Accepted Manuscript 2’-hydroxy flavanone derivatives as inhibitors of pro-inflammatory mediators: Experimental and molecular docking studies Neeraj K. Patel, Khemraj Bairwa, Rahul Gangwal, Gaurav Jaiswal, Sanjay M. Jachak, Abhay T. Sangamwar, Kamlesh K. Bhutani PII: DOI: Reference:

S0960-894X(15)00221-8 http://dx.doi.org/10.1016/j.bmcl.2015.03.025 BMCL 22513

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

2 January 2015 7 March 2015 10 March 2015

Please cite this article as: Patel, N.K., Bairwa, K., Gangwal, R., Jaiswal, G., Jachak, S.M., Sangamwar, A.T., Bhutani, K.K., 2’-hydroxy flavanone derivatives as inhibitors of pro-inflammatory mediators: Experimental and molecular docking studies, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl. 2015.03.025

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2'-hydroxy flavanone derivatives as inhibitors of pro-inflammatory mediators: Experimental and molecular docking studies

Neeraj K. Patela, Khemraj Bairwaa, Rahul Gangwalb, Gaurav Jaiswala, Sanjay M. Jachaka, Abhay T. Sangamwarb, Kamlesh K. Bhutania#

a

Department of Natural Products,

National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Mohali, Punjab - 160 062, INDIA b

Department of Pharmacoinformatics,

National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Mohali, Punjab - 160 062, INDIA

#

Corresponding Author:

K. K. Bhutani Department of Natural Products, National Institute of Pharmaceutical and Education Research (NIPER), Sector 67, S.A.S. Nagar, Mohali, Punjab - 160 062, INDIA E-mail address: [email protected], [email protected]

Abstract 2'-hydroxy flavanone (1) was previously isolated from Mimosa pudica whole plant and was found to exhibit anti-inflammatory effects in vitro. There are also reports on anti-inflammatory properties of compounds bearing flavanone/chromone nucleus. Taking this into account, fourteen derivatives of 2'-hydroxy flavanone (1) were synthesized and evaluated against pro-inflammatory mediators (TNF-α, IL-1β and NO) in in vitro and in vivo models. Results directed that among the synthesized compounds, four derivatives (11-14) showed profound inhibition of proinflammatory mediators as compared to the lead molecule. Further, 11-14 demonstrated comparable anti-inflammatory activity with ibuprofen in carrageenan-induced rat paw edema assay and appreciable inhibition of lipopolysaccharide (LPS) induced pro-inflammatory mediators (TNF-α and IL-1β) in Sprague Dawley (SD) rats. The synthesized compounds were further subjected to molecular docking analysis and in silico prediction of pharmacokinetic properties. Keywords: 2'-hydroxy flavanone, Flavanones, Anti-inflammatory activity, Nitric oxide, Tumor necrosis factor-α, Interleukin 1-β.

Pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, interleukin (IL)-6, Interferon (IFN)-γ and nitric oxide (NO) are the key mediators for the pathogenesis of osteoarthritis, inflammatory bowel diseases, psoriasis, ulcerative colitis and rheumatoid arthritis. Hence, the inhibition of pro-inflammatory cytokines and NO will be an important strategy for the treatment of these inflammatory conditions. However, most TNF-α inhibitors possess serious side effects and therefore, development of safer alternative molecules including natural products are required for the blockage of TNF-α1-7. 2'-hydroxy flavanone (1) was previously isolated from Mimosa pudica whole plant and was found to exhibit antiinflammatory effects in RAW 264.7 and J774A.1 cells (IC50=71.3 to 95.0 µg/mL for inhibition of TNF-α, IL-1 β and NO)8. There are also reports which depict the potential of flavonoids as anti-inflammatory agents which are consumed in plant food9-10. As our research program is aligned towards the discovery of potent anti-inflammatory derivatives6-8, 11-14, and the present work deals with the structural modifications on the 2'-hydroxy flavanone (1) to yield better antiinflammatory agents. The alkylation at 2'-OH position of 2'-hydroxy flavanone (1) furnished fourteen derivatives (2-15)17 which were evaluated for anti-pro-inflammatory mediators activity on RAW 264.7 and J774A.1 cells along with their cytotoxicity6. The most potent compounds (11-14) were further examined for inhibition of lipopolysaccharide (LPS)-induced TNF-α and IL-1β levels in Sprague-Dawley (SD) rats12 and also for inhibition of rat paw edema in carrageenan-induced rat paw edema model15. Furthermore, molecular docking analysis for the synthetic derivatives was carried out using GOLD program7 and pharmacokinetics parameters

