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Medicinal Chemistry Research

, Volume 27, Issue 5, pp 1504–1516 | Cite as

Dipeptides as linker for multicomponent presentation—a facile, robust, and high-bioactivity yielding strategy

  • Honnegowdanahally K. Kumara
  • Suhas Ramesh
  • Doddahindaiah M. Suyoga Vardhan
  • J. Shiva Kumar
  • Dase Channe Gowda
Original Research
  • 78 Downloads

Abstract

The need for multiple drugs arises when the response to pharmacological inflection is complicated and the disease conditions get worsened. The treatment requires a common platform to which multiple drugs are linked and this methodology is proven to be useful in drug delivery, biomaterial research, biomedicine, and vaccine development. In view of this, the present work is centered on dipeptides (KW/ KD/ KE/ kW/ kD/ kE), which are used as linkers and can potentially hold different functional group containing molecules (here, amino and carboxyl). First, we have demonstrated the incorporation of three components to the linker successfully followed by tetra component linking to those dipeptides, which contain trifunctional groups. To provide a proof of concept, these multisubstituted constructs were subjected to microbial growth suppression assay as well as anti-inflammatory assay. The biological results revealed that the multimers play a key role in enhancing the activity. Hence, the present system may be regarded as simple and straight forward which can be employed to develop various therapeutic agents as well as in different methodologies.

Keywords

Multicomponent Dipeptides Linker Bioactivity 

Abbreviations

EDCI

1-(3-Dimethylaminopropyl)-3-ethyl-carbodiimide

HOBt

1-Hydroxy benzotriazole

NMM

N-Methylmorpholine

QZN 1

3-(4-Oxo-3,4-dihydroquinazolin-2-yl)propanoic acid

QZN 2

4-(4-Oxo-3,4-dihydroquinazolin-2-yl)butanoic acid

Introduction

Multimerization is a process of assembling monomer units that is held together with non-covalent bonds and this technique has found numerous applications in biomedicine such as multidrug presentation, vaccine development, antibacterial, and antitumor drugs, etc. This concept is often found in the field of peptide science and is mainly employed to enhance the immunogenicity of the short peptide epitopes, which precludes their use in free form for purposes such as raising antisera or candidate vaccine trials. Multimerization can be performed either by simple polymerization (Borras-Cuesta et al. 1988) or by chemically better defined strategies like conjugation to sequential oligopeptide carriers (Tsikaris et al. 1996; Mezo et al. 2003) display on lysine scaffolds (Kragol and Otvos 2001; Mozdzanowska et al. 2003) or incorporation into dendrimeric systems such as the multiple antigenic peptides (MAPs) (Tam 1988). MAPs can be constructed either by solid- or solution-phase techniques and each method suffers from its own serious limitations (Wioleta et al. 2010).

Our previous experience (Suresha et al. 2011; Shantharam et al. 2013; Kumara and Gowda 2017) has shown that conjugating a heterocycle to an amino acid or peptide results in increased biological activity and this has opened up new avenue for further investigations. Our earlier work (Sharma et al. 2013a, b) showed that bis-homo heterocyclic-amino acid conjugates have exerted superior activity compared to mono-heterocyclic conjugates. This phenomenon demonstrated that an incremental increase of heterocyclic units yield superior results. To the best of our knowledge, there is no report on the multimerization of heterocyclic moieties on the amino acid or peptide core. Based on these evidences, herein we have made a first attempt to demonstrate the multisubstitution of heterocycles on dipeptide platforms and also we have provided the evidence that the multimerization improves the biological activity to a larger extent. Further, the present strategy has also been validated through docking studies which showed good receptor-binding affinity. The amino acid composition of dipeptides comprises of lysine (l and d, in order to analyze the effect of stereochemistry) as the common moiety at the N-terminus whose α- and ε-amino groups are reacted with carboxyl containing heterocycles. In the current investigation, we have demonstrated multimerization at trimer- and tetramer-levels. For trimerization, tryptophan was used at the C-terminus because of its wide biological applications (Suhas and Gowda 2012), whereas for tetramerization, aspartic acid (α- and β-carboxyl)/glutamic acid (α- and γ-carboxyl) were employed at C-terminus to which amino containing heterocyclics are connected. As far as heterocycles are concerned, we have employed carboxyl containing quinazolinone and amino containing benzisoxazole and piperazine derivatives. The criteria of choosing these heterocycles are based on our previous knowledge, their different functional groups and also their promising impact on the therapeutic actions (Suhas et al. 2011; Vardhan et al. 2013; Sharma et al. 2013a, b; Burstein et al. 2012; Keche et al. 2012).

Results and discussion

Chemistry

With the above rationale in mind, we constructed the dipeptides viz., KW, KD, KE, kW, kD, and kE wherein capital and small letters indicate l and d-amino acids, respectively. The dipeptides were synthesized in-solution using Boc chemistry employing orthogonal protecting group strategy in which Boc-Lys(Z)-OH was coupled to HCl.H-Xaa-OPg [Xaa = Trp or Asp(OBzl) or Glu(OMe); Pg = OBzl or OMe] using isobutylchloroformate/1-hydroxy benzotriazole (IBCF/HOBt) as coupling partner and N-methylmorpholine (NMM) as base, and the synthesis was confirmed by physical and spectroscopic data. The idea of using orthogonally protected Boc-Lys(Z) was to ensure safe and complete removal of Boc and Z groups without affecting each other. The dipeptides so formed were subjected to saponification using 1 N NaOH/MeOH to get C-terminus free peptides (3 and 4). One half of Boc-Lys(Z)-Trp-OH was reacted with benzisoxazole analog and the other half with piperazine moiety using 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide.HCl (EDCI)/HOBt method to obtain 5, 6, and 11, respectively. t-Butyloxycarbonyl (Boc) group was removed using trifluoroacetic acid (TFA), whereas benzyloxycarbonyl (Z) was deprotected using hydrogenolysis employing 10% Pd/C. The N-terminal Lys having αC and εC amino groups were reacted with quinazolinones, which resulted in trimers with diverse heterocycles present in a common platform (7–10, 12, and 13). Similarly, other dipeptides (KD, KE, kD, and kE) were also saponified, treated with different heterocycles, removed Boc and Z and finally treated with quinazolinones as above to furnish tetramers armed with different heterocyclic structures (26–33 and 36–39). The syntheses (Schemes 1 and 2) of final constructs and intermediates were confirmed by spectroscopic tools.
Scheme 1

Schematic representation for obtaining multicompartment trimmers. Reagents and conditions: (i) IBCF, NMM, −15 ± 1 °C to RT, overnight (ii) 1 N NaOH/MeOH, RT, 1 h (iii) EDCI/HOBt, NMM, 0 °C to RT, overnight (iv) Pd-C (10%)/HCOONH4, MeOH, RT, 1 h (v) TFA, 45 min, RT

