Discovery of Pyrido[2,3- b]indole Derivatives with Gram-Negative Activity Targeting Both DNA Gyrase and Topoisomerase IV

Article abstract of DOI:10.1021/acs.jmedchem.0c00768

The rise of multidrug resistant (MDR) Gram-negative (GN) pathogens and the decline of available antibiotics that can effectively treat these severe infections are a major threat to modern medicine. Developing novel antibiotics against MDR GN pathogens is particularly difficult as compounds have to permeate the GN double membrane, which has very different physicochemical properties, and have to circumvent a plethora of resistance mechanisms such as multiple efflux pumps and target modifications. The bacterial type II topoisomerases DNA gyrase (GyrA2B2) and Topoisomerase IV (ParC2E2) are highly conserved targets across all bacterial species and validated in the clinic by the fluoroquinolones. Dual inhibitors targeting the ATPase domains (GyrB/ParE) of type II topoisomerases can overcome target-based fluoroquinolone resistance. However, few ATPase inhibitors are active against GN pathogens. In this study, we demonstrated a successful strategy to convert a 2-carboxamide substituted azaindole chemical scaffold with only Gram-positive (GP) activity into a novel series with also potent activity against a range of MDR GN pathogens. By systematically fine-tuning the many physicochemical properties, we identified lead compounds such as 17r with a balanced profile showing potent GN activity, high aqueous solubility, and desirable PK features. Moreover, we showed the bactericidal efficacy of 17r using a neutropenic mouse thigh infection model.

Full text of DOI:10.1021/acs.jmedchem.0c00768

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Article
Discovery of Pyrido[2,3-b]indole Derivatives with Gram-negative
Activity Targeting Both DNA Gyrase and Topoisomerase IV
Yimin Hu, Houguang Shi, Mingwei Zhou, Qingcheng Ren, Wei Zhu, Weixing Zhang, Zhiwei Zhang,
Chengang Zhou, Yongqiang Liu, Xiao Ding, Hong C Shen, S. Frank Yan, Fabian Dey, Waikwong
Wu, Guanglei Zhai, Zheng Zhou, Zhiheng Xu, Ying Ji, Hua Lv, Tianyi Jiang, Wen Wang, Yunhua
Xu, Maarten Vercruysse, Xiangyu Yao, Yi Mao, Xiaomin Yu, Kenneth Bradley, and Xuefei Tan
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00768 • Publication Date (Web): 04 Aug 2020
Downloaded from pubs.acs.org on August 4, 2020
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Discovery of Pyrido[2,3-b]indole Derivatives with
Gram-negative Activity Targeting Both DNA
Gyrase and Topoisomerase IV
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†
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‡
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Yimin Hu, Houguang Shi, Mingwei Zhou, Qingcheng Ren, Wei Zhu, Weixing Zhang, Zhiwei
†
†
†
†
†
ǁ
&
Zhang, Chengang Zhou, Yongqiang Liu, Xiao Ding, Hong C. Shen, S. Frank Yan, Fabian Dey,
ǂ
ǂ
ǂ
ǂ
Δ
§
§
Waikwong Wu, Guanglei Zhai, Zheng Zhou, Zhiheng Xu, Ying Ji, Hua Lv, Tianyi Jiang, Wen
#
#

¶
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¶

Wang, Yunhua Xu, Maarten Vercruysse, Xiangyu Yao, Yi Mao, Xiaomin Yu, Kenneth Bradley,
and Xuefei Tan*, †
†
ǁ
ǂ
Δ
§
Department of Medicinal Chemistry, CADD, Lead Discovery, pCMC, Pharmaceutical Sciences,
¶Discovery Infectious Diseases, Roche Innovation Center Shanghai,Roche Pharma Research and Early
Development, Building 5, 720 Cailun Road, Shanghai, 201203 China,
&

Department of CADD, Discovery Infectious Diseases, Roche Innovation Center Basel, Roche
Pharma Research and Early Development, Grenzacherstrasse 124, CH-4070 Basel, Switzerland
#MicuRx Pharmaceuticals, Inc. (Shanghai)
Floor 3, Building B, 1976 Middle Gaoke Road, Shanghai, 201210 China
‡WuXi AppTec (Wuhan) Co., Ltd.
No. 666 Gaoxin Road, Wuhan East Lake High-tech Development Zone, Hubei, 430075 China
Abstract: The rise of multi-drug resistant (MDR) Gram-negative (GN) pathogens and the decline of
available antibiotics that can effectively treat these severe infections is a major threat to modern
medicine. Developing novel antibiotics against MDR GN is particularly difficult as compounds have to
permeate the GN double membrane, which has very different physicochemical properties, and have to
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circumvent a plethora of resistance mechanisms such as multiple efflux pumps and target modifications.
The bacterial type II topoisomerases DNA gyrase (GyrA B ) and Topoisomerase IV (ParC E ) are
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2 2
highly conserved targets across all bacterial species and validated in the clinic by the fluoroquinolones.
Dual inhibitors targeting the ATPase domains (GyrB/ParE) of type II topoisomerases can overcome
target-based fluoroquinolone resistance. However, few ATPase inhibitors are active against GN
pathogens. In this study, we demonstrated a successful strategy to convert a 2-carboxamide substituted
azaindole chemical scaffold with only Gram-positive (GP) activity into a novel series with also potent
activity against a range of MDR GN pathogens. By systematically fine-tuning the many
physicochemical properties, we identified lead compounds such as 17r with a balanced profile showing
potent GN activity, high aqueous solubility, and desirable PK features. Moreover, we showed the
bactericidal efficacy of 17r using a neutropenic mouse thigh infection model.
