- Email: [email protected]
Identification of a novel class of quinoline–oxadiazole hybrids as anti-tuberculosis agents
Accepted Manuscript Identification of a novel class of Quinoline-Oxadiazole hybrids as anti-tuberculosis agents Puneet P. Jain, Mariam S. Degani, Archana Raju, Aarti Anantram, Madhav Seervi, Sadhana Sathaye, Muktikanta Ray, M.G.R. Rajan PII: DOI: Reference:
S0960-894X(15)30265-1 http://dx.doi.org/10.1016/j.bmcl.2015.11.057 BMCL 23313
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
21 May 2015 29 October 2015 17 November 2015
Please cite this article as: Jain, P.P., Degani, M.S., Raju, A., Anantram, A., Seervi, M., Sathaye, S., Ray, M., Rajan, M.G.R., Identification of a novel class of Quinoline-Oxadiazole hybrids as anti-tuberculosis agents, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.11.057
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Identification of a novel class of Quinoline-Oxadiazole hybrids as antituberculosis agents Puneet P. Jaina, Mariam S. Degania,*, Archana Rajua, Aarti Anantrama, Madhav Seervia, Sadhana Sathayea, Muktikanta Rayb, M. G. R. Rajanb a
Institute of Chemical Technology, Nathalal Parekh Marg, Matunga, Mumbai 400019, India
b
Radiation Medicine Centre, Tata Memorial Hospital, Parel, Mumbai 400012, India
*Corresponding author: Prof. (Mrs.) Mariam S. Degani, Tel.: +91-22-3361-2213; Fax: +91-22-3361-1020; E-mail: [email protected]
Abstract: A series of novel quinoline-oxadiazole hybrid compounds was designed based on stepwise rational modification of the lead molecules reported previously, in order to enhance bioactivity and improve druglikeness. The hybrid compounds synthesized were screened for biological activity against Mycobacterium tuberculosis H37Rv and for cytotoxicity in HepG2 cell line. Several of the hits exhibited good to excellent anti-tuberculosis activity and selectivity, especially compounds 12m, 12o and 12p, showed minimum inhibitory concentration values < 0.5 µM and selectivity index >500. The results of this study open up a promising avenue that may lead to the discovery of a new class of anti-tuberculosis agents.
Keywords: Mycobacterium tuberculosis; Isosterism; Oxadiazole; Quinoline; Bedaquiline.
1
Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (Mtb), is estimated to cause latent infection in 1/3rd of the world’s population, with 5-10% lifetime risk of reactivation. It is one of the leading causes of mortality, especially in low- and middleincome countries, with approximately 1.5 million deaths and 9 million new TB cases reported in 2013. Moreover, every fourth death among HIV positive cases is a consequence of TB. Even though a sustained decline in the TB death rate has been observed since 1990, there is an alarming increase in the number of multidrug-resistant tuberculosis (MDR-TB) cases since 2009. An estimated 480,000 new MDR-TB cases were recorded in 2013, covering virtually every single country surveyed in the world. Such forms of TB do not respond to the standard six monthly treatment with first-line anti-TB drugs and can take two years or more to treat with more toxic and less effective second-line drugs.1,2 This along with recent emergence of extensively drug resistant TB (XDR-TB) and totally drug resistant TB (TDR-TB), are raising serious questions about the effectiveness of the existing therapies in the near future.3 The challenge in controlling this serious and deadly form of TB is reinforcing the need for discovery and development of new anti-TB agents.4 During the last decade, persistent efforts in this direction have led to several lead molecules progressing to clinical trials. Such efforts were successful, when after 40 years, a new drug, bedaquiline (I), from the diarylquinoline class, with a novel mechanism of action, was approved by U.S. Food and Drug Administration (US-FDA) for treatment of MDR-TB.5 Bedaquiline demonstrates potent anti-TB activity with a minimum inhibitory concentration (MIC) of 0.1 μM. It is also active against variety of strains which are resistant to several of the first-line drugs.6 Because of this success, many research groups initiated active work on modifications of the quinoline scaffold, towards the design of promising anti-tuberculosis agents (Figure 1).7–10 In 2009, Kozikowski et al, rationally designed isoxazole-quinoline hybrid compounds using molecular hybridization of mefloquine and an isoxazole containing hit obtained from a high throughput screening (HTS). This highly active series of compounds exhibited potent activity against both active as well as latent forms of tubercle bacilli, with the most active compound, II, showing MIC of 0.2 μM. Although the molecules are reported to be metabolically unstable, it is no doubt an attractive lead scaffold for rational drug designing.8
2
Figure 1: Recently reported quinoline based potent anti-TB agents
In the present work, we report the design of novel Quinoline-Oxadiazole hybrid (QOH) compounds by step wise rational modification of the lead molecule, II, to obtain a series of 19 compounds with potent activity against Mtb H37Rv (Figure 2). Earlier reports indicate Oxadiazoles, due to their electronic properties, impart metabolic stability and enhanced bioavailability to the designed molecules.11 It is also known to improve the binding affinity and the bioactivity on replacement with isoxazole.12 Also, due to its higher developability score, it can be preferred over the isoxazole ring during drug design.13 Thus, to improve the druglikeness of the designed molecules, isooxazole ring of II was isosterically replaced by 1,2,4-oxadiazole ring to get intermediate design, IV. The metabolically unstable ethyl ester functionality was replaced with diverse aryl, heteroaryl and aliphatic substitution (R) to check the effect of electronic and hydrophobic parameters on the biological activity. Likewise, the quinoline scaffold is regarded as a privileged structure due to its frequent appearance in bioactive substances displaying diverse pharmacological activities including anti-TB activity.14 In addition, newly approved anti-TB drug, bedaquiline (I) and molecules from our earlier work (III) suggest that 2-methoxy-3-substituted quinolines tend to show promising anti-TB activity.10 Accordingly, the design IV, with substitution on 4-position of quinoline ring was shifted to 3-position of 2-methoxy quinoline giving the proposed QOH design (V).
