Pengxu Wang1, Sarah M Batt2, Bin Wang3, Lei Fu3, Rongfei Qin1, Yu Lu3, Gang Li1, Gurdyal S Besra2, Haihong Huang1. 1. Beijing Key Laboratory of Active Substance Discovery and Druggability Evaluation & Chinese Academy of Medical Sciences Key Laboratory of Anti-DR TB Innovative Drug Research, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, 1 Xian Nong Tan Street, Beijing 100050, P. R. China. 2. School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom. 3. Beijing Key Laboratory of Drug Resistance Tuberculosis Research, Department of Pharmacology, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing Chest Hospital, Capital Medical University, 97 Ma Chang Street, Beijing 101149, P. R. China.
Abstract
In this study, we report the design and synthesis of a series of novel thiophene-arylamide compounds derived from the noncovalent decaprenylphosphoryl-β-d-ribose 2'-epimerase (DprE1) inhibitor TCA1 through a structure-based scaffold hopping strategy. Systematic optimization of the two side chains flanking the thiophene core led to new lead compounds bearing a thiophene-arylamide scaffold with potent antimycobacterial activity and low cytotoxicity. Compounds 23j, 24f, 25a, and 25b exhibited potent in vitro activity against both drug-susceptible (minimum inhibitory concentration (MIC) = 0.02-0.12 μg/mL) and drug-resistant (MIC = 0.031-0.24 μg/mL) tuberculosis strains while retaining potent DprE1 inhibition (half maximal inhibitory concentration (IC50) = 0.2-0.9 μg/mL) and good intracellular antimycobacterial activity. In addition, these compounds showed good hepatocyte stability and low inhibition of the human ether-à-go-go related gene (hERG) channel. The representative compound 25a with acceptable pharmacokinetic property demonstrated significant bactericidal activity in an acute mouse model of tuberculosis. Moreover, the molecular docking study of template compound 23j provides new insight into the discovery of novel antitubercular agents targeting DprE1.
In this study, we report the design and synthesis of a series of novel thiophene-arylamide compounds derived from the noncovalent decaprenylphosphoryl-β-d-ribose 2'-epimerase (DprE1) inhibitor TCA1 through a structure-based scaffold hopping strategy. Systematic optimization of the two side chains flanking the thiophene core led to new lead compounds bearing a thiophene-arylamide scaffold with potent antimycobacterial activity and low cytotoxicity. Compounds 23j, 24f, 25a, and 25b exhibited potent in vitro activity against both drug-susceptible (minimum inhibitory concentration (MIC) = 0.02-0.12 μg/mL) and drug-resistant (MIC = 0.031-0.24 μg/mL) tuberculosis strains while retaining potent DprE1 inhibition (half maximal inhibitory concentration (IC50) = 0.2-0.9 μg/mL) and good intracellular antimycobacterial activity. In addition, these compounds showed good hepatocyte stability and low inhibition of the human ether-à-go-go related gene (hERG) channel. The representative compound 25a with acceptable pharmacokinetic property demonstrated significant bactericidal activity in an acute mouse model of tuberculosis. Moreover, the molecular docking study of template compound 23j provides new insight into the discovery of novel antitubercular agents targeting DprE1.
Tuberculosis
(TB) is a chronic infectious disease caused primarily
by pathogen Mycobacterium tuberculosis (M. tuberculosis). TB is one of the
top 10 causes of death and the leading cause of mortality stemming
from a single infectious agent. In 2020, the World Health Organization
(WHO) reported that approximately 1.2 million human immunodeficiency
virus (HIV)-negative people had died and 10 million new TB cases were
identified. Globally, the TB incidence rate is falling but not fast
enough to reach the 2020 milestone of a 20% reduction between 2015
and 2020.[1] The COVID-19 pandemic threatens
to reverse the recent progress in reducing the global burden of TB
disease. The requirement for prolonged treatment with first-line drugs
coupled with often difficult-to-manage side effects routinely leads
to poor patient compliance and results in the accelerated emergence
of drug-resistant strains of M. tuberculosis. Research focusing on the development of novel small molecules with
activity against multidrug-resistant tuberculosis (MDR-TB) and extensively
drug-resistant tuberculosis (XDR-TB) remains a significant challenge.[2,3]The cell wall biosynthetic pathways have been identified as
promising
targets for the development of antitubercular agents.[4,5] Decaprenylphosphoryl-β-d-ribose 2′-epimerase
(DprE1) is crucial for mycobacterial cell wall biosynthesis.[6] DprE1 catalyzes the flavin adenine dinucleotide
(FAD)-dependent oxidation of decaprenylphosphoryl-β-d-ribose (DPR) to decaprenylphosphoryl-2′-keto-d-erythro-pentofuranose
(DPX). DPX is then reduced by decaprenylphosphoryl-d-2-ketoerythropentose
reductase (DprE2) to generate decaprenylphosphoryl-β-d-arabinofuranose (DPA), which is a unique precursor for the synthesis
of cell-wall arabinans.[7,8] Furthermore, DprE1 is specific
to mycobacteria and actinomycetes, providing inherent biochemical
selectivity over human cells and other bacterial species.[9] Therefore, DprE1 has become a vulnerable target
for treatment of drug-sensitive TB as well as MDR/XDR-TB.[10]A number of small molecules as anti-TB
agents have so far been
reported, with the benzothiazinones (BTZs) being the most well-developed
and studied DprE1 inhibitors.[11,12] Two compounds, BTZ043
and its next-generation analogue PBTZ169 (Macozinone), display potent
antimycobacterial activity (minimum inhibitory concentrations (MICs)
<0.016 μg/mL), and they are currently undergoing clinical
development.[13,14] These two small molecules likely
impart their biological activity through formation of a covalent bond
with Cys387 in the active site of DprE1. Recently, several structurally
diverse noncovalent inhibitors of DprE1 have been described in the
literature, most notably TBA-7371 and TCA1 (Figure ).[6,15−19] These compounds have the additional bonus of avoiding the nitro
group used in BTZs, a well-established structural alert, and show
the potential for further development. 1,4-Azaindole TBA-7371 has
entered clinical development, and the structure–activity relationship
(SAR) of this series has been fully explored and is well understood.[20,21] TCA1 was identified via a cell-based phenotypic
screen for inhibitors of biofilm formation in mycobacteria, which
has bactericidal activity against replicating and nonreplicating M. tuberculosis.[22,23] Inspired by
the distinct thiophenamide moiety of TCA1, we have focused on the
identification of a novel series of thiophene-arylamide compounds
with improved activity and druggability derived from lead compound
TCA1 through a scaffold hopping strategy.
Figure 1
Structures of representative
covalent and noncovalent DprE1 inhibitors.
Structures of representative
covalent and noncovalent DprE1 inhibitors.We began by analyzing the binding events displayed in TCA1 with
the DprE1 cocrystal structure (Protein Data Bank (PDB): 4KW5) shown in Figure a.[22] The thiophene moiety of TCA1 binds deeply within the bottom
of the active site in DprE1 and shows that the noncovalent binding
events between TCA1 and DprE1 are dominated by hydrophobic and van
der Waals interactions. Further to these hydrophobic interactions,
a clear hydrogen bond is formed between the thiophene moiety and His132
coupled with multiple hydrogen-bonding interactions between the carbonyl
groups of TCA1 and residues Lys418 and Ser228 (Figure a). The binding mode indicated that the 2,3-disubstituted
thiophene moiety plays an important role in maintaining key interactions
of the cocrystal structure. The benzothiazole moiety is oriented parallel
to the FAD isoalloxazine ring and forms additional hydrophobic interactions
to keep critical pharmacodynamic conformation. We speculate that the
addition of a hydrogen-bonding acceptor (HBA) in this region may enhance
binding affinity with DprE1. Moreover, the terminal carbamate moiety
at the 3-position on thiophene is considered to be metabolically labile
and modification to this region may have an advantageous effect on
overall metabolic stability.
