Discovery and SAR of benzyl phenyl ethers as inhibitors of bacterial phenylalanyl-tRNA synthetase

Article abstract of DOI:10.1016/j.bmcl.2008.12.054

A series of benzyl phenyl ethers (BPEs) is described that displays potent inhibition of bacterial phenylalanyl-tRNA synthetase. The synthesis, SAR, and select ADMET data are provided.

Full text of DOI:10.1016/j.bmcl.2008.12.054

Bioorganic & Medicinal Chemistry Letters 19 (2009) 665–669
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and SAR of benzyl phenyl ethers as inhibitors of bacterial
phenylalanyl-tRNA synthetase
*
Justin I. Montgomery , Peter L. Toogood, Kim M. Hutchings, Jia Liu, Lakshmi Narasimhan, Timothy Braden,
Michael R. Dermyer, Angela D. Kulynych, Yvonne D. Smith, Joseph S. Warmus, Clarke Taylor
Pfizer Global Research and Development, Michigan Laboratories, Ann Arbor Campus, 2800 Plymouth Road, Ann Arbor, MI 48105, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of benzyl phenyl ethers (BPEs) is described that displays potent inhibition of bacterial
phenylalanyl-tRNA synthetase. The synthesis, SAR, and select ADMET data are provided.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 17 November 2008
Revised 8 December 2008
Accepted 10 December 2008
Available online 24 December 2008
Keywords:
Phenylalanyl-tRNA synthetase
PheRS
FRS
Aminoacyl-tRNA synthetase
Benzyl phenyl ether
Antibacterial
SAR
Although numerous effective antibiotics have been developed
during the last 60 years, infectious disease remains a threat to pub-
lic health in the US and abroad. The emergence of bacterial resis-
tance to known antibiotics has inspired the search for new
antibacterial targets and novel small molecule inhibitors. Amino-
acyl-tRNA synthetases (AaRS), enzymes crucial for protein biosyn-
thesis, have received increased attention in recent years as targets
for anti-infectives.1 AaRS enzymes catalyze the synthesis of amino-
acyl-tRNAs (aa-tRNA) through a two-step process involving: (1)
reaction of the appropriate amino acid and ATP to provide an acti-
vated aminoacyl–adenylate intermediate, AaRSꢀ(aa-AMP) and (2)
transfer of the amino acid to tRNA. Inhibition of either step in this
sequence through competitive binding to the enzyme should halt
protein synthesis and cease bacterial growth.
AaRSs are conserved across prokaryotic species; therefore, broad-
spectrum activity could be attained. Moreover, the AaRSs are gen-
erally easy to handle and purify in large quantities which enables
high-throughput screening. Finally, a number of X-ray crystal
structures of bacterial AaRSs have been disclosed enabling struc-
ture-based drug design.2 To date, only one AaRS inhibitor is mar-
keted as an antibiotic—the natural product pseudomonic acid A
(mupirocin, Bactroban) which targets IleRS.3 Its use is limited to
the treatment of topical infections.
Phenylalanyl-tRNA synthetase (PheRS) is a member of subclass
IIc of the AaRSs due to its antiparallel b-sheet active site architec-
ture and
a
2b2 quaternary structure.4 Multiple small molecule
inhibitors of bacterial PheRS have been reported including sub-
strate analogs5 and inhibitors derived from high-throughput
screening hits.6 Herein, we describe the discovery and optimiza-
tion of a new class of PheRS inhibitors, benzyl phenyl ethers (BPEs)
AaRs þ aa þ ATP ꢀ AaRs ꢀ ðaa-AMPÞ þ PPi
ð1Þ
ð2Þ
AaRs ꢀ ðaa-AMPÞ þ tRNA ꢀ AaRs þ aa-tRNA þ AMP
A number of features make AaRSs particularly attractive targets
for antibiotic discovery. First, although AaRS enzymes are present
in both humans and bacteria, considerable divergence should al-
low for selective inhibition of bacterial AaRSs. Furthermore, the
central ring
H3C
O
Cl
tail group
N
NH
O
* Corresponding author. Present address: Pfizer Global Research & Development,
Groton Laboratories, Eastern Point Road, Groton, CT 06340, USA. Tel.: +1
8606869120.
head group
linker
1
Cl
Figure 1. Original HTS hit: benzyl phenyl ether 1.
E-mail address: justin.montgomery@pfizer.com (J.I. Montgomery).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.054

