Identification and Profiling of Hydantoins - A Novel Class of Potent Antimycobacterial DprE1 Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.8b01356

Tuberculosis is the leading cause of death worldwide from infectious diseases. With the development of drug-resistant strains of Mycobacterium tuberculosis, there is an acute need for new medicines with novel modes of action. Herein, we report the discovery and profiling of a novel hydantoin-based family of antimycobacterial inhibitors of the decaprenylphospho-β-d-ribofuranose 2-oxidase (DprE1). In this study, we have prepared a library of more than a 100 compounds and evaluated them for their biological and physicochemical properties. The series is characterized by high enzymatic and whole-cell activity, low cytotoxicity, and a good overall physicochemical profile. In addition, we show that the series acts via reversible inhibition of the DprE1 enzyme. Overall, the novel compound family forms an attractive base for progression to further stages of optimization and may provide a promising drug candidate in the future.

Full text of DOI:10.1021/acs.jmedchem.8b01356

Subscriber access provided by Gothenburg University Library
Article
Identification and profiling of hydantoins - a novel
class of potent antimycobacterial DprE1 inhibitors
Maciej K. Rogacki, Eleni Pitta, Olga B Balabon, Sophie Huss, Eva Maria Lopez-Roman, Argyrides
Argyrou, Delia Blanco-Ruano, Monica Cacho, Christophe M.L. Vande Velde, Koen Augustyns,
Lluis Ballell, David Barros, Robert Bates, Fraser Cunningham, and Pieter Van der Veken
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01356 • Publication Date (Web): 30 Nov 2018
Downloaded from http://pubs.acs.org on November 30, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 103
Journal of Medicinal Chemistry
1
2
3
4
5
6
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
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
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 103
1
2
3
4
5
6
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
Identification and profiling of hydantoins - a novel
class of potent antimycobacterial DprE1 inhibitors
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
Maciej K. Rogacki,†‡⊥ Eleni Pitta,†‡⊥ Olga Balabon,†‡⊥ Sophie Huss,‡ Eva Maria Lopez-Roman,‡
#
‡
‡
§
Argyrides Argyrou, Delia Blanco-Ruano, Monica Cacho, Christophe M. L. Vande Velde,
†
‡
‡
‡
‡*
Koen Augustyns, Lluis Ballell, David Barros, Robert H. Bates, Fraser Cunningham, Pieter
Van der Veken†,*
†Laboratory of Medicinal Chemistry, Department of Pharmaceutical Sciences, University of
Antwerp, Universitieitsplein 1, 2610 Wilrijk, Belgium
‡Diseases of the Developing World (DDW), Tres Cantos Medicines Development Campus
(
TCMDC), GlaxoSmithKline, Severo Ochoa 2, 28760 Tres Cantos, Madrid, Spain
#Platform Technology and Science, GlaxoSmithKline, Stevenage, SG1 2NY, UK
§Faculty of Applied Engineering, Advanced Reactor Technology, University of Antwerp,
Groenenborgerlaan 171, 2020 Antwerpen, Belgium
⊥These authors contributed equally
Keywords: tuberculosis, mycobacterium, anti-mycobacterial, hydantoin, DprE1, drug discovery,
inhibitor.
ACS Paragon Plus Environment
1

Page 3 of 103
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
ABSTRACT
Tuberculosis is the leading cause of death worldwide from infectious diseases. With the
development of drug-resistant strains of Mycobacterium tuberculosis, there is an acute need for
new medicines with novel modes of action. Herein we are reporting the discovery and profiling of
a novel hydantoin-based family of antimycobacterial inhibitors of the decaprenylphospho-beta-D-
ribofuranose 2-oxidase (DprE1). In this study we have prepared a library of more than a 100
compounds and evaluated them for their biological and physicochemical properties. The series is
characterized by high enzymatic and whole-cell activity, low cytotoxicity and a good overall
physicochemical profile. Additionally we show that the series acts via reversible inhibition of the
DprE1 enzyme. Overall, the novel compound family forms an attractive base for progression to
further stages of optimization and may provide a promising drug candidate in the future.
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
INTRODUCTION
Tuberculosis (TB) is a disease caused primarily by the pathogen Mycobacterium tuberculosis.
According to the World Health Organization, TB is the world’s most deadly infectious disease
1
with over 10 million new cases and 1.8 million TB-related deaths in 2015 alone. The current first-
choice therapy regimen still consists of drugs identified more than 60 years ago and requires
administration of medicines for at least 6 months, even in the case of less severe infections. Long
treatment regimens and pronounced side effects lead to poor patient compliance and have
accelerated the emergence of drug-resistant strains of mycobacteria. With the WHO estimation of
1
over 0.5 million multi-drug resistant cases developing in 2015 there is an acute need for new
medicines with novel modes of action. Amongst the new potential bacterial targets that recently
gained increased attention is the decaprenylphospho-beta-D-ribofuranose 2-oxidase (DprE1), a
ACS Paragon Plus Environment
2

