Design and Synthesis of a Focused Library of Diamino Triazines as Potential Mycobacterium tuberculosis DHFR Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.5b00367

We report design of a series of 2,4-diamino triazines as Mycobacterium tuberculosis (Mtb) dihydrofolate reductase inhibitors. The synthesized compounds were evaluated against Mtb (H₃₇Rv and Dormant stage H₃₇Ra), their cytotoxicity was assessed (HepG2 and A549 cell lines), and selectivity toward Mtb was evaluated by testing against other bacterial strains. Some derivatives showed promising activity along with low cytotoxicity. The most potent compound in the whole cell assay (MIC 0.325 μM against H₃₇Rv) showed selectivity in the enzyme assay and exhibited synergy with second line anti-TB agent p-amino salicylic acid. This study therefore provides promising molecules for further development as antituberculosis DHFR inhibitors.

Full text of DOI:10.1021/acsmedchemlett.5b00367

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Letter
Design and synthesis of a focused library of diamino triazines
as potential Mycobacterium tuberculosis DHFR inhibitors
Arundhati C Lele, Archana Raju, Mihir P Khambete, M.K. Ray, M.G.R. Rajan,
Manisha A Arkile, Nandadeep J Jadhav, Dhiman Sarkar, and Mariam Sohel Degani
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.5b00367 • Publication Date (Web): 17 Oct 2015
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Design and synthesis of a focused library of
diamino triazines as potential Mycobacterium
tuberculosis DHFR inhibitors
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Arundhati C. Lelea, Archana Rajua, Mihir P. Khambetea, M. K. Rayb, M. G. R. Rajanb,
Manisha A. Arkilec, Nandadeep J Jadhavc, Dhiman Sarkarc and Mariam S. Degania*
aDepartment of Pharmaceutical Sciences and Technology, Institute of Chemical Technology,
Matunga (E), Mumbai 400019, India.
bRadiation Medicine Center, Bhabha Atomic Research Centre, Tata Memorial Hospital
Annex, Parel, Mumbaiꢀ400012
cCombichemꢀBioresource Center, Organic Chemistry Division, CSIRꢀNational Chemical
Laboratory, Pune 411008, India.
Keywords: Diamino triazine, Dihydrofolate Reductase, Mycobacterium tuberculosis,
Molecular modeling, Enzyme assay, Synergy, Selectivity.
Abstract
We report design of a series of 2,4ꢀdiamino triazines as Mycobacterium tuberculosis (Mtb)
dihydrofolate reductase inhibitors. The synthesized compounds were evaluated against Mtb
(H37Rv and Dormant stage H37Ra), their cytotoxicity was assessed (HepG2 and A549 cell
lines) and selectivity towards Mtb was evaluated by testing against other bacterial strains.
Some derivatives showed promising activity along with low cytotoxicity. The most potent
compound in the whole cell assay (MIC 0.325µM against H37Rv) showed selectivity in the
enzyme assay and exhibited synergy with second line antiꢀTB agent pꢀamino salicylic acid.
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This study therefore provides promising molecules for further development as antiꢀ
tuberculosis DHFR inhibitors.
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Folate metabolism represents an important and attractive target for chemotherapy.
The importance of this pathway in chemotherapy of infectious diseases arises from its
function in DNA biosynthesis and cell replication. Tetrahydrofolate, the central component of
folate metabolism, is a critical oneꢀcarbon unit donor. Owing to this, it plays an essential role
in the biosynthesis of purines and pyrimidines and therefore in the nucleic acid biosynthesis
for all the living organisms and is thus directly or indirectly involved in the processes of cell
reproduction.1 Numerous enzymes in the cell reproduction cycle use folate either as a
cofactor or as a substrate. Differences in the enzymatic constitution of the microorganisms
and mammals, either with respect to the enzyme types or with respect to architecture of
common enzymes, have enabled devising an attack strategy towards pathogens.2 These
differences provide the basis for design of selective inhibitors devoid of toxicity to human
cells. Dihydrofolate reductase (DHFR), a key enzyme of this pathway plays an important role
in the cell growth and proliferation, as it is the sole source of tetrahydrofolate, and thus acts
as an Achilles’ heel for rapidly proliferating cells.3 DHFR is found in many pathogenic
microorganisms including Mycobacterium tuberculosis (Mtb). However, it remains relatively
unexplored in Mtb, an obligate pathogen. In the year 2014, World Health Organization has
estimated 6.1 million cases of tuberculosis (TB).4 A rigid cell wall barrier along with the
ability to remain dormant has made TB treatment difficult. The issue of subclinical
persistence and resistance development highlights the need for new molecules, particularly in
immuneꢀcompromised individuals.5 Thus, new antiꢀtuberculosis agents are required to which
could act via unique mechanisms and therefore show minimum cross resistance with the
existing drugs.
