Rational Design, Synthesis, and Biological Evaluation of Heterocyclic Quinolones Targeting the Respiratory Chain of Mycobacterium tuberculosis

Article abstract of DOI:10.1021/acs.jmedchem.6b01718

A high-throughput screen (HTS) was undertaken against the respiratory chain dehydrogenase component, NADH:menaquinone oxidoreductase (Ndh) of Mycobacterium tuberculosis (Mtb). The 11000 compounds were selected for the HTS based on the known phenothiazine Ndh inhibitors, trifluoperazine and thioridazine. Combined HTS (11000 compounds) and in-house screening of a limited number of quinolones (50 compounds) identified ～100 hits and four distinct chemotypes, the most promising of which contained the quinolone core. Subsequent Mtb screening of the complete in-house quinolone library (350 compounds) identified a further ～90 hits across three quinolone subtemplates. Quinolones containing the amine-based side chain were selected as the pharmacophore for further modification, resulting in metabolically stable quinolones effective against multi drug resistant (MDR) Mtb. The lead compound, 42a (MTC420), displays acceptable antituberculosis activity (Mtb IC₅₀ = 525 nM, Mtb Wayne IC₅₀ = 76 nM, and MDR Mtb patient isolates IC₅₀ = 140 nM) and favorable pharmacokinetic and toxicological profiles.

Full text of DOI:10.1021/acs.jmedchem.6b01718

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Article
Rational Design, Synthesis and Biological Evaluation of Heterocyclic
Quinolones Targeting the respiratory chain of Mycobacterium tuberculosis
Weiqian David Hong, Peter D Gibbons, Suet C Leung, Richard Amewu, Paul A Stocks, Andrew Valentine
Stachulski, Pedro C Horta, Maria Lurdes Santos Cristiano, Alison E. Shone, Darren Moss, Alison Ardrey,
Raman Sharma, Ashley J. Warman, Paul T.P. Bedingfield, Nicholas E. Fisher, Ghaith Aljayyoussi, Sally Mead,
Maxine Caws, Neil G. Berry, Stephen A. Ward, Giancarlo A. Biagini, Paul M. O'Neill, and Gemma Louise Nixon
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01718 • Publication Date (Web): 17 Mar 2017
Downloaded from http://pubs.acs.org on March 19, 2017
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Rational Design, Synthesis and Biological
Evaluation of Heterocyclic Quinolones Targeting the
respiratory chain of Mycobacterium tuberculosis
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a≠
a≠
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W. David Hong, Peter D. Gibbons, Suet C. Leung, Richard Amewu, Paul A. Stocks,
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Andrew Stachulski, Pedro Horta, Maria L.S. Cristiano, Alison E. Shone, Darren Moss,
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Alison Ardrey, Raman Sharma, Ashley J. Warman, Paul T. P.Bedingfield, Nicholas E.
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Fisher, Ghaith Aljayyoussi, Sally Mead, Maxine Caws, Neil G. Berry, Stephen A. Ward,
d*
a
a*
Giancarlo A. Biagini , Paul M. O’Neill and Gemma L. Nixon
a
Department of Chemistry, University of Liverpool, Liverpool, L69 7ZD, U.K.
b
Department of Chemistry, University of Ghana, P. O. Box LG56, LegonꢀAccra.
c
Department of Chemistry and Pharmacy, University of Algarve, 8005ꢀ139, Faro, Portugal.
d
Research Centre for Drugs and Diagnostics, Liverpool School of Tropical Medicine, Pembroke
Place, Liverpool, L3 5QA, U.K.
e
School of Pharmacy, Keele University, Keele, ST5 5BG, UK.
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ABSTRACT
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A Highꢀthroughput screen (HTS) was undertaken against the respiratory chain dehydrogenase
component, NADH:menaquinone oxidoreductase (Ndh) of Mycobacterium tuberculosis (Mtb).
11,000 compounds were selected for the HTS based on the known phenothiazine Ndh inhibitors,
trifluoperazine and thioridazine. Combined HTS (11,000 compounds) and inꢀhouse screening of
a limited number of quinolones (50 compounds) identified ~100 hits and four distinct
chemotypes, the most promising of which contained the quinolone core. Subsequent Mtb
screening of the complete inꢀhouse quinolone library (350 compounds) identified a further ~90
hits across three quinolone subꢀtemplates. Quinolones containing the amine based side chain
were selected as the pharmacophore for further modification, resulting in metabolically stable
quinolones effective against multi drug resistant (MDR) Mtb. The lead compound, MTC420
displays acceptable antiꢀtuberculosis activity (Mtb IC =525 nM, Mtb Wayne IC = 76 nM and
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50
MDR Mtb patient isolates IC = 140 nM) and favourable pharmacokinetic and toxicological
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profiles.
