desired product (58% yield). ¹H-NMR (500MHz, CDCl₃) δ 10.33

(s, 1H), 8.67 (d, J = 8.3 Hz, 1H), 8.20-8.11 (m, 2H), 7.79 (d, J =

1.9 Hz, 1H), 7.62 (t, J = 8.4 Hz, 1H), 7.57 (d, J = 1.8 Hz, 1H),

7.18 (t, J = 8.4 Hz, 1H), 7.14-7.07 (m, 2H), 3.71 (s, 2H). ¹³C-

NMR (125MHz, CDCl₃) δ167.9, 167.8, 165.7, 162.8, 146.7,

136.5, 136.2, 135.7, 134.5, 133.7, 133.5, 133.4, 130.8, 126.0,

124.2, 123.9, 122.2, 120.5, 118.8, 116.4, 116.2, 40.9. ESI-MS

calculated for C₂₁H₁₄Br₂FN₂O₅, 550.93 [M+H], observed 550.90.

HPLC purity 92.0%.

5.1.17. 2,4-Dibromo-6-(2-((2-nitrophenyl)amino)-2-

oxoethyl)phenyl 2-methoxybenzoate, 24.

Prepared according to the procedure provided for 2,4-dibromo-6-

(2-((2-nitrophenyl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate

using the appropriate acid chloride, affording 10 mg of the

5.1.13. 2,4-Dibromo-6-(2-((2-nitrophenyl)amino)-2-

oxoethyl)phenyl 2-iodobenzoate, 20.

1

desired product (65.6% yield). H NMR (400 MHz, CDCl₃) δ

Prepared according to the procedure provided for 2,4-dibromo-6-

(2-((2-nitrophenyl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate

using the appropriate acid chloride, affording 9.5 mg of the

desired product (77% yield). ¹H NMR (400 MHz, CDCl₃) δ 10.33

(s, 1H), 8.70 (dt, J = 8.5, 1.2 Hz, 1H), 8.23 – 8.13 (m, 2H), 8.05

(dt, J = 7.9, 1.2 Hz, 1H), 7.82 (dd, J = 2.4, 1.1 Hz, 1H), 7.65 –

7.57 (m, 2H), 7.47 – 7.39 (m, 1H), 7.26 – 7.13 (m, 2H), 3.80 (d, J

= 1.0 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 167.72, 162.75,

142.23, 141.98, 136.02, 135.78, 135.03, 134.46, 134.11, 133.74,

132.34, 132.11, 130.68, 129.74, 128.30, 125.88, 123.80, 122.38,

120.54, 118.79, 40.81. ESI-MS calculated for C₂₁H₁₄Br₂IN₂O₅,

658.83 [M+H], observed 658.90. HPLC purity 96.8%.

10.29 (s, 1H), 8.70 (dd, J = 8.5, 1.3 Hz, 1H), 8.15 (dd, J = 8.4,

1.6 Hz, 1H), 8.02 (dd, J = 7.8, 1.8 Hz, 1H), 7.80 (d, J = 2.3 Hz,

1H), 7.66 – 7.47 (m, 3H), 7.21 – 7.11 (m, 1H), 7.03 – 6.91 (m,

2H), 3.86 (s, 3H), 3.80 (s, 2H). ¹³C NMR (101 MHz, CDCl₃) δ

168.09, 162.72, 160.18, 146.93, 136.61, 135.95, 135.60, 135.16,

134.51, 133.54, 132.69, 130.81, 125.82, 123.71, 122.36, 120.40,

120.06, 119.02, 117.52, 112.24, 56.10, 40.65. ESI-MS calculated

for C₂₂H₁₇Br₂N₂O₆, 562.94 [M+H], observed 563.07. HPLC

purity 89.9%.

5.1.18. 2,4-Dibromo-6-(2-((2-nitrophenyl)amino)-2-

oxoethyl)phenyl 2-nitrobenzoate, 25.

Prepared according to the procedure provided for 2,4-dibromo-6-

(2-((2-nitrophenyl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate

using the appropriate acid chloride, affording 14.4 mg of the

5.1.14. 2,4-Dibromo-6-(2-((2-nitrophenyl)amino)-2-

oxoethyl)phenyl 2-methylbenzoate, 21.

1

Prepared according to the procedure provided for 2,4-dibromo-6-

(2-((2-nitrophenyl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate

using the appropriate acid chloride, affording 16.0 mg of the

desired product (61% yield). ¹H-NMR (500MHz, CDCl₃) δ 10.33

(s, 1H), 8.68 (d, J = 8.4 Hz, 1H), 8.16 (d, J = 8.4 Hz, 1H), 8.13

(dd, J = 8.4, 0.9 Hz, 1H), 7.80 (d, J = 2.0 Hz, 1H), 7.60 (t, J = 8.2

Hz, 1H), 7.57 (d, J = 1.9 Hz, 1H), 7.44 (t, J = 8.2 Hz, 1H), 7.27-

7.13 (m, 2H), 3.73 (s, 2H), 2.53 (s, 3H). ¹³C-NMR (125MHz,

CDCl₃) δ168.0, 163.9, 147.0, 142.4, 136.5, 136.1, 135.7, 134.5,

133.7, 133.7, 132.3, 131.8, 130.9, 126.8, 126.2, 125.9, 123.8,

122.2, 120.2, 119.1, 41.0, 22.1. ESI-MS calculated for

C₂₂H₁₇Br₂N₂O₅, 546.95 [M+H], observed 546.89. HPLC purity

96.4%.

