Time-dependent diaryl ether inhibitors of InhA: Structure-activity relationship studies of enzyme inhibition, antibacterial activity, and in vivo efficacy

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DOI: 10.1002/cmdc.201300429

Time-Dependent Diaryl Ether Inhibitors of InhA:

Structure–Activity Relationship Studies of Enzyme

Inhibition, Antibacterial Activity, and in vivo Efficacy

Pan Pan,^[a]Susan E. Knudson,^[b]Gopal R. Bommineni,^[a]Huei-Jiun Li,^[a]Cheng-Tsung Lai,^{[a, c]}

Nina Liu,^[a]Miguel Garcia-Diaz,^[d]Carlos Simmerling,^[a]Sachindra S. Patil,^[e]

Richard A. Slayden,*^[b]and Peter J. Tonge*^{[a, c]}

The diaryl ethers are a novel class of antituberculosis drug can-

didates that inhibit InhA, the enoyl-ACP reductase involved in

the fatty acid biosynthesis (FASII) pathway, and have antibacte-

rial activity against both drug-sensitive and drug-resistant

strains of Mycobacterium tuberculosis. In the present work, we

demonstrate that two time-dependent B-ring modified diaryl

ether InhA inhibitors have antibacterial activity in a mouse

model of TB infection when delivered by intraperitoneal injec-

tion. We propose that the efficacy of these compounds is relat-

ed to their residence time on the enzyme, and to identify

structural features that modulate drug–target residence time

in this system, we have explored the inhibition of InhA by

a series of B-ring modified analogues. Seven ortho-substituted

compounds were found to be time-dependent inhibitors of

InhA, where the slow step leading to the final enzyme–inhibi-

tor complex (EI*) is thought to correlate with closure and or-

dering of the InhA substrate binding loop. A detailed mecha-

nistic understanding of the molecular basis for residence time

in this system will facilitate the development of InhA inhibitors

with improved in vivo activity.

Introduction

Tuberculosis (TB) is a global infectious disease that is a serious

threat to human health due to emergence of multidrug-resist-

ant (MDR-TB) and extensively drug-resistant (XDR-TB) strains of

Mycobacterium tuberculosis.^[1]In 2008, 22% of new TB cases

were reported to be MDR-TB,^[2]however, there is a current lack

of new TB-specific chemotherapeutics to combat the spread of

these resistant organisms. In the search for novel TB lead can-

didates, we and others have developed inhibitors of InhA, the

enoyl-ACP reductase involved in the M. tuberculosis fatty acid

biosynthesis (FASII) pathway.^[3]InhA plays an essential role in

cell viability and is a target for the TB drug isoniazid (INH).^[4]

Since resistance to INH results primarily from defects in drug

activation and not from mutations in InhA,^[5]compounds that

directly inhibit InhA should be active against INH-resistant

strains. Based on this premise, we developed a series of diaryl

ethers (Figure 1) that are potent inhibitors of InhA and that

have antimicrobial activity against both INH-sensitive and re-

sistant strains of M. tuberculosis.^[3a]Unfortunately, a previous

study demonstrated that PT004 (Figure 1b) had efficacy in

a macrophage TB assay but did not show efficacy in the rapid

murine model of TB infection.^[6]Our studies on the enoyl-ACP

reductase enzymes in other pathogens revealed that the in

vivo efficacy of diaryl ether inhibitors correlates with their resi-

dence time (t_R) on the enzyme target (t_R=1/k_off) but not with

their binding affinities (K_i) values for enzyme inhibition or their

in vitro antibacterial activity (i.e., the minimum inhibitory con-

centration; MIC).^[7]Since K_iand MIC values are determined at

constant drug concentration, the studies support the impor-

tance of drug-target residence time in determining in vivo

drug activity given that the in vivo drug concentration is not

constant.^[8]Based on this hypothesis, we developed long resi-

dence time inhibitors of InhA and discovered PT070,^[9]the first

slow-onset diaryl ether inhibitor, which showed a 430-fold in-

crease in binding affinity to InhA compared with PT004 (Fig-

ure 1b). PT070 has a residence time of 24 min on InhA and

binds to the enzyme through a two-step induced-fit mecha-

nism, in which the rapid formation of the initial EI complex is

followed by the slow formation of the final enzyme–inhibitor

complex (EI*) (Figure 2).

[a] Dr. P. Pan, Dr. G. R. Bommineni, Dr. H.-J. Li, C.-T. Lai, Dr. N. Liu,

Prof. C. Simmerling, Prof. P. J. Tonge

Institute for Chemical Biology & Drug Discovery

Department of Chemistry, Stony Brook University

Stony Brook, NY 11794-3400 (USA)

E-mail: peter.tonge@sunysb.edu

[b] Dr. S. E. Knudson, Prof. R. A. Slayden

Department of Microbiology, Immunology & Pathology

Colorado State University

1619 Campus Delivery, Fort Collins, CO 80523-2025 (USA)

E-mail: richard.slayden@colostate.edu

[c] C.-T. Lai, Prof. P. J. Tonge

Biochemistry & Structural Biology, Stony Brook University

Stony Brook, NY 11794-3400 (USA)

[d] Prof. M. Garcia-Diaz

Pharmacological Sciences Graduate Program, Stony Brook University

Stony Brook, NY 11794-3400 (USA)

[e] Dr. S. S. Patil

Kanashi Biotech Pvt Ltd, A-27 H Block MIDC, Pimpri, Pune 411018 (India)

Supporting information for this article is available on the WWW under

http://dx.doi.org/10.1002/cmdc.201300429.

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Scheme 1. Previously reported synthesis of diaryl ethers.^[3a]Reagents and

conditions: a) (CuOTf)₂·PhH, Cs₂CO₃, EtOAc, [ArCO₂H], toluene, 1108C;

b) RZnCl, Pd(P(tBu)₃)₂,THF/NMP (1:10), 1308C; c) BBr₃, CH₂Cl₂, ꢀ788C.

and moisture-sensitive catalysts, and resulted in low overall

yields (10–20%). To streamline this process, we established

a new route in which intermediate 3 was prepared from vanil-

lin (Scheme 2). This involved protection of the vanillin hydroxy

with a benzyl group, attachment of a pentenyl group using

a Wittig reaction, hydrogenation to reduce the double bond,

and subsequent cleavage of the protecting group. Once com-

pound 3 was obtained, a variety of aromatic groups were cou-

pled to the phenol to provide intermediates for the target

molecules (Scheme 3–8).

Figure 1. a) The general structure of the diaryl ether scaffold. The diaryl

ethers share a scaffold consisting of an alkyl phenol A-ring and a B-ring with

various substituents. b) Lead compounds for structure–activity relationship

studies described here. c) Newly synthesized derivatives including ortho-sub-

stituted derivatives, di-substituted derivatives, and derivatives with hetero-

cyclic B-rings (2’-pyridyl or 4’-pyridyl).

Although additional steps were employed in this new ap-

proach, the overall yield of PT092, which has a similar struc-

ture to previously reported diaryl ethers, was 37% (Scheme 4).

For simple aryl halides, the reported copper(I) iodide-catalyzed

coupling reactions^[11]are generally useful for the synthesis of

aryl ethers, such as compound 4. However, this method is lim-

ited by reactivity and steric hindrance of the aryl halide, where-

as the recently published air-stable copper(I) bipyridyl com-

plex^[12]showed higher catalytic efficiency and afforded more

In order to further rationally modulate residence time in this

system, we need to understand the molecular basis that gov-

erns the interconversion of EI and EI*. The structural change

that occurs between EI and EI* has

been linked to motions of the sub-

strate binding loop (residues 198–

208), which closes over the active

site when the inhibitor is bound.^[9]

Figure 2. Kinetic scheme for

time-dependent inhibition.

The time-dependent diaryl

ether inhibitors bind through

Based on the premise that sub-

stituents on the B-ring of the diaryl

ether play a critical role in the en-

ergetics of the EI to EI* transition,

we now report structure–activity

relationship (SAR) studies on

a series of B-ring-modified diaryl

ethers that provide insight into the

role of specific interactions be-

tween the inhibitor and the sub-

strate binding loop in the time-de-

a two-step induced-fit mecha-

nism in which the rapid for-

mation of the initial enzyme–

inhibitor complex (EI) is fol-

lowed by a slow step leading

to the final enzyme–inhibitor

complex (EI*).

pendent inhibition of InhA. In addition, to further substantiate

the link between residence time and in vivo antibacterial activi-

ty, we have determined the efficacy of PT070 in a mouse

model of TB infection together with that of an additional time-

dependent InhA inhibitor, PT091.

Scheme 2. Synthesis of intermediate 3. Reagents and conditions: a) BnBr, aq

KOH, MeOH, reflux, 2 h, 91%; b) nC₅H₁₁PPh₃Br, nBuLi, THF, ꢀ788C!RT, 2.5 h,

Chemistry

Previous approaches to the preparation of diaryl ethers by us

involved a Buchwald–Hartwig cross-coupling to link the A-ring

and B-ring, and the attachment of an alkyl group using Negishi

coupling (Scheme 1).^[3a,10]This synthetic route employed air-

Scheme 3. Synthesis of PT095. Reagents and conditions: a) 1-iodo-2-(trifluor-

omethyl)benzene, (CuOTf)₂·PhH, Cs₂CO₃, 1-naphthoic acid, EtOAc, toluene,

110 8C, 24 h, 75%; b) BBr₃, CH₂Cl₂, ꢀ788C!RT, 3 h, 88%.

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82%; c) H₂, Pd/C, EtOH, 6 h, 89%.

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complex biaryl coupled products, such as compound 28. We

also modified the Ullmann protocol to use the nucleophilic ar-

omatic substitution reaction with fluoronitrobenzenes,^[10,13]

benzonitriles, picolinonitrile, pyridine N-oxide,^[14]or isonicotino-

nitrile to afford the aryl ethers 5, 15–18, 27, 29, 31, 33, 34, 36,

Scheme 4. Derivatives with mono-substituted B-rings. Reagents and condi-

tions: a) K₂CO₃, 1-fluoro-2-nitrobenzene, 18-crown-6, DMF, 1108C, 3 h, 66%;

b) H₂, Pd/C, EtOH, 6 h, 91%; c) NaNO₂, AcOH, H₂O, CuX, 08C, 30 min; d) BBr₃,

CH₂Cl₂, ꢀ788C!RT, 5 h.

Scheme 5. Derivatives with various B-rings. Reagents and conditions:

a) K₂CO₃, DMAc, 1608C, 3 h; b) Zn, HCl, EtOH/H₂O (10:1), 08C!RT, 1 h;

c) NaNO₂, H₂SO₄, Zn, EtOH/H₂O (10:1), 08C!reflux, 3 h; d) BBr₃, CH₂Cl₂,

ꢀ788C!RT, 5 h.

Scheme 6. Derivatives with di-substituted B-rings. Reagents and conditions: a) K₂CO₃, DMAc, 1608C, 4 h; b) BBr₃,CH₂Cl₂, ꢀ788C!RT, 3 h; c) K₃PO₄,Cu(bipy)₂BF₄,

1208C, DMF, 48 h, 10%; d) Zn, HCl, 08C!RT, 1 h, 41%; e) Li(TMS)₂, THF, 08C!RT, 3 h, 46%.

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tate provided 40, which was then subjected to formylation fol-

lowed by condensation to give 41. Conversion of the hydroxy

to a chloro group using phosphoryl chloride and nucleophilic

substitution by ammonia at 1308C provided 43, which was

subsequently demethylated using boron tribromide to give

the final product PT134.

Results and Discussion

We previously described the synthesis of a series of diphenyl

ether inhibitors of InhA, the most potent of which had hexyl

or octyl substituents on the inhibitor A-ring (K_i’=9.4 and

1.1 nm, respectively).^[3a]We evaluated the pharmacodynamic

properties of the hexyl analogue (PT004) in a mouse model of

TB infection, but failed to observe a significant decrease in

bacterial load.^[6]Pharmacokinetic analysis of PT004 suggested

that improvements in ClogP might result in improved in vivo

activity, and we subsequently synthesized a series of B-ring

substituted PT004 analogues.^[10]These studies, coupled with

additional SAR data on the inhibition of the enoyl-ACP reduc-

tase in other organisms,^[16]indicated that modification to the

Scheme 7. Derivatives with 2’-N-pyridyl B-rings. Reagents and conditions:

a) Cs₂CO₃, MeCN, 808C, 2 h; b) BBr₃, CH₂Cl₂, ꢀ788C!RT, 2 h; c) Fe, AcOH/

H₂O (4:1), 1008C, 30 min, 77%; d) 1. 70% HF in pyridine, 0–608C, 1 h;

2. BBr₃, CH₂Cl₂, ꢀ788C!RT, 2 h, 49%.

and 38. The ether intermediates were transformed

into the appropriately substituted diaryl ethers 6, 19–

26, 30, 32, 35, 37, and 39 using group conversions

such as reductions, deaminations,^[13,15]diazotizations,

hydrolysis, and fluorination. Final compounds were

obtained after the subsequent demethylation reac-

tion with boron tribromide.^[13,15a,c]

The synthesis of PT134 is challenging. It was first

attempted by using several metal-catalyzed coupling

conditions^[11,12]to link compound 3 with tert-butyl-

oxycarbonyl (Boc)-protected 5-bromopyrimidin-4-yl-

amine, however, none of these conditions afforded

the desired product. To address this hurdle, we con-

Scheme 9. Derivatives with a pyrimidyl B-ring. Reagents and conditions: a) ethyl bromo-

acetate, NaOEt, EtOH, 808C, 16 h, 35%; b) 1. ethyl formate, NaH, THF, 658C, 4 h; 2. forma-

structed the pyrimidine ring using a five-step synthe-

sis that employs relatively simple reaction conditions midine acetate, EtOH/MeOH (1:1), 808C, 4 h, 47%; c) POCl₃, 708C, 3 h, 46%; d) NH₄OH,

1308C, 18 h, 87%; e) BBr₃, CH₂Cl₂, ꢀ788C!RT, 3 h, 68%.

