Immunomodulatory properties of S - and N -alkylated 5-(1 H -indol-2-yl)-1,3,4-oxadiazole-2(3 H )-thione

105

Introduction

Polymorphonuclear leukocytes (PMNL) or neutrophils are the major phagocytes of the innate immune system. They play an important role in host defense against fungal and bacterial infections1. When these cells are exposed to an opsonized microbial particle, production of series of reac-tive oxygen species (ROS) is initiated in a process known as oxidative burst. Those ROS may be released from the cells and promote inflammatory responses or cause tissue damage (oxidative damage). In general, because there is a balance between the formation and the elimination of ROS in a healthy living organism, an exogenous substance that balances the physiological ROS levels may be useful to maintain homeostasis and control the inflammatory disor-ders2. On the other hand, suppression of T-cell activity and proinflammatory cytokines are required for prevention

or minimization of the immune-mediated rejection of transplanted organs and also modulation of the autoim-mune diseases3,4. Chemotherapeutic cytotoxic pharma-ceuticals such as Azathioprine (AZA), Mizoribine (MZ), Leflunomide and Bisindolylmaleimide VIII (BM VIII) (Figure 1) possess potent immunosuppressive activities that make them candidates for the treatment of diseases characterized by immunologic function disorders. Thus, Azathioprine has been used to suppress the rejection of transplanted human kidneys, anticancer and modulates autoimmune diseases5,6. Mizoribine has been marketed in Japan as an immunosuppressive agent for renal trans-plantation7. Leflunomide has been used as a drug for the treatment of rheumatoid arthritis and other autoimmune diseases8. Bisindolylmalieimide suppressed experimental allergic encephalomyelitis and adjuvant arthritis in rats9.

ReseaRch aRtIcle

Immunomodulatory properties of S- and N-alkylated 5-(1H-indol-2-yl)-1,3,4-oxadiazole-2(3H)-thione

El Sayed H. El Ashry1,2, El Sayed H. El Tamany3, Mohy El Din Abd El Fattah3, Mohamed R. E. Aly3, Ahmed T. A. Boraei1,3, M. Ahmed Mesaik4, Omer M. Abdalla4, Beenish Fatima4, Almas Jabeen4, Ahmed Shukrulla4, and Samreen Soomro4

1International Center for Chemical and Biological Sciences, H.E.J. Research Institute of Chemistry, Karachi University, Karachi, Pakistan, 2Chemistry Department, Faculty of Science, Alexandria University, Alexandria, Egypt, 3Chemistry Department, Faculty of Science, Suez Canal University, Ismailia, Egypt, and 4Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, Karachi University, Karachi, Pakistan

abstractA series of S- and N-alkylated indolyloxadiazoles 2–7 were prepared. All compounds were tested for their immunomodulatory activity against T-cell proliferation, oxidative burst and cytokine analysis. Compounds 1, 2a, 2b, 2c and 2k demonstrated highly significant (P ≤ 0.005) inhibition on PHA activated T-cell proliferation with IC50 less than 3 µg/mL concentration, while 3b exert a moderate inhibitory effect with IC50 8.6 µg/mL. Among all compounds of the series, only 2h was found to suppress phagocytes ROS production (IC50 2.4 µg/mL) in luminol-based chemiluminescence (CL) assay. Compounds 2a-k have stimulatory effect on proinflammatory cytokine predominantly IL-1β but no effect on IL-4 and NO production indicating that these compounds might have selective inhibitory effect on T-cell proliferation. Cytotoxic effect on T-cell proliferation was tested on NIH-3T3 mouse fibroblast normal cell line. All compounds were found to be free from toxic effects up to 100 μM concentration.

