Antiinflammatory and Antiproliferative Activity of Naphthoxazole, Fused Hetero-benzoxazole and Bridged Benzobicyclic Photoproducts

Biological activity of naphthoxazoles, fused hetero-benzoxazoles and benzobicyclo[3.2.1]-derivatives was investigated in proliferation and inflammation based assays. The tested compounds were prepared by photocylization or photocycloaddition reactions. Effect of compounds on proliferation of several cancer cell lines was determined by measuring cell metabolic activity through time. Lipopolysaccharide (LPS) stimulation of peripheral blood mononuclear cells (PBMC) was used to investigate antiinflammatory properties of the compounds. Several naphthoxazoles and fused hetero-benzoxazoles inhibited TNFα protein expression in LPS stimulated PBMC, indicating possible antiinflammatory role which would be interesting to further investigate. Physico-chemical properties of tested compounds have been also studied using chromatographic lipophilicity measure, chrom logD and logP was calculated as the importance of physico-chemical properties of compounds at early stage of discovery of new drugs is well established. The similarities in structure and activity of some representative compounds affirm the need to further address their antiinflammatory properties.


INTRODUCTION
ETEROCYCLIC compounds are the most widely used class in pharmaceutical and agrochemical industries.They exhibit numerous biological activities, among which are antibacterial, antifungal, antiviral, antiinflammatory, antidepressant etc. [1] For example, pyrazolone derivatives, phenazone, metamizole, aminophenazone, phenyl buta-zone and apazone are non-steroidal antiinflammatory drugs (NSAID) that exhibit antiinflammatory, analgesic and antipyretic activity due to inhibition of cyclooxygenase (COX) enzymes. [2]5] In general the oxazole moiety [6] and the bicyclo[3.2.1]-core [7] are important building blocks for the synthesis of many biologically active molecules.Some of the fused polycyclic compounds with the oxazole ring have shown antibacterial and antituberculosis [8] activities as well as anticancer activitiy [9] and some have been tested for antioxidant activity. [10]Compounds with the bicyclo[3.2.1]skeleton are potent inhibitors of dopamine and serotonin transporters and they have crutial role in treatment of central nervous system (CNS) and Alzheimer's disorders. [11][14][15][16] Naphthoxazoles as a group of compounds have been known since Fisher synthesized 2methylnaphtho [1,2-d]oxazole and 2-methylnaphtho [2,1d]oxazoles in 1906. [17]In the following years many synthetic approaches were developed for the synthesis of these compounds. [18]The synthesis of naphthoxazoles [19] and fused hetero-benzoxazoles [19,20] (Figure 1) in one photocyclization step by utilising light as a very clean reagent gave a new and easy path to this kind of polycyclic systems that are taxing to obtain via ground state organic synthesis.Syntheses of compounds containing bicy-clo[3.2.l]octane and bicyclo[3.2.l]octadiene core (Figure 1) are also documented in the literature. [21]24][25][26][27][28][29][30][31][32][33][34] Guided by all above mentioned compounds, we have selected 32 compounds from both types of photoproducts and investigated their biological activity in proliferation and inflammation based assays.Effect of compounds on proliferation of several cancer cell lines was determined by measuring cell metabolic activity through time.On the other hand, lipopolysaccharide (LPS) stimulation of peripheral blood mononuclear cells (PBMC) was used to investigate antiinflammatory properties of the compounds.
Physico-chemical properties of tested compounds have been studied using chromatographic lipophilicity measure, chrom logD and logP was calculated.[40] In this paper we analysed also the physico-chemical properties of the tested compounds as possible reason for their biological activity.

Chrom logD Determination
Chrom logD values were determined from the following equation: chrom logD = 0.0857 × CHI -2 (ref. Chromatographic Hydrophobic Index (CHI) values have been determined from gradient retention times (tR) as described by Valkó and Slegel. [41,42]These values approximately correspond to the volume percentage of organic component in the mobile phase when the compound elutes.CHI values were determined at pH 7.4.Chromatograms were obtained using an Agilent 1100 Series HPLC instruments equipped with diode array detector (DAD) coupled with Micromass Quattro API mass spectrometer.Data acquisition and processing were performed with MassLynx software version 4.1.The column used for CHI determination was Luna C18 (50mm × 3 mm i.d., 5 µm particle size, 100 Å).The aqueous part of the mobile phase was 50mM ammonium acetate adjusted with ammonia solution to pH 7.4.As an organic part of their mobile phase acetonitrile was used.The mobile phase flow rate was 1 mL min-1 for all measurements.The gradient retention times tR were measured under the following gradient condition: 0 to 3 min linear gradient from 0 to 100 % acetonitrile; 3 to 3.5 min 100 % acetonitrile; 3.5 to 3.7 min from 100 to 0 % acetonitrile; and 3.7 to 5 min reequilibration time with 100 % of aqueous part of the mobile phase.

