Bioorganic & Medicinal Chemistry Letters 24 (2014) 5181–5184

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Design, synthesis and molecular docking of substituted 3-hydrazinyl-3-oxo-propanamides as anti-tubercular agents q Arshi Naqvi, Richa Malasoni, Akansha Srivastava, Rishi Ranjan Pandey, Anil Kumar Dwivedi ⇑ Division of Pharmaceutics, CSIR-Central Drug Research Institute, B 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, India

a r t i c l e

i n f o

Article history: Received 24 June 2014 Revised 29 August 2014 Accepted 25 September 2014 Available online 2 October 2014 Keywords: Hydrazides Molecular docking Binding energy Drug likeness score Anti-tubercular

a b s t r a c t Based on the anti-mycobacterial activity of various acid hydrazides, a series of substituted 3-hydrazinyl3-oxo-propanamides has been designed. The target compounds have been synthesized from diethylmalonate using substituted amines and hydrazine hydrate in ethanol. Computational studies and anti-tubercular activity screenings were undertaken to test their inhibitory effect on protein kinase PknB from Mycobacterium tuberculosis. Binding poses of the compounds were energetically favorable and showed good interactions with active site residues. Designed molecules obey the Lipinski’s rule of 5 and gave moderate to good drug likeness score. Among the sixteen compounds (1–16) taken for in silico and in vitro studies, 3 compounds (11, 12 and 15) have shown good binding energies along with exhibiting good anti-tubercular activity and thus may be considered as a good inhibitors of PknB. Ó 2014 Elsevier Ltd. All rights reserved.

Mycobacterium tuberculosis (MTB) is considered to be the leading bacterial infection around the globe.1 With an estimated 8.7 million new tuberculosis (TB) cases (13% co-infected with HIV) and 1.4 million fatalities each year,2 MTB causes more human deaths than any other single infectious organism. The disease is one of India’s most challenging public health problems and it accounts for nearly one-third of the global burden. Approximately 2 million people acquire TB every year in India.3 Furthermore, MTB strains resistance is emerging at an alarming rate to all of the first line drugs (isoniazid, rifampicin, fluoroquinolone) and at least one of three injectable second-line drugs (i.e., amikacin, kanamycin, or capreomycin).4 Isonicotinic acid hydrazide (isoniazid, INH, Fig. 1) belongs to the group of the first line antitubercular drugs which are in clinical practice for more than 50 years. After the discovery of INH, the full therapeutic possibilities of acid hydrazides were realized. Investigations of other heterocyclic hydrazides having mono-cyclic nuclei such as furan, thiophene, pyrrole and dicyclic nuclei such as quinoline and isoquinoline was stimulated due to the remarkable clinical value of INH.5 A large number of such substances have been synthesized in pure form having differing ranges of curative effects. With a view to establish the structural requirements for antitubercular activity, Yale et al.6 reported the synthesis of a number of hydrazides of the nicotinic acid hydrazide type.

q

CDRI Communication No. 8813.

⇑ Corresponding author. Tel.: +91 522 2623405; fax: +91 522 2623938. E-mail address: [email protected] (A.K. Dwivedi). http://dx.doi.org/10.1016/j.bmcl.2014.09.080 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

A large number of hydrazides and their derivatives are reported to possess a wide array of biological activities like antifungal,7 psychotropic,7 antituberculous,8–10 antiparasite,7,11 bacteriostatic,7,12–14 antiviral,14 insecticidal15 and anti-cancer16 activities. Thus, these were found to be useful especially in the treatment of inflammatory and autoimmune diseases, osteoarthritis, respiratory diseases, tumors, cachexia, cardiovascular diseases, fever, hemorrhage and sepsis.17 Protein kinase B (PknB) plays an important role in mammalian cellular signaling. Mycobacterium tuberculosis PknB is an essential receptor-like protein kinase involved in cell growth control. The protein kinase B peptide contains two types of structural elements (VAL 95, ARG 97) and basic residue ring constituted of glycine rich residue. M. tuberculosis PknB is a trans-membrane Ser/Thr protein kinase (STPK) highly conserved in Gram-positive bacteria and apparently essential for mycobacterial viability.18 It was previously shown that PknB is regulated by auto-phosphorylation and de-phosphorylation by the Ser/Thr protein phosphatase PstP19,20 H N

O

NH2

N

Figure 1. Structure of isoniazid.