Scheme 2

Schematic representation for obtaining multicompartment tetramers (reagents and conditions are same as previous scheme)

Biological activities

Antimicrobial and anti-inflammatory activities

To validate our concept that multimerization using heterocyclics result in high activity, all the final constructs were evaluated for microbial growth inhibition assay and anti-inflammatory assay (Table 1). The microbes included in the study ranged from Gram-positive and Gram-negative bacteria to different fungal species. Multimers armed with benzisoxazole unit(s) has exhibited superior activity over piperazine counterparts, which is attributed to more lipophilic character of benzisoxazole and also may be due to the presence of fluorine (Solomon et al. 2010). Further, the effect of change of stereochemistry has also been studied which revealed that incorporation of d-Lys into the peptides found to be promising since d-amino acids are more stable and are less prone to proteolytic cleavage (Hamamoto et al. 2002). Also, side-chain alkyl length of the quinazolinone nucleus preferred to be two as increase in the carbon number decreased the antimicrobial activity, whereas the trend is opposite in case of inflammatory action, which suggested that butyl is preferred over propyl. Among the multicompartment systems, trimers resulting from KW/kW are efficient in the biological activity, which has also been proved in the docking studies (discussed below), whereas the tetramers are placed next in the series. Further, those compounds which showed high activity (trimers) by zone diffusion method were subjected to minimum inhibitory concentration (MIC) assay and the results were tabulated in Table 2. The results revealed that compounds 7, 8, and 10 exhibited excellent MIC values below 20 μg/mL against all the strains tested. The starting materials (non-conjugated compounds) showed little or no activity compared to multimers (Kumara et al. 2017). The antioxidant activity of these multimers is in progress.
Table 1

Biological evaluation and docking results of the final constructs

Entry

Antibacterial activity

Antifungal activity

Docking results with 1KZN

Anti-inflammatory activity (IC50 µg/mL)a

(Zone of inhibition in mm)a

          

E. coli

S. aureus

F. moniliforme

A. niger

Docking score

Glide energy

XP HBond

H-bond interaction

bπ-cation interaction/ cπ–π stacking interaction

7

25 ± 0.35

24 ± 0.21

26 ± 0.11

27 ± 0.47

−7.307

−76.365

−2.283

Asp 49, Asp 49, Gly 117

NF

50 ± 0.65

8

26±0.13

25±0.35

27±0.23

28±0.35

−8.059

−73.425

−1.583

Asp 49, Asp 49

bArg 76, cArg 76

45±0.90

9

18 ± 0.08

20 ± 0.42

20 ± 0.50

22 ± 0.46

−6.746

−77.708

−3.102

Asp 73, Asn 46

bArg 76

40 ± 2.65

10

19±0.27

22±0.33

21±0.13

23±0.27

−6.948

−72.631

−1.252

Asp 49

bArg 76, cArg 76

35±2.98

12

18 ± 0.65

17 ± 0.42

20 ± 0.48

21 ± 0.55

−6.541

−75.187

−1.560

Asp 49

cArg 76

55 ± 1.02

13

20 ± 0.32

19 ± 0.32

21 ± 0.37

22 ± 0.89

−6.731

−74.676

−0.998

Asp 49

bArg 76, cArg 136

50 ± 1.46

26

18 ± 0.11

17 ± 0.36

17 ± 0.32

18 ± 0.45

−6.237

−64.630

−0.527

Asp 49, Asp 49, Gly 117

NF

65 ± 0.96

27

19±0.25

17±.44

19±0.52

20±0.33

−6.334

−70.121

−2.298

Arg 136, Gly 117

NF

60±0.80

28

17 ± 0.43

15 ± 0.10

16 ± 0.56

18 ± 0.28

−6.124

−78.922

−0.799

Gly 117

cArg 136

55 ± 0.98

29

18±0.26

16±0.35

18±0.16

19±0.54

−6.204

−86.030

−1.890

Asp 49

cArg 136

50±2.16

30

15 ± 0.56

14 ± 0.46

16 ± 0.19

17 ± 0.36

−5.802

−71.025

−1.354

Asp 49, Asp 49

cArg 136

85 ± 2.65

31

16±0.09

16±0.39

17±0.20

18±0.28

−6.054

−75.549

−1.377

Asp 49

cArg 136

80±0.96

32

16 ± 0.68

14 ± 0.25

17 ± 0.25

17 ± 0.31

−5.565

−80.437

−2.148

Asp 49, Gly 117

bArg 136

75 ± 0.46

33

17±0.32

15±0.08

18±0.09

17±0.20

−5.690

−64.398

−2.804

Glu 42,

cArg 190

70±1.25

36

14 ± 0.16

15 ± 0.25

16 ± 0.65

16 ± 0.11

−5.044

−69.133

−0.483

Asp 49, Asp 49

NF

75 ± 1.90

37

16 ± 0.29

15 ± 0.36

17 ± 0.21

18 ± 0.23

−5.312

−79.552

−1.199

Asp 49, Glu 42

NF

70 ± 0.98

38

12 ± 0.22

10 ± 0.33

13 ± 0.11

12 ± 0.45

−4.792

−75.044

−0.136

Asp 49, Asp 49

NF

90 ± 1.32

39

13 ± 0.50

11 ± 0.58

15 ± 0.27

15 ± 0.35

−4.957

−75.246

−1.302

Asp 49, Asp 49

NF

85 ± 2.80

SM

14 ± 0.34

12 ± 0.14

−6.199

−44.768

−3.779

Glu 50, Asn 46, Asp 73, Arg 136

NF

BS

15 ± 0.36

13 ± 0.25

IM

60 ± 0.95

IP

65 ± 0.49

The molecules with bold values having d-Lys linker at N-terminal

SM streptomycin, BS bavistin, IM indomethacin, IP ibuprofen, NF not formed, ‘-’: no activity/ not analyzed

aValues are mean of three determinations, the ranges of which are < 5% of the mean in all cases

bπ-cation interaction

cπ-π stacking interaction

Table 2

Minimum inhibitory concentration (MIC) of the most active trimers

Entry

Antibacterial activity

Antifungal activity

(MIC in μg/mL)a

E. coli

S. aureus

F. moniliforme

A. niger

7

19 ± 1.20

18 ± 1.50

19 ± 1.25

15 ± 1.35

8

16 ± 0.85

17 ± 0.75

14 ± 0.50

12 ± 1.50

9

21 ± 1.45

23 ± 1.25

25 ± 0.95

22 ± 1.75

10

18 ± 1.10

19 ± 1.50

17 ± 1.55

18 ± 0.10

12

23 ± 1.40

25 ± 1.25

25 ± 0.75

22 ± 1.55

13

26 ± 0.95

28 ± 2.50

25 ± 0.50

24 ± 1.75

SM

25±1.50

23±0.95

BS

26±2.25

22±1.35

SM streptomycin, BS bavistin; ‘-’: not analyzed

aValues are mean of three determinations, the ranges of which are < 5% of the mean in all cases