3
Figure 2: Rational design of novel Quinoline-Oxadiazole hybrids
Prior to synthesis, an in silico ADME/Tox prediction was performed using QikProp15, a program that predicts several significant physical descriptors and relevant pharmaceutical properties of organic molecules, to check the druglikeness of the QOH compounds. The predicted properties calculated were partition coefficient, Lipinski’s rule of five, human oral absorption, CNS activity and gut-blood barrier permeability. All the QOH compounds passed the ADME/Tox analysis (supplementary information), indicating that the designed molecules have drug-like properties. In addition, an in silico site of metabolism (SoM) prediction of representative QOH compounds was performed using MetaPrint2D.16,17 The result suggests that QOH compounds must be metabolically more stable in comparison to II. The QOH compounds, 12a-12s were synthesized according to Scheme 1. The starting material, 2-chloro-3-formylquinoline (5) was synthesized by reacting acetanilide (4) with freshly prepared Vilsmeier-Haack reagent (3) under Meth-Cohn quinoline synthesis conditions.18 3 was prepared in-situ by reacting N,N-dimethylformamide (1) with excess of phosphorous oxychloride (2). Methanolysis of 5, by refluxing in methanolic potassium hydroxide solution led to synthesis of 2-methoxy-3-formylquinoline (6) in good yield.19 Subsequently, 3-(2-methoxyquinolin-3-yl)acrylonitrile (7) was synthesized by Knoevenagel condensation of 6 and cyanoacetic acid in toluene, in the presence of catalytic amount of 4
ammonium acetate and pyridine as base.20,21 Treatment of 7 with hydroxylamine hydrochloride by refluxing in 90:10 MeOH/H2O and using sodium bicarbonate as the base resulted in amidoxime (8), one of the key intermediates.22 The other key intermediates, Nacylbenzotriazoles (10a-s) were synthesized by the reaction of corresponding acids (9a-s), 1H-benzotriazole and thionyl chloride in a one-pot fashion.23
Scheme 1: Synthetic route for the synthesis of Quinoline-Oxadiazole hybrids (12as).Reagents and Conditions: (a) Vilsmeier-Haack Reagent (3) in POCl3, 80°C, 16.5h; (b) MeOH, KOH, reflux, 3-4h; (c) Cyanoacetic acid, ammonium acetate, pyridine, toluene, Dean-Stark trap, reflux, 44h; (d) NH2OH, NaHCO3, 90:10 MeOH/H2O, reflux, 8-12h; (e) SOCl2, CH2Cl2, RT, 2h; (f) N-acylbenzotriazole (10a-s), EtOH, TEA, RT, 30min; (g) TEA, EtOH/n-BuOH, reflux, 8-12h. 5
QOHs (12a-s) were synthesized by refluxing corresponding 10 and 8 in either ethanol or butanol, employing triethylamine as the mild base. The reaction proceeds via carbamate intermediate (11), which was synthesized at room temperature. These carbamate intermediates (11a-s) were isolated. When subjected to refluxing alcohol in presence of triethylamine, these intermediates led to corresponding QOHs.24 Structures of all QOH compounds (12a-s) were characterized by FTIR, NMR and HRMS analysis. The analytical results were in agreement with the proposed structures. All 19 QOH compounds (12a-s) were screened for in vitro anti-TB activity against Mycobacterium tuberculosis H37Rv by employing the Resazurin Microplate Assay (REMA) with drug concentrations from 50 µg/mL to 0.1 µg/mL (Table 1).25,26 Isoniazid was used as the standard drug. The compounds show good to excellent activity, especially the aliphatic or alkyl linked aromatic derivatives (12l - 12p) with MIC 0.4 – 1.05 µM, showing in vitro bioactivity greater than isoniazid. The compounds (12a-s) were also screened for in vitro cytotoxicity evaluation against HepG2 cell line, employing sulforhodamine B (SRB) assay using drug concentration from 10 µg/mL to 80 µg/mL. The serial microdilution technique was optimized for the toxicity screening of compounds to determine 50% cytostatic concentration (CC50).27 Adriamycin was used as the standard drug. All the compounds (12a-s) showed CC50 values >200 µM. Compound 12m displayed the highest SI of >610, indicating that this compound is very selective in its action against Mtb, exhibiting substantially low cytotoxicity towards human cells. Table 1: Biological activity of QOH compounds
Mol. ID
R
MIC (µM)a (n = 3)
CC50 (µM)b (n = 3)
SI
12a
phenyl
12.3
>240
>19.5
12b
4-methylphenyl
40.0
>230
>5.75
12c
4-methoxyphenyl
>140
>220
>1.57
12d
4-bromophenyl
9.19
>195
>21.2
6
12e
4-fluorophenyl
5.61
>230
>41.0
12f
2-methylphenyl
72.8
>230
>3.16
12g
2-methoxyphenyl
6.96
>220
>31.6
12h
2-bromophenyl
16.8
>195
>11.6
12i
3-methylphenyl
110
>230
>2.09
12j
3-methoxyphenyl
105
>220
>2.10
12k
3-chlorophenyl
>110
>220
>2.0
12l
methyl
1.05
>300
>285
12m
methoxymethyl
0.44
>270
>610
12n
phenoxymethyl
0.67
>220
>330
12o
benzyl
0.44
>230
>520
12p
4-methoxybenzyl
0.40
>215
>535
12q
(E) styryl
6.33
>225
>35.5
12r
3-pyridyl
>150
>240
>1.60
12s
2-furo
27.4
>250
>9.12
INH
-
0.73
-
-
ADR
-
-
127
-
* n = number of repetitions; INH = Isoniazid; ADR = Adriamycin a
MIC was determined with a drug concentration from 50µg/mL to 0.1µg/mL.
b
CC50 values is the concentration required to reduce the cell viability by 50%, also termed as
50% cytostatic concentration. CC50 were not evaluated for concentrations >80µg/mL due to low solubility of QOH compounds in DMSO at higher concentrations.
