Pyrrolopyrimidine inhibitors of DNA gyrase B (GyrB) and topoisomerase IV (ParE). Part I: Structure guided discovery and optimization of dual targeting agents with potent, broad-spectrum enzymatic activity

Article abstract of DOI:10.1016/j.bmcl.2012.11.032

The bacterial topoisomerases DNA gyrase (GyrB) and topoisomerase IV (ParE) are essential enzymes that control the topological state of DNA during replication. The high degree of conservation in the ATP-binding pockets of these enzymes make them appealing targets for broad-spectrum inhibitor development. A pyrrolopyrimidine scaffold was identified from a pharmacophore-based fragment screen with optimization potential. Structural characterization of inhibitor complexes conducted using selected GyrB/ParE orthologs aided in the identification of important steric, dynamic and compositional differences in the ATP-binding pockets of the targets, enabling the design of highly potent pyrrolopyrimidine inhibitors with broad enzymatic spectrum and dual targeting activity.

Full text of DOI:10.1016/j.bmcl.2012.11.032

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1529–1536
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrrolopyrimidine inhibitors of DNA gyrase B (GyrB) and
topoisomerase IV (ParE). Part I: Structure guided discovery
and optimization of dual targeting agents with potent,
broad-spectrum enzymatic activity
a
a
a
a
a
Leslie W. Tari a, , Michael Trzoss , Daniel C. Bensen , Xiaoming Li , Zhiyong Chen , Thanh Lam ,
Junhu Zhang a, Christopher J. Creighton a, Mark L. Cunningham a, Bryan Kwan a, Mark Stidham a,
Karen J. Shaw a, Felice C. Lightstone b, Sergio E. Wong b, Toan B. Nguyen b, Jay Nix c, John Finn a
⇑
a Trius Therapeutics, 6310 Nancy Ridge Dr., San Diego, CA 92121, USA
b Lawrence Livermore National Laboratory, Physical and Life Sciences Directorate, Livermore, CA 94550, USA
c Advanced Light Source, Beamline 4.2.2, 1 Cyclotron Rd., Berkeley, CA 94720, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 5 December 2012
The bacterial topoisomerases DNA gyrase (GyrB) and topoisomerase IV (ParE) are essential enzymes that
control the topological state of DNA during replication. The high degree of conservation in the ATP-bind-
ing pockets of these enzymes make them appealing targets for broad-spectrum inhibitor development. A
pyrrolopyrimidine scaffold was identified from a pharmacophore-based fragment screen with optimiza-
tion potential. Structural characterization of inhibitor complexes conducted using selected GyrB/ParE
orthologs aided in the identification of important steric, dynamic and compositional differences in the
ATP-binding pockets of the targets, enabling the design of highly potent pyrrolopyrimidine inhibitors
with broad enzymatic spectrum and dual targeting activity.
Keywords:
Bacterial topoisomerases
GyrB
ParE
Pyrrolopyrimidine
Inhibitor
Ó 2012 Elsevier Ltd. All rights reserved.
The increasing prevalence of antibiotic resistant strains of path-
ogenic bacteria continues to erode the utility of every major class
of antibiotics used in the clinic.1 To avoid cross-resistance with
established classes of antibiotics, new agents must be developed
that act via novel mechanisms of action, or against unique binding
sites on existing validated targets. Target selection is of critical
importance to preemptively minimize the probability of resistance
development. Antibacterial agents that target single enzymes can
be subject to high-level resistance resulting from single-step muta-
tions. Single agents with the capacity to interact with multiple
sites on the same target or simultaneously target enzymes from
multiple essential pathways have been demonstrated to impair
the ability of bacteria to develop resistance.1
The bacterial topoisomerases DNA gyrase (GyrA/GyrB) and
topoisomerase IV (ParC/ParE) are established antibacterial targets2
that are closely related at the structural and functional levels. Both
topoisomerases form A2B2 holoenzymes that control the topologi-
cal state of DNA during replication: DNA gyrase is primarily
responsible for the initiation of DNA replication and elongation
of nascent DNA, while topoisomerase IV is primarily responsible
for decatenation of daughter chromosomal DNA at the end of rep-
lication.3 Multiple discrete binding sites on DNA gyrase have been
identified as the targets of synthetic and natural product inhibi-
tors.4 The best characterized inhibitor classes are the fluoroquino-
lone antibiotics5, which act as topoisomerase poisons by stabilizing
a covalent GyrA-DNA complex6, and the aminocoumarins, catalytic
inhibitors of DNA gyrase which act via competitive inhibition of
GyrB ATPase activity.7 The increasing prevalence of fluoroquino-
lone resistant strains and problems with side effects are beginning
to limit the utility of this widely utilized antibiotic class.8 Amino-
coumarin antibiotics, such as novobiocin, are effective at killing
Gram-positive bacteria, but were unsuccessful in the clinic. In
addition to issues with safety, their size, poor ligand efficiency,
and weak activity against ParE (i.e., inability to dual target) have
resulted in a rapid emergence of resistance.9 A number of struc-
ture-based design efforts have been effective in generating ligand
efficient small molecule inhibitors superior to novobiocin in po-
tency and dual targeting capabilities (reviewed in2), but to date,
no synthetic or natural product GyrB/ParE inhibitors have reached
the clinic. Thus, the GyrB/ParE ATP-binding subunits represent
unexploited targets for the development of novel antibiotics. This
manuscript and the following companion manuscript highlight a
novel pyrrolopyrimidine inhibitor series with broad spectrum anti-
⇑
Corresponding author. Tel.: +1 858 452 0370; fax: +1 858 677 9975.