desired product (19.1% yield). H NMR (400 MHz, CDCl₃) δ

10.35 (s, 1H), 8.68 (dd, J = 8.6, 1.3 Hz, 1H), 8.13 (dd, J = 8.5,

1.2 Hz, 1H), 8.10 (dd, J = 7.4, 1.6 Hz, 1H), 8.01 (dd, J = 7.8, 1.4

Hz, 1H), 7.79 (d, 1H), 7.77 – 7.69 (m, 2H), 7.63 (d, 1H), 7.62 –

7.56 (m, 1H), 7.20 – 7.12 (m, 1H), 3.96 (s, 2H). ¹³C NMR (101

MHz, CDCl₃) δ 167.90, 162.39, 147.98, 145.87, 136.78, 135.86,

135.52, 134.37, 134.16, 133.47, 132.80, 131.18, 130.53, 126.30,

125.74, 124.55, 123.71, 122.51, 120.82, 118.26, 39.85. ESI-MS

calculated for C₂₁H₁₄Br₂N₃O₇, 577.92 [M+H], observed 577.79.

HPLC purity 91.1%.

5.1.19. 2,4-Dibromo-6-(2-((2-nitrophenyl)amino)-2-

oxoethyl)phenyl 2,6-dichlorobenzoate, 27.

Prepared according to the procedure provided for 2,4-dibromo-6-

(2-((2-nitrophenyl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate

using the appropriate acid chloride, affording 18.6 mg of the

desired product (78% yield). ¹H-NMR (500MHz, CDCl₃) δ 10.32

(s, 1H), 8.70 (d, J = 8.4 Hz, 1H), 8.16 (dd, J = 8.4, 1.0 Hz, 1H),

7.83 (d, J = 2.1 Hz, 1H), 7.64-7.57 (m, 2H), 7.41-7.31 (m, 2H),

7.16 (t, J = 8.4 Hz, 1H), 3.98 (s, 2H). ¹³C-NMR (125MHz,

CDCl₃) δ 167.9, 161.9, 146.1, 136.6, 136.3, 136.2, 134.5, 134.1,

133.0, 132.2, 131.2, 130.5, 128.9, 126.0, 123.9, 122.3, 121.1,

118.6, 40.5. ESI-MS calculated for C₂₁H₁₂Br₂Cl₂N₂O₅, 600.86

[M+H], observed 600.99. HPLC purity 91.2%.

5.1.15. 2,4-Dibromo-6-(2-((2-nitrophenyl)amino)-2-

oxoethyl)phenyl 3-methylbenzoate, 22.

Prepared according to the procedure provided for 2,4-dibromo-6-

(2-((2-nitrophenyl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate

using the appropriate acid chloride, affording 15.8 mg of the

desired product (72% yield). ¹H-NMR (500MHz, CDCl₃) δ 10.33

(s, 1H), 8.70 (dd, J = 8.4, 0.8 Hz, 1H), 8.13 (dd, J = 8.4, 0.9 Hz),

7.94-7.89 (m, 2H), 7.78 (d, J = 2.1, Hz, 1H), 7.61 (t, J = 8.2 Hz,

1H), 7.56 (d, J = 2.1 Hz, 1H), 7.43-7.37 (m, 1H), 7.30 (t, J = 8.4

Hz, 1H), 7.16 (t, J = 8.4 Hz, 1H), 3.71 (s, 2H), 2.33 (s, 3H). ¹³C-

5.1.20. 2-(3,5-Dibromo-2-((2,6-

dichlorobenzyl)oxy)phenyl)-N-(2-

nitrophenyl)acetamide, 28.

100%

EtOAc

to

provide

2-(3,5-dibromo-2-((2-

chlorobenzoyl)oxy)phenyl)acetic acid as a white solid (1.045g,

59%). H NMR (400 MHz, CDCl₃) δ 8.18-8.14 (m, 1H), 7.76 (d,

1

J = 2.3 Hz, 1H), 7.55-7.52 (m, 2H), 7.50 (d, J = 2.2 Hz, 1H), 7.40

(ddd, J = 7.8, 5.6, 3.0 Hz, 1H), 3.65 (s, 2H).

Prepared according to the procedure provided for 2,4-dibromo-6-

(2-((2-nitrophenyl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate

using the appropriate acid chloride, affording 33.26 mg of the

5.1.24. 2,4-Dibromo-6-(2-((3-nitrophenyl)amino)-2-

oxoethyl)phenyl 2-chlorobenzoate, 30.

1

desired product (13.7% yield). H NMR (400 MHz, CDCl₃) δ

10.13 (s, 1H), 8.62 (dd, J = 8.6, 1.3 Hz, 1H), 8.13 (dd, J = 8.5,

1.6 Hz, 1H), 7.68 (d, J = 2.4 Hz, 1H), 7.59 – 7.51 (m, 1H), 7.34

(d, J = 2.4 Hz, 1H), 7.25 – 7.21 (m, 2H), 7.17 – 7.07 (m, 2H),

5.45 (s, 2H), 3.54 (s, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 168.67,

153.71, 137.34, 136.56, 136.06, 135.91, 134.66, 133.49, 132.13,

131.17, 131.00, 128.81, 125.79, 123.52, 122.35, 118.51, 117.84,

70.16, 39.81. ESI-MS calculated for C₂₁H₁₅Br₂Cl₂N₂O₄, 586.87

[M+H], observed 586.76. HPLC purity 90.1%.

