Full text of DOI:10.1039/d0md00175a

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RESEARCH ARTICLE
Rational design, synthesis and testing of novel
tricyclic topoisomerase inhibitors for the
treatment of bacterial infections part 2†
Cite this: DOI: 10.1039/d0md00175a
*
R. Kirk,
A. Ratcliffe, G. Noonan, M. Uosis-Martin, D. Lyth, O. Bardell-Cox,
J. Massam, P. Schofield, A. Lyons, D. Clare, J. Maclean, A. Smith, V. Savage,
S. Mohmed, C. Charrier, A-M. Salisbury, E. Moyo, N. Ooi, N. Chalam-Judge,
*
J. Cheung, N. R. Stokes,
S. Best, M. Craighead, R. Armer and A. Huxley
Received 25th May 2020,
Accepted 7th August 2020
Building on our previously-reported novel tricyclic topoisomerase inhibitors (NTTIs), we disclose the
discovery of REDX07965, which has an MIC90 of 0.5 μg mL−1 against Staphylococcus aureus, favourable
in vitro pharmacokinetic properties, selectivity versus human topoisomerase II and an acceptable toxicity
profile. The results herein validate a rational design approach to address the urgent unmet medical need
for novel antibiotics.
DOI: 10.1039/d0md00175a
rsc.li/medchem
tricyclic core provides a good overlap with the moxifloxacin
quinolone core. Therefore, we reasoned that saturated
1. Introduction
The structure activity relationship (SAR) of a number of
potent, novel tricyclic topoisomerase inhibitor (NTTI)
derivatives e.g. REDX05931 (Fig. 2C) has been described in
part 1 of this work.1 Despite displaying favourable MICs
heterocycles in the C-7 position of the NTTI series could
provide similar binding interactions to our original work and
maintain activity against FQ resistant organisms.1
against
a range of Gram-positive and fastidious Gram-
negative bacterial strains, including ≤4 μg mL−1 against the 2. Results and discussion
clinically relevant FQ resistant strain of N. gonorrhoeae (WHO
2.1 SAR of R1 saturated heterocyclic amine substitution
L),2 REDX05931 and closely related analogues inhibited
multiple forms of CYP enzymes. Our SAR analysis indicated
that the 2′-methylene amino moiety was responsible for this
profile, which precluded this series from further study. As
such, a new medicinal chemistry strategy was necessary.
Multiple generations of fluoroquinolones have depended
on saturated heterocycles in the C-7 position. Optimisation of
the saturated amine heterocycle at this position of
fluoroquinolones has led to improvements in the
antibacterial spectrum of activity; covering Gram-positive,
Gram-negative and anaerobic bacteria, whilst also improving
in vitro pharmacokinetic and physicochemical properties.3 To
address the low solubility of the aryl C-7 NTTIs, and to
potentially broaden the spectrum of activity to include a
range of Gram-negative organisms, we explored the addition
of saturated heterocycles in the C-7 position (R1; Fig. 1a).
Structural superposition of REDX05931 (Fig. 2B and C) onto
the X-ray crystal structure of moxifloxacin (Fig. 1b and 2A)
bound to topoisomerase IV (PDB 2XKK) indicates that the
Matched pair analysis of all saturated heterocycles in the R1
position where X = C–Cl vs. X = C–H (Table 1, REDX06598 vs.
REDX05762, REDX05855 vs. REDX07181, REDX05840 vs.
REDX07815) clearly suggests that improved potency against
S. aureus ATCC 29213, S. pneumoniae ATCC 49619 and E. coli
ATCC 25922 (ref. 7) is observed with X = C–Cl, confirming
that the nature of the X group plays an important role in
potency. The results are consistent with the premise that the
steric bulk of the Cl, and REDX05931 consequent out of
plane distortion of the core encourages DNA breakage once
intercalated.4
Redx Anti-Infectives Ltd, Alderley Park, Macclesfield SK10 4TG, Cheshire, UK
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0md00175a
Fig. 1 a: Points of derivatisation of our NTTI series. b: Comparative
fluoroquinolone moxifloxacin, highlighting the saturated heterocycle in
the C-7 position.