Bold values represents most active multimers

Molecular docking studies

The binding efficiency and molecular interaction of the multimers were studied by molecular docking as it is considered as one of the most rational and authentic approaches in the drug design and discovery. The useful approaches for the discovery of new drugs are based on investigations of drug targets like enzymes or receptors (Collin et al. 2011; Ostrov et al. 2007). DNA gyrase is one of the attractive targets in E. coli which is involved in replication and transcription. This enzyme contains an ATPase activity, which introduces negative supercoiling of circular DNA. The enzyme belongs to a superfamily of ATPases, which is a known target for antibacterial agents since its blocking induces bacterial death (Kumar et al. 2014; Heddle and Maxwell 2002). The docking analysis was performed with active site of E. coli (PDB ID: 1KZN), which revealed that the multimers exhibited good binding interaction with receptor. The potentiality of the compounds ranks on the basis of the docking score. The multimers displayed docking score ranging from −8.059 to −4.792 and also showed various interactions like hydrogen bond interaction, π–π stacking interaction and π-cation interaction with different amino acids residues and were tabulated in Table 1. The binding modes (2D and 3D) of multimers (7, 9, 12, and 13) with protein receptors are illustrated in Fig. 1 (more in supplementary data, Supplementary Fig. S1). The quinazolinone ring is involved in all the three types of interactions and indole ring of tryptophan exhibited hydrogen bonding and π–π stacking interaction. The αNH and εNH of lysine showed hydrogen bonding interaction. Thus, the docking studies showed that multimerization enhances the binding affinity and occupied highest docking score.
Fig. 1

Docking images of the constructs 7, 9, 12, and 13 with 1KZN protein

Conclusion

In short, we have designed and developed several dipeptide cores that have exhibited the high potential to hold diverse heterocyclic systems in 1:3/ 1:4 core:cargo ratio and these types of linkers could also be substituted with different drugs/peptides/epitopes. The synthetic route has the advantage of being simple, convenient, high conversion with good yield. The constructs have shown superior biological activity over standard drugs and hence open room for new therapeutics. Further, the multimers have shown good receptor-binding affinity proving that they fit well within the enzyme pockets. Overall, the system presented in this work is found to be highly versatile, which can be used not only as therapeutics but also as a powerful strategy in multidrug presentation, vaccine development, and biomaterial research.

Experimental procedure

Materials and methods

All amino acids, EDCI, HOBt, and TFA were purchased from Advanced Chem. Tech. (Louisville, Kentucky, USA). IBCF and NMM were purchased from Sigma Chemical Co. (St. Louis, MO). All solvents and reagents used for the synthesis were of analytical grade. Silica gel (60–120 mesh) for column chromatography was purchased from Merck Pvt. Ltd., (Mumbai, India). Progress of the reaction was monitored by TLC using silica gel coated on glass plates with the solvent system comprising chloroform/methanol/acetic acid in the ratio 98:02:03 (Rfa) and 95:05:03 (Rfb). The compounds on the TLC plates were detected by iodine vapors. Melting points were determined on a Superfit melting point apparatus (India) and are uncorrected. Fourier Transform Infra red was performed using a Jasco spectrometer (Japan) using nujol media. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded on an Agilent Technologies (USA) using Dimethyl sulfoxide (DMSO)-d6 as solvent. High-resolution mass spectroscopic analysis was performed on a Bruker MicroTOF QII mass spectrometer in positive mode.

Chemistry

The syntheses of dipeptides and their conjugates were carried out according to the procedure described in our earlier reports (Kumara and Gowda 2017). The side-chain protecting group, 2-chlorobenzyloxycarbonyl (2-ClZ) was deprotected using HCOONH4/Pd-C (10%) in methanol, whereas tert-butyloxycarbonyl (Boc) group was removed using trifluroacetic acid (TFA) and further coupled with 3-(4-oxo-3,4-dihydroquinazolin-2-yl)propanoic acid and 4-(4-oxo-3,4-dihydroquinazolin-2-yl)butanoic acid to get final desired compounds in good yields. Synthesis was confirmed by IR, 1H NMR, 13C NMR and mass analysis. The chemical shift values of kW, kD, and kE are almost same as KW, KD, and KE in all final desired molecules. The physical and analytical data of final constructs were tabulated in Table 3.
Table 3

Physical and analytical data of the multimers

Entry

Yield (%)

M.P. (°C)

Rf Values

Molecular formula

R f a

R f b

7

70.2

165–167

0.35

0.45

C51H51FN10O7

8

73.4

165–167

0.34

0.45

C51H51FN10O7

9

69.8

156–159

0.37

0.50

C53H55FN10O7

10

72.8

156–159

0.37

0.51

C53H55FN10O7

12

75.2

180–182

0.42

0.54

C49H50Cl2N10O6

13

74.6

162–164

0.44

0.53

C51H54Cl2N10O6

26

75.6

175–177

0.34

0.45

C56H57F2N11O9

27

69.3

174–177

0.34

0.45

C56H57F2N11O9

28

76.4

154–156

0.35

0.46

C58H61F2N11O9

29

73.2

154–157

0.35

0.47

C58H61F2N11O9

30

87.7

178–180

0.37

0.49

C57H59F2N11O9

31

85.2

178–180

0.37

0.50

C57H59F2N11O9

32

92.6

150–152

0.38

0.53

C59H63F2N11O9

33

89.7

150–152

0.39

0.52

C59H63F2N11O9

36

72.0

206–208

0.49

0.70

C52H55Cl4N11O7

37

78.9

176–178

0.50

0.74

C54H59Cl4N11O7

38

82.0

202–205

0.52

0.75

C53H57Cl4N11O7

39

84.7

176–178

0.53

0.78

C55H61Cl4N11O7

General procedure for the synthesis of multi-heterocyclic conjugates of dipeptides (2239)

A solution of 5, 6, 11, 2225, 34, and 35 (0.42 mmol) in methanol (10 mL/g of peptide) was stirred with 10% Pd on carbon (100 mg) and HCOONH4 (2 eq.) for 4 h at room temperature. After the reaction was completed (monitored by TLC), reaction mixture was filtered through celite to remove the catalyst and the filtrate was concentrated and taken into chloroform. The organic layer was washed with 50% saturated brine solution (2 × 20 mL), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure (2-ClZ of lysine was removed) and dried. The Boc group was removed by stirring the above product with TFA (10 mL/g of peptide) for 45 min at room temperature. After the reaction was completed, TFA was evaporated, triturated with dry ether, filtered, and dried to obtain TFA salts.

