Structure guided lead generation for M. Tuberculosis thymidylate kinase (Mtb TMK): Discovery of 3-cyanopyridone and 1,6-naphthyridin-2-one as potent inhibitors

Article abstract of DOI:10.1021/jm5012947

M. tuberculosis thymidylate kinase (Mtb TMK) has been shown in vitro to be an essential enzyme in DNA synthesis. In order to identify novel leads for Mtb TMK, we performed a high throughput biochemical screen and an NMR based fragment screen through which

Full text of DOI:10.1021/jm5012947

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Article
Structure Guided Lead Generation for M. tuberculosis
thymidylate kinase (Mtb TMK): Discovery of 3-cyanopyridone
and 1,6-naphthyridin-2-one as potent inhibitors
Manoranjan Panda, Maruti Naik, Anandkumar Raichurkar, Balachandra S. Bandodkar, Varun
V. B., Shantika Bhat, Rajesh Kalkhambkar, Rani Menon, Jyothi Bhat, Beena Paul, Harini
Iyer, Syeed Hussein, Julie A. Tucker, Martin Vogtherr, Kevin J. Embrey, Helen McMiken,
Swati Prasad, Adrian Gill, Bheemarao G. Ugarkar, Janani Venkatraman, and Jon Read
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 08 Dec 2014
Downloaded from http://pubs.acs.org on December 9, 2014
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Structure Guided Lead Generation for M.
tuberculosis thymidylate kinase (Mtb TMK):
Discovery of 3ꢀcyanopyridone and 1,6ꢀ
naphthyridinꢀ2ꢀone as potent inhibitors
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Maruti Naik,† Anandkumar Raichurkar, † Balachandra S. Bandodkar, † Varun V. B. †,
Shantika Bhat, † Rajesh Kalkhambkar, † Rani Menon † Jyothi Bhat, ‡ Beena Paul, ‡ Harini
Iyer, ‡ Syeed Hussein,δ Julie A. Tucker, δ Martin Vogtherr, δ Kevin J. Embrey, δ Helen
McMiken, δ Swati Prasad,ơ Adrian Gill, δ Bheemarao G. Ugarkar, † Janani Venkatraman,‡
Jon Readδ* and Manoranjan Panda†*
†Department of Chemistry, ‡Department of Bioscience, Innovative Medicines Infection,
AstraZeneca India Pvt. Ltd., Bellary Road, Hebbal, Bangaloreꢀ560024, INDIA.
δAstraZeneca Innovative Medicines, Discovery Sciences, Alderley Park, Mereside, Cheshire,
SK10 4TG, United Kingdom.
ơ Department of Bioscience, Innovative Medicines Infection, AstraZeneca, 35 Gatehouse
Drive, Waltham, MA 02451, USA.
*corresponding authors
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M. tuberculosis thymidylate kinase (Mtb TMK) has been shown in vitro to be an essential
enzyme in DNA synthesis. In order to identify novel leads for Mtb TMK, we performed a
high throughput biochemical screen and an NMR based fragment screen through which we
discovered two novel classes of inhibitors, 3ꢀcyanopyridones and 1,6ꢀnaphthyridinꢀ2ꢀones,
respectively. We describe three cyanopyridone subꢀseries which arose during our hit to lead
campaign, along with coꢀcrystal structures of representatives with Mtb TMK. Structure aided
optimization of the cyanopyridones led to single digit nanomolar inhibitors of Mtb TMK.
Fragment based lead generation, augmented by crystal structures and the SAR from the
cyanopyridones, enabled us to drive the potency of our 1,6ꢀnaphthyridinꢀ2ꢀone fragment hit
from 500 µM to 200 nM whilst simultaneously improving the ligand efficiency.
Cyanopyridone derivatives containing sulfoxides and sulfones showed cellular activity
against M. tuberculosis. To the best of our knowledge, these compounds are the first reports
of nonꢀthymidineꢀlike inhibitors of Mtb TMK.
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Introduction
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There is an urgent need for new drugs for tuberculosis (TB) with novel modes of action to
combat the lifeꢀthreatening multiꢀdrug resistant TB.1 The complete genome sequencing of
Mycobacterium tuberculosis (Mtb) followed by a series of seminal works to determine gene
essentiality has opened up new targets for lead generation.2 Additionally, the mode of action
studies for the novel hits and leads emerged from phenotypic screenings utilizing methods
such as resistant mutant mapping, proteomics followed by biochemical studies have played
equally important role in delivering novel targets for TB drug discovery.3 Mtb thymidylate
kinase (Mtb TMK) is one of the essential targets that came from the first approach. Mtb TMK
is involved in DNA synthesis and is reported to be an essential target for the survival of the
mycobacterium.4 Furthermore, Mtb TMK is amenable to biochemical and biophysical studies
and several crystal structures of complexes with TMP and other close analogs have been
reported.5 This makes it an attractive target for structure based drug design. In Gram positive
pathogens such as Streptococcus pneumoniae and Staphylococcus aureus, inhibition of TMK
has resulted in in vivo efficacy in mouse as demonstrated in a lead optimization program.6
Lead generation efforts against TMK from Gram negative pathogens such as Pseudomonas
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aeruginosa7 and the malaria parasite, Plasmodium falciparum8 reinforces the potential of the
,
target across a variety of pathogens.
