Identification of bacteria-selective threonyl-tRNA synthetase substrate inhibitors by structure-based design

Article abstract of DOI:10.1021/jm301756m

A series of potent and bacteria-selective threonyl-tRNA synthetase (ThrRS) inhibitors have been identified using structure-based drug design. These compounds occupied the substrate binding site of ThrRS and showed excellent binding affinities for all of the bacterial orthologues tested. Some of the compounds displayed greatly improved bacterial selectivity. Key residues responsible for potency and bacteria/human ThrRS selectivity have been identified. Antimicrobial activity has been achieved against wild-type Haemophilus influenzae and efflux-deficient mutants of Escherichia coli and Burkholderia thailandensis.

Full text of DOI:10.1021/jm301756m

Article
pubs.acs.org/jmc
Identification of Bacteria-Selective Threonyl-tRNA Synthetase
Substrate Inhibitors by Structure-Based Design
Min Teng,*,† Mark T. Hilgers,‡ Mark L. Cunningham,§ Allen Borchardt,† Jeffrey B. Locke,§
Sunny Abraham,† Gregory Haley,† Bryan P. Kwan,§ Courtney Hall,‡ Grayson W. Hough,∥ Karen J. Shaw,§
and John Finn†
‡
§
∥
†Medicinal Chemistry, Structural Biology, Biology, and Analytical Chemistry, Trius Therapeutics, Inc., 6310 Nancy Ridge Drive,
San Diego, California 92121, United States
S
* Supporting Information
ABSTRACT: A series of potent and bacteria-selective
threonyl-tRNA synthetase (ThrRS) inhibitors have been
identified using structure-based drug design. These com-
pounds occupied the substrate binding site of ThrRS and
showed excellent binding affinities for all of the bacterial
orthologues tested. Some of the compounds displayed greatly
improved bacterial selectivity. Key residues responsible for
potency and bacteria/human ThrRS selectivity have been
identified. Antimicrobial activity has been achieved against
wild-type Haemophilus influenzae and efflux-deficient mutants of Escherichia coli and Burkholderia thailandensis.
antimicrobial activity due to poor bacterial cell permeabili-
ty.3f,6,8
INTRODUCTION
■
Infections caused by antibiotic-resistant pathogens have led to a
crisis in health care, especially for those due to Gram-negative
organisms. These disease-causing microbes have developed
several different classes of resistance mechanisms, one of which
is target-based mutations that reduce drug affinity, as in the case
of fluoroquinolones.1 Alternatively, as seen for the aminoglyco-
sides, enzymatic modification of a drug may lead to high-level
resistance.2 There are resistance mechanisms to all major
antibacterial drugs currently in use, and many strains are
resistant to multiple drug classes. Thus, new classes of agents
that bind to novel targets would be a significant addition to the
antimicrobial armamentarium to combat resistant pathogens.
Aminoacyl-tRNA synthetases (aaRSs) are a class of enzymes
that have been validated as antimicrobial targets.3 Mupirocin,
which inhibits bacterial isoleucyl-tRNA synthetase, is approved
for the topical treatment of impetigo due to Staphylococcus
aureus and Streptococcus pyogenes.4 With the exception of
glutaminyl-tRNA synthetase (GlnRS), absent in many Gram-
positive bacteria, all of the other 19 synthetases are highly
conserved and perform essential activities in clinically
important microorganisms.5 In addition to mupirocin, whole
cell screening of natural products has identified multiple aaRS
inhibitors with antibacterial activity.6 These include borrelidin
(ThrRS), granaticin (LeuRS), indolmycin (TrpRS), ochratoxin
A (PheRS), and cispentacin (ProRS). In recent years, other
aaRS inhibitors have emerged, including those that are mimics
of the aminoacyl adenosine monophosphate intermediate (aa-
AMP), such as 1 (Figure 1).7 Although most of the aa-AMP
mimics demonstrated low nanomolar binding affinities against
their corresponding aaRSs, they generally displayed weak
In screening marine natural products for antibacterial activity,
we identified borrelidin (2) as the active component of one of
the extracts we prioritized for structural elucidation. Borrelidin
inhibits ThrRS9 and has an interesting antibacterial profile that
includes activity on Yersinia pestis, Haemophilus influenzae, and
Francisella tularensis (unpublished results). However, the
antibacterial potency and spectrum of borrelidin need to be
significantly improved to generate a useful antibacterial agent
that targets ThrRS. In addition, borrelidin lacks selectivity for
bacterial ThrRS versus human ThrRS, raising concerns of
mammalian toxicity. Indeed, borrelidin has been shown to be a
potent inhibitor of angiogenesis.10 Although previous efforts
sought to optimize the antibacterial activity of borrelidin, none
of these efforts utilized X-ray crystallographic data to guide
optimization. To address these issues, we cocrystallized
borrelidin with Escherichia coli and human ThrRS (which will
be reported on separately).11 Comparison of the human and E.
coli enzymes reveals that the borrelidin binding site is highly
conserved between the two ThrRS enzymes and that the ATP/
substrate binding site displays more structural variability than
the borrelidin site. The E. coli and human ThrRS selectivity
ratios for a number of semisynthetic analogues of borrelidin
confirm that achieving a good bacterial selectivity with
borrelidin analogues may be a challenging task. Thus, we
explored totally synthetic inhibitors of bacterial ThrRS that
specifically target the substrate binding sites of ThrRS. In this
paper we summarizes our effort to design novel small-molecule
Received: December 20, 2012
Published: January 30, 2013
© 2013 American Chemical Society
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Figure 1. Structures of known ThrRS inhibitors: threonyl-AMP (1), borrelidin (2), threonyl-AMS (3).
antibacterial agents that selectively inhibit bacterial ThrRS
enzymes.
dioxaborolan-2-yl)isoindolin-1-one and deprotection of the Boc
(Scheme 7). The synthesis of 10h and 26−29 started with a
Heck reaction of 6-(3-bromophenyl)pyrimidin-4-amine with
vinylsulfonamide to yield 25. After coupling with a correspond-
ing acid and deprotection of the Boc and TBS under acidic
conditions, intermediate 25 furnished 10h and 26−29 (Scheme
8).
The starting point for this program was 5′-O-[N-(threonyl)-
sulfamoyl] adenosine (AMS, 3), a known competitive inhibitor
of ThrRS.12 The crystal structure of 3 bound to E. coli ThrRS
has been reported.11 As an adenosine analogue, 3 lacks
antibacterial activity due to poor (too polar) drug properties
and lacks selectivity for the bacterial enzymes (Table 1). We
aimed to address both of these issues by designing compounds
with more typical heterocyclic fragments found in drugs and by
tailoring the shape of the interaction sites of the inhibitors to
achieve bacterial selectivity.
RESULTS AND DISCUSSION
■
In the initial designs, we simplified 3 by replacing the polar
adenosine moiety with a 4-phenoxyphenyl moiety, but retained
the acylsulfonamide, as in 4. These changes resulted in loss of
potency due to the loss of the H-bonding network that is
present with adenosine. As a result, compound 4 had only
modest inhibition at 50 μM concentration. Enzymatic assays
showed that 4 was competitive with respect to both ATP and
the amino acid substrate (which will be reported on separately).
Despite the low potency, we were able to obtain a cocrystal
structure of 4 with E. coli ThrRS. This structure demonstrated
that compound 4 binds to an “open form” of the ATP binding
site that more closely resembles the apo conformation of
ThrRS than the ATP-bound conformation (Figure 2). The
threonylacylsulfonamide portion of the molecule binds to the
Zn atom, as anticipated from the Thr-AMS structure. The 4-
phenoxy group is largely disordered, suggesting that it has
negligible contributions to binding. These data revealed
opportunities for further optimization of the binding affinity.
One of the strategies to enhance the binding affinity is to
replace the distal phenoxy in 4 with a suitable group which can
induce the “closed form” of the ATP pocket. Inhibition of the
closed form of the enzyme was preferred, as it targets key highly
conserved residues that are required for enzymatic activity.
Because of their biochemical importance, these residues are
inherently difficult to mutate, thereby making target-based
resistance to the new inhibitors less likely. Furthermore, it is
well appreciated that targeting the enzymatic transition states is
a useful strategy for generating highly potent inhibitors.14
Computational modeling using Discovery Studio with
structural information from 3 suggested that compound 10a,
with an aminoquinazoline fragment, could fit the adenosine site
of the closed form, as the pyrimidine ring of 10a would bind to
the ThrRS enzyme in a fashion similar to that of the pyrimidine
ring of 3. As predicted by the modeling, compound 10a was a
strong inhibitor of E. coli ThrRS (Table 1). The cocrystal
structure of 10a bound to E. coli ThrRS showed that this
compound did indeed bind to the closed form of the ATP
pocket with the quinazoline ring sandwiched between Arg520
and Phe379 (Figure 3). In addition, the quinazoline ring
formed a number of important H-bonds with nearby residues.
The amino group formed bidentate H-bonds with Glu365 and
CHEMISTRY
■
Compound 4 was synthesized through a coupling of
sulfonamide 5 and Boc-protected L-threonine 6 followed by
deprotection of the Boc (Scheme 1). The synthesis of 10a−f
and 11a was initiated with a coupling reaction between
dioxaborolane 8a with 6 to form intermediate 9a (Scheme 2).
Suzuki coupling of 9a with a corresponding ArBr followed by
deprotection of Boc yielded 10a−f and 11a. Similarly, Suzuki
coupling of 9b with a corresponding ArB(OH)2 followed by
deprotection of Boc yielded 11c,d. Upon NCS treatment, 11d
was converted to 11b. The synthesis of 10g,i started with 12,
which was synthesized using a known procedure.13 The
synthesis of 10j started with a reaction of 7-bromo-1,2,3,4-
tetrahydroisoquinoline with sulfamoyl chloride to furnish 13. A
coupling reaction of 12 or 13 with 6 afforded 14a,b. A
conversion of Br in 14a,b to borane pinacol ester yielded 15a,b.