A representative QOH derivative, 12m, with the highest SI was subjected to metabolic stability study in rat liver microsomes.28–30 The disappearance of test compound was monitored over a period of 60 minutes. Diazepam was used an internal standard (IS). Drug to IS ratio were calculated to assess the metabolic stability of 12m at different time intervals. The resultant depletion profile (Figure 3) suggests that the compound 12m is 99% metabolically stable up to a period of 60 minutes. Thus, in accordance with the results of the 7
in silico SOM prediction, the microsomal stability study shows that the QOH derivatives are metabolically more stable in comparison to II.
Figure 3: Depletion profile of compound 12m in rat liver microsomes.
The biological results of QOH derivatives aided us to hypothesize a structure activity relationship (SAR). It was interesting to note that the highly active and selective aliphatic or alkyl linked aromatic derivatives (12l – 12p), shared a common methylene spacer at 5’position of 1,2,4-oxadiazole ring that differentiates them from the lesser active molecules. Also, the molecule with highest selectivity, 12m showed a great degree of side chain structural similarity with the lead molecule II (Figure 4), demonstrating that such similarity improves the activity. The high to moderate activity and selectivity of ligands with alkyl, alkoxy or alkene linked aromatic ring (12n - 12q), supports the previous statement and suggests that a 1 to 2 atom flexible spacer in 5’-side chain improves the activity. Amongst aromatic derivatives, halo substitution at p- and o- position (12d, 12e and 12h) seemed to contribute significantly to the activity. Replacement of aromatic substitution with a heteroaromatic ring (12r and 12s) decreased the activity.
8
Figure 4: Structure Activity Relationship (SAR) of QOH molecules
In conclusion, the present work reflects the successful design and synthesis of a novel series of QOH compounds as potential anti-tuberculosis agents via step by step rational modification of the lead molecule (II) reported previously. In vitro biological results reveal that several of the QOH compounds exhibit significant anti-TB activity and selectivity. Amongst the 19 QOH compounds, the hit molecule 12m exhibits the highest activity with MIC of 0.4µM and SI of >610, which is comparable to the lead molecule (II). The SAR results suggest that a 1 to 2 atom flexible spacer in the side chain at 5’-position and a side chain structural similarity with the lead molecule (II) is crucial for enhancement of activity. These results suggest that QOH scaffold may serve as a promising anti-TB hit/lead for further drug optimization. Further investigations on this series are ongoing in our laboratory with a view to design a potential clinical candidate.
Acknowledgements: Puneet Jain and Aarti Anantram are thankful to University Grants Commission, India and Archana Raju is thankful to Indian Council of Medical Research, India for financial support.
References: 1.
WHO Global tuberculosis report 2014 (WHO/HTM/TB/2014.08); World Health Organization: Geneva, Switzerland, 2014.
2.
WHO Multidrug and extensively drug-resistant TB (M/XDR-TB); global report on surveillance and response; World Health Organization: Geneva, Switzerland, 2010.
3.
Udwadia, Z. F.; Amale, R. A.; Ajbani, K. K.; Rodrigues, C. Clin. Infect. Dis. 2012, 54, 9
579–81. 4.
Koul, A.; Arnoult, E.; Lounis, N.; Guillemont, J.; Andries, K. Nature 2011, 469, 483– 90.
5.
Mahajan, R. Int. J. Appl. basic Med. Res. 2013, 3, 1–2.
6.
Andries, K.; Verhasselt, P.; Guillemont, J.; Göhlmann, H. W. H.; Neefs, J.-M.; Winkler, H.; Van Gestel, J.; Timmerman, P.; Zhu, M.; Lee, E.; Williams, P.; de Chaffoy, D.; Huitric, E.; Hoffner, S.; Cambau, E.; Truffot-Pernot, C.; Lounis, N.; Jarlier, V. A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis.; 2005; Vol. 307.
7.
Upadhayaya, R. S.; Lahore, S. V; Sayyed, A. Y.; Dixit, S. S.; Shinde, P. D.; Chattopadhyaya, J. Org. Biomol. Chem. 2010, 8, 2180–97.
8.
Mao, J.; Yuan, H.; Wang, Y.; Wan, B.; Pieroni, M.; Huang, Q.; van Breemen, R. B.; Kozikowski, A. P.; Franzblau, S. G. J. Med. Chem. 2009, 52, 6966–78.
9.
Thomas, K. D.; Adhikari, A. V.; Chowdhury, I. H.; Sandeep, T.; Mahmood, R.; Bhattacharya, B.; Sumesh, E. Eur. J. Med. Chem. 2011, 46, 4834–45.
10.
Jain, P. P.; Degani, M. S.; Raju, A.; Ray, M.; Rajan, M. G. R. Bioorg. Med. Chem. Lett. 2013, 23, 6097–105.
11.
Todres, Z. In Chalcogenadiazoles: chemistry and applications; CRC press: Boca Raton, 2011; p. 32.
12.
Patani, G. A.; LaVoie, E. J. Chem. Rev. 1996, 96, 3147–3176.
13.
Ritchie, T. J.; Macdonald, S. J. F.; Peace, S.; Pickett, S. D.; Luscombe, C. N. Medchemcomm 2012, 3, 1062.
14.