Keywords: Indolyloxadiazoles, reactive oxygen species (ROS), oxidative burst, T-cell proliferation, immunosuppressant

Address for Correspondence: El Sayed H. El Ashry, International Center for Chemical and Biological Sciences, H.E.J. Research Institute of Chemistry, Karachi University, Karachi 75270, Pakistan. Tel: +203-4246601. Fax: +203-4271360. E-mail: [email protected]

(Received 18 August 2011; revised 16 October 2011; accepted 24 October 2011)

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(1): 105–112© 2013 Informa UK, Ltd.ISSN 1475-6366 print/ISSN 1475-6374 onlineDOI: 10.3109/14756366.2011.636361

Journal of Enzyme Inhibition and Medicinal Chemistry

2013

28

1

105

112

18 August 2011

16 October 2011

24 October 2011

1475-6366

1475-6374

© 2013 Informa UK, Ltd.

10.3109/14756366.2011.636361

GENZ

636361

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106 E. S. H. El Ashry et al.


The relevance of indole derivatives is well known due to their diverse pharmacological activities10,11, such as antitumor12, anticonvulsant, tranquilizing13, antibacte-rial, antifungal, antiviral14 and anti-inflammatory15 activi-ties. They are also used as physiological modulators, such as inhibitors of human liver glycogen phosphorylase16, aromatase17 and human cytosolic phospholipase A

2α18.

The1,3,4-oxadiazoles have showed anti-inflammatory, analgesic19, antimicrobial20, antimalarial21, antiprolifera-tive22, and antituberculosis23 activities. Literature survey revealed that, coupling of two biodynamic heterocy-clic systems emerges conjugates of talented biological activities, for instance, indolyloxadiazoles were reported as efficient anti-inflammatory24,25 and antibacterial26 candidates.

Considering the above aspects, indoles functionalized with heterocycles had been designed and in the pres-ent study their immunomodulatory activity has been evaluated in order to find new immunosuppressants that can avoid the fatal side effects of the present drugs and also assist in alleviation of the problems associated with immune response.

Results and discussions

ChemistryThe site and/or regioselectivity during the alkylation of indolyloxadiazole thione 127 was dependent on the reac-tion conditions28. Thus, the alkylated derivatives 2 and 3 were prepared. The S→N allylic rearrangement of the allyl-thioether 2k and 5 gave 4 and 6, respectively (Figure 2).

Immunomodulatory activityIn order to study the immunomodulating potential of the synthesized compounds, the anti-proliferative effect of the tested compounds was evaluated in phytohemag-glutinin (PHA) induced T-cell proliferation assay. Results presented in Table 1 and Figure 3 indicated that com-pounds 1, 2a, 2b, 2c and 2k have immunomodulating

activities. They significantly (P ≤ 0.05) suppressed T-cell proliferation in a dose dependent manner with IC

50

ranges between 1 and 3 µg/mL, while compound 3b had a moderate inhibitory activity with IC

50 = 8.6 µg/mL. A

weak inhibitory effect was observed for compounds 2e and 5 (Table 1, Figure 3). Although this study did not ascertain the details of cellular mechanism effects of the active compounds, it demonstrated that they can affect the immune functional status and could be considered as attractive lead candidates for development of immu-nomodulatory drugs.

In order to look into effect of these compounds on the innate immune response, the whole blood phagocytes respiratory oxidative burst response was analyzed in the presence of these compounds. The luminol dependant CL assay is a sensitive indicator of phagocytes respiratory burst which is characterized by an increase in the reac-tive oxygen species (ROS) production in the presence of serum opsonize zymosan as an activator for phago-cytosis process. Results of this study showed no signifi-cant activity for most of the compounds on professional phagocytes ROS release after activation with the serum

Figure 1. Structures of immunosuppressive drugs.

Figure 2. S-Alkylated and N-alkylated indolyl-oxadiazole-thione.

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Immunomodulatory properties 107


opsonized zymosan, except for compound 2h (Table 1). This compound 2h was found to possess an inhibitory activity with an IC

50 of 2.4 µg/mL after 30 min incuba-

tion with human whole blood pahgocytes, indicating its effect on innate immune response. In addition to ROS production, effect of these compounds on Nitric Oxide (NO) generation by stimulated macrophages was also monitored. The model of LPS treated? J774.2 has been used to study the mechanism of nitric oxide synthase (NOS-2) induction which could be easily tracked down by measurement of accumulation of the nitrite ion in culture medium. Most of the compounds were found to be inactive against nitric oxide production at 25 μg/mL concentration except 2b that showed significant inhibi-tion (52%) (Table 1).