To the best of our knowledge, all of the Mtb TMK inhibitors reported so far are either
thymidine monophosphate (TMP) analogs9 or contain a thymidine core.10 This is attributable
to the fact that these inhibitors used a thymidine or pyrimidinone core as a starting point. In
order to identify novel leads that exploit active site regions that are not explored by TMP
analogs, we invested in high throughput screening (HTS) and fragment based lead generation
(FBLG) approaches against Mtb TMK. Our objective was to achieve improved potency
against the enzyme that would translate to cellular activity against mycobacteria (minimum
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inhibitory concentration, Mtb MIC). This has been one of the major challenges for lead
generation against Mtb. Our efforts resulted in the identification of two novel classes of Mtb
TMK inhibitors, 3ꢀcyanopyridones and 1,6ꢀnaphthyridinꢀ2ꢀones. Herein, we discuss the
structure guided optimization of these two series and their potential as antiꢀTB agents. To the
best of our knowledge, this is the first report of bona fide Mtb TMK inhibitors with nonꢀ
thymidine cores.
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Results and Discussion
Highꢀthroughput screening identifies 3ꢀcyanopyridones as inhibitors of Mtb TMK
SiteMap11 analyses of Mtb TMK suggest that the TMP site is highly druggable (supporting
information ꢀ S1). This was reflected in the high hit rate observed from an HTS of 120,000
compounds, a representative set of our corporate collection. Multiple leadꢀlike scaffolds were
obtained with activity in the range of 1ꢀ30 µM. Protein observed NMR studies confirmed the
binding of the inhibitors to the TMP site. Based on potency, preliminary SAR from analog
screening and opportunity to diversify the scaffold, we prioritized the 3ꢀcyanopyridone series
for our hit to lead campaign (Figure 1). The ligand efficiency (LE=0.46)12,13 and lipophilic
ligand efficiency (LLE=3.54)12,13 for compound 1, our chosen starting point, suggested that
the series is lead like and suitable for further optimization.
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Figure 1: 3ꢀcyanopyridone scaffold identified as a hit from an HTS. 2Dꢀoverlay of TMP
(black) and compound 1 (red) based on the predicted docking pose. The key Hꢀbonding
interactions are with Arg74 and Asn100. .
In the early stage of the program, publicly available complex crystal structures of Mtb
TMK with TMP analog azido TMP(AZT) (pdb ID: 1W2H)5b were used for modeling to
understand the possible binding mode of the cyanopyridones. The carbonyl oxygen and NH
of the pyridone ring form the key hydrogen bonding contact, interacting with the sideꢀchains
of Asn100 and Arg74 of Mtb TMK, whereas the pyridone ring makes a πꢀπ stacking
interaction with Phe70 (Supporting information ꢀ S2, Figure S2ꢀ1A). An overlay with bound
TMP indicated that the R1 aryl ring aligns with the C2 carbonyl of TMP (Figure 1). Thus,
this nonꢀthymidine like core gave access to a novel diversification point. We utilized this
knowledge in our design and were able to reach subꢀµM potency within ten molecules from
the starting hit (Table 1, compound 8). The predicted binding mode was later confirmed by
coꢀcrystallization of cyanopyridone (8) with Mtb TMK. Features of the Mtb TMK TMP
binding site and differentiation with respect to other bacterial TMKs and human TMK are
discussed in the following sections.
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Key derivatives that helped in understanding the structure activity relationship (SAR) for
this series are shown in Table 1. Several aryl and heteroaryl groups at R1 were tried (1-7,
Table 1) among which we found 3ꢀmethoxyphenyl as the preferred group with an IC50 of 1.5
μM (5). Keeping the 3ꢀmethoxyphenyl at R1 fixed, we next explored substituted aryl rings at
R2 (8ꢀ14). Aromatic rings at R2 showed promising results, and a limited exploration of
smaller substituents on the R2 aryl ring resulted in several compounds with subꢀµM potency.
Para substituted derivatives such as the carboxylic acid, urea and amide (8, 12 and 14, Table
1) were generally more potent.
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Table 1: SAR exploration of 3ꢀcyanopyridones.
No
R1
IC50 (µM)
3.6
Calcul
ated
logD
N
o.
R2
IC50
(µM)
Calcul
ated
logD
1.9
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0.2
1
8
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0.25
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>50
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While both pyridone and pyrimidinone cores (matched pairs: 5/15, Table 1 and Figure 2)
were active, pyridones were found to be more potent. Similarly, replacement of pyridone with
pyranone (matched pair: 1/16, Table 1 and Figure 2) led to complete loss of activity against
Mtb TMK. Replacement of cyano with other groups such as amide (matched pair: 3/17,
Table 1 and Figure 2), and methyl (matched pair: 8/18, Table 1 and Figure 2), produced
less potent or inactive compounds. This can be attributed to the electron withdrawing nature
of the nitrile group which makes the pyridine ring electron deficient and thus strengthens the
Hꢀbonds between the pyridone carbonyl and NH, and the protein. Furthermore, modeling
suggested that the nitrile group may be involved in a favorable dipolar contact with the side
chain of Arg74.