To

a

solution

of

2-(3,5-dibromo-2-((2-

chlorobenzoyl)oxy)phenyl)acetic acid (0.181g, 0.4035mmol) in

ACN (8.07mL) was added 3-nitroaniline (0.0613g, 0.4439mmol)

followed by EDC (0.1423g, 0.807mmol) and DMAP (0.0049g,

0.0403mmol). The resulting mixture was allowed to react at

room temperature overnight and was concentrated to dryness.

The residue was suspended in CHCl₃, washed with 1M HCl, sat.

NaHCO₃and brine, dried over Na₂SO₄and concentrated. The

residue was purified by flash column chromatography using a

gradient of hexanes to EtOAc, affording 1.1 mg of the desired

5.1.21. 2,4-Dibromo-6-(2-((2-nitrophenyl)amino)-2-

oxoethyl)phenyl benzoate, 29.

1

product (0.5% yield). H NMR (400 MHz, CDCl₃) δ 8.41 (t, J =

Prepared according to the procedure provided for 2,4-dibromo-6-

(2-((2-nitrophenyl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate

using the appropriate acid chloride, affording 7.3 mg of the

2.2 Hz, 1H), 8.26 (d, J = 7.4 Hz, 1H), 8.02 (s, 1H), 7.93 (d, J =

8.2 Hz, 1H), 7.86 (d, J = 8.1 Hz, 1H), 7.81 (d, J = 2.3 Hz, 1H),

7.62 (d, J = 2.3 Hz, 1H), 7.60 – 7.53 (m, 2H), 7.51 – 7.38 (m,

2H), 3.74 (s, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 167.29, 164.52,

148.63, 145.90, 138.97, 135.78, 135.38, 134.75, 133.24, 132.76,

131.92, 131.08, 129.80, 127.30, 127.00, 125.52, 121.07, 119.13,

118.63, 114.70, 39.55.. ESI-MS calculated for C₂₁H₁₄Br₂ClN₂O₅,

566.89 [M+H], observed 566.95. HPLC purity 91.3%.

1

desired product (62.1% yield). H NMR (400 MHz, CDCl₃) δ

10.34 (s, 1H), 8.70 (dd, J = 8.5, 1.3 Hz, 1H), 8.18 – 8.12 (m, 3H),

7.81 (d, J = 2.2 Hz, 1H), 7.67 – 7.57 (m, 3H), 7.49 – 7.41 (m,

2H), 7.22 – 7.15 (m, 1H), 3.74 (s, 2H). ¹³C NMR (101 MHz,

CDCl₃) δ 167.85, 163.71, 136.07, 135.65, 134.50, 134.45,

133.57, 130.85, 130.64, 128.87, 127.94, 125.86, 123.76, 122.23,

120.28, 40.83. ESI-MS calculated for C₂₁H₁₄Br₂N₂O₅, 532.93

[M+H], observed 532.98. HPLC purity 90.0%.

5.1.25. 2,4-Dibromo-6-(2-((4-nitrophenyl)amino)-2-

oxoethyl)phenyl 2-chlorobenzoate, 31.

5.1.22. 2,4-Dibromo-6-(2-((2-nitrophenyl)amino)-2-

oxoethyl)phenyl 2-bromo-4-fluorobenzoate, 26.

Prepared according to the procedure provided for 2,4-dibromo-6-

(2-((3-nitrophenyl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate

using the appropriate amine, affording 3.5 mg of the desired

product (1.4% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.26 (dd, J =

8.1, 1.5 Hz, 1H), 8.20 – 8.15 (m, 2H), 8.14 (s, 1H), 7.81 (d, J =

2.3 Hz, 1H), 7.74 – 7.67 (m, 2H), 7.64 – 7.54 (m, 3H), 7.50 –

7.42 (m, 1H), 3.74 (s, 2H). ¹³C NMR (101 MHz, CDCl₃) δ

167.37, 164.71, 145.84, 143.77, 143.73, 135.85, 135.35, 134.81,

133.17, 132.76, 131.93, 130.93, 127.32, 127.00, 125.08, 121.14,

119.31, 118.63, 39.64. ESI-MS calculated for C₂₁H₁₄Br₂ClN₂O₅,

566.89 [M+H], observed 566.86. HPLC purity 94.8%.

Prepared according to the procedure provided for 2,4-dibromo-6-

(2-((2-nitrophenyl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate

using the appropriate acid chloride, affording 9.5 mg of the

desired product (11% yield). ¹H NMR (400 MHz, CDCl₃) δ 10.34

(s, 1H), 8.69 (dd, J = 8.5, 1.3 Hz, 1H), 8.17 (dd, J = 8.5, 1.5 Hz,

1H), 7.99 – 7.92 (m, 1H), 7.83 (d, J = 2.3 Hz, 1H), 7.63 – 7.58

(m, 2H), 7.45 – 7.30 (m, 2H), 7.22 – 7.15 (m, 1H), 3.81 (s, 2H).

¹³C NMR (101 MHz, CDCl₃) δ 167.64, 161.84, 146.40, 138.59,

136.55, 136.08, 135.85, 134.37, 133.80, 131.53, 130.52, 128.88,

128.80, 127.94, 125.89, 123.88, 122.26, 120.84, 120.60, 118.68,

40.80. ESI-MS calculated for C₂₁H₁₂Br₃FN₂O₅, 628.84 [M+H],

observed 629.18. HPLC purity 87.6%.