To evaluate the effect of the synthesized compounds on the proinflammatory cytokines, the production of TNF-α and IL-1β were tested (Table 1). Most of the studied compounds showed moderate to very low level of inhibition on TNF-α (3–49%) production from THP-1 cells while compounds 2a, 2c, 2g and 2k showed signifi-cant stimulation (>50%). Only compound 1 was found to have potent stimulatory effect on TNF-α production, 163.78% at 25 μg/mL concentration. In case of IL-1β, most of the compounds found to cause stimulatory effect with seven of these compounds 1, 2b, 2c, 2g, 2i, 2k and 5 have highly significant (P ≤ 0.005) stimulatory effect in 102, 154, 183, 201, 200, and 117%, respectively. On the other hand, only 2n showed 53% inhibition whereas the rest exerted moderate to low level (1.7–32%) of inhibition.

Table 1. The IC50

effect of compounds on oxidative burst of whole blood, and T-cell Proliferation.

Compound No

IC50

(µg/mL) % Inhibition

Oxidative Burst T cell Proliferation IL-4 IL-2 IL-1β TNF-α NO1 29.8 <3.12 >25 >50 −102.33 −163.78 25.382a 43.7 ± 3.6 <3.12 >25 34.9 −74.98 −55.62 -2b >100 <3.12 >25 >50 −153.93 −4.64 52.682c >100 <3.12 >25 >50 −183.40 −78.80 7.872d >100 >50 - 32.64 32.45 1.452e >100 26.1 ± 3.7 - −95.71 13.91 −3.612f >100 >50 - −92.18 21.63 3.212g >100 >50 - −201.57 −63.35 4.482h 2.4 ± 1.3 >50 - −86.89 13.91 8.002i >100 >50 - −107.18 10.82 4.542j >100 >50 - −11.92 −42.90 1.722k >100 <3.12 >25 >50 −200.24 −78.80 8.762l >100 >50 - - - -2m >100 >50 - 14.11 30.90 -2n >100 >50 - 52.93 6.45 -3a >100 >50 - −26.91 13.91 9.503b >100 8.6 ± 0.7 - >50 −4.85 33.99 -3c >100 >50 - 1.76 3.09 −0.594 >100 >50 - 22.05 49.44 −0.995 >100 28.3 ± 2.6 - −117.32 12.36 3.036 >100 >50 - 8.82 20.09 11.537 >100 >50 - - - 3.27Control A+ 11.2 ± 1.9 - Control B++ - 0.2 < 0.5 < 0.5 - - -+Control A = Ibuprofen, ++Control B = Prednisolone.

Figure 3. Effect of eight compounds on PHA T-cell proliferation compared with the control. Control +ve = PHA activated cells with no compound. Control –ve = cells alone with no PHA or compound.

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We could state here that none of the tested compounds has potent inhibition on the proinflammatory cytokines, nevertheless, few compounds are potent stimulators.

Interleukin (IL)-4 is a cytokine able to modulate the activity of cells in all hematopoietic lineages. It is best known for its ability to support the TH2 arm of the immune response and enables B cells to produce antibodies and in particular the IgE which is known to play a prominent role in the pathogenesis of allergic reactions and in resis-tance to parasitic infections. Therefore, inhibition of IL-4 was targeted by natural and synthetic compounds. The active compounds that inhibit T cell proliferation were tested for their effect on IL-4 cytokine. However, they did not elucidate any effect on IL-4 secretion from human peripheral blood. In another set of experiment optimi-zation of the IL-2 production through T cells activation by using different mitogens was done, the optimum IL-2 production was found when combination of PHA and PMA was applied (5 µg/mL and 20 ng/mL respectively) as shown in Figure 4. Then, T-cells were challenged with 3 different concentrations of test compounds (2a, 2b, 2c, 2k, 1 and 3b) for IL-2 production after activation. Compound 2a was found to suppress the IL-2 production with an IC

50 of 34.9 µg/mL while the rest of compounds

did not exert any effect on the IL-2 production (IC50

≥ 50µg/mL). These differences between the T cell prolifera-tion and IL-2 production may be due to the difference in activating signals used for T cell proliferation (PHA alone) and the induction of IL-2 by peripheral blood mononu-clear cells used in our system. It was reported earlier that the use of PMA and PHA together allow for broad range stimulation of cytokines and multiple signaling pathways in mixed PBMC cultures29.