INTRODUCTION
In 2014 tuberculosis (TB) globally infected 9.6 million people resulting in an estimated 1.5
1
million deaths. With the emergence of multiꢀdrug resistant (MDR) and extensively drug
resistant (XDR) TB the need for new drug treatments targeting the disease has never been
2
greater. Current first line drugs for TB were developed in 1952ꢀ1966 (Figure 1). Shortcomings
of these drugs include; (i) long treatment regimens (6 to 9 months) leading to patient nonꢀ
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compliance, (ii) adverse drugꢀdrug interactions with anti HIV drugs (HIV/AIDS is a common coꢀ
infection) and (iii) limited or no activity against MDR and XDR Mycobacterium tuberculosis
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4, 5
6, 7
(
Mtb). Bedaquiline
and delamanid
are the only recently FDA approved drugs for the
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treatment of TB and their approval is currently only for MDR in cases where established
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treatments have failed (Figure 1). In order to find an effective treatment for MDR and XDR it is
believed that a drug with a novel mode of action is required in order to circumvent resistance.
Current first line drugs used to treat TB:
O
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OH
NH2
N
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Isoniazid
Pyrazinamide
Ethambutol
Rifampicin
Recently approved drugs to treat MDR TB:
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Delamanid
Bedaquiline
Figure 1. Current first line drugs used to treat tuberculosis and recently approved drugs for the
treatment of MDR TB.
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Targeting components of the Mtb respiratory chain (Figure 2) has been shown by us and other
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laboratories, to be effective in sterilizing both replicating and dormant Mtb.
The initial target
within this programme, Ndh (Rv1854c) is a single subunit 50 KDa enzyme involved in the redox
reaction of NADH oxidation with subsequent menaquinol production. Ndh has been
biochemically identified as a “choke point” and as such is essential for cell function and
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viability.
Essentiality of ndh has been shown by the inability of Mtb to tolerate insertion
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mutations in this gene and more recently in a study involving ndh knockout with subsequent
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confirmation by complementation.
The other NADHꢀdependent electron donating
dehydrogenases identified in the genome (Complex I and ndhA) have been shown not to be
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lethal.
These data are consistent with biochemical evidence that Ndh is a major source of
electrons for the ETC.
Figure 2. Schematic representation of the respiratory chain of M. tuberculosis. The chain
components are Ndh/NdhA – type II NADH:(mena)quinone oxidoreductase (two isoforms), ETF
– electron transferring flavoprotein (transfer of reducing equivalents from fatty acid βꢀoxidation
into the Qꢀpool), nuo – protonmotive NADH dehydrogenase (Complex I), bcc – cytochrome bcc
complex (note that there is no evidence for soluble cytochrome c in this organism), aa –
3
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cytochrome bcc oxidase, postulated to form a supercomplex with bcc. An alternative terminal
oxidase pathway is utilised in M. tuberculosis under conditions of low oxygen tension,
containing quinol oxidase (cytochrome bd), fumarate reductase (FRD) and nitrate reductase (nar)
components. P and n correspond to the positive and negative sides of the respiratory membrane
with respect to proton translocation. Proton movements are indicative only, and do not represent
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+
ꢀ
H /e ratios for the respective complexes.
Respiratoryꢀchain inhibitionꢀinduced death represents a fundamental shift from traditional antiꢀ
tubercular drug design that have until recently relied on drugs that selectively target the
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replication machinery of Mtb.
Antiꢀtubercular drugs developed to target the respiratory
pathways should therefore have the potential to have sterilizing activity against current MDR and
XDR Mtb strains.
Identification of hit compounds was achieved through a HTS screen of approximately 11,000
compounds that were predicted to possess activity against the Ndh enzyme. Ndh was chosen for
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the HTS due to the critical role as an important dehydrogenase during growth and pathogenicity
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and due to its tractability for heterologous expression in E.coli and HTS . The enzyme has
been observed to be sensitive to phenothiazineꢀbased inhibitors such as trifluoperazine and
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thioridazine . These inhibitors have been shown by a number of different laboratories to have
sterilizing activity against replicating and slow growing MDR Mtb (grown anaerobically) in both
1
4, 30, 31
in vitro and in vivo models.
These two compounds were used as the basis to employ a
32ꢀ35
range of ligandꢀbased chemoinformatics methods
in the rational selection of the ~11,000
compounds for the HTS campaign (selected from a commercial library of ~750,000 compounds
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6ꢀ40
(Biofocus DPI)).
Selected compounds were subject to a sequential high throughput
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screening campaign using an in vitro assay against recombinant Ndh as described previously.
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In addition to the HTS screen a limited selection of 50 quinolones were also screened inꢀhouse
against Mtb Ndh. These compounds were selected for their structural diversity from a library of
quinolones designed to target the NADH:ubiquinone oxidoreductase within the malaria parasite
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Plasmodium falciparum (PfNDH2) as described previously.
The HTS screen and inꢀhouse
screen in combination generated ~100 hits across 4 distinct templates, the most potent of which
were also tested for whole cell replicating Mtb activity. Following analysis of the in vitro
biological data, predicted DMPK properties and investigations into chemical tractability the
quinolone template was selected as the most promising for further development.