5.1.26. 2,4-Dibromo-6-(2-oxo-2-(pyridin-2-

ylamino)ethyl)phenyl 2-chlorobenzoate, 32.

Prepared according to the procedure provided for 2,4-dibromo-6-

(2-((3-nitrophenyl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate

using the appropriate amine, affording 24.9 mg of the desired

5.1.23. 2-(3,5-Dibromo-2-((2-

chlorobenzoyl)oxy)phenyl)acetic acid.

1

product (22% yield). H NMR (400 MHz, CDCl₃) δ 8.27 – 8.19

To a solution of methyl 2-(3,5-dibromo-2-hydroxyphenyl)acetate

(2.902g, 8.96mmol) in CH₂Cl₂(59mL) at room temperature was

added Et₃N (2.99mL, 21.49mmol), 2-chlorobenzoyl chloride

(1.248g, 9.85mmol) and DMAP (0.109g, 0.896mmol). The

mixture was allowed to react overnight and was diluted with

CH₂Cl₂, quenched with 1M HCl, washed with sat. NaHCO₃and

brine, dried over Na₂SO₄, concentrated and purified by flash

column chromatography to provide 2,4-dibromo-6-(2-methoxy-

2-oxoethyl)phenyl 2-chlorobenzoate (2.91g, 70%). ¹H NMR (400

MHz, CDCl₃) δ 8.19-8.16 (m, 1H), 7.74 (d, J = 2.2 Hz, 1H), 7.56-

7.72 (m, 2H), 7.50-7.48 (m, 1H), 7.44-7.39 (m, 1H), 3.64 (s, 3H),

3.62 (s, 2H). The above methyl ester, 2,4-dibromo-6-(2-methoxy-

2-oxoethyl)phenyl 2-chlorobenzoate (1.823g, 3.94mmol) was

dissolved in 1,2-dichloroethane (39.4mL) and was treated with

trimethyltinhydroxide (1.425g, 7.88mmol). The mixture was

heated to 80^oC and was monitored by TLC to confirm the

consumption of the starting material. After 2h the reaction was

diluted with CHCl₃, washed with 1M HCl and sat. NaCl, dried

over Na₂SO₄, concentrated and purified by flash column

chromatography using a gradient of 20% EtOAc/hexanes to

(m, 2H), 8.19 – 8.10 (m, 2H), 7.77 (d, J = 2.3 Hz, 1H), 7.70 –

7.64 (m, 1H), 7.58 (d, J = 2.3 Hz, 1H), 7.53 – 7.48 (m, 2H), 7.40

– 7.32 (m, 1H), 7.05 – 6.99 (m, 1H), 3.73 (s, 2H). ¹³C NMR (101

MHz, CDCl₃) δ 167.23, 162.64, 151.12, 147.96, 146.34, 138.49,

135.47, 135.01, 134.05, 133.44, 132.56, 131.66, 131.15, 127.73,

127.03, 120.48, 120.20, 118.56, 114.21, 39.73. ESI-MS

calculated for C₂₀H₁₄Br₂ClN₂O₃, 522.91 [M+H], observed

522.97. HPLC purity 98.3%.

5.1.27. 2,4-Dibromo-6-(2-((2,3-

difluorophenyl)amino)-2-oxoethyl)phenyl 2-

chlorobenzoate, 33.

Prepared according to the procedure provided for 2,4-dibromo-6-

(2-((3-nitrophenyl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate

using the appropriate amine, affording 24.9 mg of the desired

1

product (41% yield). H NMR (400 MHz, Acetone-d₆) δ 9.17 (s,

1H), 8.11 (dd, J = 7.9, 1.6 Hz, 1H), 7.80 (t, J = 7.4 Hz, 1H), 7.76

(d, J = 2.3 Hz, 1H), 7.65 (d, J = 2.3 Hz, 1H), 7.57 – 7.46 (m, 2H),

7.38 – 7.31 (m, 1H), 7.03 – 6.94 (m, 1H), 6.94 – 6.85 (m, 1H),

3.86 (s, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 167.08, 162.99,

146.01, 135.52, 135.05, 134.31, 133.18, 133.15, 132.57, 131.73,

131.12, 127.83, 127.46, 127.13, 124.21, 120.63, 118.48, 117.08,

112.62, 112.45, 39.66. ESI-MS calculated for C₂₁H₁₃Br₂ClF₂NO₃,

557.89 [M+H], observed 558.09. HPLC purity 92.3%.

The

required

phenol,

N-(3,5-dibromo-2-

hydroxybenzyl)picolinamide was prepared in two steps from (2-

(benzyloxy)-3,5-dibromophenyl)methanamine and pyridine-2-

carboxylic acid as described for N-(3,5-dibromo-2-

hydroxybenzyl)-2-nitrobenzamide, providing the desired product

5.1.28. 2,4-Dibromo-6-(2-((4,5-difluoro-2-

nitrophenyl)amino)-2-oxoethyl)phenyl 2-

chlorobenzoate, 34.

