Trisubstituted imidazoles as mycobacterium tuberculosis glutamine synthetase inhibitors

Article abstract of DOI:10.1021/jm201212h

Mycobacterium tuberculosis glutamine synthetase (MtGS) is a promising target for antituberculosis drug discovery. In a recent high-throughput screening study we identified several classes of MtGS inhibitors targeting the ATP-binding site. We now explore o

Full text of DOI:10.1021/jm201212h

Brief Article
pubs.acs.org/jmc
Trisubstituted Imidazoles as Mycobacterium tuberculosis Glutamine
Synthetase Inhibitors†
Johan Gising,‡,⊥ Mikael T. Nilsson,§,⊥ Luke R. Odell,‡ Samir Yahiaoui,‡ Martin Lindh,‡ Harini Iyer,∥
Achyut M. Sinha,∥ Bachally R. Srinivasa,∥ Mats Larhed,‡ Sherry L. Mowbray,§ and Anders Karlen*,‡
́
‡Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry, BMC, Uppsala University, Box 574, SE-751 23 Uppsala,
Sweden
§Department of Cell and Molecular Biology, Structural Biology, BMC, Uppsala University, Box 596, SE-751 24 Uppsala, Sweden
∥AstraZeneca India Private Limited, Bellary Road, Hebbal, Bangalore 560024, India
S
* Supporting Information
ABSTRACT: Mycobacterium tuberculosis glutamine synthetase (MtGS) is a promising target for antituberculosis drug discovery.
In a recent high-throughput screening study we identified several classes of MtGS inhibitors targeting the ATP-binding site. We
now explore one of these classes, the 2-tert-butyl-4,5-diarylimidazoles, and present the design, synthesis, and X-ray
crystallographic studies leading to the identification of MtGS inhibitors with submicromolar IC50 values and promising
antituberculosis MIC values.
Compound 1 also shows in vivo efficacy in a guinea pig
model, suggesting GS as a promising and druggable target in
the treatment of M. tuberculosis.10 The structure of MtGS
incubated with 1, which is phosphorylated in situ to form the
transition-state analogue L-methionine-S-sulfoximine phosphate
(MSO-P, 2, Figure 1a), has been reported showing how the
inhibitor interacts in the amino acid binding site.11
In the present study we explore a new class of MtGS
inhibitors derived from a recent high throughput screening
(HTS) study.12,13 This screen targeted the ATP-binding site of
MtGS with the goal of moving away from the amino acid
binding site,14 which is largely conserved in human GS. The 2-
tert-butyl-4-aryl-5-pyridylimidazole motif was the common
structure in the hits assigned to cluster 2, with the 4-position
substituted with various aryl groups (Figure 1b). From more
than 20 compounds in this class, the two best were chosen for
further SAR studies (3 and 7a in Figure 1c,d).
INTRODUCTION
■
Mycobacterium tuberculosis, the causative agent of tuberculosis,
is one of the world’s most deadly pathogens, leading to almost
1.7 million deaths annually despite more than 100 years of
research.1 Rapidly emerging drug-resistant and multidrug-
resistant strains have provided additional impetus to the search
for new therapeutic agents to combat the disease.2,3 M.