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In previous antimalarial discovery projects
, the inhibitors based on the quinolone core
displayed pharmacodynamics consistent of a privileged pharmacophore, with the ability to act on
multiple electron transport chain (ETC) components. For example, quinolones with a dual
mechanism of action against two respiratory enzymes, PfNDH2 and cytochrome bc have
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recently been reported. To exploit this phenomenon in this antitubercular discovery project,
further screening and SAR investigations was switched to whole cell replicating TB activity. In
order to fully establish the structure activity relationship (SAR) within the existing quinolone
library with respect to whole cell Mtb activity a further library of ~350 compounds were
screened against replicating Mtb. ~90 compounds were found to inhibit Mtb growth by >50% at
5
µM. Four subꢀtemplates were then identified as having moderate in vitro Mtb potency. The
most promising of which only had a very limited number of examples (see Table S1 –
Supporting Information) within the existing library but demonstrated significantly more potency,
as such the template based on compounds 1 and 2 was chosen for lead optimisation (Figure 3).
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Figure 3. Identification of the quinolone template for lead optimisation.
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A comprehensive medicinal chemistry SAR study around this series was then undertaken to
establish optimised leads for further development. Screening data analysis (see Table S1 –
Supplementary Information) shows NH and OAc at the 4ꢀposition are inactive for this particular
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subꢀtemplate (Table S1 ꢀ entries 20, 23 and 24) and show reduced activity for other quinolone
subꢀtemplates. Replacement of the phenyl ring with a pyridyl ring also rendered the subꢀtemplate
inactive (Table S1 ꢀ entry 20). Modification of ring C resulting in loss of in vitro Mtb potency is
a general trend that was seen across most quinolone subꢀtemplates screened. Modifications of
particular interest were therefore optimization of the side chain to optimize potency and DMPK,
the nature of the group at 3ꢀposition and the electronic/steric effect of substituents placed at the
5, 6 and 7 positions (Figure 4).
Carbonyl essential
NH and OAc
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2
Do groups at the 5, 6
inactive
Is a methyl group
optimal?
and 7 position exert
a positive electronic/
steric effect?
R2
O
R3
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R1
3
Optimise sidechain
substituents
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A
B
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N1
C
X
D
H
Pheny essential ꢀ
heterocycles/unsaturation
reduce activity
Figure 4. Known SAR and SAR to be investigated.
CHEMISTRY
Following identification of quinolones 1 and 2 as the initial hits against Mtb, our initial efforts
were focused on exploring the SAR of substituents placed in the A ring. The synthesis of these
compounds was achieved in 3 – 5 steps from commercially available starting materials (Scheme
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). Oxazoline 4 was prepared from the corresponding isatoic anhydride 3 in yields of 34 – 75%.
Where the isatoic anhydride was not commercially available, the oxazolines were synthesized inꢀ
house (see Supporting information). 4’ꢀfluoropropiophenone 5 was reacted with piperidine to
give ketone 6 in 32 – 97% yields. Reaction of oxazoline 4 with ketone 6 in the presence of triflic
acid gave the desired quinolones 1, 2, 7aꢀk in 23 – 45% yields.
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a
Scheme 1. Synthesis of Quinolones 1, 2, 7a-k.
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corresponding amine, K CO , DMF, 120 C to reflux, overnight ; (c) CF SO H, nꢀBuOH, N ,
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Table 1. Yields for the Synthesis of Compounds 1, 2, 7a-k.
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Compound
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% yield 4 % yield 6
% yield 7
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F
The nature of the group at 3ꢀposition of the quinolone was also studied. A small set of analogues
with a hydrogen at 3ꢀposition were synthesized (Scheme 2). Substituted 2ꢀaminoacetophenone 9
was converted from the respective aminobenzoic acid 8 using methyl lithium in 36% yield. 4ꢀ
fluorobenzoate 10 was reacted with piperidine in the presence of potassium carbonate to give the
piperidinyl benzoate 11 in 37% yield. Benzoate 11 was hydrolysed to benzoic acid which was
then converted to acid chloride 12 by oxalyl chloride. Acylation of 2ꢀaminoacetophenone 9 with
acid chloride 12 provided the intermediate 13 in 30 – 51% yields. Cyclisation of the intermediate
t
13 in the presence of NaOH or KO Bu gave the 3ꢀH quinolones 14a-c in 41 – 91% yields (Table
2).
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Literature precedent from the development of ETC inhibitors in the antimalarial field lead us
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then to look at the presence of a halide at the 3ꢀposition. GSK’s pyridone series demonstrated
tolerance of the presence of a chlorine at 3ꢀposition and within our own group we have shown
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the combination of 3ꢀchloroꢀ7ꢀmethoxy enhances biological activity of the quinolone core. In
order to achieve this the 3ꢀH compounds were treated with sodium dichloroisocyanurate and
sodium hydroxide to give 3ꢀCl quinolones 15a-d in 40 – 61% yields, or NBS to give 3ꢀBr
quinolones 15e-f in 55 – 63% yields.
a
Scheme 2. Synthesis of quinolones 14 a-c and 15 a-f.
a
o
Conditions and reagents: (a) MeLi, DME, 0 C, 2 h; (b) (i) K CO , DMF, reflux, overnight, (ii)
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NaOH (aq), MeOH, reflux, overnight; (c) oxalyl chloride, DCM, DMF (cat.), r.t., 2 h; (d) NEt3,
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t
o
THF, r.t., overnight; (e) NaOH (s), 1,4ꢀdioxane, 110 C, 5 h or KO Bu, BuOH, 75 C, 16 h; (f)
sodium dichloroisocyanurate, 1M NaOH (aq), MeOH, r.t., overnight (15a-d) or NBS, DCM,
DMF, r.t., overnight (15e-f).
