Mycobacterium tuberculosis Decaprenylphosphoryl-β- d -ribose Oxidase Inhibitors: Expeditious Reconstruction of Suboptimal Hits into a Series with Potent in Vivo Activity

Article abstract of DOI:10.1021/acs.jmedchem.9b01561

Decaprenylphosphoryl-β-d-ribose 2′-epimerase (DprE1) is an essential enzyme in Mycobacterium tuberculosis and has recently been studied as a potential drug target, with inhibitors progressing to clinical studies. Here we describe the identification of a n

Full text of DOI:10.1021/acs.jmedchem.9b01561

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Article
Mycobacterium tuberculosis Decaprenylphosphoryl-#-D-
ribose Oxidase inhibitors: expeditious reconstruction of
sub-optimal hits into a series with potent in vivo activity
Jennifer A Borthwick, Carlos Alemparte, Ian Wall, Benjamin C. Whitehurst, Argyrides
Argyrou, Glenn A. Burley, Paco de Dios-Anton, Laura Guijarro, Maria Candida Monteiro,
Fatima Ortega, Colin J. Suckling, Julia Castro-Pichel, Monica Cacho, and Robert J Young
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 10 Jan 2020
Downloaded from pubs.acs.org on January 11, 2020
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Mycobacterium tuberculosis Decaprenylphosphoryl-
β‑D‑ribose Oxidase inhibitors: expeditious
reconstruction of sub-optimal hits into a series with
potent in vivo activity
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†
‡
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†
Jennifer A. Borthwick *, Carlos Alemparte , Ian Wall , Benjamin C. Whitehurst , Argyrides
†
¶
‡
‡
‡
Argyrou , Glenn Burley , Paco de Dios-Anton , Laura Guijarro , Maria Candida Monteiro ,
‡
¶
‡
‡
Fatima Ortega , Colin J Suckling , Julia Castro Pichel , Monica Cacho *, Robert J.
Young†*
† Medicinal Science and Technology, GlaxoSmithKline, Gunnels Wood Road,
Stevenage SG1 2NY, UK
‡
Global Health R&D, GlaxoSmithKline, Severo Ochoa 2, Tres Cantos, 28760 Madrid,
Spain
¶ Department of Pure & Applied Chemistry, University of Strathclyde, 295 Cathedral
Street, Glasgow G1 1XL, Scotland
*jennifer.borthwick@blackbelttx.com
*monica.i.cacho@gsk.com
Rob@blue-burgundy.co.uk
*
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KEYWORDS: DprE1, tuberculosis, H2L, scaffold-hopping, fragment based design.
ABSTRACT
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Decaprenylphosphoryl-β-D-ribose 2’-epimerase (DprE1) is an essential enzyme in
Mycobacterium tuberculosis and has recently been studied as a potential drug target, with
inhibitors progressing to clinical studies. Here we describe the identification of a novel
series of morpholino-pyrimidine DprE1 inhibitors. These were derived from a phenotypic
high-throughput screening (HTS) hit with sub-optimal physicochemical properties.
Optimization strategies included scaffold-hopping, synthesis and evaluation of fragments
of the lead compounds and property-focussed optimization. The resulting optimized
compounds had much improved physicochemical properties and maintained enzyme and
cellular potency. These molecules demonstrated potent efficacy in an in vivo tuberculosis
murine infection model.
TEXT
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Introduction
Tuberculosis (TB) is the most deadly infectious disease in human history, killing over 1
billion people in the last 200 years and continues to kill at a rate of more than 1 million
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people per year. Currently treatment options for TB require patients to take a
2
combination of drugs for between 6 months and 3 years. Some of these drugs can
cause serious side-effects and many are increasingly becoming ineffective due to
3
evolving resistance patterns, including multidrug resistance (MDR), extremely (XDR)
and even totally drug resistant strains (TDR). Therefore, there is an urgent need to
develop new drug treatments for TB, especially those with novel mechanisms of action,
which would not be expected to suffer from the same resistance mechanisms as
marketed drugs.