The effect of the active compounds on PHA activated T-cells and their cytotoxic effect on the NIH 3T3 mouse embryonic fibroblast cell line was tested at concentra-tions of 1, 10 and 100 µM. None of the tested compounds revealed toxic effect on fibroblast as cells showed viabil-ity between 80–100% at the highest concentration tested (100 µM).

These biological studies has revealed that substi-tution at 2-position of the indole nucleus with the

oxadiazolyl moiety markedly enhanced its biological activity. Moreover, the alkyl moiety seems to have a role in the suppression enhancement. The introduction of the hydroxyl group in 2a, 2b 2c and the allyl group in 2k improved the activity of the parent compound 1. In addi-tion to that, among all the synthesized compounds only 2h was found to suppress phagocytes ROS (IC

50 2.4 µg/

mL) in luminol-based chemiluminescence (CL) assay and this may be attributed to the presence of the ester group.

conclusions

A series 2–7 of alkylated 5-(1H-indol-2-yl)-1,3,4-oxadiazole-2(3H)-thione 1 were tested for their effect on the activity of three immunomodulatory systems, T-cell proliferation, oxidative burst and cytokine stud-ies. The presence of the NH group in the thione 1, the hydroxyl group in 2a, 2b, 2c and the allyl group in 2k seems to be responsible for the inhibitory effect on PHA activated T-cell proliferation (P ≤ 0.005). On the other hand, these compounds did not show any effect on IL-4 secretion, NO production except compound 2b which has significant inhibition on NO and ROS suppression apart from compound 2h (IC

50 = 2.4 ± 1.3).

In case of proinflammatory cytokines only com-pound 1 found to stimulate TNF-α production, while, compounds 1, 2b, 2c, 2g, 2i, 2k and 5 were found to have potent stimulatory effect on IL-1β. This shows that these compounds enhance the immune system through the production of proinflammatory cytokines but inhibit the cellular immune response selectively by inhibiting T-cell proliferation.

experimental

SynthesesThe following compounds were prepared as reported in literature28:

5-(1H-indol-2-yl)-1,3,4-oxadiazole-2(3H)-thione (1)2-(2-Hydroxyeth-1-ylsulfanyl)-5-(1H-indol-2-yl)-1,

3,4-oxadiazole (2a)

Figure 4. Effect of three mitogens (PHA, Concanavalin-A and PMA) on the production of IL-2 by PBMNCs. Cells were activated by different doses of either mitogen or combination of each used as µg or ng/mL concentartion. Negative control represents cells without activation. Each bar represents a mean ± SD of three replicates.

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2-(3-Hydroxyprop-1-ylsulfanyl)-5-(1H-indol-2-yl)-1, 3,4-oxadiazole (2b)

2-(2-Hydroxyprop-1-ylsulfanyl)-5-(1H-indol-2-yl)-1, 3,4-oxadiazole (2c)

2-(2,2-Diethoxyeth-1-ylsulfanyl)-5-(1H-indol-2-yl)-1, 3,4-oxadiazole (2d)

5-(1H-indol-2-yl)-2-Phenacylsulfanyl-1,3,4-oxadiaz-ole (2e)

2-(t-Butyloxycarbonylmethylsulfanyl)-5-(1H-indol- 2-yl)-1,3,4-oxadiazole (2f)

2-Cyanomethylsulfanyl-5-(1H-indol-2-yl)-1,3,4-ox-adiazole (2g)

2-Ethoxycarbnylmethylsulfanyl-5-(1H-indol-2-yl)-1, 3,4-oxadiazole (2h)

