Synthesis and in vitro antimycobacterial activity of B-ring modified diaryl ether InhA inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.04.038

Previous structure-based design studies resulted in the discovery of alkyl substituted diphenyl ether inhibitors of InhA, the enoyl reductase from Mycobacterium tuberculosis. Compounds such as 5-hexyl-2-phenoxyphenol 19 are nM inhibitors of InhA and inhib

Full text of DOI:10.1016/j.bmcl.2008.04.038

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3029–3033
Synthesis and in vitro antimycobacterial activity of B-ring
modified diaryl ether InhA inhibitors
Christopher W. am Ende,a Susan E. Knudson,b Nina Liu,a James Childs,c
Todd J. Sullivan,a Melissa Boyne,d Hua Xu,a Yelizaveta Gegina,a Dennis L. Knudson,b
Francis Johnson,a Charles A. Peloquin,c Richard A. Slaydend,* and Peter J. Tongea,*
aInstitute of Chemical Biology and Drug Discovery, Department of Chemistry, Stony Brook University, Stony Brook,
NY 11794-3400, USA
bDepartment of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO 80523, USA
cInfectious Disease Pharmacokinetics Laboratory, National Jewish Medical and Research Center, Denver, CO 80206, USA
dDepartment of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1682, USA
Received 23 December 2007; revised 11 April 2008; accepted 15 April 2008
Available online 18 April 2008
Abstract—Previous structure-based design studies resulted in the discovery of alkyl substituted diphenyl ether inhibitors of InhA,
the enoyl reductase from Mycobacterium tuberculosis. Compounds such as 5-hexyl-2-phenoxyphenol 19 are nM inhibitors of InhA
and inhibit the growth of both sensitive and isoniazid-resistant strains of Mycobacterium tuberculosis with MIC90 values of 1–2 lg/
mL. However, despite their promising in vitro activity, these compounds have ClogP values of over 5. In efforts to reduce the lipo-
philicity of the compounds, and potentially enhance compound bioavailability, a series of B ring analogues of 19 were synthesized
that contained either heterocylic nitrogen rings or phenyl rings having amino, nitro, amide, or piperazine functionalities. Com-
pounds 3c, 3e, and 14a show comparable MIC90 values to that of 19, but have improved ClogP values.
Ó 2008 Elsevier Ltd. All rights reserved.
Tuberculosis (TB) is responsible for more than 1.6 mil-
lion deaths per annum with 8.8 million new cases being
reported each year. These numbers make TB one of the
leading infectious causes of death, eclipsed only by
AIDS. In addition, according to the World Health
Organization, the number of multi-drug-resistant and
extensively drug-resistant TB cases is growing with al-
most a half million new cases being reported each year.
Therefore, there is an urgent need to develop novel TB
chemotherapeutic agents.1
synthesis (FAS-II) pathway. However, before INH can
inhibit InhA, it must be activated by KatG, a catalase-
peroxidase enzyme. The activated form of INH then re-
acts with NAD+ to form the INH-NAD adduct (Scheme
1).3–7 A significant number of the strains resistant to
INH arise from mutations in KatG.8–11 Therefore, the
development of an InhA inhibitor which can bypass this
O
O
NAD(H)
NH
KatG
NAD(H)
The current front-line treatment strategy utilizes isonia-
zid (INH), a pro-drug which inhibits the synthesis of
mycolic acids that are essential components required
for the integrity of the bacterial cell wall.2 INH inhibits
InhA, the FabI enoyl reductase (ENR) in the fatty acid
N
N
NH2
INH
O
INH-NAD
OH
Cl
B
OH
O
A
Keywords: Tuberculosis; Diaryl ether; Enoyl reductase; Mycolic acids;
InhA; Isoniazid; Antitubercular drug; Diphenyl ether; FabI; Lipinski
parameter; Bioavailability.
Cl
Cl
n
n=1, 3-5, 7, 13
Alkyl diphenyl ethers
Scheme 1. Enoyl reductase inhibitors.
*
Corresponding authors. Tel.: +1 970 491 1925 (R.A.S.), tel.: +1 631
632 7907; fax: +1 631 632 7934 (P.J.T.); e-mail addresses:
richard.slayden@colostate.edu; peter.tonge@sunysb.edu
Triclosan
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.038