1

(0.10g, 35%, two steps). H NMR (400 MHz, Chloroform-d) δ

9.88 (s, 1H), 8.84 – 8.79 (m, 1H), 8.55 (d, J = 4.7 Hz, 1H), 8.20

(d, J = 7.8 Hz, 1H), 7.87 (dt, J = 7.7, 1.7 Hz, 1H), 7.59 (d, J = 2.4

Hz, 1H), 7.47 (ddd, J = 7.6, 4.7, 1.2 Hz, 1H), 7.29 (d, J = 2.4 Hz,

1H), 4.55 (d, J = 6.8 Hz, 2H). This product was reacted with 2-

chlorobenzoyl chloride according to the procedure provided for

2,4-dibromo-6-((2

chlorobenzoate, affording 11.4 mg of the desired product (38%

yield). H NMR (400 MHz, CDCl₃) δ 8.54 (dd, J = 4.7, 1.4 Hz,

1H), 8.45 (s, 1H), 8.21 (dd, J = 7.8, 1.1 Hz, 1H), 8.15 (dd, J =

7.8, 1.1 Hz, 1H), 7.84 (td, J = 7.8, 1.8 Hz, 1H), 7.74 (d, J = 2.3

Hz, 1H), 7.58 (d, J = 2.3 Hz, 1H), 7.54 (d, 2H), 7.46 – 7.39 (m,

2H), 4.66 (d, J = 6.1 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ

164.56, 162.48, 149.41, 148.35, 145.84, 137.52, 135.04, 134.94,

133.99, 132.61, 131.78, 131.65, 127.89, 127.00, 126.59, 122.51,

120.33, 118.12, 38.74. ESI-MS calculated for C₂₀H₁₄Br₂ClN₂O₅,

522.91 [M+H], observed 523.04. HPLC purity 97.7%.

Prepared according to the procedure provided for 2,4-dibromo-6-

(2-((3-nitrophenyl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate

using the appropriate amine, affording 36.7 mg of the desired

1

product (16% yield). H NMR (400 MHz, CDCl₃) δ 10.41 (s,

nitrobenzamido)methyl)phenyl

2-

1H), 8.74 (dd, J = 12.6, 7.5 Hz, 1H), 8.14 (dd, J = 7.9, 0.9 Hz,

1H), 8.07 (dd, J = 10.1, 7.9 Hz, 1H), 7.85 (d, J = 2.3, 0.7 Hz,

1H), 7.59 (d, 1H), 7.55 – 7.49 (m, 2H), 7.41 – 7.35 (m, 1H), 3.81

(s, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 167.90, 162.12, 146.58,

136.02, 135.00, 134.18, 133.71, 132.55, 131.71, 130.14, 127.47,

127.05, 120.64, 118.93, 115.14, 114.93, 111.01, 110.76, 40.75.

ESI-MS calculated for C₂₁H₁₂Br₂ClN₃O₅, 602.88 [M+H],

observed 602.77. HPLC purity 97.0%.

1

5.1.29. 2,4-Dibromo-6-(2-oxo-2-(pyrimidin-4-

ylamino)ethyl)phenyl 2-chlorobenzoate, 35.

Prepared according to the procedure provided for 2,4-dibromo-6-

(2-((3-nitrophenyl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate

using the appropriate amine, affording 15.2 mg of the desired

5.1.33. 2,4-Dibromo-6-((2,3-

difluorobenzamido)methyl)phenyl 2-chlorobenzoate,

39.

1

product (13% yield). H NMR (400 MHz, CDCl₃) δ 8.83 (s, 1H),

Prepared according to the procedure provided for 2,4-dibromo-6-

((2 nitrobenzamido)methyl)phenyl 2-chlorobenzoate from 2-

chlorobenzoyl chloride and N-(3,5-dibromo-2-hydroxybenzyl)-

2,3-difluorobenzamide, affording 7.9 mg of the desired product

(18% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.21 (dd, 1H), 7.81 –

7.73 (m, 2H), 7.62 (d, J = 2.3 Hz, 1H), 7.57 – 7.53 (m, 2H), 7.46

– 7.40 (m, 1H), 7.36 – 7.27 (m, 1H), 7.21 – 7.14 (m, 1H), 7.04 (t,

J = 11.4 Hz, 1H), 4.66 (s, 2H). ¹³C NMR (101 MHz, CDCl₃) δ

162.73, 162.47, 145.94, 135.36, 134.98, 134.44, 134.12, 132.55,

132.25, 131.69, 127.75, 127.09, 126.60, 126.57, 124.78, 124.72,

120.83, 120.65, 120.43, 118.16, 39.40. ESI-MS calculated for

C₂₁H₁₃Br₂ClF₂NO₃, 557.89 [M+H], observed 557.77. HPLC

purity 90.0%.

8.61 (s, 1H), 8.34 (s, 1H), 8.21 – 8.14 (m, 1H), 8.10 (dd, J = 5.8,

1.2 Hz, 1H), 7.80 (d, J = 2.4 Hz, 1H), 7.57 (d, J = 2.4 Hz, 1H),

7.55 – 7.52 (m, 2H), 7.42 – 7.36 (m, 1H), 3.76 (s, 2H). ¹³C NMR

(101 MHz, CDCl₃) δ 168.19, 162.98, 158.59, 158.49, 156.87,

146.25, 135.79, 135.03, 134.27, 133.32, 132.57, 131.73, 130.43,

127.54, 127.12, 120.67, 118.71, 110.42, 39.79. ESI-MS

calculated for C₁₉H₁₃Br₂ClN₃O₃, 523.90 [M+H], observed

523.92. HPLC purity 94.4%.

5.1.30. 2,4-Dibromo-6-(2-((3-nitropyridin-4-

yl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate, 36.