E-mail address: ltari@triusrx.com (L.W. Tari).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.032

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L. W. Tari et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1529–1536
(b)
(c)
(a)
(d)
H
N
N
R2
R6
N
R5
R4
Figure 1. (a) Structure of the pyrrolopyrimidine scaffold, with potential analoging vectors labeled. (b) Structure of the initial fragment hit, 1. The compound is weakly active
against the E. faecalis GyrB enzyme (20% inhibition at 100
M). (c) Crystal structure of 1, bound to E. faecalis GyrB.10 The binding pocket surface is colored by conservation of
l
active-site residues in GyrB and ParE from the following pathogens: Staphylococcus aureus, Enterococcus faecalis, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella
catarrhalis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Burkholderia pseudomallei, Yersinia pestis and Francisella tularensis. Green
regions are identical, yellow conserved, and red non-conserved. (d) Cutaway view of the binding pocket surface (semi-transparent) shown in (c), colored by sequence
conservation. The potential hydrogen-bonds between the inhibitor and conserved adenine-binding aspartate and structural water molecule are shown. The R5 and R6
analoging vectors face the active-site pocket interior, while the R2 and R4 vectors are directed towards partially solvent exposed faces of the active-site pocket.
Scheme 1. Synthesis of key pyrrolopyrimidine intermediates 7 and 8. Reagents and conditions: (a) DBU, 90%; (b) HOCH2CH2OH, pTSA, 83%; (c) NAOEt, thiourea (for synthesis
of 5), 75%; (d) NaOEt, urea (for synthesis of 6), 70%; (e) 1 M HCl (treatment of 5 yields 7), 90%; (f) 1 M HCl; POCl3 (for synthesis of 8 from 6), 93%.
Scheme 2. Synthesis of compounds 10–14 and 18–24. Reagents and conditions: (a) 3-iodopyridine, trans-cyclohexane-1,2-diamine, CuI, NMP, 90%; (b) POCl3, 75%; (c) POCl3,
DMF, 80%; (d) acetyl chloride, aluminum trichloride, DCM, 32%; (e) (diethylamino)sulfur trifluoride, DCM, 25%; (f) NCS, NMP, 92%; (g) NBS, NMP, 89%; (h) 7-iodopyrido[2,3-
b]pyrazine, trans-cyclohexane-1,2-diamine, CuI, NMP, 93%; (i) BOP, HOBt, triethylamine, NMP, 87%; (j) NCS, NMP, 85%; (k) diamine, NMP, 70–95%.
bacterial activity, including robust Gram-negative pathogens. Opti-
mization of the series was guided by two main criteria: (i) Achieve-
ment of enzyme potency, broad enzymatic spectrum and balanced
dual target activity. (ii) Empirical modulation of charge distribu-
tion and physicochemical properties to generate inhibitors that
penetrate Gram-negative cell envelopes, avoid bacterial efflux
pumps and exert antibacterial activity via specific inhibition of
the topoisomerase targets. In this first manuscript, we describe
how structure guided inhibitor design was used to optimize a frag-
ment hit into a potent, dual targeting pyrrolopyrimidine series of
inhibitors active against GyrB and ParE with broad enzymatic
spectrum.