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Keywords: Gyrase, Topoisomerase, Gram-negative, Pyrido[2,3-b]indole
Introduction
The rapid increase in bacterial resistance against all antibiotics in the clinic is an urgent global
health problem. The most severe bacterial infections are increasingly escaping first-line and even last-
resort antibiotics treatments. These life-threatening multi-drug resistant (MDR) infections are mainly
caused by the so-called ESKAPE pathogens, i.e., Enterococcus faecium, Staphylococcus aureus,
Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter
species.1,2 The last four fall into the category of Gram-negative (GN) bacteria, which are particularly
challenging to treat, as compounds with intracellular targets have to pass through both the negatively
charged outer membrane and hydrophobic inner membrane.3,4 The distinct physicochemical properties
required of a compound to cross both barriers make targeting GN bacteria more challenging than Gram-
positive (GP) bacteria, which lack an outer membrane. Also, many adaptive resistance mechanisms
developed by the bacteria such as efflux pumps, inactivation of enzymes, mutation, or biofilm further
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complicate such a challenge. As a result, no new class of antibiotic with GN activity has reached the
market in the last 40 years.5-7
The bacterial type IIA topoisomerases, i.e., DNA gyrase and topoisomerase IV, are well-known
antibacterial targets. These essential enzymes solve critical DNA-topology changes during replication,
transcription, and recombination. Specifically, DNA gyrase introduces negative supercoils into DNA
and relaxes positive supercoils that accumulate during replication and transcription. Topoisomerase IV
also removes positive supercoils as well as decatenates the replicated sister chromosomes. DNA gyrase
is a heterotetramer composed of two A (GyrA) and two B subunits (GyrB). The two GyrB subunits
hydrolyze two ATP molecules, which drive the catalytic function, i.e., binding, cleavage, and relegation
of the DNA segments. Similarly, Topoisomerase IV consists of two C subunits (ParC) and two E
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subunits (ParE). The ParE subunits hydrolyze ATP driving the enzymatic activity. Dual targeting of
both GyrB and ParE subunits is possible as both ATP binding sites are very similar and results in very
low spontaneous resistance frequencies. Also, because both enzymes are highly conserved across GP
and GN bacteria, a dual GyrB/ParE ATPase inhibitor can have broad-spectrum activity.9
Fluoroquinolones (FQ) are one of the most commonly prescribed broad-spectrum antibiotics
that target both DNA gyrase and Topoisomerase IV. However, due to its overuse and misuse for decades
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FQ resistance in hospitals has increased significantly. The resistance mechanisms include both target-
based mutations and non-target based such as plasmid-mediated resistance and efflux, which are
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difficult to overcome even by the newer generation FQ in the clinic. FQ antibiotics target the catalytic
sites in the GyrA/ParC subunits and stabilize the cleaved DNA-enzyme complex, which results in lethal
double-stranded DNA breaks. Targeting alternative binding sites such as GyrB/ParE ATPase domains,
which is distinct from the FQ mode-of-action (MoA), enables new antibacterial agents to overcome
target-based FQ resistance and to achieve broad-spectrum activity. Novobiocin previously established
the clinical proof of concept of this new MoA in the 1950s. It was, however, the first and only marketed
drug of this new class. The use of Novobiocin declined rapidly due to the later development of more
effective antibiotics. It was eventually withdrawn from the market due to the rise of resistance
development and potential safety concerns.12
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Over the last fifty years, industry and academia continued to work on finding novel scaffolds
targeting the GyrB/ParE ATP binding site. Along with the continuously improved technical
understanding of the molecular details of the MoA and factors driving resistance development against
antibiotics, numerous chemical scaffolds were identified showing gradual improvements in
physicochemical properties, safety, and antibacterial coverage compared to Novabiocin.13-15 However,
the spectrum of these GyrB/ParE dual inhibitors has been mainly limited to a GP spectrum, with only
a few common respiratory GN pathogens such as Haemophilus influenzae.
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A breakthrough scaffold targeting the GyrB/ParE ATPase site having GN activity was
discovered eventually by Trius Therapeutics. Their novel tricyclic pyrimido[4,5-b]indole series, derived
from a pyrrolopyrimidine scaffold, showed for the first time antibacterial activity effectively covering
also GN pathogens such as P. aeruginosa, A. baumannii, and K. pneumoniae (represented by GP6 in
Figure 1).16-18 The enhanced GN activity of this series is attributed to its rigidity, orientation of a critical
amino group, and ability to adopt both neutral and charged states, all of which helps to penetrate the
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GN double membranes. This series showed dual activity against both GyrB and ParE ATP binding
site, resulting in very low resistance frequencies. Additionally, Soo Yei Ho et al. recently showed that
a GyrB/ParE scaffold with only GP activity could be modified to yield broad-spectrum activity by
carefully modulating the physicochemical property of zwitterionic compounds (represented by ETC44
in Figure 1).19
In this study, we report our efforts in discovering pyrido[2,3-b]indole derivatives as a novel
class of dual GyrB/ParE inhibitors with promising GN bacteria activities and in vivo bactericidal
efficacy in a neutropenic mouse thigh infection model.
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H
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H
N
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F
N
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H
CF3
NH2
Trius GP6
GyrB Ki <1 nM ParE Ki <1 nM
MICs E. coli ATCC25922 0.344 g/mL
K. pneumoniae ATCC 700603 1.358 g/mL
A. baumannii ATCC 19606 0.678 g/mL
P. aeruginosa ATCC 27853 0.679 g/mL
ETC44
GyrB IC50 2 nM ParE IC50 3 nM
MICs E. coli 0.57 g/mL
K. pneumoniae ARC 1865 16 g/mL
A. baumannii ARC3495 0.5 g/mL
P. aeruginosa PAO1 1.14 g/mL
Figure 1. A few GyrB/ParE dual-inhibitor scaffolds. a) AZ GN-positive only azaindole ureas, AZ10
and AZ17; b) Trius’ GN-active tricyclic pyrimido[4,5-b]indole, GP6; and c) ETC’s GN-active
zwitterionic pyridine urea, ETC44.
RESULTS AND DISCUSSIONS
It has become increasingly apparent that in-house compound libraries of pharmaceutical
companies are not well suited for the identification of antibacterial starting points due to their different
physicochemical properties compared to most marketed antibiotics.20,21 Therefore, we decided not to
rely on a high-throughput screening campaign but to utilize the rich published collection of various
potent DNA Gyrase/Topoisomerase IV GP antibiotics as medicinal chemistry starting points for the
development of antibiotics against GN bacteria. A literature survey identified an azaindole scaffold
22
(
represented by AZ10 and AZ17 in Figure 1) previously discovered by AstraZeneca (AZ). This class
of compounds possesses a balanced dual enzymatic potency against both GyrB and ParE enzymes in
GP bacteria. This feature could also be retained when improving the antibacterial activity against GN
bacterial pathogens. Meanwhile, the overlay of the ATP binding sites of ParE crystalized with azaindole
AZ10 (PDB code: 4EMV) active against GP bacteria (S. pneumoniae), and GyrB crystalized with
tricyclic pyrimido[4,5-b]indole GP1 (PDB code: 4KFG) active against GN bacteria (E. coli) showed a
good structural alignment of the key ligand pharmacophores (Figure 2). Notably, first, in both cores,
the bi-dentate hydrogen bond interaction in donor-donor-acceptor pattern with strongly conserved
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aspartic acid (Asp78 in S. pneumoniae ParE, Asp73 in E. coli GyrB) and a tightly bound water molecule
were present. Comparing the scaffolds in the alignment (Figure 2) reveals a small shift. This shift is
likely due to steric reasons of the substitution at 2-position in GP1, pushing the compound further away
from the Glu-Arg salt bridge. Nonetheless, the AZ10 azaindole 5-position pyridine ring overlaps well
with the pyrimidine ring of GP1, both forming cation- stacking interactions with the neighboring
arginine side chain (Arg 76 in E. coli GyrB and Arg 81 in S. pneumoniae ParE, respectively). While the
meta carboxylate substituted R5’-pyridine of AZ10 forms a direct, GP1 engages in a water-mediated
H-bond to Arg 136. A more pronounced difference can be observed in the more hydrophobic part of
the pocket where the 2-carboxamide of the azaindole occupies the latter only partially compared to the
GP1 tricyclic core. Lastly, the substitution along the R4-vector, the position held by an amine-
containing unsaturated ring disclosed by Trius, is filled by a substituted pyrazole. While the latter mostly
engages with the floor of the pocket, the former also forms an H-bond with Asn 46.
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Figure 2. Overlay of X-ray crystal structures of ATP binding sites of GyrB with bound GP1 (PDB
code: 4KFG, protein shown with green ribbon and carbons, ligand with cyan carbons) and ParE with
bound AZ10 (PDB code: 4EMV, protein shown with grey ribbon and carbons, ligand with magenta
carbons). Hydrogen bonding interactions as red lines and residue labels shown for structure 4KFG.