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Fig. 2 A) Moxifloxacin bound to topoisomerase IV taken from PDB 2XKK (lime green). B) Overlaid superposition of REDX05931 (magenta) over
moxifloxacin (lime green). C) Chemical structure of REDX05931.
Substitution of a methylene amino group from a primary
amine to a secondary amine (REDX07948 and REDX07046)
did not improve the microbiological profiles, nor did
introduction of steric bulk around the methylene region
(REDX05848 and REDX05960; Table 1). Additionally, the N,N-
dimethyamino group (REDX07156 and REDX06885) displayed
a markedly reduced microbiological profile, indicating the
necessity of an accessible H-bond donor. Microbiological data
for the 3-amino pyrrolidine derivative (REDX05761) compared
with the methylene amino pyrrolidine derivatives suggest that
the extended amine has a beneficial effect on activity. This
trend is also seen with the six-membered counterparts. The
minimum inhibitory concentration (MIC) of the piperazine
analogue (REDX06937) was 2 μg mL−1 against methicillin-
sensitive S. aureus ATCC 29213 (MSSA); its 4-aminopiperidine
analogue (REDX07181) demonstrated an MIC of 0.25 μg
pyrrolidin-3-yl)ethan-1-amine at the C-7 position impacts the
microbiological activity.8 A crystal structure of PD0305970 in
topoisomerase IV from S. pneumoniae reveals that the
3-(aminomethyl)pyrrolidinyl is surrounded by Arg 456, Glu
474, Glu 475 and Asp 435 residues (ParE).7 Based on this
result and our computational modelling of our NTTI series;
we hypothesised that incorporation of the most active
stereoisomer ((S)-1-((R)-pyrrolidin-3-yl)ethan-1-amine) at the
C-7 position of our NTTI series would orientate the primary
amine towards GyrB-Glu466 (ref. 9) (S. aureus nomenclature).
Using this rationale in conjunction with the learnings from
part 1 (ref. 1) and the SAR described above we synthesised
REDX07965 (Fig. 3b).
REDX07965 (Fig. 3b) displayed superior activity within
the series, with a S. aureus MIC90 (ref. 14) of 0.5 μg mL−1
against clinical isolates (see Table S1†). Assessment of target
bacterial enzyme inhibition by REDX07965 showed it to be
a potent inhibitor of DNA gyrase and topoisomerase IV
displaying an IC50 of 1.82 and 6.10 μM respectively (see
Table S2†).
mL−1.
Interestingly
these
six-membered
saturated
heterocycles consistently demonstrated a loss of activity
against resistant strains (data not shown). Replacing the
terminal amino group with a hydroxy group in REDX05944
(3-hydroxypyrrolidine) resulted in a loss of activity, with an
MIC against MSSA of 32 μg mL−1. Bicyclic systems such as
REDX06876 and REDX06921 did not improve the
microbiological profile, indicating that the orientation and
lipophilicity surrounding the terminal nitrogen is important.
The latter point being accentuated with the comparative
analogues REDX06937 (piperazine) and REDX06803 (2,6-
dimethyl piperazine) with MICs of 0.25 and 8 μg mL−1,
respectively, versus MSSA. The fused core derivatives showed
a strong preference for the (S)-stereoisomer (REDX06215 vs.
REDX06415), consistent with the relative activities of
levofloxacin and dextrofloxacin.5 However, due to an eight-
fold increase in MIC against methicillin-resistant S. aureus
strains (namely MRSA NRS74), the morpholino derivative
core was not pursued as a priority.