Decaprenylphosphoryl-β-D-ribose 2’-epimerase (DprE1) was identified the target of a
series of potent bactericidal molecules, through genetic analysis of resistant strains
4
generated in vitro. DprE1 is the key enzyme in the only known biosynthetic pathway by
which mycobacteria produce arabinose, which is an essential component of the cell wall
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arabinogalactan scaffold. Furthermore, mycobacteria with reduced expression of
functional DprE1 were shown to be non-viable,6, 7 and amongst 240 clinically isolated
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strains of Mycobacterium tuberculosis (Mtb), no mutations were detected in the gene
encoding DprE1. This mechanism of arabinose biosynthesis via Decaprenylphosphoryl-
β-D-ribose is unique to mycobacteria, so there is inherent biochemical selectivity over
human cells and other bacterial species.8
A number of small-molecule inhibitors of DprE1 have now been published, reflecting the
high level of interest in this exciting target (Figure 1).4, 9-23 The covalent binders24
25
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BTZ043 and PBTZ169 (macozinone) have progressed to clinical evaluation, as have
27
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the non-covalent compounds AZ7371 and OPC-167832.
CH3
O
H3C
N
O
O
N
OH
OH
F3C
H3C
N
N
F3C
O
F
F
N
N
S
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N
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N
O
NO2
NH
NO2
N
O
O
N
F
Cl
O
H
OH
BTZ043
PBTZ169 macozinone
AZ7371
OPC-167832
Figure 1. Structures of selected published DprE1 inhibitors.4, 9, 23, 27, 28
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Pyrrolothiadiazole 1, one of 177 antitubercular compounds released by GSK was
identified as an inhibitor of DprE1 by its reduced activity in a DprE1 over-expressor
strain of M. tuberculosis.30 In this paper we describe optimization of 1 into a lead series
with an attractive physical profile and highly encouraging in vivo efficacy. We
demonstrate how the proposed “attention to decreasing lipophilicity and/or minimising
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aromatic ring count” was achieved through lead hopping. The progression and quality
of the molecules produced in the ensuing deconstruction and reconstruction process
was monitored using physicochemical measurements and efficiency metrics.31
Results and Discussion
To generate some initial structure-activity relationship (SAR) around initial hit,
pyrrolothiadiazole 1was truncated to 2, retaining reasonable ligand efficiency.
Subsequently, a key finding in the move away from the unattractive pyrrolothiadiazole
functionality was screening of compounds selected by a similarity search of the GSK
collection, based on Tanimoto scores. Of these, piperidinylpyrimidines 3 and 4 retained
DprE1 activity and showed a minimum inhibitory concentration (MIC). The most
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productive search used the piperidine-acyl-piperidine moiety as the substructure,
generating a range of alternatives for the thiadiazole, pyrrole and benzyl groups. These
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searches identified truncated compounds such as 2, which retained DprE1 activity in the
biochemical assay that translated into an MIC (Figure 2).30
N N
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Figure 2. Structure of phenotypic screening hit 1 and analogues identified by
substructure and similarity searching.
A key point in the hits to leads phase was securing DprE1 activity and an MIC with
piperidinopyrimidines 3 and 4 which had markedly improved physical properties (Table
2) and obviated perceived metabolic/toxicological risks with the pyrrole and thiadiazole
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rings (Figure 2). . The likely common SAR between the thiadiazole- and pyrimidine-
containing series was indicated by the benzyl analogues 1 and 5, but further
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physicochemical improvement was a clear priority. These data supported the notion of a
systematic exploration of the SAR and structure property relationships (SPR) in the
series, facilitated by a relatively straightforward synthetic protocol involving amide
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formation and S Ar elaboration of the aromatic heterocycle (Scheme 1). The strategy is
N
best described in terms of the five constituent rings of the structure, labelled A,B,C, D
and E for convenience; the execution including fragmentation to <20 heavy atom A-C
fragment structures to identify key binding interactions and regrowth along defined
vectors to optimize activity, property forecast index (PFI) and ligand efficiency.31
Compound
DprE1 pIC50a
Mtb H37Rv PFIc
Solubility CLND
(µM)d
MIC (µM)
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a
Table 1. Measured properties of hit 1 and analogues 2-5. DprE1 pIC is the negative