666
J. I. Montgomery et al. / Bioorg. Med. Chem. Lett. 19 (2009) 665–669
Table 1 (continued)
Table 1
Structures (1–28) and potency for HI and SP PheRS
Compound
X
Linker
Y
R
IC50
(lM)
HI
SP
O
Y
R
O
CH3
N
NH
OCH3
19
20
m-Cl
m-Cl
Cl
Cl
R4
R4
.038
2.4
1.3
23
O
R1
R2
O
O
X
linker
1-27
O
S
O
N
N
O
N
CH3
28
O
N
N
R4
R3
CH3
CH3
N
N
O
Cl
O
O O
S
N
21
m-Cl
Cl
R4
>64
>64
NH2
N
O
R5
R6
O
O O
S
22
23
m-Cl
m-Cl
CH3
Cl
R4
R4
>64
>64
>64
>64
N
H
Compound
X
Linker
Y
R
IC50
(l
M)
SP
H
N
HI
S
O O
1
2
3
4
5
6
7
p-Cl
Cl
R1
R2
R2
R2
R2
R2
R2
0.24
3.1
2.2
12
O
24
25
26
m-Cl
m-Cl
m-Cl
CH3
CH3
R5
R5
R6
0.067
0.28
0.15
3.7
11
O
m-Cl
H
O
O
O
O
O
OH
m,p-F,F
m-Cl
Cl
.0087
.034
.030
.017
3.5
0.12
CH3
Cl
0.097
0.16
0.091
6.1
CH3
CH3
ND
O
m,m-F,F
m-Cl
27
28
m-Cl
m-Cl
R6
R6
0.36
>64
46
See above
>64
Cl
m-Cl
CH3
N
H
with activity against both Gram-negative (Haemophilus influenzae,
HI) and Gram-positive (Streptococcus pneumoniae, SP) bacteria.
High-throughput screening yielded BPE 1 (Fig. 1) with nM po-
N
CH3
8
m-Cl
m-Cl
m-Cl
m-Cl
m-Cl
m-Cl
CH3
H
R2
R2
R2
R2
R2
R2
0.17
57
3.5
tency against HI and
lM potency against SP. Early work within
the series (not shown) identified preferred groups for the two aro-
matic rings (m-Cl or m,p-F,F on the tail ring and Me or Cl in position
Y of the central ring). Table 1 highlights some of the SAR for this
series around the head group and linker region. In general, modifi-
cations of the head group were designed to increase potency (espe-
cially against SP PheRS) and/or ligand efficiency,7 while linker
changes were driven by the concern that the benzylic carbon of
BPEs is a potential site of metabolism. To begin, it was found that
a simple methyl ester could replace the entire head group from
the HTS lead (2–6), a change that increased ligand efficiency (1
HI LE = 0.34, SP LE = 0.29; 6 HI LE = 0.52, SP LE = 0.47) and allowed
for the facile synthesis of analogs containing alternative linker
groups (7–15). In addition, in many cases the ratio of potencies
SP/HI was favorably impacted with the relatively small methyl es-
ter head group. Furthermore, various amides were tolerated within
the BPE series (16, 19, 24, and 26) which enabled investigation of
even more linkers. Overall, analogs with 18 alternative linker
groups were compared in a pairwise fashion with BPEs containing
identical substitution (aside from the linker region), but none
proved superior to the original ether linkage contained in HTS hit
1. However, some of the linkers did retain partial activity. Of note
are the amine linkers (7 and 8), the alkene linker (13), the reversed
ether (17), the sulfoxide (20), the alcohol (25), and the alkane (27).
Compound 28, which contains an additional ring designed to de-
crease conformational mobility, was inactive against PheRS. In
the end, it was decided to retain the ether linkage; fortunately,
concerns about metabolism were partially mitigated with low
in vitro clearance data from various BPEs (see safety/PK table later).
Having confirmed that the ether linkage is needed for high po-
tency, we turned to optimization of the head group. Table 2 dis-
plays select BPEs with amide and sulfonamide head groups as
H
N
9
>64
>64
>64
>64
0.20
CH3
N
10
11
12
13
H
>64
>64
>64
0.13
O
CH3
H
N
H
H
N
O
CH3
OH
14
m-Cl
CH3
R2
6.5
>64
OH
OH
15
16
17
18
m-Cl
m-Cl
m-Cl
m-Cl
CH3
Cl
R2
R3
R3
R3
>64
0.15
0.84
>64
>64
2.3
OH
O
O
Cl
ND
>64
HO
CH3