tuberculosis glutamine synthetase (MtGS, EC 6.3.1.2) catalyzes
the conversion of glutamate, ammonia, and ATP to glutamine,
phosphate, and ADP.4 MtGS plays a key role in controlling the
ammonia levels within infected host cells and so contributes to
the pathogen’s capacity to inhibit phagosome acidification and
phagosome−lysosome fusion.5,6 Furthermore, MtGS is believed
to be involved in cell wall biosynthesis; it is found
extracellularly in large quantities, which is related to a role in
the production of the poly-L-glutamate−glutamine that is a
major component of the cell wall in pathogenic mycobacteria.7
Treatment of M. tuberculosis with antisense oligonucleotides to
GS mRNA8 or with the GS inhibitor L-methionine-S-
sulfoximine (MSO, 1, Figure 1a)9 inhibits biosynthesis of
poly-L-glutamate−glutamine and the bacterial growth.8,9
RESULTS AND DISCUSSION
■
We first sought a synthetic route to the two most active HTS
hits in order to resynthesize them, confirm their activity, and
initiate X-ray crystallographic studies. This proved to be
problematic; despite exploration of various synthetic routes,
we were never able to resynthesize 3. However, the
trisubstituted imidazole 7a could be readily synthesized and
was therefore used in the hit expansion of this cluster. The
synthesis started from 2-bromo-6-methoxynaphthalene (4)
with two consecutive Sonogashira couplings followed by an
oxidation and cyclization to form the imidazole ring (see
Scheme 1). The ethyne was introduced by a fast microwave
assisted15 Sonogashira method16,17 using ethynyltrimethylsi-
lane, dichlorobis(triphenylphosphine)palladium, copper iodide,
Figure 1. (a) Reference GS inhibitor 1 and phosphorylated transition-
state analogue 2. These two compounds bind to the amino acid-
binding site of MtGS. (b) Common scaffold of the HTS hits of cluster
2. (c, d) Two most potent compounds from cluster 2 in the HTS
study. Compounds 3 and 7a interact with the ATP-binding site of
MtGS.12
Received: September 13, 2011
Published: February 27, 2012
© 2012 American Chemical Society
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Brief Article
a
Scheme 1. Synthetic Route to Compounds 7a−m
Table 1. Activities of Trisubstituted Imidazoles
a
Reagents and conditions: (a) ethynyltrimethylsilane, Pd(PPh3)2Cl2,
CuI, MeCN, diethylamine, microwave 120 °C, 15 min, then K2CO3,
MeOH, rt, 2 h, 85%; (b) bromoaryl/heteroaryl, Pd(PPh3)2Cl2, CuI,
MeCN, diethylamine, microwave 80−120 °C, 15 min, 22−63%; (c)
KMnO4, phosphate buffer; (d) aldehyde, ammonium acetate, n-
butanol, 50−65 °C, 0.5−5 h, 10−63%.
and acetonitrile/diethylamine (1:1) as solvent. In situ
deprotection with saturated K2CO3 in methanol gave the 2-
ethynyl-6-methoxynapthalene (5) in 85% yield. By use of the
same protocol, the 4-pyridyl moiety was incorporated in
moderate yield (6a, 47%). Oxidation of the ethyne 6a to the
diketone intermediate with potassium permanganate18 in
aqueous acetone proved to be a very sensitive reaction and
often led to oxidative cleavage of the triple bond. Changing the
standard buffer system NaHCO3/MgSO4 to NaH2PO4/
Na2HPO4, and thus lowering the pH from 7.5 to 5, provided
a more robust protocol to furnish the diketone. In our attempts
to prepare 3, the identical oxidation conditions with the
corresponding quinoline intermediate only gave overoxidation,
and no conditions were identified that suppressed oxidative
cleavage. Finally the imidazole ring was synthesized by in situ
trapping of the diketone with various aldehydes and
ammonium acetate allowing straightforward variation of the
tert-butyl group position (R2). The resynthesized HTS hit 7a
had IC50 = 3.1 μM, which is 30-fold higher than the IC50
obtained in the HTS study (0.1 μM), due to our more stringent
assay conditions.12 Unfortunately, neither 3 (from the
AstraZeneca library) nor the resynthesized 7a was active in
the mycobacterial growth assay (MIC > 32 μg/mL).
gave the inactive 7g. Clearly, there is a preference for relatively
bulky substituents in this position.
Parallel studies at this point produced the structure of MtGS
in complex with 2, 3 (obtained from AstraZeneca’s in-house
compound library), phosphate, and magnesium at 2.15 Å
resolution (crystallographic R-factor 22.5%, Figure 2a). An
overlay of this structure on that of the complex with 2, ADP,
and magnesium (PDB entry 2BVC)11 is shown in Figure 2b; an
Since numerous 4-substituted arylimidazoles of 7a (Figure 1)
had already been identified and evaluated in the HTS study, we
focused instead on optimizing positions 2 (the tert-butyl group)
and 5 (the 4-pyridyl group). The importance of the position of
the nitrogen in the 4-pyridyl ring was first examined by
synthesizing the phenyl and pyrimidine analogues (Table 1,
7b−d). The same synthetic strategy as described above was
applied, replacing the 4-pyridyl with aryl/heteroaryl groups
introduced in the second Sonogashira reaction. Cyclization with
pivalaldehyde gave the targets 7b−d in which the nitrogen was
either removed (7b) or its position altered (7c−d). The results
clearly indicate the importance of having a nitrogen in the 4-
position of the pyridine ring; all compounds lacking this feature
were inactive (IC50 > 25 μM).