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logarithm of the IC -concentration expressed in molar (M) obtained in the DprE1-
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inhibition assay. DprE1 pIC values above 7.3 are approaching tight binding limit of assay
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test occasions. Property forecast index (PFI), is the sum of the Chrom log D and the
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number of aromatic rings. Kinetic solubility measured by CLND (see Experimental
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O
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c
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R1
N
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3
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R1
2
R2
O
O
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3NH
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Cl
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B
Scheme 1. Synthesis of compounds containing replacements for left hand side
piperidine. Reagents and conditions: (a) Substituted piperidine (1.2 eq.), COMU (2 eq.),
N,N-diisopropylethylamine (3 eq.), N,N-dimethylformamide, room temperature, 2 – 20 h;
(
b) Hydrochloric acid (4 M in dioxane) (5 eq.), room temperature, 0.5 – 17 h, 91-100%
over 2 steps; (c) N,N-diisopropylethylamine (2 eq.), ethanol, microwave, 150 °C, 5 min.;
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(
d) Amine (5 eq.), N,N-diisopropylethylamine (2 eq.), ethanol, microwave, 150 °C, 1 h,
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Chemistry
The bis-piperidine amides 6 and 7 were formed by COMU-mediated amide bond
formation, followed by BOC-deprotection. Sequential nucleophilic aromatic substitutions
onto 4,6-dichloropyrimidine afforded the final products 10-16. The versatility of this route
enabled the three steps to be performed in any order, depending on where any structural
diversity was to be introduced. The two nucleophilic aromatic substitutions were carried
out in one microwave vial, with the second amine simply pipetted into the vial on
completion of the first reaction. These reactions were carried out in an appropriate volume
of solvent (0.5-0.9 mL) to allow direct injection of the crude reaction mixture onto mass-
directed prep-HPLC at the end of the reactions without the need to carry out any work-
up. The designed route allowed rapid exploration of piperidine replacements, affording
panel of compounds 10-16-.
Ring A
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A set of small branched and alicyclicamines was selected to explore potential variations
in Ring A.
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Compound
R1
Ring A = R2 DprE1
Mtb H37Rv PFIb
pIC50a
6.0
MIC (µM)
90
O
10
CH3
15.6
4.9
N
1
1
2
CH3
CH3
N(CH3)2
5.0
5.9
125
5.0
5.5
1
23.4
N
13
CH3
4.7
4.8
>125
>125
6.1
6.8
HN
14
CH Ph
H
2
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7
8
9
0
16
CH Ph
2
N
Table 2. Results of replacement of piperidine with alternative rings in compounds 10-16.
a
DprE1 pIC is the negative logarithm of the IC -concentration expressed in molar (M)
50
50
obtained in the DprE1-inhibition assay. DprE1 pIC values above 7.3 are approaching
50
b
tight binding limit of assay and should be treated with caution. Property forecast index
(
PFI), is the sum of the Chrom log D and the number of aromatic rings.
7.4
The results for ring A variation (Table 2) showed that SAR was fairly restricted and most
of the groups synthesized were less active than the parent piperidine. Only the
morpholine group (10, 16) showed a similar level of activity, within assay limits, to
piperidine, but with around a 1.5 unit reduction in chrom logD compared with the
piperidine compounds (3-5). In particular, the benzyl compound 16 represented an
important milestone in this series, being the first compound to achieve a sub-micromolar
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MIC against Mtb H37Rv. This compound was thus more fully profiled as shown in Table
3.
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
Parameter
DprE1 pIC50 a
PFIb
Compound 16
7.6
7.4
Mtb H37Rv MIC (µM)
0.6
90
c
Intracellular H37Rv MIC (µM)
0.07
>16
>128
280
90
d
DprE1 OE MIC (µM)
9
0
Antibacterial panel MIC (µg/mL)
CLND solubility (µM)e
Microsomes Clint h/m
4.4/13.5
(
ml/min/g tissue)f
a
Table 3. Full profile of compound 16. DprE1 pIC is the negative logarithm of the IC -
50
50
concentration expressed in molar (M) obtained in the DprE1-inhibition assay. DprE1
pIC values above 7.3 are approaching tight binding limit of assay and should be treated
50
b
with caution. Property forecast index (PFI), is the sum of the Chrom log D and the
7.4
c
number of aromatic rings. MIC against M. tuberculosis (H37Rv) in infected Human THP-1
d
macrophages. Overexpressor (OE) MICs are for M. tuberculosis transformed with
pMV261:dprE1.30 eKinetic solubility measured by CLND (see Experimental Section).
fMicrosomal fraction stability (see Experimental Section).