Prepared according to the procedure provided for 2,4-dibromo-6-

(2-((3-nitrophenyl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate

using the appropriate amine, affording 4.1 mg of the desired

5.1.34. 2,4-Dibromo-6-((pyrimidine-4-

carboxamido)methyl)phenyl 2-chlorobenzoate, 40.

1

product (5.8% yield). H NMR (400 MHz, CDCl₃) δ 10.50 (s,

1H), 9.33 (s, 1H), 8.67 (s, 2H), 8.13 (dd, 1H), 7.86 (d, J = 2.2 Hz,

1H), 7.59 (d, J = 2.3 Hz, 1H), 7.56 – 7.47 (m, 2H), 7.41 – 7.34

(m, 1H), 3.84 (s, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 168.31,

162.10, 156.31, 155.69, 136.16, 134.22, 133.69, 132.54, 131.72,

129.85, 129.27, 128.07, 127.41, 127.06, 120.70, 118.99, 114.62,

40.90. ESI-MS calculated for C₂₀H₁₃Br₂ClN₃O₅, 567.89 [M+H],

observed 567.82. HPLC purity 89.8%.

The

required

phenol,

N-(3,5-dibromo-2-

hydroxybenzyl)pyrimidine-4-carboxamide was prepared in two

steps from (2-(benzyloxy)-3,5-dibromophenyl)methanamine and

pyrimidine-4-carboxylic acid as described for N-(3,5-dibromo-2-

hydroxybenzyl)-2-nitrobenzamide, providing the desired product

1

(0.082g, 32%, two steps). H-NMR (400 MHz, Chloroform-d) δ

9.22 (s, 1H), 8.99 (d, J = 5.0 Hz, 1H), 8.82 – 8.74 (m, 1H), 8.10

(dd, J = 5.0, 1.4 Hz, 1H), 7.56 (d, J = 2.4 Hz, 1H), 7.32 (d, J = 2.4

Hz, 1H), 4.59 (d, J = 6.7 Hz, 2H). This product was reacted with

2-chlorobenzoyl chloride according to the procedure provided for

5.1.31. (S)-2,4-Dibromo-6-(2-oxo-2-((2-

oxotetrahydrofuran-3-yl)amino)ethyl)phenyl 2-

chlorobenzoate, 37.

2,4-dibromo-6-((2

nitrobenzamido)methyl)phenyl

2-

Prepared according to the procedure provided for 2,4-dibromo-6-

(2-((3-nitrophenyl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate

using the appropriate amine, affording 27.9 mg of the desired

product (81% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.21 (dd, J =

7.9, 1.2 Hz, 1H), 7.77 (dd, J = 2.3, 1.2 Hz, 1H), 7.59 – 7.53 (m,

3H), 7.47 – 7.40 (m, 1H), 6.18 (s, 1H), 4.55 – 4.35 (m, 2H), 4.29

– 4.14 (m, 1H), 3.61 (s, 2H), 2.76 – 2.61 (m, 1H), 2.16 – 1.99 (m,

1H). ¹³C NMR (101 MHz, CDCl₃) δ 174.76, 169.16, 135.45,

135.02, 134.32, 133.47, 132.70, 131.78, 131.23, 127.48, 127.21,

120.57, 118.45, 66.07, 49.53, 38.19, 29.81. ESI-MS calculated

for C₁₉H₁₅Br₂ClNO₅, 529.90 [M+H], observed 529.85. HPLC

purity 94.9%.

chlorobenzoate, affording 7.7 mg of the desired product (7.0%

1

yield). H NMR (400 MHz, Chloroform-d) δ 9.22 (d, J = 1.4 Hz,

1H), 8.96 (d, J = 5.0 Hz, 1H), 8.38 (t, 1H), 8.20 (d, 1H), 8.06 (d,

1H), 7.76 (d, J = 2.3 Hz, 1H), 7.61 – 7.52 (m, 2H), 7.48 – 7.38

(m, 1H), 4.66 (d, J = 6.3 Hz, 2H). ¹³C NMR (101 MHz,

Chloroform-d) δ 162.91, 162.64, 159.41, 157.96, 155.96, 146.08,

135.49, 134.27, 134.21, 132.72, 132.17, 131.77, 127.84, 127.15,

126.84, 120.46, 118.87, 118.39, 39.10. ESI-MS calculated for

C₁₉H₁₃Br₂ClN₃O₃, 523.90 [M+H], observed 523.67. HPLC purity

92.4%.

5.1.35. 2,4-Dibromo-6-(nicotinamidomethyl)phenyl

2-chlorobenzoate, 41.

5.1.32. 2,4-Dibromo-6-(picolinamidomethyl)phenyl

2-chlorobenzoate, 38.

The

required

phenol,

N-(3,5-dibromo-2-

2H), 2.20 – 2.01 (m, 2H). ¹³C NMR (101 MHz, Chloroform-d) δ

175.30, 171.77, 170.42, 169.82, 134.90, 134.56, 134.06, 131.72,

120.02, 66.36, 49.73, 37.50, 36.68, 31.16, 30.62, 30.51, 23.69.