2.2 Profiling of REDX07965
REDX07965 demonstrated excellent potency against a range
of S. aureus strains, including fluoroquinolone and
multidrug-resistant (MDR) strains. Despite reduced activity
versus Enterococcus faecium, REDX07965 displayed good
activity against the Gram-negative organisms E. coli, H.
influenzae and N. gonorrhoeae.7 A microbiological profile of
REDX07965 is shown in Table 2.
A
thorough investigation of related topoisomerase
inhibitors revealed several studies of an quinazolinedione
series,6,7 cumulating in the discovery of PD0305970 (Fig. 3a).
A published SAR examination of aminoalkyls at the C-7
revealed the absolute stereochemistry of the (S)-1-((R)-
Fig. 3 a: Chemical structure of PD0305970. b: Chemical structure of
REDX07965.
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Table 1 Selected microbiological and solubility data of saturated R1 analogues. MICs data are based on at least two experimental replicates; data in μg
mL−1
MIC S. aureus
MIC S. pneumoniae
MIC E. coli ATCC
MIC H. influenzae
TD solb
(mg L−1
)
#
R1
X
ATCC 29213a
ATCC 49619a
25922a
ATCC 49247a
REDX05761
H
2
8
1
1
n.d.
REDX05762
REDX05840
REDX05848
H
H
H
0.5
1
0.5
0.5
1
2
2
4
8
8
4
n.d.
1403
1350
1
REDX05855
REDX05944
H
H
1
1
2
1
4
1403
n.d.
32
64
64
REDX05960
H
2
2
8
16
1353
REDX06215
REDX06415
REDX06598
REDX06803
FC1
FC2
Cl
0.5
64
0.5
128
0.12
16
2
4
ND
ND
950
543
64
1
128
n.d.
8
0.12
8
Cl
4
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Table 1 (continued)
MIC S. aureus
MIC S. pneumoniae
MIC E. coli ATCC
MIC H. influenzae
TD solb
(mg L−1
)
#
R1
X
ATCC 29213a
ATCC 49619a
25922a
ATCC 49247a
REDX06876
Cl
2
4
4
4
2
4
8
8
1061
n.d.
REDX06885
Cl
REDX06921
REDX06937
H
2
2
4
4
4
2
1195
885
Cl
0.5
0.5
REDX07046
REDX07156
Cl
Cl
0.25
1
0.25
2
1
4
1
1181
1194
16
REDX07181
REDX07815
Cl
Cl
0.25
0.12
0.5
0.5
0.5
1
1
922
601
0.12
REDX07948
REDX07965
Cl
Cl
0.25
0.06
0.25
2
1
1
—
0.015
0.5
n.d.
a
Strain description – see ref. 13. b TD sol = thermodynamic solubility.
REDX07965 demonstrated an acceptable in vitro ADMET
profile (Table 3). However, in vitro investigation of cardiac
ion channel activity revealed inhibition of hERG, Ca2+ and
Na+ channels (IC50 28.7, 21.5 and 93.6 μM, respectively).
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Table 2 Microbiological profile of REDX07965
Straina
MIC (μg mL−1
)
Strain
MIC (μg mL−1
)
S. aureus ATCC 29213
S. aureus NRS70
S. aureus NRS100
S. aureus NRS106
S. aureus NRS107
S. aureus NRS108
S. aureus NRS384
S. aureus ATCC 43300
S. aureus NRS74
S. aureus NRS1
0.06
0.06
0.12
0.12
0.12
0.12
0.12
0.12
0.12
0.25
0.5
S. aureus NRS271
S. aureus VRS8
0.5
0.5
0.12
0.06
1
E. faecalis ATCC 29212
S. epidermidis ATCC 12228
S. epidermidis NRS101
E. faecium ATCC 19434
E. faecium ATCC 700221
S. pneumoniae ATCC 49619
S. pyogenes ATCC 19615
E. coli ATCC 25922
8
8
0.015
0.015
0.5
1
0.12
0.03
S. aureus NRS127
S. aureus VRS1
S. aureus NRS482
H. influenzae ATCC 49247
N. gonorrhoeae ATCC 49226
N. gonorrhoeae ATCC 700825
0.5
0.12
a
Strain description – see note 13.