Welsch, M. E.; Snyder, S. a; Stockwell, B. R. Curr. Opin. Chem. Biol. 2010, 14, 347– 61.
15.
Schrodinger Release 2012: QikProp, Version 3.5; Schrodinger, LLC: New York, NY, 2012.
16.
Boyer, S.; Arnby, C. H.; Carlsson, L.; Smith, J.; Stein, V.; Glen, R. C. J. Chem. Inf. Model. 2007, 47, 583–90.
17.
http://www-metaprint2d.ch.cam.ac.uk/metaprint2d.
18.
Meth-Cohn, O.; Narine, B.; Tarnowski, B. J. Chem. Soc. Perkin Trans. 1 1981, 1520.
19.
Kuethe, J. T.; Wong, A.; Qu, C.; Smitrovich, J.; Davies, I. W.; Hughes, D. L. J. Org. Chem. 2005, 70, 2555–67.
20.
Montgomery, J. a; Niwas, S.; Rose, J. D.; Secrist, J. a; Babu, Y. S.; Bugg, C. E.; Erion, M. D.; Guida, W. C.; Ealick, S. E. J. Med. Chem. 1993, 36, 55–69.
10
21.
Stokker, G. E.; Hoffman, W. F.; Alberts, A. W.; Cragoe, E. J.; Deana, A. A.; Gilfillan, J. L.; Huff, J. W.; Novello, F. C.; Prugh, J. D.; Smith, R. L. J. Med. Chem. 1985, 28, 347–58.
22.
Kaboudin, B.; Navaee, K. Heterocycles 2003, 60, 2287.
23.
Katritzky, A. R.; Zhang, Y.; Singh, S. K. Synthesis (Stuttg). 2003, 6, 2795–2798.
24.
Katritzky, A.; Shestopalov, A.; Suzuki, K. Arkivoc 2005, 2005, 36–55.
25.
Palomino, J.-C.; Martin, a.; Camacho, M.; Guerra, H.; Swings, J.; Portaels, F. Antimicrob. Agents Chemother. 2002, 46, 2720–2722.
26.
Martin, a.; Camacho, M.; Portaels, F.; Palomino, J. C. Antimicrob. Agents Chemother. 2003, 47, 3616–3619.
27.
Vichai, V.; Kirtikara, K. Nat. Protoc. 2006, 1, 1112–6.
28.
Iyer, K.; Walawalkar, P.; Serai, P. Indian J. Pharm. Sci. 2006, 68, 262.
29.
Masimirembwa, C. M.; Bredberg, U.; Andersson, T. B. Clin. Pharmacokinet. 2003, 42, 515–528.
30.
Riley, R. J.; Grime, K. Drug Discov. Today. Technol. 2004, 1, 365–72.
List of Figures and Tables
Figure 1: Recently reported quinoline based potent anti-TB agents Figure 2: Rational design of novel Quinoline-Oxadiazole hybrids Figure 3: Rat liver microsomal stability study of compound 1 Figure 4: Structure Activity Relationship (SAR) of QOH molecules Scheme 1: Synthetic route for the synthesis of Quinoline-Oxadiazole hybrids (12a-s). Table 1: Biological activity of QOH compounds
11
Figures:
Figure 1: Recently reported quinoline based potent anti-TB agents
Figure 2: Rational design of novel Quinoline-Oxadiazole hybrids
12
Figure 3: Depletion profile of compound 12m in rat liver microsomes.
Figure 4: Structure Activity Relationship (SAR) of QOH molecules
13
Scheme 1: Synthetic route for the synthesis of Quinoline-Oxadiazole hybrids (12as).Reagents and Conditions: (a) Vilsmeier-Haack Reagent (3) in POCl3, 80°C, 16.5h; (b) MeOH, KOH, reflux, 3-4h; (c) Cyanoacetic acid, ammonium acetate, pyridine, toluene, Dean-Stark trap, reflux, 44h; (d) NH2OH, NaHCO3, 90:10 MeOH/H2O, reflux, 8-12h; (e) SOCl2, CH2Cl2, RT, 2h; (f) N-acylbenzotriazole (10a-s), EtOH, TEA, RT, 30min; (g) TEA, EtOH/n-BuOH, reflux, 8-12h.
14
Table 1: Biological activity of QOH compounds
Mol. ID
R
MIC (µM)a (n = 3)
CC50 (µM)b (n = 3)
SI
12a
phenyl
12.3
>240
>19.5
12b
4-methylphenyl
40.0
>230
>5.75
12c
4-methoxyphenyl
>140
>220
>1.57
12d
4-bromophenyl
9.19
>195
>21.2
12e
4-fluorophenyl
5.61
>230
>41.0
12f
2-methylphenyl
72.8
>230
>3.16
12g
2-methoxyphenyl
6.96
>220
>31.6
12h
2-bromophenyl
16.8
>195
>11.6
12i
3-methylphenyl
110
>230
>2.09
12j
3-methoxyphenyl
105
>220
>2.10
12k
3-chlorophenyl
>110
>220
>2.0
12l
methyl
1.05
>300
>285
12m
methoxymethyl
0.44
>270
>610
12n
phenoxymethyl
0.67
>220
>330
12o
benzyl
0.44
>230
>520
12p
4-methoxybenzyl
0.40
>215
>535
12q
(E) styryl
6.33
>225
>35.5
12r
3-pyridyl
>150
>240
>1.60
12s
2-furo
27.4
>250
>9.12
INH
-
0.73
-
-
ADR
-
-
127
-
* n = number of repetitions; INH = Isoniazid; ADR = Adriamycin a
MIC was determined with a drug concentration from 50µg/mL to 0.1µg/mL. 15
b
CC50 values is the concentration required to reduce the cell viability by 50%, also termed as
50% cytostatic concentration. CC50 were not evaluated for concentrations >80µg/mL due to low solubility of QOH compounds in DMSO at higher concentrations.