(Scheme 9). The alkylation of 3 with ethyl bromoace-

Scheme 8. Derivatives with 4’-N-pyridyl B-rings. Reagents and conditions: a) NaOH, MeCN, 808C, 2 h, 60%; b) Fe, AcOH, H₂O, 808C, 2 h, 57%; c) BBr₃, CH₂Cl₂,

ꢀ788C!RT, 5 h; d) K₂CO₃, DMAc, 1608C, 7 h, 69%; e) KOH, MeOH, 808C, 2 h, 82%.

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B-ring might also further improve the affinity of this

inhibitor series for InhA, leading to the synthesis of

an ortho-methyl-substituted analogue with signifi-

cantly improved affinity for InhA (PT070).^[9]PT070

was found to be slow-onset inhibitor of InhA with

a residence time of 24 min on the enzyme. Based on

the knowledge that drug–target residence time

could have a dramatic impact on in vivo drug activi-

ty,^[8a,b,f]we set out to explore the effect of B-ring sub-

stituents on the time-dependent inhibition of InhA

and on in vivo activity. We show here that time-de-

pendent inhibition is sensitive to the substitution

pattern. We also show that PT070, together with an

analogue bearing an ortho-chloro group (PT091) de-

crease bacterial load in the spleens of mice infected

with M. tuberculosis.

Table 1. Derivatives with ortho-substituent groups.

Compd

R

IC₅₀^[a][nm]

MIC^[e]

[mgmL^ꢀ1

K_i[nm]

Slow

onset?

t_R[min]

]

PT004

PT010

PT013

PT070

PT091

PT092

PT095

PT096

PT113

PT114

PT119

H

11ꢁ1^[b]

2.1

12.50

3.13

3.125

1.56

3.125

50.00

25

9.4ꢁ0.5

N.D.

No

Yes

No

Yes

No

Yes

No

Yes

–

27ꢁ6

–

24ꢁ2

21ꢁ3

30ꢁ3

–

NO₂

NH₂

CH₃

Cl

Br

CF₃

I

F

OH

CN

182ꢁ2.0^[c]

61.9ꢁ4.5^[c]

50.7ꢁ4^[c]

N.D.

0.044ꢁ0.005

0.96ꢁ0.14

0.20ꢁ0.05

N.D.

49.5ꢁ2.2^[c]

10.0ꢁ0.8^[d]

29.7ꢁ1.2^[d]

44.6ꢁ7.5^[c]

12.1ꢁ4.8^[d]

48ꢁ3^[c]

3.72ꢁ5.13

0.09ꢁ0.02

15.9ꢁ3.7

2.14ꢁ0.35

–

1.56

12.5

2.5

9ꢁ3

–

80ꢁ12

235.6ꢁ10.0^[c]

[a] The half maximal inhibitory concentration (IC₅₀) is defined as the inhibitor concen-

tration required for 50% inhibition of enzyme activity. IC₅₀values were determined at

an enzyme concentration of [b] 1 nm, [c] 100 nm and [d] 20 nm. [e] The minimum in-

hibitory concentration (MIC) is the lowest inhibitor concentration required to inhibit

visible growth of bacteria. MIC values are the mean of three independent experi-

ments. IC₅₀, K_i, and residence time (t_R) values are the mean ꢁstandard deviation of

three independent experiments. N.D.=not determined.

Ortho-substituted diaryl ethers

Compounds PT004,^[3a]PT010,^[10]PT013,^[10]and

PT070^[9]have been reported in previous SAR studies.

The improvement in binding affinity of PT070 for

InhA compared with PT004 is thought to result from

decreased freedom of rotation about the ether

bond, together with increased hydrophobic contacts

between the B-ring and A198, M199, I202, and V203 in the

substrate binding loop based on the structural data.^[9]Intro-

duction of an ortho-methyl group on the B-ring also resulted

in an additional interaction between the inhibitor and A198.

These increased contacts are thought to be critical for the for-

mation of the EI* complex in which helix-6 of the substrate

binding loop has closed over the active site.^[9,17]Replacement

of the methyl group with an amino group resulted in an ana-

logue with similar IC₅₀and MIC values, but slow-onset inhibi-

tion was not detected for this compound, supporting the im-

portance of B-ring ortho-substitution for time-dependent inhib-

ition. Consequently, to better understand the mechanism of

the time-dependent kinetics and further modulate the resi-

dence time, analogues with various ortho substituents were

designed and synthesized (Table 1).

Compared with PT070, compounds PT113 (F), PT091 (Cl),

and PT092 (Br) all have sub-nanomolar K_ivalues with similar or

shorter residence times. In contrast, under the assay conditions

employed, the only analogue with a longer residence time,

PT119 (t_R=80 min), had a binding affinity that is decreased by

approximately 50-fold compared with PT070.

In an attempt to rationalize these observations, we deter-

mined the crystal structures of InhA bound to PT092^[17]and

PT119 (Figures 3 and 4). These compounds form a ternary

complex with the enzyme and the oxidized cofactor (NAD⁺),

and in both ternary complexes, the substrate binding loop of

InhA forms an a-helix that closely interacts with the B-ring,

similar to the enzyme–inhibitor complexes formed by other

time-dependent inhibitors.^[9,17,18]This is in contrast to the struc-

tures of complexes formed with rapid reversible inhibitors in

which the substrate binding loop is either disordered^[3a]or

forms an a-helix in a much more open conformation.^[17]The re-

Figure 3. Superimposed structures of InhA complexes involving PT070,

PT092 and PT119. a) Overlay of PT070 (magenta; PDB: 2X23) and PT092

(lime; PDB: 4OHU) bound to InhA. b) Overlay of PT070 (magenta; PDB:

2X23) and PT119 (cyan; PDB: 4OIM) bound to InhA. The graphics were

generated using PyMol.^[34]

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PT119 complex could represent a snapshot along the binding

coordinate from EI to EI*. Nevertheless, the decreased overall

binding affinity of PT119 compared with PT070 indicates that

EI* for PT119 is destabilized relative to PT070. Since the overall

energy barrier between EI and EI* has increased for PT119, it

follows that the transition state between EI and EI* is even

more destabilized (DDG^TS>DDG^EI*) (Figure 5). In the crystal

structure, PT119 appears to make reduced van der Waals con-

tacts with helix-6 residues (Figure 4). In addition, the bulkier

cyano substituent approaches the adjacent NAD and InhA

backbone within an unfavorably distance of 3.4 ꢁ. These less

than optimal interactions provide a plausible explanation for

the proposed destabilization.

Figure 5. Free energy diagram for the interaction of PT070 (c) and PT119

(a) with InhA. These inhibitors bind through a two-step mechanism in

which the energy barrier between enzyme–inhibitor complex (EI) and final

enzyme–inhibitor complex (EI*) is assumed to control the rate of formation

and breakdown of EI*.

Figure 4. Interactions in the PT070 and PT119 enzyme–inhibitor complexes.

a) Hydrophobic interactions between the PT070 B-ring and the substrate

binding loop (magenta; PDB: 2X23). b) Hydrophobic and hydrogen-bonding

interactions of the PT119 B-ring with the substrate binding loop and cofac-

tor (cyan; PDB: 4OIM). The graphics were generated using PyMol.^[34]

sults support the correlation between slow-onset inhibition

and ordering coupled to closure of the substrate binding loop

previously suggested for the FabI class of enoyl-ACP reductas-

es.^[19]

The results of the binding studies are consistent with a previ-

ous proposal that B-ring substituents should generally be small

in order to be accommodated in the InhA active site.^[10]How-

ever, here we observe that binding affinity is even more sensi-

tive to substituent size than we originally recognized. In partic-

ular, introduction of trifluoromethyl or cyano groups into the

ortho position of the B-ring resulted in compounds with 50–

100-fold weaker binding affinities compared with PT070, while

the introduction of polar groups such as a hydroxy (PT114)

also had a significant effect on the binding kinetics, which indi-

cates that hydrophobic interactions between the ortho group

and the protein are critical. Further studies with compounds

containing ortho halogens showed no correlation between the

binding affinity and electronegativity. These results confirm

that a small (containing one or two heavy atoms) ortho sub-

stituent is necessary for high-affinity binding and that this

group should not be a hydrogen-bond donor.

The substrate binding loop in the PT092 complex exhibits

a very similar conformation to that observed in the PT070

complex (Figure 3a), which is expected from their similar resi-

dence times and binding affinities. However, in one of the four

subunits in the asymmetric unit, a different binding mode of

PT092 relative to helix-6 is observed: A201 and I202, instead

of I202 and V203, make van der Waals contacts with the inhibi-

tor, which is accompanied by a twist of the helix-6 backbone

and displacement of the adjacent helix-7. Interestingly, the al-

tered position of the substrate binding loop in this InhA–

PT092 subunit is found exclusively in the structure of PT119

bound to InhA (Figures 3b and 4b). We speculate that this al-

tered helix-6 conformation rationalizes the increase in resi-

dence time of PT119 (t_R=80 min) relative to PT070 (t_R=

24 min) and propose that replacement of the B-ring methyl

group with a cyano group raises the energy barrier between EI

and EI* on the binding reaction coordinate.

Derivatives with di-substituted B-rings

One hypothesis for the improved binding affinity of PT070

compared with PT004 is that the additional methyl group re-

stricts rotations around the ether linkage and stabilizes the

Owing to the crystallization conditions and the potential

impact from crystal packing, the observed structure for the

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fied in Table 1, and included methyl, halogen, and

cyano groups. Compound PT107, an analogue of

PT070, was also synthesized to examine the toler-

ance for an additional para group.

Table 2. Derivatives with di-substituted B-ring.

Unfortunately, none of these derivatives had im-

proved activity compared with their parent com-

pounds (Table 2). To rationalize these results, the

docked structure of the dichloro analogue PT109

was compared with the corresponding docked struc-

ture of PT091 (monochloro) bound to the active site

of InhA (Figure 6). This analysis demonstrated that

PT109 has a higher Grid score, van der Waals, and in-

ternal energy than PT091, which explains the de-

creased binding affinity of this analogue for the

enzyme (Table S1 in the Supporting Information).

The extra steric hindrance has resulted in unfavora-

ble van der Waals interactions and thus lowered the

binding affinity of the inhibitor.

Compd

R¹

R²

R³

IC₅₀^[a][nm]

MIC^[b]

K_i[nm]

[mgmL^ꢀ1

]

PT004

PT070

PT107

PT108

PT109

PT110

PT111

PT131

PT133

H

CH₃

Cl

CH₃

F

H

CH₃

Cl

NH₂

CN

F

H

NO₂

H

11ꢁ1

50.7ꢁ4

50ꢁ5

2.1ꢁ0.9

3.125

6.25

9.4ꢁ0.5

0.044ꢁ0.005

0.13ꢁ0.03

N.D.

1570ꢁ200

86ꢁ6

100

25

>100

25

N.D.

N.I.

100ꢁ9

N.I.

79.7ꢁ24.4

C(NH)NH₂

F

N.D.

25

Cl

N.D.

[a] The half maximal inhibitory concentration (IC₅₀) is defined as the inhibitor concen-

tration required for 50% inhibition of enzyme activity. IC₅₀values were determined at

an enzyme concentration of 1 nm. N.I.=no inhibition observed at 2000 nm. [b] The

minimum inhibitory concentration (MIC) is the lowest inhibitor concentration required

to inhibit visible growth of bacteria. MIC values are the mean of three independent

experiments. IC₅₀and K_ivalues are the mean ꢁstandard deviation of three independ-

ent experiments. N.D.=not determined.

Enzyme inhibition and antibacterial growth assays

revealed that the compounds have similar SAR to

compounds with ortho-substituted B-rings—protic or

large groups are not tolerated at the ortho position

of the B-ring. Replacing a methyl group in PT108

conformation observed in the structure of the diphenyl ether

bound to the enzyme.^[9]Consequently, a series of di-ortho-sub-

stituted B-ring analogues were designed with two groups

ortho to the ether bond (Table 2). If hindered rotation about

the ether bond is important for binding, these compounds are

expected to have increased affinity compared with the ana-

logues with a single ortho substituent (Table 1). The two ortho

groups were chosen from the most potent compounds identi-

with an amino group (PT110) resulted in a total loss of binding

affinity and inhibition of bacterial growth. Introduction of the

large carbamimidoyl pharmacophore (PT131) also led to

a total loss of inhibitory activity, compared with smaller groups

such as cyano or chloro (PT111 and PT133, respectively). Thus,

substituents that include halogen or cyano groups are pre-

ferred. Although MIC values for the di-ortho analogues are

raised by 4–15-fold compared with PT070, the ortho,para-di-

substituted B-ring analogue PT107 has similar IC₅₀and MIC

values, implying that small para groups on the B-ring are well

tolerated.