5-(1H-indol-2-yl)-2-propylsulfanyl-1,3,4-oxadiazole (2i)2-Butylsulfany-5-(1H-indol-2-yl)-1,3,4-oxadiazole (2j)2-Allylsulfanyl-5-(1H-indol-2-yl)-1,3,4-oxadiazole (2k)2 - B e n z y l s u l f a n y l - 5 - ( 1 H- i n d o l - 2 - y l ) - 1 , 3 , 4 -

oxadiazole (2l)5-(1H-indol-2-yl)-2-(undec-1-ylsulfanyl)-1,3,4-

oxadiazole (2m)2-Carboxymethylsulfanyl-5-(1H-indol-2-yl)-1,3,4-

oxadiazole (2n)5 - ( 1 H- i n d o l - 2 - y l ) - 3 -p ro p y l - 2 - t h i o x o - 1 , 3 , 4 -

oxadiazole (3a)3 - B u t y l - 5 - ( 1 H- i n d o l - 2 - y l ) - 2 - t h i o x o - 1 , 3 , 4 -

oxadiazole (3b)5-(1H-indol-2-yl)-2-thioxo-3-(undec-1-yl)-1,3,4-

oxadiazole (3c)3-Allyl-5-(1H-indol-2-yl)-2-thioxo-1,3,4-oxadiazole (4)5-(1-Allyl-1H-indol-2-yl)-2-(allylsulfanyl)-1,3,4-

oxadiazole (5)3-Allyl-5-(1-allyl-1H-indol-2-yl)-2-thioxo-1,3,4-

oxadiazole (6)5-(1-Benzyl-1H-indol-2-yl)-2-benzylsulfanyl-1,3,4-

oxadiazole (7)

Biological assayReagents, chemicals, and equipmentsLuminol (3-aminophthalhydrazide) was purchased from Research Organics, while Hanks balance salts solution (HBSS), Dulbecco’s Modified Eagle’s Medium (DMEM) and Phytohemagglutinin (PHA) were purchased from Sigma-Aldrich (Steinheim, Germany). RPMI-1640 and Lymphocytes separation medium (LSM) were purchased from MP Biomedicals, Inc., (Solon, Ohio, USA). Zymosan-A (Saccharomyces cerevisiae origin), dimethylsulfoxide (DMSO) Diacolone kit for IL-4 was purchased from France. The Luminometer used was Luminoskan RS, from Labsystem (Helsinki, Finland), the β-Scintillation counter-LS 6500 was from Beckman Coulter, (Fullerton, CA, USA), the cell harvester was from Inotech 1H

280, (Dottikon, Switzerland) and Spectra

Max plus 340 from Molecular Devices (CA, USA). THP-1 (Human monocytic leukemia cells) and J774.2 (mouse macrophages) were purchased from ECACC (European Collection of Cell Cultures, UK). Lipopolysaccharide B of E. coli 0111:B4 was purchased from DIFCO Laboratories

(michigon, USA), Phorbol myristate acetate (PMA) was obtained from SERVA (Heidelberg, Germany). Human TNF-α, and IL-1β Duo set ELISA Kits were purchased from R&D Systems (Minneapolis, USA), Griess reagent from (Leicestershire, UK).

Human peripheral blood mononuclear cells isolationHuman mononuclear cells were isolated from hepa-rinized peripheral blood of healthy volunteers by Histopaque-1077 density gradient centrifugation. They were adjusted to 2 × 106 cells/mL in RPMI-1640 medium supplemented with 5% heat-inactivated FBS, 2 mM glu-tamine and 50 μg/ml of gentamicin (designated thereaf-ter as culture medium).

T-Cell proliferation assayThe T-cell proliferation assay was performed as described by Nielson et al.30. In this assay, isolated lymphocytes were stimulated by adding the Phytohemagglutinin (PHA) in the culture. The rate of proliferation and survival was mea-sured by radio-labelled thymidine incorporation method. The lymphocytes were isolated by ficoll gradient centrifu-gation. A 50 μL of cell suspension (106/mL) was added to each well of a 96 were is the dimension well round bottom tissue culture plate. 50 μL of PHA was added in each well giving a final concentration of 5 μg/mL. The plates were incubated at 37C for 72 h (90% humidity, 5% CO

2/air).