Figure 2
Optimization of TCA1 based on analysis of the
cocrystal structure.
(a) Noncovalent interactions of TCA1 with DprE1 and modification regions.
(b) Design and optimization of thiophene-arylamide compounds as DprE1
inhibitors.
Optimization of TCA1 based on analysis of the
cocrystal structure.
(a) Noncovalent interactions of TCA1 with DprE1 and modification regions.
(b) Design and optimization of thiophene-arylamide compounds as DprE1
inhibitors.Herein, we designed and synthesized
a series of novel thiophene-arylamide
derivatives to explore their structure–activity relationships
(SARs) guided by the aforementioned crystal structure. These structural
modifications were concentrated on the arylamide motif and carbamate
moiety of TCA1 based on an evaluation of their antimycobacterial activities
as well as preliminary druggability scoring (Figure b). A representative DprE1 inhibitor 25a with acceptable pharmacokinetic (PK) properties demonstrated
significant bactericidal activity in an acute mouse model of tuberculosis.
Furthermore, molecular docking studies of the template compound 23j with the benzamide moiety provide new insight into the
discovery of novel anti-TB agents targeting DprE1.
Results and Discussion
Molecular
Docking Study of Template Compound 23j in DprE1
We began our study with molecular modeling to
understand the binding mode of the template compound 23j in the active site of DprE1 (Figure ; PDB: 4KW5). The highest docking score conformation derived from
the CDOCKER protocol was selected as the best binding pose. The overlay
of the 23j model with the crystal structure of TCA1 in
complex with DprE1 showed similar interactions in the active site
(Figure a). Hydrogen-bonding
interactions were found between compound 23j and residues
Ser228, Lys418, and His132, which were consistent with the binding
mode of TCA1. Furthermore, the phenyl ring was oriented roughly parallel
to the isoalloxazine of FAD and formed hydrophobic interactions with
residues Gln334 and Cys387. Interestingly, the acyl of benzamide formed
a key hydrogen bond with Tyr60, and the terminal piperidine interacted
with residues Leu317, Arg325, and Asn324 (Figure b). This additional reinforced interaction
of acyl piperidine may enhance the binding affinity and therefore
improve the antimycobacterial activity. This predicted model, which
was in line with our design strategy, prompted us to more closely
explore the SAR exhibited by the thiophene compounds containing this
arylamide moiety.
Figure 3
Molecular docking studies of compound 23j in DprE1.
(a) Overlay of the 23j (yellow colored) model with the
crystal structure of TCA1 (cyan) in complex with DprE1. (b) Docking
model of 23j in the binding site of M.
tuberculosis DprE1 (PDB: 4KW5).
Molecular docking studies of compound 23j in DprE1.
(a) Overlay of the 23j (yellow colored) model with the
crystal structure of TCA1 (cyan) in complex with DprE1. (b) Docking
model of 23j in the binding site of M.
tuberculosis DprE1 (PDB: 4KW5).
Chemistry
The synthesis of aryl carboxylic acids with
various amide motifs 4a–p and 7a–l is outlined in Schemes and 2. The substituted
aminothiophene intermediates 11a–d, 16a, 16b, 19, and 22a–h were synthesized following the procedures
summarized in Schemes –5. The general
synthetic procedures of target compounds 23a–p, 24a–l, and 25a–q through condensation reactions are illustrated
in Schemes and 7.
Scheme 1
Synthesis of Benzoic Acid with Different Amide Substituents 4a–p
Reagents and conditions:
(a)
Et3N, CH2Cl2, room temperature (rt),
3 h; (b) 1 mol/L LiOH aqueous solution, CH3OH, rt, 3 h.
Scheme 2
Synthesis of Aryl Carboxylic Acid with Piperidinamide
Substituents 7a–l
Reagents and conditions:
(a)
HATU, Et3N, DMF, rt, 12 h; (b) 1 mol/L LiOH aqueous solution,
CH3OH, reflux, 2 h; (c) EDCI, HOBt, Et3N, DMF,
rt, 24 h.As shown in Scheme , intermediates 3a–p were obtained via amidation of commercially
available methyl 4-(chlorocarbonyl)benzoate
(1) with the corresponding amines 2a–p in the presence of triethylamine. The obtained 3a–p were converted to intermediates 4a–pvia hydrolysis with aqueous
lithium hydroxide solution.According to Scheme , the condensation reactions of various aryl
carboxylic acids 5a–l with piperidine
afforded corresponding
intermediates 6a–l in the presence
of 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU). The hydrolysis of 6a–l led to key intermediates 7a–l.As shown in Scheme 3, methyl 2-aminothiophene-3-carboxylate
(8) was reacted with (Boc)2O to form intermediate 9, and subsequent hydrolysis led to thiophene carboxylic acid 10. Key intermediates 11a and 11b were obtained via condensation of 10 with alicyclic amines and routine N-Boc deprotection.
In addition, intermediates 11c and 11d were
prepared from 8via a two-step hydrolysis/condensation
strategy without the need for N-Boc protection and
deprotection.The synthesis of aminothiophene intermediates 16a, 16b, 19, and 22a–h is summarized in Schemes and 5. 2-Cyanoacetic
acid (13) was treated with carbamates 14a, 14b in
the presence of phosphorus oxychloride to form intermediates 15a and 15b. Aminothiophene intermediates 16a and 16b were obtained by heterocyclization
of 15a, 15b with 2,5-dihydroxy-1,4-dithiane via a Gewald reaction.[23] 2-Cyanoacetamide
(17) was reacted with oxalyl chloride under reflux to
provide the isocyanate, and a subsequent reaction with ethylamine
gave intermediate 18 without further purification, which
followed the Gewald reaction to afford aminothiophene intermediate 19. The condensation reactions of 2-cyanoacetic acid (13) with various aryl amines 20a–h delivered the corresponding intermediates 21a–h in the presence of EDCI with DMAP at room
temperature. The Gewald heterocyclization of 21a–h with 2,5-dihydroxy-1,4-dithiane produced aminothiophene
intermediates 22a–h.
Scheme 4
Synthesis of Aminothiophene Intermediates 16a, 16b, and 19
Reagents
and conditions: (a)
phosphorus oxychloride, DMF, toluene, 80 °C, 3 h; (b) 2,5-dihydroxy-1,4-dithiane,
Et3N, CH3OH, 50 °C, 2.5 h; (c) oxalyl chloride,
1,2-dichloroethane, reflux, 4 h; (d) ethylamine, CH3CN,
−10 °C, 3 h.
As illustrated
in Schemes and 7, target compounds 23a–p, 24a–l, and 25a–q were conveniently obtained through
the condensation reaction with aryl carboxylic acids and aminothiophenes.