A Suzuki coupling reaction of 15a,b to a corresponding ArCl
followed by Boc deprotection furnished 10g,i,j (Scheme 3). A
coupling reaction of 6 with (3-(aminomethyl)phenyl)boronic
acid by HOAT and EDCI furnished 16. After a Suzuki coupling
reaction with 7-bromo-2-chloroquinazolin-4-amine followed by
deprotection of the Boc, 16 was converted to 17 (Scheme 4).
Compound 19 was synthesized through (3-bromophenyl)-
hydrazine. Conversion of the hydrazine to threonyl hydrazide
18a was effected by threonyl chloride generated in situ.
Conversion of Br in 18a to borane pinacol ester yielded 18b.
After a Suzuki coupling followed by deprotection of Boc, 18b
was converted to 19 (Scheme 5). The synthesis of 22 started
with a conversion of 6 into its primary amide, which was
subsequently treated with LAH to form 20. This intermediate
was treated with 3-bromobenzenesulfonyl chloride under basic
conditions to form 21a. A conversion of Br 21a into borane
pinacol ester yielded 21b (Scheme 6). Suzuki coupling of 21b
with 7-bromo-2-chloroquinazolin-4-amine followed by depro-
tection of Boc yielded 22. The synthesis of 24 started with
methylation of the acylsulfonamide 9b with iodomethane
followed by a Suzuki coupling with 5-(4,4,5,5-tetramethyl-1,3,2-
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interactions was that compound 10a is a highly potent inhibitor
of E. coli ThrRS (Ki of 2.9 nM).
Table 1. Binding Affinities and Bacterial ThrRS Selectivity of
3, 4, and 10a−j
Additional quinazoline analogues 10b−f, together with a few
rearranged skeletons, 10g−j, were prepared to explore the
structure−activity relationship (SAR) of the ATP binding
pocket. Table 1 shows the ThrRS enzymatic binding activity
and bacterial selectivity for these analogues. The addition of a
C2 amino group, as in 10b, resulted in weaker E. coli ThrRS
binding. The cocrystal structure of 10b with E. coli ThrRS
showed that although this new amine group forms a hydrogen
bond to the carbonyl of Leu373, this is a weak hydrogen bond
due to poor geometry and length. Furthermore, compound 10b
may be paying a desolvation penalty. The incorporation of a
chlorine atom at C2, as shown in 10c, produced a significant
improvement in potency. The distance from Cl to the carbonyl
of Leu373 is 2.8 Å. This is shorter than the sum of the van der
Waals radii of Cl and O (3.3 Å), indicating a halogen bond
formed (Figure 4).15 Compound 10d, with a methyl group at
C2, lost potency. The cocrystal structure of 10d in E. coli
ThrRS demonstrated that the quinazoline ring was rotated by
180°, avoiding a steric clash between the methyl group and
Leu373. To accommodate the size of the quinazoline ring of
10d, Glu365 was found in an alternative orientation (Figure 4).
The binding affinity of 10d could be partially attributed to the
donor−acceptor H-bonds with Val376. More dramatic loss of
potency was observed when a quinazoline was changed into an
isoquinoline. Isoquinolines 10e and 10f lost 30−100-fold
binding affinity, at least in part due to the loss of a H-bond with
Ser517. Lastly, other nonquinazolines, 10g−j, were examined,
in which an aminopyrimidine or pyrrolopyrimidine replaced the
aminoquinazoline ring, and a naphthyl-, a styryl-, or a
tetrahydroisoquinolinyl moiety replaced the phenyl ring.
These compounds retained good enzymatic potency, which
indicates that different chemical scaffolds can be developed to
fit tightly to the ATP binding pocket.
It is worth mentioning that 10e and 10f, though less potent,
showed good selectivity for all bacterial ThrRS enzymes tested
versus the human (cytoplasmic-1) ThrRS enzyme. These data
suggest that the H-bond to Ser517 is more critical to binding to
human ThrRS than to bacterial ThrRS and that a subtle change
in the structure can achieve bacteria/human selectivity in the
ATP binding site.
To explore the opportunity for selective bacterial ThrRS
inhibitors and to fully understand the structural components
necessary to achieve such a level of selectivity, we examined
11a−d. These compounds, missing the critical H-bond to
Ser517, were designed to have weaker potency against human
ThrRS. Table 2 shows that compounds 11a−d exploit human/
bacterial differences in a unique and dramatic way. The
cocrystal structure of 11d bound to E. coli ThrRS shows that
11d induced the expected conformational changes in the E. coli
enzyme that are typically seen upon substrate binding,
specifically the collapse of regions around both the threonine
and ATP binding sites (Figure 5a). (The same collapse has
been verified with the cocrystal structure of 11d bound to the
B. thailandensis enzyme.) The structure of 11d−human ThrRS
enzyme, however, showed that the human enzyme undergoes
the comparable collapse around the threonine site, but not in
the ATP site (Figure 5b).16 These results demonstrate that the
human and bacterial ATP sites are distinct when bound to this
class of compounds. In particular, the conformation and lack of
flexibility of Phe379, immobilized by Leu361, and therefore the
inability of the polypeptide backbone (around Val376) to
Val376, similar to the amino group in the adenine. In addition,
atoms N1 and N3 formed H-bonds with Ser517 and Val376,
respectively. The threonyl group bound to the Zn atom, as
observed for Thr-AMS. The acylsulfonamide formed a H-bond
network with its nearby residues, as predicted from the
threonyl-AMS binding mode. The net result of all these
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a
Scheme 1. Synthesis of 4
a
Reagents and conditions: (a) EDCI, DBU, HOAT, DCM, rt, 2 h, 65%; (b) 4 N HCl in dioxane, rt, 30 min, 90%.
a
Scheme 2. Synthesis of Analogues 10a−f and 11a−d
a
Reagents and conditions: (a) 6, EDCI, DBU, HOAT, DCM, rt, 2 h, 55−95%; (b) (i) PdCl2(TPP)2, ArBr (for 10a−f and 11a) or ArB(OH)2 (for
11c,d), Na2CO3, microwave 140 °C, 5 min; (ii) 4 N HCl in dioxane, 15−79%; (c) NCS, CH3CN, 70 °C, 1 h, 68%.
a
Scheme 3. Synthesis of 10g,i,j
a
Reagents and conditions: (a) sulfamoyl chloride, DMA, 24 h, 43%; (b) 6, EDCI, DBU, HOAT, DCM, rt, 2 h, 69.7%; (c) bis(pinacolato)diboron,
PdCl2(dppf)CH2Cl2, KOAc, DMF, 80 °C, 1 h, 60−80%; (d) (i) ArCl, Pd(TPP)4, Na2CO3, dioxane, microwave 10 min, 130 °C; (ii) 4 N HCl in
dioxane, 13−46% (two steps).
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a
a
Scheme 4. Synthesis of 17
Scheme 7. Synthesis of 24
a
a
Reagents and conditions: (a) 6, EDCI, DBU, HOAT, DCM, rt, 12 h,
Reagents and conditions: (a) MeI, Cs2CO3, 65 °C, 6 h, DMF, 54%;
(b) (i) PdCl2(TPP)2, ArB(OH)2, Na2CO3, microwave 140 °C, 5 min,
55%; (ii) 4 N HCl in dioxane, 47%.
99%; (b) (i) PdCl2(TPP)2, 7-bromo-2-chloroquinazolin-4-amine,
Na2CO3, microwave 140 °C, 6 min; (ii) 4 N HCl in dioxane, 12%
(two steps).
a
Scheme 8. Synthesis of 10h and 26−29
a
Scheme 5. Synthesis of 19
a
Reagents and conditions: (a) vinylsulfonamide, Pd(OAc)2, TPP,
DMF, microwave, 120 °C, 0.5 h, 69%; (b) (i) a corresponding acid,
EDCI, DBU, HOAT, DCM, rt, 2 h; (ii) 4 N HCl in dioxane, rt, 30
min, 10−68% (two steps).
a
(a) 6, ethyl chloroformate, N-methylmorpholine, THF, 15 min, 66%;
(b) bis(pinacolato)diboron, PdCl2(dppf)CH2Cl2, KOAc, DMF, 80 °C,
1 h, 44%; (c) (i) PdCl2(TPP)2, 7-bromo-2-chloroquinazolin-4-amine,
Na2CO3, microwave 140 °C, 6 min; (ii) 4 N HCl in dioxane, 31%
(two steps).
hydrogen bond with the indazole portion of the molecule in
this context forced 11d to adopt a much less energetically
favorable binding mode in the human enzyme (Figure 6). As a
consequence, 11d is selective for bacterial ThrRS with a Ki >50
μM for the human ThrRS enzyme.
a
Scheme 6. Synthesis of 22
The same explanations account for the observed selectivity
for 10e,f. When the H-bond between N1 and Ser517 is
replaced by the repulsive interactions between CH and Ser517
as in 10e,f, the isoquinoline ring will rotate to minimize the
undesired interactions. As anticipated, bacterial ThrRS enzymes
can accommodate such a rotation better than the human
ThrRS enzyme. These results represented an important proof-
of-concept that bacterial ThrRS can be targeted with good
selectivity, and that the ATP site is likely the best region to
achieve this.
