Activity-directed expansion of a series of antibacterial agents

Article abstract of DOI:10.1039/d0cc02361b

The feasibility of using activity-directed synthesis to drive antibacterial discovery was investigated. An array of 220 Pd-catalysed microscale reactions was executed, and the crude product mixtures were evaluated for activity against Staphylococcus aureus. Scale-up of the hit reactions, purification and evaluation, enabled expansion of a class of antibacterial quinazolinones. The novel antibacterials had MICs from 0.016 μg mL-1 (i.e. 38 nM) to 2-4 μg mL-1 against S. aureus ATCC29213. This journal is

Full text of DOI:10.1039/d0cc02361b

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
ChemComm
View Article Online
View Journal
COMMUNICATION
Activity-directed expansion of a series of
antibacterial agents†
Cite this:DOI: 10.1039/d0cc02361b
bc
ab
ab
Abbie Leggott,ab Justin E. Clarke,
Shiao Chow,
Stuart L. Warriner,
bc
ab
Received 1st April 2020,
Accepted 9th June 2020
Alex J. O’Neill
*
and Adam Nelson
*
DOI: 10.1039/d0cc02361b
rsc.li/chemcomm
The feasibility of using activity-directed synthesis to drive antibac- [MICs] as low as 0.003 mg mLÀ1) against multiple strains of
terial discovery was investigated. An array of 220 Pd-catalysed S. aureus.6a
microscale reactions was executed, and the crude product mixtures
We envisaged that Pd-catalysed reactions may complement
were evaluated for activity against Staphylococcus aureus. Scale-up the metal carbenoid chemistry that we have previously har-
of the hit reactions, purification and evaluation, enabled expansion nessed in ADS (Fig. 1). Specifically, we noted its potential to
of a class of antibacterial quinazolinones. The novel antibacterials yield alternative scaffolds from a common substrate (such as 1).
had MICs from 0.016 lg mLÀ1 (i.e. 38 nM) to 2–4 lg mLÀ1 against For example, Pd-catalysed carbonylation, followed by reaction
S. aureus ATCC29213.
with an amine and cyclisation, could yield 2,3-disubstituted
quinazolinones such as 2. Alternatively, Pd-catalysed amina-
The discovery of bioactive small molecules is generally driven tion, without carbonylation, could yield anilines (e.g. 3) or, with
through iterative design-make-purify-test cycles. The process is subsequent cyclisation, benzimidazoles (e.g. 4). Finally, intra-
generally underpinned by a remarkably narrow toolkit1 of molecular Heck reaction could yield quinolinones (e.g. 5).
reliable reaction classes which limits the diversity of explored
Initially, we adapted a Pd-catalysed carbonylation reaction7
chemical space.2 We have recently introduced activity-directed for execution on a 300 ml scale (100 mmol substrate) in micro-
synthesis3 (ADS), a structure-blind and function-driven discov- scale vials in 96-well plate format (Scheme 1). Accordingly, the
ery approach. In ADS, inherently promiscuous chemistry is har- o-iodo anilide 6a (final concentration: 333 mM), the aniline
nessed in which arrays of reactions are performed, and each (1.2 eq.), Mo(CO)6 (1 eq.), Pd2(dba)3 (0.1 mol%), P(tBu3)ÁHBF4
reaction has multiple possible outcomes. The crude reaction (0.6 mol%) and NEt3 (2.5 eq.) were dissolved in o-xylene; after
mixtures are then directly screened for biological activity. We
have previously harnessed metal carbenoid chemistry to drive the
activity-directed discovery of diverse series of androgen receptor
agonists and protein–protein interaction inhibitors.3
Here, we demonstrate the generality of ADS by showing that
other reaction classes (here, Pd-catalysed carbonylation4) and
assay types (here, whole-cell antibacterial activity against the
ESKAPE5 pathogen S. aureus) may be harnessed. We show that
the approach may be used to expand a series of quinazolinones6
that was developed to target penicillin-binding proteins, and
displays promising activity (minimum inhibitory concentrations
a School of Chemistry, University of Leeds, LS2 9JT, Leeds, UK.
E-mail: a.s.nelson@leeds.ac.uk
b Astbury Centre for Structural Molecular Biology, University of Leeds,
Leeds, LS2 9JT, UK. E-mail: a.j.oneill@leeds.ac.uk
c School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK
† Electronic supplementary information (ESI) available: Experimental procedures
and NMR spectra. CCDC 1991665. For ESI and crystallographic data in CIF or
other electronic format see DOI: 10.1039/d0cc02361b
Fig. 1 Potential of Pd-catalysed chemistry to yield alternative scaffolds
from common substrates (such as 1), amines and, in some cases, a carbon
monoxide source. Groups derived from an amine co-substrate (blue) and
carbon monoxide (red) are indicated.
Chem. Commun.
This journal is © The Royal Society of Chemistry 2020