were determined using commercially available Molinspiration online property calculation toolkit16. For the preparation of derivatives, 1 was procured from Sigma Chemical Co. (St. Louis, MO, USA) and 2'-O-alkylated derivatives was prepared by using alkylating reagents and K2CO3 in acetone in good yields (55-87%)17 (Supplementary Table 1) and characterized by using MS and NMR spectroscopic data (Supplementary file). All the synthetic derivatives except 218 and 1119 are found to be new (as per report of Scifinder and Reaxys databases). Scheme 1 describes the synthesis of derivatives 2-15. The NO inhibitory effects of all the derivatives of 1 was compared with the specific inhibitor, L-NAME (IC50=69.21 and 73.18 µM on RAW 264.7 and J774A.1 cells, respectively) (Table 1). In comparison to L-NAME and native compound (1), all the derivatives were found to be more active in inhibiting NO production with an IC50 of 13.2 to 48.7 µM in both cells. Interestingly, 14 (3-fluorobenzyl) > 11 (benzyl) >13 (4-fluorobenzyl) >12 (4-isopropylbenzyl) were found to be most potent NO inhibitors with half maximal concentration ranging from 13.2 to 28.6 µM. The results revealed that mostly substitution of bulky groups such as benzyl moieties containing fluoro/isopropyl leads to the potent NO inhibition. Most of the compounds depicted cell viability of more than 90% when tested at 50 µM concentration. In case of TNF-α inhibition, among the tested derivatives, 13 (4-fluorobenzyl, IC50=17.7 and 18.2 µM) > 14 (3-fluorobenzyl, IC50=19.8 and 20.1 µM) > 11 (benzyl, IC50=17.7 and 18.2 µM) > 12 (4-isopropylbenzyl, IC50=23.7 and 23.2 µM) were also found to be most potent TNF-α inhibitors when tested on RAW 264.7 and J774A.1 cells respectively. Apparently, inhibition (37.3 to 48.7%) was found to be more prominent when there is an increase in straight chain

length up to four carbons (2-5) at concentration of 30 µM (Table 2). Also, 15 (methoxymethyl) depicted significant inhibition of TNF-α ranging from 45.2 to 48.6%. With respect to IL-1β, inhibition follows the same trend as that for TNF-α except for 13. Three derivatives, 12 (4-isopropylbenzyl, IC50=25.1 and 23.1 µM), 11 (benzyl, IC50=25.2 and 24.5 µM) and 14 (3-fluorobenzyl, IC50=27.4 and 30.2 µM) were witnessed to be most potent IL-1β inhibitors when tested on RAW 264.7 and J774A.1 cells respectively (Table 2). Compounds 11-14 exhibited significant pro-inflammatory inhibitory activities. Therefore, they were further investigated for anti-inflammatory activity in carrageenan rat paw edema assay and for reduction of LPS-induced TNF-α and IL-1β levels in SD rats. Among the tested compounds, 13 exhibited the highest anti-inflammatory activity with 50.5% reduction in the carrageenan rat paw edema followed by 14 (39.5% reduction) after 5 h as compared to standard drug, ibuprofen (41.4% reduction) (Table 3). The anti-inflammatory effects were in the order: 13 > 14 > 11 > 12. For reduction of LPS-induced TNF-α and IL-1β levels in SD rats, tested compounds followed the same trend as that for edema reduction. For instance, 13 exhibited the reduction of TNF-α and IL-1β levels by 55.4 and 43.6 % followed by 14 (49.6 and 39.2 % reduction) respectively. However, none of the tested compounds were discovered as better inhibitors when compared to standard drug, dexamethasone (73.4 and 62.5% reduction for TNF-α and IL-1β respectively) (Table 3). The GOLD program was utilized to dock 2'-hydroxy flavanone and its derivatives into the active sites of TNF-α and IL-1β and the total scores of complexes of 2'-hydroxy flavanone and its derivatives with TNF-α and IL-1β are given in Table 4. Molecular docking analysis7 revealed that all semi-synthesized derivatives have greater gold fitness scores than 1 and share more or less similar interactions in comparison to co-crystallized inhibitor (PDB code: 2AZ5) in case of