The SAR around the threonylsulfonamide moiety has been
studied. Cocrystal structures of a number of our inhibitors
indicated that the acylsulfonamide, engaged in several H-bonds
with nearby residues, appeared indispensable to the binding of
the inhibitors (Figure 7). The relative contribution of each of
the acylsulfonamide components to binding was examined with
the synthesis of 17, 19, 22, and 24 (Table 3). Using 10c and
11a as reference compounds in this study, we replaced the SO2
group with a CH2 group as in 17 and with an NH as in 19.
Both changes caused >100-fold reduction in enzymatic potency
compared to that of 10c. Removal of the threonine carbonyl
group as in 22 abolished all activities. The NH of the
acylsulfonamide was “masked” with a methyl group as in 24.
This change also abolished all activity seen in 11a. These data
a
Reagents and conditions: (a) (i) ethyl chloroformate, N-methyl-
morpholine, ammonium hydroxide, THF, rt, overnight, 96%; (ii)
LAH, THF, 50 °C, 4 h, 96%; (b) 3-bromobenzenesulfonyl chloride,
TEA, DCM, 2 h, 25%; (c) bis(pinacolato)diboron, PdCl2(dppf)-
CH2Cl2, KOAc, DMF, 80 °C, 1 h, crude; (d) (i) PdCl2(TPP)2, 7-
bromo-2-chloroquinazolin-4-amine, Na2CO3, microwave 140 °C, 6
min, 22%; (ii) 4 N HCl in dioxane, 39%.
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Figure 2. Cocrystal structures of 3 (left) and 4 (right) in E. coli. ThrRS. The cocrystal structure of 3 in E. coli ThrRS reveals that the side chain
containing Arg520 folds down to be closer to the adenosine moiety to form sandwiched interactions together with Phe379 (the “closed form”). In
contrast, the apo form of the enzyme adapts a more open form in which Arg520 is engaged in H-bonds with Ile524 and Gln479. The 4-phenoxy
moiety in 4, due to a lack of H-bond interactions as in adenine, is largely disordered upon binding to the enzyme, which allows the ATP binding site
to remain “open”.
Table 2. Binding Affinity and Improved Bacterial Selectivity
of 11a−d
Figure 3. Part of the H-bond networks of compound 10a in E. coli
ThrRS. Residues that interact with the quinazoline ring are shown in
bold.
particularly critical, possibly due to its roles in mediating the
pKa of both threonine and acylsulfonamide.
Finally, analogues that outline the importance of the Zn
chelating portion of our ThrRS inhibitors were prepared.
Although it is generally believed that ThrRS enzymes recognize
L-threonine as a substrate, in our study we have expanded the
SAR surrounding the methyl group of L-threonine. The
corresponding diamine and diol had also been evaluated.
Table 4 summarizes the results for these compounds. These
data showed that there is a limitation in the Zn binding pocket
to accommodate size, with threonine being the optimal
followed by ethylthreonine 26 and isopropylthreonine 27.
The human (cytoplasmic-1) and B. thailandensis ThrRS
enzymes are particularly sensitive to the size of the head
piece, which offers an opportunity for selectivity optimization.
The recognition of threonine analogues by ThrRS appeared to
be specific in this study, as compounds 28 and 29 showed no
appreciable enzymatic activity.
Figure 4. Overlay of cocrystal structures 10c (aqua) and 10d (cyan).
The Glu365 (shown bold) residue moves in the presence of 10d. The
halogen bond is indicated with a dashed line.
reiterated the importance of acylsulfonamide to the binding
affinities of our ThrRS inhibitors. The carbonyl group appeared
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Figure 5. Phe379 is more flexible in the bacterial ThrRS enzymes such that it can shift slightly away from the active site and accommodate 11d (a).
In the human ThrRS orthologue (b), Phe379 is unable to shift, and the inhibitor is forced to adopt a very different and less energetically favorable
conformation.
Figure 6. Bacterial selectivity is due to differences in underlying ThrRS residues that prevent Phe379 movement to accommodate 11d. The tilted
conformation of selective inhibitors, such as 11d, presses tightly against the sides of the ATP pocket (compared with the conformation of the
nonselective 10a). In the bacterial enzymes, Phe379 can accommodate this tilt due to the presence of a less rigid underlying residue, Cys361. In the
human orthologue, however, the underlying residue Leu361 prevents movement of Phe379 and the inhibitor must find an alternate (less
energetically favorable) conformation.
Table 3. Binding Affinities for 10c, 11a, 17, 19, 22, and 24
ThrRS Ki (nM)
ID
E. coli
0.8
B. thailandensis
Y. pestis
human
1.2
Figure 7. Acylsulfonamide H-bond network.
10c
17
0.3
0.7
329
140
436
935
19
136
34
70
399
Through these SAR studies, we were able to evaluate
antimicrobial activity for a few potent ThrRS inhibitors. This
was carried out through measurement of antimicrobial activity
against a panel of Gram-negative wild-type strains. In addition,
antibacterial activity was measured versus E. coli imp, E. coli
tolC, and a B. thailandensis pump knockout strain to measure
the impact of permeability and efflux pumps on antimicrobial
activity. Table 5 shows the minimal inhibitory concentration
(MIC) values against clinically important pathogens and BSL2
surrogate strains of important biodefense pathogens. Compared
22
>50 μM
7.7
>50 μM
7.4
>50 μM
3.9
>50 μM
91.0
11a
24
>50 μM
>50 μM
>50 μM
>50 μM
to threonyl-AMS (3), the newly identified ThrRS inhibitors
showed improved antibacterial activity against wild-type H.
influenzae, E. coli tolC, and B. thailandensis pump mutant strains,
although all compounds had poor antimicrobial activity against
wild-type strains of E. coli and B. thailandensis. As these
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Table 4. ThrRS Inhibitors Bearing Different Head Pieces
ThrRS Ki (nM)
B. thailandensis
ID
X
Y
Z
E. coli
2.7
Y. pestis
human
10h
26
NH2
NH2
NH2
OH
OH
OH
OH
OH
NH2
CH3
C2H5
i -Pr
CH3
CH3
1.1
1.8
4.1
4.3
14.1
2.7
32
27
32.4
588
22.5
601
28
>50 μM
>50 μM
>50 μM
2262
>50 μM
>50 μM
>50 μM
12576
29
NH2
a
Table 5. MIC Values of Representative Compounds
a
Values that are ≤32 μg/mL are shaded light blue.
indicated otherwise, reagents and solvents were obtained from
commercial sources and used as received.
compounds have significantly increased activity on the pump-
deficient strains, they are likely efficient substrates for efflux
pumps and are more limited in this respect than in their ability
to permeate cellular membranes. Further structural modifica-
tions will be needed to enhance the antimicrobial activity of the
inhibitors.
In summary, we have identified a series of ThrRS synthetase
inhibitors using SBDD. These compounds bind to the substrate
binding site of ThrRS synthetase and show excellent binding
affinities for the bacterial enzymes. Several compounds possess
greatly improved bacterial selectivity. Key residues responsible
for potency and bacteria/human ThrRS selectivity have been
identified. Improvements in antimicrobial activity, as compared
to AMS, have been achieved against H. influenzae. Further work
is directed toward the improvement of potency and the
expansion of the antibacterial spectrum. These results will be
reported in due course.
General Method A for the Synthesis of Acylsulfonamide. A
solution of 6 (1.65 g, 6.0 mmol), HOAT (0.817 g, 6.0 mmol), and
EDC (1.15 g, 6.0 mmol) in DCM (20 mL) was stirred at room
temperature for 30 min, followed by the addition of sulfonamide (4.0
mmol) and DBU (1.81 mL, 12 mmol). Reaction was monitored by
LC/MS and was completed in 2 h. The reaction mixture was diluted
with EtOAc, washed with 1.0 N HCl and water, dried over Na2SO4,
concentrated, and either used as is or purified by flash chromatography
(0−100% ethyl acetate/hexane). Yield: 55−95%.
General Method B for Boc Deprotection. A solution of Boc-
protected threonylacylsulfonamide (0.063 mmol) was added to 4.0 N
HCl in dioxane (0.5 mL) at room temperature. The reaction was
stirred for 2 h, then stripped of volatiles, and purified by RPLC (10−
50% ACN in water) to get the desired compound as a TFA salt. Yield:
30−97%.
General Method C for the Suzuki Coupling Reaction. A flask
was charged with 9a (108 mg, 0.20 mmol), ArCl (0.20 mmol),
bis(triphenylphosphine)palladium(II) chloride (7.02 mg, 0.010
mmol), and aq sodium carbonate (0.2 mL, 1.0 M aq), vacuumed,
flushed with N2 five times, and then heated in the microwave at 140
°C for 6 min. The crude mixture was neutralized with acetic acid (1.0
N), concentrated, and purified by RPLC (0−90% ACN in water).
Yield: 15−87%.
(2S,3R)-2-Amino-3-hydroxy-N-((4-phenoxyphenyl)sulfonyl)-
butanamide (4). Using methods A and B, the title compound was
synthesized in 58% yield in two steps from 6. LC: 98%. 1H NMR (600
MHz, DMSO): δ 8.01 (br, 3H), 7.85 (t, J = 16.6 Hz, 2H), 7.45 (t, J =
7.7 Hz, 2H), 7.23 (dd, J = 18.1, 10.8 Hz, 1H), 7.09 (t, J = 9.8 Hz, 2H),
7.03 (t, J = 13.0 Hz, 2H), 3.95 (dd, J = 22.0, 16.0 Hz, 1H), 3.47 (dd, J
= 21.2, 16.9 Hz, 1H), 1.07 (d, J = 6.3 Hz, 3H). HRMS (ESI): m/z
calcd for C16H18N2O5S (M + H)+ 351.1014, found 351.1013.