Journal of Medicinal Chemistry
Page 4 of 103
1
2
3
4
5
6
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
2
periplasmic protein involved in the biosynthesis of the mycobacterial cell wall. The enzyme is a
flavoprotein catalyzing the first step in the redox epimerization of decaprenylphosphoryl-D-ribose.
This epimerization process produces decaprenylphosphoryl-arabinose (DPA). DPA in turn is the
biochemical donor of arabinofurnanose residues during the biosynthesis of critical constituents of
the mycobacterial cell wall. More specifically, biosynthesis of lipoarabinomannan and the
peptidoglycan-arabinogalactone-mycolic acid complex depends on the availability of DPA and,
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
3
hence, on DprE1 activity. DprE1 gained recognition during investigation of the benzothiazinone
4,5
series (BTZ), a class of covalent suicide inhibitors. Several groups have later on contributed to
the validation of DprE1 as an attractive drug target, generated insight in the binding mode and
have reported multiple, structurally distinct compound series with either a covalent/irreversible or
a non-covalent binding mode. A very exhaustive review covering all relevant DprE1-inhibitor
6
,7
literature to date was recently published in this journal. Noteworthy, the most advanced DprE1
inhibitors (benzothiazinones BTZ043, PBTZ-169/macozinone and the azaindole AZ7371) have
recently entered the clinical development phase.8,9,10 (Figure 1) Sparked by the promising
antimycobacterial profile of reported compounds, a target-based high-throughput screening
campaign for inhibitors of DprE1 was performed by GlaxoSmithKline, leading to identification of
a novel hydantoin-based hit scaffold. This report for the first time discloses the structure of the
scaffold, wich is unrelated to other known DprE1-ligands. In addition, research in the report aims
at optimization of the hits, at generating insight in the Structure-Activity-Relationships (SARs)
governing antimycobacterial potency, and at providing evidence that activity is effected through
DprE1-inhibition. Finally, with the extensive biological and physicochemical profiling data we
report, we wish to underscore the potential of the hydantoin scaffold for antimycobacterial drug
discovery .
ACS Paragon Plus Environment
3

Page 5 of 103
Journal of Medicinal Chemistry
1
2
3
4
5
6
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
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
Figure 1. Selected reported DprE1 inhibitors.2, 8, 10-12
RESULTS AND DISCUSSION
Chemistry and SAR. The hydantoin hit compound, 1 (Figure 2), appeared especially interesting
due to high enzymatic inhibition (DprE1 pIC50 = 7.0) and good whole-cell activity (MIC = 8.3
µM). In addition to a promising activity profile, the compound was characterized by good
solubility, acceptable lipophilicity and no detectable cytotoxicity (Table 1). Overall, 1 presented a
good starting point for hit-to-lead optimization. Subsequent similarity-based clustering revealed
1
0 additional analogues showing detectable inhibition of the enzyme (see Table 1 and supporting
information: Table S1). In order to organize further exploration of the series, the general structure
common for all of the compounds was divided into 5 main parts as shown in Figure 2. During our
initial assessment of 1 we focused on identifying the moieties and structural features of the scaffold
that might represent potential liabilities for the series. Structure-activity relationship (SAR) data
provided by this cluster was limited due to the small number of closely related structures. However,
several compounds retained significant inhibitory activity (pIC50 > 6, Table 1), suggesting some
possibility of modifications around rings A and B as well as the side-chain. All hit compounds
preserved the hydantoin core as well as the acetyl linker indicating the potential importance of
those fragments. At this stage the hit 1, as well as all other analogues, were tested as racemates.
Therefore, in order to better understand if one or both of the enantiomers contributed to the potency
we prepared the racemic mixture of 1 via the Bucherer-Berg hydantoin cyclisation13 and
ACS Paragon Plus Environment
4

Journal of Medicinal Chemistry
Page 6 of 103
1
2
3
4
5
6
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
subsequent alkylation with the corresponding alpha-chloroketone 6 as shown in Scheme 1. Under
these conditions alkylation occurred selectively on the N -nitrogen, providing the desired
3
regioisomer as confirmed by crystal structure analysis (Figure 3). The enantiomers 7 and 8 were
then separated by chiral HPLC. We were pleased to see that only the R-isomer contributed to both
the enzymatic and whole-cell activity (Table 1). Although, with this information in hand, for
procedural simplicity most compounds were prepared and tested as racemates.
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
Figure 2. Representative hits from the hydantoin cluster.
Scheme 1. Preparation of hit 1 and its R and S enantiomers 7 and 8a
ACS Paragon Plus Environment
5