Derivatives with heterocyclic B-rings

In order to improve hydrophilicity of the diaryl ethers, we also

explored analogues with pyridyl B-rings. Previous studies

showed that although introduction of an ortho nitrogen atom

into the B-ring decreased activity significantly, para and meta

nitrogen atoms were well tolerated.^[10]However, the position

of the meta nitrogen is ambiguous on the unsubstituted B-

ring. To differentiate 2’- and 4’-N (Figure 1c), 1’-substituted pyr-

idyl derivatives were synthesized and evaluated (Table 3). The

substituents included fluoro and cyano groups, which were

found to be preferred in studies with the phenyl B-ring ana-

logues, together with an amino group. In addition, a carboxyl-

ate group was also introduced to explore the effect of an ion-

izable group on activity. The pyridyl B-rings were found not to

alter the preference for specific ortho substituents: the fluorine

substituent resulted in the compound with the lowest IC₅₀

value (PT161) within the 2’-pyridyl derivatives, as did the

cyano group (PT115) within the 4’-pyridyl derivatives. Impor-

tantly, introduction of fluoro or cyano groups led to com-

pounds that displayed slow-onset inhibition (PT164, PT161), in

contrast to the parent compound PT077, suggesting that

Figure 6. Superimposed modelled structures of InhA complexes involving

PT091 and PT109. PT091 (yellow) and PT109 (magenta) were docked into

the InhA active site (lime) as described in the text. Also shown is the NAD⁺

cofactor (gray). The graphic was generated with PyMol.^[34]

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to identify potential differences in the interaction of

2’- and 4’-N analogues with the enzyme. This analysis

revealed that although the 2’- and 4’-N atoms have

similar hydrophobic contacts with I202 and M161,

the 2’-N position is able to form additional hydrogen-

bonding interactions with the backbone amide of

G96 (Figure 7). The poor MIC values for the 4’-pyridyl

compounds are consistent with the relatively weak

affinities of these inhibitors for InhA, while the 2’-pyr-

idine compounds had MIC values that were generally

better than compounds with phenyl B-rings (Table 1)

suggesting that the pyridine B-ring may aid uptake

into the bacterium.

Table 3. Derivatives with heterocyclic B-rings.

Compd

R

IC₅₀^[a][nm]

MIC^[b]

[mgmL^ꢀ1] onset? [min]

Slow

t_R

PT077

PT164

PT161

H

CN

F

190ꢁ12

308ꢁ4.2

40ꢁ20

3.13

0.313

0.3125

No

Yes

–

N.D.

PT112

PT115

PT116

NH₂

CN

69000ꢁ700

50

N.D.

Yes

N.D.

–

>20

–

890.6ꢁ105.8 12.5

COOH >12000

25

PT134

238.8ꢁ46.8

3.125

N.D.

–

Rapid model of in vivo efficacy

[a] The half maximal inhibitory concentration (IC₅₀) is defined as the inhibitor concen-

tration required for 50% inhibition of enzyme activity. IC₅₀values were determined at

an enzyme concentration of 100 nm. [b] The minimum inhibitory concentration (MIC)

is the lowest inhibitor concentration required to inhibit visible growth of bacteria.

MIC values are the mean of three independent experiments. IC₅₀and K_ivalues are the

mean ꢁstandard deviation of three independent experiments. N.D.=not determined.

PT004 decreased the bacterial load in the rapid mac-

rophage model of TB infection,^[6]demonstrating that

it is able to enter the macrophage and kill M. tubercu-

losis bacteria under altered growth conditions. How-

ever, PT004 did not significantly decrease the bacteri-

al load in the lung or spleen in the rapid murine

model of TB.^[6]In the present study, the time-depen-

these substituents have an important effect on time-depen-

dent kinetics. Experiments with the carboxylic group (PT116)

indicated that an ionizable group, presumably bearing

a formal negative charge, is not tolerated at the ortho position.

We found that in 1’-substituted B-rings, the 2’-N analogues

generally had lower MIC and IC₅₀values compared with the

4’-N compounds (PT164 vs PT115). Although the amino group

previously resulted a fivefold increase in the IC₅₀value com-

pared with the fluoro group (PT013 vs PT113), this difference

increased to 1700-fold (PT112 vs PT161), very likely caused by

the different scaffolds. The benefit of the 2’-N atom was also

seen in PT134, which has both 2’- and 4’-N. Improved IC₅₀and

MIC values were observed for PT134 compared with the corre-

sponding 4’-N analogue PT112, which has a 300-fold lower IC₅₀

value. The X-ray structure of PT070 bound to InhA was used

dent inhibitors PT070 and PT091 were evaluated for efficacy

when delivered via intraperitoneal injection in the same mouse

model.

Compared with untreated controls, PT070 decreased the

bacterial counts in the spleen 0.57ꢁ0.26 log₁₀with a p value

of 0.007, while PT091 decreased the bacterial counts in the

spleen 0.69ꢁ0.22 log₁₀with a p value of 0.0002 (Figure 8a).

Importantly, the spleen is a secondary site of TB infection, and

if a compound can decreased the bacterial burden in the

spleen, it is indicative of an ability to control dissemination

and disease progression. In contrast, PT004 is a rapid reversi-

ble InhA inhibitor that does not demonstrate efficacy in the

murine model of TB. Importantly, PT070 and PT091 are slow-

onset inhibitors having residence times of 24 and 21 min, re-

spectively. The improved efficacy of diaryl ethers that have in-

creased residence time is consistent with observations in the

Francisella tularensis infection model,^[7]and highlights the po-

tential importance of drug–target residence time for modulat-

ing in vivo drug efficacy.^[8e,f,20]

Conclusions

Twenty compounds were synthesized based on the diaryl

ether scaffold, with a hexyl-substituted A-ring and various

modifications to the B-ring. All of the compounds were evalu-

ated for enzyme inhibition of InhA and cell-based antibacterial

activity against M. tuberculosis. Crystallographic data demon-

strated that the slow-onset inhibitors PT092 and PT119 result-

ed in a more ordered and closed InhA substrate binding loop,

in agreement with our hypothesis that motions of this loop

are related to the structural change that accompanies the EI to

EI* transition for slow-onset inhibitors. The relatively large

cyano group in PT119 creates steric hindrance, introduces con-

formational change of helix-6, and preferentially destabilizes

Figure 7. Interactions between the PT070 B-ring and InhA. Possible hydro-

phobic and hydrogen-bonding interactions are shown for the 2’- and 4’-po-

sitions of the PT070 (green) B-ring. The graphic was generated from crystal

structure 2X23 using PyMol.^[34]

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For all the diaryl ether compounds, slow-onset inhibitors

always have higher binding affinity than rapid reversible inhibi-

tors. In contrast to the previously reported lack of efficacy for

the rapid onset inhibitor PT004, the slow-onset inhibitors

PT070 and PT091 have demonstrated efficacy in a rapid

animal model of TB infection, causing a decrease in bacterial

colony-forming units (CFUs) in the spleen. Together, these re-

sults support the importance of slow-onset kinetics for in vivo

drug efficacy.^[16]

Experimental Section

Chemistry

All commercially available chemicals and solvents were used as

supplied without further purification. All new compounds gave sat-

isfactory spectroscopic and/or analytical data. ¹H and ¹³C NMR

spectra were recorded at 300 or 400 MHz (Varian INOVA), and

chemical shifts (d) are reported in parts per million (ppm) down-

field from the internal standard, tetramethylsilane (TMS). Mass

spectra (MS) were obtained using electrospray (ES) ionization tech-

niques (Agilent Technologies, 1100 Series LC/MSD). HPLC purifica-

tion was performed using a controller (Shimadzu CBM-20A), a sol-

vent delivery unit (Shimadzu LC-20A), and a detector (Shimadzu

SPD-20A), with a PFP column (Phenomenex lunar, 3.5 mm, 4.6ꢂ

100 mm). Compounds were eluted with H₂O/CH₃CN (15:85 at

0 min, 0:100 at 22 min, flow rate: 1.0 mLmin^ꢀ1) and detected at

254 and 278 nm. The purity of all target compounds is >98% as

Figure 8. a) Histogram and b) scatterplot of colony-forming unit (CFU) data

from the lung and spleen of animals infected with M. tuberculosis (Erdman)

and either untreated (control) or drug-treated. Open bars or symbols repre-

sent CFU data from the lung, whereas shaded bars and symbols represent

CFU data from the spleen. All compounds were formulated in 15% EtOH,

20% propylene glycol, 40% polyethylene glycol 400, phosphate-buffered

saline and delivered via intraperitoneal injection. Animals were treated with

PT070, 50 mgkg^ꢀ1BID Day 2, 25 mgkg^ꢀ1BID Day 3–10, or with PT091,

25 mgkg^ꢀ1BID Day 1–4, 12.5 mgkg^ꢀ1BID Day 5–6, 12.5 mgkg^ꢀ1SID and

25 mgkg^ꢀ1SID Day 7–10. The significance between control and treatment

groups was determined via an unpaired, one-tailed t-test (t-test 1,2) with

95% confidence intervals from mean CFU values for the organs (GraphPad

Software version 5, San Diego, USA; http://www.graphpad.com).

1

determined by H NMR or HPLC.

General procedure of diazotization (for 7, 8, 9, 10): The appropri-

ate aniline (1 mmol) was dissolved in AcOH (10 mL) and H₂O

(100 mL), and the solution was cooled to ꢀ108C. NaNO₂(103.9 mg,

1.5 mmol) was added slowly, followed by the appropriate halide

(1.5 mmol) after 30 min. The ice bath was removed, and the reac-

tion was stirred for approximately 2 h. The reaction mixture was

then diluted with CH₂Cl₂(250 mL) and washed with H₂O (150 mL).

The organic layer was dried over MgSO₄, filtered and concentrated

in vacuo to give crude product. The crude compound was purified

by column chromatography or used in the next step without pu-

rification.

the transition state between EI and EI* compared with the EI*

ground state, thus resulting in a longer residence time. The ac-

tivity of the diaryl ether compounds is highly sensitive to the

size of the ortho substituents on the B-ring. Once the group

has more than two heavy atoms, the activity is significantly de-

creased. In addition, within the acceptable size limits for this

group, non-hydrogen bond donors are preferred.

General procedure for the aromatic substitution reaction (for 5,

15, 16, 17, 18, 27, 29, 31, 38): The appropriate nitrobenzene

(1.24 mmol), K₂CO₃(1.03 mmol), and a catalytic amount of 18-

crown-6 ether were added to a stirred solution of the correspond-

ing phenol (1.03 mmol) in DMF (3 mL) at RT. The solution was then

heated at 1108C for 3 h. After completion of the reaction, as

shown by TLC, the reaction mixture was diluted with water (20 mL)

and extracted with EtOAc (2ꢂ20 mL). The combined organic

phases were washed with brine (30 mL), dried over anhydrous

Na₂SO₄, filtered and concentrated in vacuo. The crude product was

purified by column chromatography to afford the substituted ni-

trobenzene.

Introduction of groups on both ortho positions resulted in

a loss of activity that, according to docking studies, leads to

a conformation in which there are unfavorable van der Waals

interactions with the enzyme. The ortho,para-di-substituted

compound PT107 showed similar activity to that of the parent

compound, suggesting that para groups on the B-ring are well

tolerated. We were able to distinguish 2’-N and 4’-N pyridyl

B-ring analogues by introducing ortho substituents into the

B-ring. The 2’-N pyridyl B-rings had significantly better activity

than the 4’-N analogues, which may reflect the improvement

in cell penetration of these compounds and suggests that the

pyridyl B-ring is a better scaffold than the phenyl B-ring.

General procedure for reduction of nitrobenzene to aniline (for

6, 19, 20, 21, 22, 30): Concd HCl (1.3 mL) was added dropwise to

a stirred solution of the nitrobenzene (0.78 mmol) in EtOH (15 mL)

at 08C, and the mixture was stirred for 5 min. Zn powder

(17.3 mmol) was then added slowly, and the reaction mixture was

allowed to come to RT. The reaction was monitored by TLC, and

after completion (~1 h), the reaction was quenched with Et₃N and

filtered. The filtrate was concentrated in vacuo, and the crude

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product was purified by column chromatography to afford the cor-

responding aniline.

4-Hexyl-2-methoxyphenol (3): Activated Pd/C (40 mg) was added

to a solution of 2 (800 mg, 2.7 mmol) in EtOH (100 mL). The reac-

tion was stirred under 1 atm of H₂for 6 h and then filtered

through Celite. The solvent was removed in vacuo to give the

crude product. Flash chromatography (EtOAc/petroleum ether, 5%)

General procedure for deamination of anilines (for 23, 24, 25,

26): Concd H₂SO₄(5 mL) was added dropwise to a stirred solution

of the aniline (4.3 mmol) in EtOH (25 mL) at 08C. A saturated solu-

tion of NaNO₂in H₂O (8.6 mmol, 5 mL) was added very slowly, and

the reaction mixture was stirred at 08C for 30 min. The solution

was allowed to come to RT, and then Zn powder (43 mmol) was

added. After heating at reflux for 0.5 h, the remaining Zn powder

was added, and the mixture was heated at reflux for an additional

2.5 h. After completion of the reaction, the mixture was filtered

and neutralized with saturated aq NaHCO₃(pH 7–8). The organic

solvent was removed by evaporation in vacuo, and the aqueous

solution was then extracted with EtOAc (2ꢂ50 mL). The combined

organic layers were washed with water (50 mL), dried over Na₂SO₄,

filtered and concentrated in vacuo. The crude material was subse-

quently purified by column chromatography to afford the appro-

priate deaminated product.

