Synthesis, in silico screening and bioevaluation of dispiro-cycloalkanones as antitubercular and mycobacterial  NAD+ -dependent DNA ligase inhibitors

Rama P. Tripathi; Jyoti Pandey; Vandana Kukshal; Arya Ajay; Mridul Mishra; Divya Dube; Deepti Chopra; R. Dwivedi; Vinita Chaturvedi; Ravishankar Ramachandran

doi:10.1039/C0MD00246A

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DOI: 10.1039/C0MD00246A (Concise Article) Med. Chem. Commun., 2011, 2, 378-384

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Synthesis, in silico screening and bioevaluation of dispiro-cycloalkanones as antitubercular and mycobacterial NAD⁺-dependent DNA ligase inhibitors†

Rama P. Tripathi *^a, Jyoti Pandey‡ ^a, Vandana Kukshal‡ ^c, Arya Ajay ^a, Mridul Mishra ^a, Divya Dube ^c, Deepti Chopra ^c, R. Dwivedi ^b, Vinita Chaturvedi ^b and Ravishankar Ramachandran *^c
^aMedicinal and Process Chemistry Division, P.O. Box 173, Mahatma Gandhi Marg, Lucknow-226001, India. E-mail: rpt.cdri@gmail.com; rp_tripathi@cdri.res.in; r_ravishankar@cdri.res.in; Fax: +91 522 2623405/2623938/2629504; Tel: +91 0522 2612411
^bDrug Target Discovery and Development Division, P.O. Box 173, Mahatma Gandhi Marg, Lucknow-226001, India
^cMolecular and Structural Biology Division, Central Drug Research Institute Chattar Manzil, P.O. Box 173, Mahatma Gandhi Marg, Lucknow-226001, India

Received 1st December 2010 , Accepted 21st January 2011

First published on 28th February 2011

Abstract

A series of dispiro-cycloalkanones were synthesized using the “Corey Chaykovsky” reaction of α,α′-(E,E)-bis(benzylidene)-cycloalkanones/methanone in good yields. The compounds were evaluated for their in vitro antituberculosis activity against M. tuberculosis H37Rv and screened in silico. Some selected compounds were screened for mycobacterial NAD⁺-dependent DNA ligase inhibitory activity. Two of the compounds showed good in vitro antitubercular and NAD⁺-dependent DNA ligase inhibitory activity along with good correlation to in silico results.

Introduction

Despite current multidrug therapy and ongoing drug development,^1–3 tuberculosis continues to be a major public health concern today. With more than 1.6 million deaths and 9.2 million new cases being reported each year, it is a leading infectious disease claiming millions of death globally.^4,5Mycobacterium tuberculosis has the ability to survive for extended periods of time in a human host and thus requires prolonged drug treatment (six to nine months), resulting in low compliance. Moreover, the evolution of multidrug-resistant (MDR) and extremely drug resistant (XDR, recent mortality rate >98%) tuberculosis,^6–10 and the AIDS epidemic,^11–13 further makes the situation worse. In Mycobacterium tuberculosis drug resistance is not due to a common mechanism for all drugs, but different mechanisms for different classes of drugs.^14,15 Almost all the conventional targets and drugs have become inadequate to control resistant TB infection and therefore the discovery of novel, sensitive and selective targets or new chemical entity is needed for development of new generation of antitubercular drugs.

DNA ligases are vital enzymes in replication and repair of DNA. They catalyze the formation of a phosphodiester linkage between adjacent termini in double stranded DNA.¹⁶ Two types of DNA ligase viz. NAD⁺-dependent and ATP-dependent ligases are known based on their respective co-factor specificities.¹⁷ NAD⁺ ligases, commonly called LigA, occur almost exclusively in bacteria while ATP-dependent ligases are more ubiquitous and occur additionally in different viruses, archaea, eukaryotes and higher organisms.^18,19 Although there is little sequence homology between the eubacterial and eukaryotic enzymes, they exhibit some structural homology in specific domains and the mechanistic steps are broadly conserved.^20,21 Different steps involved in the action of DNA ligases involve large conformational changes as well as encircling and partial unwinding of the nicked DNA substrate.^22–24M. tuberculosis codes for at least three different types of ATP-dependent ligases and one LigA. Gene knockout and other studies have shown LigA to be indispensable in several pathogens including E. coli, S. typhimurium, S. aureus and B. subtilis in contrast to ATP-dependent ligases which are dispensable in M. tuberculosis.^25–30 To find new prototypes as specific inhibitors for NAD⁺ ligase is one of the approaches for anti-TB drug research as no drug is known to act against this enzyme so far. NAD⁺ ligase specific inhibitors including aryl amines³¹ and pyridochromanones³² have been reported. We have also shown that glycosyl ureides³³ and glycosyl amines³⁴ inhibit the Mycobacterial NAD⁺ ligase and have shown bactericidal activity (Fig. 1). As a part of our continuing efforts in tuberculosis chemotherapy^35–39 we have recently shown potent antitubercular activities in bis-benzylidene cycloalkanones,⁴⁰ phenylcyclopropyl methanones⁴¹ and alkylaminoaryl phenyl cyclopropyl methanones⁴² (Fig. 2). The compounds were designed as possible inhibitors of FAS-II but the enzyme inhibition by these compounds was not very significant although few of them showed very promising antitubercular activities.