Further residues and water molecules omitted for clarity.
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We decided to first introduce further rigidity into the azaindole scaffold, a strategy that had
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been shown in several cases to be successful in obtaining anti-GN activity. After a few attempts, we
were successful in replacing the latter by a fused ethyl aniline, which not only lowered the strain in the
high energy bioactive conformation (H-bond donors facing each other) of the azaindole analog
(
AZ10)22 but also generated a rigid scaffold that could still maintain the same hydrogen-bonding
network with the protein and the structural water (Figure 3). As such, a new type of tricyclic pyrido[2,3-
b]indole analogs exemplified by 10a-d were synthesized (Table 1). A 6-fluorine substitution was
incorporated in the tricyclic core based on modeling, to optimally fill the hydrophobic pocket and form
additional van der Waals interactions.
Asp78
Asp78
H O
2
H O
2
H
N
H
H
N
H
N
N
N
N
Cylization and replacement
with fused ethyl aniline
N
'
'
3
R5
R5
O
4
1
) Led to a more rigid scaffold;
2) Release the strain energy of
high energy conformer of
6
F
N
N
N
N
N
2
-carboxamide substituted
CF3
CF3
azaindole scaffold
GP-positive only scaffold
GN-active series
Figure 3. Design of novel tricyclic pyrido[2,3-b]indole scaffold.
To our delight, these analogs showed superior enzymatic potency in both GyrB and ParE
biochemical assays (10b-d, GyrB Ki < 1 nM, ParE Ki < 5 nM). The carboxylic acid analog 10a is the
least active compound with GyrB Ki = 45 nM and ParE Ki = 118 nM, respectively. Furthermore, despite
the high lipophilicity (predicted LogD > 3), these analogs were not only potent against GP bacteria
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(
represented by S. aureus ATCC 29213, MIC < 0.25 g/mL), but also considerably active against GN
bacteria such as E. coli ATCC 25922 (10b-d, MICs ~ 0.5 g/mL). However, we noticed that a hybrid
molecule composed of an R4 non-aryl ring substitution, such as 3-amino proline analog was completely
’
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inactive (data not shown). Both pyridine 5 -carboxamide 10b and 5’-nitrile analog 10d also
demonstrated a good potential for broad-spectrum antibacterial coverage as indicated in Table 2, and
to our delight, displayed activity against GN MDR strains. In a small panel of selected GN bacteria
intended for an initial assessment of GN bacteria coverage, compounds 10b and 10d showed MICs < 6

g/mL, with consistently better potencies against E. coli and A. baumannii pathogens, but less active
against K. pneumoniae and P. aeruginosa species. Except for A. baumannii ATCC 51432 (10d only),
there was no significant potency shift between the GN bacteria wild-type and MDR stains indicating
structural features and physicochemical properties favorable for GN double membrane permeation and
devoid of strong efflux. Encouraged by these findings, we then explored the structure-activity
relationship (SAR) of substitution at the 3-position of the pyrido[2,3-b]indole scaffold (Table 1).
Based on the analysis of our new analogs and the X-ray co-crystal structure of AZ10 (PDB
code: 4EMV), it was reasonable to assume that the R3-pyridine ring formed cation- stacking
interactions with Arg 81 (Arg 76 in E. coli GyrB), and a suitable substitution on the 5’-position of the
pyridine ring could potentially interact with the adjacent amino acid Arg 140 (Arg 136 in E. coli GyrB).
Both amino acids had been reported before as being critical for good enzymatic activity. Thus,
additional substituents capable of forming optimal hydrogen-bonding interaction with Arg 81 and/or
Arg 140 were evaluated. Table 1 shows binding affinities of these analogs against both GyrB and ParE
enzymes as well as their antibacterial activities in both GP bacteria (S. aureus ATCC 29213) and GN
bacteria (e.g., E. coli ATCC 25922). The majority of substitutions were well tolerated, as indicated by
their potent Ki values of either GyrB or ParE. The least potent compound 10k (GyrB Ki = 53 nM and
ParE Ki = 239.5 nM) still showed good GP activity (MIC = 0.5 g/mL) in S. aureus ATCC 29213 and
weak GN activity (MIC = 8 g/mL) in E. coli ATCC 25922. Similar to the carboxylic acid analog 10a,
strongly acidic and basic compounds like 10g, 10k, 10n, and 10p were much less active in E. coli ATCC
2
5922 (MICs ≥ 8 g/mL). Interestingly, the basic analog 10l (pKa = 8.62) was the exception with E.
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1
1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
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3
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3
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5
6
coli ATCC 25922 MIC = 1 g/mL. A potential reason for the latter could be related to the strong
2
4
compound accumulation propensity that free amino groups can induce. Compared with 10b and 10d,
more recent analogs 10h, 10i, and 10t demonstrated 2-fold improvement of potency against E. coli
ATCC 25922 (MICs = 0.25 g/mL). Additionally, the R3 group optimization led to not only new
compounds with improved anti-GN bacterial activity in E. coli ATCC 25922 but also an improved GN
bacteria coverage (Table 2, pyrimidine 10h and pyrazolo[1,5-a]pyrimidine 10t, MICs ≤ 2 g/mL).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. Enzymatic (Ki) and antibacterial potency (MIC) and physicochemical property of pyrido[2,3-
b]indole derivatives, 10a-u.
H
N
H
N
N
N
R3
N
F
CF3
MICb
S. aureus
ATCC
MICb
E. coli
ATCC
25922
GyrB Kia
ParE Kia
pKac
mlogD7.4d
Cmpd
R3
(nM)
(nM)
2
9213
(g/mL)
(g/mL)
O
O H
10a
10b
10c
45
1.3
<1
<1
118
<1
1
>16
0.5
0.5
0.5
3.57
1.9
N
N
N
O
NH2
<0.016
<0.25
3.25
3.42
3.63
O
N
H
4.3
<1
CN
10d
10e
<0.016
N
O
O H
N
H
N
nd
nd
nd
nd
<0.125
<0.125
2
8.15e
3.05
3.41
O
O
N
H
1
0f
N
0.5
NH
N
H
10.2e,f
1
0g
3.71
<1
0.5
8
2.28
N
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N
10h
< 1
<1
< 1
32
<0.016
<0.016
<0.25
0.5
0.25
0.25
1
3.48
3.44
3.64
2.8
N
NH2
1
0i
4.84f
N
O
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0j
1.13
53
68.6
239.5
22.4
11.2
N
NH
N
8.85e,f
1
0k
8
N
NH2
8.62f
1
0l
7.1
<1
<0.25
<0.016
1
2.94
3.56
N
O H
1
0m
0.5
N
O H
1
0n
N
N
N
N
O
nd
nd
nd
nd
0.25
>8
1
3.51
4.78
2.02
3.1
NH2
10o
O
<0.125
0.444
N
N
1
1
0p
0q
HN
N
nd
nd
>11.4
0.71
0.73
2.83
0.25
1.93
3.66
3.38
3.62
3.54
2.54
N
O
nd
nd
<0.056
<0.056
<0.057
0.031
N
NH
O
O
7.59e
1
0r
N
nd
nd
N
9.85ef
1
0s
N
HN
nd
nd
N
N
10t
0u
N
1.23
71
4.74
31
O
O
O H
1
N
N
<0.125
1
5.44e
a
b
c
Ki: inhibition constant; MIC: minimum inhibitory concentration; pKa: acid dissociation constant,
d
measured in a photometric titration assay; mlogD : machine learning prediction of intrinsic
7.4
distribution coefficient between octanol and aqueous buffer (pH 7.4) based on in-house training set
data, which measured in a CAMDIS assay;25 epredicted from MoKa (version 2); ionization constant
of the conjugate acid
f
Table 2. Antibacterial GN broad-spectrum coverage of selected compounds 10, 15, and 17.