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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. Yields for the Synthesis of Compounds 14a-c and 15a-f.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compound
X
R
Y
% yield % yield % yield % yield % yield
9
11
13
51
50
30
51
50
30
60
51
60
14
70
41
68
70
41
68
91
70
91
15
1
4a
14b
4c
5a
5b
H
OMe
OMe
H
H
H
F
H
H
ꢀ
ꢀ
ꢀ
36
36
ꢀ
ꢀ
ꢀ
1
H
37
ꢀ
ꢀ
1
H
H
F
Cl
Cl
Cl
Cl
Br
Br
40
61
52
58
63
55
1
OMe
OMe
H
36
36
ꢀ
ꢀ
15c
15d
37
37
ꢀ
F
1
5e
H
H
F
ꢀ
1
5f
H
ꢀ
37
Having identified 3ꢀmethyl and 5, 7ꢀdifluoro quinolone (followed by 6ꢀfluoroꢀ7ꢀmethoxy and 7ꢀ
methoxy quinolone) to be optimal for Mtb activity (see Table 8), the focus of SAR explorations
moved to the terminal ring of the side chain to further improve Mtb activity and optimise
DMPK. Additional small groups, such as Me, F and CF attached at different positions on the
3
5
1
terminal piperidine ring were investigated. In addition the effect of chirality was explored.
Synthesis of compounds 17a-k was achieved using chemistry described in Scheme 3.
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a
Scheme 3. Synthesis of Quinolones 17a-k.
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
o
Conditions and reagents: (a) corresponding amine, K CO , DMF, 120 C to reflux, overnight ;
2
3
o
(b) CF SO H, nꢀBuOH, N , 130 C, 24 h.
3
3
2
Table 3. Yields for the Synthesis of Compounds 17a-k.
Compound
7a
R
% Yield 16
% Yield 17
1
48
32
1
7b
7c
7d
7e
73
48
40
64
54
34
57
27
1
1
1
1
7f
7g
17h
7i
84
74
45
43
1
75
72
40
39
1
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3
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3
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4
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5
5
5
5
5
5
5
5
6
1
7j
69
41
51
17k
ꢀNHCH
2
Ph
28
0
1
2
3
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7
8
9
0
1
2
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7
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9
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9
0
1
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8
9
0
1
2
3
4
5
6
7
8
9
0
Incorporation of different amino groups into the side chain as an alternative to the potentially
metabolically labile piperidine ring was also investigated. To incorporate a diethylamine group
an alternative methodology was used to synthesise the side chain, commercial available 4ꢀ
bromoꢀN,Nꢀdimethylaniline 18 was treated with butyllithium for a lithiumꢀhalogen exchange and
the intermediate was quenched with N,Nꢀdimethylpropionamide to form the side chain 19 in 78%
yield, reaction with oxazoline 4h was then carried out to give quinolone 17l in 46% yield
(
Scheme 4).
a
Scheme 4. Synthesis of quinolone 17l.
a
o
Conditions and reagents: (a) (i) nBuLi, Et O, ꢀ78 C, 30 min; (ii) N,Nꢀdimethylpropionamide, ꢀ
2
o
o
78 C to r.t., 2 h; (b) CF SO H, nꢀBuOH, N , 130 C, 24 h.
3
3
2
Extension of the side chain with a phenyl or benzyl group at the 2ꢀposition was also investigated
using the synthetic methodologies shown in Scheme 5. In addition, replacement of piperidine by
piperazine was investigated. This was to further explore the length of side chain that could be
tolerated and to improve the solubility.
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Scheme 5. Synthesis of Quinolones 21a-g.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
o
Conditions and reagents: (a) corresponding amine, K CO , DMF, 120 C to reflux, overnight;
2
3
o
(b) CF SO H, nꢀBuOH, N , 130 C, 24 h.
3 3 2
Table 4. Yields for the Synthesis of Compounds 21a-g.
Compound
1a
X
H
6ꢀF
7ꢀOMe
H
6ꢀF
A
CH
CH
CH
N
N
N
N
B
% Yield 20 % Yield 21
2
CH
2
2
2
2
2
Ph
Ph
Ph
Ph
Ph
64
64
64
58
58
64
58
33
40
42
30
28
30
38
21b
21c
CH
CH
CH
CH
2
2
2
2
1d
1e
1f
1g
7ꢀOMe
7ꢀOMe
Ph
CH Ph
2
In addition, the quinolone with a piperidine ring at the metaꢀposition 24 was also synthesised by
reacting the 3ꢀbromopropiophenone 22 with piperidine using Buchwald coupling to yield the
ketone intermediate 23, which was coupled with oxazoline 4h to give the quinolone in 45% yield
(Scheme 6).