Figure 2: Limited SAR exploration of core ring and nitrile replacement
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Crystal structures of Mtb TMK in complex with 3ꢀcyanopyridones rationalise SAR and drive
improvements in potency
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We solved the crystal structure of the complex of Mtb TMK with compound 8 (Figure 3),
and were gratified to note that the binding mode predicted from docking was in agreement
with the crystallographic pose to within 1.0 Å rmsd (Supporting information S2, Figure S2ꢀ
1B). Figures 3A and 3B show the key interactions of TMP and 8 in the Mtb TMK active site.
The πꢀπ stacking of the pyridone ring with Phe70, and the interactions of the NH and C=O of
the pyridone ring with Asn100 and Arg74 side chains, respectively, are the key interactions
for both compounds. The nitrile group, while not making an ideal hydrogen bond, is close to
the sideꢀchain of Arg74 and is involved in favorable polar contacts with this residue. The
hydrophobic pocket into which the methoxyphenyl projects, and which is comprised of the
side chains of Leu52, Ala53, Val63, Met66, Ala67, Phe70, Tyr165 and Leu171, is unique to
the Mtb TMK active site. This is an important factor in the search for selective inhibitors. The
paraꢀbenzoic acid at R2 makes critical interactions with Arg95. In addition to a salt bridge
between the carboxylate anion and the guanidine cation, the aromatic ring stacks against the
πꢀsystem of the Arg95 sideꢀchain. This interaction may be responsible for the marked
improvement in potency observed for cyanopyridones carrying this substituent.
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A
B
Figure 3: Crystal structures of Mtb TMK in complex with TMP5a (A, PDB ID: 1G3U) and
compound 8 (B, 2.5 Å resolution). A. Key interactions exhibited by TMP. B. Key interactions
made by compound 8 (PDB ID: 4UNN). Figures were prepared using PyMol v1.2. Protein
is shown in yellow cartoon representation with selected sideꢀchains labelled and shown as
sticks with carbon atoms coloured yellow. Bound ligand is drawn in stick representation with
carbon atoms in green (TMP) or cyan (compound 8). Hꢀbond interactions are shown as
dotted green lines. Electron density for compound 8 is contoured at 1.0 sigma using 2fofc
coefficients and is displayed as a grey mesh.
Further analysis of the coꢀcrystal structure of Mtb TMK with 8 suggested that the
aforementioned hydrophobic pocket could be exploited to improve the potency (Figure 4).
Of several medicinal chemistry designs investigated, two ideas led to single digit nanomolar
potency. Firstly, extension of the OMe group of 8 using a larger hydrophobic group (19)
resulted in dramatic improvement in IC50. Secondly, the torsion angle between the pyridone
ring and the phenyl ring at R1 in the coꢀcrystal structure was noted to be 18º. Thus,
introduction of an ethylene bridge between the two rings, and hence conformationally locking
the rings, was predicted to lead to an improvement in potency due to a reduction in the
entropic penalty combined with additional hydrophobic contacts with the ethylene group.
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This design led to compound 20, a fused cyanopyridone, which showed a similar leap in
potency as seen for 19, and had excellent ligand efficiency (Figure 4). Hence, we focused on
the exploration of fused cyanopyridones as a means to overcoming one of the major
challenges for bacterial TMK inhibitors, that of translating enzymatic activity into cellular
activity.
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Figure 4: Molecular surface for the active site of Mtb TMK in complex with 8. The color of
the surface indicates the nature of the active site, where red and blue are hydrophilic areas
and grey is the hydrophobic surface. Structure and potency of compounds 19 and 20.
Table 2: SAR exploration for fused cyanopyridones.
Mtb
MIC
(µg/mL)
IC50
(µM)
Calculated pKa
No
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R2
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C
logD
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OH
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> 32
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OCH3
C
C
0.005
0.002
> 32
> 32
0.27
0.36
4.4
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C
C
0.001
0.017
> 32
> 32
2.9
3.0
4.4
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25
OCH3
neutral
H
H
O
O
0.002
0.005
> 32
> 32
0.11
5.0
3.9
26
27
ꢀ1.06
Figure 5: Coꢀcrystal structure at 2.0 Å resolution of compound 23 with Mtb TMK (PDB ID:
4UNR). Protein is shown in yellow cartoon representation with selected sideꢀchains labeled
and shown as sticks with carbon atoms coloured yellow. Bound ligand is drawn in stick
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representation with carbon atoms in magenta. Hꢀbond interactions are shown as dotted green
lines. Electron density for compound 23 is contoured at 1.0 sigma using 2fofc coefficients and
is displayed as a grey mesh.
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We were successful in obtaining a coꢀcrystal structure of the fused cyanopyridone, 23, with
Mtb TMK which indicated that this subꢀseries holds promise to drive the potency further to
the picomolar range likely required for adequate cellular activity. (Figure 5). The position,
orientation and interactions of the core ring and substituents at R1 and R2 are similar to those
observed for the open form of the cyanopyridone as predicted. The improvement in potency
observed for the fused cyanopyridones can, therefore, be attributed to optimal interaction
with the hydrophobic pocket and entropic advantage from the conformationally locked
system.