	Fig. 1 Different classes of potential NAD⁺ ligase inhibitors.


	Fig. 2 Potent antitubercular compounds.

In continuation of this programme we have synthesized a series of dispiro-cycloalkanones by reacting bis benzylidene cyloalkanones with trimethylsulfoxonium iodide (TMSOI) for methylene insertion in the presence of NaOH as base and TBAB as phase-transfer catalyst (Corey Chaykovsky reaction).⁴³In silico screening results indicated NAD⁺ ligase as targets of these compounds and the compounds were also evaluated in vitro against M. tuberculosis H37Rv and full length NAD⁺-dependent DNA ligase from M. tuberculosis.

Results and discussion

Chemistry

The starting substrates α,α′-(E,E)-bis(benzylidene)-cycloalkanones/methanones (1a–1r)⁴⁰ were prepared by simple condensation of two equivalents of aromatic aldehydes with one equivalent of cycloalkanones/methanone in the presence of KOH (5 mol%) in ethanol as earlier reported by us. Cyclopropanation of the double bonds in α,α′-(E,E)-bis(benzylidene)-cycloalkanones was carried out with TMSOI to give the respective disipro-cycloalkanones. In order to establish suitable reaction conditions a model reaction of α,α′-(E,E)-bis(benzylidene)-cyclohexanone (1a) with TMSOI in the presence of tetrabutylammonium bromide (TBAB) was carried out in different solvent and bases to give 2,6-bis-(phenyl)-dispiro[2.1.2.3]decan-4-one (2) (Scheme 1) and the results are shown in Table 1.


	Scheme 1 Optimization of the cyclopropanation reaction using different solvent and base.

Table 1 Optimization of the cyclopropanation reaction using different solvent and base

Entry	Base (Conc.)	Solvent	Time (h)	Temp (°C)	Yield (%)
1	NaH (2eq.)	DMSO	20	100	30
2	NaH (2eq.)	DMF	24	100	25
3	aq. NaOH (50%)	DMSO	20	100	30
4	aq. NaOH (50%)	DMF	24	100	20
5	aq. NaOH (30%)	CH₂Cl₂	18	80	20
6	aq. NaOH (40%)	CH₂Cl₂	18	80	35
7	aq. NaOH (50%)	CH₂Cl₂	18	80	60
8	aq. NaOH (60%)	CH₂Cl₂	18	80	60
9	aq. NaOH (70%)	CH₂Cl₂	18	80	60

According to the results shown in Table 1, 50% aq. NaOH in CH₂Cl₂ (entry 7) is the most suitable protocol for the reaction. The success of this method is based on the solvation of the ionic species formed during the reaction which favors the reaction rate. Since solvation of ionic species is better in an aqueous NaOH/CH₂Cl₂ combination than in DMSO or DMF as solvent, the reaction yield is accordingly enhanced in aqueous NaOH/CH₂Cl₂ rather than in NaOH/DMSO or DMF combination. ⁴⁴ The structure of compound 2 was established on the basis of its spectroscopic data and microanalysis (see ESI†). The trans geometry of the cyclopropyl rings in compound 2 was established on the basis of literature precedents where cyclopropanation of trans-propenones with TMSOI under basic conditions (Corey-Chaykovsky reaction) is always reported to result in trans products.^45–47