A fragment-based crystallographic screening effort was used to
identify prospective inhibitor scaffolds with adjacent hydrogen-
bond donor–acceptor moieties that engage the conserved ade-
nine-binding aspartate and structural water in the active-site
pocket.10 Based on an analysis of the binding modes of these scaf-
folds to Enterococcus faecalis GyrB, a pyrrolopyrimidine scaffold
was selected as a candidate for optimization—as shown in Figure 1,
it projects optimization vectors towards all the highly conserved
sub-pockets of the GyrB and ParE active-sites.
Schemes 1–4 outline the synthetic steps and key intermediates
used to generate all the compounds highlighted in this manuscript;
detailed synthetic procedures are described elsewhere.11 All R2

L. W. Tari et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1529–1536
1531
Scheme 3. Synthesis of compounds 41–55. Reagents and conditions: (a) K2CO3, NaIO4, water, 98%; (b) BOP, (R)-tert-butyl pyrrolidin-3-ylcarbamate, triethylamine, NMP, 91%;
(c) (i) PPh3, NMP; (ii) aryl iodide⁄, trans-cyclohexane-1,2-diamine, CuI, NMP, 67–93%; (d) NaOH, NMP/MeOH/water, 85–94%; (e) NCS, NMP, 56–92%; (f) TFA, DCM, 81–98%.
⁄The aryl iodides corresponding to the R2 groups in Table 3 were used. Acid moieties were used in form of their methyl or ethyl esters. ⁄⁄Only compounds containing ester
functionalities (47, 48 and 50) were subjected to the hydrolysis step.
Scheme 4. Synthesis of compound 61⁄. Reagents and conditions: (a) Et3N, (R)-N-(pyrrolidin-3-yl)acetamide, 98%; (b) K2CO3, pyridin-3-ol, NMP, 80%; (c) NaOH, MeOH, 86%;
(d) NCS, NMP, 90%. ⁄Compounds 62 and 63 were synthesized in an analogous fashion using N-(3-azabicyclo[3.1.0]hexan-6-yl)acetamide and pyrido[2,3-b]pyrazin-7-ol,
respectively.
Table 1
S-linked compounds were synthesized starting from the ethyl pyr-
rolopyrimidine intermediate 7, and two synthetic strategies were
R5 SAR
devised to facilitate analoging at key scaffold positions. To incorpo-
rate diverse diamines in the presence of a fixed R2 group (e.g.
18–24), a copper mediated coupling reaction between 7 and the se-
lected aryl iodide moiety was used to install the R2 group
(Scheme 2). The hydroxyl at R4 was then converted into the
activated OBt analog. After successful chlorination at R5 using N-
chlorosuccinimide, diamines were used to displace the OBt group
at the last step with high (70–95%) yields. To explore diversity at
R2 in the presence of a fixed R4 group (e.g. 41–55), a Boc-protected
diamine intermediate (25) was formed first. After cleavage of the
disulfide, the selected aryl iodide was coupled to R2 followed by
deprotection of the amine (Scheme 3). R2 O-linked inhibitors were
synthesized starting from the dichloro-pyrrolopyrimide intermedi-
ate 8 (Scheme 4). The R4 chlorine was selectively displaced by an
acetamide protected diamine followed by substitution of the R2
chlorine by a phenolic derivative of the desired R2 aryl moiety,
NaOH deprotection of the amine and chlorination at R5 using
N-chlorosuccinimide.
Compd/route R5
Ft GyrB
1.2
Ki
l
M
MIC lg/mL
Ef GyrB Ft ParE Ef ParE S. aureus
9/2
H
>50
0.8
2.0
1.7
0.4
0.3
>64
64
64
64
4
10/2
11/2
12/2
13/2
14/2
CHO
COCH3 6.6
CHF2
Cl
Br
8.7
>20
0.4
3.7
1.0
1.2
3.5
3.5
0.5
0.5
0.4
2.0a
1.2
2
Ft = F. tularensis, Ef = E. faecalis.
a
Data is versus A. baumannii ParE enzyme.
The inhibitor scaffold was optimized by building substituents
into three distinct regions of the active site pocket: (i) The lipo-
philic active-site interior, along the R5 and R6 vectors, (ii) the
salt-bridge pocket, along the R2 vector, and (iii) the residues and
ordered solvent network at the mouth of the lipophilic pocket,
along the R4 vector.