To a cooled solution of 3-(4-oxo-3,4-dihydroquinazolin-2-yl)propanoic acid and 4-(4-oxo-3,4-dihydroquinazolin-2-yl)butanoic acid (0.66 mmol) and HOBt (1 eq.) in DMF separately (10 mL/g of peptide) added NMM (1 eq.). EDCI (1.2 eq.) was added under stirring while maintaining the temperature at 0 °C. The reaction mixture was stirred for an additional 10 min and pre-cooled solution of TFA salts (0.33 mmol each) in NMM (4 eq.) in DMF were added slowly. The reaction mixture was stirred for 8 h slowly warming to room temperature. The solvent DMF was removed under reduced pressure and the residue was poured into about 20 mL ice-cold 90% saturated KHCO3 solution and stirred for 15 min. The precipitated compound was extracted into chloroform and washed sequentially with 5% NaHCO3 solution (2 × 20 mL), water (2 × 20 mL), 0.1 N cold HCl solution (2 × 20 mL) followed by brine. The organic layer was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. The crude product was purified on column (silica gel 60–120 mesh, Merck) using a mixture of chloroform and methanol in the ratio 9:1 as eluting system to get desired products.

Het1: 6-fluoro-3-(piperidin-4-yl)benzo[d]isoxazole

Het2: 2,3-dichlorophenyl piperazine

QZN1: 3-(4-oxo-3,4-dihydroquinazolin-2-yl)propanoic acid

QZN2: 4-(4-oxo-3,4-dihydroquinazolin-2-yl)butanoic acid

7. Nα,Nε-bis-QZN1-Lys-Nα-Het1-Trp

1H NMR (DMSO-d6, 400 MHz) δ ppm: QZN1 = 1.09–1.24 (4H, m, –CH2), 2.47–2.57 (4H, m, –CH2), 7.09–7.89 (8H, m, ArH), 12.15 (2H, s, NH); Lys = 1.29–1.71 (6H, m, –β,γ,δCH2), 3.02–3.07 (2H, m, –εCH2), 4.29 (1H, d, –αCH), 8.02–8.16 (1H, m, –αNH), 8.24–8.28 (1H, m, –εNH); Trp = 3.19 (2H, d, –βCH2), 4.95 (1H, m, –αCH), 6.73 (1H, d, –αNH), 6.77–7.99 (5H, m, ArH), 10.83 (1H d, NH); Het1 = 2.47–2.66 (4H, m, –CH2), 2.73–2.81 (1H, m, –CH), 3.07–3.57 (4H, m, –CH2), 6.77–7.99 (3H, m, ArH).

13C NMR (DMSO-d6, 100 MHz) δ ppm: 171.67 (C, C-9), 171.21 (C, C-9’), 170.01 (C, C-6), 169.62 (C, C-28), 167.86 (C, C-34), 165.34 (C, C-36), 163.52 (C, C-14), 162.05 (C, C-14’), 157.17 (C, C-13), 149.22 (C, C-32), 136.42 (C, C-15), 134.62 (C, C-22), 133.89 (CH, C-18), 127.75 (CH, C-19), 126.11 (CH, C-17), 124.09 (CH, C-38), 123.12 (CH, C-21), 121.32 (CH, C-26), 118.76 (C, C-16), 117.21 (CH, C-25), 111.75 (CH, C-24), 110.11 (C, C-33), 109.84 (CH, C-27), 107.57 (C, C-20), 107.35 (CH, C-37), 104.81 (CH, C-35), 49.45 (CH, C-1), 47.02 (CH, C-7), 45.23 (CH2, C-29), 44.77 (CH2, C-5), 43.78 (CH2, C-2), 32.23 (CH2, C-12), 31.99 (CH2, C-30), 31.71 (CH2, C-4), 30.32 (CH2, C-8), 29.27 (CH2, C-10), 28.27 (CH2, C-31), 21.00 (CH2, C-3).

Mass value: 938.3864.

9. Nα,Nε-bis-QZN2-Lys-Nα-Het1-Trp

1H NMR (DMSO-d6, 400 MHz) δ ppm: QZN2 = 1.09–1.30 (8H, m, –CH2), 2.51–2.60 (4H, m, –CH2), 7.10–7.74 (8H, m, ArH), 12.11 (2H, s, NH); Lys = 1.33–1.69 (6H, m, –β,γ,δCH2), 3.10–3.32 (2H, m, –εCH2), 4.25 (1H, d, –αCH), 8.03–8.06 (1H, m, –αNH), 8.08–8.16 (1H, m, –εNH); Trp = 3.55 (2H, d, –βCH2), 4.96 (1H, m, –αCH), 6.74 (1H, d, –αNH), 6.72–7.74 (5H, m, ArH), 10.80 (1H d, NH); Het1 = 1.92–1.94 (4H, m, –CH2), 2.91–2.93 (1H, m, –CH), 3.04–3.10 (4H, m, –CH2), 6.72–7.74 (3H, m, ArH).

13C NMR (DMSO-d6, 100 MHz) δ ppm: 172.09 (C, C-9), 171.84 (C, C-6), 170.04 (C, C-28), 169.65 (C, C-34), 165.36 (C, C-36), 162.21 (C, C-14), 157.42 (C, C-13), 149.34 (C, C-32), 136.48 (C, C-15), 134.61 (C, C-22), 133.89 (CH, C-18), 127.73 (CH, C-19), 126.33 (CH, C-17), 124.19 (CH, C-38), 124.08 (CH, C-21), 121.31 (CH, C-26), 118.76 (C, C-16), 117.17 (CH, C-25), 111.84 (CH, C-24), 110.10 (C, C-33), 109.83 (CH, C-27), 107.57 (C, C-20), 107.35 (CH, C-37), 104.80 (CH, C-35), 52.87 (CH, C-1), 49.27 (CH, C-7), 44.83 (CH2, C-29), 34.97 (CH2, C-5), 34.75 (CH2, C-2), 32.22 (CH2, C-12), 32.10 (CH2, C-30), 31.37 (CH2, C-4), 29.32 (CH2, C-8), 29.05 (CH2, C-10), 28.74 (CH2, C-31), 23.20 (CH2, C-3), 22.48 (CH2, C-11).

Mass value: 966.3017.

12. Nα,Nε-bis-QZN1-Lys-Nα-Het2-Trp

1H NMR (DMSO-d6, 400 MHz) δ ppm: QZN1 = 2.50–2.60 (4H, m, –CH2), 3.03–3.52 (4H, m, –CH2), 6.94–7.72 (8H, m, ArH), 12.13 (2H, s, NH); Lys = 1.17–1.33 (6H, m, –β,γ,δCH2), 2.72–2.82 (2H, m, –εCH2), 4.25–4.30 (1H, m, –αCH), 7.70–8.04 (1H, m, –εNH), 8.06–8.25 (1H, m, –αNH); Trp = 2.63–2.72 (2H, m, –βCH2), 4.97–4.99 (1H, m, –αCH), 6.71–6.78 (1H, m, –αNH), 6.93–7.86 (5H, m, ArH), 10.86 (1H d, NH); Het2 = 2.80–3.72 (8H, m, –CH2), 6.90–7.73 (3H, m, ArH).