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
View Article Online
Communication
ChemComm
Table 1 Configuration of antibacterial assay to be suitable for ADS
Entrya Substrate Co-Substrateb Reagentsc
Conc.d/mM Activitye
1
6b
7b
All
50
2
3
4
5
6a
6b
6a
—
7b
—
—
7b
All
All
All
All
50
50
50
50
6
—
—
P(tBu3)ÁHBF4 50
7
8
9
—
—
6b
—
—
7b
Mo(CO)6
Pd2(dba)3
All
50
50
100
10
6b
6b
7b
7b
All
All
25
10
11
a
Reactions performed in microscale vials at 105 1C; crude reaction
mixtures were filtered through silica, evaporated and dissolved in DMSO
(total product concentration: 333 mM) before evaluation. b 1.2 eq. c Mo(CO)6
(1 eq.), 0.1 mol% Pd2dba3, 0.6 mol% P(tBu3)ÁHBF4, NEt3 (2.5 eq.), o-xylene.
d Total product concentration based on limiting substrate. e Activity against
S. aureus ATCC29213 (see Fig. 2 for colour scale); a tick indicates visual growth
inhibition (see ESI). f LC-MS revealed that a quinazolinone had been formed.
Scheme 1 Configuration of Pd-catalysed chemistry for use in ADS. aLC-MS
analysis of the reaction performed in a microscale vial revealed that the product
had been formed successfully. bPrepared using a precedented method (ref. 6a).
(and no co-substrate). The array of 220 reactions was designed to
sealing, the vial was heated at 105 1C for 48 h. It was shown that enable formation of a diverse range of quinazolinones, some
the yield of 8a, determined by NMR spectroscopy using an internal related to known analogues,6 as well as other products of Pd-
standard, was broadly similar to that obtained on a preparative catalysed processes. The substrates were mainly analogues of S1
scale in a crimp-top vial. At this stage, the quinazolinones 8b and (e.g. dehomologated, constrained), whilst the aniline and amine
8c6a were also prepared. Crucially, we had shown that Pd-catalysed co-substrates bore structurally-diverse aryl, hetaryl and other
chemistry was viable in a format suitable for ADS, and that stirring substituents. To expand the possible reaction outcomes, some
and an inert atmosphere were not necessary.
substrates and co-substrates bore alternative (e.g. S2 and S9) or
We adapted standard susceptibility testing methodology8 to additional (e.g. C6, C12 and C16) nucleophiles. The substrates and
assess antibacterial activity against S. aureus ATCC29213. Quina- co-substrates were all inactive against S. aureus ATCC29213 at
zolinone solubility was improved by using Iso-Sensitest Broth 50 mM. The array was performed in a 96-well OptiBlock and
(ISB)9 as the growth medium, and MICs were determined for assembled from stock solutions using multi-channel pipettes.
8b–c [8b: 0.5–1 mg mLÀ1 (1.45–2.90 mM); 8c: 0.125–0.25 mg mLÀ1 After 48 h at 105 1C, the crude reaction mixtures were filtered
(0.34–0.68 mM)]. The reaction of 6b and 7b (which would yield the through silica, evaporated and dissolved in DMSO (total product
antibacterial 8b) was then repeated in a microscale vial, the concentration: 333 mM). Randomly-selected product mixtures
reaction filtered through silica, evaporated, and the crude reaction (n = 23) were analysed by LC/MS; 16 of these contained carbonyl-
mixture dissolved in DMSO (total product concentration: 333 mM). ated intermolecular products, 13 of which had been dehydrated.
The stock solution of the crude product was screened at multiple In addition, one product mixture (S3 + C1) contained the product
total product concentrations (100 mM, 50 mM, 25 mM and 10 mM; of an intramolecular reaction of S3, later shown to stem from a
entries 1 and 9–11, Table 1): visual growth inhibition was only reductive Heck reaction10 (see ESI†).
detected at the two highest concentrations. Crucially, the reaction
The crude reaction mixtures were screened in duplicate against
of 6a and 7b did not yield an active crude product at 50 mM total S. aureus ATCC29213 (final total product concentration of 50 mM
product concentration (entry 2). In addition, the reactions of the in 1% DMSO in ISB) (Fig. 2). Six of the 220 reactions displayed
individual reactants 6a, 6b and 7b (entries 3–5), as well as the significant antibacterial activity in both replicate cultures: sub-
individual components P(tBu3)ÁHBF4, Mo(CO)6 and Pd2(dba)3 strate S1 (with C1 or C18); substrate S7 (with C18); and substrate
(entries 6–8), also did not result in detectable activity. The assay S10 (with C1, C6 or C18). Reassuringly, the combination of S1 and
was deemed suitable for activity-directed antibacterial discovery C1 had been previously validated in our method development
since activity was only observed for reactions that could have work (Table 1). The remaining five hit reactions were scaled fifty-
yielded the positive control 8b.
fold, and the products purified by mass-directed HPLC (Table 2).
Having established appropriate synthetic and biological meth- In each case, quinazolinones with an extended hydrophobic
ods for ADS, we designed a reaction array based on ten substrates substituent in the 2-position, and a meta-substituted phenyl group
S1–S10 (and no substrate) and nineteen co-substrates C1–C19 in the 3-position, were obtained. The products had MIC values
Chem. Commun.
This journal is © The Royal Society of Chemistry 2020