1

gave 3 as a colorless oil (500 mg, 89%): H NMR (400 MHz, CDCl₃):

d=6.80 (d, J=6.0 Hz, 1H), 6.65–6.63 (m, 2H), 5.42 (s, 1H), 3.85 (s,

3H), 2.50 (t, J=5.7 Hz, 2H), 1.56–1.54 (m, 2H), 1.30–1.27 (m, 6H),

0.86 ppm (t, J=5.1 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): d=146.5,

143.7, 135.2, 121.1, 114.3, 111.1, 56.1, 35.9, 32.0, 32.0, 29.2, 22.8,

14.3 ppm; MS (ESI+, 70 eV): m/z (%): 209.0 (100) [M+H]⁺; MS

(ESIꢀ, 70 eV): m/z (%): 207 (100) [MꢀH]^ꢀ.

4-Hexyl-2-methoxy-1-(2-(trifluoromethyl)phenoxy)benzene (4):

1-Iodo-2-(trifluoromethyl)benzene (2.0 g, 7.35 mmol),

3 (1.10 g,

7.35 mmol), Cs₂CO₃(10.5 g, 32.3 mmol), (CuOTf)₂·PhH (185.0 mg,

0.37 mmol, 5.0 mol% Cu), and 1-naphthoic acid (5.56 g, 32.3 mmol)

were dissolved in EtOAc (1 mL) and toluene (75 mL) in an oven-

dried 150 mL two-necked round-bottomed flask. Molecular sieves

(1.8 g, 4 ꢁ) were added to the flask under a continuous stream of

N₂, and then the flask was sealed with a septum and heated to

110 8C under N₂. After 24 h, upon cooling to RT, CH₂Cl₂(50 mL) was

added, and the mixture was filtered. The filtrate was washed with

5% aq NaOH (20 mL), and the aqueous layer was back-extracted

with CH₂Cl₂(2ꢂ25 mL). All organic layers including the original fil-

trate were combined and washed with brine (50 mL), dried over

Mg₂SO₄, filtered and concentrated in vacuo to give the crude prod-

uct. Purification by flash chromatography (EtOAc/hexane, 4%) gave

4 as a light yellow oil (1.94 g, 75%): MS (ESI+, 70 eV): m/z (%):

353.0 (100), 354.2 (21) [M+H]⁺.

General procedure of demethylation (for PT091–92, PT095–96,

PT107–116, PT119, PT131, PT133–134, PT161, PT164): The me-

thoxybenzene (0.627 mmol) was dissolved in dry CH₂Cl₂(50 mL),

and BBr₃(0.94 mL, 2m in CH₂Cl₂, 1.88 mmol) was added dropwise

at ꢀ408C. The reaction was gradually warmed to RT and stirred for

3 h. When TLC showed completion, the reaction was cooled to

ꢀ408C and quenched with MeOH. The solution was concentrated

in vacuo to give the crude product. Purification by chromatogra-

phy gave pure compounds.

4-(Benzyloxy)-3-methoxybenzaldehyde

(1):

Vanillin

(10 g,

65.7 mmol), BnBr (12.4 g, 72.3 mmol) and KOH (4.1 g, 72.3 mmol)

were dissolved in MeOH/H₂O (1:1, 150 mL) and heated at reflux for

2 h. After TLC showed completion, the reaction was diluted with

H₂O (300 mL) and extracted with CH₂Cl₂(2ꢂ100 mL). The com-

bined organic layers were dried with MgSO₄, filtered and concen-

trated in vacuo to give the crude product. Purification by flash

chromatography (EtOAc/petroleum ether, 8%) gave 1 as a white

4-Hexyl-2-methoxy-1-(2-nitrophenoxy)benzene (5): A solution of

3

(800 mg, 3.8 mmol), 1-fluoro-2-nitrobenzene (536.2 mg,

3.8 mmol), and 18-crown-6 (50.2 mg, 0.19 mmol) in dry DMF

(40 mL) was heated to 1108C under N₂and stirred for 3 h. The re-

action was cooled to RT and partitioned between H₂O (200 mL)

and CH₂Cl₂(200 mL). The organic layer was separated, dried over

MgSO₄, filtered and concentrated to give the crude product. Purifi-

cation with flash chromatography (EtOAc/petroleum ether, 5%)

gave 5 as a light yellow oil (826 mg, 66%): ¹H NMR (300 MHz,

CDCl₃): d=7.94 (dd, J=8.4, 1.5 Hz,1H), 7.40 (td, J=7.5, 1.8 Hz, 1H),

7.08 (td, J=7.5, 1.5 Hz, 1H), 6.99 (d, J=7.8 Hz, 1H), 6.81 (dd, J=

8.4, 1.2 Hz, 1H), 6.80 (dd, J=8.1, 1.8 Hz, 1H), 6.76 (d, J=2.1 Hz,

1H), 3.77 (s, 3H), 2.61 (t, J=7.8 Hz, 2H), 1.62 (m, 2H), 1.32 (m, 2H),

0.89 ppm (t, J=6.9 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): d=152.3,

151.3, 141.8, 141.3, 134.1, 125.8, 121.9, 121.2, 118.1, 113.6, 56.2,

36.1, 31.9, 31.8, 29.2, 22.8, 14.3 ppm; HRMS-ES+: m/z [M+H]⁺

calcd for C₁₉H₂₃NO₄: 330.1705, found: 330.1703.

1

crystalline solid (14.5 g, 91%): H NMR (400 MHz, CDCl₃): d=9.81 (s,

1H), 7.42–7.29 (m, 7H), 6.96 (d, J=6.3 Hz, 1H), 5.22 (s, 2H),

3.92 ppm (s, 3H); ¹³C NMR (100 MHz, CDCl₃): d=191.1, 153.8, 150.3,

136.2, 130.5, 128.9, 128.4, 127.4, 126.8, 112.6, 109.6, 71.09,

56.3 ppm; HRMS-ES+: m/z [M+H]⁺calcd for C₁₅H₁₅O₃: 243.1021,

found: 243.1022.

1-(Benzyloxy)-4-(hex-1-en-1-yl)-2-methoxybenzene (2): nBuLi

(2.45 mL, 4.9 mmol, 2m in cyclohexane) was added dropwise to

a

solution of n-pentyl-triphenylphosphonium bromide (2 g,

4.9 mmol) in dry THF (100 mL) at ꢀ788C. After 30 min, a solution

of 1 (1.0 g, 4.1 mmol) in dry THF (50 mL) was added dropwise.

After an additional 30 min, the cooling bath was removed, and the

reaction was stirred for another 1.5 h. When TLC showed comple-

tion, the reaction was quenched with 1m aq HCl (5 mL) and dilut-

ed with H₂O (100 mL). The aqueous mixture was extracted with

CH₂Cl₂(3ꢂ25 mL), and the combined organic layers were dried

over MgSO₄, filtered and concentrated in vacuo to give the crude

product. Purification by flash chromatography (EtOAc/petroleum

2-(Hexyl-2-methoxyphenoxy)aniline (6): Activated Pd/C (25 mg)

was added to a solution of 5 (500 mg, 1.5 mmol) in EtOH (100 mL).

The reaction was stirred under 1 atm of H₂for 6 h and then filtered

through Celite. The crude product was obtained by removal of the

solvent in vacuo and used in the next step without further purifica-

1

tion (413 mg, 91%): H NMR (400 MHz, CDCl₃): d=6.88 (td, J=5.7,

1.2 Hz, 1H), 6.78–6.75 (m, 3H), 6.72 (dd, J=6.0 Hz, 1.2 Hz, 1H), 6.65

(dd, J=5.1, 1.5 Hz, 1H), 6.62 (dd, J=6.0, 1.2 Hz, 1H), 3.86 (s, 2H),

3.84 (s, 3H), 2.55 (t, J=6.0 Hz, 2H), 1.59–1.56 (m, 2H), 1.32–1.28 (m,

6H), 0.87 ppm (t, J=5.4 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): d=

150.5, 144.9, 143.5, 139.2, 138.1, 124,0, 120.8, 119.1, 118.7, 118.4,

116.4, 113.2, 56.9, 36.0, 31.9, 31.5, 29.2, 22.8, 14.3 ppm; HRMS-ES+:

m/z [M+H]⁺calcd for C₁₉H₂₅NO₂: 300.1964, found: 300.1963.

1

ether, 5%) gave 2 as a white crystalline solid (1.0 g, 82%): H NMR

(400 MHz, CDCl₃): d=7.27–7.43 (m, 5H), 6.91 (s, 1H), 6.79 (s, 1H),

6.29 (d, J=11.7 Hz, 2H), 6.08 (td, J=11.7, 5.1 Hz, 1H), 5.13 (s, 2H),

3.89 (s, 3H), 2.18 (dd, J=5.1, 4.8 Hz, 2H), 1.46–1.29 (m, 4H),

0.91 ppm (t, J=5.4 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): d=150.0,

147.6, 137.5, 131.98, 129.7, 129.5, 28.7, 128.0, 127.5, 118.9, 114.5,

109.5, 71.4, 57.2, 32.9, 31.9, 22.5, 14.2 ppm; HRMS-ES+: m/z

[M+H]⁺calcd for C₂₀H₂₄O₂: 297.1855, found: 297.1855.

1-(2-Chlorophenoxy)-4-hexyl-2-methoxybenzene (7): Compound

6 (300 mg, 1.0 mmol) and CuCl were reacted according to the gen-

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eral procedure for diazotization to give 7 as a light yellow solid

(188 mg, 59%). The crude material was used in the next step with-

out purification. MS (ESI+, 70 eV): m/z (%): 319.2 (100) [M+H]⁺.

procedure for deamination to give 23 as a yellow oil (406 mg,

85%): MS (ESI+, 70 eV): m/z (%): 315.2 (100), 316.1 (22) [M+H]⁺.

1-(2,6-dichlorophenoxy)-3-hexyl-2-methoxybenzene (24): Com-

pound 16 (800 mg, 2.0 mmol) was treated according to the general

procedure for reduction of nitrobenzene to give 20 as a yellow

solid (577 mg, 78%). Crude compound 20 (577 mg, 1.6 mmol) was

further reacted according to the general procedure for deamina-

1-(2-Bromophenoxy)-4-hexyl-2-methoxybenzene (8): Compound

6 (300 mg, 1.0 mmol) and CuBr were reacted according to the gen-

eral procedure for diazotization, and purification by flash chroma-

tography (EtOAc/petroleum ether, 5%) gave 8 as a yellow solid

(233 mg, 64%): ¹H NMR (300 MHz, CDCl₃): d=7.26 (dd, J=6.9,

1.5 Hz, 1H), 7.18 (dd, J=4.8, 1.5 Hz, 1H), 6.96–6.64 (m, 5H), 3.82 (s,

3H), 2.60 (t, J=7.5 Hz, 2H), 1.65–1.60 (m, 2H), 1.33–1.22 (m, 6H),

0.90 ppm (t, J=6.3 Hz, 3H); MS (ESI+, 70 eV): m/z (%): 363.1 (100),

364.0 (21), 365.4 (100) [M+H]⁺.

1

tion to give 24 as a yellow oil (486 mg, 86%): H NMR (400 MHz,

CDCl₃): d=7.38 (m, 2H), 7.16 (t, J=8.5 Hz, 1H), 6.82 (s, 1H), 6.58 (d,

J=8.1 Hz, 1H), 6.32 (d, J=7.6 Hz, 1H), 3.80 (s, 3H), 2.56 (t, J=

7.5 Hz, 2H), 1.62–1.54 (m, 3H), 1.38–1.24 (m, 5H), 0.94–0.86 ppm

(m, 3H); HPLC: t_R=11.8 min (96% purity).

4-Hexyl-2-methoxy-1-(2-methoxy-4-nitrophenoxy)benzene (15):

Compounds 3 (1.0 g, 4.8 mmol) and 11 (1.0 g, 5.8 mmol) were

treated according to the general procedure for aromatic substitu-

tion to give 15 as a yellow solid (1.7 g, 98%): ¹H NMR (300 MHz,

CDCl₃): d=7.83 (d, J=2.4 Hz, 1H), 7.74 (dd, J=2.7, 6.0 Hz, 1H),

6.98 (d, J=7.8 Hz, 1H), 6.80 (td, J=7.8, 1.8 Hz, 2H), 6.60 (d, J=

9.0 Hz, 1H), 4.02 (s, 3H), 3.76 (s, 3H), 2.62 (t, J=7.8 Hz, 2H), 1.66–

1.57 (m, 2H), 1.38–1.28 (m, 6H), 0.89 ppm (t, J=6.9 Hz, 3H); MS

(ESI+, 70 eV): m/z (%): 360.3 (100), 361.1 (22) [M+H]⁺.