13C NMR (DMSO-d6, 100 MHz) δ ppm: 171.74 (C, C-9), 171.64 (C, C-9’), 171.51 (C, C-6), 171.41 (C, C-28), 170.11 (C, C-14), 162.06 (C, C-14’), 157.17 (C, C-13), 149.23 (C, C-31), 148.83 (C, C-15), 136.46 (C, C-22), 134.62 (CH, C-18), 133.00 (C, C-35), 130.66 (CH, C-33), 129.30 (CH, C-19), 128.77 (C, C-36), 127.89 (C, C-23), 126.61 (CH, C-17), 125.20 (CH, C-34), 124.39 (CH, C-21), 121.33 (CH, C-26), 120.04 (C, C-16), 118.90 (CH, C-25), 118.71 (CH, C-24), 116.27 (CH, C-32), 111.82 (CH, C-27), 109.93 (C, C-20), 52.84 (CH, C-1), 50.93 (CH, C-7), 50.74 (CH2, C-30), 49.36 (CH2, C-29), 42.08 (CH2, C-5), 31.90 (CH2, C-2), 30.34 (CH2, C-12), 29.40 (CH2, C-4), 29.25 (CH2, C-8), 28.59 (CH2, C-10), 23.23 (CH2, C-3).

Mass value: 945.2166, 947.2095, 949.2046.

13. Nα,Nε-bis-QZN2-Lys-Nα-Het2-Trp

1H NMR (DMSO-d6, 400 MHz) δ ppm: QZN2 = 1.45–2.22 (4H, m, –CH2), 2.57–2.61 (4H, m, –CH2), 3.07–3.57 (4H, m, –CH2), 6.93–7.71 (8H, m, ArH), 12.14 (2H, s, NH); Lys = 1.18–1.34 (6H, m, –β,γ,δCH2), 2.75–2.81 (2H, m, –εCH2), 4.28–4.31 (1H, m, –αCH), 7.72–8.03 (1H, m, –εNH), 8.05–8.24 (1H, m, –αCH); Trp = 2.64–2.73 (2H, m, –βCH2), 4.98–4.99 (1H, m, –αCH), 6.72–6.77 (1H, m, –αNH), 6.90–7.82 (5H, m, ArH), 10.84 (1H d, NH); Het2 = 2.81–3.68 (8H, m, –CH2), 6.93–7.71 (3H, m, ArH).

13C NMR (DMSO-d6, 100 MHz) δ ppm: 172.82 (C, C-9), 171.9 (C, C-9’), 171.65 (C, C-6), 171.55 (C, C-28), 169.60 (C, C-14), 163.08 (C, C-14’), 156.17 (C, C-13), 149.55 (C, C-31), 148.53 (C, C-15), 135.44 (C, C-22), 134.46 (CH, C-18), 133.96 (C, C-35), 131.60 (CH, C-33), 129.30 (CH, C-19), 128.92 (C, C-36), 128.50 (C, C-23), 127.20 (CH, C-17), 126.50 (CH, C-34), 125.56 (CH, C-21), 123.30 (CH, C-26), 121.04 (C, C-16), 119.80 (CH, C-25), 118.70 (CH, C-24), 116.20 (CH, C-32), 111.72 (CH, C-27), 108.80 (C, C-20), 51.80 (CH, C-1), 50.90 (CH, C-7), 50.78 (CH2, C-30), 49.30 (CH2, C-29), 41.10 (CH2, C-5), 31.80 (CH2, C-2), 30.90 (CH2, C-12), 30.50 (CH2, C-4), 29.41 (CH2, C-8), 29.25 (CH2, C-10), 28.59 (CH2, C-3), 23.23 (CH2, C-11).

Mass value: 973.2425, 975.2235, 977.2090.

26. Nα,Nε-bis-QZN1-Lys-Nα,Nβ-bis-Het1-Asp

1H NMR (DMSO-d6, 400 MHz) δ ppm: QZN1 = 1.16–1.37 (4H, m, –CH2), 2.53–2.71 (4H, m, –CH2), 7.41–7.95 (8H, m, ArH), 12.18 (2H, s, NH); Lys = 1.48–1.75 (6H, m, –β,γ,δCH2), 3.10–3.12 (2H, m, –εCH2), 4.17–4.31 (1H, m, –αCH), 8.02–8.05 (1H, m, –αNH), 8.32–8.40 (1H, m, –εNH); Asp = 2.71–2.85 (2H, m, –βCH2), 5.12 (1H, br s, –αCH), 6.77 (1H, d, –αNH); Het1 = 1.72–2.06 (8H, m, –CH2), 2.34–2.70 (2H, m, –CH), 2.92–3.14 (8H, m, –CH2), 6.72–7.81 (6H, m, ArH).

13C NMR (DMSO-d6, 100 MHz) δ ppm: 182.82 (C, C-10), 175.54 (C, C-11), 176.40 (C, C-11’), 173.73 (C, C-6), 172.92 (C, C-27), 172.74 (C, C-29), 167.41 (C, C-16), 166.96 (C, C-16’), 154.07 (C, C-15), 146.09 (C, C-17), 140.38 (C, C-25), 138.74 (CH, C-20), 131.97 (CH, C-21), 131.08 (CH, C-19), 129.82 (CH, C-31), 126.04 (C, C-18), 122.03 (C, C-26), 112.35 (CH, C-30), 108.34 (CH, C-28), 57.40 (CH, C-1), 54.12 (CH, C-7), 48.46 (CH2, C-22), 47.51 (CH2, C-5), 42.56 (CH2, C-8), 39.72 (CH2, C-2), 39.34 (CH2, C-14), 38.60 (CH2, C-23), 38.10 (CH2, C-4), 34.13 (CH2, C-12), 33.14 (CH, C-24), 27.02 (CH2, C-3).

Mass value: 1066.2468.

28. Nα,Nε-bis-QZN2-Lys-Nα,Nβ-bis-Het1-Asp

1H NMR (DMSO-d6, 400 MHz) δ ppm: QZN2 = 1.15–1.38 (8H, m, –CH2), 2.55–2.70 (4H, m, –CH2), 7.40–7.97 (8H, m, ArH), 12.17 (2H, m, NH); Lys = 1.47–1.76 (6H, m, –β,γ,δCH2), 3.12–3.14 (2H, m, –εCH2), 4.18–4.31 (1H, m, –αCH), 8.01–8.03 (1H, m, –αNH), 8.34–8.41 (1H, m, –εNH); Asp = 2.70–2.86 (2H, m, –βCH2), 5.10 (1H, br s, –αCH), 6.75 (1H, d, –αNH); Het1 = 1.76–2.09 (8H, m, –CH2), 2.35–2.72 (2H, m, –CH), 2.95–3.14 (8H, m, –CH2), 6.70–7.80 (6H, m, ArH).