After establishing the standard reaction conditions, we explored the scope of different substrates in the cyclopropanation. Thus, we carried out the cyclopropanation of different α,α′-(E,E)-bis(benzylidene)-cyclohexanones (1a–1h) with TMSOI to get the desired products 2–9 in moderate to good yields (Table 2, Scheme 2). To see the effect of ring size of the cycloalkanone moiety on this cyclopropanation reaction, the study was extended with α,α′-(E,E)-bis(benzylidene)-cyclopentanones (1i–1m) and α,α′-(E,E)-bis(benzylidene)-cycloheptanones (1n–1q). The reaction of different α,α′-(E,E)-bis(benzylidene)-cyclopentanones/cycloheptanones with TMSOI under similar reaction condition yielded compounds 10–18 in good yields (Scheme 2) and results are shown in Table 2. All the synthesized prototypes were well characterized by their spectroscopic data and microanalysis (see ESI†).


	Scheme 2 Synthesis of dispiro compounds 2–19 from different α,α′-(E,E)-bis(benzylidene)-cycloalkanones/methanone (1a–1r).

Table 2 Synthesized dispiro-cycloalkanones (2–19) and their in vitro antitubercular activity

Compd. No.	n		C logP^a	MIC^b (μM) M. tuberculosis H37Rv
a C logP was determined by OSIRIS Property Explorer Programme which is available at http://www.organic-chemistry.org/prog/peo/. b MIC= Minimum inhibitory concentration, the lowest concentration of the compound which inhibits the growth of mycobacterium >90%; MIC of the drugs used as control, INH 4.7 and ethambutol 15.9 μM against M. tuberculosis H37Rv.
2	3	Phenyl	5.20	>30
3	3	4-Fluorophenyl	5.32	>30
4	3	4-Chlorophenyl	6.43	16.8
5	3	4-Bromophenyl	6.60	13.5
6	3	4-Methoxyphenyl	4.99	>30
7	3	3,4-Dimethoxyphenyl	4.78	29.6
8	3	3,4,5-Trimethoxyphenyl	4.57	25.9
9	3	4-Benzyloxyphenyl	7.73	24.3
10	2	4-Fluorophenyl	5.00	>30
11	2	4-Bromophenyl	6.28	28.0
12	2	4-Methoxyphenyl	4.68	>30
13	2	3,4-Dimethoxyphenyl	4.47	>30
14	2	4-Benzyloxyphenyl	7.41	25.0
15	4	4-Chlorophenyl	6.75	>30
16	4	4-Methoxyphenyl	5.31	>30
17	4	3,4-Dimethoxyphenyl	5.10	28.6
18	4	3,4,5-Trimethoxyphenyl	4.89	25.2
19	0	4-Benzyloxyphenyl	6.81	26.3

Biological activities

The in vitro antitubercular activity against M. tuberculosis H37Rv was determined using agar microdilution method.⁴⁸ The in silico docking studies were carried out on the above synthesized dispiro-cycloalkanones using autodock tool^49,50 and some of the active hits were screened for their mycobacterial NAD⁺-dependent DNA ligase inhibitory activity against the full length of NAD⁺-dependent enzyme from M. tuberculosis, the major Human DNA ligase I and bacteriophage T4 DNA ligase. The compounds were assayed for their antibacterial activity via in vivo assay against S. typhimurium LT2 strain as per earlier reported protocols.^51,52

(A) In vitro antitubercular evaluation

The above synthesized dispiro-cycloalkanones 2–19 were evaluated against virulent strain M. tuberculosis H37Rv. The MIC values were determined using the agar microdilution method.⁴⁸ As evident from Table 2, among all the compounds screened compounds 4 and 5 were found to possess good activity with MIC 16.8 μM and 13.5 μM against virulent strain. However, compounds 7–9, 11, 14, 17–19 displayed a moderate antitubercular activity with MIC of 25–30 μM against virulent strain, while other compounds 2, 3, 6, 10, 12, 13, 15 and 16 possess MIC values >30 μM.

The activity results suggest that the conformation as well as the substitution pattern in the aromatic ring both govern the biological activity in the synthesized molecules. The conformational changes in the central alicyclic ring system have more impact on activity than the substitution pattern in the aromatic ring system. All these anti-TB results have good correlation with the in silico as well as in vitro enzymatic assays.