Table 3 Selected ADMET properties of REDX07965
In vitro assay (units)
4. Chemistry
4.1 Chemistry of saturated LHS analogues
Result
HepG2 cytotoxicity CC50 (μg mL−1
Human PPB (% free)
)
32–64
24.4
27.1
461.9
3.0
2.2
20.7
30
16.4
30
30
17.5
30
>100
28.7
21.5
93.6
Non-toxic
Toxic
The synthesis of the saturated C-7 derivative series began
with the synthesis of a late stage versatile intermediate (6).
Trifluorobenzoic acid (1) was activated with oxalyl chloride
followed by addition and ring closure with ethyl
isocyanoacetate under basic conditions to give a C–H oxazole
(2).11 This can be hydrolytically cleaved with aqueous HCl to
give the hydroxylamine, followed by ring closure with
triethylorthoacetate to give the methyl oxazole (3). The ester
was hydrolysed under basic conditions and activated/
functionalised with oxalyl chloride-cyclopropylamine to give
amide (5). Intramolecular SnAr gave the versatile tricyclic
intermediate (6). This advanced intermediate allowed for
derivatisation at the C-7 position via microwave-assisted
intermolecular SnAr to give intermediate (7). At this point,
the tricyclic core can be further functionalised by the
introduction of a chlorine in the C-8 position, by addition of
1 equivalent of 1,3-dichloro-5,5-dimethyl-imidazolidine-2,4-
dione (DCDMH) in DCM at room temperature.12 The
saturated heterocycles were then deprotected with TFA in
DCM (if the saturated heterocycle contains a Boc protected
amine) to give compounds 9a/b.
% LBF (predicted from Hheps)
Hhep predicted half life (min)
Hhep clint (μL min−1 per 106 cells)
Caco2 efflux ratio
Caco 2 Papp (10−6 cm s−1
CYP1A2 IC50 (μM)
CYP2C9 IC50 (μM)
CYP2C19 IC50 (μM)
CYP2D6 IC50 (μM)
CYP3A4 IC50 (μM)
CYP3A4T IC50 (μM)
)
Kinetic solubility pH 7.4 (μM)
hERG patch IC50 (μM)
NaV1.5 patch IC50 (μM)
CaV1.2 patch IC50 (μM)
Genotox BlueScreen (−S9) at 200 μM
Genotox BlueScreen (+S9) at 200 μM
The low IC50 for the hERG ion channel suggested a risk of
QT prolongation at a therapeutic dose. Additionally, despite
an acceptable cytotoxicity profile (CC50 32–64 μg mL−1 in a
HepG2 cytotoxicity assay), the compound proved to be
genotoxic with the addition of S9 at 200 μM (BlueScreen
assay10). For these reasons, further development of this
compound was terminated (Table 3).
The fused core intermediate was synthesised in an
analogous procedure to that previously described1 using
tetrafluorobenzoic acid (10). The tetracyclic core (11) was
then derivatised using standard SnAr conditions described in
Scheme 1, procedure f (Scheme 2).
3. Conclusion
Associated content
Using a rational, structure-guided drug design strategy, we
have identified a promising lead compound (REDX07965),
that inhibits bacterial DNA gyrase and topoisomerase IV with
broad-spectrum antibacterial potency and has favourable
in vitro pharmacokinetic properties. However, the risk of
cardiac and genetic toxicology, revealed by the hERG and
BlueScreen assays respectively, precludes the further
development of REDX07965.
Table S1 and experimental data are available in ESI.†
Funding
This study was funded by Redx Pharma Plc in collaboration
with the Royal Liverpool and Broadgreen University Hospitals
NHS Trust.