13C NMR (DMSO-d6, 100 MHz) δ ppm: 183.87 (C, C-10), 176.57 (C, C-11), 176.44 (C, C-11’), 173.71 (C, C-6), 173.12 (C, C-27), 172.70 (C, C-29), 168.42 (C, C-16), 166.94 (C, C-16’), 154.06 (C, C-15), 147.20 (C, C-17), 139.34 (C, C-25), 138.77 (CH, C-20), 131.99 (CH, C-21), 131.06 (CH, C-19), 130.83 (CH, C-31), 126.05 (C, C-18), 122.01 (C, C-26), 112.38 (CH, C-30), 109.62 (CH, C-28), 57.32 (CH, C-1), 54.15 (CH, C-7), 49.42 (CH2, C-22), 48.52 (CH2, C-5), 43.52 (CH2, C-8), 39.72 (CH2, C-2), 39.36 (CH2, C-14), 39.05 (CH2, C-23), 38.94 (CH2, C-4), 34.09 (CH2, C-12), 33.19 (CH, C-24), 27.95 (CH2, C-3), 22.56 (CH2, C-13).

Mass value: 1100.3230.

30. Nα,Nε-bis-QZN1-Lys-Nα,Nγ-bis-Het1-Glu

1H NMR (DMSO-d6, 400 MHz) δ ppm: QZN1 = 1.20–1.23 (4H, m, –CH2), 2.57–2.70 (4H, m, –CH2), 7.04–7.86 (8H, m, ArH), 12.25 (2H, s, NH); Lys = 1.27–1.31 (6H, m, –β,γ,δCH2), 2.70–2.80 (2H, m, –εCH2), 4.18–4.37 (1H, m, –αCH), 8.01–8.02 (1H, m, –αNH), 8.00–8.03 (1H, m, –εNH); Glu = 2.30–2.47 (2H, m, –βCH2), 2.57–2.67 (2H, m, –γCH2), 4.78–4.82 (1H, q, –αCH), 6.78 (1H, d, –αNH); Het1 = 1.48–1.83 (8H, m, –CH2), 2.30–2.80 (2H, m, –CH), 2.95–3.38 (8H, m, –CH2), 7.04–7.86 (6H, m, ArH).

13C NMR (DMSO-d6, 100 MHz) δ ppm: 181.50 (C, C-10), 174.79 (C, C-11), 171.23 (C, C-11’), 167.93 (C, C-6), 165.42 (C, C-27), 163.59 (C, C-29), 162.04 (C, C-16), 157.16 (C, C-16’), 156.04 (C, C-15), 149.20 (C, C-17), 147.42 (C, C-25), 134.61 (CH, C-20), 133.92 (CH, C-21), 127.15 (CH, C-19), 126.30 (CH, C-31), 126.10 (C, C-18), 121.33 (C, C-26), 117.25 (CH, C-30), 107.61 (CH, C-28), 59.13 (CH, C-1), 55.20 (CH, C-7), 43.78 (CH2, C-22), 42.90 (CH2, C-5), 32.01 (CH2, C-8), 31.86 (CH2, C-2), 30.34 (CH2, C-14), 29.27 (CH2, C-23), 28.55 (CH2, C-4), 28.20 (CH2, C-9), 27.60 (CH2, C-12), 27.65 (CH, C-24), 23.64 (CH2, C-3).

Mass value: 1086.3115.

32. Nα,Nε-bis-QZN2-Lys-Nα,Nγ-bis-Het1-Glu

1H NMR (DMSO-d6, 400 MHz) δ ppm: QZN2 = 1.21–1.53 (8H, m, –CH2), 2.54–2.72 (4H, m, –CH2), 7.08–7.84 (8H, m, ArH), 12.24 (2H, s, NH); Lys = 1.28–1.33 (6H, m, –β,γ,δCH2), 2.78–2.84 (2H, m, –εCH2), 4.20–4.38 (1H, m, –αCH), 8.02–8.05 (1H, m, –αNH), 8.01–8.03 (1H, m, –εNH); Glu = 2.28–2.40 (2H, m, –βCH2), 2.58–2.62 (2H, m, –γCH2), 4.77–4.83 (1H, q, –αCH), 6.76 (1H, d, –αNH); Het1 = 1.48–1.84 (8H, m, –CH2), 2.23–2.78 (2H, m, –CH), 2.94–3.40 (8H, m, –CH2), 7.05–7.87 (6H, m, ArH).

13C NMR (DMSO-d6, 100 MHz) δ ppm: 176.25 (C, C-10), 174.80 (C, C-11), 172.32 (C, C-11’), 168.62 (C, C-6), 166.12 (C, C-27), 163.65 (C, C-29), 162.24 (C, C-16), 158.24 (C, C-16’), 156.08 (C, C-15), 149.22 (C, C-17), 146.42 (C, C-25), 134.66 (CH, C-20), 132.87 (CH, C-21), 127.18 (CH, C-19), 126.95 (CH, C-31), 125.15 (C, C-18), 121.28 (C, C-26), 118.35 (CH, C-30), 107.70 (CH, C-28), 59.25 (CH, C-1), 55.28 (CH, C-7), 44.81 (CH2, C-22), 42.82 (CH2, C-5), 33.03 (CH2, C-8), 31.45 (CH2, C-2), 30.74 (CH2, C-14), 28.78 (CH2, C-23), 28.50 (CH2, C-4), 28.11 (CH2, C-9), 27.28 (CH2, C-12), 27.08 (CH, C-24), 23.46 (CH2, C-3), 21.37 (CH2, C-13.

Mass value: 1108.2460.

36. Nα,Nε-bis-QZN1-Lys-Nα,Nβ-bis-Het2-Asp

1H NMR (DMSO-d6, 400 MHz) δ ppm: QZN1 = 1.50–2.10 (4H, m, –CH2), 2.12–2.64 (4H, m, –CH2), 6.92–7.80 (8H, m, ArH), 12.13 (2H, s, –NH); Lys = 1.18–1.35 (6H, m, –β,γ,δCH2), 2.99–3.05 (2H, m, –εCH2), 4.21–4.25 (1H, q, αCH), 8.01–8.06 (1H, m, –εNH), 8.01–8.06 (1H, m, –αNH); Asp = 2.57 (2H, t, –βCH2), 5.14–5.16 (1H, m, –αCH), 6.78–6.89 (1H, m, –αNH); Het2 = 2.55–3.03 (8H, m, –CH2), 3.34–3.70 (8H, m, –CH2), 6.98–7.94 (6H, m, ArH).