A limited set of substituents were tested at R1 to explore the possibility of picking up
interactions with residues such as Ala60, Val63 and Arg107 (Table 2). The complex crystal
structure of Mtb TMK with compound 23 suggests that the pyrazole ring NH forms an Hꢀ
bond with the backbone carbonyl of Ala60. Compound 24, a fused cyanopyridone with a
longer aryl ether substituent at R1, was the most potent compound in the series. This suggests
that hybridization of designs based on the limited SAR obtained for compounds 19 and 20 is
a promising approach and can be further exploited to optimize this series.
Despite high in vitro enzyme potency, the fused cyanopyridones did not exhibit any
cellular activity even at the highest concentration tested in an Mtb whole cell assay (Mtb MIC
> 32 µg/mL) (Table 2). We hypothesize that poor cellular permeability could be one of the
reasons for the lack of Mtb MIC, and the ionic nature of the compounds (21ꢀ24; carboxylic
acids) may be responsible for this. Thus, compounds with varying pKa such as acyl
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sulfonamides, ureas and amides (25ꢀ27) were synthesized and tested. Unfortunately, these
compounds also failed to show any inhibition in a whole cell assay (Mtb MIC > 32 µg/mL)
despite their potent enzymatic activity.
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Fragment based lead generation identifies alternative core scaffolds
In our pursuit of alternate cores and novel scaffolds with the potential for cellular activity,
we screened a fragment library against Mtb TMK using a 2DꢀTROSY based NMR method. A
set of 1200 fragments was screened in mixtures of 6. Hit mixtures were deꢀconvoluted and
the Kd’s of the individual hits determined. This effort led to the identification of multiple
fragment hits (FRITs) with LE of 0.25 or above. We generated coꢀcrystal structures of a
subset of the FRITs with Mtb TMK, in order to understand their binding modes. In
combination with the IC50 of close analogs this allowed us to understand the FRIT SAR and
prioritize scaffolds for FRIT to hit optimization.
1, 6ꢀnaphthyridinꢀ2ꢀone (28, Figure 6B) was one of the most promising scaffolds resulting
from this campaign. The chemical shifts observed when this ligand binds to Mtb TMK are
shown in Figure 6A.
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A
C
B
Figure 6: Identification of 1, 6ꢀnaphthyridinone fragment hit, 28, from NMR based fragment
screening. A. Overlaid 2DꢀNMR spectra showing the chemical shift perturbation at 0.2, 0.6,
1.2 and 2.4 mM compound 28. B. Crystal structure at 2.3 Å resolution showing the binding
of FRIT 28 drawn in stick representation with carbon atoms in magenta in the TMP site of
Mtb TMK (PDB ID: 4UNP). The crystallographic pose cyanopyridone (8, cyan) have been
overlaid for comparison. Protein from 4UNP is shown in yellow cartoon representation with
selected sideꢀchains labeled and shown as sticks with carbon atoms coloured yellow. Hꢀbond
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interactions for 28 with the protein are shown as dotted green lines. Electron density for
compound 28 is contoured at 1.0 sigma using 2fofc coefficients and is displayed as a grey
mesh. C. 2Dꢀinteraction diagram. NHAC = number of heavy atom counts. LE= Ligand
efficiency.
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To gain insight into the FRIT binding mode, 1,6ꢀnaphthyridinone, 28, was coꢀcrystallized
with Mtb TMK (Figures 6B and 6C). The carbonyl oxygen and NH hydrogen of the
naphthyridinone ring make the critical hydrogen bond contacts with Arg74 and Asn100,
respectively. The naphthyridinone ring is involved in a πꢀπ stacking interaction with Phe70.
An overlay with TMP and the cyanopyridone, 8, indicated that the Cꢀ4 and Cꢀ7 positions of
the ring can be exploited for further expansion.
We used the knowledge from this coꢀcrystal structure, combined with the coꢀcrystal
structures and SAR for the cyanopyridone series, for the FRIT expansion campaign based on
1,6ꢀnaphthyridinone, 28. A virtual library was designed via enumeration with suitable
substituents at R1 and R2. The predicted binding modes of the virtual library and synthetic
feasibility were used to prioritize compounds for synthesis. Encouragingly, we could achieve
subꢀmicromolar potency for the 1,6ꢀnaphthyridinone series together with improved ligand
efficiency with a limited set of compounds (Table 3).
Table 3: SAR exploration for 1,6ꢀnaphthyridinone
Compo
und #
IC50
Ligand
Structure
Calculated logD
0.8
(µM) Efficiency (LE)
634
0.31
29
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133
0.31
0.40
2.4
2.8
30
31
0.35
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1.0
0.2
0.36
0.34
3.3
1.9
32
33
2.1
0.28
2.4
34
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C
Figure 7: A. Coꢀcrystal structure at 2.2 Å resolution of compound 33 with Mtb TMK (PDB
ID: 4UNS). B. 2Dꢀinteraction diagram. Protein is shown in yellow cartoon representation
with selected sideꢀchains labeled and shown as sticks with carbon atoms coloured yellow.
Bound ligand is drawn in stick representation with carbon atoms in magenta. Hꢀbond
interactions are shown as dotted green and yellow lines. Electron density for compound 3 is
contoured at 1.0 sigma using 2fofc coefficients and is displayed as a grey mesh. C. Overlay of
bound ligands and active site residues from coꢀcrystal structures of Mtb TMK with
cyanopyridone (8, cyan) and 1,6ꢀnaphthyridinone (33, magenta).