Cmpd
MICa
MIC
MIC
MIC
1
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E. coli strains
(g/mL)
ATCC
K. pneumoniae strains
(g/mL)
A. baumannii strains
P. aeruginosa strains
(g/mL)
(g/mL)
ATCC
ATCC
BAA-
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
19606b
ATCC
51432b
PAO-1b
ATCC
27853b
2
5922b
35218b
BAA-
BAA-
BAA-
1
0031b
700603b
340c
2146c
747c
2113c
2
1
1
1
0b
0d
0h
0.5
0.5
0.76
1.45
0.34
2.85
1.47
0.75
1
0.76
2.91
1.38
2.85
1.47
0.75
2
0.09
0.73
0.09
0.76
1.45
0.69
1.42
1.47
0.75
4
3.02
5.82
1.38
5.7
0.76
0.73
0.17
1.42
073
0.38
1
3.02
>23.3
1.38
5.7
3.02
1.45
0.69
2.85
>23.5
0.75
0.5
2
2
0.76
5.82
1.38
2.85
>23.5
1.5
0.76
1.45
0.34
1.42
1.47
0.75
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0.25
0.25
0.5
2
1
0i
0.178
0.183
0.38
2
1
0m
>23.5
1.5
>23.5
1.5
4
1
0t
5r
7r
0.25
2
1
<0.25
0.31
<0.03
<0.02
0.09
8
1
2
na
1
3.13
5.56
12.5
2.78
6.26
22.2
b
12.5
22.2
0.78
5.56
0.78
5.56
0.39
2.78
1.3
10
1.56
11.1
1.56
11.1
1
7x
0.81
a
MIC: minimum inhibitory concentration; representative GN wide-type bacteria strains;
crepresentative GN MDR bacterial strain.
Among these newly identified R3 modifications, further modification on the pyrimidine ring of
1
0h seemed attractive. The 2’-position of the pyrimidine was envisioned as a prominent site for
derivatization because this vector is pointing toward the solvent-exposed area, therefore presumably,
various groups could be accommodated without a loss of good inhibitory activity. Also, variation in
this region was highly feasible due to many building blocks commercially available. Table 3 lists the
SAR of a selection of 2’-position modified R3-pyrimidine analogs. As expected, the majority of
compounds maintained strong binding affinities to both enzymes, with ParE binding activity more
strongly affected by these modifications. A protein overlay between GyrB and ParE, however, did not
provide any reason for the observed potency difference. Except for compound 12g, all analogs had
potent GP activity in S. aureus ATCC 29213 (MIC ≤ 0.25 g/mL), likely due to their favorable inner
membrane permeability. In terms of GN activity against E. coli ATCC 25922, for the neutral analogs,
except 12k, analogs 12a-d, 12h-j, and 12v all showed comparable antibiotic potency to compound 10h.
Additionally, introduction of small polar groups such as alcohols, amides, or aniline amino groups,
likely improved the analogs’ ability to better penetrate outer membranes of E. coli ATCC 25922 as
indicated by the smaller potency shift from GP to GN stains (1~4-fold compared to 16-fold potency
shift for analog 10h between S. aureus ATCC 29213 and E. coli ATCC 25922). The comparable
antibacterial potency was also maintained for the two weakly basic compounds 12p and 12q in E. coli
1
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ATCC 25922. Concerning charged molecules, the basic secondary amino compounds (12l-n and 12r-
u) had weaker anti-GN activity, which could be due to poor penetration through the inner membrane,
as indicated by the decreased S. aureus ATCC 29213 potency when compared to compound 10h. The
GN activity varied for the few acid analogs (12e-g, 12o, and 12w). As these acids had similar pKa
values and identical GP activity against S. aureus ATCC 29213, it is reasonable to assume the particular
charged form of these compounds could have different GN outer membrane permeability profiles.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compound 10h suffered from high microsomal instability. Although the 2’-positon of
pyrimidine is not the predicted site of metabolism from Metasite (version 6.0.1), to our surprise, the
majority of new analogs listed in Table 3 yielded improved microsomal stability. Among those, three
analogs 12j, 12p, and 12v achieved relatively high metabolic stability in mouse microsomal assay.
Due to the high lipophilicity (predicted LogD 3.48), compound 10h also had very low aqueous
solubility (LYSA < 0.2 g/mL). For the newly synthesized analogs (12e, 12f, 12s, 12t, and 12w), all of
which contained a charged group, marginal to good improvement of solubility profiles (LYSA 8~133
g/mL) were observed. The acidic analog 12f displayed better solubility with an LYSA of 67 g/mL,
and it also exhibited potent anti-GN activity with E. coli ATCC 25922 MIC = 0.375 g/mL.
Table 3. Pyrido[2,3-b]indole GyrB/ParE dual inhibitors, 10h and 12a-x.
H
N
H
N
N
N
3
N
N
N
R2'
F
CF3
MICb
S.
GyrB ParE
Kia Kia
(nM) (nM)
MICb
E.coli
ATCC
LMc
LYSAd
pKae
Cmpd
R2’
aureus
ATCC
(mL/min/mg) (g/mL)
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
9213
25922
(g/mL) (g/mL)
1
0h
2a
-H
<1
<1
<1
<0.016
<0.016
0.25
0.5
76.1
52.8
<0.2
<0.2
1
-CH3
11.3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O H
1
2b(S)
<1
<1
<0.25
0.5
1
2c(R)
12d
<1
<1
1.1
<1
<1
<1
nd
<1
9.7
2.6
21.1
<1
<0.25
<0.25
<0.016
<0.016
4
0.5
1
57.2
58.7
44.0
43.8
<1
<1
9
O H
O H
CO H
2
1
2e
2f
2g
2h
1
3.83
3.8
CO2H
O H
1
0.375
16
67
1
3.90
O
NH2
1
<1
<0.25
<0.25
<0.25
<0.25
55.5
<0.2
O
NHCH3
12i
12j
nd
O
-OCH3
<1
<1
29.1
40.4
<0.016
<0.016
0.5
2
20.2
37.0
<1
1
2k
2l
2m
-OCH CH
<0.3
2
3
NH
1
1
62.1
2.3
0.063
0.063
1
1
8.62f
O
H
N
9.06 f
1
<1
62.2
<1
O
NH
1
2n
2.6
<1
3
0.063
1
2
9.34 f
4.71
O
CO2H
O
12o
12p
5.6
<0.016
43.5
<1
-NH2
<1
<1
<1
0.063
1
25.9
53.1
7.6
<1
4.39 f
1
2q
-NHCH3
7.0
<0.016
0.25
2.78f,g
NH
12r
<1
7.8
<0.25
4
9.35f
N
H
H
N
12s
12t
8.4
<1
<1
22
<1
8.7
0.25
0.063
0.031
2
1
1
60.1
57.9
61.4
8.3
7.6
<1
9.55 f
9.52 f
8.6 f
N
H
NH
N
H
N
1
2u
NH
1
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N
12v
<1
49.3
<0.016
1
24.2
22.9
<1
O
CO2H
12w
N
H
<1
2
2.9
24
<0.016
<0.016
4
133
148
5.25
3.8
CO2H
1
2xh
0.75
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
b
c
Ki: inhibition constant; MIC: minimum inhibitory concentration; LM: scaled intrinsic clearance of
d
compound in mouse liver microsomes; LYSA: solubility determined by lyophilization solubility assay
e
f
g
(
LYSA) at pH 6.5; predicted from MoKa (version 2); ionization constant of conjugate acid; pKa: acid
h
dissociation constant, measured in a photometric titration assay; 12x is a methyl aniline analog of
compound 12f (also see Figure 5).