TNF-α. Interestingly, four derivatives (11-14) have great gold fitness scores than the cocrystallized inhibitor towards binding of TNF-α. However, in case of IL-1β, gold fitness scores are lesser than the parent compound (1). Interestingly, in most of the compounds activity follows the same pattern as depicted from the molecular docking analysis. As shown in Figure 1, the 4fluro of 13 has shown the hydrogen bonding interaction with Tyr151 of A chain and Π–Π stacking interactions between the phenyl ring and Tyr59 of both A and B chains. Similarly, all the compounds were also docked into the active site of IL-1β (Table 4). Previously, we estimated that for TNF-α inhibitory activity, solvent accessible surface interaction with Tyr119 form both chain is mandatory7. The observed lesser gold fitness scores for all compounds in case of IL-1β suggest that these do not bind to the active sites of IL-1β and specifically bind to the TNF-α which corroborate with the results obtained for inhibition of pro-inflammatory cytokines in vitro and in vivo (Table 2-4). Derivatives of 2'-hydroxy flavanone were also passed through pharmacokinetic filters. A computational study of all compounds (2-15) was performed for prediction of ADME properties such as absorption (% ABS), polar surface area (TPSA), miLog P, number of rotatable bonds, and violations of Lipinski’s rule of five by using Molinspiration online property calculation toolkit. Topological polar surface area (TPSA), i.e., surface belonging to polar atoms, is a descriptor that was shown to correlate well with passive molecular transport through membranes and, therefore, allows prediction of transport properties of drugs in the intestines and blood-brain barrier crossing. Absorption (% ABS) was calculated by: % ABS =109-(0.345×TPSA). Most of the derivatives possessed all desired parameters for good oral bioavailability except 7 and 12. From all these parameters, it can be observed that all compounds exhibited a great % ABS ranging from 92.94 to 96.74% (Table 5). However, 7 and 12 violated Lipinski’s parameters.

In conclusion, on the basis of activity of the reported flavonoids for anti-inflammatory, 14 derivatives of 2'-hydroxy flavanone (1) were synthesized and evaluated for the inhibition of proinflammatory mediators in macrophages. All these compounds depicted prominent inhibition towards release of pro-inflammatory mediators. Among them, fluoro substituted benzyl derivatives (13 and 14) have potent inhibition of pro-inflammatory markers as compare to parent compound (1). Compounds 11-14 were also found to suppress appreciably the release of TNF-α and IL-1β and reduced the paw edema in SD rats at dose of 150 µmol/Kg. Further, molecular docking analysis revealed that all semi-synthesized compounds have greater gold fitness scores than 1 and share more or less similar interactions in comparison to co-crystallized inhibitor (PDB code: 2AZ5) in case of TNF-α. Interestingly, four derivatives (11-14) have great gold fitness scores than the co-crystallized inhibitor towards binding of TNF-α. In case of IL-1β, gold fitness scores are lesser than the parent compound (1). The activity pattern follows the same trend as depicted from the molecular docking analysis in most of the compounds. Also, most of the derivatives possessed all desired parameters for good oral bioavailability. Our results supports the previous findings which suggests that small-molecule inhibitors of TNF-α should be hydrophobic and large enough in size to interact with both subunits of the TNF-α dimer simultaneously to prevent the binding of the third subunit, which forms the biologically active trimer complex7,

20

. Furthermore, fluoro substituted benzyl derivatives (13 and 14) can be

considered as lead molecules for development of better anti-pro-inflammatory agents. Acknowledgements Financial support in the form of fellowship was received from Department of Science and Technology (DST)-INSPIRE, India. Authors acknowledged the research support provided by the Director, NIPER, SAS Nagar, India.