Ethyl(2-aminothiophene-3-carbonyl)carbamate (16a) was
subjected to the condensation reaction with various 4-carbamoylbenzoic
acids 4a–p in the presence of HATU
to afford the corresponding products 23a–p. In the same way, the target products 24a–l were obtained from 16a and the corresponding
carbamoyl aromatic acid or carbamoyl benzoyl chloride.4-(Piperidine-1-carbonyl)benzoic
acid (4j) was reacted
with 16b, 19, 11a–d, and 22a–h to afford the
corresponding products 25a, 25c, 25e–h, 25i–j, and 25l–q under the standard condensation
conditions. The subsequent hydrolysis of 25a with aqueous
lithium hydroxide provided compound 25d. In addition,
3-fluoro-4-(piperidine-1-carbonyl)benzoic acid (7f) was
condensed with 16b or 22b to form the desired
products 25b or 25k, respectively.
SAR Optimization
Strategy
The target compounds were
evaluated for their activities against M. tuberculosis H37Rv using the microplate Alamar blue assay (MABA).
Minimum inhibitory concentration (MIC) was defined as the lowest concentration
resulting in a reduction in fluorescence of ≥90% relative to
the mean of replicate bacterium-only controls. The compounds with
MIC less than 1 μg/mL were further tested for mammalian cell
cytotoxicity using Vero cells measured by the concentration required
for inhibiting 50% cell growth (half maximal inhibitory concentration
(IC50)) as compared to the no-treatment control. Tables –9 summarize the biological data including in vitro and in vivo anti-TB activity,
toxicity, metabolic stability, PK, and target validation for these
novel thiophene-arylamide derivatives. TCA1, isoniazid (INH), and
rifampicin (RFP) were used as reference compounds for the anti-TB
activity assay.
Table 1
SAR of Thiophene-arylamide
Compounds
at R1 and R2 Sites
MIC against M. tuberculosis H37Rv.
SI =
selectivity index, IC50/MIC.
Table 9
Inhibition
of DprE1 and Cellular Potency
by Thiophene-arylamides
compounds
MICOE (μg/mL)a (DprE1)
MICOE (μg/mL)b (DprE2)
MICWT (μg/mL)c
MIC
foldshiftd
IC50 DprE1 (μg/mL)e
23j
>1.28
0.32
0.32
>4
0.3 ± 0.1
24f
>0.51
0.13
0.064
>8
0.2 ± 0.04
24j
8.96
0.56
0.28
32
0.3 ± 0.1
25a
>12.16
1.52
0.76
>16
0.9 ± 0.2
25b
>1.98
0.25
0.12
>16
0.4 ± 0.1
25l
12.8
1.60
1.60
8
0.4 ± 0.1
TCA1
15.36
0.96
0.48
32
0.1 ± 0.01
Overexpressor (OE)
MICs are for Mycobacterium bovis bacillus
Calmette-Guerin (BCG)
Pasteur transformed with pMV261:Mt-dprE1.
Overexpressor (OE) MICs are for M. bovis BCG Pasteur transformed with pMV261:Mt-dprE2.
MICs against M. bovis BCG Pasteur strain transformed with pMV261.
Ratio of MIC values against
the
DprE1-overexpressing strain and wild-type strain.
All in vitro assays
were performed using Mt-DprE1.
MIC against M. tuberculosis H37Rv.SI =
selectivity index, IC50/MIC.MIC against M. tuberculosis H37Rv.SI = selectivity index, IC50/MIC.Keeping 2,3-disubstituted
thiophene as the key core, initial MIC-based
SAR studies against M. tuberculosis around the lead compound TCA1 led to the first thiophene-benzamide
series. The analysis of noncovalent interactions of TCA1 with DprE1
and modification sites (Figure ) indicated that the binding pocket in this active site is
deep and not fully occupied by the terminal benzothiazole moiety of
the 2-position side chain on the thiophene ring. Therefore, a variety
of benzamides with different physical and chemical properties were
introduced to further improve the antimycobacterial activity and druggability
profile. As listed in Table , to our delight, compound 23a with the terminal
propylamine exhibited good potency with improved MIC compared with
TCA1 (0.2 vs 0.48 μg/mL). Furthermore, compound 23a displayed lower cytotoxicity against Vero cells with IC50 > 64 μg/mL (SI > 320). Encouraged by the promising
results, we evaluated a series of secondary amines and the bulky cyclic
amines at the terminal of the side chain. The subsequent results revealed
that diethylamine (23c, MIC = 0.28 μg/mL) was tolerated
at the R1 and R2 positions, whereas the slightly
smaller ethyl(methyl) amine (23b, MIC = 0.95 μg/mL)
showed a decrease in activity. Encouraged by this result, large substituents,
such as amantadine, cyclohexylamine, and aniline moieties were introduced
to the side chain, aiming to explore the optimum volume at the R1 and R2 sites. Compounds 23d and 23g bearing amantadine and aniline moieties, respectively,
showed improved antimycobacterial activities as compared to the reference
compound TCA1 (0.12 vs 0.48 μg/mL) but showed
a certain level of cytotoxicity. Additionally, increasing the overall
volume through the addition of gem-dimethyl to the cyclohexylamine
led to lower potency (23e and 23f). Based
on the above results, conformationally restricted secondary amines
were embedded at the terminal of the side chain to further investigate
the most favorable size for optimal occupancy of hydrophobic pockets
of DprE1. The results indicated that smaller substituents such as
azetidine (23h, MIC = 0.84 μg/mL) led to lower
potency, while the larger pyrrolidine (23i, MIC = 0.20
μg/mL), piperidine (23j, MIC = 0.02 μg/mL),
and azepane (23k, MIC = 0.22 μg/mL) moieties showed
improved antimycobacterial activities. In particular, compound 23j showed a significant improvement in MIC, corresponding
to a 24-fold enhancement of potency relative to TCA1 (0.02 vs 0.48 μg/mL). In addition, 23j also
displayed high selectivity index (SI > 3200), indicating a good
safety
profile. Subsequently, the addition of methyl to the piperidine generated
compound 23l, which showed a MIC of 0.03 μg/mL,
demonstrating its equivalency in antimycobacterial activity to 23j. The replacement of methyl (23l, MIC = 0.03
μg/mL) with methoxyl (23m, MIC = 0.12 μg/mL)
or difluoro (23n, MIC = 0.25 μg/mL) substituents
resulted in lower potency. The bioisosteric replacement strategy to
replace methylene (23j, MIC = 0.02 μg/mL) with
an oxygen (23o, MIC = 1.66 μg/mL) or a sulfur (23p, MIC = 0.33 μg/mL) atom caused a significant decrease
in antimycobacterial activity.Based on the results from Table , coupled with the
molecular docking studies, and keeping
the privileged acyl piperidine fragment, we decided to explore the
effect of the aryl moiety on antimycobacterial activity. Compared
to compound 23j, substitution at the meta-position of
the phenyl ring instead of the para-position led to a marked decrease
in potency, as exemplified by compound 24a (MIC = 3.70
μg/mL). We next explored the introduction of halo, nitro, and
methyl substituents on the phenyl ring, which have the potential to
form π–π interactions in the hydrophobic pocket
of DprE1. Introducing fluoro, chloro, and bromo substituents to the
phenyl ring afforded compounds 24b–d, which displayed good activity with a range of MICs 0.12–0.49
μg/mL. The involvement of the strongly electron-withdrawing
nitro group (24e, MIC = 1.86 μg/mL) led to lower
potency. Compound 24f exhibited a significant increase
in potency by altering the position of the fluoro group, corresponding
to a 10-fold enhancement of potency relative to 24b (MIC
< 0.016 vs 0.12 μg/mL). The introduction
of a methyl group (24g, MIC = 0.12 μg/mL) at the
same position resulted in equal potency compared to 24b. However, replacement with naphthalene (24h, MIC =
3.13 μg/mL) and biphenyl (24i, MIC = 7.76 μg/mL)
caused an obvious decrease in activity. Subsequently, introduction
of smaller five-membered aromatic heterocycles, such as thiophene
(24j, MIC = 0.14 μg/mL), furan (24k, MIC = 0.37 μg/mL), and pyrrole (24l, MIC = 1.77
μg/mL) resulted in moderate to good antimycobacterial activity.