J. I. Montgomery et al. / Bioorg. Med. Chem. Lett. 19 (2009) 665–669
667
Table 2
Table 3
Structures (29–40) and potency for HI and SP PheRS
Structures (41–55) and potency for HI and SP PheRS
Cl
R
R
O
Cl
X
N
O
Cl
N
Cl
X
O
Compound
X
R
IC50 (nM)
SP
HI
Compound
X
R
IC50 (nM)
HI
SP
O
O
29
30
CH
N
57
30
393
50
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
CH
N
CH
CH
CH
N
CH
CH
N
CH
CH
CH
CH
CH
CH
H
H
11
12
6.6
11
156
97
95
76
42
50
28
81
60
126
110
87
81
36
53
N
H
CH3
CH2CH3
NH2
CH3
2.3
ND
1.3
2.6
2.7
1.7
11
3.1
5.5
1.5
2.1
31
32
CH
N
29
4.6
483
43
OH
NH2
N
H
NHAc
CONH2
CONH2
CH2CONH2
CH2CO2H
CH2CH2OH
SCH3
O
O
33
34
CH
N
22
16
254
34
OH
O
N
H
SOCH3
SO2CH3
35
36
CH
N
14
8.0
438
65
N
N
H
O
O
Table 4
Select PK and safety data
N
N
37
38
N
N
24
34
28
N
H
Compound HLM Clint, appa
(mL/min/kg)
PAMPA Peb
Dofetilidec
Ki ( M)
CYP inhd (>25%
CH3
(ꢂ 10ꢃ6 cm/s)
l
at 3
lM)
NDe
89% at
10 nMf
37.2
23.9
>100
>100
NDe
OH
1
9.63
1.5
N
H
19
24
33
42
<7.60
55.1
<7.60
<10.1
16.4
10.8
1.22
5.31
None
None
None
O
O
S
NEt2
39
40
CH
CH
1.9
1.9
79
12
N
CH3
1A2 (38%)
a
Apparent intrinsic clearance from human liver microsomes.
Parallel artificial membrane permeation assay.
Measures competitive binding to HEK-hERG membrane homogenates.
CYPs 1A2, 2C9, 2D6, and 3A4 measured.
Not determined.
b
c
d
e
f
O
S
O
CH3
N
N
Single point determination.
O
Table 5
well as various analogs in which a pyridine replaces the BPE central
phenyl ring. The smaller secondary amides 29, 31, 33, and 35 dem-
onstrate respectable potency and illustrate the benefit of incorpo-
rating additional heteroatoms extending from the BPE core. Their
pyridine analogs (30, 32, 34, and 36) show a marked increase in
activity, especially against SP where the average increase in po-
tency is ꢁ8ꢂ, and this change also substantially decreases clogP,
a design goal of the team in hoping to benefit ligand-lipophilicity
efficiency8 and favorably impact bacterial efflux. Amides 37 and
38 also have high activity against both PheRS enzymes and illus-
trate that many scaffolds and functional groups are tolerated in
this position. Finally, the sulfonamides 39 and 40 are single-digit
nanomolar inhibitors of HI PheRS and 40 has very high activity
against SP PheRS (12 nM).
A number of bioisosteres of the methyl ester group were also
investigated and it was found that oxadiazole 41 (Table 3), which
should be more stable than methyl ester 6 in vivo, maintained high
potency against HI and SP PheRS. Extending a methyl (43) or ethyl
(44) group from the oxadiazole led to increased SP activity. As was
previously observed in the amide series, replacing the central phe-
nyl ring with a pyridine ring also increased potency against SP
Minimum inhibitory concentrations for select compounds against HI and SP
Compound
MIC (l
g/mL)a
HI (AcrA-)b
HI (WT)c
SP (WT)c
31
32
36
55
4
2
1
1
>32
32
32
32
32
8
32
>32
a
All MICs determined in defined media with [Phe] = 100
Minus efflux pump AcrA.
Wild-type.
lM.
b
c
PheRS (42). A range of functional groups was tolerated extending
from the oxadiazole scaffold including amino (45–46), acetamide
(47), amide (48–50), acid (51), alcohol (52), thioether (53), sulfox-
ide (54), and sulfone (55). Many of these substituents led to even
higher activity including some with single-digit nanomolar po-
tency against HI and <50 nM potency against SP (45, 47, and 54).
Aminooxadiazole 45 is the most ligand efficient oxadiazole re-
ported (HI LE 0.53, SP LE 0.45).