Figure 4. X-ray structure of P. aeruginosa GyrB in complex with compound 12o (PDB code: 6M1S).
Key protein residues shown as sticks with grey and ligand with magenta carbons. All water molecules
1
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2
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5
5
5
5
6
except for structural water are hidden for clarity. Interactions are shown as color-coded dashed lines
(H-bonds in red, vdW in yellow, cation-π stacking in blue and salt bridge in light pink).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
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0
1
2
3
4
5
6
7
8
9
0
The X-ray co-crystal structure of compound 12o (PDB code: 6M1S) in complex with P.
aeruginosa GyrB protein is shown in Figure 4. As expected, there is a strong hydrogen-bonding
network formed between the ligand and amino acid Asp 75 and the structural water molecule. The
tricyclic pyrido[2,3-b]indole scaffold adopts a hydrogen-bond donor-donor-acceptor pattern that is the
same as that seen for the Trius tricyclic scaffold. Compared to the original azaindole scaffold, there is
a minor dihedral angle change between the tricyclic ring and the R3-pyridine. The cation- stacking
interaction between Arg 78 and the pyridine ring was maintained. Meanwhile, the 2’-oxypropanoic
acid-containing substituent formed a salt bridge interaction with Arg 78. Albeit the inherent flexibility
of the latter substituent, it is well defined in the electron density, which is partially stabilized by the
adjoining molecule of the next asymmetric unit (not shown). As mentioned before, both Arg 78 and
Arg 138 are important for good enzymatic activity. Whereas no direct interaction with Arg 138 was
observed, there is room for a water molecule to form a bridged H-bond interaction between the latter
and one of the pyrimidine nitrogens similar to the GP1 structure. In addition, an H-bond of the polarized
C4’ pyrimidine hydrogen to the backbone carbonyl oxygen of Gly 79 is observed. Due to the steric
demand of the phenyl ring of the pyrido[2,3-b]indole and the R3 pyrimidine substituent, the R4 3’-CF3
o
o
pyrazole substitution adopted a marginally bigger out of plane twist (58 C in 12o vs. 53.1 C in AZ10).
The 3’-CF group sits on top of the hydrophobic groove formed by amino acids Pro 81 and Ile 80 and
3
9
6. The 4’ and 5’-position of the pyrazole ring point toward a solvent-exposed area opposite which two
conserved amino acids (Asn 48 and Asp 51, omitted for clarity, see Figure 5) are located. These
residues can be utilized to improve binding affinity further. Overlay with the Trius X-ray co-crystal
structure (GP1, PDB code: 4KFG) revealed a shift of the scaffold partially due to the ethyl vs. methyl
difference. The limited room available in the back of the pocket results in the former being pushing
slightly towards the opening of the pocket, i.e., along the Asp 75 acceptor and water donor while
maintaining these key interactions. However, once a methyl aniline analog such as 12x was used, the
1
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two tricyclic cores overlaid more closely (Figure 5). Nonetheless, a small shift of the cores is still
apparent, which is - as discussed for the AZ analog before - due to the change from the R2- to the R3-
position substitution. The latter yielding shorter interaction distances for the H-bonds due to tighter
packing of the core with the protein. Conversely, the Trius compound has a closer - stacking to Arg
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
8 (76 in E. coli GyrB) than the R3-pyrimidine ring and trying to replace R4-pyrazole with non-aryl
groups such as Trius R4 proprietary amines failed to give better antibacterial potency indicating
different SAR in this region. Nonetheless, it was apparent that one could take advantage of two
additional conserved amino acids Asn 48 and Asp 51 for R4 group optimization.
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5
6
Figure 5. X-ray co-crystal structures overlay between GP1 (PDB code: 4KFG, protein shown with
green ribbon and carbons, ligand with cyan carbons) and compound 12x (PDB code: 6M1J, protein
shown with grey ribbon and carbons, ligand with magenta carbons). Further residues and water
molecules omitted for clarity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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8
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0
1
2
3
4
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8
9
0
1
2
3
4
5
6
7
8
9
0
Therefore, having explored the R3 area and its impact on physicochemical properties, we then
focused on the R4 group refinement. Table 4 lists several R4 variations keeping the R3 pyridine-3-
carbonitrile group fixed. Due to a balanced physicochemical property, the pyrazole 3’-CF substitution
3
not only had an excellent fit in the hydrophobic pocket (Ki < 1 nM), but also showed a MIC of 0.5

g/mL against E. coli ATCC 25922. A change to 3’-CF (15i) resulted in 2-fold of activity decrease.
2
Furthermore, an effort to explore alternative hydrophobic substitutions such as Me (15e), Et (15f),
isopropyl (15g), and tert-butyl (15h) all decreased anti-GN activities (4~16 folds). The pyrazole 3’-CN
substitution (15a), however, showed a surprisingly 2-fold improvement in anti-GN potency (E. coli
ATCC 25922 MIC = 0.125 g/mL) and an 8-fold of GP to GN potency shift. The few polar substituents
like carboxylic acid (15b) and amine (15d) were completely inactive (MICs > 16 g/mL), which might
be due to both the incompatibility with the hydrophobic floor of the pocket and the less favored
physicochemical properties at R4. A few heterocycle alternatives to the pyrazole were explored as well.
Although an acceptable anti-GP potency was observed (S. aureus ATCC 29213 MIC = 0.063 g/mL),
the phenyl 3’-CF (15j) showed much decreased anti-GN activity (MIC > 16 g/m) in E. coli ATCC
3
2
5922, and the imidazole 4’-CF (15k) was even inactive. For the two other replacements, pyrazole 4’-
3
CF (15l) and thiazole 3’-CF (15m), their MICs were maintained or slightly decreased. It is evident
3
3
that specific heterocyclic rings at R4 position are ideal for GN activity, although the precise reason
remained elusive.
Table 4. Pyrido[2,3-b]indole GyrB/ParE dual inhibitors, 10d and 15a-m.