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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
The highly potent compound 16 translated its enzyme activity into low MIC values both in
the cultured mycobacteria assay, and the intracellular assay in macrophages. Target
engagement was demonstrated with 16 in a DprE1 over-expressor strain of Mtb; its MIC
was shown to increase substantially. Importantly, wider profiling of compound 16
supported the potential of the series, whereby it was inactive against an antibacterial
panel of 19 common bacterial species, including Escherichia coli and Staphylococcus
aureus, in line with expectations of this unique mycobacterial process. Encouraged by
these findings, further optimization was pursued to address shortcomings such as the
high intrinsic clearance in microsomes.
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
Encouraged by these data a systematic investigation was initiated with tricyclic structures,
which, with generally <20 heavy atoms were treated as fragments. These were
synthesised to explore the minimum pharmacaphore required for measurable DprE1
inhibition. The ligand efficiency (LE) of the qualified hits suggested that with maintenance
of LE, measurable activity would be expected in the DprE1 assay for these fragments.
The initial set is exemplified in Table 4. Removal of rings A and B (18) or A, B and E (17),
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Page 15 of 114
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7
8
9
by capping 6 or 7 using acetic anhydride, gave no demonstrable activity, unlike the A,B,C
analogues 19-21 which showed weak activity, but retaining reasonable LE. An oxidised
core was also synthesised (22), which demonstrated similar activity to pyrimidine, along
with good LE.
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
Compound
LEc
pIC50a
O
O
N
N
<3.0b
H C
3
CH3
17
O
O
N
N
H C
3
<
4.0
18
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Journal of Medicinal Chemistry
Page 16 of 114
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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
N
N
N
N
N
4
.1
0.31
0.36
0.31
N
N
N
N
19
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
CH3
N
3.9b
H C
3
N
20
O
O
N
N
4
.1
.5
21
N
N
4
0.32
NH
O
22
a
Table 4. Compounds synthesised to identify efficient binding fragments. DprE1 pIC50 is
the negative logarithm of the IC50-concentration expressed in molar (M) obtained in the
DprE1-inhibition assay. DprE1 pIC50values above 7.3 are approaching tight binding limit
b
of assay and should be treated with caution. FPR assay data (see Experimental section
c
for details). Ligand efficency (LE) = pIC *1.37/number of heavy atoms.
50
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9
These results supported growth from the 4-morpholino pyrimidine fragment to find the
optimal structure for ring C. The SAR in this region of the molecule appeared to be fairly
restricted (Table 5 shows key exemplars), with most of the synthesized compounds tested
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
(44 fragment compounds were synthesized) showing no quantifiable activity in the DprE1
assay, indicating that a piperidine linker was optimal.
O
N
R
N
N
Compound
Ring C = R
DprE1
pIC50a
LE/LLEAtb
CH3
2
3
4
<4.0
4.1
N
H C
3
CH3
F F
2
0.30/0.32
N
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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
O
2
5
<4.0
N
N
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
26
27
4.6
0.32/0.25
0.26/0.33
CH3
N
CH3
4.1
O
N
28
<4.0
N
a
Table 5. Results of fragments tested to explore SAR of central piperidine ring. DprE1
pIC50 is the negative logarithm of the IC50-concentration expressed in molar (M)
obtained in the DprE1-inhibition assay. DprE1 pIC50values above 7.3 are approaching
b
tight binding limit of assay and should be treated with caution. Ligand efficency (LE) =
pIC *1.37/number of heavy atoms. Ligand lipophilicity efficiency Astex (LLE ) = 0.111
50
AT
+ 1.36*LLE/number of heavy atoms.
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9
4
-Piperidinyl was demonstrated to be the optimum C-ring through evaluation of a series
of analogues of 16 (data not shown) with pyrrolidine, piperidine and piperazine rings that
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
showed little or no activity.
Alternatives to ring D were then explored. These analogues (Table 6) all showed reduced
activity at DprE1 relative to compound 16, with only the pyrrolidine analogue 36 showing
similar potency, again indicating the likely primacy of a 4-substituted, achiral, piperidine
connectivity.