To investigate the importance of steric bulk in position 2 of
the imidazole ring, compounds with smaller alkyl chains were
synthesized. Cyclizing the diketone with propionaldehyde,
acetaldehyde, and formaldehyde gave ethyl, methyl, and
hydrogen, respectively, at position 2 of the imidazole ring
(7e−g in Table 1). Removal of two methyl groups generated a
slightly more active compound (7e, 2.2 μM vs 7a, 3.1 μM).
Removal of yet another methyl group yielded 7f, which was
inactive. Likewise, complete removal of the tert-butyl group
Figure 2. (a) MtGS (yellow carbons) in complex with 2 (MSO-P), 3
(gray carbons), phosphate, and magnesium. Hydrogen bond between
3 and Ser280 is shown as a black dashed line. Nearby magnesium ions
are green spheres. (b) Superposition of the MtGS structure with 3 on
that with bound ADP (PDB entry 2BVC) allows comparison to
nucleotide (pink carbons) binding. Protein/ADP hydrogen bonds are
pink dashed lines.
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a
rmsd of 0.2 Å is obtained when the Cα's of residues 4−478 in
the A chains are compared. Compound 2 occupies exactly the
same position in the amino acid binding site in each case; all
three of the metal ions are also in equivalent locations. The 4-
pyridyl moiety of 3 is at essentially the same place as the six-
membered pyrimidine ring of ADP, with the inhibitor’s ring
nitrogen providing the only hydrogen bond to protein (via the
side chain of Ser280) in an interaction equivalent to that of N1
of ADP’s adenine ring. The interaction with this serine explains
why a nitrogen at the 4-position of the pyridine ring is so
important for binding affinity (vide supra). The imidazole ring
and tert-butyl moieties of 3 occupy roughly the same space as
the ribose of the nucleotide. Thus, the R2 group is found at the
end of the ribose proximal to its linkage with the phosphates. A
phosphate ion assumes the position of the β-phosphate of ADP.
The quinoline moiety extends out toward the solvent, making
van der Waals interactions with the protein only near Ala362
(not shown). Correspondingly, the electron density of this
group is poorer than that of the rest of the ligand, although
density in the maps averaged over all six subunits is clear (see
Supporting Information). Efforts to cocrystallize 7a with MtGS
were unsuccessful. However, we hypothesize that the binding
modes of 3 and 7a are very similar, which is supported by our
docking studies. The comparisons in Figure 2b led us to make
7i; docking studies indicated that the hydroxyl group of this
compound could occupy the same position as O3′ of ADP and
form an equivalent hydrogen bond to the backbone carbonyl of
Tyr230 (Figure 2b). In the original synthetic route to 7i, the
hydroxyl was benzyl protected (7h) to ease the synthesis.
However, all attempts to deprotect the corresponding alcohol
by hydrogenation or oxidative protocols were unsuccessful.
Compound 7h was evaluated in the MtGS assay but did not
show any inhibitory activity at 25 μM. An alternative protective
group strategy was then employed in which 2-(tert-
butyldimethylsilyloxy)acetaldehyde was instead used in the
imidazole ring cyclization. The silyloxy group allowed smooth
deprotection with tetrabutylammonium fluoride to yield 7i.
Disappointingly, 7i was also inactive in the enzymatic assay.
The remaining compounds (7j−m) in Table 1 were similarly
suggested to reach into the sugar-binding site of the ATP
pocket. In the final step of the synthesis leading up to these
compounds, aldehydes are used as reagents (see Scheme 1).