Page 7 of 103
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
aReagents and conditions: (a) KCN, (NH
8h; (c) separation via chiral HPLC.
)
CO
, EtOH, H
O, 55°C, 18h; (b) K
CO , acetone, rt,
4
2
3
2
2
3
1
Table 1. Biological and biochemical profile of hits 1-4 and enantiomers 7 and 8.
Compound
1
2
3
4
7
8
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
DprE1 pIC50 [a]
MIC (µM) [b]
7.0
8.3
>100
202
6.8
6.4
4.4
7.2
4.2
>80
49
40
>80
n.d. [f]
n.d. [f]
n.d. [f]
6.7
>125
>100
344
4.51
HepG2 IC50 (µM) [c]
Solubility (µM) [d]
88
>100
355
4.51
n.d. [f]
n.d. [f]
n.d. [f]
n.d. [f]
ChromlogD (pH 7.4) [e] 4.54
aDprE1 pIC50 is the negative logarithm of the IC50-concentration expressed in M (molar)
obtained in the DprE1-inhibition assay; MIC against Mycobacterium tuberculosis (H37Rv),
b
c
isoniazid was used as a reference with MIC= 1.8 M; cytotoxicity against HepG2 human
d
e
caucasian hepatocyte carcinoma; kinetic aqueous solubility (CLND); lipophilicity - chromlogD
f
at pH = 7.4; n.d. = not determined.
Figure 3. Asymmetric unit of 1. Anisotropic displacement ellipsoids drawn at the 50% level.
Hydrogens are represented by spheres of arbitrary diameter. The asymmetric unit contains two
molecules of opposite chirality, which are not related by crystallographic symmetry. They form a
hydrogen bonded pair through the NH…O hydrogen bonds between the hydantoin moieties. The
ACS Paragon Plus Environment
6

Journal of Medicinal Chemistry
Page 8 of 103
1
2
3
4
5
6
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
2
,4 fluorine substituted phenyl ring is disordered by 180° ring flipping. One molecule in the asu
has a disorder 52:48 ratio, the other 63:37, reflecting the different environments that surround
them.
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
We next moved to exploring the distinct elements of the hit scaffold. We were concerned that
the acetyl linker could be a potential liability of the series. Aromatic ketones are generally
considered to be reactive groups and can contribute to increased metabolic instability as well as
1
4
possible non-specific covalent binding. Since the initial cluster did not provide information about
the possible modifications of that part, we decided to explore the importance of the linker. For this
purpose we envisaged modulating the linker length, as well as removal of the carbonyl group or
introduction of known bioisosteric substitutions. Since hits 2 and 3 preserved good, albeit lower
inhibitory activity, for some of the analogues we chose to include para-methoxyphenyl, and para-
chlorophenyl substituted hydantoin counterparts in addition to the main para-cyanophenyl to
probe the effect of alternative substitution and electron donation/withdrawal on the activity and
physicochemical properties of the series.
ACS Paragon Plus Environment
7

Page 9 of 103
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Scheme 2. Synthetic approach to analogues with linker modificationsa
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
aReagents and conditions: (a) K
c) K CO , DMF, 100°C, 2d; (d) EtOH, H
DMF, rt, overnight; (f) BH -THF, THF, rt, overnight; (g) (I) n-BuLi, Ts-Cl, THF, 0°C-rt, 1h20min,
(II) n-BuLi, THF, 0°C-rt, 40min, (III) 65°C, 3.5h, (IV) n-BuLi, Ts-Cl, THF, 0°C-rt, 1h20min; (h)
CO , DMF, 80°C-100°C, 48h.
CO
, acetone or DMF, rt, 18-48h; (b) DAST (neat), rt, 1 week;
SO , 80°C, overnight (e) paraformaldehyde, t-BuOK,
2
3
(
2
3
2
4
3
K
2
3
We prepared a number of analogues (24-40) with linker modifications via the alkylation of the
previously prepared compound 5 or available hydantoins 22 and 23, as shown in Scheme 2. The
starting alkyl halides or pseudohalides were either commercially available (6, 9-12) or prepared
15
16
using standard literature conditions (13-15). Additionally, geminal difluorination of the ketone
1
6, obtained by standard bromination of the 1-(2,4-difluorophenyl)ethan-1-one, led to 17, which
was further used to alkylate hydantoin 5 to provide the analogue 41. The oxetane 42 was prepared
17
following a 5-step synthesis. Briefly, esterification of 18, followed by hydroxymethylation and
ACS Paragon Plus Environment
8

Journal of Medicinal Chemistry
Page 10 of 103
1
2
3
4
5
6
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
in-situ deprotection gave the acid 19, which was reduced to the alcohol 20 with borane-THF
1
8
19
complex. One-pot mono-tosylation, ether formation and subsequent second tosylation of the
alcohol gave the oxetane 21, which was used to alkylate the hydantoin 5 and provide the final
product 42.
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
Scheme 3. Preparation of analogues 43a-b and 44a
aReagents and conditions: (a) HONH
HCl, MW, 100°C, 30min; (b) Red-Al, toluene, 120°C, 1h.
2
Alternatively, the ketone of the parent compounds 1 and 24 could be modified as shown on
Scheme 3. Thus, microwave irradiation of a solution of 1 and hydroxylamine led to formation of
2
0
21
two isomeric oximes (43a and b). Lastly, Red-Al mediated reduction of 24 provided alcohol
44 as a diastereoisomeric mixture.
Throughout the synthesis of this series of analogues, we noticed that the alkylation of hydantoins
consistently provided 3-substituted products (24-42) in moderate to good yields. However, inverse
regioselectivity was observed in reactions of the acyl chloride 45 with hydantoins, leading to N -
1
ACS Paragon Plus Environment
9

Page 11 of 103
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
substituted isomers 46 and 47 (Scheme 4). The desired 3-acylated analogue could be prepared by
deprotonation of the hydantoin prior to the addition of the acyl chloride, however the imide 48
proved unstable in solution over time.
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
Scheme 4. Preparation of acylated hydantoinsa
aReagents and conditions: (a) pyridine, rt, overnight; (b) NaH, DMF, rt, overnight.
ACS Paragon Plus Environment
1
0