Supplementary data S1 (Experimental and characterization part are provided in supplementary file). References and notes

1.

Boniface, K.; Guignouard, E.; Pedretti, N.; Garcia, M.; Delwail, A.; Bernard, F.-X.; Nau, F.; Guillet, G.; Dagregorio, G.; Yssel, H.; Lecron, J.-C.; Morel, F. Clin Exp Immunol. 2007, 150, 407.

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Kapoor, M.; Martel-Pelletier, J.; Lajeunesse, D.; Pelletier, J.-P.; Fahmi, H. Nature Rev. Rheumatol. 2011, 7, 33.

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Strober, W.; Fuss, I.J. Gastroenterology 2011, 140, 1756.

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Sharma, J.N.; Al-Omran, A.; Parvathy, S.S. Inflammopharmacology 2007, 15, 252.

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Agrawal, B.B.; Gupta, S.C.; Kim, J.H. Blood 2012, 119, 651.

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Bhandari, P.; Patel N.K.; Bhutani, K.K. Bioorg. Med. Chem. Lett. 2014, 24, 3596.

7.

Bhandari, P.; Patel N.K.; Gangwal, R.; Sangamwar, A.T.; Bhutani, K.K. Bioorg. Med. Chem. Lett. 2014, 24, 4114.

8.

Patel, N.K.; Bhutani, K.K. EXCLI J. 2014, 13, 1011.

9.

Rathee, P.; Chaudhary, H.; Rathee, S.; Rathee, D.; Kumar, V.; Kohli K. Inflamm. Allergy Drug Targets 2009, 8, 229.

10.

Serafini, M.; Peluso, I.; Raguzzini, A. Proc. Nutr. Soc. 2010, 69, 273.

11.

Patel, N.K.; Pulipaka, S.; Dubey, S.P.; Bhutani, K.K. Nat. Prod. Commun. 2014, 9, 661.

12.

Patel, N.K.; Bhutani, K.K. Phytomedicine 2014, 21, 946.

13.

Patel, N.K.; Ramandeep, Bhutani, K. K. Nat. Prod. Commun. 2014, 9, 1749.

14.

Patel, N.K.; Galipalli, S.; Prasanna, K.; Bhutani, K. K. Nat. Prod. Res. 2014. http://dx.doi.org/10.1080/14786419.2014.989846

15.

Bairwa, K.; Singh I.N.; Roy, S.K.; Grover, J.; Srivastava, A.; Jachak, S.M. J. Nat. Prod. 2014, 76, 1393.

16.

Molinspiration

Cheminformatics,

Bratislava,

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Republic,

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from:

http://www.molinspiration.com/services/properties.html (accessed on 13.02.14) 17.

Gautam, R.; Jachak, S.M.; Kumar, V.; Gopi Mohan, C. Bioorg. Med. Chem. Lett. 2011, 21, 1612.

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Huang, S.-H.; Wu, T.M.; Tsai, F.-Y. Appl. Organomet. Chem. 2010, 24, 619.

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Sun, H.; Yost, G. S. Chem. Res. Toxicol. 2007, 21, 374.

Scheme 1: Synthetic scheme of 2'-hydroxy flavanone (1) derivatives

Figure 1. The 2D interaction diagram of 13 at the active site of TNF-α

Legends for Tables and Figures: Table 1. NO inhibitory effects and cell viability of 2'-hydroxy flavanone derivatives on RAW 264.7 and J774A.1 cells Table 2. TNF-α and IL-1β inhibitory effects of 2'-hydroxy flavanone derivatives on RAW 264.7 and J774A.1 cells Table 3. Anti-inflammatory activity of 2'-hydroxy flavanone derivatives Table 4. Gold fitness scores of 2'-hydroxy flavanone derivatives for TNF-α and IL-1β Table 5. Pharmacokinetic filters for good oral bioavailability of 2'-hydroxy flavanone derivatives Scheme 1: Synthetic scheme of 2'-hydroxy flavanone (1) derivatives Figure 1. The 2D interaction diagram of 13 at the active site of TNF-α