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H
H
N
N
N
CN
R4
N
F
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MICb
MICb
S. aureus
E. coli
ATCC
25922
GyrB Kia ParE Kia
LYSAc
Cmpd
R3
ATCC
9213
pKad
(
nM)
(nM)
(g/mL)
2
(g/mL)
(g/mL)
N
N
1
0d
<1
1
<1
<0.016
0.5
<0.3
<1
CF3
N
N
1
5a
< 1
<0.016
0.125
CN
N
N
15b
5c
nd
< 1
nd
nd
65.5
nd
>16
0.125
2
>16
8
4.08
2.02e
8.09
CO2H
N
N
1
NH2
N
N
1
5d
5e
5f
15g
16
2
NH2
N
N
1
< 1
<1
25.7
<1
<0.25
<0.25
<0.25
<0.25
<1
N
N
1
1
<0.3
<0.7
<0.3
N
N
< 1
28.4
157.4
>16
>16
N
N
1
5h
1.02
4.7
N
N
1
5i
59.8
nd
<0.25
1
<1
F
F
0
.063
1
5j
nd
nd
>16
>16
CF3
0.25
N
1
5k
N
nd
CF3
N
N
1
5l
<1
1.1
<0.016
<0.016
1
<1
<1
CF3
S
N
1
5m
8.3
156
0.5
CF3
1
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5
5
5
5
6
a
b
c
Ki: inhibition constant; MIC: minimum inhibitory concentration; LYSA: solubility determined by
d
lyophilization solubility assay (LYSA) at pH 6.5; pKa: ionization constant predicted from MoKa
e
(
version 2); conjugate acid.
0
1
2
3
4
5
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9
0
1
2
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4
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9
0
With pyrazole as the preferred heterocycle, we then evaluated the possibility to substitute the
latter further. As indicated in Table 5, among the compound prepared, the 3’,4’-bis-substituted free
amino compound 15r maintained excellent anti-GN activity (E. coli 25922 MIC = 0.25 g/mL).
However, an attempt to further rigidify this molecule by ring cyclization gave decreased antibacterial
potency as shown by analog 15u (E. coli ATCC 25922 MIC = 1 g/mL). Meanwhile, it was also noticed
that the impact on potency for the CF substitution on the R4 bicyclic core was not as significant as on
3
the 3’-mono-substituted pyrazole ring (e.g., 15u vs. 15x). The aqueous solubility of two amino analogs
1
5r and 15x achieved LYSA = 90 g/mL and 34 g/mL, respectively. We attributed such good
solubility improvement to the molecules’ effective disruption of the intermolecular - stacking
interaction that is partially responsible for the low solubility of this series of analogs.
Table 5. Pyrido[2,3-b]indole GyrB/ParE dual inhibitors, 10d and 15n-z.
H
N
H
N
N
N
CN
R5'
N
N
F
R4'
CF3
MICa
E. coli
ATCC
25922
LMc
LYSAd
pKab
Cmpd
10d
R4’ / R5’
(
mL/min/mg) (g/mL)
(
g/mL)
N
N
0.5
61.8
<0.3
CF3
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N
N
15n
15o
>16
3.55
HO2C
CF3
N
N
2
>16
4
nd
nd
CH3O2C
CONH2
HO
CF3
N
N
N
N
1
5p
15q
5r
5s
5t
5u
5v
CF3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
nd
nd
90
CF3
N
8.08e
1
<0.25
47.0
H2N
CF3
N
N
1
8
>16
1
CF3
N
N
HO
1
CF3
N
N
6.57e
9.44
8.27
1
HN
73.2
nd
<0.3
<1
CF3
N
N
1
HN
2
CF2
N
N
1
5w
4
nd
nd
N
H
N
N
N
7.62e
8.33
8.11
1
1
5x
5y
2
2
4
70.3
20.2
52.1
34
nd
HN
HN
N
N
N
15z
200
N
H
a
b
MIC: minimum inhibitory concentration; pKa: ionization constant predicted from MoKa (version 2);
c
measured in a photometric titration assay; LM: scaled intrinsic clearance of compound in mouse liver
d
microsomes; LYSA: solubility determined by lyophilization solubility assay (LYSA) at pH 6.5;
econjugate acid dissociation constant, measured in a photometric titration assay.
The subsequent combination of several newly identified R4 groups and promising R3
modifications, such as the 2-methoxypyrimidine led to the syntheses of analogs 17r, 17u, and 17x. To
our delight, not only did the mono amino compound 17r maintain the same good potency (E. coli ATCC
2
5922 MIC = 0.312 g/mL), but the bicyclic compound 17x improved its activity (E. coli ATCC 25922
MIC = 0.812 g/mL) and with a good solubility of LYSA = 350 g/mL, the highest solubility among
the whole series.
2
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1
2
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2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The good anti-GN activity, acceptable aqueous solubility, and metabolic stability of compounds
1
5r, 17r, and 17x then supported their further evaluation in vivo.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 6. Comparison between pyrido[2,3-b]indole GyrB/ParE dual inhibitors, 15 and 17.
H
N
H
N
H
N
H
N
N
N
CN
N
R4
R4
N
N
OMe
F
F
1
5
17
MICa
d
Cl in
d
Cl in liver
E. coli
LYSAb
hepatocyte
fuc
microsomed
Cmpd
R4
ATCC 25922
(g/mL)
(mL/min/kg)
(
mL/min/kg)
(
g/mL)
N
N
1
5r
5u
5x
<0.25
90
1.2
1.2
47.0
73.2
48.6
nd
H2N
CF3
N
N
1
HN
1
2
<0.3
CF3
N
N
1
34
4.4
70.3
74.6
HN
N
N
17r
7u
0.312
1
15
4
2.7
1.6
<20.2
77.4
45.3
nd
H2N
CF3
N
N
1
HN
CF3
N
N
1
7x
0.8
350
11
21.8
53.9
HN
b
a MIC: minimum inhibitory concentration; LYSA: solubility determined by lyophilization solubility
c
assay (LYSA) at pH 6.5; fu: percentage of compound free fraction in a plasma protein binding assay;
dCl: scaled mouse intrinsic clearance of compound.
Table 7 shows mouse SDPK IV study results of compounds 15r, 17r, and 17x. All three basic
compounds showed medium in vivo clearances (Cl ), which correlate well with the in vitro hepatic
iv
clearances. The half-lives are short, around 1~2 hr, and the area under the curve (AUC) falls into the
range of 320~340 g*hr/mL. These compounds demonstrated a medium to high volume of distribution
2
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with the plasma protein binding in the range of 89~99%. Although not displaying optimal PK profiles,
these compounds nonetheless proceeded into an animal efficacy study for the initial PK/PD relationship
exploration.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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9
0
1
2
3
4
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9
0
1
2
3
4
5
6
7
8
9
0
A neutropenic mouse thigh infection model with E. coli ATCC 25922 was used to evaluate the
in vivo efficacy of compounds. Exemplified by compound 17r, Figure 6 shows the corresponding log
unit of CFU reductions under different dose regimens. A good dose-response was observed for
compound 17r, and at 45 mpk b.i.d., the bactericidal efficacy that corresponds to one log unit of CFU
reduction (vs. vehicle) was achieved. Free AUC/MIC mostly predicts the PK/PD relationship of other
Topoisomerase antibiotics like FQ. Even though no detailed PKPD study was performed, fAUC/MIC
was still used as PK index in this project at the early screening stage. So, in the mouse thigh infection
model, 17r showed bactericidal efficacy at fAUC/MIC ≥ 6, which also accounts for a total of fAUC ~
30 g*hr/mL (See SI).