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and work showed that PknB is predominantly expressed during exponential growth, where it’s over expression causes morphological changes linked to defects in cell wall synthesis and cell division.21 Therefore, there is an urgent need to develop new drugs against MTB. To achieve this endeavor, we have synthesized some substituted malonamic acid hydrazides (1–16). The molecular docking studies of newly synthesized compounds were carried out. Oral bioavailability is a desirable property of compounds under investigation in the drug discovery process. Lipinski’s rule-of-five is a simple model to forecast the absorption and intestinal permeability of a compound.22,23 The designed molecules obey the Lipinski rule of five along with obeying some additional parameters.24 Compounds which were found promising in the docking study were evaluated for their anti-mycobacterial activity by resazurin micro-titre assay (REMA) method25 against M. tuberculosis H37Rv.

O

R

+

H2N

O

O

All the compounds were prepared by Scheme 1. For synthesis, substituted amines (I–XVI) were first refluxed with ethanolic solution of diethyl malonate (XVII) for 4 h. Further refluxing with hydrazine hydrate for another 5 h gave compounds 1–16 (58–88% yield, Table 1, Scheme 1). All the compounds (1–16) were characterized spectroscopically. The base peak in ESI-MS was found at M++1. In IR spectra, a strong band from 1538 to 1681 cm 1 indicated the presence of carbonyl of amide group. Bands from 2806 to 2969 indicated the presence of methylene group. In 1H NMR spectra of compounds 1–16, singlet peak between 3.52 and 3.32 ppm, indicated two protons of the methylene group (>CH2). Broad singlet between 4.32 and 4.20 ppm was found indicating two protons attached to nitrogen atom (–NH–NH2). Peaks between 8.91 and 6.60 ppm indicated the presence of protons of aromatic ring of respective compounds. Two broad singlets between 9.36 and 9.21 ppm were found indicating –CONH– proton. 13C spectra clearly supported the results obtained from 1H NMR spectra and also justified the number of carbon atoms in corresponding compounds. Table 2 Molecular docking results of the target compounds (1–16)

O

XVII

I-XVI

Mol. No.

i

O

O

H2NHN

N H R

1-16 Scheme 1. Reagents and conditions: (i) [a] C2H2OH, reflux, 4 h. [b] NH2NH2
HCl, reflux, 5 h.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Binding energy (kcal/mol) 7.44 6.33 6.69 6.78 7.09 6.98 6.87 7.03 7.20 6.66 6.24 6.88 6.98 7.01 7.48 6.46

Inhibition constant (lM) 3.53 22.87 12.48 10.79 6.38 7.68 9.21 7.01 5.25 13.13 26.51 9.04 7.59 7.32 3.86 18.46

C log P 0.80 0.56 0.10 0.10 0.63 1.37 1.25 0.89 0.89 0.82 1.55 1.02 3.18 0.26 0.36 1.20

Table 1 Characterization & anti-tubercular activity of substituted 3-hydrazinyl-3-oxo-propanamides (1–16)

O S. No.

O

H2NHN

N H

Molecular formula

Mp (°C)

% Yield

MICa (lg/mL) H37Rv

C9H9N3O2FCl C9H10N4O4 C9H10N4O4 C9H10N4O4 C9H9N5O6 C9H9N3O2Cl2 C9H9N3O2Cl2 C11H15N3O4 C11H15N3O4 C13H20N4O2 C13H19N3O2 C12H17N3O2 C9H11N3O5S C10H11N3O4 C10H11N3O4

162–164 156–159 152–154 185–186 154–156 169–171 183–185 111–114 105–106 150–152 160–163 156–157 152–153 143–146 158–159

79.20 58.51 85.96 71.49 75.17 78.61 74.29 69.38 73.42 79.70 81.33 78.37 85.51 85.53 88.51

50 >100 >100 >100 >100 >100 >100 >100 >100 100 12.5 12.5 50 50 12.5

C8H11N3O3

151–153

60.58

>100

R 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

3-Cl-4-F 2-NO2 3-NO2 4-NO2 2,4-(NO2)2 3,5-(Cl)2 3,4-(Cl)2 2,5-(OCH3)2 2,4-(OCH3)2 4-N(C2H5)2 4-C4H9 4-C3H7 4-SO3H 2-COOH 4-COOH O

16

a

O

H2NHN

MIC INH: 0.02 lg/mL, RIF: 0.01 lg/mL.

N H

H2 C

O

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a b c d e

Mol. No.

Mol. wt

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

245 238 238 238 283 262 262 253 253 264 249 235 273 237 237 197

log Pa 0.10 1.21 1.09 1.09 1.54 0.66 0.54 0.70 0.70 0.29 0.94 0.35 2.61 1.18 1.06 1.64

log Sb [in log (moles/L)] 2.61 1.45 2.10 2.08 2.25 3.07 2.91 1.39 1.34 3.21 2.68 2.47 0.59 1.10 1.52 0.48

PSAc

HBAd

HBDe

72.08 104.46 105.46 105.46 137.84 72.08 72.08 86.56 86.56 74.82 72.08 72.08 114.49 99.79 100.49 81.15

3 5 5 5 7 3 3 5 5 3 3 3 6 5 5 4

4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 4

Solubility parameter. Calculated lipophilicity. Polar surface area (Å2). Number of hydrogen bond acceptor. Number of hydrogen bond donor.