EXPERIMENTAL SECTION
■
General Procedures. Flash chromatography was performed on
1
silica gel (280 mesh). H NMR spectra were obtained with a Bruker
300 MHz spectrometer equipped with a 5 mm QNP probe or a Bruker
600 MHz spectrometer equipped with a Bruker BioSpin TCI 1.7 mm
MicroCryoProbe. Chemical shifts are reported in parts per million
relative to tetramethylsilane, and the following multiplicity abbrevia-
tions were used: s, singlet; d, doublet; t, triplet; m, multiplet; br, broad.
HRES-LC/MS was conducted on a Micromass Q-TOF Premier
(quadrupole time-of-flight) mass spectrometer (Beverly, MA) coupled
with an Agilent 1100 (Santa Clara, CA) HPLC system equipped with a
UV diode array detector monitoring 254 nm. HPLC was equipped
with a Phenomenex kinetex-C18 column (100 × 3.0, 2.6 μm). Unless
1755
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tert-Butyl ((2S,3R)-3-(tert-Butoxy)-1-oxo-1-(3-(4,4,5,5-tetra-
methyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamido)butan-
2-yl)carbamate (9a). Following general method A, 9a (1.05 g) was
tert-Butyl ((2S,3R)-3-(tert-Butoxy)-1-oxo-1-(7-(4,4,5,5-tetra-
methyl-1,3,2-dioxaborolan-2-yl)naphthalene-2-sulfonamido)-
butan-2-yl)carbamate (15a). A solution of bis(pinacolato)diboron
(0.373 g, 1.5 mmol), PdCl2(dppf)CH2Cl2(cat.), KOAc (0.360 g, 3.67
mmol), and 14a (0.665 g, 1.224 mmol) in DMF (7.0 mL) was flushed
with argon and heated at 80 °C for 2.5 h in a sealed tube. The reaction
mixture was diluted with ethyl acetate and washed with water. The
organic layer was separated and concentrated to a solid (0.58 g, 80%)
which was used directly in the next reaction.
1
produced from 8a (0.99 g, 55%). H NMR (300 MHz, DMSO): δ
12.05 (br, 1H), 8.23 (s, 1H), 8.16 (s, 1H), 8.06 (s, 2H), 7.64 (br, 2H),
7.37 (s, 1H), 6.06 (s, 1H), 3.93 (m, 1H), 3.34 (m, 1H), 1.35 (m,
21H), 1.10 (s, 3H), 0.91 (s, 9H).
tert-Butyl ((2S,3R)-1-(3-Bromobenzenesulfonamido)-3-(tert-
butoxy)-1-oxobutan-2-yl)carbamate (9b). Following general
1
method A, 9b (1.87 g) was produced from 8b (0.94 g, 95%). H
(2S,3R)-2-Amino-N-((7-(6-aminopyrimidin-4-yl)naphthalen-
2-yl)sulfonyl)-3-hydroxybutanamide (10g). Using methods C and
NMR (300 MHz, DMSO): δ 12.21 (br, 1H), 8.04 (s, 1H), 7.94 (d, J =
8.0 Hz, 2H), 7.87−7.47 (t, J = 7.0 Hz, 1H), 6.13 (d, J = 9.2 Hz, 1H),
4.35−3.61 (m, 1H), 3.21 (m, 1H), 1.37 (s, 9H), 0.97 (m, 12H).
HRMS (ESI): m/z calcd for C19H29BrN2O6S (M + H)+ 493.1008,
found 493.1029.
1
B, 10g was produced from 15a (45.8%). LC: 94%. H NMR (300
MHz, DMSO): δ 8.89 (s, 1H), 8.75 (m, 2H), 8.64 (br, 1H), 8.27 (t, J
= 7.9 Hz, 2H), 8.18 (m, 3H), 8.05 (dd, J = 8.7, 1.7 Hz, 1H), 7.21 (s,
1H), 4.10−3.94 (m, 1H), 3.72 (m, 1H), 1.10 (d, J = 6.4 Hz, 3H).
HRMS (ESI): m/z calcd for C18H19N5O4S (M + H)+ 402.1236, found
402.1230.
(E)-2-(3-(6-Aminopyrimidin-4-yl)phenyl)ethenesulfonamide
(25). Triphenylphosphine (0.03 equiv), 6-(3-bromophenyl)pyrimidin-
4-amine (0.1 g, 1.0 equiv), diacetoxypalladium (0.03 equiv),
ethenesulfonamide (1.2 equiv), and TEA (1.2 equiv) were mixed in
DMF (1.5 mL) and degassed. The reaction was microwaved at 120 °C
for 1 h. The reaction mixture was concentrated and purified by column
chromatography, eluting with ethyl acetate, to give the title compound
as a white solid (69%). 1H NMR (DMSO-d6): δ 8.60 (s, 1H), 8.13 (s,
1H), 7.93 (d, J = 9 Hz, 1H), 7.83 (d, J = 7.5 Hz, 1H), 7.65 (t, J = 7.8
Hz, 1H), 7.63 (br, 2H), 7.50 (d, J = 15.6 Hz, 1H), 7.25 (d, J = 15.0 Hz,
1H), 7.10 (s, 1H). 6.03 (br, 2H).
((2S,3R)-2-Amine-N-((3-(4-aminoquinazolin-7-yl)phenyl)-
sulfonyl)-3-hydroxybutanamide (10a). Using methods C and B,
1
10a was produced from 9a (79%). LC: 99%. H NMR (300 MHz,
DMSO): δ 9.81 (br, 2H), 8.89 (s, 1H), 8.55 (d, J = 8.7 Hz, 1H), 8.27
(s, 1H), 8.13 (d, J = 8.7 Hz, 1H), 8.07−8.01 (m, 2H), 7.99 (d, J = 8.0
Hz, 1H), 7.84 (br, 2H), 7.72 (t, J = 7.8 Hz, 1H), 4.01−3.87 (m, 1H),
3.25 (m, 1H), 1.10 (d, J = 6.4 Hz, 3H). HRMS (ESI): m/z calcd for
C18H19N5O4S (M + H)+ 402.1235, found 402.1245.
(2S,3R)-2-Amino-N-((3-(2,4-diaminoquinazolin-7-yl)phenyl)-
sulfonyl)-3-hydroxybutanamide (10b). Using methods C and B,
1
10b was produced from 9a (41%). LC: 96%. H NMR (600 MHz,
DMSO): δ 9.10 (s, 1H), 8.87 (s, 1H), 8.49−8.28 (m, 1H), 8.17 (s,
1H), 7.86 (m, 2H), 7.82−7.67 (m, 3H), 7.63 (s, 1H), 7.48 (s, 1H),
7.35−7.06 (m, 1H), 3.94 (m, 1H), 1.08 (d, J = 6.4 Hz, 3H). HRMS
(ESI): m/z calcd for C18H20N6O4S (M + H)+ 417.1345, found
417.1350.
(2S,3R)-2-Amino-N-(((E)-3-(6-aminopyrimidin-4-yl)styryl)-
sulfonyl)-3-hydroxybutanamide (10h). Following general meth-
ods A and B, the title compound was produced as a white solid from
1
25 (68%). HPLC: 99%. H NMR (CD3OD): δ 8.66 (s, 1H), 8.15 (s,
(2S,3R)-2-Amino-N-((3-(4-amino-2-chloroquinazolin-7-yl)-
1H), 7.94 (t, J = 9 Hz, 2H), 7.8 (d, J = 15 Hz, 1H), 7.70 (t, J = 6.0 Hz,
2H), 7.50 (d, J = 15.6 Hz, 1H), 7.07 (s, 1H), 4.18 (m, 1H), 3.72 (d, J =
4.5, 1H), 1.34 (d, J = 6.0 Hz, 3H). HRMS (ESI): m/z calcd for
C16H19N5O4S (M + H)+ 378.1235, found 378.1232.
phenyl)sulfonyl)-3-hydroxybutanamide (10c). Using methods C
1
and B, 10c was produced from 9a (15%). LC: 93%. H NMR (300
MHz, DMSO): δ 8.45 (br, 1H), 8.38 (d, J = 9.0 Hz, 1H), 8.27 (s, 1H),
8.05 (d, J = 8.2 Hz, 1H), 7.84 (m, 3H), 7.69 (t, J = 7.7 Hz, 1H), 4.05−
3.77 (m, 1H), 3.5 (m, 1H), 1.10 (d, J = 6.4 Hz, 3H). HRMS (ESI): m/
z calcd for C18H18ClN5O4S (M + H)+ 436.0846, found 436.0839.
((2S,3R)-2-Amino-N-((3-(4-amino-2-methylquinazolin-7-yl)-
phenyl)sulfonyl)-3-hydroxybutanamide (10d). Using methods C
and B, 10d was produced from 9a (55%). LC: 99%. 1H NMR
(CD3OD): δ 8.34 (m, 1H), 8.3 (d, J = 7.2 Hz, 1H), 8.01 (d, J = 6 Hz,
1H), 7.89 (m, 2H), 7.81 (dd, J1 = 9 Hz, J2 = 3 Hz, 1H), 7.63 (t, J = 9
Hz, 1H), 4.13 (m, 1H), 3.56 (m, 1H), 2.50 (s, 3H), 1.25 (d, J = 6 Hz,
3H). HRMS (ESI): m/z calcd for C19H21N5O4S (M + H)+ 416.1392,
found 416.1390.