Accordingly, we drew the conclusion that a phenyl ring bearing the
electron-withdrawing fluoro substituent was the best tolerated at
the Ar site.Since the thorough SAR at the 2-position side chain
of thiophene
has been investigated, our attention shifted to the exploration of
the 3-position with substituents at the R3 site. As listed
in Table , compounds 25a and 25b with imide methyl ester showed better
antimycobacterial activity than TCA1 (MIC 0.19 and 0.03 μg/mL vs 0.48 μg/mL). Among them, fluoro-substituted phenyl
derivative (25b) showed a MIC of 0.03 μg/mL and
an SI value above 2133. Replacing the ethyl ester (23j) with ethyl amide (25c) resulted in a dramatic loss
of antimycobacterial activity. Removing the terminal acyl ester also
led to an inactive compound (25d). Next, keeping the
terminal acyl ester moiety, rigid alicyclic amines (25e–h) were introduced to further evaluate the size
differential of substituents at the R3 site; however, all
sterically encumbered substituents were not tolerated, resulting in
a large loss of potency. When we replaced the carbamate with various
rigid aromatic heterocycles aiming to enhance metabolic stability,
compounds with meta-substituted pyrimidine (25j, 25k, and 25l) showed good antimycobacterial activity,
especially 25l with a MIC of 0.2 μg/mL. The introduction
of thiazole, pyridine, and benzothiazole led to the total loss of
potency (25i, 25n, 25o, and 25q).
Table 3
SAR of Thiophene-arylamide Compounds
at X and R3 Sites
MIC against M. tuberculosis H37Rv.
SI =
selectivity index, IC50/MIC.
MIC against M. tuberculosis H37Rv.SI =
selectivity index, IC50/MIC.
In Vitro Anti-XDR-TB Activity and Intracellular
Antimycobacterial Activity
Considering that most thiophene-arylamide
derivatives displayed potent activity against M. tuberculosis H37Rv, some representative compounds were further tested
against two XDR-TB isolated clinical strains (Table ). It is gratifying to note that the selected
compounds, together with reference TCA1, demonstrated potent activities
against XDR-TB strains. In particular, compounds 23j, 24f, and 25b displayed very potent activity against
both drug-susceptible and drug-resistant tuberculosis in vitro, compared to TCA1. Furthermore, we conducted an additional assay
to assess the MIC against PBTZ169- and bedaquiline-resistant strains
of M. tuberculosis for the representative
compounds. The results revealed that the thiophene-arylamide derivatives
showed no significant cross resistance to the covalent DprE1 inhibitor
PBTZ169 and ATP synthase inhibitor bedaquiline. The results indicated
that these novel thiophene-arylamide derivatives are noncovalent DprE1
inhibitors and represent promising additions in the combination regiment
for the treatment of drug-resistant TB.
Table 4
Activity
of Representative Compounds
against Clinical Isolates of M. tuberculosis
MIC (μg/mL)
compounds
H37Rv
13946a
14862b
PBTZ169-resistant strain
bedaquiline-resistant strain
23j
0.055
0.082
0.059
0.14
24f
0.020
0.031
0.031
0.062
0.054
24j
0.059
0.12
0.12
24k
0.15
0.44
0.23
25a
0.12
0.24
0.24
0.48
25b
0.030
0.062
0.12
25l
0.20
0.94
0.48
TCA1
0.47
0.95
0.48
1.23
INH
0.019
2.46
>10
RFP
0.015
>10
9.24
PBTZ169
0.0001
0.005
bedaquiline
0.036
2.24
Resistance to isoniazid
(INH), streptomycin
(SM), rifampicin (RFP), ethambutol (EMB), rifabutin (RBT), paza-aminosalicylate
(PAS), and ofloxacin (OFLX).
Resistance to INH, SM, RFP, EMB,
PAS, prothionamide (1321), and capreomycin (CPM).
Resistance to isoniazid
(INH), streptomycin
(SM), rifampicin (RFP), ethambutol (EMB), rifabutin (RBT), paza-aminosalicylate
(PAS), and ofloxacin (OFLX).Resistance to INH, SM, RFP, EMB,
PAS, prothionamide (1321), and capreomycin (CPM).Since M. tuberculosis is an intracellular
pathogen and survives in macrophages, we investigated the selected
potent compounds for antimycobacterial activity in an intracellular
macrophage infection model. As shown in Table , 0.75–1.34 log10 CFU reduction in macrophages was observed following treatment
with selected compounds for 3 days at 10 μg/mL. Most notably,
compounds 23j, 24f, and 25l exhibited better intracellular activity with a reduction of ∼1 log10 CFU (compared to TCA1 = 0.61 log10 CFU) and similar to the positive control RFP at 5 μg/mL,
although these compounds did not display the significant dose–response
compared to the reduced colony-forming unit (CFU) data at 10 μg/mL,
which may be related to either the mechanism of action of the DprE1
inhibitor as a fast bactericidal or the permeability of the cell wall
to the specific compounds. The above encouraging data drove us to
further explore these promising thiophene-arylamides in preliminary
druggability profiles.
Table 5
Activity of Selected
Compounds in
an Intracellular Macrophage Infection Model
log10 CFU/macrophagesa
compounds
10 μg/mL
Δlog10 CFUb
5 μg/mL
Δlog10 CFUb
23j
4.45 ± 0.03
1.07
4.54 ± 0.01
0.98
24f
4.18 ± 0.04
1.34
4.41 ± 0.10
1.11
25a
4.77 ± 0.10
0.75
5.06 ± 0.03
0.46
25b
4.59 ± 0.05
0.93
4.75 ± 0.21
0.77
25l
4.54 ± 0.00
0.98
4.59 ± 0.03
0.93
TCA1
4.36 ± 0.08
1.16
4.91 ± 0.19
0.61
RFP
4.49 ± 0.11
1.03
untreated
5.52 ± 0.18
5.52 ± 0.18
log10 CFU against M. tuberculosis (H37Rv) in infected mouse
J774A.1 macrophages.
Δlog10 CFU
= log10 CFU (untreated) – log10 CFU (treated with the selected compounds).
log10 CFU against M. tuberculosis (H37Rv) in infected mouse
J774A.1 macrophages.Δlog10 CFU
= log10 CFU (untreated) – log10 CFU (treated with the selected compounds).