Journal of Medicinal Chemistry
Page 12 of 103
1
2
3
4
5
6
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
Table 2. In vitro activity, cytotoxicity and physicochemical properties of the analogues with linker
variation.
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
DprE1
pIC50
Mtb MIC HepG2 IC50 Solubility Chrom
[
a]
[b]
[c]
[d]
[e]
№
1
R
1
linker
R
2
(µM)
(µM)
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
63
(µM)
202
358
17
logD
4.54
4.78
5.54
4.26
4.53
5.28
4.56
4.84
5.68
4.50
4.72
5.59
4.40
4.60
5.35
CN
OCH
Cl
7.0
8.3
24
25
26
27
28
F
3
3
3
3
3
6.7
40
6.7
>40
CN
OCH
Cl
6.0
19.6
62
132
260
7
H
H
H
H
6.0
4.2
>125
125
2
3
3
3
3
3
3
3
3
9
0
1
2
3
4
5
6
7
CN
OCH
Cl
<4.0
<4.0
<4.0
<4.0
<4.0
<4.0
4.3
185
327
19
>125
>125
>125
>125
>125
>125
>125
>125
CN
OCH
Cl
≥404
≥381
188
≥428
≥409
222
CN
OCH
Cl
>100
>100
>100
4.4
<4.0
3
3
8
9
F
F
CN
CN
5.40
4.60
80
>100
>100
≥458
314
4.83
4.33
>80
ACS Paragon Plus Environment
1
1

Page 13 of 103
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
4
4
0
1
F
F
CN
CN
4.4
5.0
>125
>80
>100
>100
n.d.[f]
≥261
n.d.[f]
5.1
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
4
2
F
CN
CN
<4.0
>80
>100
295
4.21
4
3a
3b
4.0
4.2
>80
>80
>100
>100
≥480
≥471
3.38
3.59
F
F
4
4
4
4
4
6
7
OCH
OCH
Cl
3
4.2
>80
>100
>100
>100
287
3.96
3.43
3.72
3
<4.0
<4.0
>40
≥484
≥386
>125
aDprE1 pIC50 is the negative logarithm of the IC50-concentration expressed in M (molar)
obtained in the DprE1-inhibition assay; MIC against Mycobacterium tuberculosis (H37Rv),
isoniazid was used as a reference with MIC= 1.8 M; cytotoxicity against HepG2 human
caucasian hepatocyte carcinoma; kinetic aqueous solubility (CLND); lipophilicity - chromlogD
b
c
d
e
f
at pH = 7.4; n.d. = not determined.
As shown in the Table 2, most modifications, such as modulation of linker length or removal of
the keto group resulted in a complete loss of activity. Even methylation of the methylene group
(38) led to 40-fold decrease of enzymatic potency. Surprisingly, even closely related analogues
and bioisosteric transformations of the carbonyl moiety (41-43) did not retain activity, pointing to
specific importance of that group. It is also worth noting that 44 could be expected to be one of the
direct metabolites of the parent compound, therefore its lack of activity was particularly
interesting.
ACS Paragon Plus Environment
1
2

Journal of Medicinal Chemistry
Page 14 of 103
1
2
3
4
5
6
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
Although all applied modifications led to significant loss of potency, it was encouraging to see
the consistently low toxicity throughout the series of analogues. Except for a few more lipophilic
representatives bearing chlorine substitutions (25, 28, 31), we also observed consistently good
solubility.
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
Scheme 5. Preparation of analogues with modifications around ring A and the side-chaina
aReagents and conditions: (a) KCN, (NH
DMF, rt, 18h.
)
CO
, EtOH, H
O, 55°C, 18h; (b) K
CO , acetone or
4
2
3
2
2
3
Next, we moved to exploring substituents at the C -carbon of the hydantoin ring. Our attention
5
was drawn by the nitrile in the position 4 of the “ring A”. Carbonitrile moieties have been generally
reported and/or predicted to be able to react with cysteine22,23 and/or serine24,25 residues which
could lead to potential off-target covalent binding. Comparison of 1, 4, 24 and 25 suggested the
importance of the nitrile part, since substitution with a methoxy group (24) or a chlorine atom (25)
led to a decrease in potency and lack of any group on the phenyl ring (4) provided a completely
inactive compound. We therefore put our main focus on exploring the possible substitutions at that
position. We also decided to introduce substituents at carbons 2 and 3 of ring A to probe the space
available for further modifications. Additionally we were interested in exchanging the phenyl ring
for a heterocyclic moiety and its impact on the activity as well as physicochemical properties of
the series.
ACS Paragon Plus Environment
1
3