Although the in vivo efficacy in mice infected with E. coli ATCC 25922 was successfully
demonstrated, the lower activity of compound 17r against other GN pathogens was insufficient for
additional in vivo efficacy profiling (Table 2). Additional chemistry efforts to further improve this
chemical series’ GN broad-spectrum activity and physicochemical properties targeting other infection
models such as pneumonia and urinary tract infection (UTI) will be reported in due course.
Table 7. Single-dose intravenous pharmacokinetics of representative compounds in the mouse.
Cmax_Da
b
Clivc
t1/2d
(hr)
1.8
Vsse
(L/kg)
5.8
AUC _D
iv
Cmpd
(
kg*ng/mL/mg) (hr*kg*ng/mL/mg) (mL/min/kg)
1
1
5r
7r
305
494
792
324
341
50
48
51
1.6
3.6
17x
327
0.8
1.6
aCmax_D: dose-normalized maximal concentration; AUCiv_D: dose-normalized area under the curve; Cliv
b
c
:
plasma clearance; dt1/2: half-live of compound in plasma; Vss: volume of distribution at the steady-state.
e
2
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Figure 6: In vivo efficacy in neutropenic mouse thigh model. The plot shows the log unit CFU levels
in E. coli ATCC 25922 after twelve hours of infection with the treatment of either placebo (black dots),
ciprofloxacin control (green dots), or compound 17r (blue squares). The line for each animal group
represents the mean, mpk is mg/kg, 2x is two doses with three hours interval. Statistical significance
(Dunnett’s multiple comparison test) vs. placebo, one asterisk represents adjusted P values 0.01-0.05
and two asterisks P <0.001.
CHEMISTRY
All the final compounds were synthesized via the key pyrido[2,3-b]indole tricyclic intermediate
, of which the preparation is illustrated in Scheme 1. 2,4-Difluoro-1-nitro-benzene was reacted with
7
ethyl amine in EtOH via nucleophilic aromatic substitution to afford nitroaniline 2, which was then
deprotonated by NaH and reacted with di-tert-butyl carbonate to give the Boc-protected nitroaniline 3.
The nitro group of the aniline 3 was reduced by H with palladium on carbon, to afford aniline 4.
2
Subsequent Buchwald-Hartwig cross-coupling reaction of the aniline 4 with 2,3,5-trichloropyridine was
carried out in the presence of palladium acetate, cesium carbonate, and BINAP to yield compound 5.
The tricyclic pyrido[2,3-b]indole 6 was obtained by cyclization of 5 via an intramolecular direct
arylation using Pd (dba) catalyst and Xantphos ligand. Finally, the bi-chloro substituted tricyclic
2
3
2
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intermediate 7 was obtained from MCPBA oxidation of the tricyclic pyridine followed by POCl3
chlorination.
Boc
NH
F
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NO2
NO2
NO2
a
b
c
F
F
F
1
2
3
Boc
Boc
H
N
6
N
N
R
Boc
N
NH2
N
H
N
d
e
f
N
F
F
Cl
Cl
F
Cl
4
5
6
Boc
N
H
N
N
F
Cl
Cl
7
o
Scheme 1. Synthesis of key intermediate 7. a) EtNH , EtOH, 0 C, 3 hr, 63% yield; b) NaH, Boc O,
2
2
o
THF, 0 C then rt, 16 hr, 86% yield; c) H 50 psi, Pd/C, MeOH, rt, 18 hr, 92% yield; d) 2,3,5-
2
o
trichloropyridine, Pd(OAc) BINAP, Cs CO , 1,4-dioxane, 120 C, 16 hr, 74% yield; e) Pd (dba) , X-
2
2
3
2
3
o
o
Phos, DBU, DMA/o-xylene, 130 C, 4 hr, 51% yield; f) m-CPBA, DCM, 30 C; 12 hr, then POCl ,
3
o
o
DMF, -5 C to 0 C, 1 hr, 42% yield.
The general preparation of final compounds 10a-u and 12a-w is depicted in Scheme 2. The key
intermediate 7 was first reacted with 3-(trifluoromethyl)-1H-pyrazole through base promoted
nucleophilic aromatic substitution to give compound 8. For the syntheses of compounds 10a-u,
compound 8 was reacted with various boronic acids or esters under palladium catalyzed Suzuki
coupling reaction condition to give compounds 9. In some cases, additional chemical transformations
were carried out before the Boc-protecting group was removed by trifluoroacetic acid treatment to give
the final compounds 10a-u. For the syntheses of pyrimidine compounds 12a-w, intermediate 8 was
2
4
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
reacted with a variety of pyrimidine R3’ substituted boronic acids or esters under Suzuki coupling
condition to afford compounds 11; similarly, when necessary, appropriate chemical transformations
were applied before the Boc-protecting group was removed by the treatment of trifluoroacetic acid to
afford the final compounds 12a-w.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Boc
Boc
N
N
NH
H
N
H
N
H
N
Boc
N
H
N
N
N
N
a
b
c
F
F
F
N
F
3
3
N
Cl
N
R
N
R
N
N
N
Cl
Cl
CF3
CF3
CF3
7
8
9
10a-u
d
Boc
N
NH
H
N
H
N
N
N
e
F
F
N
N
N
N
N
N
N
N
3'
3'
CF3
R
CF3
R
11
12a-w
Scheme 2. Examplar synthesis of compounds 10a-u and 12a-w. a) 3-(trifluoromethyl)pyrazole, K CO ,
2
3
o
DMSO, 120 C, 12 hr, 86% yield; b) 9d, 3-cyanopyridine-5-boronic acid pinacol ester, Pd db , X-Phos,
2
3
o
K PO , 1,4-dioxane/H O, 100 C, 12 hr, 66% yield; c) 10d, TFA, DCM, rt, 2 hr, 81% yield; d) 11j, (2-
3
4
2
o
methoxypyrimidin-5-yl)boronic acid, Pd db , XPhos, K PO , 1,4-dioxane/H O, 100 C, 12 hr, 64%
2
3
3
4
2
yield; e) 12j, TFA, DCM, rt, 1 hr, 40% yield.