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Researchers have identified nitrogen heterocycles6ꢀ8, boron containing carboranes9
and a tripeptide as Mtb DHFR inhibitors.10 Our research group has been actively involved in
the search of DHFR inhibitors and has synthesized several novel, diverse inhibitors of DHFR
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for Mtb11,12 and opportunistic pathogens13ꢀ15, including Mycobacterium avium16,17
.
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Additionally, we have isolated Mtb DHFR enzyme from recombinant Saccharomyces
cerevisiae strains and purified it using affinity chromatography with a novel epoxy bound
resin linked to the inhibitor, methotrexate.18,19
In our earlier efforts to identify Mtb DHFR inhibitors, various diamino triazines had
been synthesized. These studies resulted in hits with Mtb inhibition in the micromolar
range.12 This encouraged us to focus our efforts towards strategically modifying the diamino
triazine scaffold, using various molecular modeling techniques, with the goal to achieve subꢀ
micromolar activity. The current work therefore deals with identification of pharmacophoric
features using the interactions of known DHFR inhibitors and alignment of our earlier
developed analogues. Further virtual screening was carried out based on the obtained
pharmacophoric features to generate hits. On the basis of these molecular modeling studies,
we report a new series of diamino triazines as Mtb DHFR inhibitors. These derivatives were
evaluated for whole cell Mtb inhibition (active and dormant) and cytotoxicity on liver and
lungs cell lines to evaluate their toxicity. These were also tested against other bacterial strains
to assess selectivity towards Mtb. The most active derivative was further evaluated for
inhibition of DHFR in an enzyme assay and synergy with the second line agent, para amino
salicylic acid (PAS), which acts upstream to DHFR in the folate pathway.
In the present work, docking interactions of known DHFR inhibitors methotrexate
(MTX) and trimethoprim (TMP) with Mtb DHFR (PDB: 1DF7) were studied which revealed
necessary interactions such as hydrogen bonding interaction with Ile94, Ile5 and Asp27 along
with presence of a tertiary nitrogen containing heterocycle (Figure 1a). Therefore diamino
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substituted ring features were shortꢀlisted for manual pharmacophore generation (Figure 1c).
Simultaneously, Pharmacophore Alignment and Scoring Engine (PHASE) module of
Schrödinger suite was used to align the 26 inꢀhouse Mtb DHFR inhibitors, having 2,4ꢀ
diamino triazine scaffold,12 and identify additional features which could enhance the activity.
It was observed that the phenyl ring, which is directly attached to the triazine ring, shows a
good overlap in the alignment (Figure 1b).
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Figure 1. Design of 5aꢀ5p.
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The alignment revealed the scope for substituting the oxygen atom attached to the
phenyl ring with various hydrophobic groups. Therefore the phenyl ring and the oxygen atom
were selected as ring and acceptor feature respectively and combined with the earlier
identified features to generate the final manual pharmacophore hypothesis (Figure 1c). The
manual pharmacophore generated was used for virtual screening, on Mtb DHFR (PDB:
1DF7), using the Phase Database to obtain virtual hits (Figure 1d). On the basis of the
obtained hits, 2,4ꢀdiamino triazines were designed as potential Mtb DHFR inhibitors (Figure
1e). The designed derivatives were docked into Mtb DHFR active site to investigate the in-
silico binding interactions. All the designed diamino triazines showed favourable binding
interactions with crucial active site residues of Mtb DHFR. The amino group of the triazine
ring showed Hꢀbond interaction with the hydrophilic residues Asp27, Ile5 and Ile94 at the
bottom of the active site cavity, while the distal aromatic ring exhibited hydrophobic
interactions with residues at the mouth of the active site tunnel. Additional πꢀπ stacking
interactions with amino acid residue Phe31 were observed in many derivatives. Docking
interaction of a representative molecule is depicted in Figure 2.
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Figure 2. Ligand interaction diagram of a representative derivative 5i.
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The designed molecules were subjected to in silico ADME prediction and solubility
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estimation to assess drug likeness and were found to be drug like.