1-(2-Fluorophenoxy)-4-hexyl-2-methoxybenzene (25): Compound

17 (800 mg, 2.3 mmol) was treated according to the general proce-

dure for reduction of nitrobenzene to give 21 as a yellow solid

(599 mg, 82%). Crude compound 21 (599 mg, 1.9 mmol) was fur-

ther reacted according to the general procedure for deamination

1

to give 25 as a yellow oil (448 mg, 78%): H NMR (400 MHz, CDCl₃):

d=7.17–7.12 (m, 1H), 7.02–6.99 (m, 2H), 6.88–6.81 (m, 3H), 6.72–

6.69 (dd, J=8.2, 2.2 Hz, 2H), 3.84 (s, 3H), 2.58 (t, J=7.4 Hz, 2H),

1.64–1.58 (m, 2H), 1.37–1.32 (m, 6H), 0.89 ppm (t, J=6.6 Hz, 3H);

HRMS-ES+: m/z [M+H]+ calcd for C₁₉H₂₉FO₂: 303.1760, found:

300.1758; HPLC: t_R=13.5 min (98% purity).

1,3-Dichloro-2-(hexyl-2-methoxyphenoxy)-5-nitrobenzene (16):

Compounds 3 (1.0 g, 4.8 mmol) and 12 (1.2 g, 5.8 mmol) were

treated according to the general procedure for aromatic substitu-

1-Chloro-3-fluoro-2-(hexyl-2-methoxyphenoxy)benzene

(26):

1

tion to give 16 as a yellow solid (1.7 g, 88%): H NMR (400 MHz):

Compound 22 (500 mg, 1.4 mmol) was treated according to the

general procedure for deamination to give 26 as a yellow oil

(407 mg, 85%): ¹H NMR (400 MHz, CDCl₃): d=7.26 (d, J=7.6 Hz,

1H), 7.18–7.04 (m, 2H), 6.82 (s, 1H), 6.62 (d, J=8.2 Hz, 1H), 6.42 (d,

J=7.9 Hz, 1H), 3.94 (s, 3H), 2.59 (t, J=6.9 Hz, 2H), 1.68–1.54 (m,

3H), 1.42–1.24 (m, 6H), 1.89 ppm (t, J=6.6 Hz, 3H); MS (ESI+,

70 eV): m/z (%): 337.3 (100), 338.0 (21), 339.4 (33) [M+H]⁺; HPLC:

t_R=11.3 min (95.4% purity).

d=8.24 (s, 2H), 6.82 (s, 1H), 6.62 (d, J=9.0 Hz, 1H), 6.42 (d, J=

2.2 Hz, 1H), 3.94 (s, 3H), 2.60 (t, J=7.6 Hz, 2H), 1.64–1.58 (m, 3H),

1.38–1.26 (m, 5H), 0.87 ppm (t, J=6.7 Hz, 3H).

2-Fluoro-1-(hexyl-2-methoxyphenoxy)-4-nitrobenzene (17): Com-

pounds 3 (1.0 g, 4.8 mmol) and 13 (0.92 g, 5.8 mmol) were treated

according to the general procedure for aromatic substitution to

1

give 17 as a yellow solid (1.0 g, 60%): H NMR (400 MHz,): d=8.00

(d, J=10.2 Hz,1H), 7.82 (d, J=9.2 Hz, 1H), 6.96 (d, J=8.2 Hz, 1H),

6.80–6.6.70 (m, 2H), 6.68 (t, J=8.1 Hz, 1H), 3.74 (s, 3H), 2.58 (t, J=

6.7 Hz, 2H), 1.64–1.56 (m, 3H), 1.38–1.22 (m, 5H), 0.87 ppm (t, J=

6.6 Hz, 3H).

4-Hexyl-2-methoxy-1-(2-methyl-4-nitrophenoxy)benzene

(27):

Compound 3 (1.0 g, 4.8 mmol) and 1-fluoro-2-methyl-4-nitroben-

zene (894 mg, 5.8 mmol) were reacted according to the general

procedure for aromatic substitution to give 27 as a yellow solid

(857 mg, 52%): MS (ESI+, 70 eV): m/z (%): 344.1 (100), 345.0 (22)

[M+H]⁺. The crude material was used in the next step without

further purification.

1-Chloro-3-fluoro-2-(hexyl-2-methoxyphenoxy)-5-nitrobenzene

(18): Compounds 3 (1.0 g, 4.8 mmol) and 14 (1.1 g, 5.8 mmol) were

treated according to the general procedure for aromatic substitu-

tion to give 18 as a yellow solid (1.4 g, 77%): ¹HNMR (400 MHz,

CDCl₃): d=8.18 (s, 1H), 7.91–7.90 (m, 1H), 6.80 (s, 1H), 6.72–6.64

(m, 2H), 3.82 (s, 3H), 2.58–2.52 (m, 2H), 1.62–1.54 (m, 3H), 1.38–

1.24 (m, 5H), 0.94–0.84 ppm (m, 3H); HPLC: t_R=8.4 min (95%

purity).

1-(2,6-Dimethylphenoxy)-3-hexyl-2-methoxybenzene (28): 2-

Bromo-1,3-dimethylbenzene (0.667 g, 3.6 mmol) and K₃PO₄(1.0 g,

4.8 mmol) were added to a stirred solution of 3 (0.5 g, 2.403 mmol)

in DMF (5 mL). The reaction mixture was degassed for 30 min, after

which Cu(bipy)₂BF₄(0.112 g, 0.24 mmol) was added, and the mix-

ture was stirred at 1208C for 48 h. After TLC showed that the reac-

tion was complete, the solution was diluted with EtOAc (40 mL)

and washed with water (2ꢂ40 mL). The organic layer was further

washed with brine (60 mL), dried over Na₂SO₄, filtered and concen-

trated in vacuo. Purification by flash chromatography (EtOAc/

hexane, 3%) gave 28 as a yellow solid (0.075 g, 10%): ¹H NMR

(400 MHz, CDCl₃): d=7.10–7.02 (m, 2H), 6.80 (s, 1H), 6.54 (d, J=

7.4 Hz, 1H), 6.20 (d, J=8.0 Hz, 2H), 3.98 (s, 3H), 2.54 (t, J=7.5 Hz,

2H), 2.18 (s, 3H), 1.64–1.48 (m, 6H), 1.38–1.24 (m, 5H), 0.89 ppm (t,

J=6.7 Hz, 3H).

4-(Hexyl-2-methoxyphenoxy)-3-methoxyaniline (19): Compound

15 (800 mg, 83%) was treated according to the general procedure

for reduction of nitrobenzene to give 19 as a yellow oil (609 mg,

1.8 mmol): MS (ESI+, 70 eV): m/z (%): 330.1 (100), 331.2 (22)

[M+H]⁺.

3-Chloro-5-fluoro-4-(hexyl-2-methoxyphenoxy) aniline (22): Com-

pound 18 (800 mg, 2.1 mmol) was treated according to the general

procedure for reduction of nitrobenzene to give 22 as a yellow

1

solid (545 mg, 74%): HNMR (400 MHz, CDCl₃): d=6.80 (s, 1H), 6.70

(s, 1H), 6.62–6.56 (m, 2H), 6.44 (d, J=7.4 Hz, 1H), 3.94 (s, 3H), 2.58

(t, J=7.4 Hz, 2H), 1.62–1.54 (m, 2H), 1.38–1.22 (m, 6H), 0.94–

0.84 ppm (m, 3H); MS (ESI+, 70 eV): m/z (%): 352.3 (100), 353.1

(21), 354.4 (33) [M+H]⁺; HPLC: t_R=7.3 min (96% purity).

1-(2-Methyl-6-nitrophenoxy)-3-hexyl-2-methoxybenzene

(29):

Compound 3 (1.0 g, 4.8 mmol) and 2-fluoro-1-methyl-3-nitroben-

zene (908 mg, 5.8 mmol) were reacted according to the general

procedure for aromatic substitution to give 29 as a yellow solid

(692 mg, 42%): ¹H NMR (400 MHz, CDCl₃): d=7.80 (d, J=8.1 Hz,

1H), 7.51 (d, J=2.1 Hz, 1H), 7.28–7.22 (m, 2H), 6.82 (s, 1H), 6.58 (d,

4-Hexyl-2-methoxy-1-(2-methoxyphenoxy)benzene (23): Com-

pound 19 (500 mg, 1.5 mmol) was treated according to the general

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J=2.1 Hz, 1H), 6.38 (m, 1H), 3.88 (s, 3H), 2.56 (t, J=7.2 Hz, 2H),

2.22 (s, 3H), 1.62–1.54 (m, 3H), 1.38–1.22 (m, 5H), 0.89 ppm (t, J=

6.7 Hz, 3H); MS (ESI+, 70 eV): m/z (%): 366.1 (100) [M+Na]⁺;

HPLC: t_R=12.3 min (99% purity).

over anhydrous Na₂SO₄, filtered and concentrated in vacuo to give

35 (92 mg, 77%): MS (ESI+, 70 eV): m/z (%): 301.2 (100), 302.0 (20)

[M+H]⁺. The crude material was used in the next step without

further purification.

2-(Hexyl-2-methoxyphenoxy)-3-methylbenzenamine (30): Com-

pound 29 (500 mg, 1.5 mmol) was treated according to the general

procedure for reduction of nitrobenzene to give 30 as a dark

yellow solid (187 mg, 41%): ¹H NMR (400 MHz, CDCl₃): d=6.99–

6.94 (t, J=8.4 Hz, 1H), 6.78 (d, J=7.6 Hz, 2H), 6.64 (m, 1H), 6.59

(m, 1H), 6.42 (m, 1H), 3.89 (s, 3H), 2.56 (t, J=7.5 Hz, 2H), 2.18 (s,

3H), 1.62–1.54 (m, 2H), 1.42–1.24 (m, 5H), 0.89 ppm (t, J=6.5 Hz,

3H); MS (ESI+, 70 eV): m/z (%): 314.0 (100), 315.3 (22) [M+H]⁺;

HPLC: t_R=11.7 min (99% purity).

3-(Hexyl-2-methoxyphenoxy)-4-nitropyridine 1-oxide (36): Com-

pound 3 (0.5 g, 2.4 mmol) was added to a solution of NaOH

(0.385 g, 9.6 mmol) in H₂O (1 mL), and the mixture was stirred for

5 min at RT. The water was removed in vacuo to afford a white

solid, which was taken up in CH₃CN (7 mL), and the resultant solu-

tion was treated with 3-bromo-4-nitropyridine 1-oxide (0.63 g,

2.8 mmol). The mixture was heated at reflux for 2 h and, after the

reaction was shown to be complete by TLC, the reaction mixture

was filtered, and the filtrate was concentrated in vacuo. Purification

by flash chromatography (EtOAc/hexane, 50%) gave 36 as a dark

2-(Hexyl-2-methoxyphenoxy)-3-fluorobenzonitrile (31): Com-

pound 3 (1.0 g, 4.8 mmol) and 2-bromo-3-fluorobenzonitrile (1.2 g,

5.8 mmol) were reacted according to the general procedure for ar-

omatic substitution to give 31 as a yellow solid (629 mg, 40%):

¹H NMR (400 MHz, CDCl₃): d=7.38–7.22 (m, 3H), 6.98 (d, J=8.2 Hz,

1H), 6.82–6.78 (m, 2H), 6.72 (d, J=7.8 Hz, 1H), 3.78 (s, 3H), 2.63 (t,

J=7.4 Hz, 2H), 1.72–1.64 (m, 3H), 1.42–1.24 (m, 6H), 0.92 ppm (t,

J=6.8 Hz, 3H); HPLC: t_R=11.0 min (95% purity).

1

yellow solid (0.5 g, 60%): H NMR (400 MHz, CDCl₃): d=7.86 (d, J=

2.2 Hz, 1H), 7.84 (d, J=8.0 Hz, 1H), 7.74 (s, 1H), 7.04 (d, J=7.8 Hz,

1H), 6.82 (m, 2H), 3.78 (s, 3H), 2.60 (t, J=7.6 Hz, 2H), 1.64–1.58 (m,

3H), 1.38–1.26 (m, 5H), 0.89 ppm (t, J=6.7 Hz, 3H); MS (ESI+,

70 eV): m/z (%): 347.2 (100), 348.1 (20) [M+H]⁺.

3-(Hexyl-2-methoxyphenoxy)pyridin-4-amine (37): A stirred solu-

tion of 36 (0.5 g, 1.4 mmol) in AcOH/H₂O (8:2, 100 mL) was heated

to 808C, and Fe powder (4.0 g, 72 mmol) was added portionwise

at 808C. The mixture was stirred at the same temperature for 2 h.

After the reaction was shown to be complete by TLC, the reaction

mixture was cooled to RT, quenched with water and extracted with

CH₂Cl₂(2ꢂ20 mL). The combined organic layers were washed with

brine (15 mL), dried over Na₂SO₄, filtered and concentrated in

vacuo. Purification by flash chromatography (MeOH/CH₂Cl₂, 5%)

gave 37 as a dark yellow oil (0.25 g, 57%): HRMS-ES+: m/z

[M+H]⁺calcd for C₁₈H₂₄N₂O₂: 301.1916, found: 301.1906.