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7
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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
a
Scheme 6. Synthesis of Quinolone 24.
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
t
o
Conditions and reagents: (a) Piperidine, Pd(OAc) , XPhos, NaO Bu, Toluene, 110 C, 24 h; (b)
2
o
CF SO H, nꢀBuOH, N , 130 C, 24 h.
3
3
2
A series of analogues with a pyrrole heterocycle in the side chain were also synthesized to
further explore the side chain SAR and enhance the metabolic stability. The synthetic route to
these compounds is illustrated in Scheme 7. Utilizing Copper and transꢀN,N’ꢀDimethylꢀ
1
,2cyclohexanediamine catalyzed Nꢀarylation with 4ꢀbromopropiophenone the side chain ketone
5
2, 53
intermediate 31 was formed in 30 – 62 % yields.
quinolones 32a-g in 35 – 57% yields.
Final cyclisation with oxazoline gave
a
Scheme 7. Synthesis of quinolones 32a-g.
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23
24
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35
36
37
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43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
o
Conditions and reagents: (a) EtMgBr, THF, 0 C, 1h; (b) PCC, DCM, r.t., 2h; (c) 5mol% CuI,
o
20mol% transꢀN,N’ꢀDimethylꢀ1,2cyclohexanediamine, K PO , Toluene, 110 C, 24 h; (d)
3
4
o
CF SO H, nꢀBuOH, N , 130 C, 24 h.
3
3
2
Table 5. Yields for the Synthesis of Compounds 32a-g.
Compound
X
Y
ꢀ
R
% yield 31
% yield 32
NC
N
32a
5ꢀF,7ꢀF
38
55
32b
5ꢀF,7ꢀF
ꢀ
30
57
32c
32d
5ꢀF,7ꢀF
ꢀ
46
35
5ꢀF
ꢀ
62
62
32
30
32e
5ꢀF,7ꢀF
ꢀ
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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
2f
5ꢀF,7ꢀF
5ꢀF,7ꢀF
mꢀCl
oꢀF
49
52
39
39
32g
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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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
Using fluorine to block metabolism and improve oral absorptions was further explored. Research
by Smith has shown that gemꢀdifluorinated piperidine compounds exhibited a significant
54
improvement in metabolic stability. This led to the design and synthesis of fluorinated
quinolones 38a-f as well as the alcohol side chain quinolones 38g-i. The chemistry used in the
synthesis of these compounds is shown in Scheme 8.
a
Scheme 8. Synthesis of Quinolones 38a-l.
a
o
Conditions and reagents: (a) corresponding amine, K CO , DMF, 120 C to reflux, overnight;
2
3
o
(
b) CF SO H, nꢀBuOH, N , 130 C, 24 h.
3 3 2
Table 6. Yields for the Synthesis of Compounds 38a-l.
Compound
8a
X
R
% Yield 37 % Yield 38
3
5ꢀF,7ꢀF
38
45
38b
5ꢀF,7ꢀF
37
47
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3
8c
8d
8e
8f
8g
38h
5ꢀF,7ꢀF
25
32
33
30
3
5ꢀF,7ꢀF
7ꢀOMe
3
32
32
69
32
30
48
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
6ꢀCl,7ꢀOMe
5ꢀF,7ꢀF
3
5ꢀF,7ꢀF
54
50
3
8i
8j
5ꢀF,7ꢀF
5ꢀF,7ꢀF
32
41
43
20
3
38k
5ꢀF,7ꢀF
5ꢀF,7ꢀF
43
41
25
37
3
8l
Removal of the benzyl group from the chiral proline derivatives 38j-l was achieved using
hydrogenation (Scheme 9) in good yields.
Scheme 9. Synthesis of compounds 39a-c.
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1
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
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3
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3
3
3
3
4
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4
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4
4
4
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4
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5
5
5
5
5
5
5
5
5
6
For the gemꢀdifluoro analogues (42a (MTC420) and 42b), 4ꢀbromopropiophenone was first
converted to a more reactive 4ꢀiodopropiophenone by an aromatic Finkelstein reaction catalysed
5
5
by copper(I) iodide in combination with N,Nꢀdimethylꢀ1,2ꢀdiaminoethane. A subsequent
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
BuchwaldꢀHartwig amination using Pd (dba) and Xantphos with the gemꢀfluorinated amine
2
3
5
6
gave the ketone side chain 41a-b in 12 – 28% yields. Reaction with oxazoline gave quinolones
2a-b in 47 – 56% yields (Scheme 10).