O
O
R
N
N
N
N
DprE1 pIC50a
Mtb H37Rv
PFIb
Compound
Ring D = R
MIC (µM)
90
2
9
5.4
125
7.2
N
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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
3
0
1
N
N
N
N
N
6.2
5.8
6.5
5.2
32
7.6
5.5
6.3
6.3
6.3
3
10
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
OH
32
10
3
3
4
>125
20
O
N
3
6.2
F
F
35
36
N
N
4.6
>80
4
4.9
6.6
N
O
6.9
a
Table 6. Results of replacements of distal piperidine ring. DprE1 pIC is the negative
50
logarithm of the IC -concentration expressed in molar (M) obtained in the DprE1-
50
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inhibition assay. DprE1 pIC values above 7.3 are approaching tight binding limit of assay
50
b
and should be treated with caution. Property forecast index (PFI), is the sum of the Chrom
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
log D and the number of aromatic rings.
7.4
Removal or replacement of ring E offered opportunities to further optimize properties,
improve potency or alleviate likely metabolic liabilities of an unsubstituted phenyl ring
(Table 7).
Replacement of the benzylic methylene group with a heteroatom was well-tolerated
(Table 7, compounds 37 and 38) and led to reduction in PFI and improved intrinsic
clearance. Blocking likely sites of metabolism with halide substituents, such as in
compound 39 retained activity and gave an improved microsomal clearance.
Replacement of the phenyl ring with heterocyclic alternatives (compounds 40-41),
resulted in fairly restricted SAR, with only pyridine rings retaining any activity against
DprE1. Interestingly, the phenyl ring could be replaced entirely with a range of alkyl
groups (compounds 42-44). Unfortunately, it was not possible to lower logD by
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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
introducing heteroatoms into alkyl analogues (e.g. with compounds 45-46). All such
attempts resulted in complete loss of DprE1 activity.
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 further iteration of optimization combined the ether linkage with the improved phenyl
replacements. This strategy was very successful in identifying compounds such as 47-51
which had excellent DprE1 potencies, low MIC values and very low microsomal clearance
values. Within the set available there were highly potent molecules with encouraging
profiles worthy of progression into in vivo studies.
O
O
N
N
N
R
N
N
Compound
Ring E = R
DprE1
pIC50a
Mtb H37Rv PFIb
Microsomes Clint
h/m (ml/min/g
tissue)c
MIC (µM)
90
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Page 23 of 114
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9
H
N
3
7
6.4
10
6.3
O
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
3
8
9
8.1
7.6
2
6.8
7.4
7.4/<0.5
4.6/3.1
3
0.6
F
40
N
7.0
6.6
3.3
8.8
5.2
5.2
3.1/7.2
N
41
42
CH3
6.8
7.1
8.0
4.0
5.6
6.2
CH3
CH3
43
44
3.3/5.1
2.1/3.1
7.6
3.0
7.9
2.2
4
5
6
N
N
CH3 <4.0
>125
CH3
4
4.1
>125
2.1
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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
O
O
4
7
7.3
7.5
7.5
1.7
1.5
1.0
6.7
7.6
6.9
<0.5/0.7
0.9/1.2
0.6/0.5
F
48
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
Cl
F
F
N
49
50
51
O
F
7.7
6.2
0.5
16
7.6
6.5
1.8/2.9
O
O
Cl
CH3
a
Table 7. Results of replacements of benzyl group. DprE1 pIC is the negative logarithm
50
of the IC -concentration expressed in molar (M) obtained in the DprE1-inhibition assay.
50
DprE1 pIC values above 7.3 are approaching tight binding limit of assay and should be
50
b
treated with caution. Property forecast index (PFI), is the sum of the Chrom log D and
7.4
c
the number of aromatic rings. Microsomal fraction stability (see Experimental Section).
To complete the systematic optimization, variation of alternatives to the pyrimidine (ring
B) offered means of modulating the physicochemical properties of the molecule, by
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varying nitrogen count and basicity. This work followed the A.C,D and E ring optimizations
and key substituents from the SAR investigations (Table 8). There were interesting SAR
effects observed in these alternative cores: heterocycles containing two nitrogen atoms
such as the isomeric pyrimidine 52 or pyrazine 56 retained good potency, but the more
basic pyridine compounds 53-55 had much reduced potency. Small substituents on the
pyrimidine ring in compounds 57-59 were well tolerated and offered alternative means of
lipophilicity modulation, e.g. the pyrimidinone compound 59, maintained good activity and
MIC levels with lower logD, which may account for its improved metabolic stability.