16
Graphical abstract
17
S0960-894X(15)30265-1 http://dx.doi.org/10.1016/j.bmcl.2015.11.057 BMCL 23313
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
21 May 2015 29 October 2015 17 November 2015
Please cite this article as: Jain, P.P., Degani, M.S., Raju, A., Anantram, A., Seervi, M., Sathaye, S., Ray, M., Rajan, M.G.R., Identification of a novel class of Quinoline-Oxadiazole hybrids as anti-tuberculosis agents, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.11.057
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Identification of a novel class of Quinoline-Oxadiazole hybrids as antituberculosis agents Puneet P. Jaina, Mariam S. Degania,*, Archana Rajua, Aarti Anantrama, Madhav Seervia, Sadhana Sathayea, Muktikanta Rayb, M. G. R. Rajanb a
Institute of Chemical Technology, Nathalal Parekh Marg, Matunga, Mumbai 400019, India
b
Radiation Medicine Centre, Tata Memorial Hospital, Parel, Mumbai 400012, India
*Corresponding author: Prof. (Mrs.) Mariam S. Degani, Tel.: +91-22-3361-2213; Fax: +91-22-3361-1020; E-mail: [email protected]
Abstract: A series of novel quinoline-oxadiazole hybrid compounds was designed based on stepwise rational modification of the lead molecules reported previously, in order to enhance bioactivity and improve druglikeness. The hybrid compounds synthesized were screened for biological activity against Mycobacterium tuberculosis H37Rv and for cytotoxicity in HepG2 cell line. Several of the hits exhibited good to excellent anti-tuberculosis activity and selectivity, especially compounds 12m, 12o and 12p, showed minimum inhibitory concentration values < 0.5 µM and selectivity index >500. The results of this study open up a promising avenue that may lead to the discovery of a new class of anti-tuberculosis agents.
Keywords: Mycobacterium tuberculosis; Isosterism; Oxadiazole; Quinoline; Bedaquiline.
1
Tuberculosis (TB), a disease caused by Mycobacterium tuberculosis (Mtb), is estimated to cause latent infection in 1/3rd of the world’s population, with 5-10% lifetime risk of reactivation. It is one of the leading causes of mortality, especially in low- and middleincome countries, with approximately 1.5 million deaths and 9 million new TB cases reported in 2013. Moreover, every fourth death among HIV positive cases is a consequence of TB. Even though a sustained decline in the TB death rate has been observed since 1990, there is an alarming increase in the number of multidrug-resistant tuberculosis (MDR-TB) cases since 2009. An estimated 480,000 new MDR-TB cases were recorded in 2013, covering virtually every single country surveyed in the world. Such forms of TB do not respond to the standard six monthly treatment with first-line anti-TB drugs and can take two years or more to treat with more toxic and less effective second-line drugs.1,2 This along with recent emergence of extensively drug resistant TB (XDR-TB) and totally drug resistant TB (TDR-TB), are raising serious questions about the effectiveness of the existing therapies in the near future.3 The challenge in controlling this serious and deadly form of TB is reinforcing the need for discovery and development of new anti-TB agents.4 During the last decade, persistent efforts in this direction have led to several lead molecules progressing to clinical trials. Such efforts were successful, when after 40 years, a new drug, bedaquiline (I), from the diarylquinoline class, with a novel mechanism of action, was approved by U.S. Food and Drug Administration (US-FDA) for treatment of MDR-TB.5 Bedaquiline demonstrates potent anti-TB activity with a minimum inhibitory concentration (MIC) of 0.1 μM. It is also active against variety of strains which are resistant to several of the first-line drugs.6 Because of this success, many research groups initiated active work on modifications of the quinoline scaffold, towards the design of promising anti-tuberculosis agents (Figure 1).7–10 In 2009, Kozikowski et al, rationally designed isoxazole-quinoline hybrid compounds using molecular hybridization of mefloquine and an isoxazole containing hit obtained from a high throughput screening (HTS). This highly active series of compounds exhibited potent activity against both active as well as latent forms of tubercle bacilli, with the most active compound, II, showing MIC of 0.2 μM. Although the molecules are reported to be metabolically unstable, it is no doubt an attractive lead scaffold for rational drug designing.8
2
Figure 1: Recently reported quinoline based potent anti-TB agents
In the present work, we report the design of novel Quinoline-Oxadiazole hybrid (QOH) compounds by step wise rational modification of the lead molecule, II, to obtain a series of 19 compounds with potent activity against Mtb H37Rv (Figure 2). Earlier reports indicate Oxadiazoles, due to their electronic properties, impart metabolic stability and enhanced bioavailability to the designed molecules.11 It is also known to improve the binding affinity and the bioactivity on replacement with isoxazole.12 Also, due to its higher developability score, it can be preferred over the isoxazole ring during drug design.13 Thus, to improve the druglikeness of the designed molecules, isooxazole ring of II was isosterically replaced by 1,2,4-oxadiazole ring to get intermediate design, IV. The metabolically unstable ethyl ester functionality was replaced with diverse aryl, heteroaryl and aliphatic substitution (R) to check the effect of electronic and hydrophobic parameters on the biological activity. Likewise, the quinoline scaffold is regarded as a privileged structure due to its frequent appearance in bioactive substances displaying diverse pharmacological activities including anti-TB activity.14 In addition, newly approved anti-TB drug, bedaquiline (I) and molecules from our earlier work (III) suggest that 2-methoxy-3-substituted quinolines tend to show promising anti-TB activity.10 Accordingly, the design IV, with substitution on 4-position of quinoline ring was shifted to 3-position of 2-methoxy quinoline giving the proposed QOH design (V).