Filling the lipophilic pocket interior via installation of an ethyl
group at R6 and small substituent at R5 generated inhibitors like
10–14 in Table 1, with 2–3 log improved potencies compared with
1.12 Selection of R5 and R6 moieties was guided by careful analysis

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L. W. Tari et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1529–1536
Figure 2. (Left, top) Table comparing active-site interior pocket volumes for GyrB and ParE. All measurements were derived from crystal structures of inhibitor complexes
with the protein orthologs described in the table.13 The differences in pocket volumes are consistent with the sequence diversity observed in the GyrB and ParE ATP-binding
pockets and suggest the following general trend in interior pocket size: Gram-positive ParE >Gram-positive GyrB ꢀ Gram negative GyrB >Gram negative ParE. (Left, bottom)
Enzyme inhibition profile for compound 10a. (Right) Identical, semi-transparent views of the ATP-binding pockets of F. tularensis ParE and E. faecalis ParE from crystal
structures of the ATP-binding domains of both enzymes complexed with the R4 methoxy analog of 10 (10a).
of the active site interior using crystal structures of several GyrB
and ParE orthologs13, chosen to encompass maximal sequence
diversity in active site pocket residues from a structural bioinfor-
matics analysis.10 An ethyl group was found to be the optimal R6
substituent. As shown in Table 1 and Figure 2, the decreased pock-
et volume along the R5 vector of the scaffold in Gram-negative ParE
enzymes restricts the size of R5 substituents that are tolerated. For
example, an acetyl substituent at R5 was accommodated by the
pockets of the Gram-positive enzymes and Gram-negative GyrB,
but not by the smaller Gram-negative ParE interior pocket. The
Cl substituent at R5 was chosen for additional optimization work:
Cl substituted inhibitors demonstrated broad spectrum enzyme
potency that was well correlated with antimicrobial activity (first
in Gram-positive organisms) as inhibitor optimization proceeded.
When bound to GyrB or ParE, the R4 analoging vector (see Fig. 3)
resides at the interface between solvent and the lipophilic interior
pocket; one side of the inhibitor faces a polar anionic pocket and an
ordered solvent network, while the other side of the inhibitor oc-
cludes a lipophilic shelf (the ‘pocket floor’, residues 80 and 96 in
Fig. 3) that is sequence diverse between GyrB and ParE. To opti-
mize dual targeting many of the reported GyrB inhibitor discovery
efforts have utilized planar inhibitor scaffolds with flat, lipophilic
substituents in the region of the pocket occupied by the R4 substi-
tuent in our series.2 To improve the physicochemical profile of our
series and minimize the potential for serum binding and off-target
activity, R4 groups were chosen that avoid extensive interactions
with the pocket floor while engaging the conserved polar residues
and ordered solvent network on the opposite face of the inhibitor
scaffold. Optimal activity was obtained with diamine substituents,
including those shown in Figure 3 and Table 2. The diamines adopt
low energy conformations that do not impinge on the pocket floor
while extending basic amines out of the pyrrolopyrimidine plane
to interact via direct or water mediated hydrogen-bonds with a
conserved asparagine at the mouth of the interior pocket (N48 in
Fig. 3). The basic amine complements the negative electrostatic
potential of the active-site pocket in the vicinity of the conserved
asparagine, aspartate and glutamate residues shown in Figure 3;
the same anionic pocket engages the terminal amine from a con-
served lysine residue observed in the dimeric complex of Esche-
richia coli ParE with ADPNP.14 Additionally, the amines engage or
displace waters from an ordered solvent network above the pocket
floor. It is important to note that a similar constellation of ordered
solvent molecules was observed in apo-GyrB and ParE structures
Figure 3. View of the active-site pocket (rendered as a semi-transparent surface)
from the structure of E. faecalis GyrB complexed with 19.13 Potential hydrogen-
bonds are depicted as dotted lines. The relative positions of the conserved Asn and
acidic residues (48, 51 and 52, respectively, in the E. faecalis structure) are shown.
The R4 amine participates in a complex hydrogen-bonding network with N48 and
the ordered constellation of solvent molecules. The solvent network is conserved in
apo-enzyme structures. The amine does not impinge on the pocket floor (I80 and
V96), where sequence diversity is observed between GyrB and ParE enzymes.