3030
C. W. am Ende et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3029–3033
initial activation step should have activity against INH-
resistant strains of Mycobacterium tuberculosis (MTB).
tent inhibitors of InhA, with Ki values as low as 1 nM
and MIC90 values of 1–2 lg/mL against M. tuberculosis
22
H37Rv
.
Importantly the alkyl diphenyl ethers display
The diphenyl ether triclosan (Scheme 1) is a potent
inhibitor of ENR’s from many organisms including
Escherichia coli and Plasmodium falciparium.12–20 How-
ever, this compound only inhibits InhA with a Ki value
of 0.2 lM.21 Using structure-based drug design we
developed a series of alkyl diphenyl ethers that are po-
similar MIC values against INH-resistant strains of
MTB.22 However, despite their promising in vitro activ-
ity, these compounds have relatively low solubility and
have ClogP values greater than 5, which is likely one
reason why they have limited in vivo efficacy.23 Based
on the observed relationship between lipophilicity and
OMe
OMe
OH
O
a or b
Y
Y
N
N
X
X
Y =
N
1a-f
X = Cl, Br
a
c
b
c
N
N
N
OMe
N
N
OH
OMe
N
e
d
O
O
OMe
f
Y
d
5
5
3a-f
2a-f
Scheme 2. Reagents and conditions: (a) K2CO3, DMAc, D, Y; (b) (CuOTf)2ÆPhH, Cs2CO3, EtOAc, toluene, 120 °C, Y; (c) hexyl ZnCl, Pd(P(t-Bu)3)2,
THF/NMP, 130 °C; (d) BBr3, DCM, 0 °C to rt.
OMe
OMe
OMe
O
OH F
+
O
a
e
b
NO2 o,m,p
NO2 o,m,p
NO2 o,m,p
Br
Br
5
4a-c
5a-c
e
OH
O
c
NO2 o,m,p
5
13a-c
OMe
OH
OMe
O
O
O
O
O
d
N
H
R o,m,p
NH2 o,m,p
N
H
R o,m,p
5
5
5
6a-c
e
10a-c R = Me
11a-c R = COOH
12a-c R = Isoxazole
7a-c R = Me
OH
8a-c R = COOH
O
9a-c R = Isoxazole
NH2 o,m,p
5
14a-c
Scheme 3. Reagents and conditions: (a) K2CO3, DMAc, D; (b) hexyl ZnCl, Pd(P(t-Bu)3)2, THF/NMP, 130 °C; (c) Zn, HCl, EtOH, rt; (d) acyl
chloride, NEt3, DCM; (e) BBr3, DCM, 0 °C to rt.
N
OMe
OMe
OMe
O
OH F
O
O
Ho,p
O
H
N
o,p
a
b
+
o,p
Br
Br
Br
15a,b
16a,b
c
N
N
OH
OMe
O
O
N
o,p
N
o,p
d
5
5
18a,b
17a,b
Scheme 4. Reagents and conditions: (a) K2CO3, DMAc, D; (b) N-methyl piperazine, NaBH(OAc)3, DCE; (c) hexyl ZnCl, Pd(P(t-Bu)3)2, THF/NMP,
130 °C; (d) BBr3, DCM, 0 °C to rt.

C. W. am Ende et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3029–3033
3031
in vivo efficacy, especially as it pertains to antibacterial
compounds,24,25 we synthesized a series of analogues
that incorporated functionalities designed to increase
the polarity of the parent diphenyl ether InhA inhibi-
tors. The effect on compound polarity was estimated
by calculating the logP value (ClogP) for each com-
pound synthesized.
The synthesis of the heterocyclic diaryl ether compounds
was initiated either by nucleophilic aromatic substitu-
tion or by Buchwald–Hartwig cross-coupling of the
appropriate nitrogen heterocycle with 4-bromo or
chloro-2-methoxy phenol producing 1a–f (Scheme
2).26,27 This was followed by palladium catalyzed Negi-
shi coupling of the diaryl ethers with hexyl zinc chloride
to give 2a–f.28 Boron tribromide cleavage of the methyl
ether was used subsequently to generate the respective
phenols, 3a–f.29 Structural characterization of all com-
In this study we describe two classes of molecules in which
alterations have been made to the diphenyl ether ‘B’ ring.
In one series of compounds we have replaced the B ring
with isosteric heterocycles that incorporate nitrogen
atoms within the ring, thereby causing little steric pertur-
bation to the overall structure of the molecule (Scheme 2).
The second series of compounds have nitro, amino,
amide, and piperazino functionalities incorporated at
the ortho, meta, or para positions of the B ring (Schemes
3 and 4). This second series ofcompounds was synthesized
not only to improve solubility but also to systematically
identify positions on the B ring which could be substituted
without diminishing biological activity.
1
pounds was performed using H NMR and ESI/MS.
The synthesis of the nitro, amino, and amide-substituted
compounds was performed using the series of reactions
shown in Scheme 3. Nucleophilic aromatic substitution
reactions with fluoronitrobenzenes were first used to
generate compounds 4a–c.27 This was followed by Negi-
shi coupling giving 5a–c followed by boron tribromide
cleavage to give compounds 13a–c or zinc-mediated
reduction giving anilines 6a–c.28–30 Cleavage of the
methyl ether gave 14a–c while acylation of the anilines
Table 1. Inhibition and solubility data for B-ring heterocycles
a
IC50 (nM)
À1
b
Compound
Structure
MIC90 (lg mL
)
ClogP
logP
OH
O
O
O
O
O
O
19
11 1c
2.1 0.9
6.47
5.76
5
OH
N
3a
3b
3c
3d
3e
11,500 1160
50
4.97
4.97
4.97
4.01
4.01
5.06
5
OH
N
N
236 31
3.13
3.13
5
OH
160 16
4.93
4.46
4.764
5
OH
N
8200 980
100.0
6.25
0
N
5
OH
N
N
650 60
0
5
OH
O
O
N
OMe
3f
NId
100
5.50
4.50
N
N
5
OMe
N
OMe
2d
>100,000
75
0
5
a Enzyme concentration is 100 nM.
b ClogP values determined using ChemDraw 8.0.
c Enzyme concentration is 1 nM.
d No inhibition.