3
Figure 2: Rational design of novel Quinoline-Oxadiazole hybrids
Prior to synthesis, an in silico ADME/Tox prediction was performed using QikProp15, a program that predicts several significant physical descriptors and relevant pharmaceutical properties of organic molecules, to check the druglikeness of the QOH compounds. The predicted properties calculated were partition coefficient, Lipinski’s rule of five, human oral absorption, CNS activity and gut-blood barrier permeability. All the QOH compounds passed the ADME/Tox analysis (supplementary information), indicating that the designed molecules have drug-like properties. In addition, an in silico site of metabolism (SoM) prediction of representative QOH compounds was performed using MetaPrint2D.16,17 The result suggests that QOH compounds must be metabolically more stable in comparison to II. The QOH compounds, 12a-12s were synthesized according to Scheme 1. The starting material, 2-chloro-3-formylquinoline (5) was synthesized by reacting acetanilide (4) with freshly prepared Vilsmeier-Haack reagent (3) under Meth-Cohn quinoline synthesis conditions.18 3 was prepared in-situ by reacting N,N-dimethylformamide (1) with excess of phosphorous oxychloride (2). Methanolysis of 5, by refluxing in methanolic potassium hydroxide solution led to synthesis of 2-methoxy-3-formylquinoline (6) in good yield.19 Subsequently, 3-(2-methoxyquinolin-3-yl)acrylonitrile (7) was synthesized by Knoevenagel condensation of 6 and cyanoacetic acid in toluene, in the presence of catalytic amount of 4
ammonium acetate and pyridine as base.20,21 Treatment of 7 with hydroxylamine hydrochloride by refluxing in 90:10 MeOH/H2O and using sodium bicarbonate as the base resulted in amidoxime (8), one of the key intermediates.22 The other key intermediates, Nacylbenzotriazoles (10a-s) were synthesized by the reaction of corresponding acids (9a-s), 1H-benzotriazole and thionyl chloride in a one-pot fashion.23
Scheme 1: Synthetic route for the synthesis of Quinoline-Oxadiazole hybrids (12as).Reagents and Conditions: (a) Vilsmeier-Haack Reagent (3) in POCl3, 80°C, 16.5h; (b) MeOH, KOH, reflux, 3-4h; (c) Cyanoacetic acid, ammonium acetate, pyridine, toluene, Dean-Stark trap, reflux, 44h; (d) NH2OH, NaHCO3, 90:10 MeOH/H2O, reflux, 8-12h; (e) SOCl2, CH2Cl2, RT, 2h; (f) N-acylbenzotriazole (10a-s), EtOH, TEA, RT, 30min; (g) TEA, EtOH/n-BuOH, reflux, 8-12h. 5
QOHs (12a-s) were synthesized by refluxing corresponding 10 and 8 in either ethanol or butanol, employing triethylamine as the mild base. The reaction proceeds via carbamate intermediate (11), which was synthesized at room temperature. These carbamate intermediates (11a-s) were isolated. When subjected to refluxing alcohol in presence of triethylamine, these intermediates led to corresponding QOHs.24 Structures of all QOH compounds (12a-s) were characterized by FTIR, NMR and HRMS analysis. The analytical results were in agreement with the proposed structures. All 19 QOH compounds (12a-s) were screened for in vitro anti-TB activity against Mycobacterium tuberculosis H37Rv by employing the Resazurin Microplate Assay (REMA) with drug concentrations from 50 µg/mL to 0.1 µg/mL (Table 1).25,26 Isoniazid was used as the standard drug. The compounds show good to excellent activity, especially the aliphatic or alkyl linked aromatic derivatives (12l - 12p) with MIC 0.4 – 1.05 µM, showing in vitro bioactivity greater than isoniazid. The compounds (12a-s) were also screened for in vitro cytotoxicity evaluation against HepG2 cell line, employing sulforhodamine B (SRB) assay using drug concentration from 10 µg/mL to 80 µg/mL. The serial microdilution technique was optimized for the toxicity screening of compounds to determine 50% cytostatic concentration (CC50).27 Adriamycin was used as the standard drug. All the compounds (12a-s) showed CC50 values >200 µM. Compound 12m displayed the highest SI of >610, indicating that this compound is very selective in its action against Mtb, exhibiting substantially low cytotoxicity towards human cells. Table 1: Biological activity of QOH compounds
Mol. ID
R
MIC (µM)a (n = 3)
CC50 (µM)b (n = 3)
SI
12a
phenyl
12.3
>240
>19.5
12b
4-methylphenyl
40.0
>230
>5.75
12c
4-methoxyphenyl
>140
>220
>1.57
12d
4-bromophenyl
9.19
>195
>21.2
6
12e
4-fluorophenyl
5.61
>230
>41.0
12f
2-methylphenyl
72.8
>230
>3.16
12g
2-methoxyphenyl
6.96
>220
>31.6
12h
2-bromophenyl
16.8
>195
>11.6
12i
3-methylphenyl
110
>230
>2.09
12j
3-methoxyphenyl
105
>220
>2.10
12k
3-chlorophenyl
>110
>220
>2.0
12l
methyl
1.05
>300
>285
12m
methoxymethyl
0.44
>270
>610
12n
phenoxymethyl
0.67
>220
>330
12o
benzyl
0.44
>230
>520
12p
4-methoxybenzyl
0.40
>215
>535
12q
(E) styryl
6.33
>225
>35.5
12r
3-pyridyl
>150
>240
>1.60
12s
2-furo
27.4
>250
>9.12
INH
-
0.73
-
-
ADR
-
-
127
-
* n = number of repetitions; INH = Isoniazid; ADR = Adriamycin a
MIC was determined with a drug concentration from 50µg/mL to 0.1µg/mL.
b
CC50 values is the concentration required to reduce the cell viability by 50%, also termed as
50% cytostatic concentration. CC50 were not evaluated for concentrations >80µg/mL due to low solubility of QOH compounds in DMSO at higher concentrations.