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The synthesis of these designed compounds was carried out according to the
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reactions depicted in Scheme 1.
Scheme 1: Synthesis of designed diamino triazine derivatives 5aꢀ5p
4ꢀfluoro benzaldehyde 1 (4g, 0.0032mol) was dissolved in THF; ammonia (30%) (20 mL)
was added and reaction mixture was stirred at room temperature for 25ꢀ30mins. To the white
suspension formed, iodine (4.92g, 0.00384mol) was added in small portions which resulted in
dark colouration of the reaction mass. The reaction mixture decolourized in 30ꢀ60 mins hr
and it was further stirred at room temperature for 2ꢀ3 hours. After completion of the reaction,
as indicated by TLC, saturated solution of sodium thiosulphate followed by 15 mL EtOAc
was added. The organic layer was separated and the aqueous layer was extracted further with
2 x 10 mL of EtOAc. The combined organic layers were washed with brine (15 mL), dried
over sodium sulphate and concentrated in-vacuo to obtain 4ꢀfluoro benzonitrile 2.2 (2g,
0.00165 mol) and various phenols (3aꢀ3p) (0.0198 mol) were dissolved in 15 mL DMSO.
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K2CO3 (0.34g, 0.0025 mol) was added and the reaction mixture was subjected to three cycles
of microwave irradiation, power 120 Watts, temperature 100°C for 15 mins with an
intermittent cooling cycle. After completion, the reaction mass was poured into 20 mL of ice
water and extracted thrice with 10 mL of EtOAc. The combined organic layer was treated
with brine (15 mL), dried over sodium sulphate and concentrated in-vacuo to obtain
derivatives 4aꢀ4p. Dicyandiamide (DCDA, 0.15g, 0.0018 mol), KOH (0.13 g, 0.00225 mol)
were added to solution of 4aꢀ4p, (0.0015mol) in EtOH (10 mL). The resultant reaction
mixture was refluxed in an oil bath for 10ꢀ18 hours. After completion of reaction, precipitated
solid was filtered, washed with EtOH (5 mL) followed by hot water (25 mL) and dried to
afford the corresponding diamino triazine derivatives 5aꢀ5p. The purity of the synthesized
derivatives was determined by HPLC, melting point and appropriate spectral characterization
using IR, NMR and Mass spectrometry.
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The synthesized derivatives were tested for their antiꢀTB activity against both
active20,21 and latent forms.22 The objective was to assess the selectivity towards
mycobacteria as use of broad spectrum antiꢀinfective agents may result in development of
resistance and cross resistance. Therefore, the synthesized derivatives were tested against S.
aureus and E. coli as representative gram positive and gram negative bacteria respectively.
(Table 1)
The derivative 5i showed better activity than both the known DHFR inhibitors MTX
and TMP. Furthermore, it showed superior activity compared to the first line antiꢀTB drugs
isoniazid (INH 0.73µM), ethambutol (MIC 19.58µM)23 and streptomycin (MIC
3.44µM)23and near equivalent activity with rifampicin (MIC 0.24µM).23 Derivative 5k
showed activity comparable to MTX. Additionally, derivatives 5j, 5l, 5m and 5o exhibited
activity between the range of MTX and TMP (Table 1).
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Table 1: Biological activity studies of the synthesized derivatives
% inhibition
MIC
MIC
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9
MIC
of Mtb
H37Ra
against
against
against Mtb
H37Rv (µM)
Comp.