(2S,3R)-N-((7-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)naphthalen-
2-yl)sulfonyl)-2-amino-3-hydroxybutanamide (10i). Using the
same procedure as for the synthesis of 10g, 10i was produced from
1
15a (13%). HPLC: 99%. H NMR (600 MHz, DMSO): δ 9.05 (s,
1H), 8.93 (d, J = 5.0 Hz, 2H), 8.54 (d, J = 8.4 Hz, 1H), 8.24 (t, J = 9.5
Hz, 2H), 8.15 (br, 3H), 8.00 (d, J = 8.4 Hz, 1H), 7.76 (s, 1H), 7.21 (s,
1H), 5.5 (br, 1H), 4.07−3.97 (m, 1H), 3.73 (m, 1H), 1.09 (d, J = 6.3
Hz, 3H). HRMS (ESI): m/z calcd for C20H19N5O4S (M + H)+
426.1236, found 426.1236.
7-Bromo-3,4-dihydroisoquinoline-2(1H)-sulfonamide (13). A
solution of 7-bromo-1,2,3,4-tetrahydroisoquinoline (0.424 g, 2.0
mmol) in DMA (3 mL) was cooled to 0 °C. To this was added
sulfamoyl chloride (0.254 g, 2.2 mmol) dissolved in DMA (2.0 mL).
The reaction mixture was stirred at room temperature overnight and
(2S,3R)-2-Amino-N-((3-(1-amino-3-chloroisoquinolin-6-yl)-
phenyl)sulfonyl)-3-hydroxybutanamide (10e). Using methods C
1
and B, 10e was produced from 9a (15%). LC: 85%. H NMR (300
MHz, DMSO): δ 8.36 (d, J = 9.0 Hz, 1H), 8.30 (s, 1H), 8.18 (m, 3H),
8.02 (m, 2H), 7.81 (d, J = 6.0 Hz, 2H), 7.10 (s, 1H), 4.05 (m, 1H),
3.76 (m, 1H), 1.11 (d, J = 6.3 Hz, 3H). HRMS (ESI): m/z calcd for
C19H19ClN4O4S (M + H)+ 435.0894, found 435.0905.
1
was purified by RPLC as a white solid (43%). HPLC: 89%. H NMR
(300 MHz, DMSO): δ 7.43 (s, 1H), 7.41−7.30 (d, J = 9.0 Hz, 1H),
7.14 (d, J = 9.0 Hz, 1H), 6.94 (s, 2H), 4.21 (s, 2H), 3.26 (t, J = 5.9 Hz,
2H), 2.94−2.79 (t, J = 6.0 Hz, 2H). HRMS (ESI): m/z calcd for
C9H11BrN2O2S (M + H)+ 290.9803, found 290.9805.
tert-Butyl ((2S,3R)-1-(7-Bromo-1,2,3,4-tetrahydroisoquino-
line-2-sulfonamido)-3-(tert-butoxy)-1-oxobutan-2-yl)-
carbamate (14b). Using procedure A, 14b was synthesized from 13
(69.4%). This compound was used without further handling.
tert-Butyl ((2S,3R)-3-(tert-butoxy)-1-oxo-1-(7-(4,4,5,5-tetra-
methyl-1,3,2-dioxaborolan-2-yl)-1,2,3,4-tetrahydroisoquino-
line-2-sulfonamido)butan-2-yl)carbamate (15b). Using the same
procedure as for the synthesis of 15a, 15b was synthesized (59.7%)
and was used without further handling.
(2S,3R)-2-Amino-N-((3-(1-aminoisoquinolin-6-yl)phenyl)-
sulfonyl)-3-hydroxybutanamide (10f). Using methods C and B,
1
10f was produced from 9a (36%). LC: 98%. H NMR (300 MHz,
DMSO): δ 13.54 (s, 1H), 9.33 (s, 2H), 8.79 (d, J = 8.7 Hz, 1H), 8.37
(s, 2H), 8.19 (m, 4H), 8.05 (d, J = 8.0 Hz, 1H), 7.87−7.66 (m, 2H),
7.33 (t, J = 11.5 Hz, 1H), 4.17−3.92 (m, 1H), 3.76 (br, 1H), 1.14 (d, J
= 6.6 Hz, 3H). HRMS (ESI): m/z calcd for C19H20N4O4S (M + H)+
401.1283, found 401.1292.
tert-Butyl ((2S,3R)-1-(7-Bromonaphthalene-2-sulfonamido)-
3-(tert-butoxy)-1-oxobutan-2-yl)carbamate (14a). Following
general method A, 14a (0.192 g) was produced from 12 (0.145 g,
69.7%). LC: 90%. 1H NMR (300 MHz, DMSO): δ 12.19 (s, 1H), 8.64
(s, 1H), 8.55 (s, 1H), 8.17 (d, J = 8.8 Hz, 1H), 8.03 (d, J = 8.8 Hz,
1H), 7.95 (d, J = 8.7 Hz, 1H), 7.85 (dd, J = 8.8, 1.9 Hz, 1H), 6.08 (d, J
= 9.3 Hz, 1H), 4.04−3.91 (m, 1H), 3.91−3.71 (m, 1H), 1.33 (s, 9H),
0.95 (d, J = 5.7 Hz, 3H), 0.89 (s, 9H). HRMS (ESI): m/z calcd for
C23H31BrN2O6S (M + H)+ 543.1164, found 543.1174.
(2S,3R)-N-((7-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-3,4-dihydroi-
soquinolin-2(1H)-yl)sulfonyl)-2-amino-3-hydroxybutanamide
(10j). Using methods C and B, 10j was produced from 15b as a HCl
1
salt (17%). HPLC: 77%. H NMR (300 MHz, DMSO): δ 13.08 (s,
1H), 12.46 (s, 1H), 9.01 (s, 1H), 8.39 (br, 3H), 8.00 (d, J = 7.6 Hz,
2H), 7.92 (s, 1H), 7.49 (d, J = 8.1 Hz, 1H), 7.13 (s, 1H), 4.71 (q, J =
15.7 Hz, 2H), 4.17−3.96 (m, 1H), 3.84 (br, 1H), 3.75−3.51 (m, 2H),
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Journal of Medicinal Chemistry
Article
1.20 (t, J = 6.0 Hz, 3H). HRMS (ESI): m/z calcd for C19H22N6O4S
(M + H)+ 431.1501, found 431.1498.
trile/water). The yield of the light yellow foam was 66% (292 mg).
HPLC: 95%. H NMR (300 MHz, DMSO): δ 9.64 (s, 1H), 8.05 (s,
1
(2S,3R)-2-Amino-3-hydroxy-N-((3-(1-oxoisoindolin-5-yl)-
1H), 7.06 (t, J = 6.0 Hz, 1H), 7.00 (s, 1H), 6.83 (d, J = 9 Hz, 1H),
6.78 (d, J = 6.0 Hz, 1H), 6.30 (d, J = 8.4 Hz, 1H), 3.99 (dd, J = 8.5, 4.1
Hz, 1H), 3.92−3.80 (m, 1H), 1.42 (s, 9H), 1.18 (s, 9H), 1.06 (d, J =
6.2 Hz, 3H). HRMS (ESI): m/z calcd for C19H30BrN3O4 (M + H)+
444.1498, found 444.1506.
phenyl)sulfonyl)butanamide (11a). Using methods C and B, 11a
1
was produced from 9a as a HCl salt (36%). HPLC: 99%. H NMR
(300 MHz, DMSO): δ 8.67 (s, 1H), 8.25 (m, 4H), 8.09 (d, J = 7.8 Hz,
1H), 7.99 (d, J = 7.9 Hz, 1H), 7.92 (s, 1H), 7.87−7.65 (m, 3H), 4.47
(s, 2H), 4.16−3.94 (m, 1H), 3.84 (s, 1H), 1.11 (d, J = 6.4 Hz, 3H).
HRMS (ESI): m/z calcd for C18H19N3O5S (M + H)+ 390.1123, found
390.1127.
tert-Butyl ((2S,3R)-3-(tert-Butoxy)-1-oxo-1-(2-(3-(4,4,5,5-tet-
ramethyl-1,3,2-dioxaborolan-2-yl)phenyl)hydrazinyl)butan-2-
yl)carbamate (18b). A septum-capped vial charged with 18a (178
mg, 0.4 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bis(1,3,2-dioxabor-
olane) (112 mg, 0.440 mmol), and KOAc (118 mg, 1.20 mmol) in dry
DMSO (1.2 mL) was vacuum flushed with nitrogen five times, then
treated with bis(diphenylphosphino)ferrocene]dichloropalladium(II)
complex with dichloromethane (9.8 mg, 0.012 mmol), and placed in
an 80 °C heating block. LC/MS after 60 min showed ∼50%
conversion. After 2 h, the reaction was diluted with ether, filtered
through Celite, concentrated, and purified by reversed-phase liquid
chromatography (5−100% acetonitrile/water), giving 86 mg (44%
(2S,3R)-2-Amino-3-hydroxy-N-((3-(3-methyl-1H-indazol-5-
yl)phenyl)sulfonyl)butanamide (11c). Using methods C and B,
1
11c was produced from 9b as a TFA salt (18%). LC: 99%. H NMR
(300 MHz, DMSO): δ 8.21 (br, 1H), 8.05 (d, J = 5.2 Hz, 2H), 8.03 (s,
1H), 7.88 (d, J = 8.3 Hz, 1H), 7.72 (s, 1H), 7.70 (s, 1H), 7.67 (dd, J =
4.3, 2.2 Hz, 1H), 7.65 (m, 1H), 7.61 (s, 1H), 7.58 (s, 1H), 4.08−3.91
(m, 1H), 3.61 (m, 1H), 2.57 (s, 3H), 1.10 (d, J = 6.4 Hz, 3H). HRMS
(ESI): m/z calcd for C18H20N4O4S (M + H)+ 389.1283, found
389.1281.
t e r t - B u t y l ( ( 2 S , 3 R ) - 1 - ( 3 - ( 1 H - I n d a z o l - 5 - y l ) -
benzenesulfonamido)-3-(tert-butoxy)-1-oxobutan-2-yl)-
carbamate (11d). Using methods C and B, 11d was produced from
9b as a TFA salt (29%). HPLC: 98%. 1H NMR (600 MHz, DMSO): δ
8.13 (s, 2H), 8.04 (s, 1H), 7.86 (br, 3H), 7.91−7.70 (m, 2H), 7.65 (s,
2H), 7.58 (t, J = 7.6 Hz, 1H), 4.01−3.84 (m, 1H), 3.48 (d, J = 11.0 Hz,
1H), 1.14−0.95 (d, J = 6.0 Hz, 3H). HRMS (ESI): m/z calcd for
C17H18N4O4S (M + H)+ 375.1127, found 375.1138.