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
View Article Online
ChemComm
Communication
Fig. 2 Activity-directed antibacterial discovery (see Table 1 for reaction conditions). Panels A and B: Structures of substrates (A) and co-substrates (B).
Panel C: Activity against S. aureus ATCC29213 (total product concentration: 50 mM), with active combinations that were scaled up shown (yellow).
Product mixtures that displayed activity against only one colony (pink) were re-assayed, but none of these combinations were validated as hits (see ESI†).
against S. aureus ATCC29213 ranging from 0.016 mg mLÀ1 (i.e. yield antibacterial products. In some cases, the meta OH or NHMs
38 nM) for 8d to 2–4 mg mLÀ1 (i.e. 6–12 mM) for 8g. Notably, 8d was group (in 8f and 8h) could be replaced by a NH2 group (in 8g);
B10-fold more active than the positive control6a 8c. The products however, none of the co-substrates that lacked such a m-substituted
8d–h had comparable activity against the methicillin-resistant phenyl group ever yielded antibacterial products. Crucially, the
USA300 JE2 strain, but were significantly less active against products of reactions that had not been identified as hits were
laboratory strain SH1000. In addition, we purified the products substantially less (or not) active. Although the quinazolinone 8i is
of three reactions that were not identified as hits: S7 + C1; S7 + matched pair of 8a, 8e and 8h (MICs: 0.5–2 mg mLÀ1), it is
aniline; and S4 + C9; two of these reactions also yielded quinazo- significantly less active (MIC: 4–8 mg mLÀ1). The quinazolinones
linones, and the third an uncyclised amide 9, and, crucially, all of 8j and the amide 9 were not active, even at 128 mg mLÀ1
.
these products displayed significantly lower (or no detectable)
antibacterial activity.
In conclusion, we have shown that ADS is feasible with a
novel reaction class (Pd-catalysed chemistry) and with a phe-
Our experiments enabled the SAR of the quinazolinones to be notypic assay (rather than, as previously,3 a biophysical assays).
significantly expanded (Fig. 3).6 We found that a –CH2CH2– linker An array of 220 reactions enabled efficient exploration of the
(as in 8d and 8f–h) could be replaced by a trans-disubstituted function of products based on several alternative product
cyclopropane (as in 8e); however, reactions involving dehomolo- scaffolds. Based on our analysis of reaction outcomes by LC-
gated (e.g. S6) or truncated (e.g. S4) substrates, or those offering MS, and the range of substrates used, we estimate that most of
alternative reaction pathways (e.g. S2, S3, S5, S8 and S9), did not these reactions productively yielded previously unexplored
Chem. Commun.
This journal is © The Royal Society of Chemistry 2020