In Vitro ADME/T Assay and In Vivo Pharmacokinetic Property Evaluation
To
identify the metabolic
and toxic liabilities of our thiophene-arylamide scaffold, the selected
compounds, along with the reference TCA1, were evaluated for their
hepatocyte stability, cytotoxicity against mammalian HepG2 cells,
and human ether-à-go-go related gene (hERG) liability. As presented
in Table , we were
pleased to find that all selected compounds exhibited no cytotoxicity against HepG2 cells
with IC50 > 64 μg/mL. In addition, low inhibition
profiles of the hERG channel (IC50 > 20 μM) across
the series indicated a low risk of blocking the cardiac potassium
channel and causing QT prolongation. Although the selected compounds
showed some metabolic liability in mouse hepatocytes compared to TCA1,
they displayed significant superior stability in human hepatocytes
(t1/2 = 20.3–72.5 vs 5.97 min).
Table 6
Hepatocyte Stability, Cytotoxicity,
and hERG Inhibition of Selected Compounds
compounds
hepatocyte
stability
cytotoxicity (HepG2)
hERG K+
mouse
human
t1/2 (min)
%remaininga
t1/2 (min)
%remaininga
IC50 (μg/mL)
IC50 (μM)
23j
56.4
69.1
50.8
66.4
>64
23.7
24f
43.7
62.1
38.1
58.0
>64
>30
24j
19.3
34.0
72.5
75.1
>64
28.3
25a
27.1
46.5
20.3
35.9
>64
22.6
25b
12.5
19.0
32.6
52.8
>64
>30
TCA1
85.3
74.8
5.97
3.1
46.1
18.3
Substrate concentrations were determined
in incubations after 30 min and normalized to concentrations at time
zero.
Substrate concentrations were determined
in incubations after 30 min and normalized to concentrations at time
zero.Spurred on by the
pronounced anti-TB activity and structural diversity,
compounds 24f and 25a were chosen for further
evaluation of their metabolic features in Balb/c mice. Pharmacokinetic
(PK) studies for compounds 24f, 25a, and
TCA1 were performed in Balb/c mice, following a single oral and an
intravenous dose (Table ). Compound 25a exhibited high plasma exposure (area
under the curve (AUC)0–∞ = 657 ng·h/mL)
and high maximum plasma concentration (Cmax = 486 ng/mL) after oral administration, compared to compound 24f with AUC0–∞ = 57.9 ng·h/mL
and Cmax = 25.4 ng/mL. In addition, the
oral bioavailability of 24f was very low (F = 2.3%) and clearance was high, which was dropped off in further
studies. Moreover, TCA1 produced a much higher exposure concentration
than 25a and possessed a 5-fold enhancement of oral bioavailability.
Based on the results of PK profiles, compound 25a with
acceptable oral bioavailability (F = 7.9%) was deemed
worthy of further evaluation in in vivo efficacy
studies.
Table 7
Mouse PK Properties of Compounds 24f, 25a, and TCA1
In vivo Efficacy Study on Compound 25a in a Mouse Model of
TB
The in vivo efficacy
of compound 25a and TCA1 was conducted in Balb/c mice
in an acute TB infection model. Compound 25a and reference
TCA1 were orally administered at 100 mg/kg, whereas the positive control,
INH, was given at 25 mg/kg. The same formulation, 0.5% carboxymethylcellulose
(CMC) in water, was used for all compounds tested. After three weeks
of treatment, the mice were sacrificed and the number of colony-forming
units (CFUs) in the lungs were counted and compared with those in
the untreated control group. As shown in Table , compound 25a showed potent in vivo activity, reducing the bacterial burden in the lungs
by 2.02 log10 CFU compared with the untreated
control group. In another batch, TCA1 displayed similar in
vivo bactericidal activity compared to 25a,
which resulted in a reduction of 2.86 log10 CFU
in the lungs. These results showed that compound 25a exhibited
similar potency even at low bioavailability compared to TCA1, which
indicates that further optimization of improving the PK profiles and
therefore enhancing the in vivo efficacy is required.
Finally, to
identify and confirm the biological target for this novel thiophene-arylamide
series, the selected compounds with potent antimycobacterial activities
and diversified side chains were measured against an overexpressed
Mt-DprE1 in M. bovis BCG (Table ). Compounds 23j, 24f, 24j, 25a, 25b, and 25l showed
a decrease in potency against only DprE1-overexpressing strains but
not against DprE2-overexpressing and wild-type strains. These compounds,
as well as positive control TCA1, displayed a 4-fold higher shift
in the MIC when Mt-DprE1 was overexpressed in M. bovis BCG. The promising results indicate that DprE1 could be the target
for this novel thiophene-arylamide series.Overexpressor (OE)
MICs are for Mycobacterium bovis bacillus
Calmette-Guerin (BCG)
Pasteur transformed with pMV261:Mt-dprE1.Overexpressor (OE) MICs are for M. bovis BCG Pasteur transformed with pMV261:Mt-dprE2.MICs against M. bovis BCG Pasteur strain transformed with pMV261.Ratio of MIC values against
the
DprE1-overexpressing strain and wild-type strain.All in vitro assays
were performed using Mt-DprE1.To further verify that thiophene-arylamides inhibit the catalytic
activity of DprE1, representative compounds were tested against M. tuberculosis DprE1. Potent IC50 values
in the range of 0.2–0.9 μg/mL were obtained, showing
a good correlation with the MIC for the compounds tested. These results
confirm a similar mode of action of the thiophene-arylamides described
here to TCA1. Thus, our scaffold hopping design strategy has resulted
in the successful identification of thiophenes containing the key
arylamide moiety as potent DprE1 inhibitors.
Conclusions
DprE1 has emerged as a promising target for the treatment of tuberculosis,
and previous studies have been indicated that the inhibition of DprE1
causes loss of its ability to construct the bacterial cell wall. Based
on the crystal structure of the TCA1–DprE1 complex, we have
reported the design, synthesis, and SAR study of a series of novel
thiophene-arylamide compounds. Molecular docking studies of template
compound 23j with DprE1 indicated that the hydrogen bond
interaction with Tyr60 provided additional binding affinity in reinforcing
the interaction of thiophene containing the critical benzamide moiety.
Subsequent scaffold hopping from the benzothiazole to arylamide moiety
led to a new series of lead compounds with improved antimycobacterial
activity and low cytotoxicity. In particular, the representative compounds
displayed very potent activity against both drug-susceptible and drug-resistant
tuberculosis compared to the target compound TCA1. In addition, the
selected compounds also displayed good inhibition of intracellular
TB growth in infected macrophages. Furthermore, the preliminary druggability
study demonstrated that the selected compounds exhibited good hepatocyte
stability and low hERG liability. Further biological studies revealed
that these novel thiophene-arylamide compounds targeted DprE1, like
representative compounds 23j, 24f, 25a, and 25b directly bound to DprE1, and displayed
good to excellent DprE1 inhibition. Importantly, compound 25a with acceptable PK profiles demonstrated significant efficacy in vivo in an acute mouse model of TB. Our efforts are ongoing
to improve the druggability profiles of this series of thiophene-arylamide
compounds by maintaining good antituberculosis activity with the aim
of developing more promising candidates as anti-TB agents targeting
DprE1.