1
yield) of the title compound. H NMR (300 MHz, DMSO): δ 10.06
(s, 1H), 8.19 (s, 1H), 7.91 (s, 1H), 7.16−7.07 (m, 2H), 7.04 (t, J = 7.0
Hz, 1H), 6.93 (d, J = 7.9 Hz, 1H), 6.21 (d, J = 9.1 Hz, 1H), 4.00 (m,
1H), 3.86 (m, 1H), 1.42 (s, 9H), 1.29 (s, 12H), 1.16 (s, 9H), 1.07 (d, J
= 5.4 Hz, 3H). HRMS (ESI): m/z calcd for C25H42BN3O6 (M + H)+
492.3245, found 492.3275.
(2S,3R)-2-Amino-N′-(3-(4-amino-2-chloroquinazolin-7-yl)-
phenyl)-3-hydroxybutanehydrazide (19). Using general methods
C and B, the title compound was synthesized from 18b in 31%
combined yield in two steps. HPLC: 98%. 1H NMR (300 MHz,
DMSO): δ 10.30 (s, 1H), 8.30 (m, 3H), 8.12 (s, 1H), 7.91−7.81 (m,
1H), 7.79 (s, 1H), 7.76−7.72 (m, 1H), 7.54 (t, J = 7.4 Hz, 1H), 7.33
(t, J = 8.0 Hz, 1H), 7.25−7.13 (m, 2H), 6.90 (d, J = 8.7 Hz, 1H), 5.70
(s, 1H), 4.00 (br, 1H), 3.66 (br, 1H), 1.25 (d, J = 6.2 Hz, 3H). HRMS
(ESI): m/z calcd for C18H19ClN6O2 (M + H)+ 387.1336, found
387.133.
(2S,3R)-2-Amino-N-((3-(3-chloro-1H-indazol-5-yl)phenyl)-
sulfonyl)-3-hydroxybutanamide (11b). A solution of 11d (0.083
mmol) and NCS (0.014 g, 0.11 mmol) in ACN (3 mL) was heated to
70 °C for 1 h. The reaction was cooled to room temperature and was
concentrated to an oil. The product was purified by RPLC to give the
1
desired product as a TFA salt (68%). HPLC: 99%. H NMR (300
MHz, DMSO): δ 13.43 (s, 1H), 8.16 (s, 1H), 7.89 (d, J = 10.6 Hz,
2H), 7.83 (m, 4H), 7.72 (d, J = 9.0 Hz, 1H), 7.58 (t, J = 7.7 Hz, 1H),
4.01−3.82 (m, 1H), 3.5 (m, 1H), 1.10 (d, J = 6.4 Hz, 3H). HRMS
(ESI): m/z calcd for C17H17ClN4O4S (M + H)+ 409.0737, found
409.0733.
tert-Butyl ((2R,3R)-1-Amino-3-(tert-butoxy)butan-2-yl)-
carbamate (20). A solution of 6 (826 mg, 3.0 mmol) and ethyl
chloroformate (1.2 equiv) in THF (15 mL) was added to TEA (1.2
equiv) slowly. After the solution was stirred at room temperature for 5
min, aq ammonium hydroxide (5 equiv) was added. The reaction was
stirred overnight. LC/MS revealed the product as M + Na (297).
Workup: the reaction was concentrated and purified by RPLC (0−
100% ACN in water, over 15 min). The yield of the colorless foam was
787 mg (96%). To a round-bottom flask charged with the above-
synthesized foam (490 mg, 1.79 mmol) dissolved in dry THF (4.5
mL) at 0 °C was added LAH (5.36 mL, 5.36 mmol, 1.0 M solution in
THF). The reaction was allowed to warm to room temperature and
stirred for a total of 4 h. Reaction progress was monitored by TLC
(10% MeOH/DCM with ammonia, visualized with ninhydrin stain).
Workup: the reaction was cooled in an ice bath and then slowly
quenched with NaOH (aq, 1.0 N) until a thick white precipitate/
emulsion was formed. The emulsion was gently extracted with ether (5
× 10 mL) and concentrated to a colorless oil (445 mg, 96%) that was
(3-(((2S,3R)-3-(tert-Butoxy)-2-((tert-butoxycarbonyl)amino)-
butanamido)methyl)phenyl)boronic acid (16). To a round-
bottom flask charged with 6 (716 mg, 2.6 mmol), (3-(aminomethyl)-
phenyl)boronic acid hydrochloride (375 mg, 2.00 mmol), HOAT (272
mg, 2.00 mmol), and diisopropylethylamine (1.4 mL, 8.0 mmol) in
DCM (6.7 mL) was added EDCI (767 mg, 4.00 mmol) in one portion
at room temperature. LC/MS after 12 h showed complete conversion.
Workup: the reaction was concentrated and purified by reversed-phase
liquid chromatography (5−100% acetonitrile/water). The yield of the
1
product as a colorless foam was 99% (810 mg). H NMR (300 MHz,
DMSO): δ 8.21 (t, J = 5.6 Hz, 1H), 7.98 (s, 2H), 7.74−7.62 (m, 2H),
7.38−7.18 (m, 2H), 6.05 (d, J = 8.9 Hz, 1H), 4.33 (dd, J = 14.8, 5.9
Hz, 1H), 4.21 (dd, J = 14.8, 5.2 Hz, 1H), 3.92 (d, J = 9.4 Hz, 2H), 1.40
(s, 9H), 1.07 (s, 9H), 1.02 (d, J = 5.8 Hz, 3H). HRMS (ESI): m/z
calcd for C20H33BN2O6 (M + H)+ 409.2510, found 409.2523.
(2S,3R)-2-Amino-N-(3-(4-amino-2-chloroquinazolin-7-yl)-
benzyl)-3-hydroxybutanamide (17). Using general methods B and
C, the title compound was synthesized from 16 as a HCl salt in 12%
combined yield for two steps. HPLC: 94%. 1H NMR (300 MHz,
DMSO): δ 9.06 (s, 1H), 8.71 (s, 1H), 8.37 (m, 1H), 8.16 (s, 2H), 7.88
(m, 1H), 7.84 (m, 2H), 7.49 (m, 3H), 5.61 (br, 1H), 4.48 (m, 1H),
3.96 (m, 1H), 3.51 (m, 2H), 1.19 (d, J = 6.0 Hz, 3H). HRMS (ESI):
m/z calcd for C19H21ClN5O2 (M + H)+ 386.1384, found 386.1390.
tert-Butyl ((2S,3R)-1-(2-(3-Bromophenyl)hydrazinyl)-3-(tert-
butoxy)-1-oxobutan-2-yl)carbamate (18a). A round-bottom
flask charged with 6 (275 mg, 1.00 mmol) and ethyl chloroformate
(115 μL, 1.2 mmol) was treated with 4-methylmorpholine (132 μL,
1.2 mmol) dropwise over 5 min. After the solution was stirred for 5
min at room temperature, (3-bromophenyl)hydrazine hydrochloride
(223 mg, 1.00 mmol) was added followed by 4-methylmorpholine
(264 μL, 2.4 mmol). After 15 min, LC/MS showed consumption of
the starting material. Workup: the reaction was concentrated and
purified by reversed-phase liquid chromatography (5−100% acetoni-
1
used without further purification. H NMR (300 MHz, DMSO): δ
7.15 (s, 2H), 5.96 (d, J = 8.9 Hz, 1H), 3.95−3.74 (m, 2H), 3.33 (m,
2H), 1.40 (s, 9H), 1.13 (s, 9H), 1.02 (d, J = 6.2 Hz, 3H). HRMS
(ESI): m/z calcd for C12H28N2O3 (M + H)+ 261.2178, found
261.2179.
tert-Butyl ((2R,3R)-1-(3-Bromobenzenesulfonamido)-3-(tert-
butoxy)butan-2-yl)carbamate (21a). To a round-bottom flask
charged with crude 20 (445 mg, 1.71 mmol) and 3-bromobenzene-1-
sulfonyl chloride (480 mg, 1.88 mmol) in DCM (5.7 mL) was added
TEA (310 μL, 2.22 mmol) dropwise over 5 min. LC/MS, after 1 h,
showed complete conversion to the product. Workup: the reaction was
concentrated, filtered, and purified by reversed-phase liquid
chromatography (5−100% acetonitrile/water). The desired product
was obtained as a colorless oil (194 mg, 25%) and was used without
further handling.
tert-Butyl ((2S,3R)-3-(tert-Butoxy)-1-oxo-1-(3-(4,4,5,5-tetra-
methyl-1,3,2-dioxaborolan-2-yl)benzenesulfonamido)butan-
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2-yl)carbamate (21b). A Teflon-capped vial charged with 21a (112
mg, 0.234 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bis(1,3,2-dioxa-
borolane) (65 mg, 0.257 mmol), and KOAc (69 mg, 0.70 mmol) in
dry DMF (0.584 mL) was vacuum flushed with nitrogen five times,
treated with bis(diphenylphosphino)ferrocene]dichloropalladium(II)
complex with dichloromethane (5.7 mg, 7.0 mmol), and placed in an
80 °C heating block. After 1 h, LC/MS showed complete consumption
of the starting material. Workup: the reaction mixture was
concentrated, diluted with ethyl acetate, filtered through Celite,
concentrated to dryness, and used in the next step without further
purification.