13C NMR (DMSO-d6, 100 MHz) δ ppm: 172.20 (C, C-11), 171.82 (C, C-10), 169.45 (C, C-6), 168.75 (C, C-29), 162.54 (C, C-16), 157.56 (C, C-15), 151.24 (C, C-24), 149.08 (C, C-17), 134.60 (CH, C-20), 129.40 (CH, C-26), 127.26 (CH, C-21), 126.65 (CH, C-19), 124.55 (CH, C-27), 121.62 (C, C-18), 116.25 (CH, C-25), 53.06 (CH, C-1), 51.95 (CH, C-7), 51.27 (CH2, C-23), 45.87 (CH2, C-22), 38.54 (CH2, C-5), 35.02 (CH2, C-8), 34.45 (CH2, C-2), 34.25 (CH2, C-14), 32.27 (CH2, C-4), 29.32 (CH2, C-12), 23.36 (CH2, C-3).

Mass value: 1086.1563, 1088.1540, 1090.1830.

37. Nα,Nε-bis-QZN2-Lys-Nα,Nβ-bis-Het2-Asp

1H NMR (DMSO-d6, 400 MHz) δ ppm: QZN2 = 1.48–2.09 (8H, m, –CH2), 2.11–2.58 (4H, m, –CH2), 6.90–7.74 (8H, m, ArH), 12.11 (2H, s, –NH); Lys = 1.17–1.34 (6H, m, –β,γ,δCH2), 2.98–3.04 (2H, m, –εCH2), 4.20–4.21 (1H, q, –αCH), 8.04 (1H, d, –εNH), 8.04 (1H, d, –αNH); Asp = 2.56 (2H, t, –βCH2), 5.13–5.14 (1H, m, –αCH), 6.76–6.90 (1H, m, –αNH); Het2 = 2.56–3.04 (8H, m, –CH2), 3.35–3.69 (8H, m, –CH2), 6.99–7.94 (6H, m, ArH).

13C NMR (DMSO-d6, 100 MHz) δ ppm: 172.18 (C, C-11), 171.85 (C, C-10), 169.32 (C, C-6), 168.70 (C, C-29), 162.20 (C, C-16), 157.42 (C, C-15), 151.17 (C, C-24), 149.04 (C, C-17), 134.59 (CH, C-20), 129.38 (CH, C-26), 127.25 (CH, C-21), 126.68 (CH, C-19), 124.65 (CH, C-27), 121.52 (C, C-18), 116.23 (CH, C-25), 53.03 (CH, C-1), 51.40 (CH, C-7), 51.18 (CH2, C-23), 45.80 (CH2, C-22), 38.78 (CH2, C-5), 35.42 (CH2, C-8), 34.65 (CH2, C-2), 34.32 (CH2, C-14), 32.20 (CH2, C-4), 29.34 (CH2, C-12), 23.36 (CH2, C-3), 23.22 (CH2, C-13).

Mass value: 1114.1906, 1116.1932, 1118.1832.

38. Nα,Nε-bis-QZN1-Lys-Nα,Nγ-bis-Het2-Glu

1H NMR (DMSO-d6, 400 MHz) δ ppm: QZN1 = 1.49–1.62 (4H, m, –CH2), 1.95–2.43 (4H, m, –CH2), 7.05–8.02 (8H, m, ArH) 12.13 (2H, s, NH); Lys = 1.17–1.33 (6H, m, –β,γ,δCH2), 2.80–2.84 (2H, m, –εCH2), 4.21–4.22 (1H, q, –αCH), 8.02 (1H, d, –εNH), 8.04–8.09 (1H, m, –αNH); Glu = 2.56–2.75 (4H, m, –β,γCH2), 4.80 (1H, q, –αCH), 7.01–7.02 (1H, m, –αNH); Het2 = 2.84–3.08 (8H, m, –CH2), 3.37–3.75 (8H, m, –CH2), 7.05–7.86 (6H, m, ArH).

13C NMR (DMSO-d6, 100 MHz) δ ppm: 172.13 (C, C-11), 171.63 (C, C-11’), 171.28 (C, C-10), 170.77 (C, C-6), 170.18 (C, C-29), 161.99 (C, C-16), 157.08 (C, C-15), 151.17 (C, C-24), 149.22 (C, C-17), 134.57 (CH, C-20), 129.36 (CH, C-26), 127.15 (CH, C-21), 126.68 (CH, C-19), 126.28 (CH, C-27), 121.45 (C, C-18), 116.27 (CH, C-25), 53.22 (CH, C-1), 51.65 (CH, C-7), 51.50 (CH2, C-23), 45.45 (CH2, C-22), 42.18 (CH2, C-5), 41.77 (CH2, C-8), 32.03 (CH2, C-2), 31.91 (CH2, C-14), 29.24 (CH2, C-9), 28.40 (CH2, C-4), 27.94 (CH2, C-12), 23.30 (CH2, C-3).

Mass value: 1100.1779, 1102.1680, 1104.1746.

39. Nα,Nε-bis-QZN2-Lys-Nα,Nγ-bis-Het2-Glu

1H NMR (DMSO-d6, 400 MHz) δ ppm: QZN2 = 1.50–1.70 (8H, m, –CH2), 1.94–2.44 (4H, m, –CH2), 7.04–8.02 (8H, m, ArH) 12.13 (2H, s, –NH); Lys = 1.18–1.35 (6H, m, –β,γ,δCH2), 2.78–2.85 (2H, m, –εCH2), 4.25–4.20 (1H, q, –αCH), 8.04 (1H, d, –εNH), 8.06–8.09 (1H, m, –αNH); Glu = 2.58–2.76 (4H, m, –β,γCH2), 4.80 (1H, m, –αCH), 6.98–7.01 (1H, m, –αNH); Het2 = 2.84–3.09 (8H, m, –CH2), 3.38–3.76 (8H, m, –CH2), 7.06–7.84 (6H, m, ArH).

13C NMR (DMSO-d6, 100 MHz) δ ppm: 172.12 (C, C-11), 171.79 (C, C-11’), 171.56 (C, C-10), 170.84 (C, C-6), 170.24 (C, C-29), 161.24 (C, C-16), 157.10 (C, C-15), 151.18 (C, C-24), 149.45 (C, C-17), 134.52 (CH, C-20), 129.30 (CH, C-26), 127.95 (CH, C-21), 126.93 (CH, C-19), 126.35 (CH, C-27), 121.46 (C, C-18), 116.45 (CH, C-25), 53.26 (CH, C-1), 51.69 (CH, C-7), 51.53 (CH2, C-23), 45.56 (CH2, C-22), 42.20 (CH2, C-5), 41.70 (CH2, C-8), 32.16 (CH2, C-2), 31.70 (CH2, C-14), 29.86 (CH2, C-9), 28.56 (CH2, C-4), 27.24 (CH2, C-12), 23.98 (CH2, C-3), 23.21 (CH2, C-13).