4
a
Scheme 10. Synthesis of quinolones 42a-b.
a
o
Conditions and reagents: (a) CuI, N,Nꢀdimethylꢀ1,2ꢀdiaminoethane, NaI, 1,4ꢀdioxane, 110 C,
t
o
24 h; (b) Pd (dba) , Xantphos, NaO Bu, 1,4ꢀdioxane, 110 C, 24 h; (c) CF SO H, nꢀBuOH, N ,
2
3
3
3
2
o
1
30 C, 24 h.
42a was identified as the lead compound in the series as it exhibited good potency and metabolic
stability (See Table 11 and Table 12), further investigation of the pyrrolidine side chain was
undertaken to improve solubility and potency. Further modifications have included adding
chirality and introducing amide functionality to rapidly ascertain if it is tolerated within the
template. Quinolones 45a-h were therefore synthesised using chemistry described in Scheme 11.
To incorporate the amide group, Ullmann coupling of 4ꢀbromopropiophenone with Dꢀproline
gave the carboxylic acid intermediate 43a-b. Crosslinking the carboxylic acid by EDC/NHS to
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respective amine provided the ketone side chain 44 in 52 – 90% yields. This was subsequently
coupled with oxazoline in 12 – 34% yields to afford quinolones 45a-g.
a
Scheme 11. Synthesis of Quinolones 45a-g.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
o
Conditions and reagents: (a) Dꢀproline, CuI, K CO , DMF, 140 C, 24 h; (b) (i) EDC, Nꢀ
2
3
hydroxysuccinamide, CHCl , NEt , amine, r.t., 6 h; (ii) amine, NEt , r.t., 2h; (c) CF SO H, nꢀ
3
3
3
3
3
o
BuOH, N , 130 C, 24 h.
2
Table 7. Yields for the Synthesis of Compounds 45a-g.
Compound
R
n
2
2
2
% yield 44
% yield 45
t
45a
ꢀNH Bu
ꢀNEt
52
80
90
20
34
25
45b
2
45c
4
5d
5e
5f
2
2
1
70
18
24
15
4
65
a
4
ꢀNMe
2
45
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1
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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
t
b
4
5g
ꢀNH Bu
1
ꢀ
12
a
b
Alternative methodology used please see supporting information. Used crude.
Incorporation of an amide moiety largely resulted in reduced antiꢀtuberculosis activity (Table 7).
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
As such our attention returned to 42a and improving its pharmacokinetic profile. Use of a proꢀ
5
7
drug strategy, previously used successfully within other quinolone development programs was
investigated leading to the synthesis of compound 46. Compound 46 was synthesized by reacting
4
2a with potassium tertꢀbutoxide and acetyl chloride to give the acetate proꢀdrug in high yield.
a
Scheme 12. Synthesis of proꢀdrug 46.
a
t
(
a) i. BuOK, THF, r.t., 1h. ii. Acetyl chloride, r.t., 3h.
RESULTS AND DISCUSSION
Structure Activity Relationships (SAR) ꢀ Initial SAR investigations around the hit compounds 1
and 2 focused on establishing the optimal Aꢀring substituents (X). Compounds 1, 2 and 7a-7h
demonstrate the most favorable X groups are 5ꢀF, 7ꢀF closely followed by 6ꢀF, 7ꢀOMe and 7ꢀ
OMe. Compounds 7i-7k were synthesised with a view to reducing the potential metabolism of
the piperidine ring. Pleasingly a good level of potency was maintained. Concomitantly the
potential for replacing the methyl group at Y was also investigated. When Y=H activity is lost as
demonstrated by compounds 14a-c. Halogenation was also investigated; again this largely
resulted in reduced antiꢀtuberculosis activity (15a-f). The one exception to this being 15e
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possessing a Br at Y. This affect appeared to be compound specific rather than a general trend
across all brominated analogues and as such it was decided that the methyl group was the
optimal group at this position.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 8. Mtb IC values for compounds 1, 2, 7a-k, 14a-c and 15a-f.