Compound 33, the most potent in the series, was successfully coꢀcrystallized with Mtb
TMK (Figure 7). The position, orientation and key interactions of the core ring were retained
with respect to the starting FRIT, 28. The aryl substituent at Cꢀ4 of the napthyridinone
extends into the ribose and phosphate binding region, where it makes a number of new polar
contacts. The NH of the sulfonamide group makes a hydrogen bond interaction with Asp9.
The oxygen atoms of the sulfonamide group make waterꢀmediated interactions with Tyr39
and Arg95. The ring nitrogen (6ꢀaza) is involved in a waterꢀmediated hydrogen bond with
Arg107.
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Exploration of substitution at Cꢀ7 indicated that an ethyl group is optimal (Table 3).
Further extension led to a drop in potency (matched pairs: 31/32 and 33/34). Overlay of the
docking poses of these matched pairs in the TMP binding site (not shown) showed a shift of
the core ring for propyl derivatives so as to better accommodate the propyl group which
clashes with residues in the hydrophobic pocket. Overlay of the complex crystal structures of
compound 28 (propyl group at Cꢀ7) and 33 (ethyl group at Cꢀ7) confirmed the docking based
predictions (supporting information S2; Figure S2ꢀ2), thus explaining the observed drop in
potency upon homologation at this position. The relatively potent compounds (31-34, Table
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3) when tested in the whole cell assay, did not exhibit any activity (Mtb MIC > 32 ꢀg/mL).
Thus, our FBLG campaign was able, with the application of limited synthetic chemistry, to
deliver a novel chemical class, the 1,6ꢀnaphthyridinones, which show sub micromolar
potency and improved ligand efficiency (0.35ꢀ0.40). However, further exploration is required
to improve the potency and establish the potential of this chemical class as an antiꢀtubercular
agent. The observed SAR and experimental binding modes lay the foundation for further
investment in this series. For example, probing the Cꢀ5 and Cꢀ7 diversification points of the
naphthyridinone ring so as to optimize the hydrophobic contacts in the unique hydrophobic
pocket of Mtb TMK may lead to improvements in potency. Furthermore, substitution at the
para position of the phenyl at Cꢀ4 can be further extended by applying the SAR knowledge
from the cyanopyridones. No further work was done on the 1,6ꢀnaphthyridinone series as our
focus shifted to the cyanopyridones which were in an advanced stage of lead generation.
Sulfoxide and sulfone subꢀseries of the cynanopyridones exhibit Mtb MIC
During the course of initial exploration of SAR at the R1 and R2 positions of the 3ꢀ
cyanopyridone ring, we found that the oxidised form of compound 1, the sulfoxide 35,
exhibited moderate Mtb MIC (12µg/mL, Table 4). This encouraged us to investigate this
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subꢀseries further. We synthesized and tested a selected set of sulfoxides (36, 37, 39 and 41)
and sulfones (38 and 40). Our SAR knowledge from earlier exploration of the
cyanopyridones was used to select appropriate R1 substituents. We were encouraged to see
that both sulfoxide and sulfone subꢀseries of the cyanopyridones exhibited cellular activity,
and that the Mtb MIC improved concurrently with optimization of the IC50 against Mtb TMK
(Table 4). Extension of the aryl ring at position R1, in a similar manner to compound 19, led
to a 10 fold improvement in Mtb TMK IC50 (40 and 41) with retention of Mtb MIC. The
sulfone 42, which lacks the critical nitrile group, did not show any inhibition in our
biochemical assay up to 25 ꢀM and thus, unsurprisingly, had no measurable Mtb MIC (> 32
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ꢀg/mL). In contrast to the equivalent aryl derivatives, compound 43, the sulfone derivative of
the fused cyanopyridone, exhibits weak activity against the enzyme (Table 2). Overall, we
noted a reasonable correlation between IC50 and MIC within our limited dataset, suggesting
an onꢀtarget basis for the observed Mtb MIC.
Table 4. Limited SAR exploration of sulfoxides and sulfones
Structure
IC50 (µM) Mtb
(µg/mL)
MIC Calculated
logD
0.82
12
0.3
35
36
0.24
8
1.6
0.14
0.1
4
8
1.2
1.2
37
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0.33
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2.0
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0.022
0.032
4
2
2.6
3.1
40
41
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>25
30
>32
32
1.4
0.2
42
43
In order to understand the mode of binding of this subꢀseries, we coꢀcrystallized compound
36 with Mtb TMK. Our structure revealed that the sulfoxide subꢀseries binds in a similar
manner as observed for other cyanopyridones (Figure 8). The sulfoxide moiety lies adjacent
to the guanidine moiety of Arg95 suggesting they may form a cation/dipole interaction.
A
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Figure 8: Coꢀcrystal structure at 2.3 Å resolution of compound 36 with Mtb TMK (PDB
ID: 4UNQ). B. 2Dꢀinteraction diagram. Protein is shown in yellow cartoon representation
with selected sideꢀchains labeled and shown as sticks with carbon atoms coloured yellow.
Bound ligand is drawn in stick representation with carbon atoms in cyan. Hꢀbond
interactions are shown as dotted green lines. Electron density for compound 36 is contoured
at 1.0 sigma using 2fofc coefficients and is displayed as a grey mesh.