2-(Hexyl-2-methoxyphenoxy)-3-fluorobenzamidine (32): Li(TMS)₂

(0.18 g, 1.18 mmol) was added to a stirred solution of 31 (0.2 g,

0.61 mmol) in THF (2 mL) at 08C, and the reaction was stirred at RT

for 3 h. After the reaction was shown to be complete by TLC, the

mixture was diluted with H₂O (15 mL) and extracted with CH₂Cl₂

(2ꢂ30 mL). The combined organic layers were washed with brine

(15 mL), dried over Na₂SO₄, filtered and concentrated in vacuo. Pu-

rification by flash chromatography (MeOH/CH₂Cl₂, 1%) gave 32 as

1

a yellow solid (0.096 g, 46%): H NMR (400 MHz, CDCl₃): d=8.96 (br

s, 1H), 7.98 (s, 2H), 7.42 (d, J=8.0 Hz, 1H), 7.12–7.03 (m, 2H), 6.90–

6.81 (m, 2H), 6.74 (d, J=7.6 Hz, 1H), 3.80 (s, 3H), 2.60 (t, J=7.2 Hz,

2H), 1.66–1.60 (m, 2H), 1.42–1.38 (m, 6H), 0.98–0.92 ppm (m, 3H);

MS (ESI+, 70 eV): m/z (%): 345.0 (100) [M+H]⁺; HPLC: t_R=9.4 min

(64% purity).

3-(Hexyl-2-methoxyphenoxy)isonicotinonitrile (38): Compound 3

(1.0 g, 4.8 mmol) and 3-chloroisonicotinonitrile (0.8 g, 5.8 mmol)

were reacted according to the general procedure for aromatic sub-

stitution to give 38 as a dark yellow oil (1.0 g, 69%): ¹H NMR

(400 MHz, CDCl₃): d=8.38 (d, J=1.5 Hz, 1H), 8.14 (s, 1H), 7.46 (d,

J=8.0 Hz, 1H), 7.12 (d, J=7.8 Hz, 1H), 6.82 (d, J=7.5 Hz, 2H), 3.78

(s, 3H), 2.62 (t, J=7.4 Hz, 2H), 1.66–1.62 (m, 2H), 1.42–1.34 (m,

6H), 0.91 ppm (t, J=6.6 Hz, 3H); HPLC: t_R=10.5 min (98% purity).

3-(Hexyl-2-methoxyphenoxy)-2-nitropyridine (34): Compound 3

(0.5 g, 2.4 mmol) was added to 5% aq NaOH (7.68 g, 9.61 mmol)

and stirred for 10 min. The solution was concentrated in vacuo,

and the residue was redissolved in CH₃CN (5 mL). The solution was

treated with 3-bromo-2-nitropyridine (0.455 g, 2.88 mmol), and the

reaction mixture was heated at reflux for 2 h. After the reaction

was shown to be complete by TLC, the mixture was filtered, and

the filtrate was partitioned between H₂O (15 mL) and CH₃CN

(50 mL). The organic layer was separated, dried over Na₂SO₄, fil-

tered and concentrated in vacuo. Purification by flash chromatog-

raphy (EtOAc/hexane, 10%) gave 34 as a yellow solid (0.13 g,

3-(Hexyl-2-methoxyphenoxy)isonicotinic acid (39): KOH pellets

(0.025 g, 0.45 mmol) were added to a stirred solution of 38 (0.07 g,

0.15 mmol) in MeOH (2 mL) at >RT. The reaction mixture was then

heated at reflux for 2 h. After the reaction was shown to be com-

plete by TLC, the reaction mixture was neutralized with 1n aq HCl

(pH 1–2) and extracted with CH₂Cl₂(2ꢂ20 mL). The combined or-

ganic layers were washed with H₂O (10 mL), dried over Na₂SO₄, fil-

tered and concentrated in vacuo. Purification by HPLC gave 39 as

1

16%): H NMR (400 MHz, CDCl₃): d=8.37 (d, J=8.1 Hz, 1H), 8.31 (d,

a dark yellow oil (61 mg, 82%): H NMR (400 MHz, [D₆]DMSO): d=

J=5.0 Hz, 1H), 7.10–7.09 (m, 2H), 6.82 (d, J=6.0 Hz, 2H), 3.71 (s,

3H), 2.62 (t, J=8.0 Hz, 2H), 1.64–1.44 (m, 2H), 1.35–1.32 (m, 6H),

0.90 ppm (t, J=6.5 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): d=156.1,

151.8, 150.9, 141.8, 139.3, 135.2, 133.9, 122.4, 120.7, 117.7, 113.1,

55.9, 35.9, 31.6, 31.2, 29.0, 22.5, 14.0 ppm; HRMS-ES+: m/z [M+

H]⁺calcd for C₁₈H₂₂N₂O₄: 331.1658, found: 331.1652; HPLC: t_R=

10.1 min (76% purity).

13.56 (s, 1H), 8.38 (d, J=2.1 Hz, 1H), 7.98 (s, 1H), 7.62 (d, J=

2.2 Hz, 1H), 7.00 (s, 1H), 6.94 (d, J=8.0 Hz, 1H), 6.78 (d, J=7.8 Hz,

1H), 3.78 (s, 3H), 2.60 (t, J=7.4 Hz, 2H), 1.60 (t, J=6.8 Hz, 2H),

1.38–1.24 (m, 6H), 0.89 ppm (t, 3H).

Ethyl 2-(hexyl-2-methoxyphenoxy) acetate (40): A solution of 3

(2 g, 9.6 mmol) in EtOH (5 mL) was added to a solution of NaOEt

(4.6 mL, 14.4 mmol) in EtOH (15 mL) at RT. Ethyl bromoacetate

(1.6 mL, 14.4 mmol) was added dropwise, and the reaction mixture

was heated at reflux for 16 h. After the reaction was shown to be

complete by TLC, the solvent was removed in vacuo, H₂O was

added, and the mixture was extracted with EtOAc (2ꢂ50 mL). The

combined organic layers were washed with brine (50 mL), dried

over Na₂SO₄, filtered and concentrated in vacuo. Purification by

flash chromatography (EtOAc/hexane, 10%) gave 40 as a yellow

3-(Hexyl-2-methoxyphenoxy)pyridin-2-amine (35): Fe powder

(81 mg, 3.1 mmol) was added to a stirred solution of 34 (128 mg,

0.38 mmol) in AcOH/H₂O (4:1, 5 mL), and the mixture was then

heated at reflux for 30 min. After the reaction was shown to be

complete by TLC, the mixture was filtered, and the filtrated was

first diluted with water and then extracted with CH₂Cl₂(40 mL).

The organic layer was separated, washed with brine (50 mL), dried

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solid (0.98 g, 35%): ¹H NMR (400 MHz, CDCl₃): d=6.78 (d, J=

8.2 Hz, 1H), 6.72 (s, 1H), 6.64 (d, J=7.8 Hz, 1H), 4.70 (s, 2H), 4.26 (t,

J=7.4 Hz, 2H), 3.82 (s, 3H), 2.57 (t, J=7.2 Hz, 3H), 1.62–1.54 (m,

4H), 1.38–1.24 ppm (m, 6H); MS (ESI+, 70 eV): m/z (%): 295.4

(100), 296.1 (19) [M+H]⁺; HPLC: t_R=9.2 min (97% purity).

thylation procedure. Purification by flash chromatography (EtOAc/

petroleum ether, 5%) gave PT092 as a light yellow oil (236 mg,

82%): ¹H NMR (400 MHz, CDCl₃): d=7.58 (dd, J=7.8, 1.6 Hz, 1H),

7.36 (td, J=8.0, 1.2 Hz, 1H), 7.02 (td, J=7.8, 1.6 Hz, 1H), 6.93 (dd,

J=8.0, 1.2 Hz, 1H), 6.80 (d, J=2.0 Hz, 1H), 6.75 (d, J=8.2 Hz, 1H),

6.68 (dd, J=8.2, 2.0 Hz, 1H), 5.32 (s, 1H), 2.60 (t, J=8.0 Hz, 2H),

1.62–1.58 (m, 2H), 1.32–1.21 (m, 6H), 0.80 ppm (t, J=9.0 Hz, 1H);

¹³C NMR (100 MHz, CDCl₃): d=154.5, 152.1, 143.2, 138.7, 131.9,

126.3, 125.1, 124.6, 123.5, 117.1, 113.0, 110.4, 35.9, 31.8, 31.1, 28.8,

22.5, 14.2 ppm; HRMS-ES+: m/z [M+NH₄]⁺calcd for C₁₈H₂₁BrO₂:

348.0725, found: 348.0732.

5-(Hexyl-2-methoxyphenoxy) pyrimidin-4-ol (41): Compound 40

(1 g, 3.4 mmol) and ethyl formate (2.5 g, 34 mmol) were added to

a stirred solution of NaH (0.146 g, 6.1 mmol) in THF (10 mL) at RT.

The mixture was then heated at 658C for 4 h. After the reaction

was shown to be complete by TLC, the solvent was removed in

vacuo, and the crude material was redissolved in MeOH/EtOH (1:1,

12 mL). Formamidine acetate (0.353 g, 3.4 mmol) was added, and

the reaction was stirred at 808C for 4 h. After the reaction was

shown to be complete by TLC, the solvent was removed in vacuo,

and the crude was purified by flash chromatography (MeOH/

CH₂Cl₂, 8%) to give 41 as a yellow solid (0.47 g, 47%): ¹H NMR

(400 MHz, CDCl₃): d=7.88 (s, 1H), 7.38 (s, 1H), 6.99 (d, J=8.0 Hz,

1H), 6.82–6.78 (m, 2H), 3.82 (s, 3H), 2.60 (t, J=7.2 Hz, 2H), 1.68–

1.48 (m, 4H), 1.38–1.22 (m, 4H), 0.89 ppm (t, J=6.8 Hz, 3H); HRMS-

ES+: m/z [M+H]⁺calcd for C₁₇H₂₂N₂O₃: 303.1709, found:

303.1708; HPLC: t_R=7.3 min (99% purity).

5-Hexyl-2-(2-(trifluoromethyl)phenoxy)phenol (PT095): Com-

pound 4 (1.0 g, 2.8 mmol) was treated according to the general de-

methylation procedure. Purification by flash chromatography

(EtOAc/petroleum ether, 5%) gave PT095 as a light yellow oil

(844 mg, 88%): ¹H NMR (300 MHz, CDCl₃): d=7.67 (dd, J=7.8,

1.5 Hz, 1H), 7.44 (td, J=7.8, 1.2 Hz, 1H), 7.16 (tt, J=8.1, 0.9 Hz,

1H), 6.93 (d, J=8.4 Hz, 1H), 6.90 (d, J=2.1 Hz, 1H), 6.86 (d, J=

8.4 Hz, 1H), 6.69 (dd, J=8.1, 1.8 Hz, 1H), 5.43 (s, 1H), 2.57 (t, J=

7.8 Hz, 2H), 1.64–1.56 (m, 2H), 1.37–1.28 (m, 6H), 0.90 ppm (m,

3H); HRMS-ES+: m/z [M+Na]⁺calcd for C₁₉H₂₁F₃O₂: 361.1391,

found: 361.1396.

4-Chloro-5-(hexyl-2-methoxyphenoxy) pyrimidine (42): POCl₃

(0.3 mL) was added dropwise to a stirred solution of 41 (25 mg,

0.08 mmol) in CHCl₃(5 mL). The reaction was heated to 708C and

stirred for 3 h. After the reaction was shown to be complete by

TLC, the solvent was removed in vacuo, and the residue was dis-

solved in CH₂Cl₂(20 mL). The organic phase was washed with

water (2ꢂ20 mL) and brine (20 mL), dried over Na₂SO₄, filtered and

concentrated in vacuo. Purification by flash chromatography

(MeOH/CH₂Cl₂, 5%) gave 42 as a dark yellow solid (12 mg, 46%):

¹H NMR (400 MHz, CDCl₃): d=7.93 (s, 1H), 7.38 (s, 1H), 6.96 (d, J=

7.8 Hz, 1H), 6.88–6.81 (m, 2H), 3.80 (s, 3H), 2.58–2.54 (t, J=7.2 Hz,

2H), 1.62–1.54 (m, 2H), 1.38–1.24 (m, 6H), 0.98–0.92 ppm (m, 3H).

5-Hexyl-2-(2-iodophenoxy)phenol (PT096): Compound 6 (300 mg,

1.0 mmol) and CuI (286 mg, 1.5 mmol) were reacted according to

the general procedure for diazotization to give 9 (269 mg, 68%).

Crude 9 was then reacted further according to the general deme-

thylation procedure, and purification of the product by flash chro-

matography (EtOAc/petroleum ether, 5%) gave PT096 as a light

yellow oil (334 mg, 84%): HRMS-ES+: m/z [M+Na]⁺calcd for

C₁₈H₂₁IO₂: 419.0484, found: 419.0479.