Page 15 of 103
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Most compounds were synthesized following the Bucherer-Berg hydantoin cyclisation and
subsequent alkylation (Scheme 5), starting from ketones that were either commercially available
or were prepared using standard literature procedures (see Supporting Information). Synthesis of
hydantoin 139 was also attempted using the same approach, however the sulfonamide moiety
appeared to be reactive during the alkylation step. We therefore decided to prepare 139 and 140
from the bromo-analogues 109 and 123 respectively, by palladium-catalyzed introduction of
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
2
6
methanesulphonamide as shown on Scheme 6. Anticipating similar reactivity problems, aniline
1
1
41 was obtained by acid catalyzed hydrolysis of the amide 119. Finally, nitrile analogues 142 and
2
7
43 were prepared by microwave-promoted, palladium-catalyzed cyanation of the corresponding
bromopyridines 130 and 131.
ACS Paragon Plus Environment
1
4

Journal of Medicinal Chemistry
Page 16 of 103
1
2
3
4
5
6
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
Scheme 6. Preparation of analogues with modifications around ring A and the side-chaina
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
aReagents and conditions: (a) CH
EtOH, HCl, 65°C, 3d; (c) Zn(CN) , Pd
SO
(dba)
NH
, K
CO
, [Pd(allyl)Cl] , t-BuXPhos, 80°C, 4h; (b)
3
2
2
2
3
2
2
2
3
, XantPhos, TMEDA, DMF, MW, 160°C, 5min.
Although initial substitutions at “ring A” led to less active compounds (24, 25), we were pleased
to observe that the nitrile group is not necessary and in fact can be exchanged for a variety of
groups providing similarly or even more potent analogues, such as 114, 116 or 139 (Table 3). It
ACS Paragon Plus Environment
1
5

Page 17 of 103
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
was also interesting to note that the high activity is maintained in the case of slightly bulkier
substituents, such as a tetrazole or a morpholine (116, 117), which suggests that the enzymatic
pocket could accommodate further expansion of the molecule in that direction. The obtained SAR
data also showed that although some modifications are permitted at position 2 of the phenyl ring
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
(
125, 126, 131, 143), substitutions at position 3 (124, 127, 130, 140, 142) generally gave a more
pronounced loss of activity (Table 4), suggesting rather limited space around the aromatic ring.
We were especially encouraged by the results obtained from the pyridine analogues 131 and 143,
which indicated that the phenyl ring A can be exchanged for a heterocyclic analogue, improving
physicochemical properties of the compound. However the possibility of manipulation may prove
to be limited, since substitution with a 5-membered heterocyclic analogue 132 led to a complete
loss of both enzymatic as well as whole-cell activity.
Although some of the most potent compounds (109, 113 and 114) were unfortunately linked to
higher lipophilicity (and subsequently reduced solubility), good enzymatic and whole-cell
potencies were also observed for the soluble and less lipophilic analogues such as the tetrazole 116
and the sulfonamide 139.
It should be emphasized, that even for analogues with increased lipophilicity, no appreciable
cytotoxicity was found throughout the series of modifications, indicating a promising safety profile
of the investigated series.
ACS Paragon Plus Environment
1
6

Journal of Medicinal Chemistry
Page 18 of 103
1
2
3
4
5
6
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
Table 3. In vitro activity, cytotoxicity and physicochemical properties of the analogues with
varying substituents at the 4-position of ring A.
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
DprE1 Mtb MIC HepG2 IC50 Solubility Chrom
[a]
[b]
[c]
[d]
[e]
№
R
pIC50
(µM)
8.3
40
(µM)
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
(µM)
202
358
17
logD
4.54
4.78
5.54
5.68
5.41
5.43
6.01
6.05
5.36
3.84
3.78
4.45
1
-CN
-OCH
-Cl
7
2
2
1
1
1
1
1
1
1
1
1
4
5
3
6.7
6.7
7.1
6.1
7.0
6.9
7.3
7.4
6.5
7.3
7.1
>40
20
09 -Br
10 -CH
11 -OC
4
3
2
80
213
167
43
H
5
20
12 -CF
13 -OCF
14 -OCHF
3
20
3
35
4
2
10
85
15 -(imidazol-1-yl)
16 -(tetrazol-1-yl)
17 -(N-morpholine)
20
280
379
277
3.1
10
1
18
6.6
5.6
>100
359
3.94
1
1
1
1
19 -NHCOCH
20 -SO CH
39 -NHSO
41 -NH
3
5.6
6.8
7.0
4.1
40
>100
>100
>100
>100
≥489
≥461
≥487
≥436
3.28
3.64
3.57
3.38
2
3
10
2
CH
3
2.5
>80
2
aDprE1 pIC50 is the negative logarithm of the IC50-concentration expressed in M (molar)
b
obtained in the DprE1-inhibition assay; MIC against Mycobacterium tuberculosis (H37Rv),
c
isoniazid was used as a reference with MIC= 1.8 M; cytotoxicity against HepG2 human
d
e
caucasian hepatocyte carcinoma; kinetic aqueous solubility (CLND); lipophilicity - chromlogD
at pH = 7.4.
ACS Paragon Plus Environment
1
7