We therefore created a set of virtual compounds by merging
aldehydes found in-house and in the ACD database, using the
Legion software.19 The compounds were then docked to the
nucleotide-binding site using Glide, and the fit was evaluated
based on the Glide score and visual inspection. Despite their
promising docking poses, 7j−m all lacked inhibitory activity at
25 μM.
Scheme 2. Synthetic Route to Compounds 11a−d
a
Reagents and conditions: (a) EtOH, Pd(OAc)2, Xantphos, DBU,
Mo(CO)6, microwave 120 °C, 30 min, 93%; (b) 2-fluoro-4-
methylpyridine, NaHMDS, THF, 0 °C, 2 h, 64%; (c) HBr, DMSO,
70 °C, 2 h, then pivalaldehyde, ammonium acetate, n-butanol, 50 °C, 2
h, 71%. (d) 11a: (i) diphenylmethanamine, dioxane, microwave 200
°C, 10 h, (ii) Pd/C, NH4OAc, MeOH, microwave 120−140 °C, 2 h,
28%. 11b: methylamine (2.0 M in THF), microwave 150 °C, 17 h,
18%. 11c: ammonium hydroxide, DMF, microwave 150 °C, 12 h, 85%.
11d: AcOH, H2O, microwave 190 °C, 2 h, 91%.
2-fluoro-4-methylpyridine with NaHMDS via nucleophilic
substitution of 8. As an alternative to creating the imidazole
ring from the diketone, an α-bromination was performed on 9
followed by cyclization with ammonium acetate and
pivalaldehyde. Attempts to resynthesize the HTS hit 3 by this
route again failed because we were never able to substitute the
ethoxy of ethyl quinoline-3-carboxylate with 4-methylpyridine.
At this point, it was encouraging to see that the fluorine in 10
contributed to an approximate 2.5-fold increase of affinity
compared to the hit 7a (IC50 of 1.2 μM vs 3.1 μM, Table 1).
The aminopyridine 11a was synthesized via microwave-assisted
nucleophilic substitution of the fluorine of 10 with
diphenylmethanamine followed by deprotection through
catalytic hydrogenation. Remarkably, 11a was over 60 times
better than the reference 7a (0.049 μM vs 3.1 μM, Table 1),
suggesting an important new interaction with the enzyme. For
comparison, the methylamino and the dimethylamino groups
were incorporated in a similar way. Compound 11b, having one
methyl group, showed a 100-fold loss in activity compared to
11a, making it even less potent than 7a. Substituting the
fluorine with a dimethylamino group resulted in 11c, which was
inactive. This suggested that having a hydrogen bond donating
group in the 2-position of the pyridine ring was advantageous.
As an alternative donating group, we introduced the 2-pyridone
ring (11d), which we believed would bind in its 2-hydroxy
tautomer. However, on the basis of the lack of activity of this
compound, one can speculate that it predominantly exists in
the pyridone form.
At this time, we succeeded in obtaining a crystal structure of
11a bound to MtGS together with 2, phosphate, and
magnesium at 2.26 Å resolution (R-factor 19.5%, Figure 3a).
The complexes with 3 and 11a are overlaid in Figure 3b; again,
the protein structures are highly similar, with an rmsd of only
0.1 Å when the Cα's of residues 4−478 are compared. Ser280
of MtGS was seen to interact with both nitrogens of the 2-
aminopyridine-4-yl moiety of 11a (only the hydrogen bond
with the ring nitrogen was present in the complex with 3),
which shifts the ligand ∼0.5 Å deeper into the binding site.
Inspection of the 11a complex indicates that the distance is too
great (∼3.5 Å) to support a hydrogen bond between the 2-
amino group and the backbone carbonyl oxygen of Lys361.