L. W. Tari et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1529–1536
1533
Table 2
R4 diamine SAR
Compd./Route
R4
Ki nM
Ef GyrB
4
Ft GyrB
7
Ef ParE
Ft ParE
4
Ec ParE
18/2
19/2
65
39
11
2
<1
23
4
20/2
21/2
<1
<1
3
69
9
6
1
36
2
<1
22/2
6
<1
19
9
6
23/2
24/1
9
<1
<1
29
4
10
<1
17
<1
<1
Ef = E. faecalis, Ft = F. tularensis, Ec = E. coli.
from multiple protein orthologs solved in our laboratory (Bensen,
D. C. and Tari, L. W., unpublished results). Thus, the ordered solvent
network has been treated as a conserved structural element during
inhibitor optimization. Significant structural diversity is tolerated
in the diamine with good retention of dual targeting activity and
enzymatic spectrum; for most of the diamines tested, the Ki values
fall within a 10- to 20-fold range against an enzyme panel selected
to encompass maximal target diversity (Table 2).
To acheive enzyme potencies in the low-/sub-nanomolar range,
inhibitors were also designed to address the unique structural and
electronic features of the active-site pocket in the vicinity of the R2
moiety (Fig. 4), namely: (i) Groups capable of p-cation interactions
with the salt-bridge Arg that also electrostatically complement the
Arg-Glu salt-bridge. (ii) A correctly positioned hydrogen-bond
acceptor to interact directly or via a water molecule with a con-
served basic residue (generally an Arg) on the outer rim of the
salt-bridge pocket. (iii) Sufficient lipophilic character to interact
with the conserved proline residue that defines the face of the
salt-bridge pocket opposite the Glu-Arg pair.
As shown in Table 3, a number of diverse chemical groups at the
R2 position were installed that complement the salt-bridge pocket
features to generate inhibitors with broad enzymatic spectrum and
dual target activity. With a sulfur atom used as a linker between
the inhibitor scaffold and the R2 group, bicyclic heteroaryl R2
groups were shown to be superior to single rings at capturing
the key interactions in the salt-bridge pocket (e.g. compare the po-
tency of 41 to the other compounds in Table 3). Empirical explora-
tion of alternate charge distributions in several aromatic R2
moieties revealed, as expected, that enzyme potency is profoundly
influenced by the electronic complementarity between the Glu-Arg
salt-bridge residues and the R2 ring systems. For example, chang-
ing the R2 moiety from a quinoline (42) to a napthyridine (44) im-
proves inhibitor potency by 1–2 logs across all the enzymes
studied. In general, aromatic bicyclic R2 groups with a nitrogen
meta- to the S-linker were found to demonstrate superior potency
when compared to inhibitors lacking the heteroatom. Additionally,
Figure 4. Detailed view of the ‘salt-bridge’ pocket from the crystal structure of F.
tularensis ParE complexed with 51.13 The pocket is rendered as a semi-transparent
surface and the key residues comprising the pocket are highlighted. Potential
hydrogen-bonds are depicted as dotted lines. The most potent pyrrolopyrimidine
inhibitors utilize R2 moieties that engage the salt-bridge Arg via
p-cation
interactions, cover the lipophilic floor and hydrogen-bond with the basic residue
on the salt-bridge pocket rim.
a number of successful strategies were employed to capture direct
or water mediated hydrogen-bonds with the conserved Lys/Arg at
the salt-bridge pocket rim. The best potency was obtained with R2
groups that directly engage the basic residue with multiple hydro-
gen-bond acceptors, and/or charge assisted hydrogen-bonds, like

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L. W. Tari et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1529–1536
Table 3
R2 SAR
Compd./
R2
Ki nM
Route
Ef
Ft
Ef
Ft
Ec
GyrB
GyrB
ParE
ParE
ParE
19/2
2
<1
23
4
11
41/3
42/3
4
4
8
157
148
36
27
127
80
10
Figure 5. Close up view of the ‘salt-bridge’ pockets from the overlaid crystal
structures of F. tularensis ParE (gold ligand, tan protein) and E. faecalis GyrB (green
ligand, grey protein) complexed with 54.13 The proline residues in the two
structures adopt different conformations due to sequence differences in proximal
residues, resulting in a widening of the pocket in the ParE structure. The flexible
linker between the pyrrolopyrimidine scaffold and R2 group allows the R2 group to
adapt to the differences in the pocket structure without altering the overall binding
mode of the ligand.