Table 1. NO inhibitory effects and cell viability of 2'-hydroxy flavanone derivatives on RAW 264.7 and J774A.1 cells Compounds

IC50 (µM) for inhibition of

Cell viabilitya (% of control)

NO production RAW 264.7

J774A.1

RAW 264.7

J774A.1

b

L-NAME

69.21 ± 2.65

73.18 ± 1.70

-

-

b

Curcumin

11.02 ± 2.40

18.52 ± 2.89

-

-

2

47.3 ± 3.47

44.5 ± 3.07

84.30 ± 2.98

89.27 ± 3.38

3

38.5 ± 1.73

38.8 ± 1.56

82.60 ± 3.79

92.72 ± 2.05

4

46.0 ± 2.84

44.6 ± 1.40

91.72 ± 2.80

93.64 ± 0.87

5

48.7 ± 1.38

47.7 ± 2.88

78.30 ± 3.43

95.47 ± 1.24

6

36.7 ± 1.67

33.2 ± 1.28

83.35 ± 3.82

84.51 ± 2.87

7

39.2 ± 2.78

39.3 ± 3.97

90.29 ± 2.67

92.13 ± 3.82

8

39.2 ± 1.48

42.6 ± 1.36

81.56 ± 2.06

88.64 ± 4.75

9

32.7 ± 3.45

34.3 ± 2.27

94.83 ± 3.10

92.34 ± 2.19

10

37.8 ± 2.93

38.6 ± 3.89

89.30 ± 4.18

84.02 ± 1.39

11

18.9 ± 3.17

23.2 ± 1.98

91.24 ± 2.29

96.46 ± 7.20

12

27.1 ± 1.76

28.6 ± 2.56

92.47 ± 1.84

99.74 ± 2.95

13

28.2 ± 1.38

25.5 ± 2.60

95.20 ± 1.65

91.08 ± 3.07

14

15.6 ± 2.45

13.2 ± 2.10

90.27 ± 2.85

92.74 ± 1.19

15

32.6 ± 2.75

35.2 ± 2.89

92.23 ± 2.05

99.42 ± 1.03

Values are represented as mean ± S.D. of three different experiments in triplicates; aCell viability was measured at 50 µM; bStandards used for the present study.

Table 2. TNF-α and IL-1β inhibitory effects of 2'-hydroxy flavanone derivatives on RAW 264.7 and J774A.1 cells Compounds

TNF-α

IL-1β

RAW 264.7

IC50

J774A.1

IC50

RAW 264.7

IC50

J774A.1

IC50

(%inhibitionb)

(µM)

(%inhibitionb)

(µM)

(%inhibitionb)

(µM)

(%inhibitionb)

(µM)