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
O
N
N
R1
R2
Compound R1
R2
DprE1
pIC50a
Mtb
PFIb
Microsomes Clint
h/m (ml/min/g
tissue)c
H37Rv
MIC90
(µM)
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1
1
1
1
1
1
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1
1
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2
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2
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5
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5
5
5
5
6
CH Ph
6.8
5.0
5.2
5.1
7.4
7.2
7.3
16.0
10.0
8.3
5.6
5.6
8.0
8.4
7.1
2
5
5
5
5
5
2
3
4
5
6
N
N
N
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
O
O
O
CH(CH
3
3
3
)
2
)
2
)
2
>125
>125
>125
3.0
N
N
CH(CH
CH(CH
N
N
N
N
N
CH
CH
CH
Ph
Ph
Ph
5.4/63.7
2.8/8.3
2
2
2
N
N
O
O
F
1.3
57
58
N
N
N
N
N
N
3.5
3.4/11.6
NH2
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O
CH
2
Ph
7.4
3.0
5.6
<0.5/4.9
59
N
HN
N
O
1
1
1
1
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1
1
1
1
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4
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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
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7
8
9
0
1
2
3
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8
9
0
1
2
3
4
5
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7
8
9
0
a
Table 8. Results of replacements of pyrimidine core. DprE1 pIC is the negative
50
logarithm of the IC -concentration expressed in molar (M) obtained in the DprE1-
50
inhibition assay. DprE1 pIC values above 7.3 are approaching tight binding limit of assay
50
b
and should be treated with caution. Property forecast index (PFI), is the sum of the
c
Chrom log D7.4 and the number of aromatic rings. Microsomal fraction stability (see
Experimental Section).
As a result of the property-focussed optimization of the series described here, a number
of compounds had potent DprE1 inhibition combined with low MIC values and much
improved in vitro DMPK properties, which together supported progression into in vivo
efficacy studies. Compounds 39 and 47 (Table 9) were thus progressed into a mouse
model of tuberculosis infection.
Compound
DprE1 pIC50a
PFI
39
7.6
7.4
47
7.3
6.7
60f
7.4
6.6
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6
Mtb H37Rv MIC90
0
.6
1.7
4
(µM)
Intracellular H37Rv
MIC (µM)
0.21
0.62
0.62
>125
<4
90
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
0
1
2
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9
0
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3
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8
9
0
1
2
3
4
5
6
7
8
9
0
DprE1 OE MIC
µM)b
1
5
62
(
Cytotoxicity HepG2
pIC50
.3c
4.5d
4
CLND solubility
160
≥364
26
(
µM)
FaSSIF solubility
µg/mL)
4
0
74
ND
(
Microsomes Clint
h/m (ml/min/g
tissue)
4.6/3.
<0.5/0.7
23.5
<0.5/4.2
1
Cl
(ml/min/kg 38.9
)
In vivo
mouse
PKe
Vss (L/kg)
t (h)
1.3
0.45
1.7
1.0
1/2
%F
100%
78.7%
a
Table 9. Profiles of compounds 39 and 47 in a range of in vitro and in vivo assays. DprE1
pIC values above 7.3 are approaching tight binding limit of assay and should be treated
50
b
c
with caution. Compound tested inactive (pIC = 4.0) on 6 out of 10 test occasions.
50
d
Compound tested inactive (pIC < 4.0) on 2 out of 4 test occasions. Compound tested
50
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e
inactive (pIC < 4.0) on 2 out of 7 test occasions. Clearance (Cl), volume of distribution
50
at steady state (Vss) and half-life (t ) obtained after intravenous administration to CD-1
1/2
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
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
male mice at 1 mg/kg; Bioavailability (%F) obtained after oral administration to CD-1 male
f
18
mice at 5 mg/kg. AZ DprE1 inhibitor benchmark (compound 9 in reference).
34
In this acute model of TB infection, mice were subjected to an intratracheal infection of
M. tuberculosis and then were treated every day for eight days beginning on the day after
infection with either compound 39 or 47 at varying doses. AZ DprE1 inhibitor 60 was
profiled as a direct comparison, both with and without 1-aminobenzotriazole (ABT), a CYP
18
P450 inhibitor. At the end of the experiment, the animals were sacrificed and the number
of colony forming units (CFUs) in the lungs were counted and compared with untreated
animals.
a
Compound
Structure
ED (mg/kg)
9
9
60
164
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