Apart from very small changes at the side chain of Phe232 and
the main chain near residue Ala362, the protein structures are
Going back to the superimposed X-ray structures, we decided
to substitute the 4-pyridyl group of 7a with a 2-aminopyridine-
4-yl, which would possibly allow the formation of a hydrogen
bond to the backbone carbonyl oxygen of Lys361, similar to the
interaction seen for N6 of ADP (Figure 2b). Another synthetic
approach was employed in the synthesis of the aminopyridines
11a−d, avoiding the sensitive oxidation of the triple bond
(Scheme 2). Ethyl 6-methoxy-2-naphthoate (8) was synthe-
sized in 93% yield by a palladium-catalyzed carbonylation of 2-
bromo-6-methoxynaphthalene (4) utilizing ethanol as the
nucleophile.20 In the next step, the fluorine of 2-fluoro-4-
methylpyridine was incorporated as a handle to allow
nucleophilic aromatic substitutions in later steps. Compound
9 was produced in acceptable yield (64%) by deprotonation of
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1H at 399.9 MHz and for 13C NMR at 100.5 MHz. Analytical HPLC−
UV/MS analysis of pure products were performed on a Gilson HPLC
system with a Chromolith SpeedROD RP-18e column (50 mm × 4.6
mm) equipped with a Finnigan AQA quadrupole mass spectrometer
using a 4 mL/min CH3CN/H2O gradient (0.05% HCOOH) and
detection by UV (DAD) and MS (ESI+). All compounds were
determined to be >95% pure by HPLC−UV at 254 nm.
4-(2-tert-Butyl-4-(6-methoxynaphthalen-2-yl)-1H-imidazol-
5-yl)pyridin-2-amine (11a). To a microwave vial (2−5 mL) were
added 10 (50 mg, 0.13 mmol), diphenylmethanamine (1.5 mL), and
dioxane (1.5 mL). The vial was then sealed under air and heated at
200 °C by microwave irradiation for 10 h. After cooling, the mixture
was filtered through a plug of silica, eluted with EtOAc/isohexane
(1:1) and the filtrate concentrated in vacuo. The crude mixture was
then taken up in MeOH (2.0 mL) and transferred to a microwave vial
(2−5 mL) loaded with Pd/C (10%, 5 mg) and ammonium acetate
(100 mg, 1.3 mmol). The vial was then sealed under air and heated at
120 °C by microwave irradiation for 2 h. After the mixture was cooled,
Pd/C (10%, 10 mg) and ammonium acetate (200 mg, 2.6 mmol) were
added, and the mixture was heated by microwave irradiation for a
further 20 min at 140 °C. After cooling, the mixture was diluted with
EtOAc and saturated NaHCO3 (10 mL each) and the two layers were
separated. The aqueous layer was washed twice with EtOAc (10 mL),
and the combined organic phases were concentrated in vacuo. The
crude product was thereafter purified by silica column flash
chromatography, eluting with EtOAc/methanol/triethylamine
Figure 3. (a) MtGS (yellow carbons) in complex with 2, 11a (cyan
carbons), phosphate, and magnesium. Hydrogen bonds are shown as
cyan dashed lines. (b) Superposition of the protein allows comparison
of binding of 3 (gray carbons) and 11a.
essentially identical. The compounds that inhibited MtGS were
also evaluated for their activity against the M. tuberculosis strain
H37Rv. The hit 7a and 7e lacked antibacterial activity (MIC >
32 μg/mL), while the 2-pyridyl substituted compounds had
promising MIC values; the most potent compound as
measured by the enzymatic inhibition, 11a, also gave the best
MIC (2 μg/mL; see Table 1). To assess the potential
cytotoxicity of 11a, we performed a standard mammalian cell
proliferation assay22 using A549 cells; 11a had an IC50 of 24.7
μm in this test, which is almost 5-fold greater than the MIC of
5.4 μM (2 μg/mL). This moderate level of cytotoxicity is not
unusual among compounds in the early stages of drug
development, and the situation is expected to improve as
more effective enzyme inhibition and antibacterial activity are
attained.