Biological Activity
Effect of compounds on cell proliferation was determined in cancer cell lines THP-1, Jurkat and HL-60.The assay is based on ability of metabolically active cells to convert MTS tetrazolium compound to formazan product detectable by absorbance at 490 nm.The quantity of formazan product is proportional to the number of metabolically active cells.In this assay staurosporine was used as reference compound since it is a protein kinases inhibitor which induces apoptosis.Furthermore, antiinflammatory activity of compounds was determined in LPS stimulated PBMC assay.LPS is one of the components of Gram-negative bacteria cell wall and is recognized by Toll-like receptor 4 (TLR4).The activation of TLR4 signaling induces release of cytokines among which are interleukin-8 (IL-8) and tumor necrosis factor α (TNFα), therefore leading to inflammatory response.In this assay, a widely used corticosteroid dexamethasone was used as reference compound because of it's antiinflammatory activity.

Cell Proliferation Assay
Effect of compounds on proliferation of THP-1, Jurkat and HL-60 cells was determined using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega).
96-well plates were seeded with 30 000 HL-60, THP-1 or Jurkat cells per well, followed by addition of three fold serial dilutions of test compounds using Mosquito instrument (TTP labtech).Staurosporine (Sigma) was used as reference compound.Final testing concentrations of test compounds were 30 µM to 13.72 nM, while of staurosporine was from 1 µM to 0.46 nM.Vehicle contained 0.3 % DMSO.Cells treated with compounds or vehicle were incubated for 24h, 48h and 72h in CO2 incubator (5 % CO2, 95 % humidity, 37 °C) after which CellTiter 96 Aqueous One Solution Cell Proliferation Assay substrate was added following manufacturer's instructions.After 4h of incubation with the substrate, absorbance at 490 nm was measured using EnVision 2104 instrument (Perkin Elmer).

LPS Stimulation Assay
Human peripheral blood mononuclear cells (PBMC) were isolated from healthy volunteer's blood obtained from the Croatian Institute for Transfusion Medicine.Blood was diluted 1:1 with PBS and PBMCs were isolated by centrifugation in Lymphoprep (Axis-Shield) density gradient.96-well plates were seeded with 200 000 PBMCs per well in RPMI 1640 (Lonza) medium supplemented with 10 % FBS (Sigma).Cells were incubated with three fold serial dilutions of test compounds for 1h in CO2 incubator (5 % CO2, 95 % humidity, 37 °C), followed by overnight stimulation with 1 ng/mL of LPS from E. coli 0111:B4 (Sigma) in CO2 incubator.Final testing concentrations of test compounds were 30 µM to 13.72 nM.Dexamethasone was used as reference compound and it was tested from 1 µM to 0.46 nM.0.3 % DMSO was used as vehicle.After overnight incubation, concentrations of TNFα and IL8 were determined in cell supernatants using 384-well format sandwich Elisa assay.Lumitrac 600 microplates (Greiner) were coated with 1 µg/mL of TNFα capture antibody (R&D Systems) or 2.5 µg/mL of IL8 capture antibody (R&D Systems).1% BSA (Sigma), 5 % sucrose (Kemika) in PBS (Gibco) was used as blocking buffer, while cell supernatants were diluted either 4x for TNFα analysis or 150x for IL8 analysis in 1 % BSA in PBS.Furthermore, 250 ng/mL of TNFα detection antibody (R&D Systems) or 25 ng/mL of IL8 detection antibody (R&D Systems) were used in Elisa assay, while Chemiluminiscence ELISA Substrate (Roche) was used for developing the signals.Luminescent signals were measured using EnVision 2104 Multilabel Reader (Perkin Elmer).

Data Analysis
Measured absorbance at 490 nm was used to calculate percentage of cell proliferation for each compound in comparison to vehicle cells using Micorsoft Excel software v2013.Concentrations of TNFα and IL8 were calculated by extrapolation from their standard curves using Micorsoft Excel software v2013.Obtained concentrations were used to calculate percentages of inhibition of each compound by first subtracting average value of vehicle samples from all other samples.Furthermore, percentage of inhibition was calculated using formula: (1 − (compound / average of LPS triggered controls)) × 100.
In both assays, logarithm of tested compound concentration in M was plotted against calculated percentage of inhibition using GraphPad Prism v7.04 software.

Biology
As it was mentioned before, 32 compounds were selected belonging to different types of heteropolycyclic photoproducts (Figures 2 and 3) and investigated their biological activity in proliferation and inflammation based assays (Figures 4-8).Amongst the tested compounds, naphtha-  Both TNFα and IL8 are mainly produced by macrophages, they promote inflammation and act as neutrophil chemoattractants, while TNFα is also one of the most notable proinflammatory factors in cancer.Furthermore, related compounds 18, 19 and 20 with similar structure to compounds 2, 5, 9 and 11 were also tested in   The effect of compounds on proliferation of several cancer cell lines (THP-1, Jurkat and HL-60) was also investigated.Tested concentrations of compounds did not exhibit any toxic or antiproliferative activity in these assays, during 72 h of incubation (Figures 4, 5 and 6).Some inhibi-   tion of proliferation up to 24 % was observed at the highest concentrations of compounds 9 and 11, however only in THP1 cells (Figure 6.).Overall, these findings further support that observed inhibition of TNFα expression is due to compounds activity, rather than the result of toxicity.