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
View Article Online
Communication
ChemComm
Table 2 Scale-up of reactions and evaluation of the activity of products against three S. aureus strains (ATCC29213, USA300 JE2, SH1000). The range of
MICs observed obtained in duplicate on three different days is shown for each strain. Penicillin G displayed MIC values as expected in all experiments
MIC (mg mLÀ1
Hit? Substrates Producta (yieldb) ATCC29213 USA300 JE2 SH1000
)
|
|
|
|
|
|
‘
S1, C1
8b (19%)
8d (4%)
8e (2%)
8f (8%)
8g (8%)
8h (14%)
8i (21%)
9 (5%)
0.5–1
0.016
1–2
1
2–4
0.5
4–8
4128
4128
0.5
0.016
2
0.5–1
2–4
0.5
4–8
64
4
S1, C18
S7, C18
S10, C1
S10, C6
S10, C18
S7, C1
0.5–1
32–64
4–8
16
1–2
16
32–64
4128
‘
S4, C9
S7, aniline 8j (18%)
‘
4128
a
b
Co-Substrate (1.2 eq.), Mo(CO)6 (1 eq.), 0.1 mol% Pd2bda3, 0.6 mol% P(tBu3)ÁHBF4, NEt3, o-xylene. After mass-directed HPLC. All compounds
were also screened against yeast (Candida albicans, Ca6) and were found to be inactive at 16 mg mLÀ1
.
J. Med. Chem., 2016, 59, 4443–4458; (c) T. W. Cooper, I. B. Campbell
and S. J. Macdonald, Angew. Chem., Int. Ed., 2010, 49, 8082–8091.
2 (a) N. Schneider, D. M. Lowe, R. A. Sayle, M. A. Tarselli and
G. A. Landrum, J. Med. Chem., 2016, 59, 4385–4402; (b) W. P. Walters,
J. Green, J. R. Weiss and M. A. Murcko, J. Med. Chem., 2011, 54,
6405–6416.
3 (a) G. Karageorgis, S. Warriner and A. Nelson, Nat. Chem., 2014, 6,
872–876; (b) G. Karageorgis, M. Dow, A. Aimon, S. Warriner and
A. Nelson, Angew. Chem., 2015, 127, 13742–13748; (c) A. Green,
F. Hobor, C. Tinworth, S. Warriner, A. Wilson and A. Nelson, Chem.
– Eur. J., 2020, DOI: 10.1002/chem.202002153.
Fig. 3 SAR expanded using ADS.
4 L. Åkerbladh, L. R. Odell and M. Larhed, Synlett, 2019, 141–155.
5 J. N. Pendleton, S. P. Gorman and B. F. Gilmore, Expert Rev. Anti-Infect.
Ther., 2013, 11, 297–308.
6 (a) R. Bouley, D. Ding, Z. Peng, M. Bastian, E. Lastochkin, W. Song,
M. A. Suckow, V. A. Schroeder, W. R. Wolter, S. Mobashery and
M. Chang, J. Med. Chem., 2016, 59, 5011–5021; (b) S. Gatadi, T. V.
Lakshmi and S. Nanduri, Eur. J. Med. Chem., 2019, 170, 157–172;
(c) Y. Qian, et al., J. Med. Chem., 2020, 63, 5287–5296.
products. Scale-up of the identified hit reactions, and purifica-
tion, yielded a range of antibacterial quinazolinones and,
thereby, expansion of their SAR. We envisage that, by harness-
ing alternative reaction classes to explore diverse chemical
space, ADS may be exploited in the discovery of novel series of
antimicrobials not subject to existing antibiotic resistance
mechanisms.11
7 J.-B. Peng, H.-Q. Geng, W. Wang, X. Qi, J. Ying and X.-F. Wu,
J. Catal., 2018, 365, 10–13.
8 F. R. Cockerill III, M. A. Wikler, J. Alder, M. N. Dudley, G. M. Eliopoulos,
M. J. Ferraro, D. J. Hardy, D. W. Hecht, J. A. Hindler, J. B. Patel,
M. Powell, J. M. Swenson, R. B. Thomson Jr., M. Traczewski,
J. D. Turnidge, M. P. Weinstein and B. L. Zimmer, CLSI, M07-A9:
Methods for dilution antimicrobial susceptibility tests for bacteria that grow
aerobically, 9th edn, Clinical and Laboratory Standards Institute, 2012.
9 D. J. Farrell, M. Robbins, W. Rhys-Williams and W. G. Love, Anti-
microb. Agents Chemother., 2010, 55, 1177–1181.
We thank EPSRC (EP/N025652/1) for funding and Luiza Galar-
ion, Merianne Mohamad and Adam Green for useful discussions.
Conflicts of interest
10 (a) S. Mannathan, S. Raoufmoghaddam, J. N. H. Reek, J. G. de Vries
and A. J. Minnaard, ChemCatChem, 2017, 9, 551–554; (b) A. Minatti,
X. Zheng and S. L. Buchwald, J. Org. Chem., 2007, 72, 9253–9258.
11 (a) P. Vikesland, E. Garner, S. Gupta, S. Kang, A. Maile-Moskowitz and
N. Zhu, Acc. Chem. Res., 2019, 52, 916–924; (b) N. A. Turner, B. K. Sharma-
Kuinkel, S. A. Maskarinec, E. M. Eichenberger, P. P. Shah, M. Carugati,
T. L. Holland and V. G. Fowler, Nat. Rev. Microbiol., 2019, 17, 203–218.
There are no conflicts to declare.
Notes and references
¨
1 (a) J. Bostrom, D. G. Brown, R. J. Young and G. M. Kereru¨, Nat. Rev.
¨
Drug Discovery, 2018, 17, 709–727; (b) D. G. Brown and J. Bostrom,
Chem. Commun.
This journal is © The Royal Society of Chemistry 2020