43/3
<1
<1
15
3
7
44/3
45/3
<1
<1
<1
<1
2
7
<1
2
<1
5
46/3
47/3
48/3
<1
<1
<1
<1
<1
<1
5
1
4
6
5
2
7
4
19
O-linker: 123.1°
S-linker: 113.4°
49/3
50/3
51/3
52/3
<1
<1
7
<1
<1
9
7
<1
<1
2
2
6
<1
11
6
Figure 6. Comparison of the molecular conformations of 56 (green) and 62 (gold)
from overlaid crystal structures of their complexes with E. faecalis GyrB.13 The
molecular conformations differ most significantly in the vicinity of the R2 linker
atom, where the bond angle at the linker elbow is opened by almost 10° when
comparing the O-linker to the S-linker. The torsion angles around the bond between
the linker and pyrimidine also differ for the two bound inhibitors, with values of
À42° and À54° for 56 and 62, respectively. The bound conformation of 62 is
approximately 2 kcal/mol closer to its minimum energy conformation than 56.
19
24
6
5
4
43, 46–50 and 55. Because it is partially solvent exposed, the R2
vector is also an important site for incorporation of small groups
into the solvent space to improve the physicochemical properties
and antimicrobial activity of inhibitors without compromising po-
tency. Compounds 48 and 49 are examples of compounds that pro-
ject groups into the solvent space while retaining good enzymatic
potency.
The intervening linker atom between the pyrrolopyrimidine
scaffold and the R2 moiety was a key variable to explore during li-
gand optimization. The linker atom occupies an electron deficient
pocket in the active site, and controls the trajectory of the R2 group
53/3
<1
<1
14
2
4
54/3
55/3
<1
<1
1
13
<1
6
2
11
<1
<1
Ef = E. faecalis, Ft = F. tularensis, Ec = E. coli.

L. W. Tari et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1529–1536
1535
Table 4
S-linker versus O-linker SAR
Compd./Route
X
R2
R4
Ki nM
Ef GyrB
4
Ef GyrB
8
Ef ParE
Ft ParE
36
Ec ParE
41/3
56/3
57/3
61/4
62/4
63/4
S
157
79
10
84
10
3
127
36
1
S
5
2
15
2
S
1
<1
3
O
O
O
<1
<1
<1
15
2
38
4
<1
<1
<1
<1
Ef = E. faecalis, Ft = F. tularensis, Ec = E. coli.
with respect to the inhibitor scaffold. Additionally, it was discov-
ered during optimization that the presence of a flexible linker be-
tween the scaffold and R2 moiety was advantageous for balanced
dual target activity; receptor plasticity was observed in the vicinity
of the R2 pocket that is unique to the Gram-negative ParE enzymes.
As shown in Figure 5, the conserved proline residue comprising the
lipophilic pocket floor in the vicinity of R2 frequently adopts a un-
ique conformation in the structures of E. coli and Francisella tular-
ensis ParE. The proline residue in the Gram-negative ParE
enzymes tilts away from the salt-bridge residues, widening the
R2 pocket in the Gram-negative ParE enzymes as compared to its
counterparts in the GyrB and Gram-positive ParE enzymes. The
flexible linker allows the R2 group to adapt to the conformational
differences in the salt-bridge pocket without severely altering the
binding mode of the pyrrolopyrimidine scaffold. As described pre-
viously,10 we observed that inhibitor scaffolds with more conform-
ationally constrained linkages between the scaffold and salt-bridge
interacting group generally demonstrated inferior activity against
the Gram-negative ParE enzymes when compared to Gram-posi-
tive ParE and all GyrB enzymes.
Because of superior synthetic tractability, all early ligand opti-
mization and SAR analysis was conducted on inhibitors with thio-
ether R2 linkers. However, analysis of the geometries of the
thioether R2 linkages in the crystal structures described herein re-
veals that pyrrolopyrimidine inhibitors adopt moderately strained
conformations upon binding to GyrB or ParE. To alleviate steric
strain between the R2 moiety and the pyrrolopyrimidine scaffold,
the thioether bond angle opens by more than 10° from its ideal va-
lue (100°)15 to 111–114° in the bound conformation. Replacement
of the R2 sulfur linker by oxygen largely alleviates this strain; the
ether bond angles in the crystal structures of O-linked inhibitors
are close to their ideal values at 123–124° (Fig. 6).15 Using quan-
tum mechanical calculations,15 it is estimated that inhibitors with
a sulfur linker and aryl/heteroaryl moiety at R2 absorb approxi-
mately 2 kcal/mol of strain energy to adopt the bound conforma-
tion in the active site pocket, versus less than 0.2 kcal/mol for
inhibitors with an oxygen linker. A comparison of the potencies
of identically decorated S- and O-linked inhibitors (Table 4), sug-
gest that the relief of conformational strain with O-linkers contrib-
utes to improved potencies; the Ki values of 56 and 62 differ by
nearly an order of magnitude versus many of the enzymes in the
panel. The potencies of 62 against the enzymes in Table 4 rival
those of the more potent S-linked inhibitors in Table 3, despite
the fact that 62 utilizes a smaller R2 group.