Experimental Section
All
reagents and solvents were purchased
from commercial suppliers and used without further purification. Reactions
were monitored by thin-layer chromatography (TLC) with visualization
of components by UV light (254 nm) or exposure to I2. Flash
column chromatography was conducted on silica gel (300–400
mesh). Melting points were determined on a Yanaco MP-J3 microscope
melting point apparatus, which is uncorrected. 1H NMR and 13C NMR spectra were recorded on Varian-400 and Mercury-500/600
spectrometers in CDCl3 or dimethyl sulfoxide (DMSO)-d6. Electrospray ionization-high-resolution mass
spectrometry (ESI-HRMS) data were measured on a Thermo Exactive Orbitrap
plus spectrometer.All target compounds were purified by chromatography
and have a purity of ≥95% as determined by high-performance
liquid chromatography (HPLC)/MS analysis conducted on a Thermo Exactive
Plus system using a reversed-phase C18 column with 5–95% CH3CN in water (0.1% HCOOH) for 5 min at a flow rate of 0.4 mL/min.
General
Procedure for the Synthesis of Intermediates 3a–p
To a solution of methyl 4-(chlorocarbonyl)benzoate 1 (1 equiv) in anhydrous CH2Cl2 were
added the corresponding amines 2a–p (1.5 equiv) and Et3N (5 equiv) cooled with an ice bath.
The reaction mixture was stirred at room temperature for 3 h under
argon, then quenched with water, and extracted with CH2Cl2 twice. The combined organic phase was washed with
1 N HCl, H2O, saturated NaHCO3, and brine in
turn. The obtained organic phase was dried over anhydrous Na2SO4, filtered, and evaporated in vacuo to give intermediates 3a–p.
General Procedure for the Synthesis of Intermediates 4a–p
To a solution of 3a–p (1 equiv) in CH3OH was added 1
mol/L LiOH aqueous
solution (2 equiv). The reaction mixture was stirred at room temperature
for 3 h and then evaporated in vacuo. The residue
was diluted with H2O, and the aqueous solution was acidified
with 6 N HCl to pH 6–7. The precipitated solid was filtered
to afford intermediates 4a–p.
General Procedure for the
Synthesis of Intermediates 6a–l
To a solution of aryl carboxylic
acids 5a–l (1 equiv) in DMF were
added HATU (2 equiv), piperidine (1.5 equiv), and Et3N
(3 equiv). The reaction mixture was stirred at room temperature for
10 h and then concentrated in vacuo. The residue
was diluted with CH2Cl2, washed with water and
brine, dried over anhydrous Na2SO4, filtered,
and evaporated in vacuo. The residue was purified
by silica gel column chromatography (petroleum ether (PE)/ethyl acetate
(EA) = 100/30) to afford intermediates 6a–l.
To a solution of compound 8 (1.00
g, 6.36 mmol) and
(Boc)2O (1.50 g, 9.54 mmol) in CH2Cl2 (100 mL) were added DMAP (0.08 g, 0.64 mmol) and Et3N
(1.29 g, 12.72 mmol). The reaction mixture was stirred under argon
at room temperature for 6 h and then quenched with water (100 mL).
The organic phase was washed with brine, dried over anhydrous Na2SO4, filtered, and evaporated in vacuo. The residue was purified by silica gel column chromatography (PE/EA
= 100/20) to give intermediate 9.
To a solution
of 9 (2.00 g, 7.77 mmol) in CH3OH (100 mL)
was added 1 mol/L NaOH aqueous solution (20 mL). The
reaction mixture was heated to reflux for 4 h. After cooling to room
temperature, the solution was acidified with 1 N HCl aqueous solution
to pH 2 at 0 °C, and the precipitated solid was filtered to afford
intermediate 10.
General Procedure for the Synthesis of Intermediates 11a and 11b
To a solution of compound 10 (1 equiv) and alicyclic amines (1.1 equiv) in anhydrous
DMF were
added EDCI (1.1 equiv), HOBt (1.1 equiv), and Et3N (1.1
equiv) in turn. The reaction mixture was stirred under argon at room
temperature for 12 h. The reaction mixture was quenched with water
and extracted with ethyl acetate twice. The combined organic phase
was washed with brine, dried over anhydrous Na2SO4, filtered, and evaporated in vacuo. The residue
was purified by silica gel column chromatography (CH2Cl2/MeOH = 100/1) to give intermediates with N-Boc. The above intermediates (1.36 mmol) and trifluoroacetic acid
(4 mL) were dissolved in CH2Cl2 (12 mL). The
reaction mixture was stirred at room temperature for 2.5 h and then
poured into water (15 mL). The aqueous phase was separated and basified
with K2CO3 to pH 7–8. The precipitated
solid was filtered to obtain intermediates 11a and 11b.
General Procedure for the
Synthesis of Intermediates 11c and 11d
To a solution of compound 12 (1 equiv) and alicyclic
amines (1.1 equiv) in anhydrous DMF were
added EDCI (1.1 equiv), HOBt (1.1 equiv), and Et3N (1.1
equiv) in turn. The reaction mixture was stirred under argon at room
temperature for 12 h. The reaction mixture was quenched with water
and extracted with ethyl acetate twice. The combined organic phase
was washed with brine, dried over anhydrous Na2SO4, filtered, and evaporated in vacuo. The residue
was purified by silica gel column chromatography (CH2Cl2/MeOH = 100/1) to give intermediates 11c and 11d.
To a solution of methyl 2-aminothiophene-3-carboxylate 8 (1.22 g, 7.77 mmol) in CH3OH (25 mL) was added
1 mol/L
NaOH aqueous solution (15 mL). The reaction mixture was heated to
reflux for 4 h. After cooling to room temperature, the solution was
acidified with 1 N HCl solution to pH 2 at 0 °C and the precipitated
solid was filtered to afford intermediate 12.
General
Procedure for the Synthesis of Intermediates 15a and 15b
To a solution of 2-cyanoacetic acid 13 (15.0 g, 176.35 mmol) and carbamate 14a or 14b (176.35 mmol) in the mixed solvents of toluene (90 mL)
and DMF (5.4 mL) was added POCl3 (8.22 mL, 88.18 mmol)
cooled with an ice bath. The reaction mixture was stirred at 80 °C
for 3 h under argon and then slowly poured into ice-cold water (500
mL). The precipitated solid was filtered and washed with saturated
NH4Cl and water to afford intermediates 15a and 15b, respectively.
General
Procedure for the Synthesis of Intermediates 16a and 16b
To a solution of 15a or 15b (102.3 mmol) in CH3OH (150 mL) were added 2,5-dihydroxy-1,4-dithiane
(7.77 g, 51.0 mmol) and Et3N (15.6 mL, 112.3 mmol) in turn
cooled with an ice bath. The reaction mixture was stirred at 50 °C
for 2.5 h under argon and then concentrated. The residue was diluted
with CH2Cl2 (100 mL) and filtered. The obtained
solid was washed with saturated NH4Cl solution (100 mL)
and water to afford intermediates 16a and 16b, respectively.
To a magnetically stirred solution of 2-cyanoacetamide 17 (2.0 g, 23.79 mmol) in 1,2-dichloroethane (50 mL) was added
oxalyl
chloride (6.0 mL, 71.36 mmol). The reaction mixture was heated to
reflux for 4 h under an argon atmosphere. The solvent was evaporated
under reduced pressure. To a solution of the residue in anhydrous
acetonitrile (30 mL) was added ethylamine (20.0 mL) in anhydrous acetonitrile
(80 mL) dropwise keeping the reaction under −10 °C. The
reaction mixture was stirred for additional 3 h at −10 °C
and then concentrated. The residue was washed with water, filtered,
dried, and used in the next step without further purification.