5
0
Compound
X
H
7ꢀOMe
6ꢀF
Y
R
H
H
H
H
H
H
H
H
H
H
F
Mtb IC (µM)
50
1
2
7a
Me
Me
Me
1.50 ± 0.19
0.73 ± 0.01
1.83 ± 0.22
>10
7
b
6ꢀOMe, 7ꢀOMe Me
6ꢀCl, 7ꢀOMe
6ꢀF, 7ꢀOMe
7
c
Me
Me
>10
7
d
0.52 ± 0.06
>10
0.27 ± 0.08
>10
7
e
5ꢀOMe, 7ꢀOMe Me
7
f
5ꢀF, 7ꢀF
7ꢀF
7ꢀCl
H
7ꢀOMe
5ꢀF, 7ꢀF
H
7ꢀOMe
7ꢀOMe
H
Me
Me
Me
Me
Me
Me
H
H
H
Cl
Cl
7g
7
h
>10
>10
7
i
7
j
F
F
1.32 ± 0.10
0.94 ± 0.12
>10
7
k
14a
4b
4c
5a
5b
H
H
F
H
H
F
1
>10
>10
1
1
1.56 ± 0.22
2.82 ± 0.21
>10
1
7ꢀOMe
7ꢀOMe
H
15c
15d
Cl
Cl
F
>10
1
1
5e
5f
H
H
Br
Br
H
F
0.60 ± 0.09
>10
With 5ꢀF, 7ꢀF and 3ꢀmethyl confirmed as optimal for antiꢀtuberculosis activity optimising the
side chain then became the focus of the SAR studies (Table 9). Initial investigations into
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piperidine ring substituents at the 4ꢀposition revealed that in addition to 4ꢀF 7k, a methyl group
is also tolerated as demonstrated with compound 17b. It rapidly became apparent that there was a
size limitation to the group tolerated at the 4ꢀposition with larger groups such as CF3,
cyclopropyl and gemꢀdifluoro resulting in loss of potency. Movement of the F and Me groups to
the 3ꢀposition resulted in improvements in antiꢀtuberculosis activity as demonstrated by
compounds 17e-h. Interestingly racemic and enatiomerically pure analogues of the 3ꢀmethyl
derivative 17f showed little variation in potency, which is in direct contrast to the pyrrolidine
analogues discussed later. Replacement of the piperidine ring with a number of alternative
amines was also investigated. Increasing ring size (17j) and use of dimethyl amine (17l) retained
good potency. Incorporation of secondary amines (17k) and more polar groups such as Nꢀmethyl
piperazine (17i) reduced antiꢀtuberculosis activity. Moving the piperidine group from the para to
the metaꢀposition (24) also resulted in loss of 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
Table 9. Mtb IC values for compounds 17a-l and 24.
50
Compound
7a
R
Mtb IC (µM)
Compound
R
Mtb IC (µM)
5
0
50
1
>10
17h
0.47 ± 0.02
>
10
17b
7c
0.61 ± 0.05
>10
17i
17j
0
.49 ± 0ꢀ07
1
1
7k
>10
1
7d
7e
>10
ꢀNHCH Ph
2
0
.41 ± 0.002
1
0.31 ± 0.03
17l
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meta
>10
1
7f
0.37 ± 0.04
0.47 ± 0.03
24
N
1
7g
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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28
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31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The size limitation and unfavorable incorporation of piperazine was further confirmed by our
concomitant investigation in to extended side chain analogues (Table 10). The aim of this series
was to explore the space available and to improve solubility with the incorporation of piperazine
to facilitate salt based formulation.
Table 10. Mtb IC values for compounds 21a-g.
5
0
Compound
1a
1b
1c
1d
21e
1f
1g
X
H
6ꢀF
7ꢀOMe
H
6ꢀF
A
CH
CH
CH
N
N
N
N
B
Mtb IC (µM)
50
2
2
2
2
CH
2
2
2
2
2
Ph
>10
>10
>10
CH
CH
CH
CH
Ph
Ph
Ph
Ph
5.74 ± 0.66
>10
2
2
7ꢀOMe
7ꢀOMe
Ph
CH Ph
>10
>10
2
With this information in hand several small heterocyclic, fluorinated, chiral and amide analogues
were synthesized to investigate SAR and improve DMPK (Table 11). Compounds 32a-g are
pyrrole derivatives. An unsubstituted pyrrole moiety is well tolerated in the 5ꢀF (32d) and 5ꢀF, 7ꢀ
F (32e) analogues, however increasing the size of the pyrrole group by addition of a fused
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2
2
2
2
2
2
2
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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
benzene ring (32b) again results in loss of potency. Incorporation of a halogen on the aromatic
ring was also investigated but reduced potency.
Table 11. Mtb IC values for compounds 32a-g, 38a-j, 39a-c, 42a-b and 45a-g.
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compound
X
R
Mtb
IC50
Compound
X
R
Mtb
IC50
(µM)
>10
(µM)
NC
N
32a
5ꢀF,7ꢀF
5ꢀF,7ꢀF
>10
38i
38j
5ꢀF,7ꢀF
5ꢀF,7ꢀF
32b
>10
0.96 ±
0.06
3
2c
2d
2e
5ꢀF,7ꢀF
>10
39a
5ꢀF,7ꢀF
1.59 ±
.05
0
3
5ꢀF
0.71 ±
39b
39c
5ꢀF,7ꢀF
5ꢀF,7ꢀF
0.32 ±
0.04
0
.05
0.44 ±
.02
3
5ꢀF,7ꢀF
>10
0
3
2f
5ꢀF,7ꢀF
Y= mꢀCl
>10
42a
42b
5ꢀF,7ꢀF
5ꢀF,7ꢀF
0.53 ±
0.08
32g
5ꢀF,7ꢀF
Y= oꢀF
>10
0.36 ±
0.04
38a
38b
5ꢀF,7ꢀF
5ꢀF,7ꢀF
0.23 ±
45a
45b
5ꢀF,7ꢀF
5ꢀF,7ꢀF
0.96 ±
0.05
0
.003
1.80 ±
0.09
>10
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8c
5ꢀF,7ꢀF
1.53 ±
.04
45c
5ꢀF,7ꢀF
>10
0
38d
5ꢀF,7ꢀF
7ꢀOMe
>10
>10
45d
45e
5ꢀF,7ꢀF
5ꢀF,7ꢀF
>10
>10
10
11
12
13
14
15
16
17
18
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21
22
23
24
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38
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45
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48
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53
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55
56
57
58
59
60
38e
3
8f
6ꢀCl,7ꢀOMe
5ꢀF,7ꢀF
>10
45f
5ꢀF,7ꢀF
5ꢀF,7ꢀF
>10
>10
38g
5.01 ±
45g
0
.03
38h
5ꢀF,7ꢀF
>10
Fluorinated analogues were synthesized in order to improve metabolic stability (see Table 11).