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Scheme 1 Reagents and conditions: (a) (1) oxalyl chloride, DCM, 0 °C-rt, 2 h, quant; (2) ethyl isocyanoacetate, THF, NEt3, 0 °C-rt 16 h, 48%. (b) (1)
1 M HCl, 1,4 dioxane, rt, 72 h, quant; (4) triethylortho acetate, 110 °C, 2 h, quant. (c) LiOH, 1,4-dioxane, rt, 16 h, 98%; (d) (1) oxalyl chloride, DMF,
DCM, rt, 1 h, quant; (2) NEt3, cyclopropylamine, DCM, rt, 18 h, 79%. (e) K2CO3, 18-crown-6, DMSO, 140 °C, 50 min, 64%. (f) Amine, DIPEA, MW,140
°C, 80 min, 71–91% (g) DCDM, DCM, rt, 20 min, 43–59%. (h) TFA, DCM, rt, 18 h, 85–99%.
Scheme 2 General synthesis of tetracyclic core.
Conflicts of interest
Notes and references
All authors were or are employees of Redx Pharma and may
hold shares or share options in the company.
1 R. Kirk, A. Huxley, A. Ratcliffe, G. Noonan, M. Uosis-Martin,
D. Lyth, O. Bardell-Cox, J. Massam, P. Schofield, S. Hindley,
D. Jones, A. Lyons, D. Claire, J. Maclean, A. Smith, V. J.
Savage, S. Mohmed, C. Charrier, A.-M. Salisbury, E. Moyo, R.
Metzger, N. Chaffer-Malam, J. Cheung, N. R. Stokes, S. A.
Best, M. W. Craighead and R. Armer, RSC Med. Chem.,
2020, DOI: 10.1039/D0MD00174K.
2 V. J. Savage, C. Charrier, A.-M. Salisbury, E. Moyo, H.
Forward, N. Chaffer-Malam, R. Metzger, A. Huxley, R. Kirk,
M. Uosis-Martin, G. Noonan, S. Mohmed, S. A. Best, A. J.
Ratcliffe and N. R. Stokes, J. Antimicrob. Chemother.,
2016, 71, 1905–1913.
Acknowledgements
We are grateful for the provision of bacterial strains by the
Network on Antimicrobial Resistance in Staphylococcus aureus
(now part of BEI Resources, Manassas, USA), to IHMA Europe
Sarl (Epalinges, Switzerland) for performing the clinical
isolate MIC90 studies, to Inspiralis Ltd (Norwich, UK) for
performing the DNA supercoiling, decatenation and cleavage
assays, to Apconix for performing the multi-ion channel
assessment (K+, Ca2+, Na+) and to Gentronix for performing
the genotoxicity assessments.
3 N. H. Rogers and P. C. T. Hannan, Use of quinolone
derivatives for the treatment of mycolasmal pneumonia in
pigs, WO1986006630A1, 1986, pp. 1–41.
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4 J. Heim, Safe drugs for bad bugs, http://docplayer.net/
143901303-Safe-drugs-for-bad-bugs.html.
5 A. L. Barry, P. C. Fuchs, S. D. Allen, S. D. Brown, J. H.
Jorgensen and F. C. Tenover, J. Antimicrob. Chemother.,
1996, 37, 365–369.
6 E. L. Ellsworth, T. P. Tran, H. D. Hollis Showalter, J. P.
Sanchez, B. M. Watson, M. A. Stier, J. M. Domagala, S. J.
Gracheck, E. T. Joannides, M. A. Shapiro, S. A. Dunham,
D. L. Hanna, M. D. Huband, J. W. Gage, J. C. Bronstein, J. Y.
Liu, D. Q. Nguyen and R. Singh, J. Med. Chem., 2006, 49,
6435–6438.