A representative QOH derivative, 12m, with the highest SI was subjected to metabolic stability study in rat liver microsomes.28–30 The disappearance of test compound was monitored over a period of 60 minutes. Diazepam was used an internal standard (IS). Drug to IS ratio were calculated to assess the metabolic stability of 12m at different time intervals. The resultant depletion profile (Figure 3) suggests that the compound 12m is 99% metabolically stable up to a period of 60 minutes. Thus, in accordance with the results of the 7
in silico SOM prediction, the microsomal stability study shows that the QOH derivatives are metabolically more stable in comparison to II.
Figure 3: Depletion profile of compound 12m in rat liver microsomes.
The biological results of QOH derivatives aided us to hypothesize a structure activity relationship (SAR). It was interesting to note that the highly active and selective aliphatic or alkyl linked aromatic derivatives (12l – 12p), shared a common methylene spacer at 5’position of 1,2,4-oxadiazole ring that differentiates them from the lesser active molecules. Also, the molecule with highest selectivity, 12m showed a great degree of side chain structural similarity with the lead molecule II (Figure 4), demonstrating that such similarity improves the activity. The high to moderate activity and selectivity of ligands with alkyl, alkoxy or alkene linked aromatic ring (12n - 12q), supports the previous statement and suggests that a 1 to 2 atom flexible spacer in 5’-side chain improves the activity. Amongst aromatic derivatives, halo substitution at p- and o- position (12d, 12e and 12h) seemed to contribute significantly to the activity. Replacement of aromatic substitution with a heteroaromatic ring (12r and 12s) decreased the activity.
8
Figure 4: Structure Activity Relationship (SAR) of QOH molecules
In conclusion, the present work reflects the successful design and synthesis of a novel series of QOH compounds as potential anti-tuberculosis agents via step by step rational modification of the lead molecule (II) reported previously. In vitro biological results reveal that several of the QOH compounds exhibit significant anti-TB activity and selectivity. Amongst the 19 QOH compounds, the hit molecule 12m exhibits the highest activity with MIC of 0.4µM and SI of >610, which is comparable to the lead molecule (II). The SAR results suggest that a 1 to 2 atom flexible spacer in the side chain at 5’-position and a side chain structural similarity with the lead molecule (II) is crucial for enhancement of activity. These results suggest that QOH scaffold may serve as a promising anti-TB hit/lead for further drug optimization. Further investigations on this series are ongoing in our laboratory with a view to design a potential clinical candidate.
Acknowledgements: Puneet Jain and Aarti Anantram are thankful to University Grants Commission, India and Archana Raju is thankful to Indian Council of Medical Research, India for financial support.
References: 1.
WHO Global tuberculosis report 2014 (WHO/HTM/TB/2014.08); World Health Organization: Geneva, Switzerland, 2014.
2.
WHO Multidrug and extensively drug-resistant TB (M/XDR-TB); global report on surveillance and response; World Health Organization: Geneva, Switzerland, 2010.
3.
Udwadia, Z. F.; Amale, R. A.; Ajbani, K. K.; Rodrigues, C. Clin. Infect. Dis. 2012, 54, 9
579–81. 4.
Koul, A.; Arnoult, E.; Lounis, N.; Guillemont, J.; Andries, K. Nature 2011, 469, 483– 90.
5.
Mahajan, R. Int. J. Appl. basic Med. Res. 2013, 3, 1–2.
6.
Andries, K.; Verhasselt, P.; Guillemont, J.; Göhlmann, H. W. H.; Neefs, J.-M.; Winkler, H.; Van Gestel, J.; Timmerman, P.; Zhu, M.; Lee, E.; Williams, P.; de Chaffoy, D.; Huitric, E.; Hoffner, S.; Cambau, E.; Truffot-Pernot, C.; Lounis, N.; Jarlier, V. A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis.; 2005; Vol. 307.
7.
Upadhayaya, R. S.; Lahore, S. V; Sayyed, A. Y.; Dixit, S. S.; Shinde, P. D.; Chattopadhyaya, J. Org. Biomol. Chem. 2010, 8, 2180–97.
8.
Mao, J.; Yuan, H.; Wang, Y.; Wan, B.; Pieroni, M.; Huang, Q.; van Breemen, R. B.; Kozikowski, A. P.; Franzblau, S. G. J. Med. Chem. 2009, 52, 6966–78.
9.
Thomas, K. D.; Adhikari, A. V.; Chowdhury, I. H.; Sandeep, T.; Mahmood, R.; Bhattacharya, B.; Sumesh, E. Eur. J. Med. Chem. 2011, 46, 4834–45.
10.
Jain, P. P.; Degani, M. S.; Raju, A.; Ray, M.; Rajan, M. G. R. Bioorg. Med. Chem. Lett. 2013, 23, 6097–105.
11.
Todres, Z. In Chalcogenadiazoles: chemistry and applications; CRC press: Boca Raton, 2011; p. 32.
12.
Patani, G. A.; LaVoie, E. J. Chem. Rev. 1996, 96, 3147–3176.
13.
Ritchie, T. J.; Macdonald, S. J. F.; Peace, S.; Pickett, S. D.; Luscombe, C. N. Medchemcomm 2012, 3, 1062.
14.
Welsch, M. E.; Snyder, S. a; Stockwell, B. R. Curr. Opin. Chem. Biol. 2010, 14, 347– 61.
15.