5-Hexyl-2-(2-methyl-4-nitrophenoxy)phenol (PT107): Compound

27 (200 mg, 0.59 mmol) was treated according to the general de-

methylation procedure, and purification of the product by flash

chromatography (EtOAc/hexane, 15%) gave PT107 as a yellow

5-(Hexyl-2-methoxyphenoxy)pyrimidin-4-amine (43): NH₄OH

(1.5 mL, 27 mmol) was added dropwise to a stirred solution of 42

(55 mg, 0.17 mmol) in CH₂Cl₂(10 mL) at RT, and then the mixture

was heated at 1308C for 18 h. After the reaction was shown to be

complete by TLC, the solvent was removed in vacuo, and the

crude product purified by column chromatography (MeOH/CH₂Cl₂,

8%) to give 43 as a dark yellow oil (45 mg, 87%): ¹H NMR

(400 MHz, CDCl₃): d=8.30 (s, 1H), 7.70 (s, 1H), 6.90 (d, J=8.2 Hz,

1H), 6.80 (s, 1H), 6.75 (d, J=7.9 Hz, 1H), 5.20 (br s, 2H), 4.80 (s,

3H), 2.64 (t, J=7.4 Hz, 2H), 1.70–1.50 (m, 2H), 1.40–1.20 (m, 6H),

0.85 ppm (t, J=6.7 Hz, 3H); HRMS-ES+: m/z [M+H]⁺calcd for

C₁₇H₂₃N₃O₂: 302.1869, found: 302.1868; HPLC: t_R=6.9 min (96%

purity).

1

solid (169 mg, 88%): H NMR (300 MHz, CDCl₃): d=8.10 (dd, J=0.6,

1.8 Hz, 1H), 7.94 (ddd, J=0.6, 3.0, 9.3 Hz, 1H), 6.91 (d, J=2.1 Hz,

1H), 6.75 (m, 2H), 6.71 (d, J=1.8 Hz, 1H), 5.41 (s, 1H), 2.58 (t, J=

7.8 Hz, 2H), 2.44 (s, 3H), 1.61 (m, 2H), 1.32 (m, 6H), 0.89 ppm (t,

J=6.9 Hz, 3H); HRMS-ES+: m/z [M+Na]⁺calcd for C₁₉H₂₃NO₄:

352.1525, found: 352.1528.

2-(2,6-Dimethylphenoxy)-5-hexylphenol (PT108): Compound 28

(200 mg, 0.64 mmol) was treated according to the general deme-

thylation procedure, and purification of the product by flash chro-

matography (EtOAc/hexane, 15%) gave PT108 as a dark yellow oil

(153 mg, 80%): ¹H NMR (400 MHz, CDCl₃): d=7.11–7.07 (m, 3H),

6,86 (s, 1H), 6.48 (d, J=8.2 Hz, 1H), 6.20 (d, J=7.8 Hz, 1H), 5.78 (br

s, 1H), 2.51 (t, J=7.8 Hz, 2H), 2.14 (s, 6H), 1.58 (t, J=6.9 Hz, 2H),

1.30–1.26 (m, 6H), 0.88–0.86 ppm (t, 3H); HRMS-ES+: m/z

[M+Na]⁺calcd for C₂₀H₂₆O₂: 321.1831, found: 321.1832; HPLC: t_R=

12.6 min (94% purity).

2-(2-Chlorophenoxy)-5-hexylphenol (PT091): Compound

7

(300 mg, 0.94 mmol) was treated according to the general deme-

thylation procedure. Purification by flash chromatography (EtOAc/

petroleum ether, 5%) gave PT091 as a light yellow oil (255 mg,

89%): ¹H NMR (400 MHz, CDCl₃): d=7.45 (dd, J=7.9, 1.6 Hz, 1H),

7.20 (td, J=8.0, 1.0 Hz, 1H), 7.07 (td, J=7.9, 1.5 Hz, 1H), 6.96 (dd,

J=8.0, 1.5 Hz, 1H), 6.88 (d, J=2.0 Hz, 1H), 6.71 (d, J=8.2 Hz, 1H),

6.64 (dd, J=8.2, 2.0 Hz, 1H), 5.50 (s, 1H), 2.55 (t, J=7.9 Hz, 2H),

1.61–1.58 (m, 2H), 1.37–1.26 (m, 6H), 0.89 ppm (t, J=9.0 Hz, 1H);

¹³C NMR (100 MHz, CDCl₃): d=152.5, 147.7, 141.1, 140.3, 130.8,

128.0, 124.9, 124.6, 120.5, 119.2, 118.0, 116.3, 35.5, 31.7, 31.4, 28.9,

22.6, 14.1 ppm; HRMS-ES+: m/z [M+NH₄]⁺calcd for C₁₈H₂₁ClO₂:

322.1230, found: 322.1572.

2-(2,6-Dichlorophenoxy)-5-hexylphenol (PT109): Compound 24

(300 mg, 0.85 mmol) was treated according to the general deme-

thylation procedure, and purification of the product by flash chro-

matography (EtOAc/hexane, 10%) gave PT109 as a dark yellow oil

(274 mg, 95%): ¹H NMR (400 MHz, CDCl₃): d=7.40 (d, J=9.0 Hz,

2H), 7.26–7.20 (t, J=8.5 Hz, 1H), 6.87 (d, J=2.2 Hz, 1H), 6.54–6.52

(dd, J=8.8, 1.8 Hz, 1H), 6.33 (d, J=8.2 Hz, 2H), 5.72 (br s, 1H), 2.51

(t, J=7.5 Hz, 2H), 1.59–1.55 (m, 2H), 1.36–1.24 (m, 6H), 0.87 ppm

(t, J=6.6 Hz, 3H); HRMS-ES+: m/z [M+Na]⁺calcd for C₁₈H₂₀Cl₂O₂:

361.0738, found: 361.0746; HPLC: t_R=10.5 min (99% purity).

2-(2-Bromophenoxy)-5-hexylphenol (PT092): Compound

8

(300 mg, 0.83 mmol) was treated according to the general deme-

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2-(2-Amino-6-methylphenoxy)-5-hexylphenol (PT110): Com-

pound 30 (200 mg, 0.64 mmol) was treated according to the gen-

eral demethylation procedure, and purification of the product by

flash chromatography (EtOAc/hexane, 10%) gave PT110 as a dark

ES+: m/z [M+H]⁺calcd for C₁₈H₂₀N₂O₂: 297.1603, found:

297.1599; HPLC: t_R=9.8 min (96% purity).

3-(4-Hexyl-2-hydroxyphenoxy)isonicotinic acid (PT116): Com-

pound 39 (200 mg, 0.61 mmol) was treated according to the gen-

eral demethylation procedure, and purification of the product by

flash chromatography (EtOAc/hexane, 10%) gave PT116 as a dark

yellow oil (144 mg, 75%): ¹H NMR (400 MHz, [D₆]DMSO): d=8.32

(d, J=1.8 Hz, 1H), 8.02 (s, 1H), 7.58 (d, J=2.2 Hz, 1H), 6.98 (d, J=

9.1 Hz, 1H), 6.78 (s, 1H), 6.62 (d, J=8.8 Hz, 1H), 2.58–2.44 (m, 2H),

1.58–1.46 (m, 2H), 1.36–1.24 (m, 6H), 0.94–0.84 ppm (m, 3H);

HRMS-ES+: m/z [M+H]⁺calcd for C₁₈H₂₁NO₄: 316.1549, found:

316.1541; HPLC: t_R=7.7 min (93% purity).

1

yellow oil (122 mg, 64%): H NMR (400 MHz, [D₆]DMSO): d=9.11 (s,

1H), 6.83 (t, J=8.7 Hz, 1H), 6.69 (s, 1H), 6.62 (d, J=8.2 Hz, 1H),

6.46–6.41 (m, 2H), 6.22 (d, J=7.6 Hz, 1H), 4.71 (s, 1H), 2.42 (t, J=

7.8 Hz, 2H), 1.98 (s, 3H), 1.54–1.48 (m, 2H), 1.34–1.22 (m, 6H),

0.94–0.82 ppm (m, 3H); HRMS-ES+: m/z [M+H]⁺calcd for

C₁₉H₂₅NO₂: 300.1964, found: 300.1959; HPLC: t_R=10.5 min (99%

purity).

3-Fluoro-2-(4-hexyl-2-hydroxyphenoxy)benzonitrile

(PT111):

Compound 31 (200 mg, 0.61 mmol) was treated according to the

general demethylation procedure, and purification of the product

by flash chromatography (EtOAc/hexane, 10%) gave PT111 as

2-(4-Hexyl-2-hydroxyphenoxy)benzonitrile (PT119): Compound 6

(300 mg, 1.0 mmol) and CuCN were reacted according to general

procedure for diazotization to give 10 (216 mg, 73%). Crude prod-

uct 10 was then further treated according to the general demethy-

lation procedure, and purification with flash chromatography

(EtOAc/petroleum ether, 5%) gave PT119 as a white solid (244 mg,

1

a yellow solid (103 mg, 54%): H NMR (400 MHz, CDCl₃): d=7.35–

7.34 (d, J=2.2 Hz, 1H), 7.27–7.23 (m, 1H), 7.00 (d, J=9.2 Hz, 1H),

6.83 (d, J=1.8 Hz, 1H), 6.70 (d, J=8.5 Hz, 1H), 6.63–6.62 (d, J=8.5,

Hz, 2H), 5.24 (br s, 1H), 2.49 (t, J=7.4 Hz, 2H), 1.53–1.51 (m, 2H),

1.26–1.18 (m, 6H), 0.82–0.80 ppm (m, 3H); HPLC: t_R=10.4 min

(99% purity).

1

86%): H NMR (300 MHz, CDCl₃): d=7.64 (br d, J=7.8 Hz, 1H), 7.46

(br t, J=8.1 Hz, 1H), 7.12 (br t, J=7.5 Hz, 1H), 6.90–6.83 (m, 3H),

6.72 (br d, J=8.1 Hz, 1H), 5.55 (s, 1H), 2.57 (t, J=7.5 Hz, 2H), 1.60

(br t, J=6.6 Hz, 2H), 1.36–1.24 (m, 6H), 0.88 ppm (t, J=6.6 Hz, 3H);

HRMS-ES+: m/z [M+H]⁺calcd for C₁₉H₂₁NO₂: 296.1651, found:

296.1647.

2-((4-Aminopyridin-3-yl)oxy)-5-hexylphenol (PT112): Compound

37 (300 mg, 1.0 mmol) was treated according to the general deme-

thylation procedure, and purification of the product by flash chro-

matography (EtOAc/hexane, 10%) gave PT112 as a brown solid

3-Fluoro-2-(4-hexyl-2-hydroxyphenoxy)benzimidamide (PT131):

Compound 32 (200 mg, 0.58 mmol) was treated according to the

general demethylation procedure, and purification with flash chro-

matography (EtOAc/petroleum ether, 5%) gave PT131 as a dark

1

(97 mg, 34%): H NMR (400 MHz, [D₆]DMSO): d=9.37 (s, 1H), 7.80

(d, J=1.8 Hz, 1H), 7.60 (s, 1H), 6.75 (d, J=8.1 Hz, 1H), 6.73 (d, J=

2.2 Hz, 1H), 6.64 (d, J=8.2 Hz, 1H), 6.58–6.56 (dd, J=8.1,

2.1 Hz,1H), 5.90 (s, 2H), 2.47 (t, J=7.8 Hz, 2H), 1.54–1.50 (m, 2H),

1.27–1.22 (m, 6H), 0.86 ppm (t, J=6.7 Hz, 3H); HRMS-ES+: m/z

[M+H]⁺calcd for C₁₇H₂₂N₂O₂: 287.1760, found: 287.1761; HPLC:

t_R=7.4 min (98% purity).

1

yellow solid (119 mg, 62%): H NMR (400 MHz, CDCl₃): d=9.12 (br

s, 1H), 8.30 (s, 2H), 7.58–7.52 (m, 1H), 7.32–7.18 (m, 2H), 6.88 (s,

1H), 6.76 (d, J=8.1 Hz, 1H), 6.58 (d, J=7.8 Hz, 1H), 2.44 (t, J=

7.8 Hz, 2H), 1.56–1.44 (m, 2H), 1.36–1.22 (m, 6H), 0.86 ppm (t, J=

6.7 Hz, 3H); HRMS-ES+: m/z [M+H]⁺calcd for C₁₉H₂₃FN₂O₂:

331.1822, found: 331.1825; HPLC: t_R=8.9 min (91% purity).

2-(2-Fluorophenoxy)-5-hexylphenol (PT113): Compound 25

(300 mg, 1.0 mmol) was treated according to the general demethy-

lation procedure, and purification of the product by flash chroma-

2-(2-Chloro-6-fluorophenoxy)-5-hexylphenol (PT133): Compound

26 (300 mg, 0.89 mmol) was treated according to the general de-

methylation procedure, and purification with flash chromatography

tography (EtOAc/hexane, 10%) gave PT113 as

a yellow oil

(126 mg, 44%): ¹H NMR (400 MHz, CDCl₃): d=7.20–7.18 (m, 1H),

7.17–6.99 (m, 3H), 6.87 (d, J=7.8 Hz, 1H), 6.72 (d, J=2.4 Hz, 1H),

6.64–6.62 (dd, J=7.8, 2.2 Hz, 1H), 5. 65 (s 1H), 2.54 (t, J=7.5 Hz,

2H), 1.58 (t, J=7.8 Hz, 2H), 1.35–1.25 (m, 6H), 0.88 ppm (t, J=

6.5 Hz, 3H); HRMS-ES+: m/z [M+Na]⁺calcd for C₁₈H₂₁FO₂:

311.1423, found: 311.1433; HPLC: t_R=12.2 min (98% purity).