ESI-MS calculated for C₂₀H₁₉Br₂N₂O₇, 556.96 [M+H], observed

556.81. HPLC purity 92.5%.

hydroxybenzyl)nicotinamide was prepared in two steps from (2-

(benzyloxy)-3,5-dibromophenyl)methanamine and nicotinic acid

as

nitrobenzamide, providing the desired product (0.048g, 17%, two

steps). H-NMR (400 MHz, Chloroform-d) δ 9.03-8.91 (m, 1H),

8.81-8.71 (m, 1H), 8.15 (d, J = 8.1 Hz, 1H), 7.61 (d, J = 2.3 Hz,

1H), 7.42 (dd, J = 7.8, 5.0 Hz, 1H), 7.31 (d, J = 2.4 Hz, 1H), 7.03

(bs, 1H), 4.59 (d, J = 6.4 Hz, 2H). This product was reacted with

2-chlorobenzoyl chloride according to the procedure provided for

2,4-dibromo-6-((2

chlorobenzoate, affording 4.2 mg of the desired product (6.5%

described

for

N-(3,5-dibromo-2-hydroxybenzyl)-2-

1

5.1.39. 2,4-Dibromo-6-(2-oxo-2-((2-

oxotetrahydrofuran-3-yl)amino)ethyl)phenyl 3-(2,5-

dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate, 44.

Prepared according to the procedure provided for 2,4-dibromo-6-

(2-((2-nitrophenyl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate

nitrobenzamido)methyl)phenyl

2-

from

(S)-2-(3,5-dibromo-2-hydroxyphenyl)-N-(2-

1

oxotetrahydrofuran-3-yl)acetamide, affording 1.8 mg of the

desired product (17% yield). ¹H NMR (400 MHz, Chloroform-d)

δ 7.68 (d, J = 2.2 Hz, 1H), 7.53 (d, J = 2.2 Hz, 1H), 6.75 (s, 1H),

4.60 – 4.48 (m, 1H), 4.46 (t, J = 9.1 Hz, 1H), 4.33 – 4.21 (m,

1H), 4.15 – 3.91 (m, 2H), 3.62 – 3.47 (m, 2H), 3.12 – 2.97 (m,

2H), 2.83 – 2.71 (m, 0H), 2.75 (s, 1H), 2.25 – 2.09 (m, 1H), 1.38

– 1.23 (m, 2H), 0.89 (d, J = 12.6 Hz, 1H). ¹³C NMR (101 MHz,

Chloroform-d) δ 175.23, 170.84, 169.52, 168.71, 146.18, 135.12,

134.60, 133.71, 131.50, 120.34, 118.19, 66.27, 49.62, 37.96,

33.58, 32.69, 31.85, 30.08, 29.87. ESI-MS calculated for

C₁₉H₁₇Br₂N₂O₇, 542.94 [M+H], observed 542.92. HPLC purity

91.8%.

yield). H NMR (400 MHz, CDCl₃) δ 8.97 (s, 1H), 8.71 (s, 1H),

8.21 (dd, 1H), 8.07 (d, J = 8.1 Hz, 1H), 7.77 (d, J = 2.3 Hz, 1H),

7.62 (d, J = 2.3 Hz, 1H), 7.58 – 7.50 (m, 2H), 7.47 – 7.39 (m,

1H), 7.39 – 7.33 (m, 1H), 6.80 (t, 1H), 4.63 (s, 2H). ¹³C NMR

(101 MHz, CDCl₃) δ 165.40, 163.37, 152.55, 148.24, 146.10,

135.58, 135.21, 135.00, 134.32, 132.79, 132.64, 131.74, 127.52,

127.17, 123.63, 120.52, 118.19, 39.50. ESI-MS calculated for

C₂₀H₁₄Br₂ClN₂O₅, 522.91 [M+H], observed 522.97. HPLC purity

89.5%.

5.1.36. 2,4-Dibromo-6-(picolinamidomethyl)phenyl

4-(2, 5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoate,

42.

5.2. LasR Bioassay

Prepared according to the procedure provided for 2,4-dibromo-6-

((2 nitrobenzamido)methyl)phenyl 2-chlorobenzoate from N-

(3,5-dibromo-2-hydroxybenzyl)picolinamide, affording 35.7 mg

of the desired product (25.1% yield). ¹H NMR (400 MHz,

CDCl₃) δ 8.53 (dd, J = 4.8, 1.3 Hz, 1H), 8.41 (s, 1H), 8.19 (dd, J

= 7.8, 1.2 Hz, 1H), 7.86 (td, J = 7.7, 1.7 Hz, 1H), 7.66 (d, J = 2.3

Hz, 1H), 7.53 (d, J = 2.3 Hz, 1H), 7.47 – 7.39 (m, 1H), 6.72 (s,

2H), 4.59 (d, J = 6.4 Hz, 2H), 3.68 (t, J = 6.7 Hz, 2H), 2.70 (t, J =

7.3 Hz, 2H), 2.10 – 1.98 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ

170.94, 170.02, 164.39, 149.49, 148.32, 146.02, 137.56, 134.94,

134.82, 134.31, 132.05, 126.56, 122.53, 119.96, 118.15, 38.75,

36.98, 31.20, 23.79. ESI-MS calculated for C₂₁H₁₈Br₂N₃O₅,

549.96 [M+H], observed 549.92. HPLC purity 89.7%.