Methyl-3H thymidine (Amersham Pharmacia Biotech) 0.5 μCi was added in each well and incubated for further 18 h. Cells were harvested on a filter mat (Type G-7) using cell harvester (Inotech−IH-280, Switzerland). The radioactiv-ity was measured using a β-scintillation counter (LS 6500, Beckman Coulter, USA). The concentration of compound dose giving 50% of inhibition of [3H] Thymidine incorpo-ration was calculated by comparing the arithmetic mean of counts per minute (CPM) of compound treated cells with that of the non-treated cells (control cells).

Oxidative burst studyLuminol-enhanced chemiluminescence assay was per-formed as reported31. Briefly, 25 μL diluted whole blood (1:50 dilution in sterile HBSS++) was incubated with 25 µL of serially diluted compounds with concentration ranges between 3.1 – 100 μg/mL. Control wells received HBSS++ and cells but no compounds. Tests were per-formed in white 96? well plates, which were incubated at 37°C for 30 min in the thermostated chamber of the luminometer. Opsonized zymosan-A, 25 μL, followed by 25 µL luminol (7 × 10−5 M) along with HBSS++ were added to each well to obtain a 100 µL volume/well. The luminometer results were monitored as chemilumi-nescence relative high unit (RLU) with peak and total integral values set for repeated scans at 30 s intervals and 1 s points measuring time.

Measurement of proinflammatory cytokines (TNF-α and IL-1β)THP-1 cells were grown in RPMI–1640 supplemented with 10% FBS, 2 mmol/L L-glutamine, 5.5 mmol/L

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glucose, 50 µmol/L mercaptoethanol, 1 mmol/L sodium pyruvate, and 10 mmol/L HEPES, until they attained 70% confluency. Cells were then plated in 24-well tis-sue culture plates at a concentration of 2.5 × 105 cells/mL and were differentiated into macrophage like cells by using phorbol myristate acetate (PMA) at a final concentration of 20 ng/mL and incubated for 24 h at 37°C in 5% CO

2. Cells were then stimulated with LPS

(bacterial lipopolysacchride) at a final concentration of 50 ng/mL and treated with compounds at a concentra-tion of 25 µg/mL and incubated for 4 h at 37°C in 5% CO

2. The supernatants collected after 4 h incubation

were analyzed for the level of TNF-α and for 18 h incu-bation were analyzed for IL-1β level. Cytokines quan-tification in supernatants were performed by human TNF-α and IL-1β Duo set Kits and according to manu-facturer’s instructions32.

IL-4 measurementsPBMCs were resuspended at a final concentration of 1 × 106 cells/ml in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glu-tamine, and 100 U/ml penicillin/streptomycin at 37°C in a moist atmosphere of 5% CO

2. Cells were cultured alone

or in the presence of the mitogen PHA 5 μg/mL or in the presence of variable concentrations of test compounds (1, 5, and 25 µg/mL) and the PHA in a 96 well flat-bottomed plate33. The supernatant was collected for IL-4 deter-mination after 18 h of culture and measured by using a Diaclone colorimetric immunoassay kit. The test is a solid phase sandwich ELISA (Enzyme-Linked Immunosorbent Assay). It utilizes a monoclonal antibody specific for IL-4 coated on a 96-well plate. 100 μL Standards and samples were added to the wells and any IL-4 present bind to the immobilized antibody. The wells were aspirated, and washed 3 times after 2 h incubation at room temperature and 100 μL Streptavidin-horseradish peroxidase conju-gate mixed with biotinylated anti-human IL-4 antibody is added, producing an antibody-antigen-antibody “sandwich”. The wells are again aspirated, and washed 3 times after 2 h incubation, then 100 μL TMB substrate solution is added, incubated for 30 min at room tempera-ture which produces a blue color in direct proportion to the amount of IL-4 present in the initial sample. The Stop Solution changes the color from blue to yellow, and the micro well absorbances were read at 450 nm.