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The observed correlation between Mtb TMK IC50 and Mtb MIC, in conjunction with
confirmation of Mtb TMK binding in the form of complex crystal structures, is indicative of
target engagement. Further mode of action studies, including resistance mutation mapping
would be required to fully establish the target link.
Comparison of all the cyanopyridone subꢀseries indicated that sulfoxides and sulfones may
possess suitable physicoꢀchemical properties for further exploration so as to improve their
antimicrobial activity against Mtb. Some of the differentiating properties associated with
sulfoxides and sulfones are ionic nature (sulfone/sulfoxide are neutral; the potent aryl
substutited pyridones in Table 1 and 2 are mostly acidic), pKa of pyridone NH (calculated
pKa’s are 6ꢀ7 for sulfones/sulfoxides whereas for the aryl substituted derivatives the values
are in the range 8ꢀ9; Supporting information ꢀ S3, Table S3ꢀ1). Further investigation such as
experimental measurement of pKa and design of neutral cyanopyridone derivatives with the
desired pKa are required to establish this hypothesis.
Inhibitor selectivity: Comparison of the TMP binding sites in Mtb, human and bacterial
TMKs
Comparison of the Mtb TMK TMP binding site with that from other bacterial TMKs and
human TMK revealed key differences in the active site (supporting information – S4; Figure
S4ꢀ1 and Table S4ꢀ1). An overlay of the human TMKꢀTMP and Mtb TMKꢀcompound 8
crystal structures shows that the aryl ring at R1 of the cyanopyridone core may not be
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tolerated in human TMK (Figure 9). The methoxy substituted phenyl ring of 8 cannot be
accommodated within the human TMK active site without a change in either the binding
mode or the protein conformation (Figure 9B). Residues in human TMK such as Thr106,
Lys109 and Tyr151 would clash with the methoxy phenyl ring of 8, suggesting that similarly
substituted cyanopyridones will not inhibit human TMK.
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A
Figure 9: Comparison of the TMP binding sites of human and Mtb TMK. A. Molecular
surface of the TMP binding site of human TMK (pdb ID: 1E2D) is shown in grey. The
binding mode of compound 8 (cyan carbon atoms) in Mtb TMK is overlaid with TMP (green
carbon atoms) using protein atoms of the respective protein complexes (Selected human
TMK residues are coloured yellow and labelled).
Selected potent cyanopyridones (5, 12, 21, 27 and 36) were tested in a human TMK
enzyme assay to confirm the structureꢀbased prediction. None of the compounds showed any
activity up to 200 µM (Supporting information, Table S4ꢀ1), the highest concentration tested
that confirmed the selectivity of cyanopyridones and the unique nature of Mtb TMK active
site. A similar comparison of Mtb TMK with the TMP binding sites of other bacterial TMKs
(E. coli, S. aureus and H. influenzae) indicated that the cyanopyridones are likely to be
selective for Mtb TMK (supporting information S4, Figure S4ꢀ1 and Table S4ꢀ1). This
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prediction was supported by IC50 data against other bacterial TMKs obtained for selected
compounds from the series (Supporting information S5, Figure S5ꢀ1).
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8
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The aforesaid results from our hit to lead for cyanopyridones, limited exploration of 1,6ꢀ
naphthyridinones and selectivity against human TMK and other bacterial TMK clearly
demonstrated that the development of selective and potent inhibitors against Mtb TMK is
achievable. The antiꢀmycobacterial activity exhibited by sulfone and sulfoxides subseries
(Table 4) was promising and indicated that modulation of physicoꢀchemical properties is vital
in conjunction with potency against Mtb TMK to achieve cellular activity.
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Conclusion
High throughput screening and fragment based lead generation approaches against Mtb
TMK resulted in two novel chemical series, 3ꢀcyanopyridone and 1,6ꢀnaphthyridinone,
respectively. Structure guided optimization of the 3ꢀcyanopyridone hit, 1, led to compounds
with single digit nanomolar potency in vitro, albeit devoid of any cellular activity.
Interestingly, a subꢀseries containing sulfoxide or sulfone substituents showed low
micromolar Mtb MIC. While the IC50ꢀMIC correlation indicates that Mtb TMK may be one
of the targets responsible for the observed cellular activity, the target link remains to be
conclusively established. The 1,6ꢀnaphthyridinꢀ2ꢀones were optimized to subꢀmicomolar
potency within a limited number of compounds using coꢀcrystal structures and SAR from the
earlier series. Comparison of the active sites of Mtb, human and bacterial TMKs indicates
that these two classes of inhibitor are likely to be selective towards Mtb TMK, in agreement
with the limited biochemical selectivity assay data to date. The SAR for both series,
combined with the insights obtained from complex crystal structures generated during this
lead generation program, paves the way for further optimization of the cyanopyridone and
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1,6ꢀnaphthyridinone series towards identification of a candidate drug for the treatment of
tuberculosis.