Page 19 of 103
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Interestingly, we observed that within the prepared series of compounds, higher enzymatic
potency did not always translate into an improvement in MIC values, as seen in case of fluorinated
methoxy (113, 114) or bromine-bearing (109) analogues. It is worth noting that these molecules
are characterized by higher lipophilicity as opposed to compounds showing an increase also in
whole-cell potency (116, 139), with ChromLogD values of 5.30-6.05 and 3.57-3.78 respectively.
However, comparison of the pair 109 and 131 showed that lowering the ChromLogD value itself
did not lead to an improvement in whole-cell potency in case of analogues with high enzymatic
activity. This suggests that the overall lipohilicity of the molecule is not the driving factor behind
the higher than expected MIC values. One could argue that the improved MIC potencies were a
result of issues with borderline solubility of some compounds, such as 109 or 113. However, as
seen from the bromine-bearing analogues 109 and 131, significant improvement of solubility did
not translate into a substantial increase of whole-cell potency.
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
Table 4. In vitro activity, cytotoxicity and physicochemical properties of the analogues with
modifications of the ring A.
DprE1 Mtb MIC HepG2 IC50 Solubility Chrom
[
a]
[b]
[c]
[d]
[e]
№
R
pIC50
(µM)
(µM)
(µM)
logD
121
4.4
>80
>100
312
4.51
122
123
<4.0
4.5
>80
80
>100
>100
285
68
4.96
5.72
ACS Paragon Plus Environment
1
8

Journal of Medicinal Chemistry
Page 20 of 103
1
2
3
4
5
6
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
1
1
24
25
5.7
>80
>100
147
4.84
6.5
6.7
6.9
58
30
80
>100
>100
>100
104
17
5.05
5.80
5.50
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
126
127
106
1
28
<4.0
>80
>100
287
4.47
1
1
1
29
30
31
4.4
6.4
7.3
>125
40
>100
>100
>100
≥462
369
3.04
4.67
4.87
15
100
1
32
<4.0
4.2
>80
>80
>100
>100
417
4.67
3.67
140
≥369
1
1
42
43
6.0
6.6
20
10
>100
>100
≥410
≥474
3.94
3.97
aDprE1 pIC50 is the negative logarithm of the IC50-concentration expressed in M (molar)
obtained in the DprE1-inhibition assay; MIC against Mycobacterium tuberculosis (H37Rv),
b
c
isoniazid was used as a reference with MIC= 1.8 M; cytotoxicity against HepG2 human
d
e
caucasian hepatocyte carcinoma; kinetic aqueous solubility (CLND); lipophilicity - chromlogD
at pH = 7.4.
While exploring the SAR around Ring A, we also decided to use the same synthetic approach
(Scheme 5) to probe the possibility of manipulating the hydantoin side-chain (Table 5). However,
ACS Paragon Plus Environment
1
9

Page 21 of 103
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
we found that only ethyl analogues (133, 134) retained high activity as compared to their methyl-
bearing counterparts (109 and 1 respectively). Further extension of the chain (135) or introduction
of a bulky substituent (136, 137) resulted in significantly less potent compounds, suggesting very
limited chemical space available around that part of the scaffold.
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
Table 5. In vitro activity, cytotoxicity and physicochemical properties of the analogues with
modifications of the side-chain.
DprE1 Mtb MIC HepG2 IC50 Solubility Chrom
[
a]
[b]
[d]
[e]
№
R
1
R
2
pIC50
(µM)
(µM)
(µM)
logD
6.17
4.99
6.64
6.57
6.80
1
1
1
1
1
33 ethyl
-Br
6.9
10
56
31
34 ethyl
-CN 7.3
5
>100
51
154
7
35 propyl
36 isopropyl
-Br
-Br
-Br
6.2
4.7
5.1
80
>80
>80
48
11
37 -CF
3
50
1
1
38
6.7
>80
>100
<1
5.71
aDprE1 pIC50 is the negative logarithm of the IC50-concentration expressed in M (molar)
b
obtained in the DprE1-inhibition assay; MIC against Mycobacterium tuberculosis (H37Rv),
c
isoniazid was used as a reference with MIC= 1.8 M; cytotoxicity against HepG2 human
d
e
caucasian hepatocyte carcinoma; kinetic aqueous solubility (CLND); lipophilicity - chromlogD
at pH = 7.4.
Next, we turned our attention to the hydantoin core. Although attractive from the synthetic
perspective and present in marketed drugs, this heterocycle has been linked to some undesired
2
8
29
side-effects, such as the congenital hydantoin syndrome or inhibition of the hERG potassium
channel.30,31 Cardiovascular risks are considered a major liability for further development of a lead
ACS Paragon Plus Environment
2
0