1
(1:0.05:0.01). Yield: 28%, 14 mg as a pale yellow solid. H NMR
(CDCl3) δ 7.81 (d, J = 1.9 Hz, 1H), 7.72 (d, J = 8.6 Hz, 1H), 7.69 (m,
1H), 7.51 (dm, J = 6.1 Hz, 1H), 7.40 (dd, J = 8.6, 1.9 Hz, 1H), 7.16
(m, 1H), 7.14 (m, 1H), 6.91 (br s, 1H), 6.65 (dm, J = 6.1 Hz, 1H),
3.91 (s, 3H), 1.43, (s, 9H); 13C NMR (CDCl3/CD3OD, 5:1) δ 170.3,
158.6, 157.4, 156.9, 146.6, 140.5, 134.5, 129.7, 128.9, 127.8, 127.6,
127.2, 126.4, 119.8, 111.7, 107.5, 106.0, 55.6, 33.2, 29.6 (one carbon
signal was not detected).21 ESI-MS (m/z) 373 (M + H+). HRMS (M
+ H+): 373.2037, C23H24N4O requires 373.2028.
ASSOCIATED CONTENT
■
S
* Supporting Information
Additional experimental details concerning synthesis of all
compounds, protein expression, purification, activity/inhibition
studies, spectroscopic data, and structural studies. This material
is available free of charge via the Internet at http://pubs.acs.org.
CONCLUSIONS
■
A recent HTS study targeting the ATP-binding site of MtGS
produced several active classes of compounds. One of these, the
2-tert-butyl-4,5-diarylimidazoles, contained several hits, and a
complex with one of the inhibitors with MtGS could be
obtained by X-ray crystallography. On the basis of this
structure, our initial SAR explorations could be rationalized as
well as the importance of having a nitrogen, acting as a
hydrogen bond acceptor, in the 4-position of the pyridine ring.
Building out from the tert-butyl group, a series of compounds
was designed to reach into the ribose-binding site of the ATP
pocket. However, these compounds lacked inhibitory activity at
25 μM. An alternative synthetic strategy in which a 2-amino
group was introduced into the 4-pyridyl ring was then pursued.
This gave us our best inhibitor (11a) with IC50 = 0.049 μM on
MtGS and an MIC = 2 μg/mL against M. tuberculosis. We were
also able to obtain an X-ray structure of this compound bound
to MtGS, which showed that instead of forming a hydrogen
bond to the backbone carbonyl oxygen of Lys361 as predicted
from docking studies, the 2-amino group formed an additional
interaction with the hydroxyl oxygen of Ser280 in its primary
binding mode.
Accession Codes
†PDB codes for the MtGS-3 and MtGS-11a structures are
3ZXR and 3ZXV, respectively.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +46-18-4714293. Fax: +46-18-4714474. E-mail:
anders.karlen@orgfarm.uu.se.
Author Contributions
⊥J.G. and M.T.N. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Johan Gustavsson for preparative work and Dr. Aleh
Yahorau, Department of Pharmaceutical Biosciences, Uppsala
University, Sweden, for conducting HRMS analyses. The
Swedish Foundation for Strategic Research (SSF), the Swedish
Research Council (VR), and the EU Sixth Framework Program
NM4TB (Grant CT:018 923) provided financial support.
EXPERIMENTAL PROCEDURES
General Methods. Microwave-assisted reactions were performed
in sealed vials dedicated for microwave processing, using a Smith
synthesizer. NMR spectra were recorded on a Varian Mercury Plus for
■
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(15) Larhed, M.; Hallberg, A. Microwave-assisted high-speed
chemistry: a new technique in drug discovery. Drug. Discovery Today.
2001, 6, 406−416.
ABBREVIATIONS USED
■
MtGS, glutamine synthetase from Mycobacterium tuberculosis;
MIC, minimum inhibitory concentration; PDB, Protein Data
Bank; WHO, World Health Organization; HTS, high
throughput screening; MSO, L-methionine-S-sulfoximine;
MSO-P, L-methionine-S-sulfoximine phosphate; MW, micro-
wave
́
(16) Erdelyi, M.; Gogoll, A. Rapid homogeneous-phase Sonogashira
coupling reactions using controlled microwave heating. J. Org. Chem.
2001, 66, 4165−4169.
́
(17) Wannberg, J.; Sabnis, Y. A.; Vrang, L.; Samuelsson, B.; Karlen,
A.; Hallberg, A.; Larhed, M. A new structural theme in C-2-symmetric
HIV-1 protease inhibitors: ortho-substituted P1/P1′ side chains.
Bioorg. Med. Chem. 2006, 14, 5303−5315.
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