(2S,3R)-N-(((E)-3-(6-Aminopyrimidin-4-yl)styryl)sulfonyl)-2,3-
dihydroxybutanamide (28). Using the same procedure as for the
synthesis of 10h, the title compound was produced in 19% yield in two
steps using 25 and (2S,3R)-2,3-O-isopropylidene-2,3-dihydroxybutyric
acid.18 1H NMR (300 MHz, DMSO): δ 8.63 (s, 1H), 8.29 (s, 1H),
8.00 (d, J = 7.7 Hz, 1H), 7.94 (d, J = 7.9 Hz, 1H), 7.70 (d, J = 15 Hz,
1H), 7.65 (d, J = 6.0 Hz, 1H), 7.53 (d, J = 15 Hz, 1H), 7.00 (s, 1H),
3.84 (m, 2H), 1.06 (d, J = 6.0 Hz, 3H). HRMS (ESI): m/z calcd for
C16H18N4O5S (M + H)+ 379.1076, found 379.1088.
(2S,3R)-2,3-Diamino-N-(((E)-3-(6-aminopyrimidin-4-yl)-
styryl)sulfonyl)butanamide (29). Using the same procedure as for
the synthesis of 10h, the title compound was produced in 15% yield in
two steps using 25 and (2S,3R)-2,3-bis((tert-butoxycarbonyl)amino)-
butanoic acid.19 1H NMR (600 MHz, DMSO): δ 8.70 (s, 1H), 8.43 (s,
1H), 8.34 (s, 1H), 8.14 (s, 1H), 7.88 (d, J = 7.4 Hz, 1H), 7.81 (d, J =
7.1 Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H), 7.46 (d, J = 18 Hz, 1H), 7.35 (d,
J = 18 Hz, 1H), 7.35 (s, 1H), 7.26 (s, 1H), 7.18 (s, 1H), 7.09 (s, 1H),
7.05 (s, 1H), 3.81 (m, 1H), 3.59 (m, 1H), 1.18 (d, J = 6.5 Hz, 1H).
HRMS (ESI): m/z calcd for C16H20N6O3S (M + H)+ 377.1396, found
377.1414.
Enzyme Assays. The tRNA synthetases catalyze a two-step
reaction. The first step is the ATP-dependent amino acid activation,
resulting in the formation of an amino acid adenylate. In the second
step, this activated amino acid is transferred to the relevant tRNA. The
enzymatic activity of ThrRS was determined using a coupled
spectrophotometric assay in which the stoichiometric release of
pyrophosphate during the ATP-dependent amino acid adenylation
step was followed. Serine was used as a surrogate amino acid substrate
due to a significantly higher rate of turnover due to the increased rate
of seryl adenylate hydrolysis.20
In ThrRS, the amino acid adenylation reaction proceeds via a
random sequential mechanism in which either ATP or amino acid
binds first to the enzyme, forming a binary complex, followed by
binding of the second substrate to form a ternary enzyme−ATP−
amino acid complex.21 The reader is referred to the numerous texts
which describe techniques used to obtain the key kinetic parameters
required for the determination of inhibition binding constants.22
All reagents were obtained from Sigma-Aldrich unless indicated.
Assays, typically 100 μL, consisted of 60 mM Tris, pH 7.6, 10 mM
MgCl2, 20 mM KCl, 200 μM MESG (Berry and Associates), 4 mM
ATP, 200 mM L-serine, 0.5 mM DTT, 0.1 U of inorganic
pyrophosphatase, and 0.001 U of purine nucleoside phosphorylase.
DMSO concentrations were maintained at 2% (v/v) in all reactions.
Due to weak potency, the conditions used for compound 4 were
modified and contained 25 mM L-serine, 0.5 mM ATP, and 20 nM
enzyme. An enzyme concentration of 5 nM was used for all
orthologues. Data were collected using a Tecan Safire II microplate
reader. Inhibition data were normalized to controls lacking inhibitor
(maximum activity) and no enzyme (blank). Typically, IC50 values
were obtained using a 16-point, 2-fold dilution series, from 50 μM to
1.5 nM, analyzed using a 4-parameter sigmoidal log/response plot with
variable slope and maximum/minimum constraint. Ki values were
calculated for a bisubstrate enzyme as described by Cheng and Prusoff.
Bacterial Strains and Culture Conditions. E. coli imp (BAS849;
permeability mutant in the MCR106 background with Imp deletion
Thr332−Tyr354),23 E. coli tolC (efflux mutant in the BW25113
background with a TolC deletion from Leu13−Ala480) and wild type
(ATCC 25922), and B. thailandensis strain E26424 and the isogenic
ΔamrAB-oprA ΔbpeAB-oprB double knockout efflux mutant strain
(KO) (H. P. Schweizer, Colorado State University) were cultured on
Mueller−Hinton II cation adjusted agar (MHA) or in MH broth
(MHB; BD catalog no. 212322) at 37 °C. H. influenzae wild type
(ATCC 31517) was cultured on chocolate agar plates or in
Haemophilus test medium broth (Remel catalog no. R112380) at 37
°C in the presence of 5% CO2.
(2S,3R)-2-Amino-N-((3-(4-amino-2-chloroquinazolin-7-yl)-
phenyl)sulfonyl)-3-hydroxybutanamide (22). Using general
methods B and C, the title compound was synthesized from 21b in
9% combined yield for the final three steps starting from 21a. HPLC:
1
97%. H NMR (300 MHz, DMSO): δ 8.41 (br, 1H), 8.39 (d, J = 8.6
Hz, 1H), 8.25 (s, 1H), 8.18 (d, J = 7.9 Hz, 1H), 7.98 (d, J = 1.5 Hz,
1H), 7.95 (d, J = 1.7 Hz, 1H), 7.90 (d, J = 8.7 Hz, 2H), 7.80 (m, 2H),
5.41 (d, J = 6.0 Hz, 1H), 3.81 (m, 1H), 2.97 (m, 3H), 1.13 (d, J = 6.0
Hz, 3H). HRMS (ESI): m/z calcd for C18H20ClN5O3S (M + H)+
422.1053, found 422.1051.
tert-Butyl ((2S,3R)-1-(3-Bromo-N-methylbenzenesulfonami-
do)-3-(tert-butoxy)-1-oxobutan-2-yl)carbamate (23). To a sol-
ution of 9b (0.17 g, 0.35 mmol) and Cs2CO3 (0.135 g, 0.413 mmol) in
DMF (3 mL) was added iodomethane (0.15 g, 3.0 equiv). The
reaction mixture was heated to 65 °C for 12 h under argon. The
reaction mixture was added to water and sonicated. The solids were
1
filtered and collected as the desired product (0.095 g, 54.3%). H
NMR (300 MHz, DMSO): δ 8.09 (s, 1H), 7.96 (d, J = 7.8 Hz, 2H),
7.60 (t, J = 8.0 Hz, 1H), 6.42 (d, J = 8.3 Hz, 1H), 4.67 (m, 1H), 3.92
(m, 1H), 3.36 (s, 3H), 1.35 (s, 9H), 1.08 (s, 9H), 0.98 (d, J = 6.0 Hz,
3H).
(2S,3R)-2-Amino-3-hydroxy-N-methyl-N-((3-(1-oxoisoindo-
lin-5-yl)phenyl)sulfonyl)butanamide (24). Using the same
procedure as for the synthesis of 11a, intermediate 23 produced
tert-butyl ((2S,3R)-3-(tert-butoxy)-1-(N-methyl-3-(1-oxoisoindolin-5-
yl)benzenesulfonamido)-1-oxobutan-2-yl)carbamate in 55.4% yield.
1H NMR (300 MHz, DMSO): δ 8.60 (s, 1H), 8.19 (s, 1H), 8.11 (d, J
= 7.8 Hz, 1H), 8.01 (d, J = 7.9 Hz, 1H), 7.94 (s, 1H), 7.85 (d, J = 8.3
Hz, 1H), 7.79 (m, 2H), 6.29 (d, J = 8.7 Hz, 1H), 4.82 (m, 1H), 4.46
(s, 2H), 3.98 (m, 1H), 3.33 (s, 3H), 1.30 (s, 9H), 1.06 (s, 9H), 1.02
(d, J = 6.0 Hz, 3H). This intermediate was treated with 4.0 N HCl in
1
dioxane at room temperature to afford product 24 in 46.8% yield. H
NMR (600 MHz, DMSO): δ 8.67 (s, 1H), 8.23 (s, 1H), 8.13 (d, J =
7.4 Hz, 1H), 8.10 (d, J = 7.1 Hz, 1H), 7.95 (s, 1H), 7.82 (m, 3H), 5.76
(br, 1H), 4.61 (s, 1H), 4.45 (s, 2H), 4.13 (m, 1H), 3.33 (s, 3H), 1.14
(d, J = 6.5 Hz, 3H). HRMS (ESI): m/z calcd for C19H21N3O5S (M +
H)+ 404.1280, found 404.1286.