1

(EtOAc/petroleum ether, 5%) gave PT133 (242 mg, 84%): H NMR

(400 MHz, CDCl₃): d=7.28 (d, J=8.1 Hz, 1H), 7.18–7.10 (m, 2H),

6.84 (s, 1H), 6.56 (d, J=7.8 Hz, 1H), 6.42 (d, J=8.0 Hz, 1H), 5.68 (s,

1H), 2.54 (t, J=7.5 Hz, 2H), 1.62–1.54 (m, 2H), 1.36–1.24 (m, 6H),

0.89 ppm (t, J=6.6 Hz, 3H); HRMS-ES+: m/z [M+Na]⁺calcd for

C₁₈H₂₀ClFO₂: 345.1034, found: 345.1032; HPLC: t_R=10.0 min (95%

purity).

5-Hexyl-2-(2-hydroxyphenoxy)phenol (PT114): Compound 23

(300 mg, 1.0 mmol) was treated according to the general demethy-

lation procedure, and purification of the product by flash chroma-

2-((4-Aminopyrimidin-5-yl)oxy)-5-hexylphenol (PT134): Com-

pound 43 (200 mg, 0.66 mmol) was treated according to the gen-

eral demethylation procedure, and purification with flash chroma-

tography (EtOAc/petroleum ether, 5%) gave PT134 as a dark

yellow oil (130 mg, 68%): ¹H NMR (400 MHz, CDCl₃): d=8.18 (s,

1H), 7.38 (s, 1H), 6.98 (d, J=8.1 Hz, 1H), 6.82 (s, 1H), 6.72 (d, J=

7.8 Hz, 1H), 2.58 (t, J=7.6 Hz, 2H), 1.66–1.58 (m, 2H), 1.38–1.24 (m,

6H), 0.94 ppm (t, J=6.7 Hz, 3H); HRMS-ES+: m/z [M+H]⁺calcd

for C₁₆H₂₁N₃O₂: 288.1712, found: 288.1716; HPLC: t_R=6.0 min (98%

purity).

tography (EtOAc/hexane, 20%) gave PT114 as

a yellow oil

1

(152 mmol, 53%): H NMR (300 MHz, CDCl₃): d=7.03–7.00 (m, 2H),

6.87 (d, J=2.4 Hz, 1H), 6.82–6.80 (m, 2H), 6.76 (d, J=8.4 Hz, 1H),

6.65 (dd, J=2.4, 8.4 Hz, 1H), 6.00 (s, 2H), 2.54 (t, J=7.8 Hz, 2H),

2.03 (s, 2H), 1.59–1.57 (m, 2H), 1.36–1.27 (m, 6H), 0.91 ppm (t, J=

6.6 Hz, 3H); HRMS-ES+: m/z [M+Na]⁺calcd for C₁₈H₂₂O₃:

309.1467, found: 309.1466.

3-(4-Hexyl-2-hydroxyphenoxy)isonicotinonitrile (PT115): Com-

pound 38 (200 mg, 0.64 mmol) was treated according to the gen-

eral demethylation procedure, and purification of the product by

flash chromatography (EtOAc/hexane, 10%) gave PT115 as a dark

2-((2-Fluoropyridin-3-yl)oxy)-5-hexylphenol (PT161): A stirred so-

lution of 35 (607 mg, 2.0 mmol) in HF/pyridine (70%, 1 mL) was

cooled to ꢀ788C. After addition of NaNO₂(158.7 mg, 2.3 mmol),

the solution was kept at 08C for 30 min and then at 608C for 1 h.

After the reaction was shown to be complete by TLC, the mixture

was poured into cold water (10 mL) and neutralized by saturated

1

yellow oil (80 mg, 42%): H NMR (400 MHz, CDCl₃): d=8.46 (d, J=

1.8 Hz, 1H), 8.34 (s, 1H), 7.56 (d, J=2.1 Hz, 1H), 6.92 (d, J=8.2 Hz,

1H), 6.78 (d, J=8.0 Hz, 1H), 5.38 (s, 1H), 2.62–2.58 (m, 2H), 1.64–

1.54 (m, 3H), 1.38–1.24 (m, 5H), 0.94–0.84 ppm (m, 3H); HRMS-

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aq NaHCO₃. The aqueous mixture was extracted with CH₂Cl₂(2ꢂ

10 mL), the organic layer was dried over MgSO₄, filtered and con-

centrated in vacuo to give crude intermediate, which was further

treated according to the general demethylation procedure. Purifi-

cation by flash chromatography (EtOAc/hexane, 15%) gave PT161

as a light yellow solid (284 mg, 49%): ¹H NMR (300 MHz, CDCl₃):

d=7.86 (td, J=1.5, 4.8 Hz, 1H), 7.28 (td, J=9.6, 1.5 Hz, 1H), 7.10

(dd, J=4.8, 7.8 Hz, 1H), 6.91 (d, J=2.1 Hz, 1H), 6.78 (d, J=8.1 Hz,

1H), 6.68 (dd, J=2.1, 8.1 Hz, 1H), 2.55 (t, J=7.8 Hz, 2H), 1.59 (m,

2H), 1.30 (m, 6H), 0.89 ppm (t, J=3.3 Hz, 3H); ¹³C NMR (75 MHz,

CDCl₃): d=155.9, 152.7, 147.1, 141.4, 140.6, 140.3, 140.3, 140.0,

139.7, 127.5, 127.5, 122.0, 122.0, 120.7, 118.9, 117.0, 35.4, 31.7, 31.3,

28.9, 22.6, 14.1 ppm; HRMS-ES+: m/z [M+H]⁺calcd for

C₁₇H₂₀FNO₂: 290.1494, found: 290.1488.

In vivo efficacy

All experiments performed with animals at Colorado State Universi-

ty are conducted under AAALAC approved guidelines and has an

OLAW number of A3572-01.

Bacterial strain: The M. tuberculosis strain used in the mouse stud-

ies was Erdman TMCC 107. Working stocks of bacteria were grown

in Proskauer–Beck medium to mid-logarithmic phase growth

(OD₆₀₀=0.3–0.5), dispensed in 1 mL aliquots, and stored at ꢀ808C.

The bacteria used in all the in vivo experiments were from these

stocks.

Mouse studies: In vivo efficacy against M. tuberculosis Erdman was

assessed in the rapid M. tuberculosis animal model of efficacy,

which utilizes gamma-interferon gene-disrupted (GKO) C57BL/6

mice (Jackson Laboratories, Bar Harbor, ME, USA).^[26]Treatment

started five days post-infection, with all compounds being deliv-

ered via intraperitoneal injection in a formulation of 15% EtOH,

20% propylene glycol, 40% polyethylene glycol 400, phosphate-

buffered saline.

3-(4-Hexyl-2-hydroxyphenoxy)picolinonitrile (PT164): Compound

3 (1.0 g, 4.8 mmol) and 3-bromopicolinonitrile (1.1 g, 5.8 mmol)

were reacted according to the same procedure as described for 34

to give 33 as a light yellow solid (0.43 g, 27%): MS (ESI+, 70 eV):

m/z (%): 311.0 (100), 312.2 (23) [M+H]⁺. Crude compound 33 was

then reacted further according to the general demethylation pro-

cedure, and purification of the product by flash chromatography

(EtOAc/hexane, 10%) gave PT164 as a light yellow solid (583 mg,

41%): ¹H NMR (400 MHz, CDCl₃): d=8.43 (d, J=4.5 Hz, 1H), 7.47

(dd, J=8.7, 4.5 Hz, 1H), 7.29–7.28 (m, 2H), 6.94 (d, J=7.0 Hz, 1H),

6.72 (d, J=9.0 Hz, 1H), 5.69 (br s, 1H), 2.61 (t, J=7.5 Hz, 2H), 1.64–

1.59 (m, 2H), 1.39–1.33 (m, 6H), 0.91 ppm (t, J=6.2 Hz, 3H);

¹³C NMR (100 MHz, CDCl₃): d=156.9, 145.1, 143.7, 143.6,129.0,

128.1, 123.6, 118.8, 118.8, 114.4, 108.0, 107.7, 31.6, 29.9, 22.6,

14.1 ppm; HRMS-ES+: m/z [M+H]⁺calcd for C₁₈H₂₀N₂O₂:

297.1603, found: 297.1607.

Control animals were treated with formulation alone. PT070 was

given 50 mgkg^ꢀ1BID day 1–4, 25 mgkg^ꢀ1BID day 5–10, and

PT091 was given 25 mgkg^ꢀ1BID day 1–4, 12.5 mgkg^ꢀ1day 5–6,

12.5 mgkg^ꢀ1SID and 25 mgkg^ꢀ1SID day 7–10. Animals were sacri-

ficed after 10 days of treatment, and the spleen was homogenized

in sterile saline (0.85%) and homogenates were diluted in saline

and plated on 7H11 agar supplemented with 10% OADC, 0.01%

asparagine, and antibiotics (10 mgL^ꢀ1cycloheximide, 50 mgL^ꢀ1

carbenicillin).

Bacterial burden in the spleen was determined by enumeration of

bacterial colonies after three weeks of incubation. The significance

between control and treatment groups was determined via an un-

paired, one-tailed t-test (t-test 1,2) with 95% confidence intervals

from mean colony-forming units (CFUs) (GraphPad Software ver. 5,

San Diego CA USA, www.graphpad.com).

Crystallization and structure determination

Crystals of the InhA–NAD⁺–PT119 ternary complex were obtained

by the hanging drop, vapor diffusion method.^[9]To co-crystallize

PT119, InhA (5 mgmL^ꢀ1) was incubated with 2 mm NAD⁺and

2 mm PT119 in 4% DMSO for 2 h at RT before mixing 1 mL of

which with 1 mL of reservoir solution containing 2.4m sodium ace-

tate (pH 5.0), 200 mm NaCl, 14% PEG3350 and 4% DMSO. The

crystals were allowed to grow for 20 d before soaking in a solution

containing 2.8m sodium acetate, 75 mm NaCl, 21% DMSO, 2 mm

NAD⁺and 2 mm PT119, and cryo-cooling in liquid nitrogen.

Steady state kinetics and progress curve assays

Kinetic assays using trans-2-dodecenoyl-Coenzyme A (DD-CoA) and

wild-type InhA were performed as described previously to deter-

mine IC₅₀values and residence times.^[9,19b]IC₅₀values were deter-

mined by varying the concentration of inhibitor in 150 mm pipera-

zine-N,N’-bis(2-ethanesulfonic acid) (PIPES) buffer containing

250 mm NADH, 25 mm DD-CoA, and 20, 50, or 100 nm InhA. Prog-

ress curves were obtained in reaction mixtures containing InhA

(5 nm), glycerol (8%, v/v), bovine serum albumin (0.1 mgmL^ꢀ1),

DMSO (2%, v/v), DD-CoA (300 mm), NADH (250 mm) and inhibitor

(0–1000 mm). Inhibition constants for rapid reversible and slow-

onset inhibitors were determined using previously reported meth-

ods.^[9,27]Data fitting was performed using Grafit 4.0 (Erithacus Soft-

ware Ltd, Horley, UK).

Datasets were collected at the National Synchrotron Light Source

at Brookhaven National Laboratory (Upton, New York, USA) on

beamline x12c. The reflections were indexed, integrated, and

scaled using HKL2000,^[21]and structures were solved using

MolRep^[22]with the crystal structure, taken from the RCSB Protein

Data Bank (PDB), of InhA as a model (PDB: 2X23^[9]). NAD⁺and

PT119 were built into the difference map calculated from the ini-

tial solutions. Structural refinement restraints for NAD⁺and PT119

were generated using eLBOW.^[23]Coordinates and atomic displace-

ment parameter (ADP) refinement including individual atom and

correlated anisotropic grouped rigid-body displacement refine-

ment by Translation-Libration-Screw (TLS) parameterization, simu-

lated annealing, and water picking were performed using

Phenix.^[24]Model building and real-space refinement were per-

formed in Coot.^[25]Data collection and refinement statistics are

given in Table S2 in the Supporting Information.

Docking experiments

A model for the complex formed between PT109 and InhA was

obtained using the DOCK 6 suite of programs.^[28]The partial atomic

charges of cofactor and ligand were computed based on the semi-

empirical AM1-BCC^[29]method using AMBER 10.^[30]The partial

atomic charges of amino acids in the protein were assigned based

on the AMBER ff99SB force field.^[31]The binding site was prepared

as described previously.^[32]Briefly, a molecular surface for InhA was

computed using the program DMS.^[33]The program SPHGEN in

The atomic coordinates and experimental data for the crystal struc-

ture of InhA–NAD⁺–PT119 have been deposited in the RCSB Pro-

tein Data Bank under ascension code 4OIM and are available via

http://www.rcsb.org/pdb.

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ChemMedChem 2014, 9, 776 – 791 790

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Received: October 30, 2013

Published online on March 11, 2014

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ChemMedChem 2014, 9, 776 – 791 791

Article Doi

Time-dependent diaryl ether inhibitors of InhA: Structure-activity relationship studies of enzyme inhibition, antibacterial activity, and in vivo efficacy

DOI: 10.1002/cmdc.201300429

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