Figure 2. Docking of active compounds (11, 12 and 15) into active site of PknB of Mycobacterium tuberculosis.

Drug likeness score 0.17 1.04 1.47 1.02 1.56 0.91 0.01 0.56 0.84 0.34 0.59 0.60 0.37 0.34 0.72 1.13

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After the achievement of synthesis and characterization of the compounds, we performed in silico studies. In order to expose the specificity of the Protein Kinase PknB from Mycobacterium tuberculosis towards the target compounds, docking approach was carried out. Docking was used to predict the binding orientation of drug to their protein target in order to in-turn predict the affinity & activity of drug which includes docking of ligand to a set of grids describing the target protein. The target compounds were docked into the nucleotide-binding pocket of the M. tuberculosis PknB structure (PDB: 2FUM). A Lamarckian genetic algorithm method, implemented in the program AutoDock 4.0, was employed. The docking of the ligand molecules (1–16) reveals that all the inhibitor compounds are exhibiting the bonding with one or the other amino acids in the active pockets. Theoretically all the sixteen molecules showed very good binding energy ranging from 6.24 kcal/mol to 7.48 kcal/mol (Table 2). Compound nos. 11 and 12 had shown minimum binding energies of 6.24 and 6.88 kcal/mol with inhibition constant 26.51 and 9.04, respectively. The C log P value of compound 11 is 1.55 and that of compound 12 is 1.02. The compound 15 had shown minimum binding energy of 7.48 kcal/mol with inhibition constant 3.86 and C log P value of 0.36. The drug candidates had shown favorable drug-like properties and follow the Lipinski’s rule of 5 with additional parameters predicted by Molsoft (Table 3). Thus, they are potentially interesting for further optimization. Their drug likeness score ranges from 1.56 to 0.72. Out of them the drug likeness score of 11 and 12 is 0.59 and 0.60, respectively, with almost same PSA value of 72.08, 3 hydrogen bond acceptors and 4 hydrogen bond donors. The drug likeness score of compound no. 15 is 0.72 with approximate molecular weight of 237, log P value of 1.06, log S value of 1.52, PSA value of 100.49, 5 hydrogen bond acceptors and 5 hydrogen bond donors. With in silico results in hand, it was thought worth-while to do in vitro studies to support the in silico studies. The anti-mycobacterial activity of the compounds (1–16) was tested and the minimum inhibitory concentration (MIC) was determined using REMA (resazurin microtitre assay) method.25 Among the tested compounds, N-(4-butylphenyl)-3-hydrazinyl-3-oxopropanamide(11), 3-Hydrazinyl-3-oxo-N-(4-propylphenyl)propanamide(12) and 4-(3-hydrazinyl-3-oxo-propanamido)benzoic acid (15) have shown good activity, N-(3-chloro-4-fluorophenyl)-3-hydrazinyl-3-oxopropanamide (1), 4-(3-Hydrazinyl-3-oxopropanamido) benzene sulfonic acid (13) and 2-(3-Hydrazinyl-3-oxopropanamido)benzoic acid (14) have shown moderate activity while rest of the compounds have MIC P100 lg/ml (Table 1). Compound no. 11 and 12 are structurally very similar and so their drug likeness scores, that is, 0.59 and 0.60, respectively with almost same PSA value, hydrogen bond acceptors and donors. The compound bearing butyl moiety has shown minimum binding energy of 6.28 kcal/mol whereas the minimum binding energy is 6.88 kcal/mol in case of isopropyl chain. Thus substitutions with alkyl chain (butyl and iso propyl) resulted in the increased antimycobacterial activity. Presence of a –COOH at para position of the aromatic ring also increased its activity both in silico and in vitro. Binding poses of compound nos. 11, 12 and 15 are shown in Figure 2. In summary, a series of substituted 3-hydrazinyl-3-oxo-propanamides were designed and synthesized. Their inhibitory effects on PknB of Mycobacterium tuberculosis were evaluated using in silico followed by in vitro studies. Molecular docking studies has