3032
C. W. am Ende et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3029–3033
with acyl chlorides afforded compounds 7, 8, and
9a–c.29,31 Boron tribromide cleavage then gave the final
compounds 10, 11, and 12a–c.29
The two most active compounds, 3c and 14a, have
MIC90 values of 3.13 lg/mL, similar to that of 19,
and have ClogP values of 4.97 and 5.24, respectively,
compared to 6.47 for the parent compound (Tables 1
and 2). In addition it is also worth noting that the
pyrazine derivative 3e has a ClogP value that is more
than one log lower than 19, but still only shows a 3-
fold increase in MIC90 compared to the parent (Table
1). In general the MIC values correlated with the IC50
values for enzyme inhibition. Thus ortho and para
amino substituents (14a,c) were well tolerated in addi-
tion to the meta nitro substituent (13b). In these three
cases the IC50 values obtained using 100 nM InhA ap-
proached 50% of the enzyme concentration, indicating
that these compounds are tight-binding enzyme inhib-
itors. Additional IC50 values were determined using 10
and 50 nM InhA in the enzyme assays. Subsequent
linear regression analyses of the IC50 values as a func-
The piperazine derivatives were synthesized in a similar
fashion starting with nucleophilic aromatic substitution
with the 2- or 4-fluorobenzaldehyde to give 13a and b
(Scheme 4).27 Subsequently, reductive amination with
methyl piperazine and sodium triacetoxyborohydride
produced 14a and b,32 whereas Negishi coupling fol-
lowed by boron tribromide cleavage gave the final com-
pounds 16a and b.28,29
The in vitro activities of the ultimate products were
evaluated using enzyme inhibition and whole cell anti-
bacterial assays as described previously (Tables 1–
3).22,33,34 In general, addition of a bulky substituent
at either the ortho, meta, or para position of the B
ring of 19 or incorporation of the most aromatic
nitrogen heterocycles resulted in a significant reduction
in both enzyme inhibition and antibacterial activity
(Tables 1 and 3). In contrast, introduction of either
amino or nitro substituents at the ortho and para posi-
tions had only a minimal effect on activity (Table 2).
tion of enzyme concentration yielded estimates for
app
Ki
of 21 3 nM (13b), 16 12 nM (14a), and
40 3 nM (14c). Thus, introduction of a meta nitro
(13b) or an ortho amino (14a) group into the B ring
of the parent compound 19 has only aa minor effect
on the affinity of the inhibitor for the enzyme. Com-
Table 2. Inhibition and solubility data for nitro and aniline compounds
ClogPb
logP
a
IC50 (nM)
À1
Compound
Structure
MIC90 (lg mL
)
OH
13a
13b
13c
180 20
48 6c
90 10
12.50
12.50
6.21
6.21
6.21
5.50
O
O
NO2 o,m,p
25.0
0
5.64
5.27
5
OH
14a
14b
14c
62
5
3.13
100
5.24
5.24
5.24
1090 90
55 6c
0
NH2 o,m,p
5
12.50
4.93
a Enzyme concentration is 100 nM.
b ClogP values determined using ChemDraw 8.0.
c Actual IC50 may be lower than this value.
Table 3. Inhibition and solubility data for amide and piperazine compounds
MIC90 (lg mLÀ1
)
ClogP b
logP
a
IC50 (nM)
Compound
Structure
OH
10a
10b
10c
1550 460
5600 770
1300 200
>200
100
0
4.90
4.90
4.90
5.28
O
O
O
N
H
5
o,m,p
50.0
0
OH
11a
11b
11c
12a
12b
12c
2360 200
580 40
100.00
4.24
4.24
4.24
5.76
5.76
5.76
O
OH
O
130 58
N
H
O
5
1930 90
3220 550
1220 60
130 34
>200
0
0
0
0
o,m,p
OH
>200
>200
>200
5.60
5.22
5.15
O
O
O
N
H
N
5
o,m,p
OH
N
18a
18b
1315 256
306 46
6.66
6.66
N
>100
o,p
5
a Enzyme concentration is 100 nM.
b ClogP values determined using ChemDraw 8.0.

C. W. am Ende et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3029–3033
3033
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Acknowledgment
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This work was supported by NIH Grant AI70383.
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Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2008.04.038.
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