Finally, the general synthesis for the preparation of final compounds 15a-z, 17r, 17u, and 17x
is depicted in Scheme 3. Similar to the Scheme 2, the intermediate 7 was first reacted with a variety of
derivatized pyrazole reagents under either base promoted aromatic nucleophilic substitution or
Buchwald-Hartwig cross-coupling reactions to afford compounds 13a-z. Subsequent Suzuki coupling
reaction with 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine-3-carbonitrile then yielded
compounds 14a-z. Whenever needed, additional chemical transformations were carried out before the
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Boc-protecting group was removed to give final compounds 15a-z. Meanwhile, intermediates 13r, 13u,
and 13x were reacted with 2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine
individually via Suzuki coupling reactions to give compounds 16r, 16u, and 16x. After the removal of
Boc-protecting group, final compounds 17r, 17u, and 17x were then obtained.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Boc
Boc
N
N
H
N
H
N
NH
Boc
H
N
N
H
N
N
N
a
b
c
N
F
F
N
F
F
R4
Cl
R4
R4
CN
Cl
Cl
CN
N
N
7
13a-z
14a-z
15a-z
d
Boc
N
NH
H
N
H
N
N
N
e
F
F
R4
R4
N
N
N
N
1
1
16x
6r
6u
OMe
17r
17u
17x
OMe
Scheme 3. Examplar synthesis of 15a-z and 17r, 17u, and 17x. a) 13r, ethyl 3-(trifluoromethyl)-1H-pyrazole-4-
o
carboxylate, K
2
CO
3
, DMSO, 120 C, 33% yield; b) 14r, 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)nicotinonitrile,
o
o
CsF, Pd
2
2 4
(dba)3, X-phos; 1,4-dioxane/H O, 110 C, 12 hr, 89% yield; NH Cl, HOBt, PyBOP, DIPEA, DMF, 30 C, 1 hr,
o
9
1
1
6% yield; BH
3
-THF, 80 C, 0.5 hr, 27% yield; c) 15r, TFA, DCM, rt, 1 hr, 75% yield; d) 16x, tert-butyl 4,6-dihydro-
o
H-pyrrolo[3,4-c]pyrazole-5-carboxylate, K
2
CO
3
, DMSO, 120 C, 12 hr, 93% yield; 2-methoxy-5-(4,4,5,5-tetramethyl-
o
3 4 2
,3,2-dioxaborolan-2-yl)pyrimidine, K PO , cataCXium A-Pd-G2, THF-H O, 70 C, 12 hr, 93% yield; e) 17x, TFA,
o
DCM, 30 C, 1 hr, 57% yield.
CONCLUSION
Due to the rapid increase of bacterial resistance to marketed antibiotics, novel antibacterial
agents are needed urgently. In particular, medicines that can treat MDR GN infections because these
infections are associated with the highest mortality rates and have limited treatment options. Dual
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
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ATPase inhibitors targeting DNA gyrase GyrB subunit and Topoisomerase IV ParE subunit have the
potential to be broad-spectrum antibiotics due to the high level of protein conservation across
pathogenic bacterial species and overcome target-based FQ resistance. Using an information-driven
approach, we converted the 2-carboxamide substituted azaindole scaffold with only GP activity into a
novel chemical series that is also very potent against the ESKAPE GN pathogens. Expanding the
activity spectrum was mainly achieved by increasing the original AZ scaffold’s rigidity via a unique
ring cyclization strategy. A systematic exploration of the physicochemical properties associated with
GN antibacterial activity enabled us to identify the lead molecule 17r, which has a balanced profile of
GN activity, aqueous solubility, and SDPK properties. Moreover, we showed the dose-dependent cidal
efficacy of 17r in a neutropenic mouse thigh infection model. To further develop this series, the
compounds need to show improved broad-spectrum in vitro coverage of GP and GN clinical isolates
and in vivo efficacy in multiple infection models (pneumonia and UTI). A novel class of GyrB/ParE
0
1
2
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9
0
1
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9
0
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9
0
1
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0
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0
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dual inhibitors that can cure the most difficult-to-treat resistant infections in the clinic would address
an urgent medical need considering the ever-increasing resistance toward existing antibiotics.
EXPERIMENTAL SECTION
General Methods for Chemistry. Reactions involving air-sensitive reagents were carried out
under an atmosphere of nitrogen or argon. The microwave assisted reactions were carried out in a
Biotage Initiator Sixty. Solvents and reagents were obtained from commercial sources and were used
without further purification unless otherwise noted. All reactions were monitored by thin-layer
chromatography (TLC) on Merck silica gel 60 F254 TLC glass plates (visualized by UV fluorescence
at  = 254 nm) or analytical LC-MS. All intermediates and final compounds were purified by either
silica gel flash chromatography or preparative HPLC (prep-HPLC) using one of the following
instruments: (i) Biotage SP1 system and the Quad 12/25 cartridge module; (ii) ISCO Combi-flash
chromatography instrument. Silica gel brand and pore size: (a) KP-SIL 60 Å, particle size 40–60 μm;
(b) CAS registry no., silica gel, 63231-67-4, particle size 47–60 μm silica gel; (c) ZCX from Qingdao
Haiyang Chemical Co., Ltd., pore 200–300 or 300–400; (iii) prep-HPLC on a reverse-phase column
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using a Waters XBridge OBD Phenyl (30 mm x 100 mm, 5 μm) or OBD RP18 (30 mm x 100 mm, 5
μm) column under acidic conditions (A, 0.1% formic acid in H O; B, 0.1% formic acid in acetonitrile)
2
or basic conditions (A, 0.01% ammonia in H O; B, acetonitrile). For SFC chiral separation, either
2
intermediates or final compounds were separated using a chiral column (Daicel Chiralpak IC, 30 mm x
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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4
5
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8
9
0
1
2
3
4
5
6
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9
0
2
50 mm, 5 μm) on a Mettler Teledo SFC-Multigram system (solvent system of 95% CO and 5% IPA
2
(
0.5% Et N in IPA), backpressure of 100 bar, UV detection at 254 nm). Optical rotation was measured
3
using a Rudolph Autopol V automatic polarimeter at a wavelength of 589 nm. The purity of final
compounds as measured by LC-MS was ≥95%. LC-MS spectra were obtained using UPLC coupled
with single quadrupole mass detector (Waters Acquity UPLC-3100 Mass Detector, Waters Acquity
UPLC-SQ Detector, Waters Acquity UPLC-SQ Detector). Standard LC-MS conditions were as follows.
Columns: Waters Acquity BEH C18, 2.1 mm  50 mm x 1.7 m and Waters Acquity CSH C18 column,
2
.1 mm  50 mm x 1.7 m. Flow rate: 0.8 mL/min. Gradient: 5-95% eluent B over 3 min under mobile
phase conditions: 1) acidic condition: A, 0.1% formic acid in H O; B, 0.1% formic acid in acetonitrile;
2
or 2) basic condition: A, 0.05% NH ·H O in H O; B, acetonitrile. Mass spectra (MS): Generally only
3
2
2
ions which indicate the parent mass are reported, and unless otherwise stated the mass ion quoted is the
+
positive mass ion (M+H) . NMR spectra were obtained using Bruker Avance 400 MHz spectrometer,
1
13
operating at 400.13 MHz ( H) and 100.62 MHz ( C). High-resolution mass spectra (HRMS) were
obtained on Xevo G2-XS-QTOF mass spectrameter equipped with an electrospray ionization source.
All of the reported yields are for isolated products and not optimized.
Synthetic procedure for the synthesis of tert-butyl N-(3,4-dichloro-6-fluoro-9H-pyrido[2,3-
b]indol-8-yl)-N-ethyl-carbamate (7).
N-Ethyl-5-fluoro-2-nitro-aniline (2). Ethylamine solution (234.26 g, 1.56 mmol, 30% in EtOH) was
added dropwise to 2,4-difluoronitrobenzene (80 g, 0.503 mol) at 0 °C over 15 min. After completion of
the addition, the reaction mixture was stirred at 0 °C for 3 h. The solution was diluted with EtOH (500
mL) and poured into 2.5 L of ice-water. The resulting precipitates were collected by filtration and dried
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