In conclusion, structure based inhibitor design against multiple
protein orthologs has been used to discover and optimize a novel
pyrrolopyrimidine inhibitor series with exquisitely potent, broad
enzymatic spectrum and dual targeting activity. The series is
highly ligand efficient, and tolerates significant chemical diversity
at two analoging vectors without compromising enzyme potency
and spectrum. In addition, many substituents at the R2 and R4 posi-
tions possess solvent facing synthetic vectors that could allow for
the incorporation of small groups to alter the charge distribution
and physicochemical properties of the series. When taken together,
these features greatly improve the prospects for developing mole-
cules with antibacterial activity and good drug-like properties. The
antibacterial activity, spectrum and SAR surrounding this series
will be described in the following manuscript.
Acknowledgements
We gratefully acknowledge Dr. Edwin Westbrook and the staff
at beamline 4.2.2 at the Advanced Light Source for their assistance
with this project. We thank Livermore Computing for the computer
time. Part of this work was performed under the auspices of the
U.S. Department of Energy by Lawrence Livermore National Labo-
ratory under Contract DE-AC52-07NA27344, LLNL-JRNL-565619.
This project has been funded with Federal funds from the National
Institute of Allergy and Infectious Diseases National Institutes of
Health, Department of Health and Human Services, under Contract
No. HHSN272200800042C.

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L. W. Tari et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1529–1536
C-terminal 6ÂHis tagged N-terminal 43 kDa ATP-binding domain. Complexes
References and notes
with inhibitors were crystallized by sitting drop vapor diffusion, from 10 to
20 mg/mL protein solutions (in 20 mM Tris, pH 8.0, 100 mM NaCl) using the
following mother liquor solutions: E. faecalis GyrB, P212121 form; 20 °C, 25% (w/
v) PEG 1500, 3% (v/v) t-butanol, 20% (v/v) glycerol, 20 mM citrate pH 5.6. No
cryoprotection required. E. coli GyrB, P21 form: 4 °C, 30% (w/v) PEG 4000,
200 mM MgCl2, 100 mM Tris pH 8.0. Crystals cryoprotected with mother liquor
supplemented with 25% (v/v) ethylene glycol.E. coli ParE, P21 form: 20 °C, 5.6%
(w/v) PEG 4000, 30% glycerol, 70 mM acetate pH 4.6. no cryoprotection
required. F. tularensis ParE, P21 form: 4 °C, 10% (w/v) PEG 4000, 10% (v/v)
isopropanol, 100 mM citrate pH 5.4. Crystals cryoprotected with mother liquor
supplemented with 25% glycerol.E. faecalis ParE, P31 form: 20 °C, 20% PEG 3350,
200 mM ammonium phosphate. Crystals cryoprotected with mother liquor
supplemented with 25% ethylene glycol. With the exception of the E. coli
GyrB complex with N-(7-(1H-imidazol-1-yl)-2-(pyridin-3-yl)thiazolo[5,4-
d]pyrimidin-5-yl)cyclopropanecarboxamide (2.6 Å), and the E. faecalis and F.
tularensis ParE complexes with 10a (2.7 Å), all other crystals studied diffracted
to 2.2 Å or better resolution on a rotating anode or synchrotron radiation
source. Structures were solved by molecular replacement. Coordinates for all
crystal structures described in the text have been deposited in the Protein Data
Bank (http://www.pdb.org) under the following accession codes: E. coli ParE
with 7-(1H-imidazol-1-yl)-2-(pyridin-3-yl)thiazolo[5,4-d]pyrimidin-5-amine
(ligand not shown in text), 4HZ0. E. coli GyrB with N-(7-(1H-imidazol-1-yl)-
2-(pyridin-3-yl)thiazolo[5,4-d]pyrimidin-5-yl)cyclopropanecarboxamide
(ligand not shown in text), 4HYP. F. tularensis ParE with 10a, 51 and 54, 4HXZ,
4HY1 and 4HYM, respectively. E. faecalis ParE with 10a, 4HZ5. E. faecalis GyrB
with 19, 54, 56, and 62, 4HXW, 4GGL, 4GFN, and 4GEE, respectively.