methicillin-sensitive Staphylococcus aureus; MRSA NRS74,
FQ-resistant methicillin-resistant Staphylococcus aureus;
MRSA NRS1, AMG- and TET-resistant (Mu50) GyrA (S84L,
E409L), GrlA (S80F); MRSA NRS70, MRSA (N315); MRSA
NRS100 COL-resistant; MRSA NRS127, LZD-resistant; MRSA
NRS271, LZD-resistant; S. aureus VRS8, MRSA VAN resistant,
MRSA NRS106, AMG-, QAC-, TMP-resistant; MRSA NRS108,
AMG-resistant, MRSA NRS107, MUP resistant (RN4220 +
pGO400); MRSA NRS384, CA-MRSA ERY-, TET-resistant, FQ
Int resistant (USA300 LAC); MRSA NRS482, FQ-resistant; E.
faecalis ATCC 29212 wild-type Enterococcus faecalis; S.
7 T. P. Tran, E. L. Ellsworth, J. P. Sanchez, B. M. Watson, M. A.
Stier, H. D. H. Showalter, J. M. Domagala, M. A. Shapiro,
E. T. Joannides, S. J. Gracheck, D. Q. Nguyen, P. Bird, J. Yip,
A. Sharadendu, C. Ha, S. Ramezani, X. Wu and R. Singh,
Bioorg. Med. Chem. Lett., 2007, 17, 1312–1320.
8 K. M. Hutchings, T. P. Tran, E. L. Ellsworth, B. M. Watson,
J. P. Sanchez, H. D. Hollis Showalter, M. A. Stier, M. Shapiro,
E. Themis Joannides, M. Huband, D. Q. Nguyen, S. Maiti, T.
Li, J. Tailor, G. Thomas, C. Ha and R. Singh, Bioorg. Med.
Chem. Lett., 2008, 18, 5087–5090.
epidermidis
ATCC
12228
wild-type
Staphylococcus
epidermidis; S. epidermidis NRS101, MRSE AMG-, ERY-
resistant; E. faecium ATCC 19434 wild-type Enterococcus
faecium; E. faecium ATCC 700221, VAN-resistant; S. pyogenes
ATCC 19615, wild-type Streptococcus pyogenes; E. coli ATCC
25922, wild-type Escherichia coli; H. influenzae ATCC 49247,
wild-type Haemophilus influenzae; N. gonorrhoeae ATCC
49226, wild-type Neisseria gonorrhoeae; N. gonorrhoeae ATCC
700825, wild-type.
14 Abbreviations used: TMP – trimethoprim, LZD – linezolid,
9 K. Drlica, A. Mustaev, T. R. Towle, G. Luan, R. J. Kerns and
J. M. Berger, ACS Chem. Biol., 2014, 9, 2895–2904.
10 K. Simpson, N. Bevan, P. Hastwell, P. Eidam, P. Shah, E.
Gogo, S. Rees and A. Brown, J. Biomol. Screening, 2012, 18(4),
441–452.
FQ
–
fluoroquinolones, TET
–
tetracyclines, VAN
–
vancomycin, MUP – mupirocin, ERY – erythromycin, AMG –
aminoglycoside. WHO – World Health Organisation, CDC –
Centers for Disease Control and Prevention, MDR
–
multidrug resistant, MSSA methicillin-susceptible S.
–
11 M. Baumann, I. R. Baxendale, S. V. Ley, C. D. Smith and
G. K. Tranmer, Org. Lett., 2006, 8, 5231–5234.
12 D. Peters, E. O. Nielsen, G. M. Olsen and S. F. Nielsen,
Heteroaryl diazacycloalkanes, their preparation and use,
US2002045618 (A1), 2002.
aureus, MRSA – methicillin-resistant S. aureus ADMET –
adsorption, distribution, metabolism, excretion, toxicity,
Physchem – physical chemical, TD – thermodynamic, PPB –
plasma protein binding, Cli – intrinsic clearance, HEP –
hepatocytes, SOL – solubility, MIC90 = minimum inhibitory
concentration required to inhibit the growth of 90% of
organisms.
13 Strain description with abbreviated known antibacterial
resistance spectrums: MSSA ATCC 29213, wild-type
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