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EXPERIMENTAL SECTION
Chemistry
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Schemeꢀ1a
R1, R2= H, CH3, OCH3, Cl
R3= OCH3, 4ꢀisopropylbenzyloxy, isopentyloxy, 3ꢀchlorobenzyloxy, trifluoromethoxy
R4= ꢀCOOH, COOCH3, NH2, Urea, Amide, Sulphonamide, Acylsulphonamide
aReagents and conditions: (a) 2ꢀcyanoꢀ3,3ꢀbis(methylthio)acrylamide, NaOH, DMSO, RT;
(b) Potassium monopersulphate triple salt, NMP, RT, 12h; (c) 2ꢀcyanoꢀ3,3ꢀ
bis(methylthio)acrylate, NaOH, DMSO, RT (d) Dil.H2SO4, 50ºC, 4h
General Procedures for the synthesis of compounds in Table 1
To the solution containing the corresponding acetyl derivative (0.599 mL, 5.55 mmol) in
DMSO (25 mL) was added 2ꢀcyanoꢀ3,3ꢀbis(methylthio)acrylamide (1044 mg, 5.55 mmol)
and powdered NaOH (444 mg, 11 mmol). The resulting reaction mixture was stirred at RT
for 12h. After the completion of the reaction, the reaction mixture was poured into water, and
neutralised with aqueous 1N hydrochloric acid. The solid obtained was filtered, washed with
water and dried.
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The crude compound was purified by flash chromatography using silica gel and Ethyl
acetate/Hexane as eluent to obtain the corresponding pyridones. Subsequently PREP HPLC
was used to provide compounds with > 95 % purity.
9
Procedure for the synthesis of compound 16
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To the solution containing the corresponding acetyl derivative (500 mg, 3.96 mmol) in
DMSO (25 mL) was added 2ꢀcyanoꢀ3,3ꢀbis(methylthio)acrylate (861 mg, 3.96 mmol) and
powdered NaOH (317 mg, 7.93 mmol). The resulting reaction mixture was stirred at RT
overnight. After the completion of the reaction, the reaction mixture was poured into water,
and neutralised with aqueous 1N hydrochloric acid. The solid obtained was filtered, washed
with water and dried.
The crude compound was purified by flash chromatography using silica gel and Ethyl
acetate/Hexane as eluent to obtain 4ꢀ(methylthio)ꢀ2ꢀoxoꢀ6ꢀ(thiophenꢀ2ꢀyl)ꢀ2Hꢀpyranꢀ3ꢀ
carbonitrile in 58% yield. Subsequently PREP HPLC was used to provide compounds with >
95 % purity.
Schemeꢀ2a
R1, R2= H, CH3, OCH3, Cl
R3= OCH3, 4ꢀisopropylbenzyloxy
R4= ꢀCOOH, COOCH3, NH2, Urea, Amide, Sulphonamide, Acylsulphonamide
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aReagents and conditions: (a) Aromatic aldehyde, NaOH, Ethanol, RT, 3h; (b) 2ꢀ(1Hꢀ
benzo[d][1,2,3]triazolꢀ1ꢀyl)propanamide, NaOH, Ethanol, Reflux, 12h; (c) Aromatic
aldehyde, Ethylcyanoacetate, Ammonium acetate, Ethanol, Reflux, 12h
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General Procedures for the synthesis of compounds in Table 2
To the solution of 1ꢀ(3ꢀmethoxyphenyl)ethanone (0.457 mL, 3.33 mmol) in ethanol (50
mL) was added the corresponding aromatic aldehyde (3.33 mmol), ethyl 2ꢀcyanoacetate (377
mg, 3.33 mmol) and ammonium acetate (1283 mg, 16.65 mmol). The resulting reaction
mixture was refluxed overnight.
After the completion of the reaction, volatiles were removed under high vacuum in a rotaꢀ
evaporator. The residue so obtained was triturated with water, filtered and dried.
The crude compound was purified by flash chromatography using silica gel and
Methanol/Dichloromethane as eluent to obtain the corresponding pyridones. Subsequently
PREP HPLC was used to provide compounds with > 95 % purity.
Procedure for the synthesis of compound 18
To a solution of (E)ꢀ4ꢀ(3ꢀ(3ꢀmethoxyphenyl)ꢀ3ꢀoxopropꢀ1ꢀenyl)benzoic acid (148 mg, 0.53
mmol) in Ethanol (15 mL) was added 2ꢀ(1Hꢀbenzo[d][1,2,3]triazolꢀ1ꢀyl)propanamide (100
mg, 0.53 mmol), NaOH (52.6 mg, 1.31 mmol). The resulting suspension was refluxed
overnight. After the completion of the reaction, the reaction mixture was cooled, and the solid
filtered and washed thoroughly with water. The crude compound was purified by flash
chromatography using silica gel and Methanol/Dichloromethane as eluent to obtain 4ꢀ(6ꢀ(3ꢀ
methoxyphenyl)ꢀ3ꢀmethylꢀ2ꢀoxoꢀ1,2ꢀdihydropyridinꢀ4ꢀyl)benzoic acid in 25% yield.