14. Bellon, S.; Parsons, J. D.; Wei, Y.; Hayakawa, K.; Swenson, L. L.; Charifson, P. S.;
Lippke, J. A.; Aldape, R.; Gross, C. H. Antimicrob. Agents Chemother. 1856, 2004,
48.
1. (a) Walsh, C. Antibiotics: Actions, Origins, Resistance; American Society for
Microbiolgy (ASM) Press: Washington, DC, 2003; (b) Silver, L. L. Nat. Rev. Drug
Disc. 2007, 6, 41.
2. Oblak, M.; Kotnik, M.; Solmajer, T. Curr. Med. Chem. 2007, 14, 2033.
3. Sissi, C.; Palumbo, M. Cell. Mol. Life Sci. 2001, 2010, 67.
4. Collin, F. E.; Karkare, S.; Maxwell, A. Appl. Microbiol. Biotechnol. 2011, 92, 479.
5. Emmerson, A. M.; Jones, A. M. J. Antimicrob. Chemother. 2003, 51, 13.
6. Laponogov, I.; Sohi, M. K.; Veselkov, D. A.; McAuley, K. E.; Fisher, L. M.;
Sanderson, M. R. Nat. Struct. Mol. Biol. 2010, 16, 667.
7. Heide, L. Nat. Prod. Rep. 2009, 26, 1241.
8. (a) Hooper, D. C. Emerg. Infect. Dis. 2001, 7, 337; (b) Wolfson, J. S. Eur. J. Clin.
Microbiol. Infect. Dis. 1989, 8, 1080.
9. (a) Lambert, H. P.; O’Grady, F. W. In Coumarins; Lambert, H. P., O’Grady, F. W.,
Eds.; Churchill Livingstone: Edinburgh, 1992; pp 140–141; (b) Maxwell, A. Mol.
Microbiol. 1993, 9, 101.
10. Tari, L. W.; Bensen, D.; Trzoss, M.; Lam, T. et al. In: 51st Annual Meeting of the
Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago,
IL, September 16–20, 2011, F2–1872.
11. Creighton, C.; Tari, L.; Chen, Z.; Hilgers, M.; Lam, T.; Li, X.; Trzoss, M.; Zhang, J.;
Finn, J.; Bensen, D. Int. Patent Appl. Pub. WO/2011/032050, 2010.
12. GyrB and ParE activity was evaluated using the coupled spectrophotometric
Enzchek™ assay in which the enzyme-dependent release of inorganic
phosphate from ATP hydrolysis was measured. The assay comprises between
20 and 100 nM GyrB or ParE (active site concentrations) in 50
buffer (pH 7.6), 2 M MgCl2, 125 M NaCl, 0.2 M 7-methyl-6-thioguanosine,
1 U/ml purine nucleoside phosphorylase. The reaction is initiated by addition
of 3 M ATP and monitored at 360 nm for 30 min at 27 °C. Inhibitor potency is
determined by incubating the target enzyme in the presence of various
concentrations of inhibitor ranging between 1.5 nM and 50 M for 10 min
lM Tris–HCl
l
l
l
l
15. Ligand strain energy due to angular deformation of the linker atom angle was
calculated by taking the difference between the energy resulting from a partial
optimization of the bound crystal ligand geometry, holding only the linker
l
prior to addition of ATP substrate. The final concentration of DMSO is kept
constant at 2.5% (v/v). Enzyme activity in the presence of inhibitor is expressed
relative to the no-inhibitor control and Ki values determined using Morrison
tight-binding equation to account for ligand depletion. All analysis is carried
out using GraphPad Prism 4.0.
angle constant (at its bound value), and the energy from
a subsequent
optimization allowing the linker angle of the ligand to relax. DFT optimizations
using the B3LYP functional and 6–311+G⁄ basis set were used to calculate the
energies. Frequency calculations verified the energy minima of full
13. Crystallization of E. faecalis GyrB, E. faecalis ParE, E. coli GyrB, and E. coli ParE
was conducted using C-terminal 6xHis tagged versions of the N-terminal
27 kDa ATP-binding domains of each protein, expressed in E. coli BL21 Star
(DE3). F. tularensis ParE was expressed in the same manner, using the
optimizations. Calculations were performed using GAUSSIAN03
Wallingford, CT).
(GAUSSIAN, Inc,