revealed that compounds 11, 12 and 15 have minimum binding energies with good drug likeness scores and may be considered as a good inhibitors of PknB. Thus compounds 11, 12 and 15 have emerged as most active amongst all the tested compounds against Mycobacterium tuberculosis H37Rv. Acknowledgments Authors are thankful to staff members of SAIF, CDRI for the spectral data, Council of Scientific and Industrial Research, University Grant Commission, Ministry of Health and Family Welfare, Government of India, New Delhi, for providing financial assistance. We are thankful to Jalma Institute of leprosy and other mycobacterial diseases, Agra, India for anti-tubercular activity and BioDiscovery-Solutions for future for docking studies. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.09.080. References and notes 1. Chhabria, M.; Patel, S.; Jani, M. Anti-Infect. Agents in Med. Chem. (Formerly ‘Curr. Med. Chem.—Anti-Infect. Agents) 2010, 9, 59. 2. WHO Global Tuberculosis Control Report, 2012. 3. Ballell, L.; Field, R. A.; Duncan, K.; Young, R. J. Antimicrob. Agents Chemother. 2005, 49, 2153. 4. Goldman, R. C.; Plumley, K. V.; Laughon, B. E. Infect. Disord. Drug Targets 2007, 7, 73. 5. Fox, H. H. J. Org. Chem. 1952, 17, 542. 6. Yale, H. L.; Losee, K.; Martins, J.; Holsing, M.; Perry, F. M.; Bernstein, J. J. Am. Chem. Soc. 1933, 1953, 75. 7. Dutta, M. M.; Goswami, B. N.; Kataky, J. C. S. J. Heterocycl. Chem. 1986, 23, 793. 8. Bernstein, J.; Lott, W. A.; Steinberg, B. A.; Yale, H. L. Am .Rev. Tuberculosis 1952, 65, 357. 9. Bernstein, J.; Jambor, W. P.; Lott, W. A.; Pansy, F.; Steinberg, B. A.; Yale, H. L. Am. Rev. Tuberculosis 1953, 67, 354. 10. Bernstein, J.; Jambor, W. P.; Lott, W. A.; Pansy, F.; Steinberg, B. A.; Yale, H. L. Am. Rev. Tuberculosis 1953, 67, 366. 11. Troeberg, L.; Chen, X.; Flaherty, T. M.; Morty, R. E.; Cheng, M.; Hua, H.; Springer, C.; McKerrow, J. H.; Kenyon, G. L.; Lonsdale-Eccles, J. D.; Coetzer, T. H.; Cohen, F. E. Mol. Med. 2000, 6, 660. 12. Erman, P. H.; Straub, H. U.S. Patent 5,318,963, 1994; Chem. Abstr. 1995, 122, 55819s. 13. Markham, P. N.; Klyachko, E. A.; Crich, D.; Jaber, M. R.; Johnson, M. E.; Mulhearn, D. C.; Neyfakh, A. A. PCT Int. Appl. WO 01 70, 213, 2001; Chem. Abstr. 2001, 135, 251941h. 14. Sengupta, A. K.; Bhatnagar, A.; Khan, S. K. J. Indian Chem. Soc. 1987, 64, 616. 15. Opie, T. R. Eur. Pat. Appl. EP 984,009, 2000; Chem. Abstr. 2000, 132, 194382p. 16. Mansour, A. K.; Eid, M. M.; Khalil, N. S. A. M. Molecules 2003, 8, 744. 17. Broadhurst, M. J.; Johnson, W. H.; Walter, D. S. PTC Int. Appl. WO 00 35,885, 2000; Chem. Abstr. 2000, 133, 58802u. 18. Sassetti, C. M.; Boyd, D. H.; Rubin, E. J. Mol. Microbiol. 2003, 48, 77. 19. Boitel, B.; Ortiz-Lombardia, M.; Duran, R.; Pompeo, F.; Cole, S. T.; Cervenansky, C.; Alzari, P. M. Mol. Microbiol. 2003, 49, 1493. 20. Villarino, A.; Duran, R.; Wehenkel, A.; Fernandez, P.; England, P.; Brodin, P.; Cole, S. T.; Zimny-Arndt, U.; Jungblut, P. R.; Cervenansky, C.; Alzari, P. M. J. Mol. Biol. 2005, 350, 953. 21. Kang, C. M.; Abbott, D. W.; Park, S. T.; Dascher, C. C.; Cantley, L. C.; Husson, R. N. Genes Dev. 2005, 19, 1692. 22. Lipinski, C. A. J. Pharmacol. Toxicol. Methods 2000, 44, 235. 23. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Deliv. Rev. 2001, 46, 3. 24. Vistoli, G.; Pedretti, A.; Testa, B. Drug Discovery Today 2008, 13, 285. 25. Jadaun, G. P.; Agarwal, C.; Sharma, H.; Ahmed, Z.; Upadhyay, P.; Faujdar, J.; Gupta, A. K.; Das, R.; Gupta, P.; Chauhan, D. S.; Sharma, V. D.; Katoch, V. M. J. Antimicrob. Chemother. 2007, 60, 152.