(B) In silico screening

Molecular interactions of LigA with the compounds. An analysis of the AutoDock predicted docking poses of all the compounds suggests that these inhibitors interact with several essential residues lining the AMP binding site, with one of the aromatic rings of the inhibitor overlapping the adenine base of AMP and the rest of the aromatic/aliphatic moieties projecting down the NAD⁺ binding tunnel (Fig. 3a). All the compounds are making polar interactions with important active site residues like E184, R211, G126, and D125. The compounds are also making hydrophobic interactions, particularly stacking with the H236 (Fig. 3a and 3b). Thus the stacking interaction seems to be the characteristic hallmark of the ligand recognition in the MtuLigA inhibition and catalysis. Among all the compounds used in the AutoDock study only four compounds 2, 3, 4 and 5 proved to be the best scored (Table 3).


	Fig. 3 (a). Compound 2 (ball and stick) occupying the same cavity as that occupied by AMP in the LigA binding pocket. The AMP is shown in yellow stick in panel A. In panel B, Compound I is shown as docked in the ligA binding cavity. The hydrogen bonding interactions are marked by dotted line. (b). Compounds 4 and 5 (ball and stick) are shown as docked in the ligA binding cavity, panels (A) and (B) respectively. The compounds are again occupying the characteristic disposition peculiar for natural ligand AMP with one of the aromatic moieties stacked with the protein’s H236 ring.

Table 3 In vitro inhibition of M. tuberculosis NAD⁺-dependent DNA ligase (MtuligA), Human DNA ligase (HuligI) and T4 DNA ligase (T4 lig)

Compounds	IC₅₀ (μM)			Docking energy (Kcal/mol)
Compounds	MtuligA	T4 lig	HuligI	Docking energy (Kcal/mol)
2	180 ± 5	210 ± 8.3	130 ± 10.5	−6.30
3	8.6 ± 0.3	33.4 ± 3.1	45.4 ± 2.2	−6.02
4	12.0 ± 0.7	20.2 ± 1.1	18.5 ± 0.7	−6.85
5	7.3 ± 0.5	70.2 ± 3.6	58.6 ± 3.2	−7.95

(C) In vitro enzymatic assays

To identify the drug target, compounds 2, 3, 4 and 5 which were sorted out based on the scoring function and fitness scores (minimum docking energy) as implemented in the AUTODOCK program, were assayed against the full length NAD⁺-dependent DNA ligase from M. tuberculosis, the major Human DNA ligase I and bacteriophage T4 DNA ligase, for the determination of in vitro inhibitory potency. Two compounds, 3 and 5, showed selective inhibition of M. tuberculosis ligase, and were further evaluated for in vivo antibacterial activities.

In vitro inhibition of nick joining activity

DNA ligase nick joining activity was done as described earlier in the presence of varying concentrations of inhibitors. A quick screening of inhibitors was carried out with DNA ligation assay at high concentration, 100μM against both MtuLigA and T4Lig. This served as a sieve for selecting compounds with the potential to distinguish between NAD⁺- and ATP-dependent ligases for detailed experiments. Based on the obtained results, subsequent efforts were focused on four compounds which we assayed for its in vitro inhibitory potency against the full length NAD⁺-dependent enzyme from M. tuberculosis, the major Human DNA ligase I and bacteriophage T4 DNA ligase, respectively. In vitro inhibition data IC₅₀ (Table 3) shows that these compounds are inhibiting MtuLigA in low micromolar range.

Out of the four compounds, compound 2 bound to the Human DNA ligase (HuligI) and T4 DNA ligase (T4 lig) with low affinities; there is no selectivity of this compound for NAD⁺ and ATP DNA ligases. On the other hand, compound 3 distinguishes between the ATP-dependent DNA ligase and NAD⁺-dependent DNA ligase of M. tuberculosis by a factor of four and between Human and M. tuberculosis enzyme by a factor of five and has high affinity for M. tuberculosis enzyme with IC₅₀ of 8.6 μM. Compound 4 also showed greater affinity for M. tuberculosis enzyme while compound 5 showed highest the affinity among all the four compounds. For M. tuberculosis enzyme the IC₅₀ value for this compound is 7.3 μM and it can distinguish between NAD⁺-dependent DNA ligase and ATP-dependent DNA ligase by a factor of 8–10 (Fig. 4). A good correlation have been observed between the MIC of in vitro antitubercular activity and IC₅₀ of in vitro inhibition of nick joining activity in compound 4, compound 5 is also in good agreement.