Procedure for the synthesis of 2ꢀ(1Hꢀbenzo[d][1,2,3]triazolꢀ1ꢀyl)propanamide
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To the reaction mixture containing sodium benzo[d][1,2,3]triazolꢀ1ꢀide (2 g, 14.17 mmol)
and 2ꢀbromopropanamide (2.154 g, 14.17 mmol) in toluene (50 mL) was added 18ꢀcrownꢀ6
(100 mg, 0.38 mmol). The reaction mixture was refluxed overnight. After the completion of
the reaction, the reaction mixture was cooled and extracted with ethyl acetate. The ethyl
acetate layer was washed with water and brine solution and dried over anhydrous sodium
sulphate. The organic layer was evaporated at high vacuum. The solid obtained was taken for
the next step without purification. The reaction mass, water and the toluene layer were
separated and evaporated to obtain 2ꢀ(1Hꢀbenzo[d][1,2,3]triazolꢀ1ꢀyl)propanamide in 74.2%
yield.
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MS (ES+): m/z =191 (m+H)
Procedure for the synthesis of (E)ꢀ4ꢀ(3ꢀ(3ꢀmethoxyphenyl)ꢀ3ꢀoxopropꢀ1ꢀenyl)benzoic acid
To a solution of 1ꢀ(3ꢀmethoxyphenyl)ethanone (0.457 mL, 3.33 mmol) in ethanol (10mL)
was added 4ꢀformylbenzoic acid (500 mg, 3.33 mmol) and sodium hydroxide (266 mg, 6.66
mmol). The reaction mixture was stirred at RT for 2hrs. After the completion of the reaction,
the volatiles were evaporated under vacuum. The solid obtained was taken up in water and
neutralised cautiously to get the solid. The solid so obtained was filtered, washed with water
and dried. Yield: 53.2%. Subsequently PREP HPLC was used to provide compounds with >
95 % purity.
MS (ES+): m/z =282.9 (m+H)
Schemeꢀ3a
O
N
O
HN
a
X
X
R2
R1
R1
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R1= H, CH3, OH, OCH3, Pyrazole,4ꢀisopropylbenzyloxy
X= C, O, N,
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8
R2= ꢀCOOH, COOCH3, NH2, Urea, Amide, Sulphonamide, Acylsulphonamide
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60
aReagents and conditions: Aromatic aldehyde, Cyanoethylacetate, NH4OAc, Reflux,
Overnight.
General Procedures for the synthesis of compounds in Table 2 (21ꢀ25)
To the corresponding 3,4ꢀdihydronaphthalenꢀ1(2H)ꢀone (0.34 mmol) in ethanol (15 mL)
was added the corresponding aldehyde (0.34 mmol), ethyl 2ꢀcyanoacetate (0.34 mmol) and
ammonium acetate (1.7 mmol). The resulting suspension was refluxed overnight. After the
completion of the reaction, volatiles were evaporated and the residue so obtained was taken
up in water, triturated and filtered. The compound was purified by chromatography using
silica gel and methanol/dichloromethane as eluent. Yield: 60ꢀ80%. Subsequently PREP
HPLC was used to provide compounds with > 95 % purity.
General Procedures for the synthesis of compounds in Table 2 (26 and 27)
To the corresponding chromanꢀ4ꢀone (1.73 mmol) in ethanol (15 mL) was added the
corresponding aldehyde (1.73 mmol), ethyl 2ꢀcyanoacetate (1.73 mmol) and ammonium
acetate (8.64 mmol). The resulting suspension was refluxed overnight. After the completion
of the reaction, volatiles were evaporated, the residue obtained was taken up in water,
triturated and filtered. The compound was purified by chromatography using silica gel and
methanol/dichloromethane as eluent. Yield: 60ꢀ80%. Subsequently PREP HPLC was used to
provide compounds with > 95 % purity.
Schemeꢀ4a: Synthesis of 1,6ꢀnaphthyridinones (Table 3; compound 31ꢀ34)
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Journal of Medicinal Chemistry
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R1=ethyl, propyl
R2=H, Br, NHSO2CH3
aReagents and conditions: Cyanoacetic acid (6 eq), T3P (20 eq), TEA (2 eq), Ethylacetate
(10 vol), 110 °C, overnight
General Procedures for the synthesis of compounds in Schemeꢀ4
To the solution of Cyanoacetic acid in ethyl acetate was added T3P and triethylamine. The
reaction mass was stirred at RT for 10 minutes. Compound (i) was added and heated at 110ºC
overnight. After completion of the reaction, the reaction mass was cooled to room
temperature and partitioned between ethyl acetate and water. The organic layer was washed
with water and brine solution. The organic layer was then dried over sodium sulphate and
evaporated under high vacuum to obtain the crude compound, which upon purification by
chromatography using silica gel and methanol/dichloromethane as eluent, gave the
compounds in 25ꢀ50% yield. Subsequently PREP HPLC was used to provide compounds
with > 95 % purity.
The compounds 28, 29 and 30 were procured from AstraZeneca fragment repository and
purity of the samples were confirmed to be > 95 % based on standard analytical method. The
syntheses of these fragments are reported in the literature.14
General Procedures for the synthesis of compounds in Table 4 (compounds 35-43)
To the solution of the corresponding 4ꢀ(methylthio)ꢀ2ꢀoxoꢀ1,2ꢀdihydropyridineꢀ3ꢀ
carbonitrile (0.29 mmol) in NMP (10 mL) was added Potassium monopersulphate triple salt
(1763 mg, 2.87 mmol). The reaction mixture was stirred at room temperature overnight.
Both sulfone and sulfoxide were formed in the reaction.
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