(2S,3R)-2-Amino-N-(((E)-3-(6-aminopyrimidin-4-yl)styryl)-
sulfonyl)-3-hydroxypentanamide (26). Using the same procedure
as for the synthesis of 10h, the title compound was produced in 15%
yield in two steps using 25 and (2S,3R)-2-((tert-butoxycarbonyl)-
amino)-3-((tert-butyldimethylsilyl)oxy)pentanoic acid (Annova
Chem). 1H NMR (300 MHz, DMSO): δ 8.40 (s, 1H), 8.16 (s,
1H), 7.98 (d, J = 6 Hz, 1H), 7.56 (d, J = 6.0 Hz, 1H), 7.48 (m, 1H),
7.36 (d, J = 15 Hz, 1H), 7.18 (d, J = 15.6 Hz, 1H), 7.09 (s, 1H), 4.17
(m, 1H), 3.75 (d, J = 4.5 Hz, 1H), 1.14 (m, 2H), 0.72 (t, J = 7.4 Hz,
3H). HRMS (ESI): m/z calcd for C17H21N5O4S (M + H)+ 392.1392,
found 392.1402.
(2S,3R)-2-Amino-N-(((E)-3-(6-aminopyrimidin-4-yl)styryl)-
sulfonyl)-3-hydroxy-4-methylpentanamide (27). Using the same
procedure as for the synthesis of 10h, the title compound was
produced in 10% yield in two steps using 25 and (2S,3R)-2-((tert-
butoxycarbonyl)amino)-3-((tert-butyldimethylsilyl)oxy)-4-methylpen-
tanoic acid.17 1H NMR (300 MHz, DMSO): δ 8.53 (s, 1H), 8.23 (s,
1H), 7.99 (d, J = 7.7 Hz, 1H), 7.80 (d, J = 7.1 Hz, 1H), 7.58 (t, J = 7.8
Hz, 1H), 7.45 (d, J = 14.0 Hz, 1H), 7.37 (m, 2H), 7.00 (s, 1H), 5.78
(d, J = 5.3 Hz, 1H), 3.94 (br, 1H), 3.58 (m, 1H), 1.72 (m, 1H), 0.93
(d, J = 5.8 Hz, 3H), 0.87 (d, J = 6.6 Hz, 3H). HRMS (ESI): m/z calcd
for C18H23N5O4S (M + H)+ 406.1549, found 406.1546.
Minimum Inhibitory Concentration (MIC) Assays. Broth
microdilution MIC assays were performed according to CLSI
guidelines with the exception that compound stocks were made up
at 50× and the end point detection was determined colorimetrically
via detection with alamarBlue (Invitrogen, catalog no. DAL1100).25
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Stock solutions of all test compounds were prepared in 100% dimethyl
sulfoxide (DMSO) at 6.4 mg/mL and then serially diluted in two
steps. Two microliters from each dilution series was added to 98 μL of
inoculated media, resulting in final test concentrations of 0.063−128
μg/mL. Serial dilutions of the starting inocula were plated to ensure
that the assay contained roughly the desired 5 × 105 CFU/mL. MIC
plates were incubated overnight at 37 °C in the appropriate conditions.
The following day, 10 μL of alamarBlue was added to each well. The
plates were shaken and incubated for another 1 h, and then the MICs
were determined as the lowest concentration of compound where no
color change was visible.
Protein Purification and Cocrystallization Conditions. The
proteins were purified by metal chelate affinity chromatography,
exploiting a vector-derived C-terminal 6 × His tag. Compounds were
cocrystallized by hanging drop vapor diffusion using a mother liquor
containing 12% (w/v) PEG 4000, 21% (v/v) MPD, and 0.1 M sodium
citrate, pH 5.9. The resulting crystals were of space group P212121 and
typically diffracted to 1.8 Å or better on a rotating anode X-ray source.
Crystals of the human enzyme (cytoplasmic isoform 1) were generated
with an analogous construct and purification scheme.
(3) (a) Finn, J.; Tao, J. In Aminoacyl-tRNA Synthetase as Drug Targets
in Aminoacyl-tRNA Synthetases; Ibba, M., Ed.; Landes BioScience
Press: Georgetown, TX, 2004; pp 405−413. (b) Jarvest, R.; Berge, J.;
Berry, V.; Boyd, H.; Brown, M.; Elder, J.; Forrest, A.; Fosberry, A.;
Gentry, D.; Hibbs, M.; Jaworski, D.; O’Hanlon, P.; Pope, A.;
Rittenhouse, S.; Sheppard, R.; Slater-Radosti, C.; Worby, A. Nano-
molar inhibitors of Staphylococcus aureus methionyl tRNA synthetase
with potent antibacterial activity against Gram-positive pathogens. J.
Med. Chem. 2002, 45, 1959. (c) Finn, J.; Mattia, K.; Morytko, M.;
Ram, S.; Yang, Y.; Wu, X.; Silverman, J.; Mak, E.; Gallant, P.; Keith, D.
Discovery of a potent and selective series of pyrazole bacterial
methionyl-tRNA synthetase inhibitors. Bioorg. Med. Chem. Lett. 2003,
13, 2231. (d) Finn, J.; Stidham, M.; Hilgers, M.; Kedar, G. C.
Identification of novel inhibitors of methionyl-tRNA synthetase
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(14), 3932−3937. (e) Yu, X.; Finn, J.; Hill, J.; Wang, Z.; Keith, D.;
Silverman, J.; Oliver., N. A series of heterocyclic inhibitors of
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aminoacyl-tRNA synthetases. J. Mol. Evol. 1995, 40, 487−498.
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based on ancient aminoacyl-tRNA synthetase gene duplications. Proc.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Full description of the crystallographic methods, protein
purification methods, enzymatic assay, and MIC conditions.
This material is available free of charge via the Internet at
http://pubs.acs.org.
Accession Codes
Coordinates for the cocrystal structures of compounds 10a,c,d
and 11d bound to E. coli ThrRS and compound 11d bound to
the human ThrRS have been deposited in the Protein Data
Bank (http://www.pdb.org) under accession codes 4HWO,
4HWS, 4HWP, 4HWR, and 4HWT, respectively.
(7) (a) Van de Vijver, P.; Vondenhoff, G. H. M.; Denivelle, S.;
Rozenski, J.; Verhaegen, J.; Van Aerschot, A.; Herdewijn, P.
Antibacterial 5′-O-(N-dipeptidyl)-sulfamoyladenosines. Bioorg. Med.
Chem. 2009, 17, 260−269. (b) Forrest, A. K.; Jarvest, R. L.; Mensah, L.
M.; O'Hanlon, P. J.; Pope, A. J.; Sheppard, R. J. Aminoalkyl adenylate
and aminoacyl sulfamate intermediate analogues differing greatly in
affinity for their cognate Staphylococcus aureus aminoacyl tRNA
synthetases. Bioorg. Med. Chem. Lett. 2000, 10, 1871−1874. (c) Brown,
P.; Richardson, C. M.; Mensah, L. M.; O’Hanlon, P. J.; Osborne, N. F.;
Pope, A. J.; Walker, G. Molecular recognition of tyrosinyl adenylate
analogues by prokaryotic tyrosyl tRNA synthetases. Bioorg. Med. Chem.
1999, 7, 2473−2485.
AUTHOR INFORMATION
Corresponding Author
*E-mail: mteng@triusrx.com. Phone: (858) 452-0370.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Mark Stidham for budget management and Dr.
Douglas Phillipson for his support in compound purifications.
This work was supported by the Transformational Medical
Technologies program (Contract HDTRA1-10-C-004) of the
Department of Defense Chemical and Biological Defense
program through the Defense Threat Reduction Agency
(DTRA).
(8) Hurdle, J. G.; O’Neill, A. J.; Chopra, I. Prospects for aminoacyl-
tRNA synthetase inhibitors as new antimicrobial agents. Antimicrob.
Agents Chemother. 2005, 49, 4821.
(9) (a) Nass, G.; Poralla, K.; Zaehner, H. Effect of the antibiotic,
borrelidin, on the regulation of threonine biosynthetic enzymes in
Escherichia coli. Biochem. Biophys. Res. Commun. 1969, 34 (1), 84−91.
(b) Poralla, K. Zaehner, Hans. Metabolic products of microorganisms.
LXII. Inhibition of the attachment of threonine to sRNA in a cell-free
system and of the synthesis of protein and nucleic acids in the cell by
the antibiotic borrelidin. Arch. Mikrobiol. 1968, 61, 143−153.
(c) Huetter, R.; Poralla, K.; Zachau, H. G.; Zehner, H. Metabolic
products of microorganisms. LI. Mechanism of action of borrelidin.
Inhibition of the incorporation of threonine into sRNA (soluble
RNA). Biochem. Z. 1966, 344 (2), 190−196.
ABBREVIATIONS USED
■
B. thailandensis (KO), B. thailandensis pump knockout; ThrRS,
threonyl-tRNA synthetase; aa-AMP, aminoacyl adenosine
monophosphate; AMS, 5′-O-[N-(threonyl)sulfamoyl]-
adenosine; TEA, triethylamine; SBDD, structure-based drug
design; ThrRS, threonyl-tRNA synthetase
(10) Wakabayashi, T.; Kageyama, R.; Naruse, N.; Tsukahara, N.;
Funahashi, Y.; Kitoh, K.; Watanabe, Y. Borrelidin is an angiogenesis
inhibitor; disruption of angiogenic capillary vessels in a rat aorta matrix
culture model. J. Antibiot. 1997, 50 (8), 671−676.
(11) Constructs encoding truncated E. coli, Y. pestis, and B.
thailandensis ThrRS enzymes, lacking the editing domains, and
comparable to that previously described were cloned and expressed
in E. coli BL21-AI using the pET28 expression vector:
Sankaranarayanan, R.; Dock-Bregeon, A. C.; Rees, B.; Bovee, M.;
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