Interleukin-2 (IL-2) assayThe effect of the synthesized compounds on IL-2 pro-duction was studied following our recently reported work34 with slight modification. In brief, the peripheral blood mononuclear cells were cultured in a 96 well flat-bottomed plate (1.0 × 105 cell/well) in the presence or absence of three concentrations of test compounds (0.5, 5, and 50 µg/mL), and phytohemagglutinin (PHA)/Phorbol myristate acetyl (PMA) in a final concentration of 5 µg/mL and 20 ng/mL respectively. After an incubation period of 18 h at 37°C in a humidified atmosphere of 5%

CO2 in air, the supernatant was collected for IL-2 deter-

mination. Interleukin-2 levels were measured by using enzyme-linked immunosorbent assay (ELISA) develop-ment kit (R&D systems, Minneapolis, MN, U.S.A.). Plates were then read at 450 nm in a plate reader (DIAReader GMBH, Wr. Neudorf, Austria) and results were analyzed using Microsoft Excel software.

Measurement of nitric oxideThe mouse macrophage cell line J774.2 was cultured in 75 cm2 flasks in DMEM that contain 10% fetal bovine serum supplemented with 1% streptomycin-penicillin. Flasks were kept at 37°C in atmosphere of humidified air containing 5% CO2. The cells were seeded in 24-well plates 1 × 106 cells/mL. Compounds at 25 µg/mL con-centration were added and the Nitric oxide synthase (NOS-2) in macrophages was induced by LPS from Escherichia coli serotype 0111:B4 at 20 µg/mL. Finally, Cell Culture supernatant was collected 48 h after LPS stimulation. Nitrite accumulation in J774.2 cell culture supernatant was measured using the Griess reagents as described previously33. Briefly, equal volume of 1% sul-phanilamide in 2.5% phosphoric acid, and 0.1% N-(1-naphtyl)-ethylene diamine in 2.5% phosphoric acid were added to culture medium. After 10 min of incu-bation at 23°C the absorbance at 550 nm was recorded. Micro molar concentration of nitrite was calculated from standard curve constructed with sodium nitrite [reference compound].

Cytotoxicity assayCytotoxic activity of compounds was evaluated in 96-well flat-bottom micro titer plates by using the stan-dard MTT (3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyl-tetrazolium bromide) colorimetric assay34. In this assay the 3T3 cell line (mouse embryo fibroblast cell) was cul-tured in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% of fetal bovine serum (FBS), 1% penicillin-streptomycin in 75 cm2 tissue culture flask and kept in 5% CO

2 incubator at 37°C. Exponentially

growing cells were harvested, counted with haemocy-tometer and diluted with DMEM. Cell culture with the concentration of 2.5 × 105 cells/mL was prepared and added in 100 µL portions to each well of the 96-well plates. After overnight incubation, the supernatant was carefully removed and 200 µL of fresh medium was added with 100, 10 and 1 μM of each compound. After 48 h, 50 µL MTT (2 mg/mL) was added to each well and incubated for further 4 h. Then 100 µL of DMSO was added to each well after proper aspiration of the MTT. The extent of MTT reduction to formazan within cells was calculated by measuring the absorbance at 540 nm; using a micro plate reader. The cytotoxicity was recorded as concentration causing 50% growth inhibition (IC

50).

Statistical analysisResults are expressed as mean values ± SEM (standard error of the mean). The statistical significance of the

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difference between control and treated samples was calculated by the Student’s t-test. The differences were considered to be highly significant at P ≤ 0.05.

acknowledgement

The authors thank Prof. Dr. Atta-Ur-Rahman and Prof. Dr. M. I. Choudary for their valuable discussions. The technical assistance of M. Asif is highly appreciated.

Declaration of interest

The support from the Higher Education commission (HEC) Pakistan Project No, 20-1444/R&D/09 2196 is highly appreciated.

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Documents

Immunomodulatory properties of S - and N -alkylated 5-(1 H -indol-2-yl)-1,3,4-oxadiazole-2(3 H )-thione