Mass value: 1128.1365, 1130.1653, 1132.1832.

Biological activities

Anti-inflammatory activity

Human erythrocyte suspension

The whole blood was collected from a healthy volunteer who had not taken any NSAIDS for 2 weeks prior to the experiment and collected in heparinized vacutainer. The blood was washed three times with 0.9% saline and centrifuged simultaneously for 10 min at 3000 rpm. The packed cells were washed with 0.9% saline and 40% v/v suspension made using isotonic phosphate buffer, which was composed of 154 mM NaCl in 10 mM sodium phosphate buffer at pH 7.4 used as Stock erythrocyte or RBC suspension.

Hypotonic solution-induced hemolysis

The membrane stabilizing activity of the compounds was assessed according to the reported method (Shinde et al. 1999) with slight modification. The test sample consisted of stock erythrocyte (RBC) suspension 0.5 mL mixed with 5 mL of hypotonic solution (50 mM NaCl in 10 mM sodium phosphate buffered saline at pH 7.4) containing different concentrations of sample (25, 50, 100, 200, and 300 µg/mL). The control consists of 0.5 mL RBC suspension mixed with 5 mL of hypotonic buffered solution alone. The standard drugs indomethacin and ibuprofen was treated similar to test concentration. The experiment was carried out in triplicate. The mixtures were incubated for 10 min at room temperature, centrifuged for 10 min at 3000 rpm and absorbance of the supernatant was measured spectrophotometrically at 540 nm. The percentage inhibition of hemolysis or membrane stabilization was calculated from the following equation.

\(\% {\mathrm{inhibition}}\,{\mathrm{of}}\,{\mathrm{haemolysis}} = \left[ {\frac{{A_1 - A_2}}{{A_1}}} \right] \times 100\) where: A1 = Absorbance of hypotonic buffered solution alone. A2 = Absorbance of test/standard sample in hypotonic solution

Antibacterial activity

In vitro antibacterial activity was evaluated against human pathogens of both Gram-negative pathogen E. coli and Gram-positive organism S. aureus by agar-well diffusion method (Perez et al. 1990) as well as a microdilution method (Rios et al. 1988) with slight modifications.

Agar-well diffusion method

The microorganisms were inoculated in to the sterilized nutrient broth and maintained at 37 °C for 24 h. On the day of testing, bacteria were subcultured separately into 25 mL of sterilized nutrient broth. Inoculated subculture broths were kept at room temperature for the growth of inoculums. Each test compound and standard drug of 10 mg was dissolved in 10 mL of DMSO to get a concentration of 1 μg/mL and further diluted to get a final concentration of 50 μg/mL. About 15–20 mL of molten nutrient agar was poured into each of the sterile plates. With the help of cork borer of 6 mm diameter, the cups were punched and scooped out of the set agar and the plates were inoculated with the suspension of particular organism by spread plate technique. The cups of inoculated plates were then filled with 0.1 mL of the test solution, streptomycin solution, and DMSO (negative control). The plates were allowed to stay for 24 h at 37 °C and zone of inhibition (mm) was then measured.

Microdilution method

All the microorganisms were grown in Muller-Hinton broth. After cultivation for 16–18 h at 37 °C, the bacteria were harvested and their density was determined by measuring O.D at A600. MIC of the compounds was determined by agar dilution method. Suspension of each microorganism was prepared to contain approximately (1 × 104 – 2 × 104 CFU/mL) and applied to the plates with serially diluted compounds (dissolved in DMSO) to be tested and also reference drug and incubated at 37 °C overnight. The MIC was considered to be the lowest concentration that completely inhibited the growth of microorganisms on the plates. Zone of inhibition (mm) was measured after 24 h and MIC values were determined.

Antifungal activity

In vitro antifungal activity was evaluated against two fungal species namely F. moniliforme and A. niger by agar-well diffusion method (Singh and Singh 2000) as well as a microdilution method (Balouiri et al. 2016) with slight modifications.

Agar-well diffusion method

The fungal strains were subculture separately into 25 mL of sterilized nutrient broth and compounds and standard drug (bavistin) of 10 mg was dissolved in 10 mL of DMSO to get a concentration of 1 mg/mL and further diluted to get a final concentration of 50 μg/mL. Molten media of sabouraud agar of 10–15 mL was poured into the petri plates and allowed to solidify. Fungal subculture was inoculated on the solidified media. With the help of 6 mm cork borer, the cups were punched and scooped out of the set agar. The cups of inoculated plates were then filled with 0.1 mL of the test solution, bavistin solution, and DMSO (negative control). The plates were allowed to stay for 3 days at room temperature and zone of inhibition (mm) was then measured.

Microdilution method

Sabouraud agar was used for the preparation of plates. A suspension of each microorganism was prepared to contain 105 CFU/mL. The agar plates were inoculated with fungal strains and serially diluted test compounds and the reference drug dissolved in DMSO. The plates were incubated at 25 °C for 48–72 h. The minimum inhibitory concentration was considered to be the lowest concentration that completely inhibited the growth of microorganisms on the plates. The zone of inhibition (mm) was measured after 48 h and MIC values were determined.

Molecular docking studies

Maestro 9.3.5 version of the Schrodinger software suite, 2011 was used to obtain binding interaction of molecules with target site. The 3D crystallographic structure of proteins (PDB ID: 1KZN) was retrieved from Protein Data Bank (www.rcsb.org/pdb). The lowest energy states of ligand with combination of all stereoisomers were achieved using LigPrep program and it was optimized by force field OPLS-2005 (Optimized Potential for Liquid Simulations). The protein structures were pre-processed, modified, and refined by Protein Preparation Wizard. Further, it was minimized by OPLS-2005 force field. The protein and ligand interaction performed by generation of receptor gridin, the target site of protein by GLIDE. Depending on the extent of interaction docking scores were produced it will determine the best fitted ligand to target protein.

Notes

Acknowledgements

We gratefully acknowledge Department of Science and Technology (DST) New Delhi for awarding Inspire Fellowship, University Grant Commission (UGC) New Delhi for awarding BSR faculty fellowship and UGC-Post doctoral Fellowship (PDFSS). We also acknowledge DST-Purse and Instrumentation facility.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

44_2018_2168_MOESM1_ESM.docx (7.7 mb)
Supplementary Information(DOCX 7906 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Honnegowdanahally K. Kumara
    • 1
  • Suhas Ramesh
    • 1
  • Doddahindaiah M. Suyoga Vardhan
    • 1
  • J. Shiva Kumar
    • 1
  • Dase Channe Gowda
    • 1
  1. 1.Department of Studies in ChemistryUniversity of MysoreMysuruIndia

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