Procedure
for the Synthesis of Intermediate 19
To a solution
of compound 18 (20.0 mg, 1.24 mmol)
in CH3OH (20 mL) were added 2,5-dihydroxy-1,4-dithiane
(98.0 mg, 0.65 mmol) and Et3N (0.27 mL, 1.94 mmol) cooled
with an ice bath. The reaction mixture was stirred at 50 °C for
2.5 h under argon and then concentrated. The residue was purified
by silica gel column chromatography (CH2Cl2/MeOH
= 100/1) to give compound 19.
General Procedure for the Synthesis of Intermediates 21a–h
To a solution of compounds 20a–h (1 equiv) and cyanoacetic acid 13 (3 equiv) in anhydrous DMF were added EDCI (2 equiv) and
DMAP (0.5 equiv). The reaction mixture was stirred under argon at
room temperature for 24 h. The reaction mixture was quenched with
water and extracted with ethyl acetate twice. The combined organic
phase was washed with brine, dried over anhydrous Na2SO4, filtered, and evaporated in vacuo. The
residue was purified by silica gel column chromatography (CH2Cl2/MeOH = 100/1) to give compounds 21a–h.
General Procedure for the
Synthesis of Intermediates 22a–h
To a solution of compound 21a–h (1 equiv) in methanol were added 2,5-dihydroxy-1,4-dithiane
(0.5 equiv) and Et3N (1.1 equiv). The reaction mixture
was stirred for 7 h under argon at 50 °C and then evaporated in vacuo. The residue was purified by silica gel column
chromatography (CH2Cl2/MeOH = 100/1) to give
compounds 22a–h.
General Procedure for the
Synthesis of Target Compounds 23a–p
To a solution of benzoic
acids 4a–p (1.2 equiv) and 2-aminothiophene 16a (1 equiv) in DMF were added HATU (2 equiv) and Et3N (3 equiv) in turn. The reaction mixture was stirred at room
temperature for 12 h, then quenched with water, and extracted with
CH2Cl2 thrice. The combined organic phase was
washed with brine, dried over anhydrous Na2SO4, filtered, and evaporated in vacuo. The residue
was purified by silica gel column chromatography (CH2Cl2/MeOH = 100/1) to give target compounds 23a–p.
General Procedure for the Synthesis of Target
Compounds 24a, 24c, 24d, 24f,
and 24h–l
The target compounds
were prepared from 16a and corresponding aryl carboxylic
acids 7 in the same manner as described for 23a–p.
General Procedure for the Synthesis of Target
Compounds 24b, 24e, and 24g
To a
magnetically stirred solution of the benzoic acids 7b, 7e or 7g (1 equiv) in CH2Cl2 were added thionyl chloride (2 equiv) and a drop of DMF.
The reaction mixture was heated to reflux for 3 h under an argon atmosphere.
The solvent was evaporated under reduced pressure. The residue was
dissolved in anhydrous THF. To a solution of 2-aminothiophene 16a (0.7 equiv), DMAP (0.07 equiv), and Et3N (3.5
equiv) in anhydrous THF was slowly added the above THF solution cooled
with an ice bath. The reaction mixture was stirred for additional
3 h at room temperature and then concentrated. The residue was purified
by silica gel column chromatography (CH2Cl2/MeOH
= 100/1) to give target compounds 24b, 24e, and 24g, respectively.
Procedure
for the Synthesis of Target Compound 25d
To
a solution of 25a (50 mg, 0.12 mmol) in
30 mL of CH3OH was added 1 mol/L LiOH aqueous solution
(0.6 mL). The reaction mixture was heated to reflux for 2 h and then
concentrated. The residue was diluted with CH2Cl2 (30 mL), washed with water (30 mL) and brine (30 mL), dried over
anhydrous Na2SO4, filtered, and concentrated.
The residue was washed with n-hexane and dried to give target compound 25d.
General Procedure for the
Synthesis of Target Compounds 25e–h
Target compounds 25e–h were prepared from 4j (1.6 equiv) and the corresponding
aminothiophene intermediates 11a–d (1 equiv) in the same manner as
described for 23a–p.
General Procedure for the
Synthesis of Target Compounds 25i–q
To a solution of compounds 22a–h (1 equiv) in anhydrous DMF were
added the corresponding benzoic acid 4j or 7f (1.2 equiv), EDCI (1.3 equiv), HOBt (1.3 equiv), and Et3N (2.5 equiv) in turn. The reaction mixture was stirred for 24 h
under argon at room temperature, then quenched with water, and extracted
with EA thrice. The combined organic phase was washed with brine,
dried over anhydrous Na2SO4, filtered, and evaporated in vacuo. The residue was purified by silica gel column
chromatography (CH2Cl2/MeOH = 100/1) to give
target compounds 25i–q.
MICs against replicating M. tuberculosis H37Rv or clinical isolates
were determined by the microplate Alamar blue assay (MABA) following
the protocol as described previously.[24,25] RFP, INH,
and TCA1 were included as positive controls. M. tuberculosis H37Rv or isolated clinical strains were grown to the
late log phase (70–100 Klett units) in Difco Middlebrook 7H9
Broth (Seebio) supplemented with 0.2% (v/v) glycerol, 0.05% Tween
80, and 10% (v/v) albumin–dextrose–catalase (Seebio)
(7H9-ADC-TG). Cultures were centrifuged, washed twice, and then resuspended
in phosphate-buffered saline. Suspensions were then passed through
an 8 μm pore size filter to remove clumps, and aliquots were
frozen at −80 °C. Twofold dilutions of compounds were
prepared in 7H9-ADC-TG in a volume of 100 μL in 96-well clear-bottom
microplates (BD). M. tuberculosis (100
μL containing 2 × 105 CFU) was added to yield
a final testing volume of 200 μL. The plates were incubated
at 37 °C; on day 7 of incubation, 12.5 μL of 20% Tween
80 and 20 μL of Alamar blue were added to all wells. After incubation
at 37 °C for 16–24 h, the fluorescence was read at an
excitation of 530 nm and an emission of 590 nm. The MIC was defined
as the lowest concentration resulting in a reduction in fluorescence
of ≥90% relative to the mean of replicate bacterium-only controls.
Cytotoxicity Assay[24,25]
Vero cells
and HepG2 cells were cultured in Roswell Park Memorial Institute (RPMI)
1640 medium supplemented with 10% fetal bovine serum (FBS). The cells
were incubated in a humidified atmosphere of 5% CO2 at
37 °C. Stocks of cells were cultured in 25 cm2 tissue
culture flasks and subcultured two to three times per week. Cytotoxicity
testing was performed in a transparent 96-well microplate. Outer perimeter
wells were filled with sterile water to prevent dehydration in experimental
wells. The cells were incubated at 37 °C under 5% CO2 until confluency and then diluted with the culture medium to 4 ×
105 cells/mL. Threefold serial dilutions of the stock solutions
resulted in final concentrations of 64 to 0.26 μg/mL in a final
volume of 100 μL. After incubation at 37 °C for 48 h, the
medium was removed and the monolayers were washed twice with 100 μL
of warm Hanks balanced salt solution (HBSS). Warm medium (100 μL)
and 10 μL of freshly made methyl-thiazolyldiphenyl-tetrazolium
bromide (MTT) were added to each well, and then, the plates were incubated
for 4 h, after which the absorbance was determined at 492 nm.