The LasR reporter strain bioassay was performed as described

previously with modifications.²³Briefly, the assays were

performed in E. coli strain BL21 DE3 Gold (Agilent) carrying a

plasmid containing lasR (maintained with 100 µg/mL ampicillin)

and a plasmid containing the rsaL promoter-driving expression of

gfp (maintained with 50 µg/mL of kanamycin.) The reporter

strain described above was kindly supplied by Bonnie L. Bassler

(Princeton University). This E. coli strain was grown overnight at

37^oC in Luria broth (LB) (Fisher) with the appropriate

antibiotics. The overnight culture was subcultured 1:40 for

bioassay analysis. The native LasR agonist, N-3-oxo-dodecanoyl-

L-homoseriene lactone was used as a positive control for all

agonist assays (EC₅₀=72.9 24.6nM) and was employed at a

constant concentration of 50nM for all antagonist assays. For

agonists assays, maximal % activation is reported with respect to

N-3-oxo-dodecanoyl-L-homoseriene lactone set as 100%

activation. For antagonism assays, the previously described

antagonist, (S)-2-(4-bromophenyl)-N-(2-oxotetrahydrofuran-3-yl)

acetamide was used as the positive control for antagonism with

an IC₅₀of 26.31 15.7µM.⁸Maximal % inhibition is reported with

respect to (S)-2-(4-bromophenyl)-N-(2-oxotetrahydrofuran-3-yl)

acetamide set as 100% inhibition. Compounds were added to the

diluted overnight reporter strain in 96-well black microplates

with clear bottoms (Corning) at the starting concentrations

described and titrated by serial dilution. Plates were incubated

with shaking at 37^oC for 4-6h and were evaluated for

fluorescence (ex485/em538) and absorbance (A₆₀₀) using a

Molecular Devices SpectraMax M5 microplate reader, or a

FlexStation II 384 Molecular Devices for fluorescence and a

MultiSkan Spectrum for absorbance measurements. Dose-

response curves were fit to the data using standard nonlinear

regression data fitting settings in GraphPad Prism 6, no

corrections for compound purity were made. All data points are

reported as mean with error describing the standard deviation.

5.1.37. (S)-2-(3,5-Dibromo-2-hydroxyphenyl)-N-(2-

oxotetrahydrofuran-3-yl)acetamide.

To a solution of 2-(3,5-dibromo-2-hydroxyphenyl)acetic acid

(0.750g, 2.42mmol) and L-Homoserine lactone hydrobromide

(0.881g, 4.84mmol) in DMF (12.1mL) was added HATU

(1.103g, 2.90mmol) followed by i-Pr₂NEt (1.3mL, 7.26mmol).

The resulting mixture was allowed to stir at room temperature

overnight, was diluted with EtOAc, washed with water, 1M HCl

and brine, dried over Na₂SO₄and concentrated to dryness. The

residue was purified by flash column chromatography using a

gradient from hexanes to EtOAc to provide the desired product

(0.430g, 45%). ¹H-NMR (400 MHz, Chloroform-d) δ 8.68 (s,

1H), 7.55 (d, J = 2.3 Hz, 1H), 7.21 (d, J = 2.3 Hz, 1H), 6.92 (d, J

= 6.1 Hz, 1H), 4.61-4.52 (m, 1H), 4.48 (t, J = 9.1 Hz, 1H), 4.36-

4.25 (m, 1H), 3.62 (ABq, ∆ν_AB= 11.3 Hz, J_AB= 12.3 Hz, 2H),

2.85-2.75 (m, 1H), 2.25-2.11 m, 1H).

5.1.38. 2,4-Dibromo-6-(2-oxo-2-((2-

oxotetrahydrofuran-3-yl)amino)ethyl)phenyl 4-(2,5-

dioxo-2, 5-dihydro-1H-pyrrol-1-yl)butanoate, 43.

Prepared according to the procedure provided for 2,4-dibromo-6-

(2-((2-nitrophenyl)amino)-2-oxoethyl)phenyl 2-chlorobenzoate

from

(S)-2-(3,5-dibromo-2-hydroxyphenyl)-N-(2-

5.3. Computational Modeling

oxotetrahydrofuran-3-yl)acetamide, affording 12.5 mg of the

desired product (61% yield). ¹H NMR (400 MHz, Chloroform-d)

δ 7.67 (d, J = 2.3 Hz, 1H), 7.58 (d, J = 2.3 Hz, 1H), 7.14 (s, 1H),

6.77 (s, 2H), 4.59 – 4.48 (m, 1H), 4.44 (t, 1H), 4.32 – 4.21 (m,

1H), 3.77 – 3.64 (m, 2H), 2.89 – 2.79 (m, 1H), 2.84 – 2.67 (m,

5.3.1. Ligand Preparation.

The triaryl series of ligands were prepared using first Maestro

Elements then Maestro 10.3. The 2D structures were draw using

the 2D sketcher function in Maestro Elements. The templates for

the ligands were obtained from the Perez Lab. The 2D sketches

were then converted to 3D structures. Epik (an empirical pka

prediction program) was utilized to determine the charge of the

molecule at pH 7.²⁴Finally, the 3D structures were uploaded into

the Maestro 10.3 and restrained minimization was run. This

function is used to relax and optimize the ligand structure.

References and notes

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5.3.3. Ligand Docking.

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7.

8.

The Glide XP Dock function performs

a comprehensive

systematic search for the best receptor conformations and

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pocket. The small heavy atom RMSD of 0.6Å between the

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docking method (see online supporting information, Figure S1).

9.

5.3.4. Binding energy Calculations and

decompositions methods.

10.

Molecular Mechanism-General Born Surface Area (MM-

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Article Doi

Development of a novel series of non-natural triaryl agonists and antagonists of the Pseudomonas aeruginosa LasR quorum sensing receptor

DOI: 10.1016/j.bmc.2016.10.021

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Article abstract of DOI:10.1016/j.bmc.2016.10.021

Full text of DOI:10.1016/j.bmc.2016.10.021

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