Both mono (38a and 38b) and gemꢀdifluoro (42a) substituted pyrrolidine derivatives exhibited
good to excellent potency. The gemꢀdifluoro azetidine (38c) and 3ꢀsubstituted piperidine (42b)
also demonstrated good potency. Incorporation of an alcohol group in the side chain to reduce
lipophilicity and potentially facilitate proꢀdrug approaches provided mixed results. Gemꢀmethyl,
OH analogues 38g-i were not tolerated whereas inclusion of prolinol (39a-b) gave good antiꢀ
tuberculosis activity. Benzylated analogue 38j and amide analogues 45a-g largely resulted in
loss of potency. For the pyrrolidine analogues the effect of chirality on activity was marked with
the (R)ꢀ3ꢀfluoro analogue 38a (Mtb IC50 = 0.23 µM) demonstrating significantly superior
potency over the (S)ꢀ3ꢀfluoro analogue 38b (Mtb IC = 1.80 µM). The effect of chirality was
5
0
also observed with the prolinol analogues, (S)ꢀprolinol analogue 39b (Mtb IC = 0.32 µM) being
50
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6
more active than (R)ꢀprolinol analogue 39a (Mtb IC = 1.52 µM). The overall SAR trends for
5
0
the series can be seen in Figure 5.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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5
6
7
8
9
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Overall SAR trends for the heterocyclic quinolone series.
In vitro DMPK and toxicity ꢀ Analogues demonstrating good potency were then moved through
our screening cascade and evaluated for microsomal turnover and HEPG2 cytotoxicity. None of
the compounds were found to be cytotoxic and all had good therapeutic indexes. From the earlier
analogues tested (entries 1ꢀ6 in Table 12) it was apparent that the compounds were being
metabolised quickly by liver microsomes. Resolving this issue was therefore the driving force for
a large proportion of the medicinal chemistry manipulations described in Table 11 above.
Table 12. HEPG2 and microsomal turnover t1/2 for selected analogues.
Compound
Mtb
IC50
(µM)
Mtb
IC90
(µM)
HEPG2
GLU
(µM)
Therapeutic
Index
Microsomal
Turnover (h, m,
r) t (min)
1
/2
7
f
0.270 ±
0.78
>100
>370
108
hꢀ7.31
mꢀ8.27
rꢀ8.30
hꢀ5.7
mꢀ4.4
rꢀ8.4
0.080
7
k
0.950 ±
0.120
1.83
1.93
102.2
>100
17b
0.611 ±
>164
hꢀ<10
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0
.048
mꢀ<10
rꢀ<10
hꢀ7.8
mꢀ6.8
rꢀ10.1
hꢀ7.9
mꢀ22.3
rꢀ10.8
hꢀ8.54
mꢀ7.65
rꢀ 5.72
hꢀ10.2
mꢀ20.7
rꢀ30.1
hꢀ10.2
mꢀ4.4
rꢀ10.6
hꢀ60
1
7e
0.300 ±
0.025
0.56
0.63
0.66
0.69
0.50
>10
188.1
>100
85.54
141
627
>272
223
1
7f
0.367 ±
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
.040
17h
0.400 ±
0.023
3
2e
0.432 ±
342
0.020
38a
0.231 ±
.036
150.6
ND
649
0
38d
>10
ND
mꢀ60
rꢀ60
42a
0.525 ±
0
1.10
0.83
>100
ND
>190
ND
hꢀ72.8
mꢀ114.9
rꢀ61.6
hꢀ17.4
mꢀ16.2
rꢀ13.7
.080
42b
0.361 ±
.041
0
Two strategies were employed to address the metabolic stability issues (Figure 6). The first was
to replace the piperidine ring with an alternative heterocycle. Amongst those selected pyrrole
(
32e) provided the most active compound with a modest improvement in metabolic stability.
Fluorination of the pyrrole (38d) at the 3 and 4 positions resulted in complete resolution of
metabolic instability; however antiꢀtuberculosis activity was also lost. From earlier SAR studies
we knew that replacing the piperidine ring (7f) with a pyrrolidine ring (17l) was tolerated in
terms of activity and may provide us with more opportunity to modify the ring in what we
believe to be a limited space. Monoꢀfluorination (38a) provided a very modest improvement in
stability. Subsequent synthesis of the gemꢀdifluoro analogue (42a) however provided us with a
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