Schrodinger Release 2012: QikProp, Version 3.5; Schrodinger, LLC: New York, NY, 2012.
16.
Boyer, S.; Arnby, C. H.; Carlsson, L.; Smith, J.; Stein, V.; Glen, R. C. J. Chem. Inf. Model. 2007, 47, 583–90.
17.
http://www-metaprint2d.ch.cam.ac.uk/metaprint2d.
18.
Meth-Cohn, O.; Narine, B.; Tarnowski, B. J. Chem. Soc. Perkin Trans. 1 1981, 1520.
19.
Kuethe, J. T.; Wong, A.; Qu, C.; Smitrovich, J.; Davies, I. W.; Hughes, D. L. J. Org. Chem. 2005, 70, 2555–67.
20.
Montgomery, J. a; Niwas, S.; Rose, J. D.; Secrist, J. a; Babu, Y. S.; Bugg, C. E.; Erion, M. D.; Guida, W. C.; Ealick, S. E. J. Med. Chem. 1993, 36, 55–69.
10
21.
Stokker, G. E.; Hoffman, W. F.; Alberts, A. W.; Cragoe, E. J.; Deana, A. A.; Gilfillan, J. L.; Huff, J. W.; Novello, F. C.; Prugh, J. D.; Smith, R. L. J. Med. Chem. 1985, 28, 347–58.
22.
Kaboudin, B.; Navaee, K. Heterocycles 2003, 60, 2287.
23.
Katritzky, A. R.; Zhang, Y.; Singh, S. K. Synthesis (Stuttg). 2003, 6, 2795–2798.
24.
Katritzky, A.; Shestopalov, A.; Suzuki, K. Arkivoc 2005, 2005, 36–55.
25.
Palomino, J.-C.; Martin, a.; Camacho, M.; Guerra, H.; Swings, J.; Portaels, F. Antimicrob. Agents Chemother. 2002, 46, 2720–2722.
26.
Martin, a.; Camacho, M.; Portaels, F.; Palomino, J. C. Antimicrob. Agents Chemother. 2003, 47, 3616–3619.
27.
Vichai, V.; Kirtikara, K. Nat. Protoc. 2006, 1, 1112–6.
28.
Iyer, K.; Walawalkar, P.; Serai, P. Indian J. Pharm. Sci. 2006, 68, 262.
29.
Masimirembwa, C. M.; Bredberg, U.; Andersson, T. B. Clin. Pharmacokinet. 2003, 42, 515–528.
30.
Riley, R. J.; Grime, K. Drug Discov. Today. Technol. 2004, 1, 365–72.
List of Figures and Tables
Figure 1: Recently reported quinoline based potent anti-TB agents Figure 2: Rational design of novel Quinoline-Oxadiazole hybrids Figure 3: Rat liver microsomal stability study of compound 1 Figure 4: Structure Activity Relationship (SAR) of QOH molecules Scheme 1: Synthetic route for the synthesis of Quinoline-Oxadiazole hybrids (12a-s). Table 1: Biological activity of QOH compounds
11
Figures:
Figure 1: Recently reported quinoline based potent anti-TB agents
Figure 2: Rational design of novel Quinoline-Oxadiazole hybrids
12
Figure 3: Depletion profile of compound 12m in rat liver microsomes.
Figure 4: Structure Activity Relationship (SAR) of QOH molecules
13
Scheme 1: Synthetic route for the synthesis of Quinoline-Oxadiazole hybrids (12as).Reagents and Conditions: (a) Vilsmeier-Haack Reagent (3) in POCl3, 80°C, 16.5h; (b) MeOH, KOH, reflux, 3-4h; (c) Cyanoacetic acid, ammonium acetate, pyridine, toluene, Dean-Stark trap, reflux, 44h; (d) NH2OH, NaHCO3, 90:10 MeOH/H2O, reflux, 8-12h; (e) SOCl2, CH2Cl2, RT, 2h; (f) N-acylbenzotriazole (10a-s), EtOH, TEA, RT, 30min; (g) TEA, EtOH/n-BuOH, reflux, 8-12h.
14
Table 1: Biological activity of QOH compounds
Mol. ID
R
MIC (µM)a (n = 3)
CC50 (µM)b (n = 3)
SI
12a
phenyl
12.3
>240
>19.5
12b
4-methylphenyl
40.0
>230
>5.75
12c
4-methoxyphenyl
>140
>220
>1.57
12d
4-bromophenyl
9.19
>195
>21.2
12e
4-fluorophenyl
5.61
>230
>41.0
12f
2-methylphenyl
72.8
>230
>3.16
12g
2-methoxyphenyl
6.96
>220
>31.6
12h
2-bromophenyl
16.8
>195
>11.6
12i
3-methylphenyl
110
>230
>2.09
12j
3-methoxyphenyl
105
>220
>2.10
12k
3-chlorophenyl
>110
>220
>2.0
12l
methyl
1.05
>300
>285
12m
methoxymethyl
0.44
>270
>610
12n
phenoxymethyl
0.67
>220
>330
12o
benzyl
0.44
>230
>520
12p
4-methoxybenzyl
0.40
>215
>535
12q
(E) styryl
6.33
>225
>35.5
12r
3-pyridyl
>150
>240
>1.60
12s
2-furo
27.4
>250
>9.12
INH
-
0.73
-
-
ADR
-
-
127
-
* n = number of repetitions; INH = Isoniazid; ADR = Adriamycin a
MIC was determined with a drug concentration from 50µg/mL to 0.1µg/mL. 15
b
CC50 values is the concentration required to reduce the cell viability by 50%, also termed as
50% cytostatic concentration. CC50 were not evaluated for concentrations >80µg/mL due to low solubility of QOH compounds in DMSO at higher concentrations.
16
Graphical abstract
17