Journal of Medicinal Chemistry
Page 22 of 103
1
2
3
4
5
6
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
series, we were therefore interested in finding a suitable replacement for the core. Additionally,
since the hydantoin core can serve as a platform for formation of multiple hydrogen bonds, we
planned stepwise modifications to evaluate the role of the heterocycle in interacting with the
DprE1 enzyme.
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
Some close analogues of hydantoin are succinimide, imidazolidin-2-one and γ-lactam rings. We
considered these heterocycles to be attractive alternatives since they lack one or more hydrogen
bond donors/acceptors while maintaining similar overall geometry of the original hit scaffold.
Analogues built around these cores were prepared as shown on Scheme 7. The succinimide ring
32
of 149 was constructed via the approach described by Tominaga et al., starting with a reaction of
4-bromoacetonitrile and a commercially available nitroketene dithioacetal, followed by cyclisation
to 145. Subsequent acid-catalyzed hydrolysis of the oxime and further reduction with zinc
provided the succinimide 147. Alkylation of 147 with a haloketone provided 148, which was
further methylated to yield the desired product 149. The cyclic urea was obtained from hydantoin
21
2
4 by Red-Al mediated reduction, as reported by von Kieseritzky and Lindström. Under these
conditions the carbonyl group in the linker was reduced to an alcohol, which was subsequently
3
3
oxidized using the Swern reaction. The resulting mixture was purified by chiral HPLC, providing
pure enantiomers 151a and 151b. Finally, the lactam 155 was synthesized starting from 2-(4-
bromophenyl)acetic acid. Briefly, the acid was protected with a methyl ester and alkylated first
with methyl iodide and then with bromoacetonitrile. Resulting ester 153, was cyclized to 154 under
3
4
reductive conditions with sodium borohydride in the presence of cobalt chloride. Alkylation of
1
54 yielded the final lactam 155.
To further evaluate the role of N
1
-nitrogen, we decided to prepare analogues 156 and 157 by
simple methylation or acylation of the hit 1, as shown in Scheme 8.
ACS Paragon Plus Environment
2
1

Page 23 of 103
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Finally, we wanted to probe the possibility of a more aggressive scaffold hopping approach by
replacing the original core with an aromatic heterocycle. For that purpose, we envisaged imidazole
as a direct analogue of a hydantoin cycle. The aromatic analogues were obtained as shown in
Scheme 9. The 4-substituted imidazoles 158 and 159 were constructed by cyclisation of the
corresponding bromoketones with formamide.35 Subsequent alkylation with chloroketone 6
provided analogues 160 and 161. Additionally, we also prepared an analogue in which the core is
be replaced by a pyrazole. This compound was synthesised starting from a commercially available
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
4
4
-bromopyrazole (Scheme 9). After initial trityl protection, the intermediate was coupled with a
-cyanobenzeneboronic acid under Suzuki-Miyaura conditions.36 Acid-mediated deprotection
provided 162 which was further alkylated to obtain the final pyrazole analogue 163.
ACS Paragon Plus Environment
2
2

Journal of Medicinal Chemistry
Page 24 of 103
1
2
3
4
5
6
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
Scheme 7. Preparation of analogues with modified coresa
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
aReagents and conditions: (a) NaOH, DMSO, rt, 4h; (b) MeOH, reflux, 30min; (c) HCl, MeOH,
reflux, 5d; (d) Zn, AcOH, reflux, 2.5h; (e) 16, K
acetone, rt, 2d; (g) Red-Al, toluene, 120°C, 2h15min; (h) (I) oxalyl chloride, DMSO, DCM, -78°C,
5min, (II) TEA, rt, 2h; (i) H SO , MeOH, 80°C, overnight; (j) (I) diisopropylamine, n-BuLi, THF,
78°C, 20min, (II) MeI, 0°C, 30min; (k) (I) diisopropylamine, n-BuLi, THF, -78°C, 20min, (II)
bromoacetonitrile, -78°C, 1.5h - , 0°C, 30min; (l) NaBH4, CoCl x6H O, 0°C – rt, overnight; (m)
6, NaH, THF, rt, overnight.
2
CO
3
, acetone, rt, overnight; (f) MeI, K
2
CO
3
,
1
2
4
-
2
2
1
ACS Paragon Plus Environment
2
3

Page 25 of 103
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Scheme 8. Preparation of analogues with substituted N
1
-nitrogen.
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
aReagents and conditions: (a) K
CO
, acetone, rt, 2d; (b) AcCl, TEA, dioxane, rt, 3d.
2
3
Scheme 9. Preparation of analogues with aromatic cores.
aReagents and conditions: (a) formamide, 170°C, 2-3h; (b); K
CO , DMF, 0°C – rt, overnight; (c)
2
3
Cs
2
CO
3
, DMF, 80°C, 90min; (d) TrCl, DMF, 0°C, 1h; (e) (4-cyanophenyl)boronic acid, Pd(PPh
CO , DMF, MW, 60°C, 30min.
3
4
) ,
dioxane, 85°C, overnight; (f) TFA, 70°C, 1h; (g) 6, K
2
3
Table 6 summarizes the results obtained from the modifications of the core hydantoin. It was
disappointing to observe that none of the compounds tested was able to retain potency comparable
to the parent hydantoins. Almost complete loss of enzymatic activity of both ureas 151a and 151b
suggests that the carbonyl moiety at position 4 may take part in interactions that are crucial for the
ACS Paragon Plus Environment
2
4

Journal of Medicinal Chemistry
Page 26 of 103
1
2
3
4
5
6
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
potency of the series. Modifications around position 1 were also quite noteworthy. Both placing
substituents at the nitrogen (156, 157) as well as exchanging it for a carbon (149) led to a
significant decrease of enzymatic activity. This could suggest that the N -nitrogen atom is
1
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
important for binding to the enzyme. Interestingly enough, although methylation of this nitrogen
led to a complete loss of enzymatic activity (156), amide 157 retained whole cell potency
comparable to the original hit (MIC = 10 and 8.3 µM respectively). Since 157 appears to be
chemically stable in solution, one can speculate that it could be subject to hydrolysis by the
bacterial amidases and hence act like a prodrug.
Unsurprisingly, the aromatic analogues 160, 161 and 163 also did not retain potency.
Pronounced differences in the overall geometry of the final molecules as well as lack of important
hydrogen bond donors/acceptors were the likely culprits for this loss of activity and demonstrated
that in this case the imidazole and pyrazole heterocycles are not suitable candidates for a scaffold
hopping approach.
Taken together, these results indicate that the core hydantoin cycle not only serves as a linking
scaffold for proper spatial organization of the peripheral moieties, but seems to also be involved
in interactions with the protein that are crucial for the potency of the series.
ACS Paragon Plus Environment
2
5