ID
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R
S. aureus
E. coli
(Dormant
Stage)
(µM)
(µM)
at 10 µg/ml
19.36
ꢀ
ꢀH
4ꢀCH3
109.56
>417.63
>396.03
>369.38
>447.46
>381.37
>380.14
>398.42
0.325
447.55
426.16
404.11
365.72
426.16
381.37
372.69
398.42
406.70
348.98
381.37
406.70
398.42
447.55
426.16
404.11
365.72
426.16
381.37
372.69
398.42
406.70
348.98
381.37
406.70
398.42
5a
5b
5c
5d
5e
5f
4ꢀOCH3
4ꢀClꢀ3,5ꢀdiCH3
3ꢀCH3
37.54
14.50
15.04
7.27
4ꢀClꢀ3ꢀCH3
4ꢀt butyl
4ꢀCl
42.19
81.06
16.10
83.04
87.19
96.86
27.62
5g
5h
5i
2,3ꢀdi CH3
4ꢀBr
3.56
5j
4ꢀClꢀ2ꢀCH3
2,6ꢀdi CH3
2ꢀCl
1.96
5k
5l
20.34
20.72
5m
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4ꢀF
>420.47
3.59
4.98
420.47
358.99
420.47
358.99
5n
5o
2,4ꢀdi Cl
93.62
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RꢀPhꢀOH=
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211.61
11.65
423.21
423.21
5p
RꢀPhꢀSH
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
MTX
TMP
INH
0.968
28.93
0.73
Promising in-vitro biological testing results were obtained as indicated by the activity
of derivatives 5i, 5j, 5k, 5l, 5m and 5o. It was observed that substitution at 2 position of the
terminal phenyl ring with both electron withdrawing or donating group enhances activity
against Mtb H37Rv. Methyl substitution at position 2 was preferred as observed with the
activities of derivatives 5k and 5o. Addition of another methyl group at 3 position led to the
most active compound of this series, 5i. Most of the derivatives exhibited moderate to good
inhibition of the latent tubercle bacilli at the concentration of 10 µg/ml. All the derivatives
were found to be inactive against S. aureus and E. coli indicating selectivity towards
mycobacteria.
In-vitro cytotoxicity testing was carried out using HepG2 cell line (liver)24 and A549
cell line (lungs).25 The results indicated that these derivatives were relatively nonꢀtoxic.
MTX is very potent albeit a nonꢀselective DHFR inhibitor, toxic to human cells and
therefore is not used in antiꢀinfective therapy. Hence, one of the objectives of the current
work was to assess the potency and selectivity of the most active derivative 5i by testing
against both Mtb and human DHFR. The enzyme assay26,27 revealed that 5i showed IC50
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values of 25.875 ± 0.006µM and 42 ± 0.063µM against Mtb and human DHFR respectively,
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indicating around two fold selectivity towards the pathogenic enzyme. The IC50 values for
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MTX were 0.00825 ± 0.00025µM and 0.0016
± 0.0003µM against the pathogen and host
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enzymes respectively with a selectivity of 0.194. The derivative 5i is less active than MTX
but is 8 times more selective, thus providing promising insight for design of selective, potent
Mtb DHFR inhibitors.
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Synergy studies were carried out for 5i with second line drug pꢀamino salicylic acid
(PAS)28, acting upstream of DHFR. Synergy of a molecule can be measured from its
Fractional Inhibitory Concentration Index i.e. FICI which is determined by using the
following formula.
FICI= MICa combination
MICa alone
MICb combination
MICb alone
The FICI value for 5i was found to be 0.75 indicating synergistic association with
PAS. (Table 2)
Table 2: Synergy studies of5iwith PAS
Compound
Alone (µg/ml)
In Combination (µg/ml)
0.1
1
0.025
0.5
5i
PAS
To summarize, molecular modelling was used to develop a manual pharmacophore
which was further used for virtual screening and design of diamino triazines. Some
compounds showed better or comparable activity to the first line antiꢀTB drugs and known
DHFR inhibitors (MTX and TMP). Particularly, 5i was found to have potent whole cell
activity against Mtb H37Rv (MIC: 0.325µM) along with low cytotoxicity against both liver
and lungs cell lines. Testing against gram positive and gram negative strains indicated
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selectivity towards Mtb. The enzyme assay results indicated that this derivative showed
promising DHFR inhibition and was 8 times more selective than MTX. Additionally, it
exhibited synergistic association with PAS. Thus, this study represents a successful attempt to
achieve subꢀmicromolar Mtb inhibition using a combined structure and ligand based
approach and can provide impetus for further lead development of promising preꢀclinical
candidates.
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ASSOCIATED CONTENT
Supporting Information: Molecular modelling protocols, along with spectral
characterization and HPLC purity data for compounds 5aꢀ5p and details of biological testing.
AUTHOR INFORMATION
Corresponding author
Tel.: +91 22 33612213
Fax: +91 22 33611020
ms.degani@ictmumbai.edu.in; msdegani@gmail.com
ACKNOWLEDGEMENT
Author ACL, MAA and NJJ are thankful to Council of Scientific and Industrial Research,
New Delhi, author AR is thankful to Indian Council of Medical Research, New Delhi, India
and author MPK is thankful to DSTꢀINSPIRE, New Delhi, India for financial assistance. The
authors declare that there is no conflict of interest.
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