Reference Compounds
In the investigation of the biological activity of the tested compounds 1-32, staurosporine and corticosteroid dexamethasone were used as reference compounds (Figures 10 and 11).Their effects on proliferation of HL60 (A), Jurkat (B) and THP1 (C) cells after 72 h of incubation (Figure 10) and on IL8 (A) and TNFα (B) production in LPS stimulated PBMC were presented and compared to the effects of the investigated photoproducts.Also, IC50 values determined for reference compounds in cell proliferation assay (staurosporine) and LPS stimulation assay (dexamethasone) are presented in Table 1.

Physico-Chemical Properties
As an initial attempt for better understanding and explaning the biological activity of compounds 1-32, their physico-chemical properties were also investigated.Experimental lipophilicity, as determined by RP-HPLC was expressed by using chrom logD.These values were in the range 1.89-7.50at pH 7.4.Physico-chemical properties of investigated  Experimental values show that the majority of the investigated compounds are lipophilic (Table 2), which can be favorable in CNS terapeutic area.Correlation between calculated logP and experimental Chrom logD values qualitative agreement was observed between calculated logP and experimental Chrom logD especially for fused hetero-benzoxazoles 9 and 11 (Table 2), compounds which also inhibited TNFα protein expression in LPS stimulated PBMC (Figure 8), indicating possible antiinflammatory role interesting to further investigate.In case of bicyclo[3.2.1]core correlation between calculated logP and experimental Chrom logD values did not show good precision in predicting lipophilicity, since Chrom logD is expected to account for the ionizability of investigated compounds.High-throughput chromatographic determination of lipophilicity enables quick classification of novel compounds.From the group of benzobicyclic compounds 23-32, there are no examples with promising antiinflammatory or antiproliferative activity.These physico-chemical properties and results for biological activity are the reason why these compo-unds are also tested for cholinesterase inhibitory activity and antioxidant characteristics, the benzobicyclo[3.2.1]-derivatives showed better results as potential cholinesterase inhibitors and the oxazole derivatives having better antioxidant properties. [43,44]ven though calculated logD and logP values do not show great precision in predicting lipophilicity for analised compounds, it can be used for qualitative prioritization of virtual libraries (Figure 12).Most CNS drugs have logP values less than 5 so it is easily absorbed in blood brain barrier where active diffusion of drug is likely to have most lipophilic area like lipid bilayer of membrane and if the value is less than 0 it will move toward hydrophilic compartment like blood serum.For fused hetero-benzoxazoles 9 and 11 it is achieved best correlation between Chrom logD and logP, but values are low potential CNS drugs.Compound 29 possessing the benzobicyclo[3.2.1]-octadiene structure has potential to cross BBB using active diffusion.
As the importance of physico-chemical properties of compounds at early stage of discovery of new drugs is now well established, according to the results in this study further investigatons will be performed on the amino derivatives of both groups of compounds.As it was concluded, the similarities in structure and activity of some representative compounds affirm the need to further address their antiinflammatory properties.The starting compound for the synthesis of the amino substituted naphthoxazoles at the defined position will be the chloro derivative 20, and the chloro derivative 28 for the group of benzobicyclo[3.2.1]-octadienes.The choliesterase inhibitory activity of those new functional amines will be also investigated in the light of the obtained physico-shemical properties and concerning information about the biological targets and effects predicted by Pass (in silico).

CONCLUSION
Biological activity of naphthoxazoles 1-5 and 12-22, fused hetero-benzoxazoles 6-11 and benzobicyclo[3.2.1]derivatives 23-32 was investigated in proliferation and inflammation based assays.The tested compounds were prepared by photocylization or photocycloaddition reactions.Several naphthoxazoles and fused heterobenzoxazoles inhibited TNFα protein expression in LPS stimulated PBMC, indicating possible antiinflammatory role which would be interesting to further investigate.Physicochemical properties of tested compounds have been also studied using chromatographic lipophilicity measure, chrom logD and logP was calculated as the importance of physico-chemical properties of compounds at early stage of discovery of new drugs is well established.The similarities in structure and activity of some representative compounds affirm the need to further address their antiinflammatory properties.

Figure 1 .
Figure 1.General structures of the investigated compounds.

Scheme 1 .
Scheme 1.General scheme for the synthesis of the investigated naphthoxazoles and fused hetero-benzoxazoles.

Table 1 .
IC50 values determined for reference compounds in cell proliferation assay (staurosporine) and LPS stimulation assay (dexamethasone).N/A = compound not tested.

Table 2 .
Chrom logD values determined for some investigated compounds from the following equation: chrom logD = 0.0857 × CHI -2.Chromatographic Hydrophobic Index (CHI) values have been determined from gradient retention times (tR) at pH 7.4.