Curcumin

-

3.4 ± 2.44

-

5.6 ± 0.21

-

17.5 ± 1.67

-

67.4 ± 1.34

a

-

0.04 ± 3.12

-

0.4 ± 2.26

-

4.7 ± 1.52

-

22.0 ± 3.29

DEXA

2

37.3 ± 0.34

-

34.5 ± 2.50

-

37.30 ± 1.89

-

38.27 ± 1.63

-

3

38.5 ± 0.37

-

38.8 ± 4.20

-

29.60 ± 3.29

-

27.72 ± 1.90

-

4

46.0 ± 0.28

-

44.6 ± 1.40

-

45.72 ± 2.50

-

43.64 ± 3.78

-

5

48.7 ± 1.38

-

47.7 ± 0.88

-

19.30 ± 1.43

-

42.47 ± 2.42

-

6

36.7 ± 1.67

-

33.2 ± 1.28

-

30.35 ± 3.82

-

33.51 ± 1.98

-

7

39.2 ± 0.78

-

39.3 ± 0.97

-

41.29 ± 0.67

-

40.13 ± 2.82

-

8

39.2 ± 1.48

-

42.6 ± 1.36

-

47.56 ± 2.06

-

43.64 ±1.75

-

9

41.7 ± 1.54

-

31.7 ± 1.27

-

41.83 ± 4.10

-

40.34 ± 1.19

-

10

47.8 ± 2.93

-

38.6 ± 2.89

-

49.30 ± 3.18

-

47.02 ± 2.39

-

11

86.1 ± 1.74

20.1 ± 3.71

83.1 ± 3.98

22.4 ± 2.67

93.24 ± 1.29

25.2 ± 1.74

91.46 ± 3.20

24.5 ± 3.72

12

80.6 ± 1.76

23.7 ± 1.67

78.6 ± 2.56

23.2 ± 1.35

91.47 ± 2.84

25.1 ± 4.15

91.74 ± 1.95

23.1 ± 1.67

13

92.7 ± 1.81

17.7 ± 2.81

94.1 ± 2.14

18.2 ± 1.52

45.20 ± 1.76

-

42.08 ± 2.07

-

14

91.6 ± 2.45

19.8 ± 1.89

96.2 ± 4.10

20.1 ± 3.54

93.27 ± 2.85

27.4 ± 2.69

92.74 ± 1.19

30.2 ± 2.19

15

48.6 ± 2.75

-

45.2 ± 2.89

-

35.23 ± 1.05

-

33.34± 2.03

-

Values are represented as mean ± S.D. of three different experiments in triplicates; aStandards used for the present study; b% inhibition at 30 µM; DEXA, Dexamethasone

Table 3. Anti-inflammatory activity of 2'-hydroxy flavanone derivatives Compounds

% inhibition in paw edema at

% inhibition of

3h

5h

TNF-α levels

IL-1β levels

11

9.8 ± 2.94

35.7 ± 3.50

32.1 ± 4.50

28.4 ± 2.78

12

18.0 ± 2.84

32.3 ± 2.93

30.3 ± 2.34

24.6 ± 3.61

13

18.2 ± 1.95

50.5 ± 1.65

55.4 ± 3.57

43.6 ± 2.95

14

5.2 ± 1.60

39.5 ± 2.47

49.6 ± 1.89

39.2 ± 2.90

30.5 ± 3.53

41.4 ± 1.72

-

-

73.4 ± 2.69

62.5 ± 3.48

Ibuprofen Dexamethasone

-

Data are expressed as mean ± S.D. (n = 5).

-

Table 4. Gold fitness scores of 2'-hydroxy flavanone derivatives for TNF-α and IL-1β Compounds

GOLD FITNESS SCORE TNF-α

IL-1β

1

44.48

40.94

2

47.67

34.39

3

46.62

35.59

4

51.09

36.17

5

53.90

37.60

6

55.33

37.71

7

55.72

39.27

8

56.60

35.04

9

56.25

36.54

10

52.86

39.21

11

61.31

40.84

12

59.50

40.29

13

62.81

39.55

14

62.65

36.80

15

49.31

34.18

2AZ5

57.89

NA

NA: Not available

Table 5. Pharmacokinetic filters for good oral bioavailability of 2'-hydroxy flavanone derivatives Compounds miLogPa PSAb

a

natoms MW

nON nOHNH nviolation nrot volume

1

3.123

46.533

18

240.258

3

1

0

1

214.226

2

3.191

35.539

19

254.285

3

0

0

2

231.754

3

3.567

35.539

20

268.312

3

0

0

3

248.556

4

4.070

35.539

21

282.339

3

0

0

4

265.357

5

4.629

35.539

22

296.366

3

0

0

5

282.159

6

4.314

35.539

22

296.366

3

0

0

4

281.944

7

5.134

35.539

23

310.393

3

0

1

6

298.961

8

4.844

35.539

23

310.393

3

0

0

5

298.746

9

4.868

35.539

23

308.377

3

0

0

4

292.533

10

3.836

35.539

21

280.323

3

0

0

4

259.726

11

4.786

35.539

25

330.383

3

0

0

4

303.403

12

6.298

35.539

28

372.464

3

0

1

5

353.353

13

4.950

35.539

26

348.373

3

0

0

4

308.334

14

4.926

35.539

26

348.373

3

0

0

4

308.334

15

3.163

44.773

21

284.311

4

0

0

4

257.54

Molinspiration Octanol-water partition coefficient milogP; bMolecular polar surface area (PSA)