	Fig. 4 Inhibition of growth of M. tuberculosis (ligand affinity).

(D) Antibacterial in vivo assay

To evaluate the in vivo inhibition of NAD⁺ ligases, two bacterial systems were used and the results depicted in Table 4 clearly show that the compounds inhibit more specifically the NAD⁺- dependent DNA ligases as compared to ATP-dependent DNA ligases. Cell viability assay with the compounds again showed that the wild type S. typhimurium LT2 strain is less viable as compared to the viability of the ligase deficient variant rescued with T4Lig (Fig. 5a and 5b, Table 4). The in vivo assay results demonstrate that compounds have higher specificity for NAD⁺-dependent ligases and strongly suggest that the observed antibacterial activities are due to in vivo inhibition of LigA.

Table 4 Antibacterial activity of compounds 3 and 5

Compd. No.	MIC (μM)
	S. typhimurium	S. typhimurium	E. coliGR501	E. coliGR501	E. coliGR501
	LT2	TT15151	+pTRC99A	+MtuNAD+ligase	T4 DNA ligase
3	29.5	147.9	5.9	44.3	177.5
5	43.4	76.0	8.6	52.1	65.2


	Fig. 5 (a). Bactericidal activity of compound 3. (A) S. typhimurium LT2 and (B) its DNA ligase minus (null) derivative TT15151 on their respective exposure to compound 3. (b) Bactericidal activity of compound 5. (A) S. typhimurium LT2 and (B) its DNA ligase minus (null) derivative TT15151 on their respective exposure to compound 5.

(E) Mode of inhibition

We chose compound 5 to evaluate the mode of inhibition. The standard kinetics was done with NAD⁺ in overall nick sealing reaction in vitro. When nick joining activity was measured in the presence of different concentrations of compound 5 (0–50μM) with increasing concentration of NAD⁺, the kinetics clearly indicated a competitive inhibition of NAD⁺ by compound 5 (Fig. 6). The linear regression using the apparent Km value leads to K_i value of 5.902μM.


	Fig. 6 Mode of inhibition of MtuLigA with respect to NAD⁺ by compound 5. (a) Activity of MtuLigA measured in the presence of rising concentrations of NAD⁺ (0–50 μM) and compound 2 (0–20 μM). (b) A double reciprocal plot of the data clearly indicates competitive binding between NAD⁺ and compound 5. (c) Linear regression plot of the inhibitor concentration versus the Km_app. The K_i value is marked with an arrow.

(F) DNA binding assay

In order to check whether these two active compounds (3 and 5) are generally interacting with DNA and thereby influencing the inhibitory behavior, we carried out ethidium bromide displacement assays. Compounds were added to a maximum concentration of 250 μM. Even at this high concentration, representing a 50-fold excess over ethidium bromide (5 μM) no loss in fluorescence was observed (Fig. 7). We also carried out gel shift assays where the electrophoretic mobility of plasmid DNA was checked in the presence of increasing inhibitor concentrations. The experiments did not support any general interaction of compounds 3 and 5 with DNA.


	Fig. 7 Ethidium bromide displacement assay.

Conclusion

In conclusion, a series of dispiro-cycloalkanones with conformationally different cycloalkyl ring systems was synthesized and evaluated against M. tuberculosis H37Rv in vitro and full length of mycobacterial NAD⁺-dependent DNA ligase. A few of the compounds showed moderate to significant antitubercular and DNA ligase inhibitory activities. The possible mode of action of these compounds was also evaluated by using in silico screening, in vitro and in vivo enzymatic assays.

Acknowledgements

This is a CDRI communication no 8017. The authors thank UGC and CSIR New Delhi for the fellowship. We sincerely acknowledge the financial assistance from DRDO and DBT New Delhi. SAIF CDRI is also acknowledged for providing the spectral and microanalytical data of the compounds.

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Footnotes

† Electronic supplementary information (ESI) available: Experimental procedures, characterization data and copies of ¹H NMR and ¹³C NMR spectra, protocols of biological assays See DOI: 10.1039/c0md00246a

‡ Authors having equal contribution.

Synthesis, in silico screening and bioevaluation of dispiro-cycloalkanones as antitubercular and mycobacterial COMPOUND LINKSRead more about this on ChemSpiderDownload mol file of compoundNAD+-dependent DNA ligase inhibitors†