668
J. I. Montgomery et al. / Bioorg. Med. Chem. Lett. 19 (2009) 665–669
R
N
Y
CO2Me
b
(1,16,19,24,
26,29-38)
NR2
Y
CO2Me
O
(2-6)
Br
O
a
X
+
(41-44)
O
BPE
O
N
BPE
HO
X
d
c
BPE
NH2
NHAc
O
f
e
O
NH2
N
(47)
BPE
N
N
CONH2
l
k
N
H
BPE
N
BPE
g
O
(45-46)
(50)
O
HO2C
O
N
S
CO2Et
S O
S
N
BPE
h
O
O
m
n
+
O
O
O
i
N
N
N
N
N
N
BPE
N
N
BPE
BPE
N
BPE
N
BPE
(53)
OH
(55)
(54)
(51)
O
CO2Me
N
CONH2
(48-49)
N
(52)
N
j
BPE
O
O
N
N
BPE
N
BPE
O
O
O
O
Y
S
O
Y
S
O
p
o
NR2
Cl
Y
CO2Me
S
OH
BPE
NR2
HO
HO
(39-40)
X
(14-15)
Y
O
CO2Me
OH
r
Y
CO2Me
Y
CO2Me
q
s
X
+
X
u
t
Br
X
(13)
Y
CONR2
HO
O
Y
O
N
Y
O
X
OH
NR2
(27)
Y
v
X
+
X
CONR2
(18)
X
(25)
Br
Scheme 1. Synthetic route to select analogs. Reagents and conditions: (a) Cs2CO3, CH3CN; (b) 1—LiOH; 2—HNR2, HATU, TEA, DMF; (c) 1—LiOH; 2—SOCl2; 3—hydrazine
hydrate; (d) triethyl orthoformate, orthoacetate, or orthopropionate, reflux; (e) cyanogen bromide, NaHCO3; (f) Ac2O, pyridine; (g) 1—CS2, KOH; 2—NaOH, then MeI; (h)
mCPBA; (i) TEA, methyl oxalyl chloride, then TsCl; (j) 7 N NH3 in MeOH; (k) 1—LiOH; 2—SOCl2; 3—pyridine, ethyl 1H-tetrazole-5-acetate, then
LiOH; (n) LiBH4; (o) N,O-bis(trimethylsilyl)acetamide then TEA, HNR2; (p) substituted benzyl bromide, Cs2CO3, CH3CN; (q) Pd(OAc)2, P(o-tol)3, TEA; (r) AD-mix
D
; (l) 7 N NH3 in MeOH; (m)
or b; (s)
a
mCPBA; (t) 1—BF3; 2—NaBH4; 3—LiOH; 4—HNR2, HATU, TEA, DMF; (u) 1—LiOH; 2—HNR2, HATU, TEA, DMF; 3—H2, RaNi, AcOH; (v) 1—t-BuLi then epoxide; 2—3 N HCl; 3—
HNR2, HATU, TEA, DMF.
Various lead compounds were screened in select PK and safety
assays (Table 4). HTS hit 1 had moderate clearance, but amides 19
and 33 showed improved (lower) values. Furthermore, a range of
permeabilites was observed with amide 19 displaying the highest
value.
Dofetilide displacement from the hERG channel was a problem
for early compounds (1, 19, and 24), but smaller amides (33) and
oxadiazoles (42) mitigated this risk. Finally, most compounds were
clean when screened (using CYP enzymes) for possible drug–drug
interactions.
Heck reaction between substituted styrenes and aryl bromides.
The alkene could readily be converted to diols (14 and 15) and al-
kane 27. In addition, epoxidation followed by boron trifluoride pro-
moted rearrangement and reduction of the resulting aldehyde
provided alcohol 18. Finally, analog 25 was synthesized through
epoxide opening with the anion formed from bromide/lithium ex-
change from a substituted aryl bromide. Deprotection to reveal the
corresponding carboxylic acid followed by amide formation pro-
vided 25.
In conclusion, a novel class of HI and SP phenylalanyl-tRNA syn-
thetase inhibitors, the benzyl phenyl ethers, was described. Clear
SAR trends emerged around the linker and head group regions of
the molecules, and high potency (nM) against both enzymes was
achieved. A range of values for common safety and PK assays
Select compounds were tested for antibacterial activity
against pump-knockout HI and wild-type HI and SP (Table 5).
Single-digit
lg/mL MICs for AcrA-HI were observed for amides
31, 32, and 36 as well as oxadiazole 55. Unfortunately, activity
against wild-type HI and SP was significantly lower, presumably
due to efflux.
was observed, and single-digit
lg/mL MICs against AcrA-HI were
observed, but high whole-cell activity against wild-type HI and
SP remained elusive. Finally, synthetic routes for many of these
analogs were described.
The synthesis of various analogs is detailed in Scheme 1. Treat-
ment of phenols with benzyl bromides in the presence of cesium
carbonate provided a convenient route to the benzyl phenyl ether
core with a synthetic handle in the form of a methyl ester in place
for further elaboration (2–6). Conversion to amides was straight-
forward. In addition, all the oxadiazoles were synthesized through
this template, either through the hydrazide or for analogs 50–52,
using ethyl 1H-tetrazole-5-acetate. The sulfonamides 39–40 were
formed from the corresponding phenolic sulfonyl chloride which
was protected in situ using N,O-bis(trimethylsilyl)acetamide then
converted to the BPE by phenol alkylation. Various alternative link-
ers were available from alkene 13 which was synthesized using a
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