Antituberculosis
Activity in Macrophages
The assays
were performed as described previously using mouse J774A.1 macrophages.[26] The final concentrations of selected compounds
were 10 μg/mL and 5 μg/mL. The concentration of rifampicin
(RFP) as the positive control was 5 μg/mL. All assays were performed
in triplicate in at least three separate experiments.
DprE1 Inhibition
Assay
DprE1 assays were performed
as described previously.[27] Briefly, assays
were performed at 30 °C in 384-well black plates in buffer containing
50 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (Hepes), pH 7.5, 100 mM NaCl, 1.5% (v/v)
DMSO, 100 μM Tween-20, 2 μM FAD, and 50 μM resazurin,
with variable concentrations of FPR and DprE1. Reactions were monitored
by following an increase in fluorescence intensity (λex = 530 nm, λem = 595 nm) associated with the formation
of resorufin. For inhibition studies, DprE1 (1 μM) was measured
by the resazurin assay with 1 mM FPR in the presence of different
inhibitor concentrations. The IC50 values were obtained
by plotting the initial velocities with inhibitor concentrations.
The IC50 values were calculated using the software program
GraphPad Prism.
MIC Assay against the Strain Overexpressing
DprE1[27]
Mt-DprE1 was cloned into
the plasmid
pMV261 to generate pMV261-Mt-dprE1 and introduced
into M. bovis BCG Pasteur. This host–plasmid
system permits the constitutive expression of target proteins. After
the bacteria were incubated in the presence of the compound for 7
days, cell viability can be assessed by the ability of endogenous
reductases to reduce resazurin to resorufin. As a proof of concept,
cells transformed with pMV261-Mt-dprE1 grew in the
presence of TCA1 with a MIC >16-fold higher than that when cells
were
transformed with an empty vector. Cells transformed with pMV261-Mt-dprE1 did not confer any growth advantage over cells transformed
with the vector alone when cells were grown in the presence of INH.
Hepatocyte Stability Assay
The assay was performed
with hepatocytes from male mice (BioIVT) and mixed humans (BioIVT)
following the protocol described previously.[25] The assay evaluated the metabolic stability of compounds in hepatocytes
by measuring the amount of parent compounds remaining of the test
samples.
Inhibition Evaluation on the hERG K+ Channel
Electrophysiology recording of the hERG channel
current was carried
out following the standard protocol as described previously.[28] hERG current inhibition in the presence of five
concentrations, including 30, 10, 3.0, 1.0, and 0.3 μM, was
tested for IC50 determination. Dofetilide was also included
as a positive control to ensure the accuracy and sensitivity of the
test system. All experiments were performed in duplicate for IC50 determination. The compound with IC50 > 30
μM
was generally considered to have a lower potential for hERG K+ channel inhibition.
Pharmacokinetic Studies
in Mice[29]
All animal protocols
were approved by the Institute Animal
Care and Welfare Committee of Shanghai Bioduro Biologics Co., Ltd.
The selected compounds 25a, 24f, and TCA1
were subjected to pharmacokinetic studies in Balb/c mice (male) weighing
26–27 g with three mice in the oral administration group and
three mice in the intravenous injection group. The tested compound
was formulated at a concentration of 5 mg/mL for a dose of 50 mg/kg
given orally (p.o.) and at 1 mg/mL for a dose of 5 mg/kg given intravenously
(i.v.). The tested compound was formulated with 0.5% carboxymethyl
cellulose for p.o. administration and with a mix solution (10%DMSO/50%poly(ethylene
glycol) (PEG)400/40%water) for i.v. administration. Blood samples
were collected at 5, 15, 30 min, 1, 2, 4, 7, 24 h after oral dosing
and i.v. administration. Plasma was harvested and stored at −80
°C until analyzed. The pharmacokinetic parameters were calculated
using WinNonlin software version 6.3 based on noncompartmental analysis
(Pharsight Corporation, Mountain View). The oral bioavailability was
calculated as the ratio between the area under the curve (AUC) following
intravenous administration corrected for dose (F =
(AUCp.o. × dosei.v.)/(AUCi.v. × dosep.o.)).
In Vivo TB Infection Assay[30]
All animal
protocols were approved by the Institute
Animal Care and Welfare Committee of Beijing Tuberculosis and Thoracic
Tumor Research Institute, Beijing Chest Hospital, Capital Medical
University. SPF Balb/c mice (female, 18–20 g) were used in
this study. Each treated group was composed of six mice. Mice were
infected via aerosol with a suspension of 5 ×
106 CFU/mL M. tuberculosis H37Rv using a Glas-Col inhalation system to deposit 50–100
bacilli into the lungs of each animal. The course of infection was
followed by plating homogenates of harvested organs [n = 3] on 7H11 agar plates (7H11 plates containing 10% oleic acid–albumin–dextrose–catalase
(OADC) enrichment and 50 μg/mL cycloheximide, 200 U/mL polymyxin
B, 50 μg/mL carbenicillin, and 20 μg/mL trimethoprim)
and determining CFUs on days 3, 10, and 30 postinfection. INH, TCA1,
and 25a were dissolved or suspended in 0.5% CMC and administered
by oral gavage in a maximum volume of 200 μL such that doses
of 25 and 100 mg/kg body weight were achieved. The control group received
only 0.5% CMC. Mice were treated 5/7 days per week during the acute
phase of infection, from day 10 to 30. Mice were sacrificed the day
after the last day of treatment, and lungs were removed, homogenized,
and serially diluted in 10-fold steps in HBSS. A total of 100 μL
was spread on 7H11 agar in duplicate. The plates were incubated at
37 °C for 3 weeks. Data are expressed as log10 (and
as log10 reduction) provided by the given dose of the compound
against the growth of the organism in the untreated control group.
Mean log10 values were calculated from bacterial burden
counts. Student’s t test was used to compare
means between the test and control groups. A P value
of ≤0.05 was considered significant.
Table 2
SAR of Thiophene-arylamide Compounds
at the Ar Site
Authors: Miroslav Brecik; Ivana Centárová; Raju Mukherjee; Gaëlle S Kolly; Stanislav Huszár; Adela Bobovská; Emöke Kilacsková; Veronika Mokošová; Zuzana Svetlíková; Michal Šarkan; João Neres; Jana Korduláková; Stewart T Cole; Katarína Mikušová Journal: ACS Chem Biol Date: 2015-04-29 Impact factor: 5.100
Authors: Hongyi Zhao; Bin Wang; Lei Fu; Gang Li; Haijia Lu; Yuke Liu; Li Sheng; Yan Li; Baoxi Zhang; Yang Lu; Chen Ma; Haihong Huang; Dongfeng Zhang; Yu Lu Journal: J Med Chem Date: 2020-08-05 Impact factor: 7.446
Authors: Sarah M Batt; Talat Jabeen; Veemal Bhowruth; Lee Quill; Peter A Lund; Lothar Eggeling; Luke J Alderwick; Klaus Fütterer; Gurdyal S Besra Journal: Proc Natl Acad Sci U S A Date: 2012-06-25 Impact factor: 11.205
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