Page 27 of 103
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 6. In vitro activity, cytotoxicity and physicochemical properties of the analogues with
modifications of core hydantoin.
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
DprE
pIC50 (µM)
1
Mtb MIC HepG2 IC50 Solubility Chrom
[a]
[b]
[d]
[e]
№
Core
R
(µM)
(µM)
logD
1
49
-Br
5.8
>80
>100
191
6.86
1
1
51a[f]
51b[f]
<4.0
4.5
>80
>80
>100
>100
88
4.72
4.72
-
OCH
3
177
1
55
-Br
5.1
4.9
>80
>80
>100
>100
89
6.74
5.22
156
-CN
167
1
57
-CN
4.5
10
>100
61
5.77
1
1
1
60
61
63
-CN
4.7
>125
>125
>125
>100
>100
>100
25
4.58
4.68
5.17
-OCH
3
<4.0
<4.0
352
21
-CN
aDprE1 pIC50 is the negative logarithm of the IC50-concentration expressed in M (molar)
b
obtained in the DprE1-inhibition assay; MIC against Mycobacterium tuberculosis (H37Rv),
c
isoniazid was used as a reference with MIC= 1.8 M; cytotoxicity against HepG2 human
d
e
caucasian hepatocyte carcinoma; kinetic aqueous solubility (CLND); lipophilicity - chromlogD
f
at pH = 7.4; separated enantiomers – absolute configuration was not determined.
ACS Paragon Plus Environment
2
6

Journal of Medicinal Chemistry
Page 28 of 103
1
2
3
4
5
6
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
Finally, we turned our attention to substitution around ring B. Structural features of that fragment
were not identified as liabilities, therefore we were mainly interested in probing the chemical space
available for manipulations and their contribution towards the SAR. Additionally we also
envisaged introduction of more polar groups that could help decrease the lipophilicity of the
molecule and improve the overall physicochemical profile of the series. For this exploration, in
addition to the hydantoin counterparts bearing the hit 4-cyano group, we decided to also include
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
the 4-OCHF substituted analogues. Since 114 showed the highest enzymatic activity without an
2
increase in the whole-cell potency, the difluoromethoxy analogues were prepared to further
explore this apparent disconnection.
Compounds 180-198 were prepared following the standard alkylation approach shown on
Scheme 10 from haloketones that were either commercially available or prepared using literature
procedures (see Supporting Information). Additionally, the library was expanded with molecules
199-209, that were available in GSK compound database (see Table 7).
Scheme 10. Preparation of analogues with modifications around ring B.
aReagents and conditions: (a) K
CO , acetone or DMF, rt, overnight.
2
3
As shown in Table 7, we observed that a number of modifications are permitted in that part of
molecule. Although the original 2,4-difluorophenyl ring from hit 1 remained amongst the most
active groups, single fluorine (180, 182) or methyl substituents (204) also provided analogues with
good enzymatic potency. Although introduction of more polar groups such as methoxy (184-187),
ACS Paragon Plus Environment
2
7

Page 29 of 103
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
nitro (189) or dioxole (182) usually led to less potent molecules, it was particularly interesting to
see that the phenyl ring can also be exchanged for some other aromatic heterocycles, such as
pyridines (192, 193) or a thiophene (206-207), leaving some potential for optimization of the
physicochemical properties without a significant loss of potency. It seemed however, that all
aliphatic substitutions (178-181) resulted in a more pronounced loss of activity indicating that an
aromatic moiety at ring B is important for the high potency of the compound. Taken together, these
results suggest that the SAR around Ring B is not confined to limited chemical space and ring B
can be modified during further optimization of the series.
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
Table 7. In vitro activity, cytotoxicity and physicochemical properties of the analogues with
modifications of ring B.
DprE1 Mtb MIC HepG2 IC50 Solubility Chrom
№
[
a]
[b]
[c]
[d]
[e]
pIC50
(µM)
(µM)
(µM)
logD
R
1
R
2
1
-CN
-OCHF
-CN
7
8.3
10
>100
>100
>100
>100
202
85
4.54
5.63
4.26
5.08
1
14
2
7.4
6.0
7.2
2
6
19.6
15
132
98
1
80
-OCHF
2
1
81
-OCHF
2
6.4
20
>100
144
5.13
1
1
82
83
-OCHF
2
2
7.3
7.2
20
30
>100
>100
141
180
5.23
4.97
-OCHF
1
1
84
85
-CN
5.7
6.2
>80
80
>100
>100
270
104
4.25
4.92
-OCHF
2
ACS Paragon Plus Environment
2
8